Biomass For Biofuels

BIOMASS FOR BIOFUELS
Biomass for Biofuels
Editors
Katarzyna Bukowska, Zygmunt Mariusz Gusiatin,

Ewa Klimiuk, Artur Pawowski & Tomasz Pokj
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Table of contents
Introduction 1
1 Biofuels and sustainable development 3

Ewa Klimiuk & Artur Pawowski
1 Introduction 3
1.1 Sustainable development 3
1.2 Strategies for sustainable development 3
2 Environmental aspects of biofuels production 5
2.1 Depletion of fossil fuel resources 5
2.2 Environment pollution 5
2.3 Changing the use of natural space and reducing biodiversity 8
3 Economic aspects of biofuels production 9
3.1 Cost effectiveness of biofuels production and energy balance 9
3.2 Energy security 10
3.3 Loss of government revenue 10
4 Social aspects of biofuels production 10
4.1 Rural development 10
4.2 Diversification of production 11
4.3 Risks associated with the production of biofuels 11
5 Prospects for the development of the biofuels market 12
2 Biomass for fuels classification and composition 15

Zygmunt Mariusz Gusiatin & Artur Pawowski
1 Definition and classification of biomass 15
1.1 Definition of biomass 15
1.2 Categories and types of biomass 16
2 Biomass characteristics 17
2.1 Criterion of expressing biomass composition 17
2.2 Biomass composition proximate analysis 20
2.3 Biomass composition ultimate analysis 21
2.4 Biochemical biomass composition 22
2.4.1 Characteristic of structural components in biomass 22
2.4.2 Lignin isolation from biomass and its characterization 28
3 Biomass feedstock for biofuels production 37

Katarzyna Bukowska & Artur Pawowski
1 Introduction 37
2 Biomass feedstock for the first and next generation biofuels production 38
3 Biomass feedstock for the second and third generation bioethanol production 41
3.1 Lignocellulosic biomass 41
3.1.1 Biomass from short-rotation forestry 41
3.1.2 Perennial herbaceous energy crops 44
3.1.3 Residues and waste 47
3.2 Algae biomass 49
4 Biomass feedstock for the second and third generation biodiesel production 49
4.1 Non-edible oil seed 49
V
VI Table of contents
4.2 Spent oil and animal fats 52

4.3 Algae biomass 52
4 Outlook for advanced biofuels 63

Katarzyna Bukowska, Ewa Klimiuk & Artur Pawowski
1 Introduction 63
2 Thermal processes 64
2.1 Biofuels from syngas 64
2.2 Pyrolysis 67
3 Microbial biofuels production 70
3.1 Metabolic pathways as criterion classification of advanced biofuels 70
3.2 Production of alcohols via fermentative pathways 70
3.3 Production of alcohols via non-fermentative pathways 75
3.4 Fatty acid-based biofuels 76
3.5 Isoprenoid-based biofuels 76
4 Olechemical processes 78
5 Hybrid processes 80
6 Properties and usage of advanced biofuels 81
6.1 Gasoline and alternative biofuels 81
6.2 Diesel and alternative biofuels 82
6.3 Jet fuel and alternative biofuels 84
5 Conversion of lignocellulosic biomass into sugars: the effect of the

structure of lignocellulose 95
Katarzyna Bukowska, Ewa Klimiuk, Tomasz Pokj & Artur Pawowski
1 Introduction 95
2 Recalcitrance nature of plant cell walls 96
3 Resistance of main components of lignocellulose 98
3.1 Cellulose 98
3.1.1 Structure of cellulose 98
3.1.2 Effect of crystallinity 100
3.1.3 Degree of cellulose polymerization 102
3.1.4 Accessible surface area 102
3.2 Hemicelluloses 103
3.2.1 Hemicelluloses as a barrier for accessibility of cellulose 103
3.2.2 Effect of acetyl groups 104
3.2.3 Stability of lignin-carbohydrate bonds 106
3.2.4 Stability of lignin-carbohydrate complexes 107
3.3 Lignin 109
3.3.1 Resistance of lignin to biodegradation 109
3.3.2 Lignin as a barrier for accessibility of cellulose 110
6 Pretreatment of lignocellulosic biomass 121

Katarzyna Bukowska & Ewa Klimiuk
1 Introduction 121
2 Mechanical method: milling 121
3 Chemical methods 122
3.1 Pretreatment with dilute acids 122
3.1.1 Operational condition of acid hydrolysis 122
3.1.2 Reactors 126
3.2 Pretreatment with alkaline 128
3.3 Organosolv fractination 131
Table of contents VII
3.4 Oxidative delignification 134

3.5 Ionic liquids 135
3.5.1 Pretreatment of biomass dissolution of cellulose 135
3.5.2 Pretreatment of biomass dissolution of lignin 138
3.5.3 Dissolution of biomass in ionic liquid 138
4 Physico-chemical methods 139
7 Fermentative and non-fermentative pathways of butanol and its analogues 155

Tomasz Pokj & Ewa Klimiuk
1 Introduction 155
2 Butanol production via fermentative pathway 156
2.1 Sugars and starch as substrates 156
2.2 Butanol production from lignocellulosic materials 159
2.2.1 Consolidated bioprocessing (CBP) 161
2.2.2 Inhibitory effect of hydrolysis by-products on clostridia 162
2.3 Engineering pathways to improve butanol production in solventogenic
clostridia 163
2.4 Escherichia coli as host for butanol/isopropanol production 168
2.4.1 Butanol 168
2.4.2 Isopropanol 170
3 Non-fermentative alcohol fuels 171
3.1 Production of higher-chain alcohols using the keto acid pathways 171
3.1.1 Propanol and butanol 171
3.1.2 Isobutanol 173
3.1.3 2-methyl-1-butanol and 3-methyl-1-butanol 173
About the Authors 183

Introduction
The increase in world population and economic growth are the main reasons for the growing demand
for energy. According to the forecast of the U.S. Energy Administration, during the following three
decades, world energy use will increase by 56%. One of the important sectors, with huge demand
for energy, is transport. Its share in global use of energy is about 27%. Due to decreasing resources
of fossil fuels and growing pressure on con-ducting proecological policy, covering this energy
demand will require the gradual replacement of conventional by alternative fuels, like biofuels
produced on the base of biomass.
Biomass is widely available resource, which can be characterized by high production potential,
enabling the production of different types of biofuels, which can be used in both spark-ignition
engine and compression-ignition engine. The knowledge on the subject of biofuels production
processes is extensive and still evolving. Scientists in different parts of the world are developing
technologies enabling the creation of biofuels with high calorific value and better physicochemical
properties. They are also taking into account the need to eliminate social problems arising from
the competiveness of the prices of resources used for the production of biofuels and food products,
since growing demand for biofuels could lead to rising food prices in local markets. As for now, the
biggest barrier in development of biofuels market is not lack of know-how, but economic factors,
connected with political decisions.
This book presents technological aspects of the biomass conversion into advanced biofuels.
We also discuss the influence of growing biofuels market on the natural environment and social
relations, as well as economic aspects of acquisition of biomass and its processing into biofuels.
Important part of this work is also presentation of biomass characteristics. We provide its definition,
chemical composition and properties. The focus is on lignocellulosic biomass, which complex
structure is a limiting factor for biofuels production via biological processes. Thats why we discuss
mechanical, chemical and physicochemical methods enabling the increase of its availability for the
microorganisms used for biomass conversion to biofuels.
1
Chapter 1
Biofuels and sustainable development
Ewa Klimiuk & Artur Pawowski
1 INTRODUCTION
1.1 Sustainable development

The concept of sustainable development was introduced into scientific discourse by the Our Com-
mon Future report, published in 1987 by the United Nations World Commission on Environment
and Development. The document was an attempt at a holistic view of current world problems. Sus-
tainable development was pointed to as the way to solve contemporary problems whereas caution
was advised towards the generally accepted narrow, economic only interpretation of the develop-
ment notion, as well as the equally narrow definition of the environment notion. It was defined
as development which meets the needs of the present without compromising the ability of future
generations to meet their own needs (WCED, 1987).
The need for integration of activities in three key areas was highlighted (Pawowski, 2011):
economic, the aim of which is to promote long-term economic growth promoting consumption
and production models, which protect human rights and the regenerative capacity of the Earth,
ecological, aimed at protecting natural resources and the natural environment. Its implementation
depends on developing economically viable solutions to reduce resource consumption, to stop
the degradation of the natural environment and to protect and restore biodiversity,
social, which implies safeguarding minimum needs, the maintenance of cultural wealth and
social justice.
1.2 Strategies for sustainable development

These areas are very clearly identified in the European Union documents, among whichThe renewed
EU Sustainable Development Strategy (EC, 2006) and Europe 2020: A European strategy for smart,
sustainable and inclusive growth (EC, 2010) play a key role.
The first strategy includes the following objectives:
to protect the natural environment and to preserve its potential (including the need to limit
climate change and to increase the scope of uses of clean energy),
to promote the principles of a democratic society with respect for cultural diversity,
to support a thriving economy, which will use environmental resources (including sustainable
production, consumption, and the issue of waste) in a rational manner,
to support sustainable development in both internal and external EU policies.
The Europe 2020 document extends this discussion and indicates three priorities:
smart growth: developing an economy based on knowledge and innovation,
sustainable growth: promoting a more resource efficient economy, which is more environmen-
tally friendly and more competitive,
socially inclusive growth: fostering a high-employment economy delivering social and territorial
cohesion.
3
4 Biomass for biofuels
Figure 1.1. The worlds perspective: installed electricity capacity by type (EIA, 2015).
Many of these general recommendations refer to the energy sector. Without energy our civilisa-
tion is unable to function properly, yet the impact on the environment through the use of individual
energy sources is diversified.
Currently, on a global scale, we have noted a growing interest in sources of renewable energy
as shown in the International Energy Agency (IEA) annual reports. In 2005 the individual energy
sources as a percentage of the total available capacity were as follows:
68.5% thermal power stations burning fossil fuels,
19.7% hydroelectric power stations,
9.65% nuclear power stations,
2.15% other non-water renewable energy sources.
This figures show that at that time, on a global scale, water was the only significant source of
renewable energy, the remaining renewable energy sources only played a marginal role.
However, over the next 10 years much had changed, Currently (IEA, 2015), the individual energy
sources as a percentage of the total available capacity are as follows (Figure 1.1):
64.97% thermal power stations burning fossil fuels,
20.2% hydroelectric power stations,
8.28% other non-water renewable energy sources,
6.73% nuclear power stations.
It turned out that contribution from conventional thermal power stations fell, including nuclear
(which fell to last place) in favour of renewable energy sources.
In the case of thermal power stations, a major role in the decline of their importance was played
by the energy policies of the UN and the EU, which on the one hand, assumed a reduction in the
development of this type of energy, due to its associated environmental pollution, and on the other,
encouraged development of energy production from renewable sources.
In the case of nuclear power stations, the Fukushima disaster of 2011 played a major role. It
turned out that even with advanced technology unpredictable accidents can occur. In Fukushima
the pressure pumps pumping water to cool the reactors failed, which led to a serious accident and
environmental pollution. Elevated levels of radioisotopes were found even in places far from Japan.
For example, in 2011, the average annual concentration of air-borne Caesium-137 in Europe was
nearly 8 times greater than in any other year during the period from 2001 to 2013 (GUS, 2015).
Among the renewable energy sources used in the thriving transport sector, biofuels are playing
an increasing role.
Referring to the above-mentioned three pillars of sustainable development, the following mea-
surable environmental benefits should be highlighted: (e.g. reduced air pollution, reduced fossil
fuels consumption), social benefits (e.g. cultivation start-ups of energy crops and their positive
Biofuels and sustainable development 5
impact on the labour market), and economic benefits (as exemplified by the Brazilian market, and
most recently by the US market).
Furthermore, the use of biofuels in the promotion of the use of energy from renewable sources
is in accordance with Directive 2009/28/EC dated 23rd April 2009. It defines the energy policy
principles for countries within the EU up to 2020. It sets mandatory targets for Member States to
make energy renewable sources account for 20% of EU energy, and a minimum 10% for biofuels
specifically in transport by 2020. At the same time, it does not dictate how to achieve those targets,
but requires individual countries be guided by sustainability criteria during their production and
use (Art. 17, par. 18).
Currently, work is being carried out on a new directive, with a target date even further in the
future. According to a European Commission proposal, by 2030 the reduction in greenhouse gas
emissions should reach 40%, energy efficiency should rise to 40%, and the share of energy from
renewable sources should exceed 30%.
We will now examine in more detail the conditions associated with biofuels in the context of the
key areas of sustainable development.
2 ENVIRONMENTAL ASPECTS OF BIOFUELS PRODUCTION
2.1 Depletion of fossil fuel resources

On average, every inhabitant of our planet daily consumes 16 kg of raw materials, and in the rich
Western world, this figure is much higher amounting to as much as 57 kg per citizen (WC, 2016).
Given that the Earths population is 7.4 billion this amounts to an unimaginable total of 110,400,000
tonnes of materials used on a daily basis. Such a rapid consumption of raw materials means that
many of them are now close to exhaustion. Fossil fuels occupy a special place among these raw
materials. The increased interest in biofuels is mainly due from the need to gradually reduce the
consumption of this type of fuel.
Sources of fossil fuels include: deposits currently being exploited, deposits currently not being
exploited for economic or technical reasons, and undiscovered resources.
Depletion of resources is an undisputed fact. The open question is only of time, how long before
it occurs.
If we assume that fossil fuels extraction will remain at the early 1990s level, then the documented
raw material reserves should be exhausted as follows: oil within 40 years, natural gas 60 years,
coal 197 years, and lignite 293 years.
However, when trying to come up with a reliable estimate, the problem lies in the incomplete
recognition of energy resources. In fact, not only are we consuming them all the time, but simul-
taneously we are discovering new deposits. For example, in 2011, oil deposits were discovered in
three different regions of the world (Brazil, Mexico and Norway) and estimated at approximately
7 billion barrels (Pawowski, 2011).
According to rough estimates, the worlds natural resources, together with those still to be
discovered are: coal more than 740 billion tonnes, oil approximately 320 billion tonnes, natural
gas approximately 330 billion m3 , which at the current rate of consumption should be enough to
last as follows: oil approximately 125 years, natural gas approximately 210 years, and coal
approximately 360 years (WEC data, 2010).
Although these resources appear to be significant, many of todays initiatives are aimed towards
reducing the level of their exploitation. The main reason is that the burning of fossil fuels inevitably
leads to rising pollution levels.
2.2 Environment pollution

Among the many environmental pollutants introduced into the atmosphere from the burning of
fossil fuels, a lot of attention is currently being paid particularly to the problem of greenhouse
gases (GHGs) and the greenhouse effect.
The greenhouse effect is a natural process by which the temperature of the planet is raised by
the presence of GHGs in the atmosphere. It should be regarded positively, because it increases the
Earths surface temperature by 2034 C, so that our planets average temperature is approximately
14 C. In the absence of the greenhouse effect the average temperature would be minus 19 C
(Le Treut et al., 2007).
The problem is an excessive increase in GHG emissions caused by human activity, which is
now considered to be the cause of the so-called global warming. However, it should be noted that
there are also other causes of global warming, among which a specific role is played by increased
tropical deforestation which is destabilising the climate.
GHGs are gaseous components which absorb and emit infrared radiation with a wavelength of
between 780 nm and 1 mm. GHGs include among others: carbon dioxide (CO2 ), methane (CH4 ),
carbon monoxide (CO), nitrous oxide (N2 O), ozone (O3 ), chlorinated aliphatic hydrocarbons and
hydrofluorocarbons (CFCs, HFCs), perfluorocarbons (PFCs), sulphur hexafluoride (SF6 ) and water
vapour.
The main stream of anthropogenic GHG emissions is produced by the combustion of fossil
fuels (gas, liquid and solid). On a global scale, the energy sectors share of total CO2 emissions is
approximately 69%, which means an emission of 32 GtCO2 /year (IPCC, 2014).
The transport sectors share of total GHG emissions is approximately 14% which is approximately
7 GtCO2 /year (IPCC, 2014).
Carbon dioxide emissions in road transport, represent approximately 80% of this gas in the entire
transport sector. Other GHGs are N2 O and CH4 , produced during the burning of fuels, and HFCs
escaping from vehicle air conditioning units. N2 O emission is 2.02.8% of total GHG emissions,
while that of CH4 ranges from 0.1 to 0.3%. In 2003 CFC emissions were 0.30.6 GtCO2 eq which
is equivalent to 510% of total CO2 emissions from the transport sector (Ribeiro et al., 2007).
One of the main objectives of using biofuels is to reduce CO2 emissions. Directive 2009/28/EC
dated 23rd April 2003 of the European Parliament and of the Council recommended that, in places
where they had been introduced, biofuels demonstrate at least 35% lower emissions when compared
to other fuels.
Figure 1.2 shows the GHG balance for different raw materials and biofuels technologies. From
the data it can be seen that bioethanol produced from maize (Koonin, 2006) is least favourable.
In transport, the use of bioethanol produced from wheat can reduce gas emissions by between 19
to 47%, from sugar beet by between 35 to 53% (IEA, 2004), and sugar cane by approximately
92% (Macedo et al., 2004). The emission balance for bioethanol produced from lignocellulosic
raw materials reduces CO2 emissions between 50 to 100% (IEA, 2004).
In the global carbon cycle, the amount of CO2 emitted during the production and combustion of
biofuels should be less than the amount of carbon assimilated by the biomass and retained in the
soil. A positive CO2 balance is achieved when wasteland is used to grow the energy crops. As a
result of state biofuels subsidies, the following adverse practices have become common: conversion
of forests and grasslands into arable land in temperate climate zones, and allocation of arable areas
where tropical forests were felled for this purpose in the tropics. Some authors even believe that
achieving the planned utilisation level of biofuels in the transport sector by 2020 in the EU countries
will result in additional net emissions of GHGs (Crutton et al., 2008; Bowyer, 2011).
The time taken to compensate for the amount of CO2 released from the soil by increasing the
acreage of sugar cane (for the production of bioethanol) at the expense of the Cerrado (land occupied
by the Brazilian tropical savannah ecoregion) is estimated to be 17 years (Fargione et al., 2008).
The conversion of grasslands into maize fields in Central USA extends this compensation period to
93 years, while the conversion of tropical forests into palm oil plantations for biodiesel production
in Indonesia and Malaysia can make the compensation period as long as 420 years. The solution
is to use degraded land for cultivation. For this purpose, a suitable plant is Jatropha, which, due to
its ability to store water, helps to improve soil quality and prevents further degradation.
However, the energy sector is not only responsible for emissions of CO2 and other greenhouse
gases. During the combustion of petroleum products many other pollutants are also produced such
Figure 1.2. Reduction of GHG emissions from biofuels compared to fossil fuels (based on data from US
EPA, 2002; quoted by Dufey, 2006); positive values indicate a decrease in emissions, negative values indicate
an increase in emissions.
as particulates (soot), monocyclic aromatic hydrocarbons (e.g. benzene) or polycyclic aromatic

hydrocarbons (e.g. benzopyrene), aliphatic hydrocarbons, aldehydes, nitric oxide and nitrogen
dioxide, benzopyrene, sulphur dioxide, platinum (used as a catalyst), and dioxins. Moreover,
during the combustion of coal, radioactive substances such as uranium and thorium are released
into the atmosphere (luckily in small quantities) (Gabbard, 1993).
The combustion of biofuels produces lower emissions of most pollutants in comparison to the
combustion of fuels derived from crude oil (Table 1.1). For example, the combustion of bioethanol
(E85) significantly reduces emissions of sulphur compounds by 80% and carbon monoxide by
40%. Replacing diesel with biodiesel (B100) reduces emissions of particulate matter by 70%
and carbon monoxide by 50%, as well as hydrocarbons, including carcinogenic benzopyrene by
approximately 70%.
The negative effect of burning biodiesel is an increase in the emission of nitrogen oxides by up
to 9% compared to diesel, while burning bioethanol increases emissions of acetaldehyde.
The data cited in Table 1.1 shows, that even partially replacing oil products with biofuels leads
to less pollution.
The benefits are noticeable, particularly in large conurbations, where the concentration of exhaust
gases is at its greatest. Reducing emissions from transport impacts on the environment by improving
Table 1.1. Comparison of emissions from toxic substances between selected biofuels and standard transport
fuels (based on data from US EPA, 2002, quoted by Dufey, 2006).
Bioethanol Biodiesel Biodiesel Fischer-Tropsch

E85 B20 B100 Fuel
15% reduction in 10% reduction in carbon 50% reduction in A reduction in nitrogen

emissions of volatile monoxide emissions; carbon monoxide oxide emissions due to
organic compounds 15% reduction in emissions; an increase in the
which create an particulate emissions; 70% reduction in cetane number. Greater
ozone hole; 10% reduction in particulate reduction is possible
40% reduction in carbon hydrocarbon emissions; emissions; with the addition of a
monoxide emissions; 20% reduction in 40% reduction in catalyst;
20% reduction in emissions of sulphur hydrocarbon Low or no emissions of
particulate emissions; compounds; emissions; particulate matter due to
10% reduction in nitric 2% increase in emissions 9% increase in the low content of sulphur
oxide emissions; of nitrogen oxides; emissions of and aromatic compounds;
80% reduction in No effect on methane nitrogen oxides; A reduction in hydrocarbon
emissions of sulphur emissions. No effect on and carbon monoxide
compounds; methane emissions. emissions is expected.
Less emissions of
reactive hydrocarbon;
Greater emissions of
ethanol and
acetaldehyde.
the quality of life for communities and by reducing the risk of incidence of lifestyle diseases caused
by air pollution.
2.3 Changing the use of natural space and reducing biodiversity

The use of biomass and biofuels means putting large areas under special cultivation. As already
mentioned, it does not carry negative consequences when cultivation takes place on wasteland, or
low quality soils.
The problem arises when cultivating using high quality soils and in environmentally valuable
areas.
In the first case, this means displacing traditional crops, which can increase food prices, with
significant social consequences. We shall return to this later.
In the second case, the increase in biofuels production becomes one of the reasons for the disap-
pearance of natural biocoenoses, due to an increase in the amount of land containing monocultures
at the expense of forests and grasslands. It is estimated that in Brazil alone, achieving planned bio-
fuels production targets for 2030 will require increasing sugar cane acreage by 3 million hectares,
an increase of approximately 52% compared to today (Costa, 2006). To meet the demand for bio-
fuels in the 23 EU Member States by 2020, an additional 4.1 million to 6.9 million hectares should
be earmarked for this purpose (Bowyer, 2011). Increasing soya bean acreage in the Cerrado (the
tropical savannah ecoregion of Brazil) and in areas of palm oil cultivation in the tropical forests of
Malaysia and Indonesia is particularly risky.
The ecological consequence of cultivating monocultures over large areas is to reduce species
diversity. Currently, most of the plants allocated for biofuels production are cultivated in tropical
and subtropical climates, where the best conditions for photosynthesis exist and biomass yield
is maximum. Naturally, these areas are a multi-storey tropical rainforest or savanna, with a rich
abundance of flora and fauna. Their transformation into farmland entails an irretrievable loss
of biodiversity and the extinction of many rare species of animals. For example, between 1990
and 2005, 24.1% of the forests were cut down in Indonesia, and currently, approximately 6 m
hectares of forest are cut down annually which threatens orangutans, the Sumatran tiger, and the
Asiatic elephant among others, with extinction. Since forests stabilise the climate, such massive
indiscriminate deforestation contributes to the emergence of increasingly frequent and increasingly
dangerous weather extremes and climate anomalies.
Growing demand for biofuels on a global scale cannot be a factor when encouraging land use of
natural areas of high biodiversity for monoculture cultivation. These natural areas include primary
forests, some grasslands in the temperate and tropical climates such as savannas, steppes, scrubland
and prairies. For these reasons, the European Union does not permit the introduction of incentives
for biofuels production from plants grown in such areas.
3 ECONOMIC ASPECTS OF BIOFUELS PRODUCTION
3.1 Cost effectiveness of biofuels production and energy balance

The energy derived from biomass is a commodity, which allows biofuels to be evaluated in the
economic dimension. Cost effectiveness is the basic prerequisite for the development of this type of
technology. However, until now, biofuels production costs have been higher, even double, compared
to petroleum products production costs.
The biofuels price includes raw material costs, processing costs and product distribution costs.
The basic criterion for the suitability of the raw material is biofuel yield per hectare. Maize, with
a yield of 2,800 dm3 ethanol/ha is considered to be a more expensive raw material in comparison
with sugar cane, with a yield of 4,000 dm3 ethanol/ha. The use of complex and energy-intensive
technologies increases production costs. Cost effectiveness is also determined by the local price
of oil. In 2006, on the European markets, bioethanol was competitively priced when the price of
oil reached USD 70 per barrel. During this time on the US market the price fluctuated between
USD 5060 per barrel, while in Brazil it oscillated around USD 2530 (Dufey, 2006, 2010). The
higher the price of oil, the greater the competitiveness of biofuels. On the other hand, increasing
the share of biofuels in transport, results in lower oil prices. The question of cost differences can be
resolved on different levels: political (e.g. lower taxes), economic (CO2 emissions market, cheaper
raw materials) and technical (development of more efficient technologies).
It is not coincidental that the United States and Brazil are mentioned in the above commentary.
In terms of bioethanol production, these two countries have monopolised almost the total market
with a combined share of almost 85% of world production, of which 60% is attributable to the
United States. The European Union is in third place, but its share is only a mere 6% (RFA, 2015).
An important element in assessing the cost effectiveness of biofuels is energy balance. The net
energy balance is positive if the energy yield upon combustion is greater than the energy required
to produce one unit of biofuel. Discussions on whether the energy balance of biofuels is more
advantageous than that of conventional fuels have persisted since the 1970s. In terms of energy, it
is most efficient to produce bioethanol from Brazilian sugar cane, for which the energy balance
ranges between 3.710.2 Langevin, 2005). This is due to the high-yield sugar cane in the Brazilian
climate zone and the low power consumption of ethanol production (energy from the recovery of
waste products completely covers the energy requirements for this process) (IEA, 2004; Dufey,
2010). In the US, the energy balance of bioethanol produced from maize is 2, which is associated
with higher raw material preparation and production costs. In the EU, the energy balance for
bioethanol produced from wheat is 0.811.03, while from sugar beet it is 0.560.65. The energy
balance of biodiesel has a wide range: 0.330.82 (rapeseed), 1.23.2 (soya bean), 4.6 (Jatropha),
and 5.63 (coconut palm).
Analytical results indicate that biofuels production is cost effective for sugar cane, sugar sorghum,
palm oil, castor oil and Jatropha, but requires a warm and humid tropical climate for cultivation.
However, the economic policy adopted in many industrialised countries permits subsidies for plants
that are not as energy efficient, which in turn results in biofuels funding.
An additional issue is the cost of transporting (and the associated energy expenditure) the crops
from where they are grown to where the biofuels are produced. For it to be economic, local resources
should be used, thus minimising transportation costs.
3.2 Energy security

An important economic aspect, but which is also a political element in the use of biofuels, is
associated with a countrys improved energy security, as a result of its diversification of energy
sources, i.e. differentiation of raw materials used to produce it. Energy security is dependent on a
continuous supply of fuels and energy.
Currently, energy production is mainly based on fossil raw materials, unevenly dispersed across
the globe. This results in countries, whose economy is based on the import of energy resources,
to be largely dependent on the conditions dictated by the exporters, such as Russia, the USA,
and the OPEC (Organization of Petroleum Exporting Countries) Member countries. The scale of
the problem is evidenced by the high share of energy imports in the economies of the OECD
(Organisation for Economic Cooperation and Development) Member countries. For instance, in
the year 2000, oil imports covered 52% of their energy needs. It is estimated that by 2020 this may
reach 76% (Dufey, 2006).
Countries dependent on foreign supplies of oil must therefore devote a large part of their foreign
exchange reserves to oil imports, which slows down the development of other, underfunded sectors
of the economy. Therefore, distribution of biofuels can improve market balance, and contribute
to the preservation of financial resources and their allocation for other purposes. According to
Langevin (2005), Brazil saved USD 43.5 billion between 1976 and 2000 by replacing petrol with
bioethanol.
Increased biofuels production allows many countries to become partially independent of the
energy supply giants, who are unstable politically and economically. Given the high oil prices,
the uncertain assessment of oil reserves, and the growing demand for energy in the industrialised
countries, including the strongly growing economies of China and India, it is expected that energy
security will become a priority issue in the politics of many countries (Dufey, 2006).
3.3 Loss of government revenue

The adopted policies supporting the biofuels market are not based on economic factors. For this
reason, they are the cause of additional costs incurred by the state when promoting biofuels intro-
duction into the marketplace. Exempting biofuels from excise duties results in reduced budget
revenues from the fuel sector. It was calculated that in the UK, by increasing the biofuels share of
the fuel market by 1%, additional costs will be GBP 90 million annually (IEA, 2004).
In the economic aspect, improved energy security is gained by increasing production and utilisa-
tion of biofuels. This problem is particularly important since the demand for energy carriers in the
transport sector is high and is increasing. In the current economic climate and level of technology,
economic evaluation based on profit and loss accounting is not encouraging. For this reason, new
and more efficient methods for biofuels production are being developed.
4 SOCIAL ASPECTS OF BIOFUELS PRODUCTION
4.1 Rural development

The issue of sustainability also has a social dimension in which two basic types of social envi-
ronments, urban and rural, have been identified. It is believed that biofuels distribution and
consumption will become dominant in cities, while rural areas will become a place of biofuels
production. The increase in demand for agricultural commodities will increase employment in
agriculture and in biofuels production, thereby raising prosperity in rural communities. Biofuels
production provides an opportunity for small and medium sized farms, as well as small businesses
to expand. In Brazil alone, approximately 1 million people are employed in bioethanol production
(Moreira, 2005), which is more than in the production of petroleum products. Most of the workers
are uneducated, with little employment opportunities. In China, the program for the development
of the biofuels market aims to create more than 9 million jobs (Bhojvaid, 2006). It is envisaged
that it will raise living conditions in local communities, while at the macro level, increase profits
from exports.
Supporters of biofuels argue that on Earth, there is sufficient natural land of lower quality to
grow bioenergy crops rather than use natural tropical forests or savannahs. In addition, it is accepted
that biofuels will not replace petroleum products but only complement them. Attaining a 2030%
biofuels share of the transport sector can therefore be realised in a way which is environmentally
safe and which does not stir up social tensions.
4.2 Diversification of production

It is envisaged that promoting biofuels production will create new markets for agricultural prod-
ucts. This may have a positive impact on the economic development of those countries in which
biofuels, or raw materials for their production, are produced. Increased market competitiveness
of commodities such as soya beans, cassava, and rice will not only limit their price volatility, but
will also cause a reduction of surplus goods. Profits from the export of these raw materials could
substantially strengthen a developing countrys budget and help fight poverty around the world.
It is estimated, for example, that bioethanol production from sugar cane will increase Colombias
gross domestic product by 3% (Fuel Ethanol Program in Columbia).
4.3 Risks associated with the production of biofuels

Increased demand for biofuels could lead to rising food prices in local markets. The World Banks
analysis published in 2008 shows that from 2005 there has been a sharp rise in food prices despite
high yields worldwide. In the period 20052008, the price of maize increased almost three-fold,
the price of wheat increased by 127%, while that of rice by 170%. It is believed that the cause of
these changes in 7075% of cases is increased biofuels production (Mitchell, 2008).
The demand for food will increase for two reasons: an increase in the human population and
an increase in the number of people who are being better fed. Today, 25,000 people die of hunger
and about 780 million people in developing and 27 million in developed countries suffer from
malnutrition each day. In this situation, allocating huge areas for biomass fuel cultivation raises
moral concerns (Pimentel, 2012). This particularly applies to liquid biofuels used in transport.
To make biofuels viable, European governments are propping up powerful industry and farming
lobbies with huge sums of money. For example, by 2020 biofuels will annually cost each person in
Great Britain about GBP 35 (GBP 12 billion total) and in Germany about EUR 30 (EUR 1.42.2
billion total). The EU governments pay it directly to big business without the citizens knowledge
(Oxfam Briefing Paper, 2012).
Also, due to subsidies, in the United States ethanol production as a fuel additive rapidly expanded.
In the USA, ethanol is produced mainly from corn. Allocating such a large amount of corn for
ethanol production in the period 20072012 resulted in a twofold rise in the price of corn. Large
imports of food crops for biofuels by the European Union countries led to a dramatic two-and-a-half
fold increase in the FAO food index in the period 20072010 (FAO, 2013; Elbheri, 2013).
Future price increases will depend on the volume and share of second generation biofuels,
produced from raw materials which are not in competition with food products.
But this is not the only threat.
For example, fully mechanised soya bean production increases price competitiveness, making
its cultivation less profitable for small producers, while at the same time supporting the largest
players in the market, including multinational corporations.
There are also legitimate concerns that the pursuit of larger and quicker profits may result in, or
exacerbate the existing practice of using cheap labour in developing countries. This occurs on most
sugar cane and palm oil plantations, on which poor working conditions, lack of health and safety,
and the risk of escalating conflicts about land ownership rights have repeatedly been observed. The
expansion of agricultural land for energy crop production and unregulated property rights become
the reasons for migration and social problems in many countries like Indonesia (Dufey, 2006).
It can therefore be said that in terms of social development, the biofuels market carries both
benefits and risks. Currently, it appears that those rural communities, which take on the role of
producers of raw materials should benefit from it.
5 PROSPECTS FOR THE DEVELOPMENT OF THE BIOFUELS MARKET
The accurate determination of whether, and to what extent, biofuels fulfil the requirements of
sustainable development is not a simple matter.
Biofuels should ensure energy independence and facilitate lower emissions of harmful com-
pounds, including greenhouse gases. The primary tool for assessing biofuels in terms of the
environment and economy is the Well-to-Wheel Life Cycle Assessment (WTWLCA), developed
by the European Commissions Joint Research Centre. It takes into account three elements: green-
house gas emissions, the energy obtained, and production costs. Gas emissions and energy gain
are evaluated for all the stages of the production cycle, which consists of crop cultivation, biofuel
production, transport, distribution, and combustion.
WTWLCA does not take into account social costs. Currently, the prevailing view is that, for local
communities, the production of biofuels in decentralised systems is most beneficial. This allows
local energy sources to be used, shorter transportation of raw materials and increased security of
supply. By creating decentralised systems, the development and cohesion of local communities is
supported, by providing them with additional sources of income and jobs.
The assessment is made more difficult since the biofuels market is still in the early stages of
development. It is envisaged that the next few years should bring breakthroughs in this area due to
technology improvements, the use of raw materials not in competition with food, and the creation
of integrated systems which will process by-products and waste into energy.
Worldwide, intensive research and developmental work is underway on the production of second
generation biofuels that use crops grown solely for energy purposes (e.g. Jatropha) and waste (e,g.
straw, wood chips). Thermochemical processing of lignocellulosic raw materials is already well
mastered. However, high production costs are still a barrier, despite the higher energy content of
biofuels.
According to forecasts in the Biofuels in the European Vision. A vision for 2030 and Beyond
report, development of second generation biofuels technology based on lignocellulosic raw materi-
als (wood biomass) by 2020 is predicted. Total displacement of first generation biofuels shouldnt
occur until after 2050, while from 2020 there will be a gradual increase in the use of biohydrogen
in fuel cells (Londo et al., 2007). In the longer term development of bio-refineries, analogous to
the current refinery, is predicted, which will use integrated processing of various types of biomass
for biofuels and bio-based products. The brightest future is predicted for biodiesel whose market
will be focussed on Europe (67% of world production).
Perspective directions of development of biofuel technologies in Europe were drawn up by the
European Strategic Research Agenda (ESRA) on 31st January 2008 during a members meeting of
the European Biofuels Technology Platform (EBTP). It foresees two paths for converting biomass
into biofuels:
(i) gasification or pyrolysis of biomass by a thermochemical route followed by conversion into
transportation fuels (synthetic hydrocarbons, alcohols, biodiesel) (Biomass to Liquid (BtL)
technology),
(ii) biological or thermochemical processes capable of producing hydrogen.
A secure future for the biofuels market thus appears to be assured.
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Chapter 2
Biomass for fuels classification and composition
Zygmunt Mariusz Gusiatin & Artur Pawowski
1 DEFINITION AND CLASSIFICATION OF BIOMASS
1.1 Definition of biomass

The term biomass can be applied to materials derived from both animals and vegetables. For biofu-
els, biomass usually relates to plant based material. The United Nations Framework Convention on
Climate Change (UNFCCC) defined biomass as non-fossilized and biodegradable organic mate-
rial originating from plants, animals and microorganisms (UNFCCCa ). Biomass is recognized as
natural and renewable resources. The term natural resources means materials and components
occurring in nature that can be used for economic production or consumption. The term renewable
natural resources stands for natural resources which, after exploitation, can return to their previ-
ous stock levels by natural processes of growth or replenishment in the same or lower amount of
time (OECD, 2008). Some renewable resources, such as solar energy, wind energy and geothermal
pressure have essentially and endless supply, while others, such as plants or animals are considered
renewable even though some time or effort must go into their renewal. As the energy comes from
the sun, biomass is a renewable energy source and can regrow over a relatively short period of time.
According to the UNFCCCb , biomass must meet certain criteria to be classified as renewable. In
the last decade, biomass has shown signs of revival because it is considered as renewable, storable,
substitutive, abundant and carbon neutral material (Yokoyama, 2008).
Plants absorb solar energy through the process of photosynthesis, which enables them to turn
CO2 into glucose with a release of O2 . The carbon used to produce biomass is absorbed from
the atmosphere as carbon dioxide (CO2 ) by a plant, using energy from the sun. The carbon from
plants may be transferred through the food chain to animals bodies and their waste, then to the
human using the plants as food. If plant material is not consumed, it is generally broken down by
microorganisms. During biodegradation, carbon returns to the atmosphere, mainly as either carbon
dioxide (CO2 ) in aerobic conditions or methane (CH4 ) in anaerobic conditions (Basu, 2010).
Biomass is the oldest source of fuel energy. Up to 19th century, biomass in the form of firewood
and charcoal was the main source of energy, then it was replaced by coal and oil (Yokoyama, 2008).
However, using biomass for the production of transport fuels is a relatively new application, which
increased significantly within the last 10 to 15 years. Biofuels can be a substitute for fossil derived
transport fuels, with the advantage of providing carbon from a renewable source.
The legislative definition of biomass has changed over time. The definition for biomass stated in
legislation determines what sources of material are deemed eligible as biomass and which lands are
eligible for biomass removal for inclusion in the Renewable Fuel Standard (RFS) and for treatment
in the tax code (Bracmort, 2012). The biomass definition in legislation influences the decisions
pertaining to the types of crops grown, site of their growing, and their potentially preferred energy
uses (Bracmort, 2012). In some countries the term biomass is used for any plant-derived organic
matter available on a renewable basis, including dedicated energy crops and trees, agricultural
food and feed crops, agricultural crop waste and residues, wood waste and residues, aquatic plants,
animal waste and municipal waste. In other countries, the term biomass is defined more strictly and
15
pertains only to the fuels arising from agricultural and forestry sources, using a separate category,
waste fuels, for the waste products of human, urban and industrial processes (Spliethoff, 2010).
In the EU, the Renewable Energy Directive (2009/28/EC) defines biomass as the biodegradable
fraction of products, waste and residues of biological origin from agriculture (including vegetal
and animal substances), forestry and related industries, including fisheries and aquaculture. It also
includes the biodegradable fraction of industrial and municipal waste. By 2020, 20% of all energy
used in the EU has to come from renewable sources, including biomass, bioliquids and biogas
(Kerolli-Mustafa et al., 2015).
In the United States biomass is termed as biomass resource and renewable biomass (Klass,
2004; Milbrandt et al., 2013; Santos & Falberg, 2015). A biomass resource is (i) organic matter
derived from a plant and available on a renewable basis (e.g. dedicated energy crops) and (ii)
organic waste from harvesting or agriculture (e.g. animal waste, wood waste, sewage). The term
renewable biomass is very broad, divided into 8 categories: plant material from agricultural land,
plant material from pasture land, non-hazardous vegetative matter from waste, animal waste and
byproducts, algae and vegetative matter from evacuation by a public official, residues or byproducts
of milled logs, residues from forested land (Basu, 2010). In Japan, biomass was for the first time
recognized as a new energy source in 2002 (Amano & Sedjo, 2003; Yokoyama, 2008). Formerly,
biomass had been merely considered as a kind of renewable resource, but the amended law now
sees it as an independent category of new energy. However, some waste, such as paper waste, food
waste, demolition waste, and black liquor, are considered to be recyclable resources and they are
not strictly classified. In Norway, biomass involves firewood, black liquor, bark and other forms of
wood waste, and municipal waste from households and industry used in the production of district
heating (Chen, 2004; Trmborg et al., 2008).
1.2 Categories and types of biomass

Generally, there are two ways to categorize biomass: biologically, on the basis of existing biomass
in nature (according to ecology stratification or the type vegetation) and secondly, with regard
to the use or application of resources. Territorial division into different agricultural production
zones is made on the basis of various ecological conditions, such as soil, water, and climate. Agro-
ecological stratification creates a basis for effective agro-resources utilization, while strengthening
full potential of individual regions aims to properly select agro-forestry land use type. The vegetation
classification is based on a combination of the following criteria: climate pattern, phenology and/or
growth form, and dominant species.
Categorization according to the source is important for designing biomass usage systems. The
sources of biomass are usually: agriculture (crops), forestry (wood), and waste (municipal, agri-
cultural, forestry and industrial). A wide range of sustainable feedstocks are potentially available
for the production of biofuels.
Basu (2010) distinguished two major groups of biomass: virgin (terrestrial and aquatic, e.g.
forest biomass, grasses, energy crops, algae etc.) and waste (municipal, agricultural solid waste,
forestry and industrial). Yokoyama (2008) gives biomass categorization in terms of its use and
application as:
conventional biomass resource: agriculture, forestry (woody), fishery, livestock farming, food
materials, pulp, chips etc.;
biomass waste (derivatives): agricultural, forestry, livestock residues, sawdust, sewage sludge,
black liquor;
plantation biomass: forestry (eucalyptus, poplar, willow, oil palm), herbaceous (sugarcane,
switchgrass, sorghum etc. aquatic (giant kelp, water hyacinth etc.).
Kishore (2008) divided biomass on the basis of the source, i.e. of plant- and animal-origin. Plant-
derived biomass includes forestry, energy plantations, agricultural residues, aquatic and marine.
Animal-derived biomass is represented by different waste, including municipal and industrial.
Sewage sludge was categorized as biomass of plant and animal origin.
Biomass for fuels classification and composition 17
According to Vassilev et al. (2010), biomass can be divided into several groups and sub-groups
based on their distinct biological diversity and similar source and origin:
1. Wood and woody biomass: coniferous or deciduous; angiospermous or gymnospermous; soft
or hard; stems, branches, foliage, bark, chips, lumps, pellets, briquettes, sawdust, sawmill and
other, from various wood species.
2. Herbaceous and agricultural biomass: annual or perennial and field-based or processed-based
such as grasses and flowers (e.g. alfalfa, arundo, bamboo), straws (e.g. barley, bean, flax, corn),
other residues (e.g. fruits, stalks, cobs).
3. Aquatic biomass: marine or freshwater algae; macroalgae or microalgae; seaweed, water
hyacinth etc.
4. Animal and human biomass waste: bones, meat-bone meal, chicken litter, various manures.
5. Contaminated biomass and industrial biomass waste: municipal solid waste, demolition wood,
refuse-derived fuel, sewage sludge, hospital waste, paper-pulp sludge, waste papers, wood
pellets etc.
6. Biomass mixtures: blends from the above-mentioned varieties.
Alonso et al. (2010) distinguished three types of biomass on the basis of biomass chemistry:
(i) triglycerides feedstock (TFG) (vegetable oils, animal fats, waste cooking oils and microalgal
oils), (ii) sugar and starchy feedstock (SSF), including sucrose containing biomass (e.g. sugar beet,
sweet sorghum, sugar cane etc.), starchy biomass (e.g. wheat, corn, barley, maize etc.) and (iii)
lignocellulosic feedstock (LCF) (e.g. wood, straw, grasses etc.).
Demirbas (2009) distinguished 9 major categories of biomass feedstock. These include: (1)
forest products (wood, sawdust, bark), (2) biorenewable waste (crop residues, mill and urban wood
waste), (3) energy crops (grass, starch and sugar crops), (4) aquatic plants (water hyacinth, water
weed), (5) food crops (grains, oil crops), (6) sugar crops (sugar cane, sugar beets), (7) landfill, (8)
industrial organic waste and (9) others (algae, kelps, lichens and mosses).
In the EU, biomass is divided into three categories depending on its source: (i) biomass from
agriculture, (ii) biomass from forestry and (iii) biomass from waste, followed by biomass categories
and types with general and specific definitions (Table 2.1).
Lignocellulosic biomass can be broadly classified into virgin biomass, waste biomass and energy
crops (Tan et al., 2008). Virgin biomass includes all naturally occurring terrestrial plants such as
trees, bushes and grass. Waste biomass is produced as a low value byproduct of various industrial
sectors such as agricultural (corn stover, sugarcane bagasse, straw etc.), forestry (saw mill and
paper mill discards). Non-edible lignocellulosic biomass, including residues of crops or forestry
production (corn cobs, rice husks, forest thinning, sawdust, etc.), and whole plant biomass (e.g.
energy crops such as switchgrass, poplar, and other fast growing trees and grasses) serve as a raw
material for production of second generation biofuel (Carriquiry et al., 2011).
2 BIOMASS CHARACTERISTICS
2.1 Criterion of expressing biomass composition

The main topic of research related to biomass used in biofuel production focuses on extending and
improving the basic knowledge on composition and properties and using this knowledge for the
most useful advances and environmentally safe utilization (Vassilev et al., 2010). At present, several
thermochemical and chemical processes of biomass conversion to biofuels exist, e.g. pyrolysis,
gasification, Fischer-Tropsch synthesis and biodiesel production, and biochemical processes as
bioethanol production. However, the biomass conversion pathways in each process depend on its
type and characteristics. Generally, biomass is characterized with a complex composition. Vassilev
et al. (2010) distinguished three main types of matter in biomass, i.e. organic, inorganic and
fluid. Each of them poses specific state and type of constituents. Organic matter contains solid,
non-crystalline components (cellulose, hemicelluloses, lignin, extractives) and solid, crystalline
Table 2.1. Biomass categories in the EU (Elbersen et al., 2012).
Biomass origin Biomass category Biomass type detail General definition Specific definition
Agriculture Energy crops Woody/ligno-cellulosic Biomass from agricultural production Solid (lingo-cellulosic and woody) energy crops (for
biomass activities generating electricity, heat, 2nd generation biofuels)
Sugar, starch, oil Crops for biodiesel and bioethanol
Wet biomass Energy maize and maize residues (for biogas)
Agricultural Dry manure Dry manure (poultry, sheep, goat manure)
primary residues Wet manure Pig and cattle manure
Solid agricultural Biomass from agricultural cultivation, Other solid agricultural residues (prunnings,
residues harvesting and maintenace activities orchards residues)
Biomass from permanent grasslands Grass
Biomass from agricultural cultivation Straw/stubbles
and harvesting activities
Forestry Forestry biomass Woody biomass Biomass from forestry: forests and other Stem wood production
wooded land, i.e. tree plantations and
short rotation forests
Biomass from forest and other wooded Volume of additionally harvested wood
land, i.e. tree plantations available for bioenergy
Primary forestry Cultivation and harvesting in forests and Branches, roots, woody residues from lands
residues other wooded lands, biomass from trees/ outside forests
hedges outside forests
Secondary forestry Biomass from industrial wood processing Woodchips, sawdust, black liquor
residues
Waste Primary residues Biodegradable waste Biomass from road side verges Biomass residues from maintenance activities (e.g. from
grass and woody cuttings from road side verges)
Secondary Solid and wet Processing of agricultural Processing residues (e.g. shells and husks from seed)
residues agricultural residues products, e.g. food and feed
Tertiary residues Biodegradable Biomass from private households, Woody fractions, e.g. food leftovers,
waste residential gardens waste paper, discarded furniture
Organic waste from Biomass from industry and trade, Woody fractions, e.g. bulk transport packaging,
industry and trade excluding forest industry recovered demolition wood
Waste biomass Biodegradable waste Biomass from industry and Sewage sludge
private households
Figure 2.1. Biomass analysis: a) dry basis composition, b) proximate, c) ultimate, d) biochemical (Mi
inherent moisture, Ms surface moisture).
components (organic minerals such as Ca, Mg, K, Na). On the other hand, inorganic matter
is composed of solid, crystalline components (e.g. phosphates, carbonates, silicates, chlorides
etc.); solid, semi-crystalline components (poorly crystallized silicates, hydroxides etc.) and solid,
amorphous components (mainly silicates). Fluid matter includes moisture and gases.
The mass balance per unit weight of biomass is commonly expressed in four ways: dry basis,
proximate, ultimate and biochemical analysis (Tanger et al., 2013, Fig. 2.1). The dry basis compo-
sition refers to the composition of the biomass excluding all water content. The proximate analysis
involves the heating of biomass to quantify its thermal recalcitrance via the relative proportions of
moisture, volatile matter, fixed carbon and ash. The ultimate analysis concerns the relative content
of individual elements such as C, H, O, N and S. This method was originally designed for the
characterization of coal (e.g. American Society for Testing and Materials, standard D3172).
Biochemical composition of biomass is based on the concentration analysis of various biopoly-
mers (e.g., cellulose, lignin, hemicelluloses) and extractives in the biomass. The extractives include
substances present in vegetable or animal tissue like proteins, oil, starch, sugars etc. A relatively
new method of determining the lignocellulosic composition of biomass is thermo-gravimetric anal-
ysis (TG) (Carrier et al., 2011). This method seems to be reliable and less time-consuming than
conventional methods for the determination of biomass composition. In this technique, a known
amount of biomass sample is placed in a furnace, in a controlled gaseous atmosphere at the desired
temperature. The weight loss of the sample is recorded as a function of time. This gives a continuous
record of the weight change of the biomass. TG can be coupled to a spectrometer for improving
the understanding of the thermal decomposition mechanisms. The thermo-gravimetric curves of
biomass can be divided into three regions: hemicelluloses (250300 C), cellulose (300350 C)
and lignin (300500 C). According to Carrier et al. (2011) the successful TG method cannot be
extended to the lignin content because of important deviations in the correlation curves. Thus,
by using this method it is possible to determine with a comparable or enhanced accuracy the
hemicelluloses and cellulose contents of biomass sample. The TG method is easier to implement,
and more cost-effective than the existing wet chemical techniques.
2.2 Biomass composition proximate analysis

Biomass generally contains high levels of volatile matter (VM). Taking into account the main
groups of biomass like wood and woody-biomass (WWB), herbaceous and agricultural biomass
(grasses HAG, straws HAS, other residues HAR, animal biomass AB), the content of VM decreases
in the following order: HAG (79%) > WWB (78%) > HAS (74%) = HAR (74%) >> AB (56%)
(Vassilev et al., 2010). Among grasses, the richest in VM are bamboo (80.2%), buffalo gourd
grass (81.6%), sorghastrum grass (81.6%) and miscanthus grass (81.2%). Among straws, there are
mainly oat straw (80.5%) (Thesis et al., 2006a,b), wheat straw (7185%) and rice straw (6498%)
(Tanger et al., 2013). The extremely high VM in woody biomass comes from oak and pine sawdust
(86%, on average). The content of VM in animal biomass is the lowest and can vary between 48%
and 63%.
Fixed carbon is a solid carbon in the biomass that remains in the char in the pyrolysis process after
devolatilization (Basu, 2010). Its content in biomass can range between 1% and 38% (Vassilev et al.,
2010). A relatively high content of fixed carbon is characteristic for some wood species, e.g. 26.3%
tamarack bark (Bryers, 1996), 25.5% hemlock bark (Bryers, 1996), 24.4% pine bark (Moilanen,
2006) and some residues, e.g. 37.9% walnut shells (Dembiras, 2004). Other types of biomass have
lower contents of fixed carbon, e.g. 19.3% maize stover, 7% sugarcane bagasse, 16.8% wheat straw
(Das et al., 2015).
Ash content can vary greatly between plant species, and is generally higher in agricultural
residues than in other biomass types. The ash present in plants will depend on their stage of
growth, the time of year, and their location. The major cations present in ashes from lignocellulosic
materials are calcium, potassium and magnesium. Other elements such as manganese, sulphur and
phosphorus; trace elements (such as Al, Fe, Zn, Cu, Ti, Pb, Ni, V, Co, Ag and Mo); anions (chloride,
carbonate, sulphate and silicate) are present in minor amounts (Vassilev et al., 2010).
Among different biomass types, animal biomass is characterized with high ash content, 31%
on average. Equally high or even higher contents of ash are found in sewage sludge (46%) or
greenhouse-plastic waste (32%). Herbaceous and woody biomass contain significantly less ash,
5.7% and 3.5% on average, respectively. However, some biomass from these groups can contain
relatively high ash content, e.g. rice straw (20.1%), land clearing wood (16.5%). Ash composition
changes, depending on the biomass type. For example, woody biomass is rich mainly in SiO2
and CaO, grasses and straws in SiO2 and K2 O, animal biomass in CaO and P2 O5 (Vassilev et al.,
2010). Ash is the residue remaining after the material has been incinerated. Therefore, it has no
energy value and, being made up of the inorganic elements in the biomass, is of no direct value in
hydrolysis technologies. High ash-contents can cause problems in many thermochemical processes
(e.g. pyrolysis, gasification). The content of mineral and elemental ions (e.g. Na, K, Mg, Ca, Cl,
S, Si) in ash can lower oil yields. It increases char and gas products, causes fouling of reactors
as a result of liquid slags or solid deposits formation during the combustion (Jenkins et al., 1998;
Tanger et al., 2013). In addition, it lowers the heating value of biomass and changes the distribution
of conversion products (Miles et al., 1996).
Moisture content tends to vary widely, depending on biomass species, age, geographic locations
and genetic differences. It also varies between different anatomical fractions of the same plant
and throughout the year. Surface moisture refers to moisture which is removed during biomass
drying in air, while the inherent moisture is retained. The moisture content in different types of
biomass can vary in a wide range from 3% to 63% (Vassilev et al., 2010). Large amounts of water
(up to 80%) are contained in the living cells, like the shoots, leaves and inner bark (Werkelin
et al., 2005). Generally, air-dried biomass contains 1520% moisture (Das et al., 2015). The
moisture content of biomass is a crucial parameter for its combustion, but less important in some
Table 2.2. Chemical composition of biomass based on ultimate analysis.
Ultimate analysis (%, dry and ash free)
Biomass C O H N S References
Wood biomass
Oak wood 50.6 42.9 6.1 0.3 0.1 Dembiras (2004)
Pine bark 53.8 39.9 5.9 0.3 0.07 Moilanen (2006)
Poplar 51.6 41.7 6.1 0.6 0.02 Miles et al. (1995)
Willow 49.8 43.4 6.1 0.6 0.06
Grasses
Miscanthus 49.2 44.2 6.0 0.4 0.15 Miles et al. (1995)
Sweet sorghum 49.7 43.7 6.1 0.4 0.09 Moilanen (2006)
Switchgrass 49.7 43.4 6.1 0.7 0.11 Miles et al. (1995)
Straws
Alfalfa 49.9 40.8 6.3 2.8 0.21 Miles et al. (1995)
Corn 48.7 44.1 6.4 0.7 0.08 Masia et al. (2007)
Rape 48.5 44.5 6.4 0.5 0.1
Maize 45.6 43.4 5.4 0.3 0.04 Das et al. (2015)
Wheat 46.7 41.2 6.3 0.4 0.1
Rice 41.8 36.6 4.6 0.7 0.08
lignocellulose hydrolysis technologies. Too high moisture content can decrease the effectiveness
of individual thermochemical processes. The desired moisture in biomass can range from 5% (for
combustion) to even 30% (for gasification). From logistic point of view, minimizing moisture
allows more cost effective transport of biomass (Tanger et al., 2013). Beside direct impact on
conversion performance, moisture content also influences feedstock preparation, i.e. grinding,
particle size distribution (Mani et al., 2004).
2.3 Biomass composition ultimate analysis

The elemental composition of biomass is important in performing mass balances on biomass
conversion processes (Sannigrahi et al., 2010). The elemental composition for selected woody
biomass, grasses and straws is given in Table 2.2.
The data indicate that variations in the composition of individual elements in different biomass
types are slight. Carbon, oxygen and hydrogen constitute a major part. On average, their contents
are 50.2%, 42.9% and 6.2%, respectively. The average content of nitrogen and sulphur are 0.7%,
and 0.1%. The low content of sulfur is advantageous in terms of strong environmental regulations
limiting the sulfur content of transportation fuels. A suitable H:C ratio is required for a thermo-
chemical conversion of biomass. Because biomass has a low molar H:C ratio (0.72.8), addition
of steam or H2 may be necessary for its complete conversion (Tanger et al., 2013). The elemental
composition of biomass can cause potential technical problems during thermochemical processes
like pollution effects. A possible pollution and corrosion effect can come from chlorine presence
(Stahl et al., 2004).
On the other hand, the results of proximate and ultimate analysis can be used for the deter-
mination of heating value for biomass (Yin, 2011; Motghare et al., 2016). The heating value
(also known as calorific value) is one of the most important characteristics concerning biomass
combustion. This is the energy available in the feedstock, as estimated from the heat released
during the complete combustion to CO2 , H2 O (gaseous H2 O for lower heating value, LHV,
or liquid H2 O for higher heating value, HHV) (Tanger et al., 2013). Cordero et al. (2001)
studied the predicted heating values on dry basis for lignocellulosic and carbonaceous mate-
rials, based on proximate analysis. Dulong and Boie equations are commonly used for the
determination of heating values using ultimate analysis (Gupta & Manhas, 2008). The prox-
imate and ultimate analysis could be more advantageous in determination of heating value
than the standard method using calorimetric bomb. According to Motghare et al. (2016), the
HHV for given biomass types using proximate analysis decreased in the following order: cotton
waste (19.1 MJ/kg) > soybean waste (18.8 MJ/kg) > sarcasaasoca leaf (17.7 MJ/kg) > wheat straw
(17.0 MJ/kg). The order was slightly different when HHV was calculated using ultimate analysis:
cotton waste (23.6 MJ/kg) > sarcasaasoca leaf (20.5 MJ/kg) > soybean waste (17.5 MJ/kg) > wheat
straw (15.4 MJ/kg). Among 31 analyzed biomass types, the HHV for selected plants (in MJ/kg), on
the basis of ultimate analysis, amounted to: 16.0 (rice straw), 16.2 (sorghum straw), 17.1 (barley
straw), 17.6 (corn stover), 19.1 (maize straw), 19.3 (sugar cane bagasse), 19.5 (hybrid poplar), 19.8
(oak bark and pine wood) and 20.5 (redwood) (Vallios et al., 2016). In general, the heating value
for lignocellulosic biomass is in the range 1519 MJ/kg, 1719 MJ/kg for most woody biomass
and 1517 MJ/kg for agricultural residues (Stahl et al., 2004).
2.4 Biochemical biomass composition

The lignocellulosic biomass chemical composition differs with the source of plant species. Fig. 2.2
presents the contents of lignocellulosic components and selected non-lignocellulosic components
(extractives, ash, uronic acids) in five types of biomass, i.e. aquatic plants, algae, agricultural
residues, softwood and hardwood.
In general, over 50% biomass of aquatic plants is composed of lignocellulosic components,
mainly cellulose and hemicelluloses. The lignin and ash contents depend on plant species. Also,
some algae contain a relatively high content of hemicelluloses (4060%, on average). Other species,
like Spirulina, are characterized by a low content of lignocellulosic components (below 30%),
but higher content of non-lignocellulosic components, i.e. proteins (above 60%). Agricultural
residues are rich in cellulose and hemicelluloses. They have comparable or even higher content
of hemicelluloses than woody biomass (approximately 2535%) (Demirbas et al., 2005). Selected
types of agricultural biomass, e.g. sweet sorghum, wheat straw, corn grains or barely spent grains
have a relatively high content of extractives (1223%). The share of lignin in these residues varies
from 9% to 24%, on average. The lignin content of hardwoods is usually in the range of 18
25%, whereas the lignin content of softwoods varies between 25% and 35% (Rowell et al., 2005).
Softwood and hardwood differ greatly in wood structure and composition. Hardwood contains a
greater fraction of vessels and parenchyma cells. Hardwoods have a higher proportion of cellulose,
hemicelluloses and extractives than softwoods, which have higher proportion of lignin (Demirbas,
2009). In general, lignocellulosic components in softwoods and hardwoods comprise over 80% of
total mass.
2.4.1 Characteristic of structural components in biomass

Cellulose, hemicelluloses and lignin are the major structural components of biomass (Ragauskas
et al., 2006; Pu et al., 2008; Carroll & Somerville, 2009). In composite material, known as
lignocellulose, cellulose, hemicelluloses and lignin bind strongly to each other by non-covalent
forces, as well as by covalent cross-links, giving a complex structure (Davison et al., 2013). The
characteristics of cellulose, hemicelluloses and lignin were described below.
Cellulose
Cellulose is a glucose polymer linked by -1,4 glycosidic bonds. The basic building block of this
linear polymer is cellobiose (Harmsen et al., 2010). The chemical structure of cellulose is given in
Fig. 2.3.
Many properties of cellulose depend on its degree of polymerization (DP). The DP for cellulose
varies from 5000 in native wood to 1000 in bleached wood pulp, and 5001000 in the herbaceous
cellulose. The nature of the bonding between the glucose molecules (-1,4 glycosidic) allows the
Figure 2.2. Lignocellulosic and non-lignocellulosic components in different biomass types (1Towler, 2004,
2 Energy Efficiency and Renewable Energy, 3 Roberto et al., 2003, 4 Silva et al., 2010, 5 Martin et al., 2011,
6Tamanini et al., 2004, 7 Dehnavi, 2009, 8 Ruiz et al., 2008, 9 Sung & Cheng, 2002, 10 Mosier et al., 2005,
11 Rabemanolontsoa et al., 2015, 12 Rabemanolontsoa et al., 2011, 13 Rabemanolontsoa & Saka, 2012).
Figure 2.3. Chemical structure of cellulose (Hallac & Ragauskas, 2011).
polymer to be arranged in linear chains. Cellulose has a strong tendency to form intra- and inter-
molecular hydrogen bonds by hydroxyl groups between the molecules of cellulose. The hydrogen
bonds in the linear cellulose chains promote aggregation into a crystalline structure and give
cellulose a multitude of partially crystalline fiber structures and morphologies. There are two
types of orientation chains in the crystal lattice: parallel (cellulose I) and antiparallel (cellulose II)
(Dumitriu, 2004). The raw materials of natural origin are mainly cellulose with parallel chains held
by strong hydrogen bonding interactions and van der Waals forces between the adjacent layers.
Hemicelluloses
In contrast to cellulose, which is a homopolymer, hemicelluloses represent a type of heterogeneous
polysaccharides (Amidon et al., 2011). They are short, highly branched polymers of six-carbon
(C6) sugars like glucose, mannose, galactose and five-carbon (C5) as xylose and arabinose and
glucose derivatives, such as glucuronic acid.
The major hemicelluloses in softwood are galactoglucomannan and arabinoglucuronoxylan
(Tab. 2.3, Fig. 2.4). Softwood hemicelluloses also include arabinogalactan, xyloglucan and other
glucans. In softwood, all xylans have a backbone of -(14)-linked xylopyranose units. They
contain a lot of 4-O-methyl-D-glucuronic acid, forming arabino-4-O-glucuronoxylans. Unlike
xylans, the backbone of mannans consists exclusively of mannan or of mannose and glucose in
a non-repeating manner. The mannans are often partially acetylated. The main chain of mannan
hemicelluloses in hardwoods is composed of glucose and mannose (1.52:1), while for the mannan
hemicelluloses in softwood it is composed of glucose and mannose at 3:1 ratio.
In hardwoods and herbaceous crops, xylans (O-acetyl-4-O-methylglucurono--D-xylan, also
known as glucuronoxylan) are the prevalent group of hemicelluloses (Table 2.3, Fig. 2.5)
(Spiridon & Popa, 2008; Faik, 2010). Beside glucuronoxylans (GX), hardwood contains galac-
tomannan (GM); however, its content is low. The backbone of xylans is made exclusively of xylose
which is attached to methyloglucuronic/glucuronic acid and arabinose. In addition, the xylose
residues may be acetylated on C2 or C3 (Pauly & Keegstra, 2008). On average, seven of ten sub-
units of the chain are substituted with acetyl groups at C2 or C3 positions, or in both. The DP of
xylans from hardwood is from 150 to 200 and is larger than for xylans in softwood.
In Table 2.3, the percentage content of hemicelluloses in soft- and hardwood, the hemicelluloses
units, molar ratio, linkages and degree of hemicelluloses polymerization are given.
The structure of water-soluble birch and beech xylans, extracted from hemicelluloses using
dimethyl sulfoxide, employing 1 H and 13 C NMR spectroscopy together with chemical analysis
were analyzed by Teleman et al. (2002). These polysaccharides were found to be O-acetyl-(4-O-
methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately
every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these
polymers was 0.4.
The most abundant grass hemicelluloses are mixed-linkage glucan (MLG) and glucuronoarabi-
noxylan (GAX) (Scheller & Ulvskov, 2010; Vega-Sanchez et al., 2013). The GAX is an important
type of hemicelluloses in grasses used for biofuel production found, for instance, in sugar cane
and corn. It consists of a xylan backbone with branches of mainly glucuronic acid and arabinose,
Figure 2.4. The chemical structure of hemicelluloses in softwood: (a) galactoglucomannan;

(b) arabinoglucuronoxylan. Monomer units for galactoglucomannan: -D-glucopyranose (GlcP),
-D-mannopyranose(ManP), -D-galactopyranose (GalP), R CH3 CO or H. Monomer units for
arabinoglucuronoxylan: -D-xylopyranose (XylP), O-acetyl-4-O-methylglucurono--D-xylan (GLcpA),
-L-arabinofuranose (Araf ) (Fengel & Wegener, 1989; Sjstrm, 1993; Shimizu, 2001).
whose amount and ratio varies substantially with the plant genotype (Pauly & Keegstra, 2008).
In grass xylan, ferulic acid esters are linked to O-5 position of some arabinose residues. Ferulate
esters enable cross-linking and enhance cell wall recalcitrance against enzymatic attack (Scheller &
Ulvskov, 2010).
Important aspects of the structure and composition of hemicelluloses are the lack of crystalline
structure mainly due to the highly branched structure and the presence of acetyl groups in the
polymer chain (Harmsen et al., 2010). Therefore, hemicelluloses are more susceptible to hydrolysis
than cellulose (Trajano & Wyman, 2013). Hemicelluloses extracted from plants possess a high
degree of polydispersity, polydiversity and polymolecularity (a broad range of size, shape and
mass characteristics). However, the DP does not usually exceed 300 units, whereas the minimum
limit can be around 50 monomers (Ragauskas, 2014).
Lignin
Lignin is an amorphous, cross-linked, and three-dimensional phenylpropanoids polymer with
phenyl ring (C6) and propane (C3) side chain. The lignin formation begins with the biosynthesis
of its phenylpropanoid subunits and subsequent non-enzymatic polymerization of the phenyl units
Figure 2.5. The chemical structure of hemicelluloses (glucuronoxylan) in hardwood. Monomer units for
glucuronoxylan: -D-xylopyranose (XylP), O-acetyl-4-O-methylglucurono--D-xylan (GLcpA), R CH3 CO
or H (Fengel & Wegener, 1989; Sjstrm, 1993; Shimizu, 2001).
Table 2.3. The major hemicelluloses components in softwood and hardwood (Sjstrm, 1993).
Compostion
Wood Content in Molar

type Hemicelluloses type wood (%) Unit ratio Linkage DP
Soft Galacto-glucomannan 58 -D-mannopyranose 3 14 100

-D-glucopyranose 1 14
-D-galactopyranose 1 16
Acetyl residues 1
1015 -D-mannopyranose 4 14 100
-D-galactopyranose 0.1 16
Acetyl residues 1
Arabino-glucuronoxylan 710 -D-xylopyranose 10 14 100
4-O-Me--D-glucopyranose 2 12
-L-arabinofuranose 1.3 13
Hard Glucuronoxylan 1530 -D-xylopyranose 10 14 200
4-O-Me--D-glukopyranose 1 12
Acetyl residues 7
Glucomannan 25 -D-mannopyranose 12 14 200
(monolignols). The type and amount of cross-linking bonds is determined by the type of aromatic
monomer units in the lignin. The most important monomers are coniferyl (G), p-coumaryl (H),
and sinapyl (S) alcohols (Achyuthan & Kumar et al., 2009) (Fig. 2.6). The monomers differ in the
degree of methoxylation of their carbon ring. G-units have one methoxy group at the O-3 position;
H-units lack ring methoxy groups, and S-units are methoxylated at both O-3 and O-5 ring positions
(Boerjan et al., 2003). On the basis of the relative amounts of monomers, lignins are described as
softwood lignin, hardwood lignin, or grass lignin (Glazer & Nikaido, 2007). Softwood lignin is
primarily composed of coniferyl (about 90%), hardwood lignin contains coniferyl (2550%) and
sinapyl monomers (4675%), while grass lignin all of the three monomers (Li et al., 2015).
Figure 2.6. The lignin monomers (monolignols): (a) p-coumaryl alcohol (p-hydroxyphenol (H)); (b) coniferyl
alcohol (guaiacyl (G)); (c) sinapyl alcohol (syringyl (S)).
Figure 2.7. The main types of lignin inter-unit linkages: (a) 5-5-dimer; (b) -5-dimer; (c) 5-O-4-dimer;
(d) --dimer (pinoresinol); (e) -O-4-dimer; (f) -1-dimer; (g) spirodienone.
Lignin units undergo oxidative coupling in the cell wall to form many types of dimers, including
arylglycerol--aryl ether unit (O4), phenylocoumaran unit (5), units, 55 units, diaryl
ether units (5O4), and 1 units, which significantly increases the structural heterogeneity of
lignin (Lin et al., 2015). The structure of the main types of lignin linkages is presented in Fig. 2.7.
Because the chemical structure of precursors (monolignols) is different, each of the precursors
is able to couple with the radicals in several sites. More often, however, a growing number of
oligomers is a reason of formation of a diverse network of intramolecular connections, thereby
creating a spatial structure of the lignin. Figure 2.8 shows a lignin macromolecule formed of
different monomers (S, G and H) and by various linkages (O4, 5, , 55, 5O4, 1).
2.4.2 Lignin isolation from biomass and its characterization

Lignin isolation
The lignin content of wood and pulp is generally determined as Klason lignin in accordance with
the standard method. The Klason lignin, is obtained by treating wood with sulfuric acid (Obst &
Kirk, 1988). The polysaccharides are hydrolyzed to water-soluble sugars, and lignin is recov-
ered as an insoluble residue. Among different methods, there are: MWL (Milled Wood Lignin),
CEL (Cellulotlytic Enzyme Lignin), EMAL (Ezymatic Mild Acidolysis Lignin) and LCC (Lignin
Carbohydrate Complex) (Bjrkman, 1956; Chang et al., 1975; Wu & Argyropoulos 2003; Guerra
et al., 2006; Tolbert et al., 2014). Apart from the method, milling has commonly been used
as first step to open up the entangled lignin structure, with or without a subsequent enzymatic
hydrolysis.
The MWL was widely used to elucidate the chemical lignin structure. In the MWL method,
sample is extensively milled, and next free lignin is extracted by dioxane-water azeotrope from
the lignocellulosic matrix (Bjrkman, 1956). Min et al. (2013) analyzed the influence of isolation
conditions on the structure of lignin by quantitative 13 C nuclear magnetic resonance spectroscopy.
Experiments were conducted for different milling times (4, 8 and 12 h), and extraction temper-
ature (20 C and 45 C). The criteria of an efficient lignin isolation method were yield and the
lignin content in the crude MWL. For hardwood (sweetgum), the optimal conditions of isola-
tion were 8 h milling and extraction at 20 C, while for softwood (loblolly pine), 12 h and 20 C,
respectively.
For lignin isolation from poplar and black spruce, Wu & Argyropoulos (2003) used MWL, CEL,
and enzymatic/acidolysis lignin (EAL) by 0.01 mol/L HCl. EAL gave higher yield of lignin (2.5
times greater) than the corresponding MWL preparation. However, structural analysis of lignin did
not show any marked differences between samples MWL and EAL.
The CEL is structurally similar to MWL, but it can be obtained in higher yields. Chang et al.
(1975) proposed the CEL procedure for residue after the aqueous p-dioxane extraction of ball-
milled biomass. This method involves removing a substantial amount of the carbohydrate fraction
by means of the CEL treatment. Next, the residue was washed with dioxane/water resulting in
lignin dissolution. For spruce wood, the yield of CEL was approximately 29%.
Guerra et al. (2006) compared data obtained for different wood species using MWL, CEL, and
EMAL methods. The EMAL protocol offered gravimetric lignin yields 25 times greater than
those of the corresponding MWL and CEL. The purities of the EMALs were 3.7510.6% higher
than those of their corresponding CELs, depending upon the wood species from which they were
isolated. The yields and purities of EMAL, MWL, and CEL from hardwood were greater than those
obtained for the examined softwoods.
Several methods have been reported to complete fractionation with proper preservation of the
bonding structure between the lignin and carbohydrate (Lawoko et al., 2006; Balakshin et al.,
2011; Du et al., 2013). The effect of milling time on the LCC fraction for wood and pulp of euca-
lypt (Eucalyptus globulus) was investigated by Li et al. (2011). After milling DMSO (dimethyl
sulfoxide) + TBAH (tetra-n-butylammonium hydroxide) solvent systems were used to completely
dissolve the wood or pulp meal. By applying the minimum milling time (12 h) required for
complete dissolution, structurally unaltered wood or pulp was subsequently separated into lignin-
carbohydrate fractions. Two different lignin-carbohydrate fractions were obtained from eucalypt
pulp, one glucan- and one xylan-enriched fraction, with the latter having more syringyl units in its
lignin moieties.
Lignin characterization
There is a variety of methods for lignin characterization. Wet chemical methods concern the
measurement of specific functional groups in lignin. In order to analyze lignin structure, the
chromatography in conjunction with wet chemical methods is employed. Pyrolysis gas chromatog-
raphy/mass spectrometry is also applied. Other techniques include thermogravimetric analysis
and Fourier-transform Raman spectroscopy. UV spectrophotometry provides another analytical
tool for lignin structural analysis, as lignin absorbs light in this region of the electromagnetic
spectrum. Visual lignin characterization, including its ultrastructure and molecular configuration,
is determined with atomic force and electron microscopy. Nuclear magnetic resonance (NMR)
spectroscopy offers a novel insight in lignin structure (Lupoi et al., 2015).
Lignin structure is important for feasibility of many biomass conversion processes. The ratio
between individual monomers in lignin is crucial for thermochemical processes. In turn, the ability
of biomass destruction to recover sugars is fundamental for biochemical processes. In addition,
lignin structure and inner linkages also affect formation of different derivatives, that affect the
performance of biological processes during biochemical conversion of biomass. Hardwood lignin
produces guaiacyl and syringyl derivatives. Softwood lignin produces mostly guaiacyl deriva-
tives but no hydroxyphenyl or syringyl compounds. Grass lignin uniquely produces vinylphenol,
propenyl-phenols, and p-hydroxybenzaldehyde (Lin et al., 2015). Lignin-enriched residues gen-
erated in large-scale industrial biorefineries from lignocellulosic biomass can also be valuable
(Katahira et al., 2016). Usually they can be utilized as sulphur-free solid fuel, sub-bituminous
coal or natural binder and adhesive (Kamm & Kamm, 2004). However, in modern biorefineries,
lignin after effective depolymerization can be converted into products of sufficient value and
market size. For example, low molecular weight aromatics from lignin can be upgraded to fuels
or chemicals.
Lignin structure was characterized by many authors. Del Rio et al. (2012) showed that the lignin
in wheat straw is a p-hydroxyphenyl-guaiacyl-syringyl lignin (with an H:G:S ratio of 6:64:30)
associated with p-coumarates and ferulates. 2D-NMR indicated that the main substructures were
-O-4-ethers (75%), followed by phenylcoumarans (11%), with lower amounts of other typical
units. Yank et al. (2015) characterized lignin structure of triploid of Populustomentosa Carr. The
results showed that the main substructures in the lignin were -O-4 aryl ether and resinol. The main
inter unit linkages present in wheat straw lignin and in olive tree pruning lignin were -O-ethers,
followed by resinols and phenylcoumarans (Santos et al., 2015).
Although lignin has only three basic structures, their quantity proportions vary greatly in different
plants. Vanholme et al. (2013) found that in lignin from poplar wood the ratio between G, S and
H was 55:45:1. The dioxane lignin isolated from extractive-free maize samples contained mainly
coniferyl (33.072.0 mol%) and sinapyl (29.666.4%) monomers. The content of p-coumaryl was
below 1.5% (Chazal et al., 2014). The sugarcane bagasse lignin showed similar contents of G and
S monomers and a minor content of H monomer, with an H:G:S ratio of 27:35:38. The soybean
root lignin consists of a high content of the G unit when compared with the levels of the H and S
units, and had an H:G:S ratio of 16:69:15. The lignin content in the soybean seed coat was formed
by similar levels of the H and G units and a low content of S unit, with an H:G:S ratio of 44:44:12
(Moreira-Vilar et al., 2014).
The lignin composition was characterized by Stewart et al. (2015) in five crop residues: corn
(Zea mays L., C4), sorghum (Sorghum bicolor L. Moench, C4), soybean (Glycine max L., C3),
sunflower (Helianthus annuus L., C3) and wheat (Triticumaestivum L., C3). In general, lignin in
crops of C3 type was characterized with higher content of G and H monomers than C4 crops.
The opposite trend was for H monomers. The average content of G monomers for crops of C3
type was 25.7% (the highest for soybean, 31.6%), whereas for C4 crops it was 17.6%. Lignin
from sunflower contained the highest content of S monomers, which was comparable with the
content of G monomers. Corn and sorghum had a large content of 4-vinyl phenol (30.2 and 25.9%,
respectively) belonging to H monomers. For C3 crops, the content of H monomer varied from 6.1%
(sunflower) to 17.7% (wheat). Larger concentration of these compounds affects the amount of acid-
linked lignocellulose in these C4 grasses, which could influence digestibility and decomposition
of these crops (Hatfield et al., 2009).
The lignin polymer contains more different functional groups involved in its depolymerisation
and degradation than cellulose and hemicelluloses. In contrast to S lignin monomers, the H and G
monomers are easily linked with reactive functional groups. The H and G monomers are considered
as reactive units, while the S unit as inert ones (Li et al., 2015). The most abundant functional
groups in lignin are: methoxyl groups, free phenolic hydroxyl groups, carbonyls, benzyl alcohols
and terminal aldehyde groups (Santos et al., 2013). The presence of individual monomers affects
Figure 2.8. Schematic of lignin structure including syringyl (S), guaiacyl (G), and p-hydroxyphenol (H)
phenylpropanoid moieties, and lignin-lignin linkages (Lupoi et al., 2015).
the lignin structure, which is looser with smaller molecular weight when it contains more S units.
However, it is more compact when H and G units prevail.
A good indicator of overall lignin composition and response to pulping and biomass pre-treatment
is the S/G ratio (Sannigrahi & Ragauskas, 2010). Lignins with high S/G ratio are less cross-linked
and easier to degrade in pulping process than lignin with low S/G ratio (Reina et al., 2014). For
comparison, the S/G ratio for Eucalyptus grandis lignin ranged between 2.3 and 3.6 (Reina et al.,
2014), whereas for poplar lignin it was 1.3 to 2.2 (Sannigrahi & Ragauskas, 2010). Fagerstedt et al.
(2015) found that the S/G ratio can be different in various tissues of silver birch. In cork cambium
and non-conductive phloem, the S/G ratio was the lowest (1.01.5), whereas it was the highest
for lignified xylem (S/G = 7.0). Santos et al. (2015) characterized two lignin-rich residues from
biochemical ethanol production (including steam explosion pretreatment, saccharification, and
fermentation) of wheat straw and olive tree pruning. Wheat straw lignin showed a very low S/G ratio
Table 2.4. The quantities of linkages in lignin from various plants.
Benzyl Phenyl Spiro-

Plant, lignin form ether -O-4 - -5 -esters glycoside dienone References
Eucalyptus globulus, n.a. 7783 911 38 n.a. n.a. 15 Rencoret et al., 2008
E nitens, E. maidenii,
E. grandis, and E.dunnii,
milled wood ligninb
Loblolly pine, crude milled 3.9 27.5 4.4 9.8 1.8 2.0 n.a. Balakshin et al., 2011
wood lignina
White birch, crude milled 1.4 38.4 9.4 2.2 5.3 3.5 n.a.
wood lignina
Poplar (Populustomentosa), 2.1 41.5 14.6 3.7 3.4 4.1 0.7 Yuan et al., 2011
milled wood ligninb
Cotton stalk by-product, n.a. 75.6 12.2 7.4 n.a. n.a. n.a. Kang et al., 2012
ammonia-extractable ligninb
Spruce (Piceaabies), n.a. 56 9 17 n.a. n.a. 1 Du et al., 2014
milled wood ligninb
Sugarcane bagasse, milled n.a. 2532 1.7 3.3 n.a. n.a. 1.4 Zeng et al., 2014
bagasse lignina
Sisal (Agave sisalana), n.a. 80 3.0 3.0 n.a. n.a. 4.0 Jos et al., 2016
milled wood lignina
Abaca (Musa textilis), n.a. 80 0.0 1.0 n.a. n.a. 1.0
milled wood lignina
a as amounts of linkages per 100 aromatics; b % of the total side chains; n.a. not analyzed.
associated with p-hydroxycinnamates (p-coumarate and ferulate), whereas a strong predominance

of S over G units was observed for olive tree pruning lignin.
Huang et al. (2016) examined the relationships between the lignin structure and hemicelluloses
composition in different wood species. In the 48 hardwood species the xylose/glucose, rhamnose/
glucose, and arabinose/glucose ratios increased when the syringyl ratio was higher. In the 14
softwoods, the mannose/glucose ratio increased when lignin content decreased. For both hardwoods
and softwoods, there was an affinity between lignin with a higher syringyl ratio and hemicelluloses
with higher xylan/mannan ratio. Moreover, the authors showed that both the syringyl ratio and
xylan/mannan ratio were always higher in the insoluble fraction.
The lignin structure is affected not only by individual monomers, but also by inner linkages.
The quantities of lignin linkages in lignin from various plants are given in Table 2.4. Although the
proportion of various linkages can be different, lignin originated from different plants is enriched
mainly in -O-4 alkyl-aryl ether linkages. Ether bonds may appear between allylic and aryl carbon
atoms, or between aryl and aryl carbon atoms, or even between two allylic carbon atoms (Harmsen
et al., 2010). The cleavage of the ether bond can lead to the separation of lignin from the polysaccha-
rides matrix and degradation of the polymers to monomer sugars and lignin fragments. Ester bonds
are identified between lignin and polysaccharides. Hydrogen bonds connect lignin with cellulose
and hemicelluloses (Harmsen et al., 2010).
In addition to the various types of bonds present within the lignin itself, there are also associations
between lignin and polysaccharides, forming a lignin-carbohydrate complex with benzyl-ether,
benzyl-ester, and phenyl-glycoside bonds (Kang et al., 2012).
Spruce and birch lignin have been examined using 1 H NMR spectral methods by Lundquist
(1992). Apart from 5 structures, , non-cyclic benzyl aryl ethers, phenolic and carboxylic
groups were found as well. About 7% of the units 5 were present in spruce lignin. Guaiacyl-
propane unit predominated in spruce lignin, while birch lignin was composed of nearly equal
amounts of quaiacylpropane and siringylopropane units. Crestini & Argyropoulos (1997) studied
the nature of ester bonds in wheat straw (Triticumaestivum) lignin. Milled straw lignin was found
to contain about 12 ester units per 100 phenylpropane units. Approximately 77% of the carboxyl
fraction of these ester bonds was found to be composed of p-coumaric acid, while the rest consti-
tuted other aromatic acids bound to lignin via intra- and/or intermolecular ester bonds. In contrast,
the hydroxyl fraction of the ester bonds was found to be almost exclusively aliphatic.
Sun et al. (2005) characterized sequentially extracted lignin and hemicelluloses with high
yield/purity using acidic dioxane/water solution and dimethyl sulfoxide from ball-milled wheat
straw. The acidic dioxane lignin fraction was distinguished by high -O-4 structures and low
amounts of condensed units (-5, 5-5, and -1). Hemicelluloses contained arabinoxylans as the
major polysaccharides, which were substituted by -l-arabinofuranose, 4-O-methylglucuronic acid,
acetyl group, and xylose at O-3 and/or O-2 of xylans. They found that arabinoxylans formed
cross-links with lignins through ferulates via ether bonds, glucuronic acid via ester bonds, and
arbinose/xylose via both ether and glycosidic bonds, respectively, in the cell walls of wheat straw.
The content of lignocellulosic materials in biomass can affect the conversion process and its
products. For example, cellulose and hemicelluloses contribute to the bio-oil production yield,
while lignin yields larger proportion of solid char (Akhtar & Amin, 2012). Higher lignin content
may increase the average molecular weight and viscosity but decrease the water concentration of the
bio-oils (Fahmi et al., 2008). Since lignin is less oxidized than hemicelluloses, it has a higher heating
value and this typically translates to lower heating values of herbaceous biomass as compared to
woody biomass or some agro-industrial residues, such as olive press cakes (BISYPLAN, 2012).
The lignin content also affects, to some extent, the combustion speed. For the biomass with higher
cellulose content, the pyrolysis rate became faster. On the other hand, the biomass with higher
lignin content gave slower pyrolysis rate (Gani & Naruse, 2007).
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Chapter 3
Biomass feedstock for biofuels production
Katarzyna Bukowska & Artur Pawowski
1 INTRODUCTION
The development of biofuels derived from biological materials abundant with lignocellulosic
biomass reduces the dependence of transportation systems on fossil fuels and emissions of green-
house gases (GHG) that contribute to global warming (Morales et al., 2015). However, modern
biofuels production creates new problems, in particular:
competition between the use of biomass for food or fuel production,
increased food prices,
limited land availability for cultivation of energy crops,
environmental impacts.
In general, there is a growing consensus that biofuel technologies must become more efficient
in terms of reducing net lifecycle emissions of greenhouse gases (GHG), and be socially and
environmentally sustainable (Sangeeta et al., 2014). The trends in the development of biofuel
production are influenced by the following factors (Okonko et al., 2009):
the type of feedstock used,
the properties of the biofuels, with a view to their application in existing systems of distribution
and trade of fuels,
the technical and economic acceptability of new methods of biofuel production.
The development of biofuels that does not rely on grain crops as inputs will require a diverse set of
feedstocks which can be grown sustainably and processed cost effectively (Simmons et al., 2008).
Nowadays, four different sectors: agriculture, forestry, industry and aquaculture are considered
as renewable carbon-based feedstocks (Cherubini et al., 2009). Regardless of the source, biomass
feedstocks vary in composition, having different shares of basic components.
Lignocellulose is the most abundant renewable biomass on the earth (Harmsen et al., 2010).
It contains primary metabolites such as carbohydrates (simple sugar, cellulose, hemicelluloses,
starch) and lignin in high concentration. Although different lignocellulosic crops vary among
species, they generally consist of 75% carbohydrate polymers (cellulose and hemicelluloses)
and lignin 25% (de Carvalho et al., 2015; Zhang et al., 2006; Kim, 2004; Scordia et al., 2011).
The deconstruction structure of lignocelluloses and the hydrolysis of cellulose and hemicelluloses,
leads to formation of C6 sugars and C5 sugars, which are the main intermediates linking feedstock
and final products such as bioethanol, biobutanol, higher chain alcohols, isoprenoids and others
(Anwar et al., 2014).
Recently, there has been considerable interest in developing biofuels, which can be readily
integrated with petroleum fuel in a drop-in fashion. These biofuels are distinguished by a low
concentration of oxygen, low water solubility and a high degree of carbon bond saturation, and
typically have functional characteristics similar to gasoline, diesel or jet fuel. The non-food oilseed
crops, such as crambe, camelina and jatropha, are being considered as an oil source for production
37
drop-in fuel. Likewise, algae and aquaculture can be used as sustainable, renewable bioenergy
feedstocks. Their advantage consists in the potential to provide significantly more oil per acre
of land than traditional oil seed crops. Furthermore, algae can be grown in arid climates with
fresh water or sea water. The usage of autotrophic algae allows for transformation of inorganic
carbon feedstock (CO2 /HCO 3 ) in lipids (Jajesniak et al., 2014). However, the use of algae-based
technology for oil production requires further research in the selection of appropriate species, the
optimization of culture conditions for the enhancement of lipid productivity, and the development
of an efficient lipid extraction procedure (Duong et al., 2012).
This chapter presents the characteristics of feedstocks for the production of the first and next gen-
eration of biofuels, the properties of various advanced biofuel technologies, and the characteristics
of drop-in biofuels.
2 BIOMASS FEEDSTOCK FOR THE FIRST AND NEXT GENERATION

BIOFUELS PRODUCTION
In 2014, the world production of biofuels was estimated at 24,570 millions of gallons (RFA, 2015).
The US share was 58%; Brazil 25%; and Europe 6%. According to their application, biofuels
can be divided into two categories: biodiesel used in compression ignition engines and bioethanol
in the engines with spark ignition. Although biodiesel is favored in several European countries,
ethanol dominates on the majority of the world biofuel market, including the one of the United
States (Fortman et al., 2008). Corn-based ethanol dominates domestic production in the United
States, while Brazil produces ethanol mainly from sugar cane (Eisentraut, 2010).
In North America, ethanol is produced from corn starch and cereals (wheat, barley, milo). On
the other hand, in South America and Asia it is primarily produced from sugar cane and cassava,
while in Africa from sugar cane only. In Europe, sugar beets and sugar cane are mainly used as
feedstock (Kim et al., 2004). Together, U.S. and Brazil account for 89% of the current global
bioethanol production (Limayem & Ricke, 2012). European countries are deploying extensive
efforts to increase their 5% worldwide bioethanol production (Gnansounou, 2010). The ethanol
produced from food crops like corn, wheat, barley and sweet sorghum is sometimes called grain
alcohol, whereas ethanol produced from lignocellulosic biomass such as agro residue (i.e. rice straw,
wheat straw) and grasses (switchgrass) is known as biomass ethanol. Grain ethanol is produced
through fermentation while biomass alcohol through fermentation, as well as hybrid processes
(gasification and fermentation).
The popularity of biodiesel is increasing worldwide due to higher interest and feedstock costs.
Among the edible oil plants, the highest yield of oil can be obtained from oil palm (6000 L/ha),
rape (1200 L oil/ha) and sunflower (965 L oil/ha). Due to the regional availability of feedstock, the
EU is the largest producer of biodiesel, accounting for 56% of global production. Germany is one
of the largest EU producers. Whereas most EU countries use rapeseed/canola, France and Spain
also use sunflower. The U.S. and Brazil have used soybeans, while palm oil is increasingly used
in South East Asia. Malaysia is the largest exporter of palm oil in the world. In 2008, Malaysia
produced 17.7 million tones of palm oil on 4.5 million hectares of land (Szmigielski et al., 2009)
and was the second largest producer of palm oil, employing more than 570,000 people (Chhetri
et al., 2008).
The increasing criticism of many first generation (1G) biofuels has gathered greater attention
on the potential of second generation (2G) biofuels (Naik et al., 2010). 2G feedstock comes from
dedicated energy crops, which do not compete with food crops. Apart from that, it cannot be taken
from primary forest, protected natural areas or highly biodiverse grassland (Renewable Energy
Directive 2009/28/EC). Lands with high carbon stocks, such as wetland or peatland, can only be
used under certain circumstances (i.e. if the land use has not been changed since January 2008). 2G
feedstock includes by-products and wastes from agricultural, forestry and industry, suggesting that
the new fuels could offer a considerable potential in promoting rural development and improving
economic conditions in emerging and developing regions. The list of wastes, residues and other
Biomass feedstock for biofuels production 39
feedstocks set out in the European Commissions proposal on Indirect Land Use Change (ILUC)
is presented in Kretschmer et al. (2013).
In third generation (3G) biofuels, carbon is derived from aquatic autotrophic organisms algae.
Algae constitute a vast variety of photosynthetic species inhabiting diverse environments (Mata
et al., 2010; Nigam & Singh, 2011). Algae are grouped into two categories microalgae and
macroalgae based on their morphology and size (Chen et al., 2009). Generally, they use light,
carbon dioxide and nutrients to produce the feedstock. Although most of them are phototrophic,
some groups contain members that are mixotrophic, deriving energy both from photosynthesis and
uptake of organic carbon. Algae can thrive in various aquatic environments such as fresh and saline
water, or municipal wastewater (Gouveia & Oliveira 2009; Harun et al., 2010). All of them are
capable of taking CO2 from the atmosphere to produce biomass more efficiently and rapidly than
terrestrial plants. The average photosynthetic efficiency of aquatic biomass is 68%, which is much
higher than that of terrestrial biomass (1.82.2%) (Ross et al., 2008). The microalgal cells have
a very fast productivity and harvesting cycle (110 days) compared with other feedstock (harvest
once or twice a year) and thus provide enough supplies to meet ethanol production demands (Singh
et al., 2011).
Algae can produce carbohydrates, lipids and proteins, which can then be processed into biofuels.
The real advantage of microalgae over plants lies in their metabolic flexibility, which offers the
possibility of modification of their biochemical pathways (e.g. towards protein, carbohydrate or oil
synthesis) and cellular composition (Tredici, 2010). Depending on the microalgae species, other
unique products may also be extracted, including polyunsaturated fatty acids, oils, natural dyes,
sugars, pigments, antioxidants, high-value bioactive compounds, and other fine chemicals (Li et al.,
2008a; Li et al., 2008b; Raja et al., 2008).
Production of algae is advantageous from the environmental point of view. One example of this is
the removal of CO2 from industrial flue gases by algae bio-fixation (Wang et al., 2008), reducing the
GHG emissions while producing biodiesel (Directive 2003/30/EC).A further example is wastewater
treatment for the removal of NH+ 3
4 , NO3 , PO4 , when algae feed on these contaminants, thus
growing (Wang et al., 2008). After oil extraction, the resulting algae biomass can be processed
into ethanol, methane, livestock feed, used as organic fertilizer due to its high N:P ratio, or simply
burned for energy cogeneration. As opposed to land-based biofuels produced from agricultural
feedstocks, cultivation of algae for biofuels does not necessarily use agricultural land and requires
only negligible amounts of freshwater (if any).
Successful commercial algal growth requires the development of strains and conditions for
culture that allow a rapid production of biomass with high lipid content and minimal growth of
competing strains. A number of projects and pilot plants are now identifying the best types of
algae to use and the best production technologies. The Aquatic Species Program (ASP) funded
by the Department of Energy (DoE) from 1978 to 1996 ASP was successful in demonstrat-
ing the feasibility of algal culture as a source of oil and resulted in important advances in
the technology.
However, this does not necessarily imply that 2G is always more sustainable than 1G, and 3G is
always more sustainable than 2G or 1G, as other factors relating to land use, competition with food
crops, and the efficiency of the production process, total energy balance, etc. need to be taken into
account across each specific value chain.
The sustainability of biofuels will depend on whether producers comply with criteria, like min-
imum lifecycle and GHG reductions, including land use change and social living standards. The
EU has defined a set of sustainability criteria to ensure that the use of biofuels used in transport
is done in a way that guarantees real carbon savings and protects biodiversity. Only biofuels that
comply with the criteria can receive the government support or count towards national renewable
energy targets. In order to be considered sustainable, biofuels must achieve greenhouse gas savings
of at least 35% in comparison to fossil fuels. This savings requirement will rise to 50% in 2017.
In 2018, it will rise again to 60% but only for new production plants. All life cycle emissions
are taken into account when calculating greenhouse gas savings. This includes emissions from
cultivation, processing, and transport.
Figure 3.1. Biomass as renewable feedstock for biorafineries (based on Naik et al., 2010).
Table 3.1. Summary of feedstock sustainability based on Bauen et al., 2009 and Fike et al., 2013.
Feedstock Factors affecting sustainability and potential Likelihood of impact
Energy crops High yields, low agricultural inputs, can have sustainability Mid-low
benefits. Could avoid land use impacts if grown on land not
needed for food production; Seeds cannot be commercialized
and should readily developed; Relatively low cost compared
with other lignocellulosic biomass.
Residues and Large amounts of the resource, even when limited to Low
wastes sustainable extraction levels.
Can have impacts if diverted from another use; Limiting
factor is a high recalcitrant compounds.
Conventional Direct land use change e.g. deforestation has GHG and High
oil crops biodiversity impacts. Grown on agricultural land and so risk
indirect land use change in the short term.
New oil crops Could grow on poorer quality land than conventional crops, Mid-low
potentially with lower fertiliser inputs, though concerns
remain over yields on this land.
Algae Can be grown on non-productive land, with high yields. Low
Potential impacts from GMOs and non-native species
in open ponds.
Biofuel sustainability certification processes are adapted to regulate the impact of biofuel produc-
tion on GHG emissions and on the sustainable use of natural resources. A large share of the worlds
agriculture and other natural resources based on production is located in developing countries and
exported to developed countries. Summary of the feedstock sustainability is presented in Table 3.1.
3 BIOMASS FEEDSTOCK FOR THE SECOND AND THIRD GENERATION

BIOETHANOL PRODUCTION
3.1 Lignocellulosic biomass

A large variety of feedstocks is currently available for producing ethanol from cellulosic biomass
(2G). The biomass feedstock can be categorized as: agriculture (dedicated crops and crops residues),
short rotation forestry (SRF), agricultural wastes, forestry residues, and others.
3.1.1 Biomass from short-rotation forestry

In this article, short-rotation forestry stands for sustainable plantations of fast-growing tree species
that produce woody biomass on agricultural and forest land. Trees are grown either as single stems or
as coppice systems (SRC). Biomass from short-rotation woody crops has been considered a viable
feedstock for producing biofuels and bioproducts using the forest biorefinery concept to reduce
our reliance on fossil fuel, mitigate climate change, and stimulate rural economic development
(Zhu & Pan, 2010; Zalesny et al., 2011).
Short rotation forestry/coppice includes such tree species as poplar (Populus sp.), eucalyptus
(Eucalyptus sp.), pine (Pinus sp.), spruce (Picea sp.), locusts (Robinia sp.), and willow (Salix sp.).
Poplars have been the subject of significant interest due to their potential for management under
short rotation coppice or very short harvest cycles, low nutrient demand and high biomass yield
on different types of land (Littlewood et al., 2014). Poplar breeding mainly focuses on three native
species, i.e. P. deltoides (eastern cottonwood) and P. trichocarpa (western black cottonwood),
which are a wide distributed in the western and eastern U.S. and Canada, respectively, as well as
P. balsamifera (balsam poplar); and two non-native species, namely P. maximowiczii (Asian black
poplar) and P. nigra (European black poplar) (ONeill et al., 2010; Townsend et al., 2014). Poplars
are important forestry and SRC species in Europe with about 950,000 ha of poplar plantation and
130,000 ha of natural forests with indigenous poplar (Coaloa & Nervo, 2010). Poplar can grow
from 5 to 10 feet per year, depending on the variety and location (Stanturf et al., 2001). Upon
maturity, poplar species can grow up to approximately 26 m in height and 60 cm in diameter.
In five EU countries (Sweden, Italy, Spain, Slovakia, France), poplar time rotation for SRC was
30-year with 57 year harvesting intervals and for very-short-rotation coppice (VSRC) was 30-year
rotation with 23 year harvesting intervals (Guo et al., 2014).
Poplar is sensitive to low temperatures and its yield is lower (Table 3.2) in North European
countries; for this reason, it is rarely grown in Northern European countries. In Central Europe,
its yield is about 22 Mg d.w./hayear. The nominal yield (including moisture content at harvest) of
hybrid poplar species in North America is estimated to be 14 Mg/hayear (Sannigrahi et al., 2010).
Poplar is characterized by higher yield compared with willow when harvesting is carried out every
4 years or more. It grows better in soils rich in humus, although it can also be grown on less fertile
soils.
Poplar species and hybrids have cellulose content ranging from 42 to 49%, hemicelluloses
from 16 to 23%, and total lignin contents from 21 to 29%. The cellulose content of poplar is higher
than that of switchgrass and corn stover and is comparable to other hardwood feedstock, such as
eucalyptus. In poplar, the biomass glucan concentration is from 39 to 49%, xylan from 13 to
19%, mannan from 1.7 to 3.9%, galactan from 0.6 to 1.5% and arabinan 0.41 to 0.89%
(Sannigrahi et al., 2010). Cellulose crystallinity ranges from 54 to 68% (Foston et al., 2009). Kumar
et al. (2009) estimated the cellulose degree of depolymeryzation (DP) from viscosity measurements
and obtained a DP value of 3500 for untreated poplar.
Table 3.2. Comparison of production estimates of hybrid poplar from different regions (based on Guo et al.,
2014; Townsend et al., 2014).
Production estimates Growth cycles

Region (Mg/hayear) (years) References
Europe
Sweden 7* 7 Johansson & Karacic, 2011
6.3** 3 Rytter, 2012
Italy 14* 5 Paris et al., 2011
12.6** 2 Spineli et al., 2011
Spain 14.4* 5 Gonzlez-Garca et al., 2010
12.9** 2
Slovakia 8.4* 7 Fischer et al., 2005
7.6** 3
France 10* 7 Nonhebel, 1997
9** 3
USA
Lake states 3.18.4 58 Miller & Bender, 2012
Upper Midwest 6.112.8 13 Zamora et al., 2013
Mississippi River Valey 5.07.5 810 Zalesney et al., 2011
Pacific Northwest 7.721.5 611 Berguson et al., 2010
* short-rotation coppice (SRC), ** very-short-rotation coppice (VSRC)
Analyzing the literature data, Littlewood et al. (2014) stated that using modern molecular biotech-
nology can achieve progress in reducing the lignin content in poplar and other woody species. In
comparison to conventional plant breeding techniques, improvement for chemical composition
of biomass may be attained by altered expression of genes involved in the biosynthesis of the
p-hydroxyphenyl, guaiacyl and syringyl building blocks of lignin.
Eucalyptus is widespread in tropical and subtropical areas of Africa, Asia and South America.
Eucalyptus plantations can be found in more than 90 countries on five continents and is by far the
fastest-growing hardwood forestry industry in the world, with a total plantation area estimated at
between 16 and 19 million hectares (4047 million acres) (Flynn, 2010). There are approx. 600
species from Australia, New Guinea and south-eastern Indonesia. It is a versatile tree which adapts
itself to a variety of edaphic and climatic conditions, from tropical to warm temperatures and with
annual rainfall ranging from 400 to 4000 mm. It grows well in deep, fertile, and well-drained
loamy soils with adequate moisture (Nag & Manchikanti, 2008). Eucalyptus globulus yield can
reach 24 to 30 Mg/ha. year (Pereira et al., 1989; Stricker et al., 2000; Rockwood et al., 2008). When
compared to cereal straws or biomass grasses, the eucalyptus, is characterized by a considerably
lower content of pentose sugars (Lima et al., 2013). However, an efficient conversion of woody
biomass into fermentable monomeric sugars is largely dependent on pretreatment of the cell wall,
whose formation and complexity lend itself towards natural recalcitrance, against its efficient
deconstruction (Blanch et al., 2011). The cellulose concentration of eucalyptus can vary between
44.45 and 49.90% depends on species (Table 3.3).
Spruce grows in Central and Eastern Europe. Picea abies is the most commercially important
coniferous species in Europe. If the potential productivity of Norway spruce is estimated by a
simple up-scaling, the average production of above ground biomass with 2500 trees/ha within 28
years would be a total of 97 tons of dry weight/ha (Kilpelinen et al., 2010). Table 3.3 shows the
composition of biomass spruce including cellulose, hemicelluloses, and lignin.
Willow is found primarily on moist soils in cold and temperate regions of the Northern Hemi-
sphere. The cultivation of willow in Poland is estimated to occupy an area of 50009000 ha
(Stolarski et al., 2015). The largest area occupied by willows (about 12,000 ha) is in Sweden
Table 3.3. Main chemical compositions of several biomass feedstocks.
Hemicelluloses
Cellulose Xylan Galactan Arabinan Mannan Lignin Extractives Ash

Feedstock % % % % % % % % Reference
Woody biomass (hardwood)

Black Locust Robinia 40.38 14.00 0.77 0.74 2.07 28.55 3.87 2.08 EERE
Black Locust 41.61 13.86 0.93 0.94 1.92 26.70 7.31 2.15 Hamelinck et al., 2005
Eucalyptus saligna 48.07 10.42 0.74 0.30 1.23 26.91 4.15 1.22 EERE
Eucalyptus (E. urophylla E. grandis) 49.90 12.10 1.21 0.30 0.91 27.37 n.a. n.a. de Carvalho et al., 2015
Eucalyptus, E. camaldulensis 44.45 10.53 2.24 0.82 0.28 35.18 n.a. 2.14 Zheng et al., 2006
Hybrid Poplar 39.23 13.07 0.88 0.89 1.81 25.18 6.89 2.03 EERE
Hybrid Poplar 44.70 14.56 0.97 0.82 2.20 26.44 7.12 1.71 Hamelinck et al., 2005
Salix sp. 43.00 14.90 2.00 1.20 3.20 26.60 n.a. 1.00 Sassner et al., 2006
White oak 43.60 18.00 0.40 2.40 2.90 23.20 n.a. 0.60 Kim, 2004
Red oak 43.40 18.90 n.a. 1.90 2.70 25.80 n.a. 0.40
Walnut 46.20 16.50 n.a. 1.80 2.60 21.90 n.a. 1.00
Maple 44.90 17.30 n.a. 2.80 2.90 20.70 n.a. 0.60
Woody biomass (softwood)
Pinus radiata 41.70 5.90 2.40 1.50 10.70 25.90 2.70 0.30 EERE
Athel pine, Tamarix aphylla L. 49.34 11.82 0.46 0.68 0.27 30.42 n.a. 5.43 Zheng et al., 2006
Spruce 43.40 4.90 1.80 1.10 12.00 28.10 1.00 n.a. Tenborg et al., 1998
Spruce 39.00 4.60 1.50 0.90 11.00 27.40 n.a. n.a. Soudham et al., 2013
Spruce 43.00 5.40 2.30 n.a. 12.00 27.00 n.a. n.a. Rudolf et al., 2005
Pine 44.55 6.30 2.56 1.60 11.43 27.67 2.88 0.32 Hamelinck et al., 2005
n.a. not analyzed

(Aronsson et al., 2014). The plant yield of willow can reach from 6.5 to 14.6 Mg d.m./ha. year
depending on species (Cunniff et al., 2015).
Black locust (Robinia pseudoacacia L.) is a fast-growing tree, native from the south-eastern
United States, but it has been widely planted elsewhere in temperate North America, Europe and
Asia. Gonzlez-Garca et al. (2011) show that some regions Italy and other European countries
have a high potential to increase the black locust biomass crops for the purpose the bioethanol
production. However, the cellulose concentration of black locust is similar or slightly lower in
comparison to other fast-growing and short-rotation energy crops (Table 3.3).
3.1.2 Perennial herbaceous energy crops

The major perennial herbaceous energy crops that have been selected for bioethanol production are
switchgrass (Panicum virgatum), miscanthus (Miscanthus spp. Anderss.), canary grass (Phalaris
arundinacea), giant reed (Arundo donax L.), alfalfa (Medicago sativa L.), and Napier grass (Pen-
nisetum purpureum) (Hill et al., 2006; Barnes et al., 2007; Hadar, 2013; Chandel & Singh, 2011).
The advantages are that the perennial no-food crops can be grown on marginal lands and they do
not require advanced agricultural treatments (Scordia et al., 2014).
Panicum virgatum or switchgrass is a warm-season perennial grass desirable for high productivity
across many environments, and suitable for marginal and erosive land. Moreover, it has relatively
low water and nutrient requirements and boasts positive environmental benefits (Parrish & Fike
2005; Bouton, 2007). There are also newer varieties of switchgrass with improved biomass yield
and chemical composition (Vogel et al., 1996; Burns et al., 2008a; Burns et al., 2008b). The
dry matter of switchgrass varies from 4.2 to 13.0 Mg/ha, depending of the year of cultivation,
fertilization and soil quality (Brown et al., 2015). Switchgrass has been identified as a good model
bioenergy species, due to its high yield, high nutrient-use efficiency, and broad geographical
distribution. Furthermore, it also has good attributes in terms of soil quality and stability, cover
value for wildlife, and low inputs of energy, water and agrochemicals (Nag & Manchikanti, 2008).
Fifteen times more bioethanol can be generated from switchgrass than from corn, for this reason
switchgrass may entail more profits than conventional crops for specific area (Nag & Manchikanti,
2008).
Miscanthus, a C4 grass native to Asia is viewed as a model herbaceous biomass feedstock for
Europe (Lewandowski et al., 2003). It has been evaluated as a bioenergy crop in Europe and is
grown in several European countries (Nag & Manchikanti, 2008). It is also a promising feedstock
for ethanol production (Arnoult et al., 2015). The perennial miscanthus crop is characterized by
high yield, low input and low environmental impacts. Miscanthus is adapted to a wide range of
climatic and soil conditions (Chapman, 1996) and efficiently recycles N between the above ground
biomass and storage structures (rhizomes) below ground (Lewandowski & Schmidt, 2006).
Miscanthus giganteus as a sterile hybrid genotype from Miscanthus sacchariflorus and Mis-
canthus sinensis has attracted attention and is widely used in Europe and recently also in North
America for productivity trials (Brosse et al., 2012). M . giganteus prefers sandy soil and sandy
loam, with a high content of organic matter. Heaton et al. (2008) showed that miscanthus could
provide 260% more ethanol per hectare than corn grain. However, M . giganteus can not produce
high quantities of biomass under various climates because it is sensitive to heavy frost (Zub et al.
2012) and a lack of water (Cosentino et al., 2007).
Miscanthus can be cultivated for up to 25 years, during which miscanthus biomass is produced
in two phases: a yield-building phase, where the biomass gradually increases, and an adult phase
often described as a plateau phase, where the biomass production is maintained (Lewandowski
et al., 2003; Zub & Brancourt-Hulmel, 2010; Arnoult & Brancourt-Hulmel, 2014). Miscanthus
yielded 33% more biomass than Kanlow switchgrass (18.1 vs. 14.1 Mg/ha) grown on a heavy clay
soil in south-western Germany (Boehmel et al., 2008).
The range of harvestable M. giganteus yields can be between 5 and 55 Mg/ha, making it one of the
most productive land plants in temperate climates (Heaton et al., 2010). The aboveground biomass
production of 49 Mg dm/ha was recorded for a M . giganteus clone in France under irrigated
conditions (Tayot et al., 1995). Generally the Miscanthus species differ in biomass production and
biomass components, with the average values 12 and 18 Mg d.w./ha for the winter harvest during
the second and third years, respectively. In Germany and Denmark, yields are 1330 Mg/ha for
310 years old plantation (Nag & Manchikanti, 2008).
Fischer et al. (2005) proposed six suitability classes used in the presentation of the results reflect
the performance of the best adapted species in each land unit. The highest miscanthus biomass
yields were estimated for Slovenia (27.5 Mg/ha) and Russia, west of the Ural (28.1 Mg/ha) (Fischer
et al., 2005). According to Clifton-Brown et al. (2001) in experimental plots harvested for 3 years in
Denmark, the mean was 9.1 Mg/ha, while in the case of experimental plots irrigated and harvested
for 3 years in Portugal, the mean was 25.2 Mg/ha.
M . giganteus seemed to be the best biomass producer, when compared with the M. sacchari-
florus and M. sinensis species. However, the maximum biomass production of 31.9 Mg d.w./ha was
recorded for a hybrid composed of two M. sinensis clones (EMI no. 7) in Portugal with irrigation,
compared with a 30.6 tDM/ha maximum for a M . giganteus clone (Greef et Deu) in Italy with
irrigation as well (Arnoult & Brancourt-Hulmel, 2015).
The cellulose and lignin contents in miscanthus biomass increased from 40.6 to 46.4% dry matter
and 8.0 to 9.4% dry matter on average between the autumn and winter harvests, respectively. In
contrast, the hemicelluloses content tended decreased with the averages 29.4 and 28.8% of dry
matter during the autumn and winter harvests, respectively (Hayes, 2013). High cellulose content
in M . giganteus and M. sacchariflorus species is preferred from the view point of biochemical
processes, such as hydrolysis, fermentation. However, high lignin content, can reduce efficiency
for these processes. One M . giganteus clone (EMI08) and M. sinensis species showed lower
lignin content and, therefore, may be particularly interesting for biochemical processes (Arnoult &
Brancourt-Hulmel, 2014).
Reed canary grass as a highly productive perennial grass for Northern Europe (El-Bassam, 1998;
Lewandowski et al., 2003). Reed canary grass is rhizomatous and can be cultivated in low value
areas, such as bogs after peat production, and on fields which are not needed for food production
(Kallioinen et al., 2012). It grows as a tall coarse grass to a height of 1.53.0 m (Dien et al., 2012).
Dry matter of reed canary grass content is 525 g/kg (Williams & Shinners, 2014). The yield of
reed canary grass is 1 Mg/ha in soils with low nitrogen content and unfavorable weather conditions
for plant growth. In soils with nitrogen contents of more than 0.6%, the average dry matter yield
of reed canary grass is about 67 Mg/ha (Kukk et al., 2011). Biomass yield of reed canary grass
varied considerably among harvest treatments, locations, and years, ranging up to 12.6 Mg/ha. Dry
matter percentage ranged from 37% for spring-harvested biomass to 84% for prewinter biomass
(Tahir et al., 2011).
Results from research on cropping-systems in southern Germany indicated that perennial
biomass systems based on miscanthus, switchgrass, or willows (Salix schernii E. Wolf viminalis)
could be as productive as energy maize with lower energy inputs (Boehmel et al., 2008). Nitrogen
fertilizer was the most energy-intensive input and accounted for 41 to 64% of energy inputs for
annual crops and 17 to 45% of inputs for perennials. The willow dry matter yield is about 22 Mg/ha
(harvest every three years). The productivity of willow plantations in Sweden is 89 Mg d.w./ha
year (Majtkowski, 2007). Biomass production is approx. 5 times higher than the annual growth of
wood in the forests. The composition of willow is comparable with others short rotation coppices
(Table 3.4).
Energy plants with yields in Poland (Journal of Laws 2007/55/364) are: willow 8 Mg/ha d.w.;
rosa multiflora (Rosa multiflora L.) 12 Mg/ha; black locust (Robinia pseudoacacia L.) 8 Mg/ha;
poplar (Populus spp.) 10 Mg/ha; alder (Alnus spp.) 8 Mg/ha; birch (Betula spp.) 8 Mg/ha; and
hazel (Corylus avellana L.) 8 Mg/ha. In view of the most traditional trees growing in the Polish
forests, there are no level reference yields available. This includes species such as pine (Pinus sp.),
oak (Quercus sp.), spruce (Picea sp.), beech (Fagus sylvatica L.), and fir (Abies sp.).
To sum up, Table 3.5 shows values of theoretical ethanol yield for biomass from selected perennial
grasses.
Table 3.4. Main chemical compositions of several biomass feedstocks.
Hemicelluloses
Cellulose Xylan Galactan Arabinan Mannan Lignin Extractives Ash

Feedstock % % % % % % % % Reference
Crop residues
Corn stover 36.1 21.4 2.5 3.5 1.8 17.2 n.a. 7.1 Kim et al., 2005
Corn stover (Zea mays L.) 34.61 18.32 0.95 2.54 0.40 17.69 7.74 10.24 EERE
Corn stover 37.50 21.70 1.60 2.70 0.60 18.90 n.a. 6.30 Lee et al., 2007
Corn stover (whole crop) 33.18 18.94 2.17 3.13 1.12 22.1 n.a. 3.37 Liu et al., 2010
Wheat straw 32.64 19.22 0.75 2.35 0.31 16.85 12.95 10.22 EERE
Wheat straw 37.60 19.50 1.10 2.80 0.60 14.50 n.a. 6.40 Lee et al., 2007
Rye straw 33.12 19.46 0.31 2.47 0 19.80 n.a. 6.15 Sun & Cheng, 2005
Grasses
Switchgrass 30.97 20.42 0.92 2.75 0.29 17.56 16.99 5.76 EERE
Switchgrass 37.30 22.80 1.40 3.10 0.30 19.10 n.a. 5.90 Lee et al., 2007
Switchgrass 37.8 24.9 1.1 3.4 0.4 21.4 17.0 5.8 Wiselogel, 1996
Switchgrass 31.98 21.09 0.95 2.84 0.3 18.13 17.54 5.95 Hamelinck et al., 2005
Bermudagrass 32.36 19.37 1.09 4.33 0 20.33 4.17 Sun & Cheng, 2005
Giant reed 35.7 18.6 0.6 1.6 0.2 25.0 n.a. 3.7 Scordia et al., 2011
Bagasse 39.01 22.05 0.46 2.06 0.35 23.09 3.78 3.66 EERE
Bagasse 32.00 18.00 0.50 1.60 0.20 25.10 n.a. n.a. Soudham et al., 2013
Bagasse 36.00 21.40 0.75 1.96 0.80 17.97 n.a. n.a. de Carvalho et al., 2015
Miscanthus 38.2 19.0 0.4 1.8 n.a. 25.0 5.6 2.0 de Vrije et al., 2002
n.a. not analyzed

Table 3.5. Theoretical yield of ethanol production.
Theoretical ethanol Theoretical ethanol

Substrate yield (Mg/ha) yield (L/ha) References
Arundo donax 11.13 7908 Scordia et al., 2014

Miscanthus giganteus 6.65 6210
Saccharum spontaneum 9.25 6933
White clover Trifolium Repens L. 2.38 530570 Springer & Aiken, 2015
Switchgrass Panicum virgatum L. 2980 Liu et al., 2014
Switchgrass Panicum virgatum L. 18705600 Liu et al., 2015
Sweet sorghum Sorghum bicolor 21295696 Smith et al., 1987
L. Moench
Switchgrass Panicum virgatum L. 17493691 Schmer et al., 2012
Sugarcane bagasse 8478.6 Lima et al., 2014
Panicum maximum 8571.0
Pennisetum purpureum 11529.4
Brachiaria brizantha 6231.4
Eucalyptus grandis bark 7083.5
Eucalyptus urophylla Eucalyptus 7230.3
grandis bark
Figure 3.2. (a) Quantities of wasted crops potentially available for ethanol production (Tg); (b) Potential of
ethanol production from wasted crops (GL).
3.1.3 Residues and waste

Residues can be divided into primary, secondary and tertiary residues. Primary residues are pro-
duced during the harvesting of crops or timber. They comprise of agricultural residues like straw
and stover, as well as forestry residues like treetops, branches, and stumps. Secondary residues are
created during the processing of crops into food products or the production of other biomass based
materials. Feedstocks in this category include nutshells, bagasse, presscake, and fruit bunches,
as well as sawdust, bark and scrap wood. Tertiary residues include post consumer residues that
are derived from consumption of biomass based products, e.g. municipal solid waste (Ibeto et al.,
2011).
Global potential of bioethanol production from wasted crops defined as crops (i.e. corn, bar-
ley, oat, rice) lost during handling, storage and transport and lignocellulosic biomass such as
crop residues and sugar cane bagasse for producing bioethanol was estimated by Kim & Dale
(2004). The authors show that the potential of bioethanol production from crop residues and
wasted crops (491 GL) is about 16 times higher than world ethanol production (31 GL) (Fig. 3.2b).
Figure 3.3. (a) Quantities of lignocellulosic biomass potentially available for ethanol production (Tg);
(b) Potential ethanol production from lignocellulosic biomass (GL).
Among lignocellulosic biomass, the rice straw is potentially the most favorable feedstock, which
constitutes about 50% of total dry lignocellulosic residues (1.5 Pg) available for ethanol pro-
duction (Fig. 3.3a). Crop residues are responsible for 90% of the total potential bioethanol
production.
Fruit waste is generated in large quantities from the processing of agricultural products. Over
115 million tons of citrus fruits are produced annually, and about 30 million tons are processed
industrially for juice production. After industrial processing, citrus peel waste accounts for almost
50% of the wet fruit mass (Choi et al., 2015). Fruit and citrus peel waste contains a high concentra-
tion of sugars, including sucrose, glucose, and fructose, and structural compounds, like cellulose
and hemicelluloses. The sugars are suitable for bioethanol production (Snati et al., 2015), although
the peels also contain compounds that can inhibit the process, such as D-limonene. Choi et al.
(2015) consider D-limonene to be a fermentation inhibitor, and obtained about 12 times higher
bioethanol production after removing it from fruit peels.
Municipal solid waste fractions, that can be converted into bioethanol are paper and card-
board. Cardboard is usually produced from cellulose mass after the removal of hemicelluloses
and lignin. The polysaccharides content of cardboard is about 70% of dry weight. This feedstock
easily undergoes saccharification without chemical pretreatment. Municipal solid and industrial
wastes are readily available, and in contrast to agricultural waste, they are produced year-round
and their collection and transport is usually well organized. Eleazer et al. (1997) showed that the
concentration of cellulose in used office paper was 87.4 wt%, while hemicelluloses and lignin
8.4 wt% and 2.3 wt%, respectively. Komilis & Ham (2003) reported that the office paper, which is
a fraction of municipal waste contains 65.4 wt% cellulose, 7.4 wt% hemicelluloses, and 16.8 wt%
lignin.
The bioethanol from lignocellulosic material is perceived more positively, as its production
only marginally competes with food and feed production, especially if agricultural or forest waste
products are used. The use of lignocellulose for bioethanol production requires more complex tech-
nological processes, both during the feedstock preparation and fermentation. Organic wastes and
residues may fluctuate and are affected by market growth, although climate and other factors
have influences, especially when considering the primary sources (Kang et al., 2014). How-
ever, relying on waste products for large-scale production is very challenging due to expensive
logistics. Furthermore, dedicated crops would induce long-term land use change, which causes
potential indirect competition with food production. For these reasons, the contribution of biofuels
to transportation in the long term should remain limited to a reasonable percentage.
Table 3.6. Starch content in algae.
% starch
Algal source (g/dry weight) Reference
Chlamydomonas reinhardtii UTEX 90 53.0 Kim et al., 2006

Chlorella sp. TISTR 8262 21.5 Rodjaroen et al., 2007
Green algae NKG 121701 >50.0 Matsumoto et al., 2003
Oscillatoria sp. TISTR 8869 19.3 Rodjaroen et al., 2007
Synechococcus sp. 15.0 Spolaore et al., 2006
3.2 Algae biomass

Algae constitute a potential third generation feedstock for bioethanol production. Microalgae
like Chlorella, Dunaliella, Chlamydomonas, Scenedesmus, and Spirulina are known to contain
a large amount (>50% dry weight) of starch and glycogen, which are useful as raw materials for
ethanol production (Ueda et al., 1996). Microalgae can also assimilate cellulose, which can also
be fermented to bioethanol (Chen et al., 2009).
Macroalgae can also be utilized for ethanol fermentation by converting their storage material
to fermentable sugars (Adams et al., 2009). The enzymatic hydrolysis of algal cellulose is simple
because of the complete absence or near absence of lignin. Macroalgal genera, such as Laminaria,
Saccorhiza, and Alaria belong to the brown algal group, and their main energy reserves are
laminarin and mannitol (Nobe et al., 2003; Adams et al., 2009; Horn et al., 2000a). Table 3.6 shows
the starch content in algae. Red algae, such as Gelidium amansii, which are composed of cellulose,
glucan and galactan, can also serve as a potential feedstock for bioconversion to ethanol (Wi et al.,
2009; Kim et al., 2010; Yoon et al., 2010).
There are several commercial advantages of algal bioethanol production that have interested
researchers and entrepreneurs around the world. These include the following: (i) algae bioethanol
production does not need to compete with food production in land, marine and freshwater envi-
ronments (Parker et al., 2008), (ii) the content of carbohydrate in algal cells is abundant, and
carbohydrates such as starches and sugars can be fermented to produce bioethanol, (iii) algae
have no lignin and little hemicelluloses, which increases hydrolysis efficiency and fermentation
yield (Douskova et al., 2009; Rosenberg et al., 2008); thus, the use of algae can reduce the cost
of bioethanol production. The non-carbohydrate part of algal biomass can potentially be used to
derive co-products.
4 BIOMASS FEEDSTOCK FOR THE SECOND AND THIRD GENERATION

BIODIESEL PRODUCTION
4.1 Non-edible oil seed

Among non-edible feedstocks, there are many crops and tree-borne oilseed plants, such as karanja,
neem, and jatropha, which have been underutilized due to the presence of toxic components in
their oils.
Jatropha curcas produces seeds with a high oil content (Martnez-Daz et al., 2015). The plant
is grown in India, Indonesia, Thailand, the Philippines (Demirbas, 2007; Krbitz, 1999), China,
Brazil, and North and Central America (Martnez-Daz et al., 2015). Jatropha grows in hot, dry,
tropical climates (Dorado, 2008). The tree produces fruit for 3040 years, with a yield of about
7 tons of seed per hectare. The oil content of seeds is 3040%. One hectare can produce 2.2 to
2.7 tonnes of oil, at an efficiency of cold pressing of about 91%. Approx. 4 kg of jatropha seeds
can yield 1 liter of biodiesel. Pomace is a by-product, which can be composted and used for soil
fertilization due to its high nitrogen content.
Table 3.7. Oil composition of various oils from non-edible plants.
Oil Eicosadi- Eicosa-

content Palmitic Stearic Behenic Arachidic Palmitoleic Oleic cis-Vaccenic Eicosenoic enoic acid trienoic Erucic Linoleic Linolenic Lignoceric
Type/species (%) C16:0 C18:0 20:0 22:0 C16:1 C18:1 cis 18:1 C20:1 20:2 acid (20:3) 22:1 C18:2 C18:3 C24:0 References
Jatropha
J. curcas 34.135.8 13.016.0 6.08.0 0.70.8 38.040.0 1.01.3 37.038.0 Barros et al.,
2015
J. mollissima 17.119.4 10.013.0 7.09.0 0.50.6 17.019.0 0.91.2 58.063.0
J. gossypifolia 20.523.6 8.09.0 5.06.0 0.60.9 14.015.0 0.60.7 68.072.0
Pongamia 3.77.9 2.48.9 44.571.3 9.512.4 10.818.3 Karmee &
pinnata Chadha, 2005
Camelina 5.4 2.6 0.25 1.4 14.3 16.8 2.9 14.3 38.4 Frhlich &
sativa Rice, 2005
Camelina 2.35 6.43 2.57 1.24 14.90 2.12 1.61 1.62 16.90 35.20 Abramovic &
sativa Abram, 2005
Karanja 3.77.9 2.48.9 44.571.3 10.818.3 1.13.5 Dorado, 2008
Table 3.8. Fatty acids composition of algae used for bioethanol production.
04:0 06:0 08:0 10:0 11:0 11:1 12:0 12:1 13:0 14:0 14:0 iso 15:0 15:1 16:0 16:0 iso 16:1 16:2 16:3 17:0 17:1 18:0 18:1 18:2 18:3 20:0 20:2 20:4 20:5 22:6 24:0
Type/species %FA
Chlorococcum 0.74 5.92 13.26 1.35 29.42 7.3 6.85 18.36 5.42 6.55
infusionuma
Porphyridium 0.2 0.4 0.6 0.2 26.8 2.6 0.33 0.63 ; 0.54 3.92 0.62 12.82 25.41 0.11
cruentumb
Chlorococcum 0.203 1.016 3.807 13.117 9.916 36.12 0.7013 , 0.449 2.0885 6.322 1.5924 14.036 7.077
sp.c 3.5654
Chlorella 0.2 2.77 0.26 1.39 2.17 0.87 0.41 1.03 0.69 1.7 3.53 14.42 4.04 5.34 4.9 0.12 0.27 1.57 17.62 11.972 15.791 0.14 0.301 0.22
vulgarisd
1 n 3; 2 n 6; 3 n 7; 4 n 9; 5 n 10; 6 n 9, 12; 7 n 9, 12, 15

a Karemore et al., 2013,
b Durmaz et al., 2007,
c Mahapatra & Ramachandra, 2013,
d tles & Ruhsen, 2001
Use of oil from karanja (Pongamia pinnata) has also been studied. Seeds of P. pinnata contain
about 35% oil with a high concentration of free fatty acids (up to 20%) (Naik et al., 2008). A
single tree provides 990 kg of seed, indicating that the seed yield potential of 1 hectare of culti-
vation is 9009000 kg (assuming 100 trees/ha). Approximately 25% of this mass is recoverable oil
(Karmee & Chadha, 2005).
For biodiesel production, edible oils should be replaced by lower-cost and reliable feedstocks
such as non-edible plant oils from economic and social reasons (Bankovic-Ilic et al., 2012). There
are also certain edible plants, from which biodiesel production is more economically reasonable than
from rapeseed oil or soybean oil. These low cost edible oils are cardoon oil (Cynara cardunculus),
Ethiopian mustard oil (Brassica carinata), camelina (Camelina sativa), and tigernut oil (Cyperus
esculentus).
Camelina sativa is a spring annual oilseed plant of the genus Cruciferae that grows well in
temperate climates, and matures earlier than other oilseed crops.
In some climates, biodiesel production with the oilseed crop Camelina sativa can be cheaper
than with rapeseed. Camelinas seed yield can reach 1.4 Mg/ha (Eberle et al., 2015), and the seeds
contain 3647% oil with 90% unsaturated fatty acids (Li & Sun, 2015), which is far superior to
typical oil content in soybeans (1822 wt.%).
Camelina oil yields an average of 420640 L/ha, and the protein and fiber content in its meal
byproduct is similar that in soybean meal (Retka-Schill, 2008b; Sawyer, 2008). Camelina is used
for biofuels production because it is productive on marginal land and not widely used for food; it
can be grown as a rotation or fallow crop, harvested and processed with existing equipment and
infrastructure; and it is extremely easy to transform because it is amenable to metabolic engineering
of novel traits. Oil composition obtained from various non-edible plants is given in Table 3.7.
4.2 Spent oil and animal fats

There has been a recent increase in the popularity of spent oil, known as waste cooking oil (WCO) or
waste frying oil (WFO), and animal fat wastes (AFWs), which are made up mostly of triglycerides.
In Mexico, the potential of biodiesel from waste frying oil is estimated between 1.5 and 3.3%
of petro-diesel consumption for the road transport sector and can reduce between 1.02.7% of
CO2 -associated emissions (Sheinbaum-Pardo et al., 2013). In 2010, China produced 13.74 million
tonnes (Mt) of waste oil, including 6.58 Mt of gutter oil, 1.55 Mt of acid oil, and 5.61 Mt of rice
bran oil (Liang et al., 2013).
Waste cooking oil is obtained after using edible vegetable oils, such as palm, sunflower, and
corn oils, and animals fats which have been heated and used for cooking a wide variety of food
such as meat, fish or vegetable products. Afterwards, they become a disposal problem. The United
States alone generates approximately 10 million tons of WCO annually. In the EU countries, the
total waste cooking oil production was approximately 0.71 million Mg/year (Kulkarni & Dalai
2006; Arjun et al., 2008).
One of the drawbacks of using WCO for the production of biodiesel is that it contains several
impurities, such as free fatty acid and water, which must be treated before transesterification
because of their significant adverse effects on the process.
Animal fats are primarily derived as by-products from meat animal processing facilities and by
the rendering process. The main animal fats include tallow from processing cattle, lard and choice
white grease from swine processing, and poultry fat from the processing of chicken, turkey, or
other birds (Adewale et al., 2015).
4.3 Algae biomass

Biofuels made from renewable resources could be a more sustainable alternative, particularly
if sourced from organisms, such as algae, that can be farmed without using valuable arable land
(Georgianna & Mayfield, 2012). Algal biocrude can be successfully converted into diesel, gasoline,
and/or aviation fuel with the appropriate catalysts and treatments (NAABB Final Report, 2014).
Figure 3.4. Microalgae biodiesel value chain stages (based on Mata et al., 2009 and Medipally et al., 2015).
Algae can be grown on fresh or marine water, and even in association with wastewater treatment
plants or industrial parks where their cultivation offers the additional benefit of bioremediation
(Leite et al., 2013). Algae offer many advantages and have the potential to provide orders of
magnitude more oil per acre of land than traditional oil seed crops (Chisti et al., 2007). As an
example, the palm oil containing 36% wt. yields of 5,366 L oil/ha. year, whereas biodiesel produc-
tivity is 4,747 kg biodiesel/ha. year. For microalgae with medium oil content (50%), the values are
97,800 L oil/ha. year and 86,515 kg biodiesel/ha. year, respectively. Higher values can be obtained
for microalgae with high oil content (70%), i.e. the oil yield 136,900 L oil/ha. year and biodiesel
productivity 121,104 kg biodiesel/ha. year (Medipally et al., 2015).
Most common algae accumulate lipids between 20 and 50% by weight of dry biomass. According
to Mata et al. (2010), lipid content in % dry weight biomass for marine and freshwater microal-
gae species was shown in brackets for Chlorella sp. (1048), Crypthecodinium cohnii (2051),
Dunaliella sp. (1867), Isochrysis sp. (7.133), Nannochloris sp. (2056), Nannochloropsis (12
53), Neochloris oleobundans (2965), Nitzschia sp. (1647), Phaeodactylum tricornutum (1857),
Porphyridium cruentum (919/61), and Tetraselmis sp. (1315). Microalgae can produce lipids as
a storage product in the amount 50% to 60% dry weight (Griffiths & Harrison, 2009).
Microalgae biomass and biofuel production can be developed at two major phases that involve
upstream and downstream processes (Fig. 3.4). The upstream phase involves different cultivation
technologies to maximize biomass quality and quantity, whereas the downstream stage puts empha-
sis on harvesting technologies and sustainable production of biofuel (Medipally et al., 2015).
Microalgae biodiesel value chain stages is shown in Fig. 3.4.
Different species of microalgaes have varied ability of oil production, and Table 3.8 gives the
related information. The composition of fatty acids of the different microalgae species is also sig-
nificant. These are composed of saturated and unsaturated fatty acids with 1222 carbon atoms,
some of them of w3 and w6 families. Different nutritional and environmental factors, cultiva-
tion conditions and growth phases may affect the fatty acid composition. For example, nitrogen
deficiency and salt stress induced the accumulation of C18:1.
Nascimento et al. (2013) tested 12 microalgae strains by applying, as selective criteria, the
volumetric lipid productivity and the fatty acid profiles. Volumetric lipid productivity varied
among strains from 22.61 to 204.91 mg/Lday. The highest lipid yields were observed for Chlorella
(204.91 mg/Ld) and Botryococcus strains (112.43 and 98.00 mg/Lday for Botryococcus braunii
and Botryococcus terribilis, respectively).
In the longer term, it has been suggested that some bioenergy/biofuel production (next genera-
tion) could be coupled with carbon dioxide capture and storage (Bio-CCS). However in everyday
use, next generation biofuels can be considered as more sustainable regarding the feedstock and
processes, because they offer greater levels of GHG reduction and do not compete with food
crops for land use. Out of all new generation feedstocks of biodiesel, microalgae are the most
promising one.
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Chapter 4
Outlook for advanced biofuels
Katarzyna Bukowska, Ewa Klimiuk & Artur Pawowski
1 INTRODUCTION
In recent decades the number of companies working for improvement of the existing technologies
of biofuel production or creation of the new technologies has been steadily growing. The different
types of biomass and manners of their pretreatment have been tested, as well as the methods
of feedstock conversion to biofuels. Taking the type of feedstock and the degree of complexity
technology as classification criteria, a rough division of conventional and advanced biofuels can be
made (IEA, 2011). The conventional technologies allow to commercially produce first generation
biofuels, i.e. sugar- and starch-based ethanol (Gnansounou & Dauriat, 2005; Bai et al., 2008;
Canilha et al., 2012), biodiesel produced from vegetable oil (Van Gerpen, 2005; Gupta & Bhojvaid,
2006; Tabatabaei et al., 1999), as well as biogas from silage and agriculture waste (Cave, 2013;
Blumenstein et al., 2015). The main disadvantage of the first generation biofuels is the food-versus-
fuel competition. This is the reason for rising food prices, caused by the increase in the production
of these fuels. In order to overcome this problem, non-edible biomass (lignocellulose) can be used
as feedstock. In this way the lignocellulosic material offers the potential for the development of
novel biofuels, called advanced biofuels (Sims & Taylor, 2008; Sims et al., 2010).
The European Commission defined an advanced biofuel as:
produced from feedstock that does not compete directly with food and feed crops,
having low CO2 emission or high GHG reduction,
reaching zero or low Indirect Land Use Change (ILUC) impacts of biofuels. ILUC impact relates
to the unintended consequence of releasing more carbon emissions due to land-use changes
around the world induced by the expansion of croplands for biofuels production.
Advanced biofuels are commonly referred to as the second- or third-generation (Cheng &
Timilsina, 2010, 2011; Sanna, 2014) including lignocellulotic ethanol, butanol, higher alcohols,
liquid biohydrocarbons and others. Advanced biofuel technologies are still either in the research
and development (R&D) stage, or demonstration phase (Advanced Ethanol Council, 2012-2013).
Development status is discussed in terms of technology readiness level (TRL), informing about
the maturity of technologies. TRLs are measured on a scale of 1 to 9, where TRL 1 corresponds
to basic research on a new invention or concept, and TRL 9 corresponds to a fully commercial-
ized technology (Nattrass, 2014). For example commercialization status of lignocellulosic ethanol
equals 8, while for isobutanol it varies between 7 and 8, and in the case of farnesene amounts to 7
(E4tech, RE-CORD and WUR, 2015).
Conventional transportation fuels are composed of liquid hydrocarbons with different molec-
ular weights and chemical structures for gasoline, diesel fuel or jet fuel (Lee et al., 2008). The
entire transportation infrastructure (including engines, fueling stations, distribution networks, and
storage tanks) has been developed to take advantage of the properties of these fuels. Thus, instead
of producing oxygenated biofuels from biomass (such as ethanol) an attractive alternative is to
generate fuels chemically similar to those derived from oil (Serrano-Ruiz & Dumesic, 2011). Such
biofuels are characterized with high energy content and physicochemical properties comparable
to fossil fuels, such as low oxygen content, low water solubility, and a high degree of saturation.
63
Some authors defined them as drop-in biofuels, due to the full compatibility with existing fuel
infrastructure.
There are several ways to produce drop-in biofuels, including: hydroprocessing of lipid feedstock
from oil crops, algae or tallow, the thermal conversion of lignocellulosic biomass to gas or oil and
next catalytically upgrading the products to hydrocarbon fuels (Canabarro et al., 2013; Damartzis &
Zabaniotou, 2011; Hu et al., 2012; Alonso et al., 2010; Sreekumar et al., 2015). To date, the
oleochemical-based processes have been the main supplier of the drop-in biofuels for commercial
application by the aviation sector (Cheng & Timilsina, 2011; Demirbas, 2007; Naik et al., 2010).
Production of drop-in biofuels requires more complex facilities and higher processing inputs in
particular hydrogen H2 than bioethanol and biodiesel (Karatzos et al., 2014). Consequently, one
should strive to overcome the techno-economic challenges and thus achieve competitive cost of
their production.
Microbial production of biofuels is an alternative for thermal or chemical methods. Progress
in metabolic engineering, and synthetic biology, have allowed the engineering of microbes to
produce advanced biofuels with similar properties to petroleum-based fuels, especially longer-
chain alcohols (C4) (Atsumi et al., 2008) fatty acid-based fuel molecules (alka(e)nes and fatty
alcohols) and isoprenoids (Tippmann et al., 2013; Howard et al., 2013; Lin et al., 2015; Schirmer
et al., 2010; Beller et al., 2015).
The review discusses the basic processes used for producing advanced biofuels and indicates
their status. The chapter also includes characteristics of individual types of biofuels, their properties
and potential application in transportation.
2 THERMAL PROCESSES
Thermochemical processes rely on the thermal conversion of biomass to fluid intermediates (gas or
oil). Their advantage is the possibility of using low-value biomass as feedstock. Thermochemical
processes are realized via gasification or pyrolysis (Bridgwater, 2003; Mohan et al., 2006; Goyal
et al., 2008). Next, the products are catalytically converted or upgraded to synthetic biofuels, which
can be used as transportation fuels for jet and diesel engines (Schablitzky et al., 2011).
Inconvenient biomass properties such as high oxygen content, low calorific value, hydrophilic
nature and high moisture content, can be improved by torrefaction. Torrefaction is based on the
removal of oxygen from biomass which aims to produce a fuel with increased energy density and
hydrophobicity by decomposing the reactive hemicellulose fraction (Van der Stelt et al., 2011; Chen
et al., 2015). Torrefaction is a form of pyrolysis (heating at 200300 C, in the absence of oxygen,
at atmospheric pressure) that converts biomass to bio-coal for the production of torrefied pellets.
Pellets can be used more easily as a high quality feedstock in gasification for high quality syngas
production than non-treated biomass (Uslu et al., 2008; Hu et al., 2012). The syngas produced from
sawdust pellets and torrefied pellets is tar-free and characterized by a relatively stable composition
and calorific value (LHV = 44.85.8 MJ/Nm3 ) (Dudynski et al., 2015).
2.1 Biofuels from syngas

Gasification
The thermochemical production of synthetic biofuels is integrated with gasification in biomass-to-
liquid (BTL) systems, in which syngas from gasification of biomass is converted to liquid fuels.
In the first stage gasification gas mixture rich in hydrogen and carbon monoxide is produced
by the partial oxidation of biomass at high temperature. A gasification process and characteristics
of different gasifiers are discussed in detail in the works of many authors (Basu, 2010; Hu et al.,
2012; Canabarro et al., 2013). The produced gas comprises CO and H2 and others gases such as
CO2 , CH4 , N2 in various concentrations (Bridgwater, 2003; Goyal et al., 2008; Basu, 2010). Gas
is contaminated by such constituents as particles, alkali metals, nitrogen components, tars, sulfurs
and chlorides. After the gas cleaning and conditioning, its composition is proper for production of
Outlook for advanced biofuels 65
Figure 4.1. A simplified schematic of catalytic conversion of syngas to fuels (modified: Spath & Dayton
(2003)).
synthetic biofuel (Hu et al., 2012). The synthetic biofuels can be produced in one of the following
processes: Fischer-Tropsch synthesis, methanol synthesis, ethanol synthesis, and mixed alcohols
synthesis (Fig. 4.1).
A brief characteristic of conversion process from biomass-to-liquid biofuels via the syngas route
is discussed below. The major steps in the production of Fisher-Tropsch liquid, methanol and high
mixed alcohols are showed in Fig. 4.2.
Fisher-Tropsch liquids
Among BTL, the production of Fischer-Tropsch liquids (FTL) from biomass has been given the
most attention. In Fischer-Tropsch (FT) synthesis, the hydrogen (H2 ) and carbon monoxide (CO)
in the syngas are reacted over a catalyst to form a wide range of hydrocarbon chains of various
lengths. The catalysts used are generally iron or cobalt based (Dry, 2001). The reaction is performed
at the pressure of 2040 bar and the temperature range of either 200250 C or 300350 C. The
typical FT products consist of high molecular weight paraffinic waxes as a main product and FT
fuels in the diesel and naphtha boiling range (Schablitzky et al., 2011). The products are very clean
and sulphur free and can be further converted to automotive fuels.
FT diesel can be produced directly, but a higher yield is achieved if FT waxes are produced first.
The upgrading of FT waxes to biofuels is achieved by a catalytic cracking process under the presence
of hydrogen via bifunctional catalysts (acid and hydrogenation function) (Bouchy et al., 2009). As a
result, the heavy hydrocarbons are converted into lighter products, for example naphtha, kerosene
and diesel oil. The Fischer-Tropsch naphtha is a drawback for gasoline production. It requires
aliphatic alkylation and catalytic reforming. The yield of each fraction of FT product, such as
gasoline, diesel and jet, can be modified either by process parameters or by adding additional steps
such as selective hydrocracking, isomerization, aliphatic alkylation or reforming (Fig. 4.2).
Methanol
Production of methanol from syngas involves reacting CO, H2 and a small amount of CO2 over
a copper-zinc oxide catalyst. Methanol synthesis is commercially available from ICI or Lurgi
Company. The process is carried out at 220300 C and 50100 bar (Broeren, 2013; Courty et al.,
Figure 4.2. Simplified schematic for production of fuels through the synthesis route.
1990). The basic reactions are:
The ratio of CO2 to CO should be optimized for methanol production. The synthesis of methanol
is most efficient when the feed gas contains the correct ratio of component, given by:
The CO2 /CO ratio can be adjusted via the water gas shift reaction, followed by hydrogenation of
CO2 . The water-gas-shift reaction is a catalytic process operating at 200475 C which involves
converting CO and steam to H2 and CO2 :
Shift conversion is also used to adjust the H2 :CO ratio. For methanol, the optimum ratio of hydrogen
to carbon monoxide is around 2.2. Excess CO2 may be removed by scrubbing. Formed methanol
can be dehydrated by a suitable catalyst (e.g. -Al2 O3 ) to dimethyl ether (DME).
Recently, sequential routes involved syngas (H2 , CO) production, methanol synthesis and the
subsequent upgrade to gasoline in so called methanol-to-gasoline (MTG) process or to olefins
methanol-to-olefin (MTO) process. Both processes are strongly dependent on the catalysts and/or
the process operating conditions (Galadima & Muraza, 2015). In the methanol-to-gasoline process,
methanol is partly dehydrated to produce an equilibrium mixture of methanol, DME and water,
followed by conversion to light olefins (C2 -C4 ) and in the final reaction step to higher olefins,
n/iso-paraffins, aromatics and naphthenes assisted by a zeolite catalyst (ZSM-5).
Methanol is converted to gasoline at high efficiency through the Mobil MTG process, using
zeolite ZSM-5 catalyst. This process has been commercially proven in New Zealand, where natural
gas is converted to methanol and then to gasoline.
Synthesis of higher alcohols

Higher alcohol synthesis (HAS), also known as mixed alcohols synthesis (MAS), produces a
mixture of alcohols. In this process, a substantial part of the alcohols should be longer than
methanol, which includes ethanol, propanol, butanol and some heavier alcohols (Forzatti et al.,
1991; Lu et al., 2014; Andersson, 2015).
The overall reaction can be described in the following way:
where n ranges from 1 to 8 (Forzatti et al., 1991).

This exothermic reaction requires significant amounts of cooling to keep the temperature in the
reactor at a constant level. One advantage is the ability to use syngas characterized by the lower
H2 :CO ratio than the one required for methanol synthesis. The exothermic nature of the reaction
dictates that the catalyst should be operated at high pressures and low temperatures. The key to the
development of mixed alcohol synthesis is the selective control of alcohols and efficient removal
of reaction heat via both catalyst and reactor innovation (Fang et al., 2009).
The mixed alcohol stream is degassed and dried before the alcohols are separated. Drying takes
place by adsorbing water in a molecular sieve. The adsorbed water is removed by flushing with
methanol and by depressurization. The methanol/water mixture is recycled back to the mixed
alcohol synthesis reactor. Two distillation columns are used to separate the higher alcohols from
ethanol. The first column separates methanol/ethanol from the higher alcohols. The second one
separates methanol and ethanol. The ethanol and the higher alcohols are sold as the product of the
process, whereas the methanol is recycled back to the alcohol synthesis section.
BTL technologies are still in research and development stages. Currently, there are various
commercial-scale gasification facilities that are either operational, under construction or in the
planning stages (Karatzos et al., 2014). While most of these facilities have been built for heat and
power generation, it is hoped that some of them will also be able to manufacture liquid biofuels.
In Europe the most of the biomass gasification activities are concentrated in Germany, Austria and
the Scandinavian countries.
2.2 Pyrolysis
Pyrolysis is a thermal decomposition of biomass occurring in the absence of oxygen. Fast pyrolysis
is crucial to maximizing bio-oil liquid yields at the minimizing expense of char and gas production
(Bridgwater, 2012). As a result, it is possible to obtain between 6075 wt% bio-oil, 1525 wt%
bio-char and 1020 wt% non-condensable gases.
Bio-oil is a dark brown and free flowing liquid fuel, composed of more than 300 different carbon
molecules (Mohan et al., 2006, Zhang et al., 2007). Its physical properties and the specific compo-
sition depend on the feed, the type of reactor and process conditions (Blin et al., 2007; Bardalai &
Mahanta, 2015). Among organics, hydroxyaldehydes, hydroxyketones, sugars, carboxylic acids,
esters, furans, guaiacols, and phenolics could be replaced (Elliott et al., 1990; Akhtar, 2011). An
essential ingredient of bio-oil is water, constituting 1030 wt%. The bio-oils contain about 40%
oxygen, in comparison to the typical maximum amount of 2% oxygen found in crude oil (Zhang
et al., 2007; No 2014). This affects the homogeneity, polarity, heating value, viscosity, and acidity
of the bio-oil.
The most important properties affecting bio-oil fuel quality are: incompatibility with conven-
tional fuels from the high oxygen content of the bio-oil, high solids content, high viscosity, and
chemical instability. The utilization of the oil requires a general decrease in the oxygen content,
which requires to separate the organic product from the water, increase the viscosity, and improve
the stability.
Physical upgrading of bio-oil includes filtration, reduction of the ash and alkali content in the
oil. Homogenization and lower viscosity of bio-oil can be achieved by the addition of polar solvent.
Especially methanol showed a significant effect on the oil stability. Bio-oils are not miscible with
hydrocarbon fuels, but they can be emulsified with conventional fuel with the aid of surfactants.
Such bio-oil can be used as a component for fuel production. Some work has recently been car-
ried out on homogenous blends of bio-oil, biodiesel and bioethanol (Alcala & Bridgwater, 2013).
Upgrading bio-oil to a conventional transport fuel such as diesel, gasoline, or kerosene, requires
a full deoxygenation and conventional refining. It corresponds to a large proportion of equipment
and production costs, including significant hydrogen gas (H2 ) inputs (Jones et al., 2009). There is
also an interest in partial upgrading to a product that is compatible with refinery streams (Huber
& Corma, 2007).
Two general routes for bio-oil upgrading have been considered: hydrodeoxygenation (HDO) and
zeolite cracking (Mortensen et al., 2011; Xiu & Shahbazi, 2012). HDO is a high pressure operation
where hydrogen is used to separate oxygen from the bio-oil, yielding a high grade oil product
equivalent to crude oil. Hydrotreating (i.e., treatment of the bio-oil at moderate temperatures and
high hydrogen pressures) is a commonly used method to achieve oxygen removal from bio-oils.
Several works on bio-oil HDO use catalytic systems, such as Co-Mo or Ni-Mo based catalysts
(Romero et al., 2010; Moberg et al., 2010). Bio-oils typically contain significant amounts of
lignin-derived phenols which, once transformed into aromatic hydrocarbons, are valuable gasoline
components. Full hydrotreatment yields a naphtha-like product that requires refining to derive
conventional transport fuels.
Catalytic cracking accomplishes deoxygenating occurring in the presence of zeolite catalysts
(Galadima & Muraza, 2015). Bio-oil deoxygenation is carried out at milder conditions and without
external hydrogen by processing the bio-liquid over acidic zeolites, in a route that resembles the
catalytic cracking approach used in petroleum refining. Under these conditions, bio-oil components
undergo a number of reactions involving dehydration, cracking and aromatization, and oxygen is
removed in the form of CO, CO2 and water. As a result, bio-oil is converted into a mixture of
aliphatic and aromatic hydrocarbons.
A recent concept that has attracted much interest is the decentralized production of bio-oil or
bio-oil-char slurries for transportation to a central process plant for gasification and synthesis of
hydrocarbon transport fuels, by for example Fischer-Tropsch synthesis, or alcohols. Commercial-
ization technology providers active in the supply chain include biomass gasification developers,
and syngas clean-up and catalyst providers (Bridgwater, 2013).
German-based Choren is one of the leading developers of the technology involving conversion
of biomass to liquids via the FT route. Choren has built a plant in Freiberg with 100,000 t/year BTL
fuel output, or around 1,520 odt/day biomass input. Timber, energy crops, straw, forest residues, saw
mill residues, agricultural residues and municipal solid waste are used as feedstock. Installation
operates in three-steps: Carbo-V gasification (pyrolysis of biomass and gasification pyrolysis
gas), raw gas clean up and conditioning and, finally, FT synthesis.
A number of pilot and small scale integrated demonstration plants are operating in Europe, includ-
ing REPOTEC/CTU and ECN producing bioSNG, and Chemrec and Bioliq producing bioDME.
Bioliq at Karlsruhe Institute of Technology (KIT) Air Liquide (Germany) is a complex plant in pilot
scale. The process comprises four stages: in the first stage, the dry residual biomass is subjected to
decentralized flash pyrolysis to form a substance so-called biosyncrude. It is then transported and
subjected to further central processing. In the centralized process, the high-pressure entrained flow
gasifier converts the biosyncrude into a tar-free synthesis gas at temperatures above 1200 C and
pressures of up to 80 bar. This synthesis gas is mainly composed of carbon monoxide and hydrogen.
By means of downstream hot gas cleaning, impurities such as particulate matter, chlorine, sulfur,
and nitrogen compounds are separated from the syngas. In the synthesis stage, this synthetic
gas is converted into customized fuels or basic chemical products (Luque et al., 2012). The list of
companies producing advanced biofuels in thermochemical processes is given in Table 4.1.
Table 4.1. Advanced biofuels produced in thermochemical processes (Karatzos et al., 2014; Damartzis &
Zabaniotou 2011; Lehto et al., 2013; Advanced Biofuels Project Database, 2011).
Biofuel
production
Name Feedstock(s) Conversion process Product (Mg/year)
Primus Green mixed biomass Biomass to gasoline gasoline 30

Energy (wood, miscanthus,
(Hillsborough, New switchgrass,
Jersey, USA) a) agricultural residues)
Range Fuels wood waste Biomass to methanol methanol then ether 12000
(Soperton, Georgia, via gasification
USA) a)
British municipal solid FT renewable drop-in 57000
Airways/Solena, waste fuel
East London UKa)
Choren Frieburg woodwaste/bagasse/ FT renewable diesel/jet 13000
Germany (Global)a) cane trash fuel
Choren Tech GmbH, 200000
Schwedt Germanyc)
Rentech (Rialto, FT renewable drop-in 773550
California, USA) a) fuel
CORE BioFuel wood waste Biomass-to gasoline gasoline 53000
(Houston B.C. via gasification
Canada) a)
ECN (Petten lignocellulosics Biomass to syngas syngas (SNG side 346
Netherlands)b) (clean wood and stream)
demolition wood)
ECN (Alkmaar, lignocellulosics Biomass to SNG via SNG 6500
Netherlands)b) gasification
Karlsruhe Institute lignocellulosics FT DME; gasoline-type 608
of Technology (KIT) fuel;
(Karlsruhe,
Germany)b)
Research Triangle lignocellulosics FT FT liquids; mixed 22
Institute (Research alcohols
Triangle Park,
United States)b)
Virent (Madison, pine residues, FT diesel-type 30
Wisconsin, United sugarcane bagasse hydrocarbons
States)b) and corn stover
Pyrolysis
Dynamotive Energy biomass Fast Pyrolysis, BioOil, BioOil
System (Geismar, Biomass Into GasOil Plus, CQuest
LA, USA) (BINGO) Biochar, UBA, UBB
Hydroreforming &
Hydrotreating
Ensyn Canada TBD woody biomass Pyrolysis (Fluid bio-crude
Malaysia (white wood and Catalytic Cracking)
bark), agricultural
residues (oil palm,
sugar cane) palm
waste
Continued
Table 4.1. Continued.
Biofuel
production
Name Feedstock(s) Conversion process Product (Mg/year)
KiOR Columbus woodchips pyrolysis renewable drop-in 187500

(Missisippi, Georgia, fuel
Texas) USA a)
BTG (Biomass rotating 44
Technology Group)
BioLiquids BV,
Netherlands d)
Trillium FiberFuels, mixed cellulose pyrolysis bio-oil
Inc. UK (Global)
Pyrolysis and
gasification
BTG Biofuels biomass pyrolysis and syngas
(Biomass gasification
Technology Group)
(Netherland)
a) Advanced Biofuels Project Database (2011).

b) Bacovsky et al. (2013).
c) Damartzis & Zabaniotou (2011).
d) Lehto et al. (2013).
3 MICROBIAL BIOFUELS PRODUCTION
3.1 Metabolic pathways as criterion classification of advanced biofuels

The advanced/drop-in biofuels can be produced from sugars in various biochemical pathways
(Zhang et al., 2011). These pathways can be loosely divided into: feed pathways, at which the
sugars are converted into the central metabolic intermediates as pyruvate and acetyl-CoA; and
product pathways, which converts intermediate formed to the chosen biofuels (Fischer et al., 2008;
Clomburg & Gonzales, 2010). In this first feed pathway, central intermediate is pyruvate, which
leads to the synthesis of isoprenoids and higher alcohols, while in second feed pathways acetyl-
CoA is central intermediate in ethanol, butanol or fatty acid production (Peralta-Yahya & Keasling,
2010; Rabinovitch Deere et al., 2013). Taking into consideration the product of pathways (Fig. 4.3),
advanced biofuels can be grouped into four classes (Rude & Schirmer, 2009):
1. alcohols synthesized in the traditional fermentative pathways such as ethanol, butanol and
isopropanol,
2. higher alcohols (C4-C8) produced in non-fermentative pathways,
3. fatty acid pathways as precursors of alka(e)nas, hydrocarbons and fatty acid esters,
4. isoprenoid-based biofuels produced either the mevalonate (MVA) or deoxyxylulose-5-phosphate
(DXP) pathways.
3.2 Production of alcohols via fermentative pathways

Ethanol
The commercial success of the grain ethanol industry has increased the interest in development of
fermentative alcohol production. Traditionally, ethanol is produced through fermentation of native-
producing microbes such as yeast Saccharomyces cerevisiae (Brandberg et al., 2004; Li et al.,
2015) and, to a lesser extent, the bacterium Zymomonas mobilis (Panesar et al., 2006).
Figure 4.3. A simplified schematic of advanced biofuels biosynthetic pathways (Peralta-Yahya & Keasling (2010); modified).
The advanced biochemical platform of bioethanol employs cellulose (and hemicellulose) from
plant fibers as feedstock instead of starch (Sun & Cheng, 2002; Hahn-Hgerdal et al., 2006; Lin,
Tanaka, 2006; Sanchez & Cardona 2008; Sarkar et al., 2012; Saini et al., 2015). Extensive research
on conversion of lignocellulosic materials to ethanol was conducted. The first step is pretreatment
of biomass, which involves delignification of the feedstock, using physical, physicochemical,
chemical, and biological treatment in order to make cellulose more accessible in the hydrolysis
step. Recently, the most common methods are steam explosion and dilute acid prehydrolysis. In the
second stage of hydrolysis, the cellulose is released from biomass and subsequently converted into
glucose, by acids or preferably cellulase enzymes. The conversion of cellulose and hemicelluloses
to ethanol can be expressed by the reaction of glucan (for hexoses) and xylan (for pentose) with
water:
The maximum theoretical yield of hexoses and pentoses is 1.136 kg and 1.111 kg per kg of glucan
and xylan, respectively. Genetically engineered fungi that produce large volumes of cellulase,
xylanase, and hemicellulase enzymes are under investigation.
Finally, the conversion of hexoses (C6) and pentoses (C5) to ethanol is as follows:
The maximum theoretical yield of both hexoses and pentoses is 0.511 kg ethanol and 0.489 kg
CO2 per kg sugar (Kang et al., 2014).
While most of the principles of advanced ethanol production are the same as in conventional
processes, the progress of genetic tools and metabolic engineering allowed optimization of this
productivity. S. cerevisiae, the yeast commonly used for the production of first generation ethanol,
cannot metabolize xylose. In recent times, the metabolic and evolutionary engineering strategies
have been extensively performed in constructing and enhancing the xylose fermentation capac-
ity of both laboratory and industrial S. cerevisiae strains. Additional research was conducted in
order to find microorganisms which can effectively ferment both types of sugars into ethanol with
Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis as promising candidates (He et al.,
2014). S. cerevisiae is more robust in large-scale fermentations compared to E. coli. It is relatively
tolerant to low pH and high concentrations of sugars, as well as fairly resistant to inhibitors (Hong &
Nielsen, 2012, Kim et al., 2012).
Thermophilic anaerobes are in the center of interest in regard to the biotechnological potential due
to their ability to produce ethanol from a broad range of substrates and the ability of some species
to degrade biopolymers such as cellulose, starch, and hemicelluloses including xylan (Taylor et al.,
2009; Chang & Yao, 2011; Scully & Orlygsson, 2015).
Novel developments include the simultaneous saccharification and co-fermentation (SSCF), and
the consolidated bioprocessing (CBP), which allow producing all required enzymes and ethanol
using a single type of microorganisms in a single reactor (Jouzani & Taherzadeh, 2015).
Commercialization of lignocellulosic ethanol

There are around six technology providers operating or planning full-scale demonstration plants
in Europe and around nine in the US. In addition, there are numerous technology developers at an
earlier stage of development (Nattrass, 2014). The cellulosic ethanol is produced in Canada, Brazil,
and Australia. Companies such as Iogen Corp. (Canada), POET (earlier called Broin), Du Pont
and Abengoa are building refineries that can process biomass and turn it into ethanol. Genencor,
Diversa, Novozymes and Dyadic produce enzymes which could enable manufacturing cellulosic
ethanol in the future (Menon & Rao, 2012). Many European plants plan on using wheat straw as
either the sole feedstock or along with other agricultural residues.
A worldwide leader Abongea Bioenergy owns and operates 14 bioethanol facilities throughout
United States, Europe and Brazil. One of commercial facilities which produce cellulosic ethanol
using wheat straw and corn stover is located in Hugoton KS (AEC, 2013). Prior to enzymatic
hydrolysis steam explosion pre-treatment is used (AEC, 2013). Another plant is located in Spain
(Babilafuente, Salamanca). It produces 5 million liters per year from 70 tons of agricultural residue
per day (mainly wheat straw).
In Europe, German cellulosic ethanol from agricultural residues is known as the Sunliquid
process developed by Clariant. The Sunliquid process involves: a hydrothermal pretreatment of
biomass, enzymatic hydrolysis integrated with the production of enzymes, simultaneous fermen-
tation of C5 and C6 sugars into ethanol and separation of ethanol by adsorption (Clariant, 2016).
The agricultural residues and dedicated energy crops are used as feedstocks.
In Sweden, SEKAB (Swedish Ethanol Chemistry AB) pilot plant started from the production
of 300400 liters of bioethanol per day from 2 tons of dry biomass. The feedstock is composed
mainly of forestry residues like wood chips from pine trees, also sugarcane bagasse, wheat, corn
stover and grass (Gnansounou, 2010). Danish Biotechnology and Engineering Company, BioGasol,
developed pretreatment technologies and C5 sugar fermentation for maximize ethanol production
(Langvad et al., 2010). The plant capacity is 10 Mg of ethanol per year. BioGasol has tested various
feedstocks including corn stover, corn fibre, corn cob, sugarcane bagasse, wheat straw and woody
biomass.
Mossi & Ghisolfi Group (M&G) from Crescentino (Italy) is among the companies which ethanol
on commercial scale. It is currently the worlds largest advanced biofuels refinery with a production
capacity of 75 million litres of cellulosic ethanol annually. The ethanol production is based on the
patented Proesa process, and uses Novozymes enzyme technology to convert wheat straw, rice
straw and Arundo donax to ethanol. Extracted lignin is used at a side line, where power is generated
to cover the facilitys energy needs.
The list of companies producing lignocellulosic ethanol and butanol is given in Table 4.2.
Butanol and isopropanol

Butanol and isopropanol are produced during the fermentation of mixed product in various strains of
Clostridium. Butanol is naturally produced through biological routes known as butanol fermentation
or acetone-butanol-ethanol (ABE) fermentation at typical ratios products of about 3:6:1 by weight
(Jones & Woods, 1986). Some strains produce isopropanol in addition to acetone, butanol, and
ethanol, while others produce isobutanol in place of acetone (Rabinovitch-Deere et al., 2012).
Various strains of Clostridium are able to convert a wide range of sugars in ABE fermentative
process with different titers (Zheng at al., 2015).
The metabolism of ABE producing clostridia is typically biphasic in batch culture, starting from
an acidogenic phase and followed by a solventogenic phase. In the acidogenic phase, acetic and
butyric acids are produced during the exponential growth of bacteria. The acetate and butyrate
are effluxed from the cells and accumulate in the media. As the organic acid accumulates, pH
drops to the lowest point during the fermentation. Which leads to switching of acidogenic phase to
solventogenic phase. This drop in extracellular pH significantly changes the cellular physiology.
Both acetate and butyrate are taken back into the cells, which are no longer dividing at exponential
rates and reassimilated onto coenzyme A. This influx of organic acids begins the solventogenic
phase, where the solvents (acetone, butanol and ethanol) are produced. During fermentation, carbon
dioxide and hydrogen are produced.
While most of the production principles of this advanced ethanol and butanol are the same as con-
ventional processes, the progress of genetic tools and metabolic engineering allowed optimization
of this productivity (Atsumi et al., 2008).
Commercialization of butanol
China is a leader in the effort to store-commercialize the ABE fermentation process. Over $200 mil-
lion has recently been invested in China to install 0.21 million Mg/year of solvent capacity, with
plans to further expand it to 1 million Mg/year. There are six major plants that produce per year
Table 4.2. Advanced biofuels produced in biochemical processes (Ethanolrfa, 2016; Bacovsky et al., 2013).
Fuel
Conversion process production
Name Feedstock(s) (microorganisms) (Mg/year)
Ethanol
Abengoa Bioenergy cereal straw (mostly barley and biochemical 4000
(Babilafuent, Salamanca, wheat)
Spain)
Abengoa Bioenergy Biomass of corn stover, wheat straw, switch biochemical 75000
Kansas LLC (Hugoton, USA) grass
Abengoa Bioenergy, S.A. agricultural and forest residues biochemical 40000
(Arance, France)
BioGasol (Aakirkeby, straw, various grasses, garden biochemical 4000
Bornholm, Denmark) waste
BP Biofuels (Jennings, USA) dedicated energy crops biochemical 4200
Clariant (Straubing, Mnchen, wheat straw and other biochemical 1000
Germany) agricultural residues
DuPont (Vonore, USA) lignocellulosic: corn stover, biochemical 750
cobs and fiber, switchgrass
Inbicon (DONG Energy) wheat straw biochemical 4300
(Kalundborg, Denmark)
INEOS Bio (Vero Beach, vegetative waste, waste wood, biochemical 24000
United States) garden waste
Mascoma Corporation (Rome, wood chips, switchgrass and biochemical 500
United States) other raw materials
Butanol
American Process (Alpena, biomass enzymatic hydrolysis 1410
Michigan USA, Global)
Butalco Switzerland (Global) biomass fermentation (yeast) 30
Butamax, Hull, UK (Global) sugar two-stage acid 481500
hydrolysis pretreatment
and fermentation
with native utilizes
Clostridium bacterium
Cobalt Biofuels (Sausalito, macroalgae fermentation 30
California, USA) (Clostridium sp.)
Gevo St. Joseph, Missouri, rice straw, corn stalk fermentation () 900000
USA, (Global)
Idemitsu Kosan (Japan) fermentation () 30
about 30000 Mg butanol from corn starch (Ni & Sun 2009). Most plants are located next to ethanol
plants to reduce utility and operating costs. Co-located operations tend to share effluent treatment
facilities based on anaerobic digestion (AD) (Green, 2011). Biogas, produced from the AD process,
is used for the generation of heat and power. Although not widely practiced, additional value can
be gained from the recovery of hydrogen from the fermentation exhaust gas (typically 1/10th of the
mass of produced butanol). In Brazil, HC Sucroqumica is an example of a sugarcane biorefinery
producing butanol (Mariano et al., 2013). This plant produces 8000 Mg solvent/year from sugar
cane juice and is located next to an ethanol distillery and sugar mill.
Butamax Advanced Biofuels, a joint venture of BP and DuPont, has developed an innovative
butanol production technology designed to convert the sugars from various biomass feedstocks,
Figure 4.4. Simplified pathways for biofuels production with central metabolic intermediates based on
Rude & Schirmer (2009).
including corn and sugarcane, using existing biofuel production facilities (Butamax, 2016).
Englewood-based Gevo (Colorado, USA) produces isobutanol, which is recovered from the broth
through flash evaporation. Biomass of corn, sugar, and beets is used for production of butanol.
Both Butamax and Gevo intend to build plants using lignocellulosic sugars in the future.
There are three main technology providers operating pilot or small scale demonstration plants
for the conversion of lignocellulosic feedstocks to butanol: Green Biologics, American Process
and Cobalt Biofuels (Nattrass, 2014). Cobalt Technologies utilizes Clostridium strains for con-
tinuous fermentation process using wood pulp and sugar cane bagasse as feedstock (Biobutanol,
2016). During pretreatment process C5 sugars are extracted from biomass without the use of costly
enzymes (Cobalttech, 2016). Microorganisms ferment sugars from cellulosic biomass to butanol,
including C5 sugars. Butalco in Fuerigen, Switzerland, has developed a technology to construct
strains of yeast that can metabolize C5 sugars, and proposes using only endogenous genes to
improve isobutanol production (Buelter et al., 2012; Donaldson et al., 2011; Festel et al., 2011).
3.3 Production of alcohols via non-fermentative pathways

In recent years, attention has been paid to higher chain alcohols (C 4) produced in non-
fermentative pathways call as the 2-keto acid pathways. In these pathways, 2-keto-acid intermedi-
ates are transformed to corresponding aldehydes and subsequently to alcohols by decarboxylases
and alcohol dehydrogenases, respectively (Atsumi et al., 2008). In nature, the mechanism for
aliphatic alcohol production is through the Ehrlich pathway in some yeast species. Saccharomyces
cerevisiae convert the keto acids in the amino acid pathways into fusel alcohols as a by-product of
fermentation. Propanol from isoleucine, isobutanol from valine and n-butanol from norvaline can
be produced in these pathways (Dickinson et al., 1997; Dickinson et al., 1998; Dickinson et al.,
2000). The carbon number (up to 5) of the alcohols derived from this type of pathway is limited by
the carbon number in the branched chain amino acid (Atsumi, 2008; Peralta-Yahya et al., 2010).
Atsumi et al. (2008) reported production of several higher alcohols in engineered E. coli, includ-
ing isobutanol, butanol, 2-methyl-1-butanol (2MB), 3-methyl-1-butanol (3MB). Isobutanol and
3-methyl-1-butanol are produced from 2-ketoisovalerate, which is an intermediate in valine biosyn-
thesis pathways. 2-keto-butyrate is an intermediate in other branched alcohols. Linear alcohols
ranging from 1-pentanol (C5) to 1-octanol (C8) were also produced from threonine overproducing
E. coli (Marcheschi et al., 2012). In the E. coli pathway, 2-ketoacids are converted to the correspond-
ing aldehyde with a 2-keto acid decarboxylase (KDC) and then to the alcohols using an alcohol dehy-
drogenases (ADH) by expression of promiscuous keto acid decarboxylase (kivd) from Lactococcus
lactis with alcohol dehydrogenase 2 (adh2) from S. cerevisiae (Lamsen & Atsumi, 2012).
However, unlike the fatty acid and isoprenoid syntheses, ketoacid chain extension reactions
(C > 5, C6-C8) are not naturally recursive. The recursive pathways are broadly defined as those
that catalyze a series of reactions in which the key, bond-forming functional group of the substrate
is regenerated in each cycle, allowing for a new cycle of reactions to begin. The two major enzymes
involved in these pathways are 2-isopropylmalate synthase (IPMS) in the leucine pathway and
acetohydroxy acid synthase (AHAS) in the valine pathway. An artificial pathway for 2-keto acids
elongation that uses an engineered isopropylmalate synthase to recursively condense acetyl-CoA
with 2-keto acids has been built (Felnagle et al., 2012). The carbon chain of 2-keto acids was
recursively elongated by an engineered leucine synthesis pathway from E. coli (EcLeuABCD).
Higher alcohols produced via various metabolic pathways are characterized by relatively higher
titers than other advanced biofuel molecules, even though they are considered to have better fuel
properties than ethanol. Of these, isobutanol is the closest to industrial use, and although it has a
similar energy content to butanol, its branching gives it improved properties, such as a better octane
number (a measure of a fuels resistance to knocking in spark ignition engines) (Tao et al., 2014).
Table 4.3 shows the production titer of fermentative/non-fermentative alcohols and fatty acids.
3.4 Fatty acid-based biofuels

Because of the ionic nature of their carboxyl group, fatty acids cannot be used directly as biofuel, but
they can be readily converted by microorganisms into non-ionic, hydrophobic molecules, such as
fatty alcohols, fatty-acid alkyl esters, alkenes and alkanes. Fatty acids are biosynthesized naturally
by a large, multienzyme system called fatty-acid synthase (FAS), which condenses malonyl-CoAs
into various lengths of fatty acyl esters with acyl-carrier protein (ACP). The fatty acid alcohols
could be produced by a sequential reduction of the fatty acid to the fatty alcohol, whereas alkanes
can be produced by a reduction of the fatty acid to the aldehyde followed by decarbonylation
(Tseng & Prather 2012; Kalscheuer & Steinbchel, 2003) or, alternatively, from further reduction
of the fatty aldehyde to alcohol and then to an alkane (Kalscheuer et al., 2006). Finally, fatty
acids could be converted to esters (biodiesel) via esterification with small alcohols (Runguphan &
Keasling, 2014).
The only genetically characterized alkane biosynthesis pathways pertain to the production of very
long chain alkanes (>C28) by Arabidopsis thaliana (Kunst & Samuels, 2009) and the production of
pentadecane (C15:0) and heptadecane (C17:0) by cyanobacteria (Chang et al., 2013, Kaiser et al.,
2013). New pathways for the biological production of different alkanes must therefore be developed.
Exploitation of fatty-acid metabolism is being pursued in a variety of host organisms. Fatty-acid-
derived compounds, such as FAMEs, fatty alcohols, alkanes and olefins, can be produced from
E. coli in single-step fermentation from carbohydrates.
3.5 Isoprenoid-based biofuels

Isoprenoids represent a diverse group of hydrocarbons synthesized from two universal precursors,
the isopentyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). These pre-
cursors can be produced from acetyl-coenzymeA (CoA) via the mevalonate pathway or from
glyceraldehyde-3-phosphate and pyruvate through the deoxyxylulose phosphate (DXP) (also
known as the methylerythritol pathway (MEP)). The MVA pathway has been more extensively
exploited for production of biofuel precursors such as farnesene (Rude & Schirmer 2009), bisabo-
lene (Peralta-Yahya et al., 2011), pinene (Sarria et al., 2014) and limonene (Alonso-Gutierrez et al.,
2013; Du et al., 2014). In addition to branched-chain hydrocarbons, this pathway can be used to
produce isopentanol (isoamyl alcohol) and its acetate ester, compounds proposed as additives for
spark ignition fuels (Hull et al., 2006).
Table 4.3. Production titer of fermentative/non-fermentative alcohols and fatty acids.
Higher titer
Native or heterologous reported
Class biofuels Type of biofuels Formula producers Pathway applied g/L References
Fermentative Ethanol E. coli TCS099 ABE 40 Trinh & Srienc, 2009

alcohols C 2 H6 O E. coli LY01 60 Yomano et al., 1998
Butanol E. coli CoA-dependent 30 Shen et al., 2011
C3 H9 OH
Isopropanol C. beijerinckii ABE 2.2 Survase et al., 2011

C3 H7 OH NRRL-B-593 4.9 Hanai et al., 2007
E. coli 13.6 Jojima et al., 2008
E. coli 143 Inokuma et al., 2010
E. coli 7.6 Dai et al., 2012
C. acetobutylicum Rh8
Nonfermentative Isobutanol E. coli 2-keto acids 50 Baez et al., 2011

alcohols C4 H10 O
2-methyl-1-butanol E. coli 2-keto acids 1.25 Cann et al., 2008

C5 H12 O
3-methyl-1-butanol E. coli 2-keto acids 9.8 Connor et al., 2010

C5 H12 O
3-methyl-3-butenol E. coli MVA 2.2 George et al., 2015
1-pentanol E. coli EcLeuABCD 2.22 Marcheschi et al., 2012

C5 H12 O
1-hexanol E. coli EcLeuABCD 0.3
C6 H14 O
1-heptanol E. coli EcLeuABCD 0.08
C7 H16 O
Fatty acids FAEE E. coli 3.4 Goh et al., 2014
Plants are the natural sources of isoprenoids. Previously suggested sources of isoprenoids for
fuel production include oil-producing algae such as Botryococcus braunii (Chan & Yong, 1986).
These organisms produce large amounts of isoprenoids as well as fatty acids. The disadvantage is
their slow growth and large amounts of fatty acids akin to a biocrude, which would require cracking
to form useful biofuels (Banerjee et al., 2002).
Advances in the understanding and engineering of isoprenoid biosynthesis pathways might facil-
itate the production using microorganisms. High-level production of IPP has been achieved in both
E. coli and S. cerevisiae. These different iso-prenoids have been produced with the use of these engi-
neered hosts. The increase of their production can be obtained by deregulation or over-expression
of the deoxyxylulose-5-phosphate and mevalonate pathways in native E. coli and S. cerevisiae and
introduced heterologously into these microorganisms (Martin et al., 2003, Tippmann et al., 2016).
Of all the isoprenoid-based biofuels, farnesene is the closest one to commercialization. The renew-
able products company Amyris based in Emeryville, California, uses the industrial yeast strain
S. cerevisiae PE-2 to produce farnesene. It is then chemically hydrogenated to farnesene, which is
being evaluated as a high-performance advanced biofuel (Peralta-Yahya et al., 2012).
4 OLECHEMICAL PROCESSES
The oleochemical-based processes have been the main supplier of the drop-in biofuels. These
processes require a simple hydroprocessing step to catalytically remove oxygen and eventually to
saturate C=C double bonds to produce paraffinic n-alkanes from lipid feedstock. The first stage
involves the production of vegetable oils from crops (cultivation, drying and storage, oil extraction)
or the collection of spent cooking oil or tallow, followed by the transportation of these oils to the
hydrotreatment facility. Next, impurities are removed from the oils (degumming), and then they
are heated and hydrotreated. Hydrogen is reacted with the trigylcerides under high temperature and
pressure in the presence of catalysts.
There are two principal pathways by which oxygen can be removed from the triglycerides:
hydrodeoxygenation (HDO) and catalytic decarboxylation (DCO). These pathways require different
inputs and produce different products (Fig. 4.5).
Chemical reaction of vegetable oils, animal-based waste fats, and by-products of vegetable oil
refining with hydrogen (HDO) produces hydrocarbons with properties superior to conventional
biodiesel and fossil diesel (Bacovsky, 2013). Finally, the only by-products of HDO are propane
and water. Companies applying this type of technology include NesteOil (Porvoo, Finland), which
hydrotreatments of palm oil, rapeseed oil and animal fat and Dynamic Fuels, which hydroprocesses
of animal fats, spent cooking greases, into renewable synthetic diesel.
For the decarboxylation process, crude fat feedstock is first converted into fatty acids and
glycerol. The fatty acids are then put through catalytic decarboxylation, a process which decouples
oxygen without using hydrogen. As a result, unsaturated and saturated hydrocarbons are formed. It
makes the production of renewable olefins possible. DCO produce the same amount of propane as
the HDO pathway, but it also CO2 , CO and water in amounts that depend on the extent of reverse
water gas shift and methanation. Therefore, DCO also has a higher potential for GHG emissions.
The company Alipha Jet converts any renewable oils and fats (such as waste vegetable oil, tal-
low, algal oil, and non-food oil crops like pennycress, camelina, jatropha, and pongamia), into true
drop-in hydrocarbon fuels including diesel (F-76), jet fuel (Jet-A, JP-5, JP-8), and high-octane
gasoline. If this triacylglyceride blend is hydrotreated, the main product will be diesel and the
yield of kerosene type compounds suitable for jet fuel will only constitute about 10%. In order to
increase the yield of kerosene compounds suitable for jet fuel, an additional selective cracking step
must be included. The yield of paraffinic kerosenes from this process is increased from 50 to 70%
(Holmgren, 2009).
One of the advantages of the oleochemical production biofuels is that it makes use of the existing
refining technology. Hydrotreatment units are already used in conventional refineries in order to
desulfurise fractional distillates, including diesel oil. As such, the same technology can be applied
to the hydrotreatment of renewable oils, in order to produce biofuels.
Figure 4.5. The principal pathways of hydrocarbons production in oleochemical processes from: a) fatty acids, b) triacylglycerols (Kinder and Rahmes (2009)).
5 HYBRID PROCESSES
Apart from the above, some technologies combine two or more different processes and are thus
referred to as hybrid. Hybrid technology, the bioethanol production employs the syngas platform
wherein syngas is an intermediate which link feedstocks and final products, unlike on sugars plat-
form wherein sugars acted as an intermediary product (Demirbas, 2004; Goyal et al., 2008; Tanger
et al., 2013). Hybrid process based on syngas platform involves conversion of biomass into synthetic
gas (intermediate), which is then cleaned and cooled, and subsequently transformed to ethanol
via fermentation routes (Munasinghe & Khanal, 2011; Devarapalli & Atiyeh, 2015). Although
some contaminants in syngas, such as H2 S and NH3 , can be used as nutrients by fermenting
microorganisms. High levels of NH3 can inhibit growth and enzyme activity.
In syngas fermentation, acetogens metabolize CO, CO2 , and H2 to alcohols and organic acids.
Certain acetogens such as Clostridium ljungdahlii, Clostridium carboxidivorans, Alkalibaculum
bacchi and Clostridium ragsdalei can use syngas as a source of energy and carbon (Najafpour &
Younesi, 2006; Younesi et al., 2005; Liou et al., 2005; Liu et al., 2012; Kundiyana et al., 2011).
Ethanol formed:
Acetate formed:
Most of the microorganisms which are currently known to ferment syngas to ethanol are pre-
dominantly mesophilic which operate within the temperatures in the range of 3040 C. Syngas
fermentation has several advantages over the sugar fermentation and thermocatalytic syngas conver-
sion (Daniell et al., 2012; Munasinghe & Khanal, 2010). Unlike saccharification and fermentation,
syngas platform allows utilizing lignin in addition to carbohydrate fractions of biomass. The main
disadvantage is the low solubility of CO and H2 gases in aqueous solutions, which is necessary to
microbially assimilation of gaseous substrates. In comparison to thermocatalytic syngas conver-
sion, it has also been claimed to be economical at a smaller scale, because of lower capital costs,
while proving to be less sensitive to impurities (Daniell et al., 2012).
Syngas fermentation companies such as LanzaTech, Coskata, and INEOS Bio have shown
that ethanol can be produced commercially (Liew et al., 2013; Devarapalli & Atiyeh, 2015).
Coskata, Inc. employs fermentation of syngas produced from natural gas after its reforming or gas
obtained from wood and coal gasification (Coskata, 2011). Recently, a new strain Clostridium
coskatii has been isolated and patented (Zahn & Saxena, 2012). INEOS Bio has operated the first
commercial cellulosic ethanol and power generation facility using syngas fermentation technology
in Vero Beach, Florida (USA) since July 2013 (INEOS, 2013). LanzaTech is a company from
New Zealand that utilizes CO-rich flue gases from steel making industries to produce ethanol.
LanzaTech has reported to be planning an expansion of production with more syngas fermentation
products (LanzaTech, 2015).
Another hybrid system for producing biofuels from lignocelluloses involves the hydrolytic con-
version of lignocellulosic biomass into sugar monomers, which are converted to hydrocarbons
through the catalytic processes. This technology is classified as a hybrid system, because sugars,
which are nominally a product of biomass conversion using physical and biochemical processes
form drop-in biofuels as a result of chemical processes. Sugars can be dehydrated via chemical
catalysis to yield hydroxymethylfurfural (from 6-carbon sugars like glucose) and furfural (from
5-carbon sugars like xylose). These molecules are building blocks for transformation into potentially
viable transportation fuels such as ethyl levulinate, dimethylfuran, and -valerolactone (Huber,
2008).
The company Virent Energy Systems (Virent) is currently the primary developer trying to com-
mercialize this approach. Virents BioForming platform is based on a combination of Aqueous
Phase Reforming (APR) technology with modified conventional catalytic processing. The process
has been demonstrated with conventional sugars obtained from existing sugar sources (corn wet
mills, sugarcane mills), as well as a wide variety of cellulosic biomass from non-food sources.
The aqueous phase reforming step utilizes heterogeneous catalysts at moderate temperatures and
pressures to reduce the oxygen content of the carbohydrate feedstock. As a result, a mixture of
chemical intermediates including alcohols, ketones, acids, furans, paraffins and other oxygenated
hydrocarbons is obtained from the APR. These intermediate compounds undergo further catalytic
processing to generate a cost-effective mixture of non-oxygenated hydrocarbons. The chemical
intermediates from the APR step can reacted over a Virent modified ZSM-5 catalyst to produce a
high-octane gasoline blendstock that has a high aromatic content similar to a petroleum-derived
reformate stream. Virent has trademarked this product as BioFormate. A key advantage to the
BioForming process is the ability to produce hydrogen in-situ from the carbohydrate feedstock or
utilize other sources of hydrogen such as natural gas for higher yields and lower costs. Companies
pursuing this approach include BIOGY, Cobalt and Gevo.
6 PROPERTIES AND USAGE OF ADVANCED BIOFUELS
6.1 Gasoline and alternative biofuels

Gasoline, the fuel for spark ignition engines, is a complex mixture of hydrocarbons. Linear,
branched, and cyclic alkanes account for 4060% of its composition, while aromatics make up
for 2040% (Sawyer, 1993). The carbon number of hydrocarbons in gasoline varies from 4 to 12.
Its net heating value is 43,330 kJ/kg. Among its other important properties is an octane number
>87 (Lee et al., 2008).
Ethanol
Ethanol is the most popular additive to gasoline and has an octane number of 129 (Renewable Fuels
Association, 2002). Cellulotic bioethanol is classified as an advanced biofuel and has the same
properties as ethanol, a first generation feedstock (Naik et al., 2010). However, it is not an ideal
fuel for the future because of its corrosiveness, high hygroscopicity and low energy density, which
is defined as the enthalpy of combustion per kilogram of fuel. Also, it contains only about 70% of
the energy content of gasoline. Gasoline has almost zero oxygen, whereas ethanol contains 36%
oxygen, and butanol contains 22% oxygen.
Butanol
Butanol has a 4-carbon structure and the carbon atoms can form either a straight-chain or a branched
structure (Liu et al., 2013). Because butanol is a longer-chain hydrocarbon, it resembles gaso-
line more closely. Butanol is hydrophobic and its energy content (27 MJ/L) is similar to gasoline
(32 MJ/L). The vapor pressure of butanol (4 mmHg at 20 C) is approximately 11 times lower than
that of ethanol (45 mmHg at 20 C). The properties of butanol are given in Table 4.4.
1-butanol has been proposed as a substitute for and a supplement to gasoline used in transporta-
tion. Butanol is suitable for use in road transportation as a blendstock with conventional gasoline.
Blends of up to 16% by volume are allowed in the US, as opposed to 10% maximum in the case
of ethanol (Butamax, 2016). Butanol is not suitable for use as an aviation fuel due to its low
heating value relative to a pure hydrocarbon (Hileman, 2009). Even through butanol may have
fewer hygroscopic problems than ethanol, it still cannot be classified as a drop-in replacement.
Isobutanol (2-methyl-1-propanol) has very similar properties to n-butanol with a higher octane
number (a measure of anti-knock properties) and a low melting temperature. It is currently under
investigation as a new biofuel target (Gevo, 2016).
The short-chain alcohols are good gasoline replacements or blends (Lee et al., 2008).
Table 4.4. Properties of butanol (Green, 2011).
Properties Unit Butanol
Reid vapor pressure kPa 2.3

Critical pressure hPa 48.4
Lower flammability limit
Concentration vol% 1.4
Temperature C 36
Upper flammability limit
Concentration vol% 11.2
Temperature C
Flash point C 36
Autoignition temperature C 343
Cloud point C 89.3
Boiling point C 117.7
Density kg/L 0.81
Vapor specific gravity 2.6
Kinematic viscosity mm2 /sec 3.7
Lower heating value
Mass MJ/kg 33.22
Volume MJ/L 26.9
BTU per gallon 84000
Research octane number 96
Cetane number 17
The C4 and C5 alcohols have distinct advantages over ethanol. First, they have higher energy
densities, leading to reduced distribution costs. Similarly, they are less hygroscopic and corrosive
than ethanol, facilitating their storage and transportation in existing distribution networks. However,
the C4 and C5 alcohols have a higher enthalpy of vaporization than ethanol, which means their
distillation will require more energy.
6.2 Diesel and alternative biofuels

Diesel fuel is a complex mixture of hydrocarbons including linear, branched, and cyclic alkanes
(75%) and aromatics (25%). The carbon number of hydrocarbons in petrodiesel varies from 9 to
23, with an average of 16. The net heating value is 42,640 kJ/kg. Its important properties include:
viscosity of 1.94.1 mm2 /sec at 40 C, flash temperature > 52 C, and cetane number > 40 (Demirel,
2012).
Biodiesel
Potential advanced biofuels that could supplement or replace diesel are fatty acid methyl esters
(FAMEs, biodiesel) (Lee et al., 2008). Biodiesel is generally composed of fatty acid methyl esters,
and is mostly derived from vegetable oil or animal fat. The fatty acids in FAMEs generally have
a chain length from 12 to 22, and contain zero to two double bonds. Biodiesel has a cetane rating
and energy content that are similar to those of petrodiesel (Knothe & Steidley, 2005). It offers
additional advantages, such as environmental friendliness, renewability, reduced emissions, high
combustion efficiency, improved lubricity, and high levels of safety. However biodiesel has similar
problems when transported in pipelines because its cloud and pour points are higher than those of
petroleum, and its energy content is approximately 11% lower than that of petrodiesel (BDpedia,
2016).
Table 4.5. Properties of ULSD, BIODIESEL and HDRD (renewable diesel).
No. 2 Petroleum Biodiesel Renewable

Properties ULSD (FAME) Diesel
Carbon, wt% 86.8 76.2 84.9

Hydrogen, wt% 13.2 12.6 15.1
Oxygen, wt% 0.0 11.2 0.0
Specific Gravity 0.85 0.88 0.78
Cetane No. 4045 4555 7090
T90 . C 300330 330360 290300
Viscosity, mm2 /sec. at 40 C 23 45 34
Energy Content (LHV)
Mass basis, MJ/kg 43 39 44
Mass basis, BTU/lb. 18,500 16,600 18,900
Vol. basis, 1000 BTU/gal 130 121 122
Drop-in biofuels
A new type of biofuels referred as drop-in biofuels has recently been developed. This group is
classified as:
hydrotreated vegetable oils (HVO or HEFA), produced by oleochemical processes, such as
hydroprocessing,
hydrocarbon biofuels called Fischer-Tropsch Liquids (FT liquids),
hydrotreated pyrolysis oils (HPO), produced by thermochemical conversion of biomass,
hydrotreated isoprenoids.
Alternative acronyms for HVO are HEFA (Hydroprocessed Esters and Fatty Acids), HRV
(Hydrotreated Renewable Vegetable oils), and HRO (Hydrotreated Renewable Oils). Also, the
terms green diesel or renewable diesel are often used. These are drop-in biofuels that are produced
by hydrotreating lipids derived from vegetable, algae and animal fats. They are straight-chain paraf-
finic hydrocarbons that are free of aromatics, oxygen and sulfur and have high cetane numbers
(Hilbers et al., 2015). The cold properties of HVO can be adjusted to meet the local requirements
by changing the severity of the process or by additional catalytic processing. HVO can be mixed
with petrodiesel in any proportion, but users may need to use an additive to address the lubricity
issues associated with compounds with no oxygen. HVOs do not have the detrimental effects of
ester-type biodiesel fuels, such as increased NOx emission, deposit formation, storage stability
problems, more rapid aging of engine oil or poor cold properties. They are also approved for use as
aviation fuels. Table 4.5 shows the properties of petrodiesel from biodiesel (FAME) and renewable
diesel.
Fischer-Tropsch diesel
Fischer-Tropsch diesel is similar to fossil diesel with regard to its energy content, density and
viscosity, and it can be blended with fossil diesel in any proportion without a need for engine or
infrastructure modifications. With regard to certain fuel characteristics, Fischer-Tropsch diesel is
even more favorable, i.e. it boasts a higher cetane number (better auto-ignition qualities) and lower
aromatic content, which results in lower NOx and particle emissions.
Isoprenoids
Certain isoprenoids and their associated alcohols have been reported to be potential fuel substi-
tutes or additives to diesel after hydrogenation (Liu & Khosla, 2010; Rude & Schirmer, 2009).
Naturally, isoprenoids are characterized by methyl branches, double bonds, and linear (farnasene)
ring (limonene, pinene, sabinene) structures, which improve their fluidity at lower temperatures
but lower their cetane ratings. Therefore, linear or cyclic monoterpenes (C10) or sesquiterpenes
(C15) are potential targets for partial replacement of biofuel after reduction of double bonds, which
would improve their cetane rating. For the production of farnesene a diesel equivalent the prod-
uct is treated with a conventional hydrogenation catalyst to saturate the alkene bonds to alkanes
(Renninger & McPhee, 2008).
Bisabolene is produced by engineered E. coli or S. cerevisiae, and can be chemically hydro-
genated to form bisabolane, which can serve as an alternative to D2 diesel, as shown by
Peralta-Yahya et al. (2011). Hydrogenated commercial bisabolene has a cetane number (41.9)
similar to D2 diesel. The branching found in the linear portion of bisabolane results in beneficial
cold properties, because it has a cloud point of 78 C. Finally, the ring portion of bisabolane
increases the density of the fuel, which increases the energy density per volume of fuel.
Limonene, 1-methyl-4-(1-methylethenyl)-cyclohexene, is one of the simplest monocyclic type
monoterpenes. The hydrogenated form of limonene can be used as fuel. Mixtures of diesel fuel
and up to 10% 1-isopropyl-4-methylcyclohexane were tested as diesel fuel additives. The results
showed that all tested mixtures were within the acceptable ranges specified by ASTM D975 for
diesel fuel and that the additives lowered the measured cloud point, compared to the base diesel
fuel. Saturated limonene had positive effects on viscosity as well (Tracy et al., 2009).
Isoprenoid biosynthesis provides an additional route to energy-dense C5 alcohols, namely
isopentenol (3-methyl-3- and 3-methyl-2-buten-1-ol, also known as isoprenol and prenol, respec-
tively) and isopentanol (3-methyl-1-butanol) (George et al., 2015). These alcohols have octane
numbers and combustion properties that make them potential gasoline replacements (Hull et al.,
2006). 2-phenylethanol, with a boiling point of 221 C, is at the lower end of the diesel range.
Considering that its aromatic nature gives it poor cetane qualities, this compound would likely
require finishing hydrogenation to convert it to ethylbenzene, which is a good octane enhancer for
gasoline (Yang et al., 2010). Table 4.6 shows the production titer of isoprenoids.
Single cell oils (SCOs) are fuel precursors and require subsequent processing. SCOs are com-
parable to vegetable oil. Hydrotreating SCOs produces completely deoxygenated hydrocarbons for
blending into diesel (Holmgren, 2007). This type of fuel could be used as jet fuel and road diesel.
Higher molecular weight olefins (C20) can be used as feedstock for oil refineries, like fatty
acids. Their high molecular weight precludes them from being used as diesel or gasoline. These
fuels can be catalytically cracked using existing refinery operations to make a variety of fuels.
Fischer-Tropsch Liquids (FT liquids) are hydrocarbon biofuels. They offer great potential for the
production of biopetroleum, bio-jet-fuel and other drop-in fuels which have very similar properties
to their fossil fuel counterparts. Table 4.7 shows the comparison of biodiesel and renewable diesel
production technologies.
6.3 Jet fuel and alternative biofuels

Jet fuels (Jet A, Jet A-1, JP-8, and JP-5) are also complex mixtures of hydrocarbons with a carbon
number of 816 and about 25% limit of aromatics (v/v). Jet A is a kerosene type fuel having a
maximum freezing point of 40 C, Jet A-1, is a kerosene type fuel, identical to Jet A but with
a maximum freezing point of 47 C. JP-8, or JP8 (Jet Propellant 8) is a jet fuel specified and
used widely by the US military. It is similar to Jet A-1 for commercial aviation, but also contains
corrosion inhibitor and anti-icing additives. Table 4.8 shows the summary of jet fuel requirements.
A kerosene-based fuel, JP-8 is projected to remain in use until at least 2025. Jet fuel is very
similar to kerosene or diesel fuel, but has a lower freezing point since it is used in extreme cold.
There are three main types of biofuels that could be drop-in replacements for jet fuel:
jet fuel produced via gasification of biomass followed by Fischer-Tropsch synthesis and
upgrading,
hydrotreated renewable jet fuel (HRJ). This technology is very similar to the one currently used
for producing hydrotreated vegetable-oil biodiesel for road transport,
Table 4.6. Production titer of isoprenoids.
Class Biofuels or precursors Native or heterologous Pathway Titer

biofuels of biofuels Formula producers applied (mg/L) References
Isoprenoids Limonene Escherichia coli MEP 17.4 Du et al., 2014
C10 H16
Escherichia coli MVA 100 Alonso-Gutierrez et al.,
2013
Synechococcus sp. PCC MEP 4 Davies et al., 2014
7002
Pinene Escherichia coli MVA 400 Sarria et al., 2013
C10 H16 Escherichia coli MVA 28 Sarria et al., 2014
(precursor of pinene
dimers)
Sabinene Escherichia coli MEP 2650 Zhang et al., 2014
C10 H16 BL21(DE3) MVA
Farnesene MVA NA Rude & Schirmer, 2009

C15 H24 MEP
Bisabolene Saccharomyces cerevisiae Exogenous MVA >900 Peralta-Yahya et al., 2011
(precursor of Escherichia coli
bisabolane)
Synechococcus sp. PCC MEP 0.6 Davies et al., 2014
7002
E. coli MVA 1150 Alonso-Gutierrez et al.,
2015
Isoprenol E. coli MVA 1300 Zheng et al., 2013
Prenol E. coli MVA 200 Zheng et al., 2013

Table 4.7. Comparison of biodiesel and renewable diesel production technologies.
Feedstocks: Favourable
Large scale Availability product Capital
production Process Product and price properties investments
1995 Esterification Biodiesel/FAME +

2007 Hydrotreating HDRD + +++
2015 Gasification Fischer-Tropsch Renewable diesel +++ +++
Table 4.8. Summary of jet fuel requirements.
Requirement Reason Specification
Energy content Affects aircraft range Minimum energy density by mass

Freeze point Impacts the ability to pump fuel at Maximum allowable freeze point
low temperature temperature
Thermal stability Coke and gum deposits can clog or Maximum allowable deposits in
foul fuel system and nozzles standardized heating test
Viscosity Viscosity impacts the ability of Maximum allowable viscosity
fuel nozzles to spray fuel and of
engine to relight at altitude
Combustion characteristics Creation of particulates in Maximum allowable sulphur and
combustor and in exhaust aromatics content
Lubricity Impacts the ability of fuel to Maximum allowable amount of
lubricate fuel system and engine wear in standardized test
controls
Material compatibility Fuel comes in to contact with large Maximum acidity, maximum
range of metals, polymers and mercaptan concentration, minimum
elastomers aromatics concentration (new)
Safety To avoid explosions in fuel Minimum fuel electrical
handling and tanks conductivity and minimum
allowable flash point.
synthetic hydrocarbons. There are a number of proposed routes from biomass feedstocks to jet
fuels based on novel biological or chemical processes. Linear or branched hydrocarbons with
medium carbon chain-length produced from the fatty acid or isoprenoid biosynthetic pathways
are primary targets for bio jet-fuels.
Current biosynthetic jet fuels, such as hydroprocessed esters and fatty acids (HEFA), derived
from algae triglycerides (Trimbur, 2011) and from the natural oils present in oil-seed plants, such
as Camelina (Braukus, 2013), have been used to power both military and commercial aircrafts in
50:50 blends with Jet-A fuel (American Society for Testing and Materials, 2013).
The use of isoprenoids as a jet fuel has recently been investigated, as they have low freezing
points due to their branching and cyclic structure. Pinene dimers can be generated via pinene (red)
dimerization using chemical catalysis. Current advanced biofuels have lower density and heating
value than high energy-density petroleum-based fuels such as JP-10 and RJ-5. In contrast, pinene
dimers (red) have a density and heating value similar to that of JP-10. Moreover, pinene dimers
mimic the strained ring systems found in JP-10 and RJ-5.
Amyris has a patent on a group of C15 isoprenoids (farnesene and derivatives), some forms
of which they report are suitable for use as a jet fuel that meets US specifications. For example,
farnesene has physical and performance properties that are consistent with C15 iso-paraffin and
superior in some aspects to the usual blending components for jet fuel. It has a low freezing point
(<100 C), high thermal stability (up to 380 C), and high energy content (44 MJ/kg). Farnesene
also lacks sulphur, which greatly improves its properties as a fuel.
Iso-butanol can be added directly to fuel used in road transport, and can be processed into
hydrocarbon fuels, including renewable jet and diesel. Also, for use as a gasoline substitute, it
can be dehydrated into isobutylene (Taylor et al., 2010), which can be processed into a jet fuel
(paraffinic kerosene) (Peters & Taylor, 2011).
More recently, the dehydration of butanol into butene followed by oligomerization resulted in
butene oligomers that can also be used as jet fuel (Wright et al., 2008; Peters & Taylor, 2011).
Most of the above-mentioned studies have been conducted at laboratory scale. With the exceptions
of 1-butanol and 2-propanol, 16 other biofuels are generally produced only in small amounts.
The two greatest challenges to commercialization are engineering catalysts to reach the yields
and productivities required to meet economic targets, and scaling these processes without losing
performance. In recent years, there has been a significant progress in understanding the biochemical
pathways of the microorganisms that produce advanced biofuels. These pathways exist naturally
in many different microorganisms and can be genetically manipulated or transported into a native
host to increase its capacity to biosynthesize a specific fuel product. Much attention has been
paid to improving microbes in order to increase theoretical yield, final titer (concentration) and
productivity. There have also been efforts to develop continuous fermentation processes and low
energy methods for product recovery and purification.
To summarize, biochemical conversion routes appear to be well-suited for producing fuel alco-
hols, whereas thermochemical conversion routes are more favorable for hydrocarbon fuels (Foust
et al., 2009). Although the overall economics of these two processes are similar (Foust et al., 2009),
comparative life cycle assessment suggests that biochemical conversion gives better results in terms
of greenhouse gas emissions and energy balance (Mu et al., 2010).
The future of syngas fermentation technology depends on the production of high value products
other than ethanol. Ethanols low heating value, miscibility with water and its unsuitability to be
used in the existing infrastructure for fuel transportation are just a few of the disadvantages that
led to the focus towards advanced biofuels such as butanol and hexanol.
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Chapter 5
Conversion of lignocellulosic biomass into sugars:

the effect of the structure of lignocellulose
Katarzyna Bukowska, Ewa Klimiuk, Tomasz Pokj & Artur Paowski
1 INTRODUCTION
Lignocellulosic biomass is the most abundant renewable carbon resource in the world. In 2012,
the total amount of available lignocellulosic feedstock was 341 million tons. About 70% of this
amount came from agricultural residues and 30% came from forest residues (Balan, 2014). Ligno-
cellulosic biomass can be an important source of many reactive intermediates (platforms) that link
feedstocks and final products, i.e. biofuels and chemicals (Sun & Cheng, 2002; Cherubini et al.,
2009; Chundawat et al., 2011). To date, the costs of production of biofuels from lignocellulosic
biomass (estimated based on energy equivalent) are two to three times higher than production of
petroleum fuels (Carriquiry et al., 2011). To reduce their price, the logistical, financial, politi-
cal and infrastructural barriers to using lignocellulose as a biofuel feedstock must be overcome
(Hoekman, 2009).
The conversion of lignocellulosic materials to biofuels requires their decomposition due to
their recalcitrance to biodegradability. Currently, pretreatment of biomass is usually employed
to make structural and compositional changes in lignocelluloses. Pretreatment methods include
acid and alkali hydrolysis, steam explosion, liquid hot water treatment, organosolv fractionation,
ionic liquid hydrolysis and others (Mosier et al., 2005; Kumar et al., 2009; da Costa Sousa et al.,
2009). Ultimately, they all produce more accessible cellulose that will easier undergo enzymatic
hydrolysis (Harmsen et al., 2010). The commonly known pretreatment technologies have mostly
been developed empirically (Himmel et al., 2007). There is a lack of knowledge about how the
individual components in the raw materials interact, and how they affect the decomposition of
lignocellulosic biomass (Salmn & Burgert, 2009).
Lignocelluloses are three-dimensional nanocomposites and a dynamic mixture of multifunc-
tional components. Thus, compositional analysis of lignocellulose before and after pretreatment
is not sufficient (Karimi & Taherzadeh, 2016). It is much more important to understand where
the lignin is located and how it interacts with the other components. Furthermore, there is a
need, a new approach for study the nature of the phenomenon of recalcitrance of lignocellu-
losic biomass in the aspect of its conversion technologies (McCann & Carpita, 2015; Carpita &
Sage, 2015). The authors propose that recalcitrance be viewed as the result of the interactions
of biomass and catalysts during the conversion processes. Details of the features of ligno-
cellulosic biomass that limit its breakdown and the efficiency of its bioconversion have been
presented by McCann & Carpita (2015). Analytical methods to characterize the chemical and
molecular features related to biomass recalcitrance have been reviewed by Foston & Ragauskas
(2012).
The present article highlights some of the scientific challenges to understanding the structure
of lignocellulose. Factors responsible for the recalcitrance of lignocellulosic biomass are also
discussed. In particular, the effect of cellulose crystallinity, hemicellulose structure, lignin content,
and lignin-carbohydrate complexes are given in detail.
95
2 RECALCITRANCE NATURE OF PLANT CELL WALLS
Cell wall is the major component of plant biomass. The rigid and compact structure resulting from
its structural and functional roles play in plants is controlled by the composition and organization
of individual components. The cell walls of most terrestrial plants contain cellulose, hemicellulose
and lignin as structural components. It also includes structural protein and mineral components
associated with cell wall (Vogel et al., 2008). Cellulose forms a skeleton, which is surrounded
by other polymers functioning as matrix (hemicelluloses), and by encrusting (lignin) materials.
Non-structural components are also present, including sugars, pectin and proteins, as well as, to a
lesser extent, other minerals (Raven, 1992; Lin et al., 2015). Some of the components are found
in conjunction with the main structural polymers in the cell walls (Timell et al., 1982). They are
responsible for the cohesive forces within the cell wall (Mortimer et al., 2010). As a result, the
physical and chemical interaction between the components of lignocellulose forms an extensive
three-dimensional polymeric network.
The share of various cell wall components can differ from one plant species to another (Pauly &
Keegstra, 2008; Carroll & Somerville, 2009; Allison et al., 2010). Generally, woody biomass
is more abundant in cellulose (4055%) and lignin (1835%), whereas grass biomass has higher
contents of hemicellulose (mainly xylan) (2050%), extractives (425%), and ashes (217%) (Zhao
et al., 2012) For example, poplar contains 49.9% cellulose and 18.1% lignin, whereas red oak has
43.4 and 25.8%, respectively. In the case of grass biomass, rice straw has higher concentrations of
xylan and extractives (24.5 and 17.9%, respectively) than corn cob (18.0 and 7.3%, respectively)
(Zhao et al., 2012).
The relative amounts of the constituents of lignocellulose change with the age of the plant
(o Di Nasso et al., 2010), the stage of growth (Rancour et al., 2012), and the conditions of
cultivation (Monono et al., 2013; Serapiglia et al., 2013). Mann et al. (2009) found that, in
biomass of switchgrass (Panicum virgatum L.) cv. Alamo, the content of xylose (19.4%) in the
leaf and lignin (19.1%) in the stem were higher when the plants were grown in the field (seed in
2007 and harvest in October of 2008) than in the greenhouse (38 C/25 C day/night temperature
and 16-h day/8-h night cycle) (17.5 and 16.9%, respectively). The results obtained by Allison et al.
(2011) suggested that the cell wall composition and neutral detergent fiber (NDF) concentration of
M. sinensis, M . sacchariflorus and M. giganteus are highly stable between growth years but are
affected by site location.
Although plant biomass is commonly considered as having a uniform composition, in fact the
various parts of a plant differ substantially in this regard. Jin & Chen (2006) divided rice straw into
fractions by morphological character. The lowest lignin (6.4%) content was found in the internode
fraction, while the highest lignin content (10.4%) was obtained in the panicle. The node had the
lowest content of cellulose (25.4%), and the highest content of hemicelluloses (37.1%). The highest
content of cellulose (38.5%) was in the leaf sheath, and the lowest hemicelluloses content (32.5%)
was in the leaf blade fraction. Thus, Lam et al. (2013) propose that biomass recalcitrance should
be considered with respect to either the whole plant or its anatomical regions.
Molecular structure of the cell wall in plants is shown in Figure 5.1. The cell wall of plants is
formed by the middle lamella and the primary wall. The middle lamella is largely pectic in nature
and can be considered a network of cellulose microfibrils intertwined with hemicelluloses, both of
which are embedded in pectins.
While plant cells are still elongating, they are surrounded by a primary wall. Once cell elongation
ceases, a secondary wall is deposited (Sorek et al., 2014). The secondary cell walls are composed
of cellulose microfibrils embedded in a complex noncellulosic matrix comprised mainly of hemi-
celluloses and lignin (Silveira et al., 2013). Cellulose is more abundant in secondary walls than
in primary walls, therefore the secondary wall is rigid and not readily stretched (Fig. 5.2). In the
secondary wall, unlike the primary wall, the cellulose microfibrils are aligned in parallel in layers.
Lignin is common in the secondary walls of wood cells. In mature woody tissue, moreover, the
middle lamella becomes heavily encrusted with lignin. The thickness of each layer and its con-
stituents composition vary in different types of cell walls (Albersheim et al., 2010), tissue types
Conversion of lignocellulosic biomass into sugars 97
Figure 5.1. Structure of a plant cell wall (Smith, 2001).
(Summers et al., 2003; Monti et al., 2008; Rancour et al., 2012; Sabatier et al., 2012) and plant
species (Jin & Chen 2006; Tao et al., 2012a).
There are three major types of lignocellulosic biomass: softwood, hardwood and grasses. Based
on literature data, Vogel (2008) showed that primary cell walls in grasses contain xylans and
mixed-linkage glucans with relatively minor proportions of xyloglucans, pectic polysaccharides,
and structural proteins such as arabinogalactan proteins. In contrast, dicot primary walls are char-
acterized by a higher abundance of xyloglucans and mannans as the predominant hemicelluloses,
and relatively higher abundances of pectic polysaccharides and structural proteins. In secondary
cell walls of grasses and woody dicots the predominant non-cellulosic components are xylans and
lignin. Esker et al. (2004) and Westbye et al. (2007) found that lignin induces agglomeration of
xylan and increases the affinity of xylan to the cellulose surface by formation of covalent bonds
between xylan and lignin. As a consequence, the retarded incrustation of the cell wall by lignin
precursors is more than a simple pore filling process (Boerjan et al., 2003).
According to Loqu et al. (2015), the principal components of poplar wood and grass straw
are similar, although there are differences in the abundance of cellulose, noncellulosic polysac-
charides and lignin. Moreover, there are differences the abundance of mannose rich polymers and
glucuronosylated xylans in hardwood and arabinosylated xylans, and p-hydroxy-cinnamic acids
such as p-coumaric acid and ferulic acid are present in the lignin of grass straw. The authors showed
schematic models of the structure of primary and secondary walls.
The plant cell wall architecture and molecular structure contribute to recalcitrance of lignocellu-
losic biomass. The organization and interaction among polymers of the cell wall affect its resistance
to physical and chemical agents and biological treatment (Himmel et al., 2007; Pu et al., 2013).
There are several factors that are related to the various polymers that are believed to contribute to
the recalcitrance of lignocellulosic biomass to biological and chemical deconstruction:
cellulose: crystallinity of cellulose, degree of polymerization (DP), available surface area for
enzymes, pore volume (Hu & Ragauskas, 2012; Peciulyte et al., 2015);
hemicellulose: degree of hemicellulose acetylation and hemicellulose sheathing (Buchanan et al.,
2001; Laureano-Perez et al., 2005);
Figure 5.2. Schematic models of plant cell walls. The emphasis is placed on wall composition rather than
architecture. (a) Pectinaceous primary cell walls found in dicots. (b) Lignified secondary walls (Loqu
et al., 2015). The thickness of each layer and its constituents composition vary in different cell walls types
(Albersheim et al., 2010), tissue type (Summers et al., 2003; Monti et al., 2008; Rancour et al., 2012; Sabatier
et al., 2012) and plant species (Jin & Chen, 2006; Tao et al., 2012a).
lignin: resistance of lignin to chemical and biological agents, non-productive binding of cellu-
lolytic enzymes, formation of stable lignin-carbohydrate complexes (LCC) and toxicity of lignin
derivatives to microorganisms (Zheng et al., 2014).
3 RESISTANCE OF MAIN COMPONENTS OF LIGNOCELLULOSE
3.1 Cellulose
3.1.1 Structure of cellulose
The cellulose is composed of linear -1,4-glucan chains and appears to be the core of the ligno-
cellulose complex. Cellulose has a simple chemical composition but the arrangement of cellulose
Table 5.1. Components of microfibrillated cellulose (Chinga-Carrasco, 2011; Sehaqui et al., 2011).
Diameter (nm) Biological structure Diameter (nm) Technological terms
10,000 to 50,000 tracheid 3 (plants); > 20 (algae, cellulose microfibrils

tunicates)
<1000 macrofibrils 1530 up to 5000 microcrystalline cellulose (MCC)
<100 350 cellulose nanocrystals (CNC)
<35 microfibril 25100 microfibrillated cellulose
3.5 cellulose elementary 530 nanofibrillated cellulose
fibril (CEF)
Figure 5.3. Schematic model of cellulose elementary fibril (CEF): (a) hexagonal shape, 36 chains; the model
contains 18 surface chains (blue), 12 transition chains (green), and 6 core chains (red) (Ding et al., 2014);
(b) rectangular shape, 24 chain (Fernandes et al., 2011, modified).
chains causes that structure of cellulose is complex (Peciulyte et al., 2015). In plant cell walls,
cellulose tightly aggregates into fibrils, held together via strong intra- and intermolecular hydrogen
bonds and van der Waals forces (Chundawat et al., 2011). Intra-chain hydrogen bonds raise the
stiffness of polymer, whereas an interchain hydrogen bond forms a network, which links chains to
form a two-dimensional sheet. The sheets are mainly packed together by weak van de Waals inter-
actions resulting from pyranose ring stacking (Shen & Gnanakaran, 2009). Table 5.1 shows, based
on literature data, structural components of cellulose, using both the classical terminology from
plant physiology (Chinga-Carrasco, 2011) and the terminology used in applied sciences (Sehaqui
et al., 2011).
The universal structural unit of natural cellulose is the cellulose elementary fibril (CEF) with
a diameter of approximately 3.5 nm (Ding et al., 2014). Several models have been proposed to
explain the internal structure of cellulose within the plant cell wall. According to Ding & Himmel
(2006), Ding et al. (2014), the cellulose elementary fibril (CEF) is a 36-chain, hexagonally shaped
and 3.2 5.3 nm in cross-section (Fig. 5.3).
The CEF is as long as several micrometers (Ding et al., 2012). In the other models, structure
CEF is composed of approximately 24 hydrogen-bonded chains and characterized by a rectangular
cross-section (Guerriero et al., 2010). Fernadez et al. (2011) presented a rectangular model CEF
with 24-chain of cellulose and both hydrophobic and hydrophilic surfaces exposed.
Several the elementary fibrils, with an average thickness of 3.5 nm, can associate with one
another to form macrofibrils. The macrofibrils contain two, three, four, and multiple CEFs. Both
CEF and macrofibrils, which are a bundle of CEFs, contain only cellulose (Ding et al., 2014).
The final size of the macrofibrils varies depending on the number of CEFs.
Figure 5.4. Schematic of microfibril (CEF and hemicelluloses): (a) containing one CEF (Ding 2014,
modified), (b) containing 5 CEFs (Ding et al., 2014).
The term microfibril is widely used to describe cellulose structure, but the precise definition is
sometimes unclear. Guerriero et al. (2010) defined a microfibril as the morphological entity that
corresponds to the minimum number of -(1 4) glucan chains required to form a crystalline
structure. The microfibril as a morphological unit often observed by microscopy and may contain
a single CEF or a small macrofibril, in each case associated with hemicelluloses (Ding & Himmel,
2006) (Fig. 5.4). The size of the microfibril has been reported to range from 2 to 50 nm based
on observations with various microscopic and/or spectroscopic techniques (Thomas et al., 2013).
Microfibrils associate with each other, forming larger structures with varying diameters (Donald-
son, 2007). It is worth noting that the structure of cellulose at the surface is different from its
crystalline bulk structure.
3.1.2 Effect of crystallinity

The molecular structure of cellulose is responsible for its properties: hydrophilicity, chirality,
degradability, and broad chemical variability due to the high donor reactivity of the OH groups.
Every glucosyl ring of cellulose has three active hydroxyls: one primary hydroxyl group and
two secondary hydroxyl groups. Thus, cellulose may have a series of chemical reactions related to
hydroxyl. However, these hydroxyl groups can also form hydrogen bonds between molecules, which
influences the reactivity of cellulose chains (Chen, 2014). The extensive hydrogen bond networks
give cellulose a multitude of partially crystalline fiber structures and morphologies (Klemm et al.,
2005). A characteristic of the cellulose microfibrils is their para-crystalline nature, which means that
they contain amorphous and crystalline regions. Possible organization patterns of these parts could
be either (1) an amorphous shell surrounding a crystalline core, (2) crystalline and amorphous
segments alternating along the long axis of the fibril, or (3) a combination of both (Salmn &
Bergstrom, 2009). The cellulose crystallinity of most wood-based cellulose samples seems to be
roughly 40 to 70% (Andersson et al., 2003; Thygesen et al., 2005).
The crystallinity has an important effect on the physical, mechanical and chemical properties
of cellulose (Sir & Plackett, 2010). When crystallinity is increased, tensile strength, dimensional
stability and density increase, whereas chemical reactivity and swelling decrease (Terinte et al.,
2011). When this occurs, the crystalline region of cellulose becomes more resistant to enzymes and
microbial attack than the amorphous region. This may be because of the robustness of hydrogen
bonding in cellulose microfibrils arising from extended hydrogen bonding and the difficulty of
enzymes acting on poorly hydrated cellulose surfaces.
An indicator termed the crystallinity index (CI) has been used to describe the relative amount
of crystalline material in cellulose. The CI of cellulose can be measured using several different
techniques including X-ray diffraction (XRD), solid-state 13 C nuclear magnetic resonance (NMR),
infrared (IR) spectroscopy, Raman spectroscopy and terahertz-time domain spectroscopy (THz-
TDS) (Park et al., 2010; Polletto et al., 2013; Pu et al., 2013; Vieira & Pasquini, 2014). There
are also several methods used for calculating CI from the raw spectrographic data (Terinte et al.,
2011; Ahvenainen et al., 2016). Literature suggests that the value of the CI is highly dependent
on the method of measurement of crystallinity. Thygesen et al. (2005) compared four different
analysis techniques, and reported that the CI of Avicel cellulose varied significantly from 39 to
67%, depending on the technique used.
The most popular method for estimating cellulose CI is the XRD method, which provides infor-
mation about the crystalline and amorphous parts of the cellulose. However, this method gives the
CI values, which are significantly higher than those obtained from using the other methods (Park
et al., 2010). NMR provides a more accurate measurement of cellulose crystallinity, and reveals
information about the ultrastructure of the cellulose, particularly the ratio of the interior-to-surface
of cellulose crystallites (Park et al., 2010).
Polletto et al. (2014) tested the main structural differences between six vegetal fibers (curaua,
ramie, kenaf, jute, sisal and buriti) and four wood fibers (Pinus elliottii, Eucalyptus grandis,
Mezilaurus itauba and Dipteryx odorata) commonly used as reinforced fillers in composite
materials with X-ray diffraction (XRD) analysis, fourier transform infrared (FTIR) spectroscopy
and thermogravimetric analysis (TGA). The results showed that lower quantities of extractives
and bound water were associated with higher crystallinity. The higher crystallite size slows the
degradation process and increases the thermal stability of lignocellulosic fibers.
Cellulose can be depolymerized into cellobiose and glucose by the combined action the cellu-
lases. These enzymes act on only one face of the microfibril. Some studies have indicated that
with an increase in the crystallinity the rate of cellulose hydrolysis decreases, especially in the
initial step of process (Zhu et al., 2008; Chundawat et al., 2011). This is because not all cellulase
systems are capable of hydrolyzing crystalline substrates. The extensive hydrolysis of crystalline
cellulose requires the synergistic cooperation of egzo- and endocellulases (Mansfield et al., 1998;
Mansfield et al., 1999; Nidetzky et al., 1993). The synergistic degradation of crystaline cellulose
into glucose is done by three types of cellulases: endoglucanases (EC 3.2.1.4), which randomly
cleave -1,4-glycosidic bonds in cellulose chains away from chain ends, cellobiohydrolases (EC
3.2.1.91), which produce cellobiose by attacking cellulose from chain ends, and -glucosidases (EC
3.2.1.21), which convert cellobiose to glucose (Zhang & Lynd, 2004; Bansal et al., 2012). Thus,
crystallinity probably influences reduction in cellulose hydrolysis efficiency when synergism is
lacking due to an incomplete cellulase system (Mansfield, 1999).
Cellulose crystallinity is one of the factors that influences the rate of hydrolysis. However, there
are contradictory reports regarding its varied at the time of conversion of cellulose. According
to some authors, the amorphous regions are more readily attacked by cellulases than the crys-
talline regions, and the hydrolysis of amorphous cellulose is faster than hydrolysis of crystalline
cellulose (Fan et al., 1980; Fan et al., 1981; Sasaki et al., 1979; Karimi et al., 2013; Kumar &
Wyman, 2013). This is indicated by the fact that in native cellulose the slowing down or cessa-
tion of enzymatic hydrolysis is observed when the more easily amorfic cellulose was converted to
sugars, whereas the crystalline form is more resistant to enzymatic hydrolysis (Park et al., 2010).
In this situation during hydrolysis, crystallinity of cellulose should increase as a result of a more
rapid removal of amorphous portion of cellulose (Lynd et al., 2002). However, according to some
authors no relationship between the degradability of cellulose and crystallinity was found (Bansal
et al., 2012). Earlier, Puls et al. (1991) noted that no change in the crystallinity index was detected
during enzymatic hydrolysis during the 1st to 7th day of experiments. The explained this as being
due to the simultaneous solubilization of both the crystalline and amorphous regions (Puls et al.,
1991). According to Peciulyte et al. (2015), most cellulose fibrils are part of aggregates, which
means that crystalline regions are located in the interior of the aggregates, preventing direct enzy-
matic attack during enzymatic hydrolysis, at least during the initial stages of hydrolysis. CP/MAS
13
C-NMR spectra revealed no direct relationship between the degree of crystallinity before
enzymatic hydrolysis and the degree of conversion in different samples.
Considering both the uncertainty of the methodologies for measuring the crystallinity index as
well as results in the changes of the CI during hydrolysis, it is difficult to consider crystallinity
as the key determinant of the rate of enzymatic hydrolysis (Lynd et al., 2002; Mansfield et al.,
1999). The accessibility of substrate to enzymes is affected by crystallinity, but also by other
factors such as particle size, the porosity of cell walls, and the content and distribution of lignin
and hemicelluloses. Consequently, the CI is just one of several indicators that should be considered
when assessing the likely rate of enzymatic hydrolysis of cellulose. The observed alterations in the
rate and extent of saccharification are likely governed by not only crystallinity, but also the factors
mentioned below.
3.1.3 Degree of cellulose polymerization

The degree of polymerization (DP) of different cellulosic substrates varies greatly. Depending on
the source of cellulose, the DP ranges from 100 to 10,000 (Zhang & Lynd, 2004). Agricultural
residues, such as bagasse and wheat straw, have lower cellulose DP (1000) than hardwoods and
soft woods (40005500) (Hallac & Ragauskas, 2011). The highest cellulose DP is found in cotton
(10,000) (Pu et al., 2007). Cellulose solubility decreases drastically as DP increases, due to
the presence of intermolecular hydrogen bonds. Cellodextrins, with DP 26, are soluble in water,
whereas cellodextrins, with DP 713 or greater, are somewhat soluble in hot water (Klemm et al.,
1998; Zhang & Lynd, 2003).
The effect of cellulose DP on enzymatic hydrolysis is not clear. Although, it would be expected
that exoglucanase would prefer substrates with a lower DP, there are no evidence that the rate of
chain-end creation by endoglucanase is affected by DP of substrate (Zhang & Lynd, 2004).
3.1.4 Accessible surface area

Since direct physical contact between the enzyme and the substrate is required for hydrolysis of
cellulose, the accessible surface area (ASA) is an important factor that limits the reaction rate. The
accessibility of cellulose to cellulases can be limited by two factors:
the structure of the cellulose microfibrils, whose sizes are of the order of nanometers (nm),
the anatomical structure of the plant cell wall, which may also affect accessibility to cellulases
(Yang et al., 2011).
The cellulose is fibrous material with a high surface-to-weight ratio and also a porous material
with a potentially unlimited internal surfaces (Chang et al., 1981). Typically, dry cellulosic fibers
have small size, about 15 to 40 m, and therefore they possess a considerable external specific
surface area, e.g. 0.61.6 m2 /g (Taherzadeh & Karimi, 2008). In general, this area is several orders
of magnitude larger than the external surface. At full swelling, it may range from 300 to 600 m2 /g.
The pore size of these capillaries also varies and can be as large as 200 , but many are smaller
than 30 (Chang et al., 1981). When cellulose microfibrils are associated into fibrils and further
into fiber walls, their accessibility is dramatically reduced. Due to the insoluble nature of cellulose,
large domains are not exposed to cellulases.
Cellulases can adsorb only on the accessible part of the substrate which is determined experi-
mentally by measuring its maximum adsorption capacity. However, adsorption can be sometimes
unproductive, due to lack of reactive sites, improper orientation of cellulose chain with respect to
the catalytic domain, or substrate competition between the adsorbed enzymes (Bansal et al., 2012).
Various methods have been used to measure accessible surface area. This area can be deter-
mined by adsorption measurements (the Bennet-Emmit-Teller method), which utilize an inert gas
like nitrogen (N2 ) (Kocherbitov et al., 2008). A more suitable technique for measurement of ligno-
cellulosic substrates is the solute exclusion technique, which determines the area available in the
form of pores. Mercury-intrusion porosimetry (Moura et al., 2005), cryoporometry, NMR (Nuclear
Magnetic Resonance) (Li et al., 1993; stlund et al., 2010; Foston, 2014), and differential scanning
calorimetry can be used to measure pores in the size range typically found in cellulose (Maloney
et al., 1998; Park et al., 2006). Also, solid-state NMR has been shown to be capable of determining
the specific surface area in pulp samples (Chunilall et al., 2010; Larsson et al., 2013).
Cellulosic materials are rather heterogeneous porous substrates, and their available surface area
can generally be divided into exterior and interior surfaces (Arantes & Saddler, 2011), the internal
surface area being much larger than the external surface area (Zhang & Lynd, 2004). The external
surface area is related to the size and shape of the particles. The internal surface area depends on
the capillary structure of cellulosic fibers and consist of internal pores, fissures and micro-cracks
(Rowland, 1977; Arantes & Saddler, 2011).
The enzymatic hydrolysis of cellulose could occur both on the external surface by a sequen-
tial broke down of the cellulose fibrils, or by the cellulase mixture entering pores/fissures large
enough to accommodate enzymes and then initiating the cellulose depolymerization process
(Arantes & Saddler, 2010). Several investigation showed that large particles of substrates are slowly
hydrolysable. The hydrolysis rate doubled in 10-h reaction when the average size was reduced from
82 to 38 m (Gan et al., 2003). Peciulyte et al. (2015) observed that hydrolysability is signifi-
cantly reduced when the pores are about the same size as typical enzyme molecules or smaller.
When pore volume is increased after lignin removal, the susceptibility of the substrate to hydrolysis
increases (Grethlein, 1985). Author observed linear correlation between the initial hydrolysis rate
of pretreated biomass and the pore size accessible to a molecule with a diameter of 5.1 nm, which
is about the diameter of a representative cellulase.
Decrease in particle size or increase in pore volume always leads to an increase of ASA. Strong
evidence to support a correlation between particle size and hydrolysis rates of relatively pure
cellulosic substrates has been found. However, there is little evidence to support such a correlation
for lignocellulosic substrates. M. sinensis was ball-milled and separated into four size fractions:
the 250355 mm fraction, 150250 mm fraction, 63150 mm fraction, and the <63 mm fraction.
X-ray diffraction analysis indicated that with the decrease in the size, the crystallinity of the biomass
declined. The rates of release of glucose and pentoses increased with the reduction size of particle
and crystallinity of cellulose, suggesting that the crystalline structure of M. sinensis slows down
the enzymatic hydrolysis of cellulose (Yoshida et al., 2008).
In sum, it can be stated the crystallinity is important, but not the sole factor determining the
ability of lignocellulose to hydrolyze (Karimi et al., 2013; Shafiei et al., 2015). In cases where
a higher crystallinity is accompanied by a higher digestability of cellulose, other factors, e.g.,
accessible surface area, porosity, and particle size, can also have a significant effect.
3.2 Hemicelluloses
3.2.1 Hemicelluloses as a barrier for accessibility of cellulose
Hemicelluloses are branched polysaccharides classified according to the main sugar residues in
the backbone (Wyman et al., 2005). The backbone of hemicelluloses is substituted with various
sugars or acidified forms such as glucuronic acid and galacturonic acid (Scheller & Ulvskov, 2010).
Xylans are the most abundant hemicellulose component in grasses such as switchgrass (Panicum
virgatum) and miscanthus and hardwoods, such as eucalyptus, willow, and aspen (Populus spp.).
However, mannans are the major hemicellulose component in some gymnosperm softwood species
(Sorek et al., 2014). Detailed characterization of hemicelluloses structure is given in Chapter 2 of
the present study.
In plant cell walls, the cellulose is interconnected by hemicellulosic polysaccharides, such
as xyloglucan (XG), or arabinoxylan forming a cellulose/hemicellulose network. This network
co-exists with another network consisted of pectic polisaccharides (Pauly et al., 1999).
The xyloglucan, or mannan, forms hydrogen bonds with the surface of cellulose fibrils. As the
cell expands and/or elongates, large macrofibril of cellulose may split to become smaller bundles
or individual CEFs, which are simultaneously coated with hemicelluloses to form microfibrils of
variable sizes (Ding et al., 2014). Recently, it was suggested that a small fraction of xyloglucan
is commonly entrapped in the cellulose microfibrils (Wang et al., 2012). These insertions may be
what is perceived as amorphous cellulose and may serve as initiation sites for cellulose degradation
(Sorek et al., 2014).
The microfibrils that contain one CEF are arranged nearly parallel, and the hydrophobic faces of
the CEF are perpendicular to the cell wall surface (Ding et al., 2014). Dammstrm & Gatenholm
(2006) suggest the carboxylic groups of the glucuronoxylan, which are arranged in a face-to-
face manner, generate electrostatic repelling forces to prevent the aggregation of the cellulose
Figure 5.5. Schematic of the arrangement of cellulose and xylan with positioning and spacing pattern of the
coated microfibrils due to electrostatic repulsion of the acid coat (Reis & Vian, 2004, modified).
microfibrils and to favor their parallel alignment (Fig. 5.5). This results in a strong but also flexible
connection of cellulose and hemicelluloses, as hydrogen bonds can easily be opened and reformed
(Salmn & Burgert, 2009).
Two alternative hypothetical architectures of cellulose-xyloglucan networks in primary cell walls
have been presented by Cosgrove & Jarvis (2012). In the first, the tethered network model, xyloglu-
cans fully coat the surfaces of cellulose microfibrils and additionally span the 2040 nm gap between
adjacent cellulose microfibrils as load-bearing tethers (Fig. 5.6). In an alternative model, there are
no direct microfibril-microfibril links because the xyloglucan is trapped between microfibrils.
Through this arrangement the xyloglucan glues microfibrils into a network of microfibril bun-
dles. The hemicelluloses tightly bound to the microfibrils are sheathed in a layer of less tightly
bound hemicelluloses, which in turn are embedded in the pectin matrix, filling the spaces between
microfibrils.
In this way, hemicelluloses form a physical barrier around cellulose and hence, limit the acces-
sibility of cellulose to cellulases (Himmel et al., 2007; Hu et al., 2011; hgren et al., 2007; Zhu
et al., 2008, Penttila et al., 2013). The study by Penttila et al. (2013) revealed that a certain
fraction of xylan remains tightly attached to cellulose fibrils, and certain fraction of xylan loosely
forms a three dimensional structure throughout the lignocellulosic matrix. The presence of loosely
bound xylan limits the hydrolysis of crystalline cellulose. The removal of hemicelluloses from
lignocellulosis biomass contributes to enzymatic digestibility of cellulose (Canilha et al., 2011).
This results from the fact that increasing the pore volume of the substrate makes the surface area
accessible for the enzymes (Alvira et al., 2010; Zhu et al., 2008). Therefore, Sierra et al. (2008)
suggested that moderate hemicelluloses removal (>50%) is required to significantly increase the
enzymatic digestibility of cellulose.
3.2.2 Effect of acetyl groups

O-acetyl groups occur on the backbones or branches of hemicelluloses (Gille & Pauly, 2012). In
hardwoods, the O-acetyl groups are combined with the xylose units, whereas in the softwoods, they
are combined with mannose and glucose units of glucomannan (Kim & Holtzapple, 2006). Pawar
et al. (2013) suggested that in the primary cell walls of softwoods and hardwoods the main sources
of O-acetyl groups are xyloglucan (XG) and pectins (homogalacturonan, rhamnogalacturonan I,
and rhamnogalacturonan II), whereas in the primary cell walls of grasses, the main O-acetylated
Figure 5.6. Architectures of cellulosexyloglucan networks in primary cell walls (Cosgrove & Jarvis,
2012): (a) the tethered network model; xyloglucans (black lines), cellulose microfibrils (larger red rods),
xyloglucanase-specific endoglucanase (yellow Pacman symbols); (b) a revised architecture based on the
enzyme/biomechanics analysis of Park & Cosgrove (2012a); load-bearing xyloglucan (broken black lines
highlighted by gray ellipses).
polymer is glucuronoarabinoxylan. The largest pool of acetyl residues in lignocelluloses, coming

from the secondary cell walls since they constitute the bulk of biomass (Pawar et al., 2013).
Acetylation level is the degree of acetylation (DA), which is the molecular ratio between the
total content of acetyl groups and the total content of monomers that can bear them. DA varied
from 0.60 to 0.75 in aspen wood glucuronoxylan and from 0.3 to 0.4 in (galacto)glucomannan in
the wood of aspen, birch and spruce (Teleman et al., 2000, 2003).
In glucuronoxylans, which are the main hemicellulose of hardwoods, the percentage of acetyl
groups ranges between 8 and 17% of total xylan, corresponding to 3.57 acetyl groups per 10
xylose units. Glucomannans occur in minor amounts in the secondary wall of hardwoods. The
content of acetyl groups in galactoglucomannan is around 6%, corresponding to 1 acetyl group per
34 hexoses units (Aln, 2000; Menon et al., 2010). In plant cell wall, acetyl groups (OAc) amount
to about 16% of the biomass compounds, depending on plant species (Peng et al., 2011).
In plant cell wall, acetyl groups (OAc) amount to 16% of the biomass compounds, depending
on plant species (Peng et al., 2011). Eucalyptus globules Labill. (wood) contains 3.5% OAc of d.w.
(Evtuguin et al., 2003), similar to Populus tremuloides Michx. (wood) with 3.7% OAc (Sjstrm,
1993).
Acetyl groups in hemicelluloses are considered to play an important role in the resistance mecha-
nism of plant cell wall to enzymatic hydrolysis of cellulose due to the steric hindrance of hydrolytic
enzymes. Grohmann et al. (1989) observed that as the xylan fraction becomes deacetylated, it
becomes 57 times more digestible. This, in turn, makes the cellulose fraction more accessible and
23 times more digestible.
Figure 5.7. Types of lignin carbohydrate linkages: (a) phenyl glycoside LC-bond, (b) acetal type LC-bond,
(c) benzyl ether LC-bond, (d) benzyl ester LC-bond (-ester) (Lawoko, 2005), (e) benzyl ester LC-bond
( -ester).
3.2.3 Stability of lignin-carbohydrate bonds

It is accepted that native plants possess no covalent bonds between cellulose and lignin (Koshijima &
Watanabe, 2003). Although, lignin is hydrophobic overall, due to its phenolic rings, monolignols
also contain a flexible three-carbon (C7C9) chain with hydroxyl groups. When associating with
cellulose lignin interacts mostly via its flexible chain atoms. Hemicelluloses and lignin are not
only entangled, but also covalently cross-linked. In lignocellulosic materials, hemicelluloses are
physically associated with cellulose and physically and chemically associated with lignin.
Strong interactions between cellulose, lignin and the xylan were observed in aspen holocellulose
by Dammstrm et al. (2009). There are two different fractions of xylan present in aspen wood: the
first is associated with the cellulose and second associated with the lignin. It is indicated that non-
branched xylan preferably associated with the cellulose while a more branched type is preferably
deposited together with the lignin. Thus, xylan plays a role similar to glucomannan in softwood.
The authors proposed a schematic model of the secondary cell wall in non-coniferous wood.
Lignin is associated with hemicelluloses via covalent bonds at two sites: -carbon and C4 in the
benzene ring (Fig. 5.7). p-coumaric and ferulic acid subunits in lignin might participate in benzyl
Figure 5.8. Schematic of the arrangement of cellulose, xylan and lignin in the secondary cell walls of aspen
wood (Dammstrm et al., 2009).
ester and ether linkages with hemicellulose sugars. With benzyl ethers, the -hydroxyl group of
lignin is connected to the hydroxyl group of carbohydrates. In benzyl esters, the -hydroxyl group
is linked to the carboxyl group of a glucuronic residue in a xylan (Fig. 5.7).
3.2.4 Stability of lignin-carbohydrate complexes

The lignin-carbohydrate complex (LCC) structure has chemical bonds/linkages between lignin and
carbohydrate (Koshijima & Watanabe 2003; Balakshin et al., 2008, 2014). LCC in situ forms a large
macromolecule encompassing in entire fiber due to multiple crosslinking among carbohydrate and
lignin molecules. The extent of cross-linking via lignin-carbohydrate complexes has been correlated
with increased cell wall rigidity and resistance to enzymatic digestion. Thus, these cross-links must
be broken by chemically hydrolysing the ester bonds in order to obtain an effective deconstruction
process (Grabber et al., 1998).
In softwood and hardwood, direct complexes between lignin and carbohydrates are present
(Fig. 5.8). It is thought that they are formed during lignification, when hydroxyl groups of carbo-
hydrates react with electrophilic ketone methide intermediates of the growing lignin polymer chains
(Brandt et al., 2013).
In grasses, these lignin-carbohydrate complexes contain ferulic acid (Fig. 5.9). The ferulates
have also been found in sugar beets (Beta vulgaris L.) and in pine (Pinus pinaster). The ferulic acid
is covalently attached via an ester linkage formed between the carboxylic acid group of ferulic acid
and the primary alcohol on C5 carbon of arabinosyl side-chains of arabinoxylans (Hatfield et al.,
1999). During lignification, its aromatic ring can be incorporated into the growing lignin network
by participating in the radical polymerisation reaction. Ferulic acid that was etherified to lignin
was also esterified to arabinoxylans, thus demonstrating that ferulates were indeed cross-linking
lignin and polysaccharide.
The occurrence of stable lignin-carbohydrate bonds creates significant problems in selective
separation and isolation of lignin, and in preparation of carbohydrate from lignocellulose. The
separating lignin LCC-rich fractions is complicated due to close association with the other cell
wall polymers, poor solubility and the tendency of lignin to degrade or react upon isolation when
common solvents are used (Guerra et al., 2006). Generally, two steps are required for direct
elucidation of LC linkages in plant cell walls. A first step is a fractional method to isolate LCC-rich
Figure 5.9. Grass lignin-carbohydrate complexes involving ferulic acid (Brandt et al., 2013).
fractions and a second step is an analysis of LC linkages in the LCC-rich fractions obtained by the
fractionation method.
The several methods have been reported for complete fractionation with proper preservation of
the bonding structure between the lignin and carbohydrate (Lawoko et al., 2003, 2006; Chundawat
et al., 2011; Balakshin et al., 2011; Du et al., 2013). Balashin et al. (2011) proposed a schematic
description of different methods for isolation of LCC. LCC preparations can be classified as carbo-
hydrate rich LCC (Bjorkmans LCC and similar ones enzymatic LCC fraction) and lignin rich LCC
(MWEL, CEL, MWLc). In the case of lignin rich preparation, when most of the carboxyhydrates
are decomposed by enzymatic hydrolysis and/or ball milling, the carbohydrate composition of the
residual sugars provide information on which carbohydrate can be linked to lignin (Balashin et al.,
2011).
Identification of various LCC linkages using 2D NMR methods was an important milestone in
LCC studies. This method showed various LCC linkages in preparations isolated from soft- and
hardwoods. There is little quantitative information on various types of linkages between lignin and
carbohydrates (Balashin et al., 2011).
Du et al. (2013) suggested LCC fractionation protocol for native and processed plant biomass.
This protocol includes sample disintegration by ball milling (for structural preservation), fol-
lowed by complete dissolution before fractionation into several LCCs with quantitative recovery
(Fig. 5.10). The samples of spruce wood were ball milled and dissolved in DMSO+TBAH, then
diluted with water. Glucan-lignin (GL) fraction was obtained from precipitate while glucamannan-
lignin (GML) and xylan-lignin (XL) from solution after Ba(OH)2 precipitation according to given
procedure. This fractionation was quantitative. Using mass balance, GL accounted to 49.5% of the
sample; GML, 30.9%; and XL, only 12.8%.
Lawoko et al. (2003, 2004) presented a quantitative method for isolating lignin as LCCs from
chemical pulps and spruce wood, in which LCC is systematically prepared at quantitative yield,
fractionated and qualitatively expressed. This methods involves a partial enzymatic hydrolysis of
cellulose, subsequent swelling, and quantitative dissolution, into four major fractions. The LCCs
obtained from spruce wood lignin were a galacto-glucomannan LCC (8% wood lignin); a glucane
LCC (4% wood lignin); a xylan-lignin-glucomannan network LCC (40%), and a glucomannan-
lignin-xylan network LCC (48%). The study provided conclusive evidence of covalent linkages
Figure 5.10. Isolation of LCC preparations for wood (Balakshin et al., 2014).
between lignin and carbohydrates in the native lignin in wood. It was concluded that carbohydrate-
free lignin, i.e., lignin without covalent bonds to carbohydrates, probably cannot be present in
spruce wood (Lawoko et al., 2006).
Lawoko et al. (2005) described the differences in lignin structure and reactivity within the various
LCC fractions. There are two different forms of lignin present in the wood fiber wall. These forms
are linked to glucomannan and xylan, respectively (Fig. 5.10). The xylan-linked lignin is to a large
extend degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form
more high molecular mass material.
Du et al. (2014) isolated xylan-lignin (XL), glucomannan-lignin (GML) and glucan-lignin (GL)
complexes from spruce wood, hydrolyzed them with xylanase or endoglucanase/-glucosidase,
and analyzed them by analytical pyrolysis and 2D NMR. The study provided evidence for the
existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan)
and cellulose in spruce wood.
In poplar (Populus tomentosa Carr.), NMR results indicate that lignin and carbohydrates are
directly bonded through ether linkages (Yuan et al., 2011c). The main substructures in the four
lignin fractions i.e. milled wood lignin (MWL), lignin-carbohydrate complex (LCC), cellulolytic
enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHREL) were -O-4 aryl
ether and resinol. The LCC fraction contained a high percentage of xylose (96.2%) and numerous
-O-4 aryl ether linkages (84.4 per 100Ar). The data provided evidence for ether bonds between
lignin and the C1, C5, and C6 atoms of pentoses and hexoses.
Balakshin et al. (2011) quantified the LCC linkages of benzyl ether, phenyl glycoside and
-ester types in white birch (Betula pendula) and loblolly pine (Pinus taeda) preparations by using
a combination of quantitative 2D HSQC and 13 C NMR spectroscopic techniques. They detected
different amounts of benzyl ether, -ester and phenyl glycoside LCC bonds. The pine wood showed
higher amounts of benzyl ether, but lower amounts of phenyl glycoside and -ester LCC linkages
than birch wood. Benzyl ester moieties were not detected.
3.3 Lignin
3.3.1 Resistance of lignin to biodegradation
Since lignocellulosic materials serve a structural purpose, they are, by nature, relatively resistant
to microbial attack. The softwoods, such as pine, contain (2535% of the cell wall) lignin while
hardwoods, such as poplar (1825%), or grasses (1030%) (Snchez, 2009).
The resist of lignin for degradation can be attributed to its the complex cross-linked three-
dimensional network polymeric structure (Ruiz-Dueas & Martnez, 2009). As a hydrophobic
polymer, lignin also serves as a barrier against water penetration. Unlike cellulose, lignin is irregular
polymer and has no identical, repeating subunits. The irregularity of the lignin structure requires a
complicated evolutionary pathway in order to generate a single enzyme that is capable of recognizing
and breaking all the various types of linkages found in lignin (Hatfield & Vermerris, 2001). The
bonds in lignin are very difficult to break under normal conditions. The major inter-unit linkages
within the lignin monomer (H, S, and G) are -O-4, -, -5, -1 arrangements (Ghaffar & Fan,
2013; Lupoi et al., 2015).
The bonds formed among lignin monomers are less reactive than those of most other biological
polymers and are of sufficient chemical diversity to preclude the ability of any single enzyme to
recognize and degrade them all (Weng et al., 2008; Cesarino et al., 2012). The most common
intramolecular linkages in lignin -O-aryl ethers represent 3050% of linkages in wood and up
to 90% of linkages in grasses, preferentially cleave by dilute acids (Villaverde et al., 2009). The
ester linkages, which can be high in some grasses, are broken under alkaline conditions. The
carbon-carbon linkages in lignin are the most recalcitrant (Sorek et al., 2014).
Huang et al. (2016) characterized the structural features of the milled wood lignins isolated
from green (MWLg) and yellow (MWLy) bamboo (Phyllostachys pubescens) using 13 C NMR
spectroscopy and 2D HSQC NMR spectroscopy. The phenol glycoside and benzyl ester LCC
linkages in the MWL preparations were clearly quantified, while the amount of -ester LCC was
ambiguous for quantification.
Wen et al. (2012) in situ characterized of the structural heterogeneity of lignin polymers of
bamboo during pretreatment in DMSO/NMI and enzymatic hydrolysis with 2D HSQC NMR
technique. The various lignin-carbohydrate complex linkages (benzyl ether and phenyl glycoside
linkages) can be assigned.
The ratio of S/G is considered as the parameter that informs about the degradability of lignin.
Higher S-lignin amount led to increase in removal of lignin and increase in sugar yields (Li et al.,
2010; Studer et al., 2011). The importance of the S/G ratio and lignin content in a Populus family
on the release of xylose by dilute acid hydrolysis was analyzed by Davison et al. (2006). Authors
obtained the xylose yield of 25.8% of lignin and 2.3 S/G (high lignin, high S/G) sample produced
30% of the theoretical yield, whereas the xylose yield of the 22.7% lignin and 1.8 S/G (low lignin,
low S/G) was 55% of the theoretical value.
3.3.2 Lignin as a barrier for accessibility of cellulose

In lignocellulosic materials the lignin covers hemicelluloses which cover cellulose chains. Nearly
a quarter of the total cellulose surface area is covered by lignin, significantly reducing the area
accessible to the enzymes (Igarashi et al., 2011; Lindner et al., 2013). Thus, lignin reduces cellulose
macro-accessibility indirectly and prevents the enzymatic hydrolysis of cellulose. Reis & Vian
(2004) founded that the cellulose/glukuronoxylanes composite is a charged and highly anisotropic
construction forming a sort of host structure for lignin precursors. The lignin precursors are inserted
and polymerised in the gaps extended between microfibrils (Fig. 5.11). The hemicelluloses and the
side chains of branched hemicelluloses (uronic acid and arabinose) are covalently bonded to lignin
to create enzyme-impenetrable cross-links (Chundawat et al., 2011).
The noncrystalline regions of cellulose are observed to have a lower tendency to associate with
lignin than crystalline regions, and this is found to arise from stronger hydration of the noncrystalline
chains. The results suggest that the recalcitrance of crystalline cellulose to hydrolysis arises not only
from the inaccessibility of inner fibers but also due to the promotion of lignin adhesion (Lindner
et al., 2013). Xylan forms a complex network with cellulose by coating and connecting cellulose
crystallites, and limiting the accessibility of cellulases towards cellulose (Dammstrm et al., 2009).
Vermaas et al. (2015) using multi-component simulation model containing cellulose, lignin,
and cellulases (TrCel7A), identified three types of lignin aggregates sheets, in which lignin
monolayers bind to a single cellulose fiber; piles, in which the lignin aggregates onto a single
cellulose fibril but not as a monolayer; and linkages, in which the lignin aggregates connect
cellulose fibrils. If lignin adopts an extended morphology (a sheet or linkage), more surface is
exposed, and lignins propensity to bind to enzymes is increased. The piles are the least effective
Figure 5.11. Structure of hemicelluloses-lignin co-polymers: (a) xylan-lignin co-polymer containing 40% of
the wood lignin; (b) glucomannan-lignin co-polymer containing 48% of the wood lignin (Lawoko et al., 2005).
at trapping enzymes and hence the least inhibitory to cellulase action. In presence of lignin the
access of cellulase enzymes to cellulose is difficult (Chang & Holtzapple, 2000). Therefore lignin
is believed to be a physical barrier major hidrance in hydrolysis (Laureano-Perez et al., 2005).
The hydrophobic irreversible binding of lignin to cellulase enzyme and enzyme deactivation
significantly reduces enzyme effectiveness (Karimi et al., 2013; Taherzadeh & Karimi, 2008;
Yang & Wyman, 2006; Kumar & Wyman, 2013; Wyman et al., 2005). Pareek et al. (2013)
used lignin preparations derived from different types of processes and different origin [alkali
lignin (AL), hydrolytic lignin (HL), organosolv lignin (OL), and lignosulphonic acid sodium salt
(LS), beech wood xylan (BWX), and galactomannan (GM) (Locust bean gum) procured from
SigmaAldrich such as softwood (SL, spruce lignin) and hardwood (PL, Populus lignin)] and two
enzymes Celluclast and Novozyme 188. The black cottonwood lignin had the highest adsorp-
tion capacity (141 mg protein/g lignin) for Celluclast. The adsorption capacity decreased in the
order PL > LS > SL > AL > OL > HL > GM > BWX. The highest apparent affinity and binding
strength was observed for lignosulfonate followed by alkali lignin. For Novozyme 188, the apparent
adsorption capacity of lignosulfonate was considerably higher than that of the other substrates.
Removal of lignin always results in the increase of specific area, which increases the accessibility
of cellulose to enzyme (Chang & Holtzapple, 2000; Mansfield et al., 1999; Zhao et al., 2007; Zhao
et al., 2008a; Zhao et al., 2008b; Zhao et al., 2009; Laureano-Perez et al., 2005). However pretreat-
ment of biomass produces phenolic and non-phenolic inhibitors that inactivate the carbohydrate
hydrolyzing enzymes (Nakagame et al., 2010; Tejirian & Xu, 2011; Ximenes et al., 2011; Jnsson
et al., 2013; Rahikainen et al., 2013). Recently various lignin-related inhibitory processes have
been proposed, including cellulose association with lignin blocking enzymatic access to cellulose,
and the unproductive binding of the enzymes to lignin.
Chang & Holtzapple (2000) reported correlations between enzymatic digestibility and three
structural factors: lignin content, crystallinity, and acetyl content. They concluded that (1) extensive
delignification is sufficient to obtain high digestibility regardless of acetyl content and crystallinity;
(2) delignification and deacetylation remove parallel barriers to enzymatic hydrolysis; and (3)
crystallinity significantly affects initial hydrolysis rates but has less effect on ultimate sugar yields.
These results indicate that an effective lignocellulose treatment process should remove all the acetyl
groups and reduce the lignin content to about 10% in the treated biomass.
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Zhu, L., ODwyer, J.P., Chang, V.S., Granda, C.B. & Holtzapple, M.T. 2008. Structural features affecting
biomass enzymatic digestibility. Bioresource Technology 99: 38173828.
Chapter 6
Pretreatment of lignocellulosic biomass
Katarzyna Bukowska & Ewa Klimiuk
1 INTRODUCTION
Pretreatment is the first step enabling reduce the recalcitrance of lignocellulosic biomass so that
enzymatic hydrolysis of polysascharides like cellulose takes place more rapidly and gives greater
yields (Sun & Cheng, 2002; Harmsen et al., 2002). It can be achieved by reduction of the partic-
ulate size, increase surface area and porosity, redistribute the main components of lignocellulosic
biomass, release and depolymerize hemicellulose, decrease the crystallinity of cellulose, and
modify lignin structure (Galbe & Zacchi, 2002; Mosier et al., 2005). To avoid the formation of
degradation products that inhibit enzymatic hydrolysis and fermentation, pretreatment processes
need to be optimized. Furthermore, capital and energy costs should be lowered as much as possible
(Jaiswal & Ravindran, 2015).
A number of pretreatment methods have been developed during the last few decades and then
described in detail in the literature (Mosier et al., 2005; Harmsen, 2010; Chaturvedi &Verma, 2013).
There are various schemes for classifying these methods, some of which group pretreatment strate-
gies into physical, chemical and biological methods, or combinations of these methods (Hsu, 1996;
Zheng et al., 2009; Harmsen, 2010; Agbor et al., 2011). Physical pretreatment reduces particle
size, increases pore size and accessible surface area. Chemical and physicochemical pretreatments
increase biomass solubility, decrease its degree of polymerization, partially or completely delignify
the biomass, and partially or completely hydrolyze the hemicelluloses that it contains. Chemical
pretreatment methods include acid hydrolysis, alkaline hydrolysis, ozonolysis, oxidative delignifi-
cation, organosolv process, and ionic liquid pretreatment. Physicochemical methods include steam
explosion (autohydrolysis), liquid hot water pretreatment, ammonia fiber explosion (AFEX), and
CO2 explosion. Biological pretreatment methods reduce the degree of polymerization of cellu-
lose, and partially degrade hemicelluloses and lignin; these methods use the activity of fungi and
actinomycetes.
2 MECHANICAL METHOD: MILLING
Physical pretreatment such as chipping, grinding and milling makes biomass easier to handle, due
to increasing its surface/volume ratio, reducing its particle size and crystallinity, and reducing
particle size and degree of polymerization of cellulose. The biomass powders can be produced by
different grinding processes like sieve-based grindings, ball milling, and jet milling. Silva et al.
(2012) achieved progressive particle size reduction of wheat-straw powders: from coarse (median
particle size 800 m) to fine particles (50 m) using sieve-based grindings, then ultra-fine
particles 20 m by jet milling and 10 m by ball milling.
Mechanical methods are costly in terms of energy and capital, which can significantly increase
the cost of producing biofuels. The cost depends on the type and degree of milling (Cadoche &
Lopez, 1989): for example, milling hardwood to 1.6 mm particles requires 130 kWh/t, whereas
fragmenting straw to particles of the same size needs 7.5 to 42 kWh/t, depending on the cutting
121
method. The choice of method depends on the type of biomass and the planning of further processes
like enzymatic hydrolysis.
3 CHEMICAL METHODS
3.1 Pretreatment with dilute acids

Acid hydrolysis is one of the most promising pretreatment methods for industrial implementation.
It is usually performed with mineral acids (H2 SO4 , HCl, H3 PO4 ), but organic acids and sulfur
dioxide have been also tested (Behera et al., 2014; Jung & Kim, 2014).
Dilute acid pretreatment contributes to the hydrolysis of hemicelluloses, the partial depolymer-
ization of cellulose, and the structural modification and redeposite of lignin (Chandel et al., 2012;
Jung & Kim, 2014). In acidic environments, ether or ester bonds between lignin and polysac-
charides are cleaved and converted into hydroxyl, carbonyl or carboxyl groups. Hemicelluloses
hydrolyze to oligo- and monosaccharides (Lee et al., 1999) and lignin is redeposited. In optimal
operational conditions, more than 90% of hemicelluloses can be solubilized as sugars (Wyman
et al., 2005; Xiu et al., 2011).
At lower temperatures (T < 160 C), a portion of hemicelluloses hydrolyzes rapidly while the rest
hydrolyzes more slowly (Mcmillan, 1992). The fraction of slow-reacting xylan is estimated to be
0.200.32 (Lee et al., 1999). It can explained that some part of hemicellulose is easily accessible,
whereas the other part is located deeper between cellulose chains. Moreover, xylan can be intimately
associated with the lignin matrix by being embedded within that matrix or linked to the lignin by
lignin-carbohydrate bonds (Harmsen et al., 2010).
Taking into account the above, the hydrolysis rate of the hemicelluloses can be expressed in the
form of two parallel pseudo first order reactions for the rapidly hydrolyzing fraction (H1 ), and the
slowly hydrolyzing fraction (H2 ) (Grohmann et al., 1986; Trajano & Wyman, 2013).
Degradation of both hemicellulose fraction leads to the formation of oligomers. They are defined
as water-soluble polymers (1 < DP < 10) (Lee et al., 1999). The oligomers are hydrolyzed to
monomers. The hydrolysis rates of oligomers vary with the DP value. In general, it is observed that
mild temperature led to a significant recovery of sugars while higher temperatures caused further
sugar degradation, aiding the formation of inhibitors (Yang & Wyman, 2008).
The hydroxymethylfurfural (HMF) is a toxic compound originating from hexose degradation.
During acid hydrolysis, pentose sugars can degrade to furfural, a toxic compounds (Fig. 6.1).
Moreover phenolic compounds can be released from lignin such as furans, weak acids and
others. They acted as potential inhibitors to fermentation microorganisms (Canilha et al., 2006,
2008; Chandel et al., 2007; Harmsen et al., 2010).
3.1.1 Operational condition of acid hydrolysis

The acid concentration, temperature and residence time are key parameters during biomass pre-
treatment (Taherzadeh & Karimi, 2007; Behera et al., 2014). The dilute acid pretreatment can be
conducted at high temperature (T > 160 C) during a short period of time or lower temperature
(T < 160 C) for longer retention time (3090 min) (Jung & Kim, 2014). The scheme of dilute
sulfuric acid process flow is shown in Figure 6.2. The concentration of sugars released during
pretreatment dependents on the type of lignocellulosis material, acids used to hydrolyse, acid con-
centration, solid-to-liquid ratio and the type of reactors employed in the process (Lee et al., 1999;
Pretreatment of lignocellulosic biomass 123
Figure 6.1. Acid-catalyzed dehydration of sugars to furans.
Figure 6.2. Scheme of dilute sulfuric acid process flow.
Lenihan et al., 2011; Mosier et al., 2005; Taherzadeh & Karimi, 2007; Xiang et al., 2003; Akpinar
et al., 2009).
Many authors determined the optimal parameters of dilute acids pretreatment for different
ligninocellulosis biomass including rice and wheat straws, sugarcane bagasse, eucalyptus residue
and others. Baek and Kwon (2007) obtained sugars production of 17.2 g/L xylose, 4.3 g/L glucose
and 3.3 g/L arabinose from rice straw under following conditions: 1.5% H2 SO4 , 130 C, 30 min.,
and solid:liquid ratio (S/L) of 1:10. Wheat straw was submitted to dilute acid hydrolysis of 1.85%
H2 SO4 under the conditions of 90 C, 18 h, solid:liquid ratio of 1:20, allowing to produce 12.8 g/L
xylose, 1.7 g/L D-glucose and 2.60 g/L L-arabinose (Nigam & Singh, 2011). Aguilar et al. (2002)
reported the best conditions like 122 C, and 24 min. for acid pretreatment of sugarcane bagasse
with 2% H2 SO4 . The sugar release was 21.6 g/L for xylose, 3 g/L for glucose, 0.5 g/L for furfural
and 3.65 g/L for acetic acid. Canettieri et al. (2007) evaluated the release of sugars from eucaliptus
residue by 0.65% H2 SO4 at 157 C and for 20 min. During this process 1.65 g/L glucose, 13.65 g/L
xylose, 1.55 g/L arabinose, 3.10 g/L acetic acid and 1.23 g/L furfural were produced. Zhao et al.
(2008) pretreated aspen, basam, fir, basswood, red maple and switchgrass with 0.251% H2 SO4
at 160190 C for 0240 min. During the process, the maximum yield of xylose ranged from 70%
(basam) to 94% (switch grass), glucose from 10.6% to 13.6% and other minor sugars from 8.6%
to 58.9%. Tao et al. (2013) obtained the optimal treatment conditions of sulfuric acid pretreatment
for Achnatherum splendens (needlegrass), which were 2% (w/v) sulfuric acid, reaction time of 3 h
and a temperature of 100 C. Under these conditions, 92.6% hemicelluloses were solubilized and
the content of cellulose in pretreated solids increased to 66.8%.
Laopaiboon et al. (2010) compared hydrochloric and sulfuric acids for pretreatment sugarcane
bagasse and found the maximum sugar recovery with HCl. The maximum catalytic efficiency was
10.85% under the conditions of 0.5% HCl at 100 C for 5 h, of which the main components (in
g/L) in the hydrolysate were glucose, 1.50; xylose, 22.59; arabinose, 1.29; acetic acid, 0.15 and
furfural, 1.19. Li et al. (2008) used double acid hydrolysis (via combination of hydrochloric acid
and sulfuric acid) for recovering the sugars from lignocellulosic waste. The first stage of hydrolysis
was conducted with 1% (w/w) of hydrochloric acid at 120 C for 25 min. and at L/S of 8 and the
second stage with 1% (w/w) of sulfuric acid at 165 C for 15 min. and at L/S equal 8. Under these
conditions, the concentration of xylose and glucose was 34.47 and 40.51 g/L, respectively, and
the total fermentable monosaccharide concentration and yield could attain 74.98 g/L and 76.55%,
respectively.
Although the mineral acids such as sulfuric acid and hydrochloric acid are the most commonly
used, some authors also tested H3 PO4 for hydrolysis of lignocellulosic biomass. Phosphoric acid is
less aggressive than other mineral acids. Dilute phosphoric acid, on hydrolysates from sugarcane
bagasse, has shown fermentable sugars with 21.4 g of sugar/L with less than 4 g/L of inhibitors at
operating conditions of 6% acid concentration at 100 C for 300 min. (Gmez et al., 2004). Avci
et al. (2013) treated corn stover (10%, w/w) with dilute H3 PO4 (0.02.0%, v/v). The maximum
glucose yield (85%) was obtained after enzymatic hydrolysis when corn stover was pretreated with
0.5% (v/v) acid at 180 C for 15 min. The highest yield for xylose (91.4%) was observed from
corn stover pretreated with 1% (v/v) acid at 160 C for 10 min. Nair et al. (2015) evaluated the
optimal conditions for wheat bran pretreatment with H3 PO4 at acid concentration of 1.75% (w/v),
temperature of 190 C and time of 10 min. The maximum total polysaccharide yield of 0.27 g/g dry
biomass loading, corresponding to 66% of the theoretical maximum yield observed. The effect of
the dilute acid pretreatment on the functional groups of the wheat bran cellulose was determined
with 78% reduction in the cellulose crystallinity index.
As an alternative to mineral acid, organic acid, mainly dicarboxylic acid, has been studied. So
far, several dicarboxylic acids have been found to show a high selectivity to a substrate because
dicarboxylic acids are similar to both the catalytic core of cellulase and cellulose binding molecules.
Zhang et al. (2013) evaluated that the highest total xylose yield of 84% of the theoretical maximum
was for both 0.5% oxalic and sulfuric acid pretreatment of marple wood at 160 C. Kootstra et al.
(2009) suggest that at 150 C and 2030% (w/w) dry wheat straw, the pretreatment with dilute
fumaric or maleic acid can be a serious alternative to dilute sulfuric acid pretreatment.
For pretreatment using dilute acid severity factor (SF), which combines temperature and
residence time into a single factor, presents the following equation:
where: R0 is reaction ordinate, t is pretreatment time (min.), Tr pretreatment temperature ( C),

Tb reference temperature ( C).
The purpose of introducing the severity function, referred as reaction ordinate R0 , is to trade time
and temperature of treatment in order to equivalent final effects, for example enzyme accessibility,
are obtained. The reference temperature is usually 100 C. Assuming the overall reaction following
first-order kinetics and Arrhenius relation of temperature, the empirical parameter is usually set
equal to 14.75 (Kim et al., 2014).
For pretreatment using dilute acid combined severity factor (CSF) defined as is used to substract
out the effect of pH (Lee & Jeffries, 2011).
CSF involves changes in temperature, time and acidity into a single value, which facilitates
comparisons of data from different conditions. An extremely low acid level of 0.050.1% was
applied to retain the pH near 2.5 (Lee et al., 1999). The application of a combined severity concept
using a small amount of acid and a short residence time at a high temperature is the advantage in
the dilute acid pretreatment.
Pappas et al. (2014) the pretreatment conditions of Phalaris aquatica L. expressed in a combined
severity factor, ranged from 0.13 to 1.16. The concentration of xylose and total monomeric sugars
released from hemicelluloses increased as the CSF increased. Ruiz et al. (2013) performed dilute
sulfuric acid pretreatment of sunflower stalks. The process was conducted at constant time of 5 min.
and the various range of temperature and acid concentration, which was centered at 175 C and
1.25% (w/v), respectively. Optimized results were obtained at 167 C and 1.3% of sulfuric acid.
The xylose recovery from the pretreated solid decreased as the combined severity factor increased.
It can be seen that the content in furfural and HMF (degradation products for xylose and glucose,
respectively) are almost negligible at the lowest combined severity factor (1.18).
Lee et al. (2013) showed that the furfural concentration in rape straw hydrolyzates increased
from 1.80 to 2.16 g/L according to increase CSF values. The higher concentration of furfural was
observed for rape straw in comparison to rice and barley straw. The furfural concentration in the
barley straw hydrolizate was the highest at 1.101.38 g/L, compared to only 0.40 g/L over the entire
CSF range in rice straw hydrolyzate.
Dilute acid processes usually yield sugar recoveries from hemicelluloses above 70% up to 95%
(Allen et al., 2001a; Carvalheiro et al., 2004a; Marzialetti et al., 2008; Monavari et al., 2009). The
hydrolysat contains mainly xylose (80% of the sugar content in hemicellulosic fraction) and others
sugars as arabinose, glucose, galactose, and mannose. The xylan + mannan + galactan recovery
yield of the poplar sawdust treated with 4.0% (w/w) H2 SO4 at 185 C was maximized at 88.6%
by Kim et al. (2013). The sugar content (xylan + mannan + galactan) in the treated-solid with
0.5% (w/w) and 7.0% (w/w), was 11.115.2% and 0.95.7%, respectively. Akpinar et al. (2009)
tested tobacco stalk (TS), sunflower stalk (SS), cotton stalk (CS), and wheat straw (WS). The
yield of xylooligosaccharide depends on acid concentration and hydrolysis time, but the yield of
monosaccharide depends on the structure and composition of xylan besides acid concentration and
the time. The conversion of TS, CS, SS, and WS into xylooligosaccharides was easily achieved by
0.25 M H2 SO4 for 30 min. The all xylans were hydrolyzed to a variety of oligosaccharides ranging
from xylobiose trough xylohexaose to longer chain oligosaccharides. Authors concluded that the
production of high amounts of furfural was the most important limitation of acid hydrolysis.
The effects of pretreatment conditions on lignin separation from poplar wood were reported by
Zhang et al. (2015). The water-only and 0.05% (w/w) sulfuric acid pretreatments were performed
at temperatures ranged from 160 to 270 C in a flow through reactor system for 210 min. Results
showed that water-only flow through pretreatment primarily removed syringyl (S units). Increased
temperature and/or the addition of sulfuric acid enhanced the removal of guaiacyl (G units) com-
pared to water-only pretreatments at lower temperatures, resulting in nearly complete removal of
lignin from the biomass. NMR spectra of the lignin in pretreated liquid revealed significant -O-4
Figure 6.3. Schematic of reactors: (a) co-current and counter-current; (b) temperature step-change and
(c) two-stage reverse flow (Lee et al., 1999).
cleavage, - deoxygenation to form cinnamyl-like end groups, and slight -5 repolymerization

in both water-only and dilute acid flow through pretreatments.
Solution-state two-dimensional (2D) 1 H-13 C hetero-nuclear single quantum coherence (HSQC)
nuclear magnetic resonance spectroscopy, was used to analyze 13 cultivars of rice straw before
and after dilute acid pretreatment, to characterize general changes in the lignin by Teramura et al.
(2015). Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate,
guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained
unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin
aliphatic linkages or ferulate decreased.
Lee et al. (2015) explore the feasibility of applying sequential dilute acid and alkali pretreatment
into the hydrolysis of corn stover. H2 SO4 used in the first step selectively hydrolyzed 74.677.3% of
xylan and NaOH used in the second step removed 85.989.4% of lignin, from the raw corn stover.
Compared to single dilute acid pretreatment, the proposed combined pretreatment minimized the
generation of byproducts such as acetic acid, furfural and hydroxymethylfurfural in the hydrolysates,
and enhanced the enzymatic hydrolysis of the solid residue. The overall glucose and xylose yields
finally obtained after enzymatic hydrolysis reached 89.197.9% and 71.075.9%, respectively.
3.1.2 Reactors
The reactors are an important consideration for the maximum depolimerization of hemicelluloses
during dilute acid hydrolysis. Harmsen et al. (2010) distinguishes two types of weak acid hydrolysis:
high temperature and continuous flow process for low-solids loading (T > 160 C, 510% w/w of
Figure 6.4. The schematic representation of reactors used for acid hydrolysis of lignocellulose (a) a
shrinking-bed reactor and (b) two-stage, counter-current pretreatment reactors.
substrate concentration) and low temperature and batch process for high-solids loading (T 160 C,
1040% w/w of substrate concentration).
The development of such types as the plug-flow reactors (PFR), percolation reactors and shrink-
ing bed counter current reactors have shown promising results for dilute acid mediated hydrolysis
of agro-residues (Fig. 6.3, 6.4) (Taherzadeh & Karimi, 2007; Lenihan et al., 2011). In a percola-
tion reactor high lignocellulosic solid/liquid ratio can be used. Dilute (mostly sulphuric) acid was
sprayed onto the raw material and the mixture was held at 160220 C for short periods up to a few
minutes. The monomeric sugars and soluble oligomers released from biomass into the hydrolysate
which was easily removed from the solid fraction, thereby reducing sugar decomposition.
Percolating reactors operate as co-current and counter-current mode (Fig. 6.3a). The counter-
current reactors have shown better results for the maximum hemicellulosic breakdown with fast
reaction rates, and consequently produced low concentrations of inhibitors (Lee et al., 1999).
The biphasic nature of the hemicelluloses hydrolysis led to a modified percolation process into
two types: step change and two-stage reverse flow percolation (Fig. 6.3b). In the step change
percolation, temperature change during the process, from uniform low to uniform high (Lee et al.,
1999). It involves two-stage processing of biomass, a low-temperature stage and a high-temperature
stage (Kim & Lee, 2007). Authors determined the optimum temperature difference in step-change
operation to be 30 C for a wide range of reaction temperature.
In the two-stage reverse flow percolation the biomass is first treated at a low temperature in
percolation mode. It is then treated again at a high temperature. The difference is that the stream
from the high-temperature treatment is again put through a reactor packed with fresh biomass at
low temperature. The reacted solid residue in this reactor is then treated with fresh acid at high
temperature. This process is repeated.
A shrinking-bed reactor was designed by the National Renewable Energy Laboratory (NREL)
to maintain a constant bulk packing density of cellulosic biomass (Fig. 6.4a) (Wan & Hanley,
2003). The operation principle of bed shrinking was described by Taherzdeh & Karimi (2007).
Shrinking bed reactors reduces the amount of dilute acid and results in higher sugar concentration.
The shrinking-bed reactors are a promising pretreatment reactors with the potential for scale-up
for commercial applications.
NREL also showed the concept, in which biomass is first treated in the relatively low temperature
(174 C). This allows converts the part of easy hydrolyze hemicellulose to sugars. Those liquefield
sugars are removed. Next the remaining biomass is treated in the higher temperature (204 C) at
which other fractions of hemicelluloses are hydrolyzed (Fig. 6.4b). The sugars released flow back
to the first chamber, in which degradation is minimized by the lower temperature. The pretreated
biomass is washed with hot water to rinse out the acid, that prepare the remaining cellulose for
enzymatic hydrolysis.
The use of mineral acids is an effective method in relation to many types of biomass. A disad-
vantage of the process is the production of significant quantities of by-products which can inhibit
the alcohol fermentation.
3.2 Pretreatment with alkaline

The pretreatment of the lignocellulosic biomass may be performed by alkaline, especially sodium,
potassium, calcium or ammonium hydroxides (Kaar & Holtzapple, 2000; Mosier et al., 2005;
Chang & Holtzapple, 2000). The alkaline hydrolysis allows the removal of acetyl and uronic acid
groups. The hydrolysis of ester intramolecular bond of xylan in hemicellulose and extramolec-
ular bonds between lignin and hemicelluloses also occurs (Sun & Cheng, 2002; Chaturvedi &
Verma, 2013). By treating with alkali partially lignin solubilization is achieved, while hemicel-
lulose remains mainly in the insoluble polymeric form. The alkaline pretreatment increases the
digestability of cellulose by enhancing the accessibility for cellulase enzymes and reduction of
the degree of polymerization (Bali et al., 2015). Alkali pretreatment also improves the enzymes
efficiency because it eliminates nonactive adsorption sites (Sierra et al., 2011).
The conditions of alkali pretreatment vary depending on the type and the composition of biomass
used for pretreatment. The higher alkaline pretreatment efficiency is obtained for hardwood, herba-
ceous crops and agricultural residues with low lignin content than for softwood with high lignin
content (Singh et al., 2015). However, for alkali treatment of softwood much more severe condi-
tions need to be set, making the pretreatment more like a pulp-cooking process (Galbe & Zacchi,
2002).
The most important parameters affecting pretreatment of lignocellulosic biomass are the type and
concentration of alkali, biomass loading, pretreatment temperature, and the reaction time. Alkali
pretreatment utilizes low temperatures and pressure. It may be carried out at ambient conditions,
but requires an extension of the reaction time to hours or to even days.
The cheapest alkali is Ca(OH)2 , which allows to remove lignin and hydrolyze acetyl groups
that increase the rate of enzymatic saccharification (Chang, 2007). Lime pretreatment has been
reported for various biomass, such as switch grass (Chang et al., 1997; Xu & Cheng, 2011), wheat
straw (Chang, 2007; McIntosh et al., 2011), rice straw (Cheng et al., 2010), corn stover (Karr &
Holtzapple, 2000), raw sugarcane bagasse (Grimaldi, 2015), and poplar wood (Chang et al., 2001;
Bali et al., 2015).
The lime pretreatment of raw sugarcane bagasse promoted solubilization of lignin (30%) and
hemicelluloses (5%) accompanied by a cellulose accumulation (11%) (Grimaldi et al., 2015).
Moreover considerable damaged of bagasse fibers, including rupture of the cell wall, and exposured
of the cellulose-rich areas to enzymatic action. The study Sindhu et al. (2015) revealed that the
optimum conditions of pretreatment were 0.15 g/g of biomass, pretreatment temperature of 86.8 C,
and pretreatment time of 65.6 h.
Figure 6.5. Process flow chart for alkali pretreatment of lignocellulosic biomass (Pandey et al., 2014).
Chang et al. (1998) for lime pretreatment of wheat straw found that reaction times (13 h) and
temperatures (85135 C) allowed higher sugar yields, but after longer treatment times (24 h)
and lowering temperatures (5065 C) sugar production was even more efficient. The optimal lime
loading was 0.1 g/g dry mass. Under optimal conditions, the yield of reducing sugars was 10 times
higher compared with untreated wheat straw. The lime pretreatment with oxidans of Miscanthus
giganteus was tested by Yang et al. (2015). Under selected conditions (0.2 g of lime/g of biomass,
200 psig O2 , and 150 C for 1 h), delignification was 64.7%. The pretreated biomass was then
enzymatic hydrolysis. The conversion yield of cellulose to glucose in the recovered solid was 91.7%
and hemicelluloses to xylose was 67.3%, it was 7.1 and 18.2 times higher than those obtained from
raw biomass, respectively.
NaOH is commonly used in the chemical pretreatment of lignocelluloses because of its ability to
delignify biomass. This pretreatment causes swelling of lignocellulosic biomass, which leads to an
increase in the internal surface area, reduces cellulose crystallinity, and disrupts lignin structure,
by enhancing the reactivity of the remaining carbohydrate. Figure 6.5 shows the process flow chart
for alkali pretreatment of lignocellulosic biomass.
The optimum conditions for NaOH pretreatment of sugarcane tops were 3% (w/w) NaOH with
15% (w/w) of biomass loading and pretreatment time of 60 min. (Sindhu et al., 2014). Sawdust from
Australian timber mills was treated using 310% (w/w) NaOH at temperatures of 60 C, 121 C,
and (20) C. The maximum yields were obtained at 121 C and (20) C using 7% NaOH, with
29.3% and 30.6% ethanol yields, after 0.5 and 24 h respectively (Trevorah & Othman, 2015).
Another alkali pretreatment method involves aqueous ammonia treatment at elevated tempera-
tures. This method leads to hydrolysis of hemicelluloses and destruction of lignin by ammonolysis.
The cellulose forms a complex with ammonia resulting in the breaking of hydrogen bonds. The loss
of crystallinity increases in accessibility to enzymatic hydrolysis of cellulose (Mittal et al., 2011;
Chaturvedi & Verma, 2013). Ammonia pretreatment techniques can be divided into two broad
types, i.e.: the Ammonia Recycle Percolation (ARP) (Kim et al., 2003) and Soaking in Aqueous
Ammonia (SAA) treatments (Kim et al., 2008).
Ammonia percolation recycle system (ARP, Ammonia Recycle Percolation) was developed for
the pretreatment of the leaves and stalks of corn. The 15% of ammonia solution was pumped
through the bed filled with biomass. The temperature was 170 C and the pressure 2.3 MPa. After
delignification of 85%, in a next step enzymatic hydrolysis was used, in which glucose was almost
completely recovered. However, the xylan hydrolysis took place with low efficiency. Iyes et al.
(1996) obtained the extent of delignification in the ARP process at the range of 7480% for corn
cobs/stover mixture and 7184% for switchgrass at 10% ammonia concentration.
Pretreated biomass with aqueous ammonia in a flow-through column reactor has been researched
by Kim et al. (2003) and Kim & Lee (2005). This process reduces the lignin content by 7085%,
the hemicellulose about 4060% and leaves cellulose intact. The liquid fraction is sent into a steam-
heated evaporator for ammonia recovery and separation of lignin and other sugars. Ammonia is
then recycled to the reactor inlet, whereas the separated fraction is sent into a crystallizer. After
crystallization, a washing step is carried out to extract the sugars that have been retained in the solid
matrix. Kim & Lee (2005) optimized conditions consisting as followed 0.47 g of NH3 and 2.7 g of
water at 170 C for pretreatment of 1 g of corn stover. Results indicated 73.4% delignification and
88.5% digestibility with enzyme loading of 15 FPU/g of glucan.
A two-step process combining percolation-mode ammonia pretreatment of poplar sawdust with
mild organosolv purification of the extracted lignin was conducted by Bouxin et al. (2014). This
combination produced high quality and high purity lignin in up to 31% yield and 50% recovery.
The uncondensed fraction of the isolated lignin was up to 34%, close to the native lignin (40%).
In collaboration with researchers at the Joint BioEnergy Institute and Bioenergy Science Center,
researchers in the Great Lakes Bioenergy Research Center developed a new liquid ammonia pre-
treatment called Extractive Ammonia (EA) to simultaneously convert native crystalline cellulose I
to a highly digestible cellulose III allomorph, and extract selectively up to 45% of the lignin from
lignocellulosic biomass with near-quantitative retention of all polysaccharides. EA pretreatment
involves a three-stage process: reaction, extraction, and product/solvent recovery. During reaction,
the cellulose-ammonia complex is formed, ester bonds are cleaved, and lignin is partly solubilized
in the liquid ammonia phase. The key reactions is disrupt lignin-polysaccharide crosslinks. In the
extraction stage, the pretreated biomass is filtered to separate the ammonia-soluble components
from residual solids. During this stage, lignin is extracted, and a highly digestible cellulose allo-
morph is formed from the cellulose-ammonia complex. During the recovery stage, the ammonia is
evaporated from the extractives, which are subsequently recovered as a dark brown viscous liquid.
EA pretreated corn stover yielded higher fermentable sugars compared to the Ammonia Fiber
Expansion (AFEX) process using 60% lower enzymatic loading. EA preserves extracted lignin
functionalities, offering the potential to co-produce a lignin-derived fuels and chemicals in the
biorefinery. This single-stage EA fractionation process achieves high biofuel yields (18.2 kg Et-
OH/100 kg untreated corn stover on a dry weight basis) (da Costa Sousa et al., 2016).
In the Soaking inAqueousAmmonia (SAA) method, the aqueous ammonia, at high temperatures,
causes swelling and efficient delignification of biomass (Fig. 6.6).
Kim & Lee (2005) investigated the soaking of corn waste in 29.5% (w/w) ammonia solution. The
lignin removal was more than 74%. The residue contains 85% xylan and almost all, not removed
the glucan. Kim & Lee (2007) obtained the same results for xylan and glucan and lower for lignin
removal (62%) using corn stover treated at 15% (w/w) of NH3 and 60 C. Kim et al. (2008) treated
Figure 6.6. Scheme of soaking in aqueous ammonia (SAA) process flow.
barley hull using 15% (w/w) ammonia at 75 C. The 5066% original lignin was removed, while
6576% xylan was retained without any of glucan loss. The efficiency of SAA is comparable to
the efficiency of the ARP but the process is less expensive (Wyman et al., 2005).
A novel soaking pretreatment of corn stover using NaOH and aqueous ammonia for deligni-
fication to improved enzymatic saccharification rate was evaluated by Zuo et al. (2012). Results
revealed 63.6% lignin removal while most of the carbohydrates were reserved. The optimal condi-
tions of pretreatment were 1% NaOH + 8% NH4 OH with a solid liquid ratio of 1:10 and pretreatment
temperature of 50 C for 48 h.
There are several advantages and disadvantages of alkali pretreatment. Lignocellulosic pretreat-
ment with alkaline is characterized by a lower degradation degree of the sugars in comparison to
pretreatment with acids (Alvira et al., 2010; Agbor et al., 2011). The potential need for neutral-
ization makes down-stream processing difficult and also increases the cost of scaling up alkali
pretreatment. The degradation of lignin to other soluble aromatic compounds may promote the
formation of inhibitors. This method has not been used on a large-scale plant.
3.3 Organosolv fractination

The organosolv pretreatment can occur in organic or aqueous-organic solvent systems with or
without catalysts at a temperature range of 100250 C (Muurinen, 2000; Zhao et al., 2009). A
wide range of solvents have been tested. Among them are alcohols with low boiling points such
as methanol and ethanol, alcohols with high boiling points like ethylene glycol, tetrahydrofurfuryl
alcohols and glycerol, and other classes of organic compounds like ketones, phenols and ethers
(Wen et al., 2013; Jimenez et al., 2004; Sun & Chen, 2008).
The Hildebrand solubility parameter or -value of a solvent can be used to estimate the solubility
of lignin or other polymers. Solvents which display the good lignin solubility have values around
11, with acetic acid ( = 10.1), formic acid ( = 12.1), ethanol ( = 12.9), and acetone ( = 9.7)
(Sannigrahi & Ragauskas, 2013; Quesada-Medina et al., 2010). A mixture of dioxane, ethanol, and
acetone with 25% water was also found to have -values close to lignin and exhibited the ability
to dissolve both high- and low-molecular-weight lignin fractions (Pan & Sano, 1999).
During pretreatment of lignocellulosic biomass, cellulose is recovered as solids while most of
the lignin and hemicellulose dissolve into the organic solvent, forming liquor (Zhang et al., 2016).
Organic solvent pretreatment allows separation of high-purity cellulose with only minor degrada-
tion. In further processing, lignin precipitates and hemicelluloses remain as a water-soluble fraction
(Sannigrahi & Ragauskas, 2013). As a result, the major lignocellulosic biomass components such
cellulose, lignin, and hemicelluloses can be separated into three process streams. The advantage
of this process is the easy recovery of organic solvents by distillation and their recycling for use in
pretreatment.
Pretreatment can be performed with or without a catalyst, with auto-catalyzed pretreatments
being performed at higher temperatures (185210 C). Mineral acids (hydrochloric acid, sulfuric
acid, and phosphoric acid) are good catalysts to accelerate delignification and xylan degradation,
as are organic acids (formic, oxalic, acetylsalicylic, and salicylic acid) which can also be used as
catalysts (Sun & Cheng, 2002). In addition, magnesium sulfate, magnesium, calcium chloride or
nitrate, barium chloride or nitrate, sodium bisulfate, and sodium hydroxide can also be used as
catalysts (Zhao et al., 2009).
Low boiling point alcohol pretreatment. Methanol and ethanol are commonly used in the process,
primarily due to their low cost, low boiling point and easy recovery. The combination of ethanol
as the solvent and sulfuric acid as the delignification catalyst has been used with several common
biomass feedstocks (Sannigrahi & Ragauskas, 2013). The process of methanol/ethanol pretreatment
is described in Fig. 6.7.
The insoluble fraction recovered after the organosolv process consists mostly of cellulose,
together with minor amounts of unhydrolyzed hemicelluloses and lignin. After delignification, the
pretreated solid is first washed with alcohol and then with water. From the pretreatment liquor,
the solvent is recovered by evaporation, and then condensed, which allows it to be recycled to
the reactor. The remaining concentrated black liquor is diluted with water, filtering, washing, and
drying the precipitated lignin. The filtrate contains an aqueous solution of hemicellulose sugars,
which consist mainly of xylose in the case of hardwoods or agricultural residues, the acetic acid,
furfural, xylose, and extractives.
The efficiency of pretreatment depends on many factors like temperature, ethanol concentration,
and acid dose. Optimization of enzymatic digestion of wheat straw resulted in a maximum glucose
yield of 86% without the use of a catalyst (lignin yield of 84%, organosolv at 210 C, 50% (w/w)
aqueous EtOH). Using 30 mM H2 SO4 as catalyst resulted in similar glucose and lignin yields at a
lower temperature (190 C, 60% (w/w) aqueous EtOH). Lowering the pretreatment temperature by
using an acid catalyst substantially improved the yield of the hemicellulose derivatives like xylose
and furfural (Wildschut et al., 2013).
Ethanol organosolv pretreatment was performed on loblolly pine to improve the efficiency
of enzymatic hydrolysis of cellulose to glucose. Loblolly pine sawdust was immersed in a 65%
ethanol/water solution containing 1.1% sulfuric acid and treated in a Parr pressure reactor (3.8 L) at
170 C for 1 h. Following the organosolv pretreatment, the crystallinity of the cellulose was reduced,
rendering the substrate easily hydrolyzable by cellulase (Sannigrahi et al., 2010).
The advantage of the organosolv pretreatment process is its ability to obtain high-quality lignin
which can be used as a high-value drop-in chemical for a broad range of industrial applications.
Among these applications are specific adhesives and resins for coatings, construction, plywood,
etc., concrete plasticizers for construction, friction materials for high-performance brake products,
and grease (Arato et al., 2005).
Zhu et al. (2015) treated Eucommia ulmoides Oliver (EU) wood in an integrated process combin-
ing organosolv pretreatment and autohydrolysis (50% aqueous ethanol with 1% HCl as a catalyst
at 180 C for 30 min.). NMR characterization of the lignin revealed substantial cleavage of -O-4
linkages and formation of stilbene, but resinol units (-) were resistant to degradation by organo-
solv delignification. This integrated process obtained 9.5 g xylooligosaccharides, 14.5 g lignin, and
41.0 g cellulose-rich residue from 100 g EU wood.
Triploid of Populus tomentosa Carr. chips were subjected to an auto-catalyzed ethanol organo-
solv pretreatment process (AEOP) by Guo et al. (2015). Three lignin preparations were sequentially
isolated: lignin dissolved in the pretreatment liquor (DL), lignin reprecipitated onto the pretreated
residue (PL) and residual lignin in the ethanol-water washing-residue (RL). The results demon-
strated that certain amounts of aryl- ether (-O-4 ) linkages were cleaved and stilbene units were
formed during this process, whereas the resinol (- ) and phenylcoumaran (-5 ) units were left
Figure 6.7. Schematic representation of an ethanol-organosolv pretreatment approach.
intact. In addition, the DL, PL and RL fractions contained less aliphatic hydroxyl groups and more
phenolic hydroxyl groups than lignin after enzymatic hydrolysis.
Lewis acids have been studied by Constant et al. (2015) as catalysts in the organosolv treatment
of wheat straw. Fractionation of the lignocellulosic biomass and fragmentation of lignin have
been performed in aqueous ethanol in the presence of FeCl2 , CuCl2 , FeCl3 , Ga(OTf)3 , ZrOCl2
or Sc(OTf)3 . Hard Lewis acids allow to obtain a higher degree of delignification and higher yield
of Klason lignin than that achieved with sulphuric acid. This process leads also to the formation of
aromatic monomers in very small amount, and a significant amount of soluble phenolic-derived
oligomers, issued from delignification process.
Post treatment with H2 O2 is an effective method to enhance the efficiency of organosolv pre-
treatment. Geng et al. (2012) noted that horticultural waste treated with 70% ethanol at 70 C in
the presence of 1% HCl followed by H2 O2 post-treatment demonstrated the highest sugar yield
of 57.8%.
Compared to ethanol, methanol is more toxic and must be applied at higher concentration (Zhao
et al., 2009). Gandolfi et al. (2014) removed more than 75% of total hemicellulose and 75% of
total lignin from hemp hurds under the following conditions: 165 C, 3% H2 SO4 , 20 min. reaction
time, and 45% MeOH. After pretreatment the enzymatic hydrolysis of the residual biomass yielded
up to 60% cellulose-to-glucose conversion.
Butanol is an excellent delignification agent due to its hydrophobicity. The limited miscibility
of butanol in water allows to concentrate hemicelluloses in the aqueous layer, lignin in the butanol
layer, and cellulose in the solid fraction, but the high solvent costs decrease their attractiveness (Zhao
et al., 2009). Del Rio et al. (2010) employed MgCl2 , H2 SO4 , SO2 , and NaOH, and the solvents were
ethanol and butanol for pine beetle-killed lodgepole pine (Pinus contorta) chips. Authors noted
that the use of butanol, yielded higher concentrations of sugars in the aqueous fractions. It was
caused by using the lower volume of butanol, approximately 20%, than in ethanol pretreatments
because of the limited miscibility between butanol and water. Therefore, especially in the case of
the pretreatments with H2 SO4 , the utilization of butanol resulting in higher concentration of sugars
because of the limited aqueous layer.
Polyhydroxy Alcohols. Ethylene glycol and glycerol are the most commonly employed higher-
boiling-point alcohols for organosolv pretreatments. One of the main advantages of using such
solvents is that the pretreatment can be performed at atmospheric pressure, which reduces energy
costs and the need for pressure vessels.
Acetone and Methyl Isobutyl Ketone. Araque et al. (2008) optimized the conditions for organosolv
pretreatment of Pinus radiata with aqueous acetone (50%) and 0.9% sulfuric acid. Huijgen et al.
(2010) fractionated a key agricultural residue, wheat straw, using auto-catalyzed acetone organosolv
pretreatment. Bozell et al. (2011) developed a novel organosolv biomass fractionation process
which they termed Clean Fractionation. In this method, lignocellulosic material is separated with
a ternary mixture of methyl isobutyl ketone, ethanol and water in the presence of sulfuric acid,
which selectively dissolves lignin and hemicelluloses, leaving a cellulose residue.
Klamrassamee et al. (2013) optimized the pretreatment of eucalyptus wood chips. The authors
pretreated 16.7% (w/v) biomass in a ternary mixture of methyl isobutyl ketone:methanol:water
(25:42:33) with 5% AC-H3 PO4 and incubated at 180 C for 60 min. Under these conditions, 41.2%
(w/w) cellulose was obtained in enriched solid pulp with the average glucan content of 75.9%.
The hemicelluloses was hydrolysed in the aqueous-methanol phase, which contained 17.8% (w/w)
monomeric xylose and xylooligomers while 13.7% (w/w) lignin was separated in the organic phase.
This procedure is an alternative to corrosive homogeneous acids for lignocellulose fractionation in
integrated biorefineries.
Araque et al. (2008) optimized the organosolv acetonewater pretreatment conditions for Pinus
radiata D. chips. Authors obtained the 99.5% ethanol yield (36 g/L) from an organosolv pretreated
material under following conditions at 195 C, 5 min., pH 2.0 and the acetone:water ratio of 1:1.
The lignocellulosic material of woody biomass was separated by Bozell et al. (2011) with a
ternary mixture of methyl isobutyl ketone, ethanol and water (at 16:34:50% ratio) in the presence
of an acid promoter. This procedure enabled for selective dissolution of lignin and hemicelluloses,
and leaving cellulose undissolved. The yield of the cellulose fraction averaged 47.7% (w/w) and
for the lignin fraction was 18.3% (w/w).
These processes are not commercial yet, but have been demonstrated in pilot and demonstration
scale. Organosolv pulping or fractionation of lignocellulosic biomass is nowadays one of the
selected pretreatments to produce high quality cellulose for pulp and/or biofuel production together
with a high purity lignin for materials and chemicals.
3.4 Oxidative delignification

Delignification of lignocellulose can also be achieved by treatment with an oxidizing agent such
as hydrogen peroxide, ozone, oxygen or air. Hydrogen peroxide is the most commonly employed
oxidizing agent. Application of hydrogen peroxide caused the dissolution of hemicelluloses and
lignin, while the cellulose remains intact. The remaining cellulose in the dry residue may then be
treated by enzyme. The optimum pH is 11.5. The dissolved hemicelluloses can be recovered by
ethanol. Hemicelluloses containing 50% xylose, after purification, can be directed to the enzymatic
hydrolysis or hydrolysis with acids. Studies have shown that dissolution of about 50% lignin and
most hemicelluloses has been achieved in a solution of 2% H2 O2 at 30 C (Chaturvedi & Verma,
2013).
Li et al. (2015) determined the alkaline hydrogen peroxide pretreatment catalyzed by Cu(II) 2,2 -
bipyridine complexes to substantially improve the enzymatic hydrolysis of woody hybrid poplar. The
alkali-soluble lignin content increased with time during the catalytic oxidation process, although,
the molecular weight distributions were unaltered. Yields of aromatic monomers (including pheno-
lic acids and aldehydes) were found to be less than 0.2% (w/w) on lignin. Oxidation of the benzylic
alcohol in the lignin side-chain was evident in NMR spectra of the solubilized lignin, whereas
minimal changes were observed for the pretreatment of insoluble lignin.
Bhalla et al. (2016) pretreated hybrid poplar by an alkaline extraction incorporated prior to the
copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) treatment and H2 O2 was added batch-wise
over the course of 10 h. Authors revealed that the alkaline pre-extraction improved glucose (86%)
and xylose (95%) yields following enzymatic hydrolysis. An increase in the lignin solubilization
was also observed with fed-batch H2 O2 addition relative to batch-only addition, which resulted in
increased glucose and xylose yields (77 and 93% versus 63 and 74%, respectively). Importantly,
combining these strategies led to significantly improved sugar yields (96% glucose and 94% xylose)
following enzymatic hydrolysis. In addition, the authors found that the chemical inputs (enzyme,
H2 O2 , and catalyst) could be substantially lower, while still maintaining high product yields utilizing
the improved Cu-AHP process.
Wet oxidation involves oxygen or air in combination with water (Varga et al., 2003). When the
process takes place at low temperatures, hydrolysis of lignocellulose occurs. At high temperatures,
oxidation of lignocellulose occurs with liberation of carbon dioxide and water (Chaturvedi & Verma,
2013). The advantage of the process are lower costs compared with hydrogen peroxide.
The use of ozone in the pretreatment leading to decomposition of the complex lignocellulosic
and degradation of lignin. To a lower extent, the ozonolysis process affects hemicelluloses and
cellulose (Sun & Cheng, 2002). The process is carried out at atmospheric pressure and room
temperature (Vidal & Molinier, 1988; Neely, 1984). It can be used to disrupt the structure of many
different lignocellulosic materials, such as wheat straw, bagasse, pine, peanut, cotton straw and
poplar sawdust (Sun & Cheng, 2002).
3.5 Ionic liquids

Ionic liquids (ILs) are group of low-melting molten salts that can exist in liquid form at relatively low
temperatures (less than 100 C), and frequently they are liquids at room temperature (RTILs). The
properties of ionic liquids can be tuned by appropriate selection of anions and cations. Modern ionic
liquids usually contain organic cations as: alkylimidazolium [R1R2IM]+, alkylpyridinium [RPy]+,
tetraalkylammonium [NR4] + (Pinkert et al., 2009; Wang et al., 2010). Alkylated phosphonium
and sulfonium cations are also used (Brandt et al. 2015). Ionic liquid anions can be either organic
(formate, acetate) or inorganic (Figure 6.8). Among anions halogens, formates, acetates, amides,
imides, thiocyanates, phosphates, sulfates, sulfonates, and dichloroaluminates can be selected
(Mki-Arvela et al., 2010). The number of potential ion combinations available reputedly equates
to 1012 ILs (Forsyth et al., 2005).
Unique physical and chemical properties of ILs distinguish them from molecular solvents. ILs
characterized by low melting point, high polarity, high viscosity, minimal vapor pressure, non-
flammability, high heat capacity, thermal stability, and showed excellent solubility with many
organic compounds (Sowmiah et al., 2009). Ionic liquids are used either for the pretreatment of
biomass (swelling and selective extraction of components) or for complete dissolution of biomass
(Hossain & Aldous, 2012).
3.5.1 Pretreatment of biomass dissolution of cellulose

Dissolution of cellulose is desired in biomass-to-fuels processing. Ionic liquids, typically with
1,3-dialkylimidazolium cations, are effective in dissolving cellulose (Wang et al., 2010). Swatloski
et al. (2002) demonstrated that [BMIM]Cl breaks the extensive hydrogen bonding network present
in cellulose, allowing for obtain much quicker dissolution times, and dissolve higher concentrations
Figure 6.8. Selected chemical structure of representative cations (a) and anions (b) used in ionic liq-
uids, and ionic liquids commonly used for the pretreatment of lignocellulosic biomass (c). The cations
include (from the left to right): (top row) alkylmethylimidazolium, alkylpyridinium, tetraalkylammo-
nium, tetraalkylphosphonium, trialkylsulfonium. The anions include (from the left to right): (second
row) chloride, bromide, formate, nitrate, hydrogen sulfate (HSO4 ), (third row) methylsulfate (MeOSO3 ),
methylphosphonate (MePO3 H), dimethylphosphate (MeO2 )PO2 , tetrafluoroborate (BF4 ), hexafluorophos-
phate (PF6 ), (fourth row) thiocyanate (SCN), dicyanamide (DCA). Ionic liquids include (from the left to the
right): (fifth row) 1-ethyl-3-methylimidazolium acetate [EMIM]OAc, 1-allyl-3-methylimidazolium chloride
[AMIM]Cl, 1-butyl-3-methylimidazolium chloride [BMIM]Cl, (bottom row) 1-ocyl-3-methylimidazolium
chloride [OMIM]Cl, 1-butyl-3-methylpyridinium chloride [3BMPY]Cl. Prepared according to Sowmiah et al.
(2009), Mki-Arvela et al. (2010), Hossain and Aldous (2012), da Costa Lopes et al. (2013), Hayes et al.
(2015), Elgharbawy et al. (2016).
of cellulose than the traditional solvent systems. It was observed that for imidazolium-based ILs,
the shorter the alkyl chain is, the higher the solubility will be. It has been reported that 1-allyl-3-
methylimidazolium cation, [AMIM]+ , is more powerful in dissolution of cellulose than 1-butyl-
3-methylimidazolium [BMIM]+ due to its smaller size (Zhang et al., 2005). The longer-chain
substitutes ionic liquids such as 1-hexyl-3-methylimidazolium acetate ([HMIM]OAc and 1-octyl-
3-methylimidazolium acetate [OMIM]OAc) appear to be less efficient at dissolving cellulose
(Swatloski et al., 2002).
Zhao et al. (2012) tested the effect of IL cations differing with heterocyclic structure on the disso-
lution of cellulose. The dissolution of cellulose in 1-butyl-3-methyl pyridinium chloride [BMPY]Cl
was better than that in [BMIM]Cl.
Currently, it dominates the view that ILs with strong hydrogen-bond acceptor anions are more
effective in the dissolving pretreatment of cellulosic biomass. The anions that are good hydrogen
bond acceptors such as OAc HCOO and (C2 H5 O)2 (PO2 ) have been found effective for cellulose
Figure 6.9. Dissolution mechanism of cellulose in ionic liquids (Feng & Chen, 2008).
dissolution in ionic liquids. Ethyl-3-methylimidazolium acetate [EMIM]OAc is more efficient than

[EMIM]Cl (Zavrel et al., 2009). Generally, with the same cation, the solubility of cellulose in ILs
decreases in the order: [(CH3 CH2 O)2 PO2 ] [OAc] > [SHCH2 COO] > [HCOO] > Cl >
Br [SCN] (Sun et al., 2011). To reduce the production cost and improve the thermal stability of
ILs, a series of alkylimidazolium ILs containing phosphonate-based anions have been synthesized
(Fukaya et al., 2008; Vitz et al., 2009).
The mechanism of cellulose dissolution in ILs involves the oxygen and hydrogen atoms of
cellulose hydroxyl groups, which form electron donor-acceptor complexes that interact with ILs
(Feng & Chen, 2008). Remsing et al. (2006) using 13 C and 35/37 Cl NMR relaxation measurements
demonstrate that solvation of cellulose by the ionic liquid ([BMIM]Cl) involves hydrogen-bonding
between the carbohydrate hydroxyl protons and the IL chloride ions in a 1:1 stoichiometry. Upon
interaction between celluloses hydroxyl groups and ILs, hydrogen bonds are broken, leading to
opening of the hydrogen bonds between molecular chains of the cellulose, resulting in cellulose
dissolution (Fig. 6.9) (Zheng et al., 2014).
Xu et al. (2012) showed, that both chloride anions and imidazolium cations of the IL interact
with the oligomer via hydrogen bonds, whose hydroxyl groups act as the hydrogen bond donors
and acceptors, respectively. The strength and number of hydrogen bonds and the interaction energy
of chloride anions with the oligomer are much larger than of imidazolium cations, implying that
chloride anions of the IL are mainly responsible for the observed effective dissolution of cellulose
in the IL, whereas imidazolium cations of the IL play a relatively less important role.
The imidazolium-based carboxylate characterized by high viscosity which would make cel-
lulose difficultly disperse. To overcome the disadvantages, Rinaldi (2011) developed (IL +
aprotic polar solvent including dimethylsulfoxide and dimethylformamide) systems which
have lower viscosity and higher dissolving rate than ILs. Xu et al. (2013) designed novel
[BMIM]CH3 COO/DMSO solvents by adding an aprotic polar solvent DMSO to [BMIM]CH3 COO
and found that the solvents could effectively dissolve cellulose at ambient temperature with-
out heating. In next work, Xu et al. (2015) showed, that with increasing alkyl chain length in
imidazolium cation, the cellulose solubility decreases in the order: [BMIM]CH3 COO + DMSO >
[EMIM]CH3 COO + DMSO > [HMIM]CH3 COO + DMSO > [OMIM]CH3 COO + DMSO.
When considering ionic liquids as cellulose solvents, it is important to investigate whether
any structural changes occur in the cellulose molecule during its dissolution takes place. Zhu
(2008) noted, that the regenerated sample of cellulose has the same degree of polymerization and
polydispersity as the initial cellulose. However, its macro- and micro-structures, especially the
degree of crystallinity, can be manipulated by changing regeneration process.
Solubilized cellulose can be rapidly precipitated with anti-solvents such as ethanol, methanol,
acetone, or water and easily regenerated in a variety of forms, such as flocs, films, beads (Swatloski
et al., 2002).
Zhang et al. (2005) suggests that [AMIM]Cl can be considered as suitable solvent for cellu-
lose. [AMIM]Cl is thermostable and nonvolatile, and can be easily prepared and recycled. The
cellulose materials regenerated by coagulation with water exhibited a good mechanical prop-
erties. FTIR spectra of cellulose before and after regeneration are quite similar, indicating no
chemical reaction occurred during the dissolution and coagulation processes of the cellulose.
1-butyl-3-methylimidazolium chloride, 1-methyl-3-methylpyridinium chloride and N-benzyl-N,N-
dimethyltetradecylammonium chloride were found to be non-derivatising solvents for cellulose
(Heinze et al., 2005).
3.5.2 Pretreatment of biomass dissolution of lignin

The efficient enzymatic conversion of cellulose requires the physical removal of lignin. Lignin
is more difficult to be dissolved than the other components of lignocellulosic biomass, because
of its strong covalent bonds and complex structure. The dissolution of lignin was investigated
in several imidazolium salts containing methyl-, ethyl-, allyl-, butyl-, hexyl- and benzyl groups
in the imidazolium ring coupled to a number of common anions, such as chloride, bromide,
tetrafluoroborate, acetate, trifluoromethanesulfonate and methylsulfate (Mki-Arvela et al., 2010).
Pu et al. (2007) tested solubility of lignin isolated from a southern pine kraft pulp in different
ILs. Solubility of lignin in 1,3-dimethylimidazolium methylsulfate ([MMIM]MeSO4 ) was 344 g/L,
1-hexyl-3-methylimidazolium trifluoromethanesulfonate ([HMIM]CF3 SO3 ) 275 g/L but in
1-butyl-2,3-dimethylimidazolium tetrafluoroborate ([BM2IM]BF4) only 14.5 g/L. For [BMIM]+
based ILs, the solubilities of lignin followed in the order: [MeSO4 ] > Cl > Br >> PF6.
Lee et al. (2009) used 1-ethyl-3-methylimidazolium acetate ([EMIM]CH3 COO) as a pretreat-
ment solvent to extract lignin from wood flour. When 40% of the lignin was removed, the cellulose
crystallinity index dropped below 45, resulting in > 90% of the cellulose in wood flour to be
hydrolyzed by Trichoderma viride cellulase.
For softwood Kraft lignin up to 20% (w/w) lignin could be dissolved with in ILs
based on imidazolium cations. The solubility varies with the anions in the following order:
[CF3 SO3 ] [MeSO4 ] >> [OAc] > [HCOO] >> Cl Br >> [BF4] >> [PF6] . The
strongly hydrogen-bonding anions such as [MeSO4 ] make efficient solvents for lignin. The
two most powerful solvents for lignin were 1-butyl-3-methylimidazolium trifluoromethanesul-
fonate and 1,3-dimethylimidazolium methylsulphate (Mki-Arvela et al., 2010). The large,
non-coordinating [BF4] and [PF6] anions show very limited efficiency in lignin dissolution (Pu
et al., 2007).
Tan et al. (2009) used an ionic liquid mixture containing the 1-ethyl-3-methylimidazolium cation
and a mixture of alkylbenzenesulfonates with xylenesulfonate as the main anion to extract lignin
from sugarcane plant waste at atmospheric pressure and elevated temperatures (170190 C). An
extraction yield exceeds 93%. The by-products of the extraction was a cellulose pulp.
Acidic ionic liquid water mixtures based on the hydrogen sulfate anion have also been shown
to efficiency extract lignin from lignocellulosic biomass. Brandt et al. (2015) isolated lignin from
Miscanthus giganteus after extraction with the protic ionic liquid 1-butylimidazolium hydrogen
sulfate ([HBIM]HSO4 ) followed by precipitation with the antisolvent water. The ionic liquid pre-
treatment breaks lignin-hemicellulose linkages and depolymerized the lignin through the cleavage
of glycosidic, ester and -O-4 ether bonds.
The structure of eucalyptus lignin after ionic liquid and alkaline ethanol pretreatment processes
was investigated by Xu et al. (2015). A maximum yield of 35.0% was achieved for lignin prepared
from the 1-allyl-3-methylimidazolium chloride pretreated feedstock. The lignins prepared with
ILs pretreatment contained lower amount of carbohydrates (0.782.13%) than milled wood lignin
(9.11%) and alkaline ethanol lignin (2.29%). Additionally, the integrated method pretreatment of
eucalyptus led to significant improvement of cellulose hydrolysis, and the optimal yield of glucose
was 92.6%.
3.5.3 Dissolution of biomass in ionic liquid

The ability of some ionic liquids (ILs) to dissolution of raw biomass under relatively mild conditions
makes this method particularly interesting in biorefineries (Sun et al., 2009). Overall, IL processing
of lignocellulosic biomass could have tremendous advantage over current technologies by enabling
the recovery of all three of the major natural lignocellulosic biopolymers: cellulose, hemicelluloses,
and lignin.
Fort et al. (2007) indicates that solvent systems based on [BMIM]Cl dissolve cellulose and lignin
from different sources of wood with varying hardness, including pine, poplar, eucalyptus, and oak.
However, none of the samples dissolved completely even after extended periods of time exposition.
[EMIM]OAc appeared a better solvent for wood than [BMIM]Cl under the same operation condi-
tions (Sun et al., 2009, 2011). Both softwood (southern yellow pine) and hardwood (red oak) could
be completely dissolved in [EMIM]OAc after mild grinding. The explanation of more effective of
[EMIM]OAc compared to [BMIM]Cl is that the increased basicity of the acetate anion makes it
more efficient at disrupting the inter- and intramolecular hydrogen bonding in biopolymers than
Cl (Fukaya et al., 2008). Additionally, the lower viscosity and lower melting point of [EMIM]OAc
also facilitate the dissolution Sun et al. (2011). Sun et al. (2009) observed a higher dissolution
of hardwood in ILs, compared to softwood, under the same conditions. This might be explained
higher density and hardness in hardwood. However, softwood contains more lignin resulting in a
more complex and inaccessible structure, and makes the dissolution more difficult.
The particle size of the wood chip exhibited large effects in the dissolution efficiency (Wang
et al., 2010). The dissolution rate was depend on the wood particle sizes (spruce, pine) as fol-
lows: ball-milled wood powder > sawdust thermomechanical pulp (TMP) fibers >> wood chips
(Kilpelinen et al., 2007). As the structure of wood sample is incompact, ILs are easy to diffuse into
the woods interior and break the intermolecular forces, resulting in a higher solubility of wood.
Sun et al. (2011) proposed flowchart for the process of dissolution and regeneration of wood
in the IL [EMIM]OAc. The major components can be recovered, with partial separation, from
the IL medium by proper selection and sequential addition of reconstitution solvents. Cellulose-
rich material and pure lignin can be recovered separately with the use of aqueous acetone as
reconstitution solvent (Fig. 6.10).
Li et al. (2010) compared the ionic liquid and acid pretreatments of switchgrass and stated that
the ionic liquid pretreatment is a promising alternative to the dilute acid pretreatment process in
terms of total process time to produce high yields of sugar from the recovered product. Ionic liquid
pretreatment enabled a significant enhancement in the rate of enzyme hydrolysis of the cellulose
component of switchgrass, with a rate increase of 16.7-times, and a glucan yield of 96.0% obtained
in 24 h.
Rapid dissolution of bagasse and southern yellow pine has been achieved in the ionic liquid
1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) by using a dissolution temperature above the
glass transition of lignin (ca. 150 C). Upon regeneration in acetone/water, lignin and carbohy-
drate can be partially separated as lignin and a cellulose-rich material (CRM, pulp) (Li et al.,
2011).
4 PHYSICO-CHEMICAL METHODS
The steam explosion (SE) is characterized by low using of chemicals and limited energy con-
sumption. In this process biomass is treated with high pressure steam (1 to 3.5 MPa) at high
temperature (180 to 240 C) for a few seconds or a few minutes before the biomass is subjected
rapidly depressurized. The decompression causes temperature drops and quenching the process.
There is a combination of mechanical forces after penetration of high-pressure steam into the
plant cell walls and chemical effects called autohydrolysis. The autohydrolysis occurs when the
acetic acids is released from acetyl groups linked to the hemicelluloses. Due to acidic conditions,
hydrolysis of hemicelluloses into oligosaccharides and monosaccharides occurs (Zheng et al., 2009;
Alvira et al., 2010). The lignin is melted, solibilizes and recondensed. During explosion step, plant
biomass particles are exploded into small pieces. As a result the fibrils structure in lignocelluloses
biomass is destroyed (Chen & Liu, 2015).
The ultrastructure of steam-exploded wood tested by Donaldson et al. (1988) showed, that the
enhanced digestibility of steam exploded the softwood Pinus radiata D. is attributed to three main
factors: an increase in surface area caused by fragmentation of the wood, an increase in porosity due
to lignin redistribution, and an increase in porosity due to hydrolysis and removal of hemicelluloses.
Figure 6.10. Flowchart for the process of dissolution and regeneration of wood in the IL [EMIM]OAc (Sun
et al., 2009).
The main disadvantages of the steam explosion process are the partial degradation of hemicellu-
lose and that toxic compounds are generated, which could affect the subsequent hydrolysis and
fermentations steps (Olivia et al., 2003). The steam explosion gives a dark brown product which
contains partially hydrolysed hemicelluloses easily recovered by water-washing. In water-insoluble
fraction remains cellulose, residual hemicelluloses and a chemically modified lignin. Limitation
of steam explosion includes the formation of different degradation products that may inhibit down-
stream processes (Garcia-Aparicio et al., 2006). Figure 6.11 shows the schematic fractionation of
lignocellulosic biomass with ethanol production and chemicals from lignocellulosic biomass.
SE has been successfully applied to ethanol production from several types of plant biomass.
Treatment efficiency of steam explosion on cellulose enzymatic yield has been shown on eucalyptus
(Nunes & Pourquie, 1996) pine chips (Martin et al., 1995), rice straw (Moniruzzaman, 1996), and
Miscanthus giganteus (Sorensen et al., 2008). The utilization of hardwoods (aspen chopsticks)
and softwoods (Japanese cedar) in the bioethanol production by using steam explosion were tested
by Asada et al. (2011, 2012). Results show that the softwood required higher steam conditions
than the hardwood. Poplar, salix and birch were chopped into wood chips length size of 20 mm,
210 mm and 10 mm, respectively. Uzelac (2014) compare the efficiency of steam explosion of the
hardwood birch and the softwood spruce. The slightly favorable trends were obtained for spruce.
The key factors for uncatalyzed steam explosion are time residence, temperature, particle size and
moisture content (Negro et al., 2003). Analogically, the particle size, temperature and residence
Figure 6.11. Schematic of fractionation of lignocellulosic biomass and production of some chemicals from
lignocellulosic biomass.
time on steam-explosion pretreatment of agriculture residue (Brassica carinata) was tested by

Ballesteros et al. (2002). Authors showed that the efficiency of the steam-explosion pretreatment
of herbaceous plant depended on the initial particle size of the substrate. The best hydrolysis yields
was obtained when particles dimensions were between 8 and 12 mm. Smaller particles had no
significant effect on the improvement of the hydrolysis efficiency. The best operational conditions
were 210 C, 48 min., since the highest cellulose recovery and enzymatic hydrolysis yields were
obtained under such conditions.
Conversely, Jin & Chen (2006) show that an extremely fine grinding (<1 mm) applied on rice
straw can significantly improve the hydrolysis yield. It is important that the wood chips characterized
by low humidity. When the pores are filled with water vapor, the penetration is hindered, which
reduces heat transfer and, consequently, start autohydrolysis process. Jacquet et al. (2015) the
best hydrolysis performance for all monosaccharides (glucose + xylose) obtained for a pressure of
3540 bar, temperature of 250 C and a retention time of 40 s. Generally, lower temperature and
longer residence time were more favorable than higher temperature and shorter time.
Many authors the effects of chemical hydrolysis and mechanical stress on lignocellulosic material
determined by the severity factor (S = log(R0 )). R0 is a function of residence time and saturated
steam temperature (Overend & Chornet, 1987). Its values are essential for the thermal degradation
of cellulose and hemicellulose, breaking the lignified matrix, changes in the ash content and color,
elemental compositions, and inhibitor formation (Iroba et al., 2014). The limitation of this model is
that it does not consider the moisture content in the raw material and particle size. Iroba et al. (2014)
use combined temperatures (140180 C) and time (510 min.) for pretreatment of barley straw.
The severity factor increased with temperature and time and ranged from 1.88 to 3.36. Authors
suggested that severity of the pretreatment should also incorporate an additional function (moisture
or acid or alkaline function) which will directly affect the hydrolytic mechanisms.
Moreover, a different time is required to reach the different target pressures, the treatment severity
must include the time necessary to reach the target pressure. Jacquet et al. (2015) established that
the temperature increased linearly with every increment in pressure (0.1 MPa) and authors offered
appropriate equation including this relationship.
The application of the high temperature and long pretreatment time cause the increase of the
severity factor, resulting in a high recovery of cellulose and lignin. Chornet and Overend (1991)
reported a severity factor of 2.64.2 during steam explosion treatment of Populus deltoides. The
authors observed that the yield of sugars and degree of polymerization decreased with an increase
of severity factor.
Cotana et al. (2015) tested three different severity factors (3.6, 4.0, and 4.4) to pretreated
Phragmites australis. At the lower S value, the water insoluble substrate (WIS) contained less
cellulose (48.96%) as well as a higher hemicellulose fraction (12.5%). This finding demonstrated
that the pretreatment at lower severity factor values was relatively ineffective, since only about half
of the hemicelluloses and acetyl groups were removed. At S value equal 4.4, the cellulose fraction
started to decrease since cellulose was probably degraded by severe pretreatment conditions.
Angles et al. (2003) investigated the steam explosion of softwood with combined temperatures
ranging from 176 to 231 C and time from 2.5 to 5.5 min. The severity factor ranged from 2.6 to 4.6.
The result showed the influence of the severity factor on the chemical composition of the isolated
lignin. When the severity of the pretreatment increased, the high molecular weight fraction of lignin
was observed probably because of the recondensation effect.
Jacquet et al. (2011) investigated the thermal degradation of a bleached cellulose obtained from
wood pulp and purified hemicelluloses and lignin. When the severity factor of the treatment was
below 4.0, the cellulose degradation was limited and fermentation inhibitors like 5-hydroxymethyl-
furfural (5-HMF) were not detected in the treated sample. For a severity factor between 4.0 and 5.2,
the thermal degradation mechanism was initiated. Quantities of 5-HMF in the water-soluble extract
increased with the increasing of the severity factor consecutive to an active depolymerization of
the cellulose. As a result, a theoretical diagram of cellulose degradation during the treatment was
established. This diagram allowed identification of the treatment conditions which minimized the
degradation of cellulose and limited the formation of inhibitors products like 5-HMF.
Till now steam explosion can be carried out with a great variety of plant biomass including
woody biomass (Biermann et al., 1984) forest and agricultural residues such as cassava and sug-
arcane bagasses (Rocha et al., 2012), wheat straw (Ballesteros et al., 2006), potatoes (Kobayashi
et al., 1998), corn residues (Tucker et al., 2003), hemp fibers (Vignon et al., 1996), peanut hulls,
Onopordum nervosum and Cynara cardunculus (Martinez et al., 1990), bamboo grass culms (Tsuda
et al., 1998), rice straw (Chen et al., 2011), Brassica carinata (Ballesteros et al., 2002), sunflower
stalks (Ruiz et al., 2008), olive stones (Fernandez-Bolanos et al., 2001), and cotton gin waste
(Jeoh & Agblevor, 2001).The large particle of wood shavings the retention time should be longer
and the temperature decrease to avoid overheating, which causing released of inhibitors from the
wood chips (Ballesteros et al., 2002).
Lpez-Linares et al. (2015) tested the effect of uncatalyzed steam explosion as a pretreatment
method to increase the enzymatic digestibility of rapeseed straw. Experimental statistical design
and response surface methodology were used to evaluate the influence of the temperature (185
215 C) and the process time (2.57.5 min.). According to the rotatable central composite design
applied, 215 C and 7.5 min. were confirmed to be the optimal conditions. The authors obtained
the concentrations of ethanol of 43.6 g/L (5.5% by volume) at using 20% (w/v) solid loading,
equivalent to 12.4 g ethanol/100 g biomass.
Li et al. (2015) pretreated rice straw using steam explosion technique. The effect of steam
pressure, pressure retention time, and straw moisture content on the yield of reducing sugar was
Figure 6.12. Process flow diagram of AFEX pretreatment (Wyman, 2013).
tested. All the investigated variables had significant effects (P < 0.001) on the reducing sugar
yield. The optimum yield of 30.86% was obtained under the following pretreatment conditions:
steam pressure of 1.54 MPa; pressure retention time of 140.5 sec; and straw moisture content
of 41.6%.
At present, most of the data available in the literature have been obtained lab-scale batch systems.
A developed platform for continuous processing and a detailed understanding of corresponding
changes in lignocellulosic biomass will be crucial in enabling the industrial application. The steam
explosion can be carried out in batch systems or continuous systems. In batch systems biomass is
introduced in the reactor through a pneumatic loading valve and then soaked with saturated steam.
After the elapsed time, the blow valve is opened, pressure decreases dramatically in a very short
time and the biomass is discharged in a storage tanks.
Catalyzed steam explosion is very similar to uncatalyzed steam-explosion on their action modes,
except that some acidic chemicals (gases and liquids), primarily including SO2 , H2 SO4 , CO2 , oxalic
acid, etc. are used as catalysts to impregnate the biomass prior to steam-explosion.
Ammonia fiber explosion (AFEX). AFEX utilizes both physical (high temperature and pres-
sure) and chemical (ammonia) processes to achieve effective pretreatment. AFEX increases the
surface accessibility for hydrolysis, promotes cellulose decrystallization, partial hemicelluloses
depolymerization and reduces the lignin recalcitrance in the treated biomass (Balan et al., 2009).
The unique advantages is almost complete recovery of ammonia. In order to reduce the use of
chemicals, ammonia is given to recycling, but this significantly increases the cost of the process.
During the steam explosion involving ammonia, there was no release of the compounds to be
inhibitors of alcoholic fermentation. Another advantage of the method is the possibility of the
process for large particles, which eliminates machining (Fig. 6.12).
The key variables during the AFEX process are treatment time (t), temperature (T ), ammonia-
to-biomass ratio, and moisture content. An optimal combination of these variables depends on the
recalcitrant nature of the lignocellulosic biomass. Ammonia or its aqueous solution in a weight
ratio of 1:1 is added to wet lignocellulosic material (at a pressure of 2.7 MPa). Semi-liquid mass
is heated to a temperature of about 65 C for 10 min. and then with the valve directed to the tank
where it is rapidly expanded. After the AFEX process, depending on the type of biomass and
process conditions, susceptibility to enzyme hydrolysis of lignocellulose increased 0.311 times.
In typical AFEX, the amount of consumed ammonia is in the range from 1 to 2 kg/kg dw. The
method is most efficient for crops, grasses and municipal waste. When the material contains high
concentration of lignin, the process efficiency decrease.
Table 6.1. Effect of pretreatment methods on the chemical composition and physical structure of lignocellulosic biomass.
The optimal pretreatment conditions for switchgrass pretreated by AFEX were found at tem-
perature of 100 C, 1:1 of ammonia to switch grass ratio (w/w), 80% moisture content, and 5 min.
residence time. Hydrolysis results of AFEX-treated and untreated samples showed 93% vs. 16%
glucan conversion, respectively. The ethanol yield of optimized AFEX-treated switchgrass was
about 0.2 g ethanol/g dry biomass, and was 2.5 times more than that of the untreated sample
(Alizadeh et al., 2005).
CO2 explosion. Lignocellulosic wastes are treated with a carbonic acid generated during the
operation of the lignocellulose carbon dioxide under high pressure. The process has a lower effi-
ciency compared to the explosion itself and explosion involving ammonia and is also considered
to be more expensive. In the process, there is no release of inhibitors (Zheng et al., 1998). Pretreat-
ment of lignocellulosic materials is also used carbon dioxide in supercritical state. The process is
carried out in a pressured reactor at a suitable temperature.
Kim & Hong (2001) studied the effect of pretreatment of aspen wood and pine by carbon
dioxide in supercritical state on enzymatic hydrolysis. The research were conducted at variable
moisture content (073%), temperature (112165 C), pressure (31004000 psi) and reaction time
(1060 min.).
After processing, samples of biomass were subjected to enzymatic hydrolysis involving cellulase.
The optimal conditions were temperature of 165 C, pressure of 3100 psi and reaction time of
30 min. In the case of aspen wood, hydrolysis yield of cellulose was 84% of theoretical value,
while the pine wood 27%.
Li and Chen (2014) analyzed the effect of steam explosion combined with fungal treatment.
The corn stalk was first pretreated by steam explosion and then treated by Phellinus baumii. The
results revealed that the physical changes in the structure of corn stalk caused by steam explosion
facilitated the following fungal treatment, and the modified lignin and hemicelluloses can be sig-
nificantly degraded by fungi. Phellinus baumii could selectively degrade 34.7% and 36.58% lignin
for 1.4 MPa and 1.7 MPa steam exploded corn stalk, respectively. As a result, the highest glucose
yield for 1.7 MPa steam exploded corn stalk associated with 21 d P. baumii reached 313.31 g/kg,
which was 2.88 and 1.32 times higher than that of the untreated corn stalk and the 1.4 MPa steam
exploded corn stalk, respectively.
The use of SO2 or CO2 can significantly reduce the formation of components causing inhibition
during the enzymatic hydrolysis and increase removal efficiency of hemicelluloses.
The described pretreatment methods are different in efficiency of the process. Table 6.1
gives the effect of pretreatment methods on the chemical composition and physical structure of
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Chapter 7
Fermentative and non-fermentative pathways

of butanol and its analogues
Tomasz Pokj & Ewa Klimiuk
1 INTRODUCTION
The new microbial fuels have great potential to replace or supplement petroleum-derived liquid
transportation fuels. In recent years progress has been made in the development of microbial strains
that convert simple sugars into advanced biofuel such as butanol (Green, 2011; Visioli et al., 2014)
or higher alcohols (Atsumi et al., 2008a) which can be used as substitutes for gasoline (Lee et al.,
2008a).
Today, many technological and economic barriers need to be overcome to enable microbes to
be used to produce butanol on a large scale. The major obstacles are the high cost of traditional
substrates that contain starch (Sakuragi et al., 2011) and low productivity of the processes (Tashiro &
Sonomoto, 2010). To reduce the cost of substrates, starch could be replaced by lignocellulosic
biomass, including waste and agriculture residues. To achieve this aim, new methods need to be
developed to breakdown lignocellulosic feedstocks into sugars (Mood et al., 2013; Mood et al.,
2014; Maurya et al., 2015).
Traditional ABE fermentation uses solventogenic clostridia as the producing strains. Using
molecular biology techniques, four distinct species of clostridia were identified according to their
genetic background, ie. Clostridium acetobutylicum, C. beijerinckii, C. saccharobutylicum and
C. saccharoperbutylacetonicum (Keis et al., 1995; Johnson et al., 1997; Keis et al., 2001). Among
them, C. acetobutylicum is relatively unique because of its phylogenetic characteristics and phys-
iological phenotypic properties (e.g. high amylase activity), which determine its comparatively
remote genetic relationship with the other three species. C. beijerinckii is deficient in starch uti-
lization but suitable for fermenting various monosaccharides. Therefore, as lignocellulosic biomass,
which is rich in hexoses and pentoses, has become one of the main renewable resources for future
bio-based industries, research on lignocellulosic biomass-based ABE production by C. beijerinckii
has been intensified in recent years (Gu et al., 2011).
The natural rates of microbial butanol and higher alcohols synthesis are typically too low to
support industrial-scale production (Rabinovitch-Deere et al., 2013). Traditionally, the metabolic
engineering is used to optimize metabolic flux through natural pathways in native or heterologous
host organisms. Compared to native producers, these non-native systems carry the advantages
of fast growth, simple nutrient requirements, readiness for genetic modifications, and even the
capability to assimilate CO2 and solar energy (Jin et al., 2014).
Escherichia coli has the ability to utilize a variety of carbon sources. These include glucose
and xylose which are components of biomass, as well others carbon sources such as glycerol or
fatty acids (Atsumi & Liao, 2008; Clomburg & Gonzales, 2010). Recently a number of advanced
molecular biology tools have been developed for precision engineering of yeast (Krivoruchko et al.,
2011; Da Silva & Srikrishnan, 2012). Furthermore, a lot of information about regulation of yeast
metabolism is available (Oliveira & Sauer, 2012; Oud et al., 2012).
This chapter reviews microbial production of advanced biofuels such as butanol, and its ana-
logues. In the chapter technologies of butanol production via fermentative pathways are described
155
and new approaches for the use of engineered strains are indicated. Biochemical pathways of
non-fermentative higher alcohols are also presented.
2 BUTANOL PRODUCTION VIA FERMENTATIVE PATHWAY
2.1 Sugars and starch as substrates

Butanol production via a fermentation of carbohydrates is a well-known industrial process often
referred to ABE fermentation, due to its major products, ie. acetone, butanol and ethanol (ABE
fermentation) (Drre, 1998; Ktai et al., 2013; Sreekumar et al., 2015). The fermentation process
fell into disuse in the US and Europe in the 1950s when renewable solvents could no longer
compete with their synthetic equivalents on price. Moreover, it has recently been revived because
of the potential application of butanol as an advanced biofuel (Lee et al., 2008a; Visioli et al., 2014;
Sreekumar et al., 2015).
The ABE producing clostridia strains possess a broad substrate utilization ability. They
metabolise a wide variety of sugars including glucose, fructose, mannose, lactose, sucrose, mal-
tose and arabinose. Some also metabolise xylose (Tangney et al., 1998; Shaheen et al., 2000; Ezeji
et al., 2007a). Clostridia display strong amylolytic activity, and some substrates containing starch
have been used without a need for prior hydrolysis by amylolytic enzymes. The original starch-
fermenting strains belong to C. acetobutylicum. Genomic sequence data of C. acetobutylicum 824
demonstrated the presence of more than 90 gene encoding enzymes involved in the degradation of
carbohydrate polymers, including at least 14 distinct families of glycosyl hydrolases (Ezeji et al.,
2007a). Likewise, C. beijerinckii 8052 has the genetic potential for utilization of a wide variety of
carbohydrates (Drre, 2005).
Traditionally, butanol is produced from starch and sugars (Ezeji et al., 2014). The common
industrial starches are typically derived from cereals (corn, wheat, rice, sorghum), tubers (potato,
sweet potato) or roots (cassava). The high cost of conventional starch and sugar is a major factor
affecting the economic viability of butanol production. For example, the corn starch accounts for
up to 79% of the overall solvent production cost while energy for operations including distillation,
contributes 14% to the overall cost (Pfromm et al., 2010).
In South East Asia, cassava represents an attractive carbon source of starch for fermentation
processes from both economic and geographical considerations (Thang et al., 2010). Cassava
contains circa 40% of starch. Batch fermentation by C. saccharoperbutylacetonicum N14 as a
hyperamylolytic strain resulted in 21.0 and 19.4 g/L total solvent from cassava starch and cassava
chips, respectively, as compared with 24.2 g/L total solvent production when using glucose. Solvent
productivity in fermentation of cassava starch was from 42 to 63% higher than that obtained in
fermentation using corn and sago starches in the same conditions (Thang et al., 2010).
Lepiz-Aguilar et al. (2013) produced butanol in ABE fermentation by C. beijerinckii BA101
using cassava flour (CF) after enzymatic pretreatment. Optimized fermentation conditions were
40 C and 60 g/L CF. An average of 37.01 g/L ABE was produced after 83 h, with a productivity of
0.446 g/L h. Butanol production was 25.7 g/L and productivity 0.310 g/L h. Results suggest that
the use of cassava as a substrate in ABE fermentation could be a cost-effective way of producing
butanol in tropical regions.
The main challenge of cassava-based butanol production is to enhance the simultaneous saccha-
rification and fermentation with high hyperamylolytic activity and butanol yield. Li et al. (2015)
developed a technology for enhancing butanol production with simultaneous hydrolysis of cassava
using a cofactor-dependent modulation method to maximize the production efficiency of butanol by
Clostridium sp. strain BOH3 . It was realized through supplementing CaCO3 to the medium contain-
ing cassava and applying redox modulation with l-tryptophan (a precursor to de novo synthesis of
NADH and NADPH). By combining CaCO3 and l-tryptophan, 17.8 g/L butanol with a yield of 30%
and a productivity of 0.25 g/L h was obtained with a hydrolytic capacity of 88% towards cassava
Fermentative and non-fermentative pathways of butanol and its analogues 157
in a defined medium. The metabolic patterns were shifted towards more reduced metabolites as
reflected by the higher butanolacetone ratio (76%) and butanolbioacid ratio (500%).
The sago palm has an important socioeconomic role in crop farming in Southeast Asia. There are
14 species of palms belonging to 8 genera which are exploited for sago production, but among these
only Metroxylon and Arenga pinnata are of major importance as palm starch sources. Composition
studies in various sago starch samples from Southeast Asia showed that the moisture content in
the sago samples varied between 10.6 and 20.0%, ash between 0.06 and 0.43%, crude fat between
0.10 and 0.13%, fiber between 0.26 and 0.32%, and crude protein between 0.19 and 0.25%. The
amylose content varied between 24 and 31% (Ahmad et al., 1999). Sago can compete economically
on yield and price with other crops. Growing in a suitable environment with organized farming
practices, sago palm could have a yield potential of up to 25 tons of starch per hectare per year.
Sago starch yield per unit area could be about 3 to 4 times higher than that of rice, corn, or wheat,
and about 17 times higher than that of cassava (Karim et al., 2008).
Madihah et al. (2001) fermented gelatinized sago starch by C. acetobutylicum P262. Using
sago starch at concentration of 30 g/L, the total solvent (acetonebutanolethanol) production
was 11.03 g/L and it was comparable to fermentation of corn starch and about twice higher than
fermentation of potato or tapioca starch. In the range of sago starch concentration of 1080 g/L,
the highest total solvent production (18.8 g/L) was obtained at starch concentration of 50 g/L.
Jerusalem artichoke (Helianthus tuberosus L.) as an alternative carbon source has potential as a
renewable feedstock (Yang et al., 2015). The principal storage carbohydrate of Jerusalem artichoke
is inulin, however, monomeric sucrose, glucose and fructose are also present (Matas et al., 2011).
Inulin is a polymer consisting of several fructose residues (from 10 to 36) in furanose form and
one glucose residue in pyranose form, connected through -2.1 glycoside bonds. Its concentration
is 1722%. During acid or enzymatic hydrolysis, inulin produces D-fructose and a small amount
of glucose (Barkhatova et al., 2015).
Sarchami & Rehmann (2014) studied the effect of temperature, pH and time on the acid hydro-
lysis of inulin derived from Jerusalem artichoke using three different mineral acids (HCl, H2 SO4 ,
and H3 PO4 ). H2 SO4 was found to have the greatest potential for sugar yields. The maximum
inulin conversion of 94.5% was achieved under the following conditions of hydrolysis: temperature
of 97 C, pH of 2.0, and time of 35 min, without producing inhibiting concentrations of hydro-
xymethyl furfural (HMF). An ABE yield of 0.31 g/g and an overall fermentation productivity of
0.25 g/L h were obtained. Chen et al. (2010) used C. acetobutylicum L7 for butanol production
from hydrolyzate of Jerusalem artichoke juice. It was found that at an initial sugar concentration
of 62.87 g/L, 11.2 g/L butanol was produced during 60 h of fermentation. The ratio of butanol,
acetone and ethanol was 0.64:0.29:0.05.
Agricultural residues and wastes containing starch, demonstrated to be both renewable and
economical, are available (Jones & Woods, 1986). Apple pomace is a solid agricultural waste which
contains approximately 10% (w/w) carbohydrates (67% fructose, 23% glucose and 10% sucrose).
Potatoes contain up to 19% starch. In recent years, this has focused attention on the use of wastes
from potato processing industries as a biofuels feedstock. Potato peel is a zero value waste produced
by potato processing plants (Arapoglou et al., 2010). Maiti et al. (2016) demonstrated fermentative
butanol production by C. beijerinckii B-466 from three agro-industrial wastes: suspended brewery
liquid waste (BLW), starch industry wastewater (SIW) and apple pomace ultra-filtration sludge
(APUS). The highest butanol production of 11.04 g/L was from SIW after 96 h of fermentation
using pretreated and concentrated suspension containing 62 g/L total reducing sugar including 3%
(w/v) glucose supplements. At this level of reducing sugar, the highest yield and productivity of
butanol was 0.27 g/g and 0.11 g/L h, respectively. The production of butanol from APUSH and
BLWH were 9.3 g/L and 8.06 g/L, respectively, using 60 g/L total reducing sugar.
Food wastes are the single largest component of the wastes stream. They include unconsumed
food that is discarded by food processing industries, retailers, restaurants, and consumers. Food
wastes hold several significant advantages for producing butanol. Firstly, most food wastes contain
significant amounts of sugars and starch, which can be easily utilized by the butanol produc-
ing culture (Clostridium) (Humbird et al., 2011). Secondly, food wastes comprise significant
quantities of functionalized molecules (i.e. proteins, fatty acids, minerals), which can act as nutri-
ents to support the culture growth (Lin et al., 2013). Huang et al. (2015) investigated the application
of food wastes containing mashed potatoes, sweet corn and white bread as a potential feedstock for
butanol fermentation using C. beijerinckii P260. In fermentation of 81 g/L food wastes (containing
equivalent glucose of 60.1 g/L) the culture produced 18.9 g/L ABE with an ABE productivity of
0.46 g/L h and a yield of 0.38 g/g. Fermentation of food wastes at higher concentrations (129,
181 and 228 g/L) required integration of ABE fermentation with vacuum stripping technology to
simultaneously recover butanol from the fermentation broth to relieve the culture butanol inhibi-
tion. The ABE productivity with vacuum fermentation was 0.49 g/L h, which was 109% higher
than the control fermentation (glucose based), and allowed near-complete utilization of the sugars
(98%) in the broth when the food waste concentration was as high as 129 g/L.
Cheese whey has attracted interest as an alternative substrate for ABE fermentation because
of problem of its disposal and availability in many countries. After the precipitation and removal
of casein, whey permeate contains, however, a relatively low sugar content (4 to 5% lactose).
Therefore, it has proved to be a relatively poor substrate when overall reactor productivities in
batch fermentations are considered, compared with starch and molasses. The potential of cheese
whey as feedstock for butanol production has been pointed out by several authors. Qureshi &
Maddox (1987) used immobilized C. acetobutylicum onto bone char in a packed bed reactor for
the continuous production of solvents from whey permeate. A maximum solvent productivity of
4.1 g/L h, representing a yield of 0.23 g solvent/g lactose utilized, was observed at a dilution rate of
1.0 1/h. In the following years, the authors produced acetonebutanolethanol from whey permeate
medium, supplemented with lactose, by C. acetobutylicum P262 in a batch reactor. The solvents
were produced at a yield of 0.44 g solvent/g lactose utilized (Qureshi & Maddox, 2005). Foda et al.
(2010) used as feedstock cheese whey (a dairy industry waste) contained lactose at concentration
ranging from 4.5 to 5.0% (w/w). The two strains C. acetobutylicum DSM 792 and C. acetobutylicum
AS 1.224 were tested. Preliminary lab experiments, performed in aerobic conditions on lactose
medium, have shown that C. acetobutylicum DSM 792 strain was better in the solvents production,
compared with AS 1.224 strain. Recently, unsupplemented cheese whey as renewable feedstock
was used for butanol production by C. acetobutylicum DSM 792 in a biofilm packed bed reactor
(PBR). Under optimized conditions, the performances were: butanol productivity of 2.66 g/L h,
butanol concentration of 4.93 g/L, butanol yield of 0.26 g/g. The butanol selectivity of the overall
solvents was 82% w/w (Raganti et al., 2013).
Algal biomass is considered to be a fermentation substrate, which presents some advantages for
the utilization and bioconversion as a potentially large renewable resource. According to Thang et al.
(2010), C. saccharoperbutylacetonicum N14 showed amylolytic properties toward starch based
polymers, which is essential component of many algae. Particularly, Scenedesmus and Chlorella,
are known to contain greater than 50% (dry weight) of starch, cellulose, and glycogen (Ellis et al.,
2011).
ABE fermentation by C. saccharoperbutylacetonicum N14 using algae from the Logan City
Wastewater Lagoon system as source of biomass was demonstrated by Ellis et al. (2012). Batch
fermentations were performed with 10% algae as feedstock. Acid/base pretreatment produced
8.92 g/L soluble sugars, whereas non-pretreated algae gave only 0.73 g/L soluble sugar. Fermenta-
tion of acid/base pretreated algae produced 2.74 g/L total ABE. Pretreated algae supplemented with
1% glucose allowed to get 7.27 g/L ABE. When xylanase and cellulase enzymes were introduced,
ABE concentration increased to 9.74 g/L. Supplementation of enzymes made possible to achieve
the highest yield of 0.311 g/g and volumetric productivity of 0.102 g/Lh.
Castro et al. (2015), optimized acid hydrolysis of mixed microalgae for sugar release. The
sugars were subsequently fermented to produce acetone, butanol, and ethanol by C. saccharoper-
butylacetonicum N14. The findings provided an optimal sugar yield of 166.1 g/kg dry algae, with
concentrations of butanol of 3.74 g/L.
The optimization of algae saccharification through acid hydrolysis results in increased fer-
mentable sugar yields. Moreover, acid hydrolysis of algae wastewater as method pretreatment
for ABE fermentation by Clostridium spp. is a potentially effective and low cost method. The
negligible content of lignin in microalgae reduces the costs, time, and difficulty of the conversion
process (Harun et al., 2014).
2.2 Butanol production from lignocellulosic materials

One of the strategies to decrease butanol production costs is to use cheap and renewable feedstocks,
such as lignocellulosic materials. Generally, the clostridia species, belonging to natural producers
of ABE, do not ferment lignocellulosic biomass directly due to insufficient expression of hydrolyz-
ing enzymes. The enzymatic hydrolysis of native lignocellulose produces less than 20% glucose
from the cellulose fraction (Zhang & Lynd, 2004). For this reason, in most processes an initial pre-
treatment step to increase plant polysaccharide accessibility is required (Pu et al., 2013; Akinosho
et al., 2014). The pretreatment methods should: (1) disintegrate the lignin polisaccharides com-
plex, (2) improve the sugar yield as a result of hydrolysis of cellulose and hemicelluloses, and (3)
prevent excessive degradation or loss of carbohydrate. Thereby, agricultural residues and wastes,
such as rice straw, wheat straw, wood (hardwood), by-products from the corn milling process (corn
fiber), annual and perennial crops, waste paper and switchgrass can be potential carbon sources
for butanol fermentation.
Qureshi & Ezeji (2008) analyzed the possibility of bioconversion of plant materials such as wheat
straw (WS), corn stover (CS), barley straw (BS), and switchgrass (SG) to butanol by C. beijerinckii
P260. According to the authors, the successful fermentation of low-value WS made butanol fer-
mentation economically attractive. Production of butanol from other agricultural residues including
CS, BS, and SG has been making steady progress.
There are many pretreatment methods including diluted sulfuric acid, alkali, steam explosion,
hydrothermal pretreatment, and others. Each of them has some advantages, however, none could
be recommended for all types of biomass.
To date, diluted mineral acids have been the most commonly used for pretreatment (Taherzadeh &
Karimi, 2008; Dussn et al., 2014). Bellido et al. (2014) investigated ABE fermentation of
steam-exploded and ozonated wheat straw hydrolysates by C. beijerinckii. The glucose recov-
ery was comparable for both methods, while xylose recovery was higher for steam-exploded
than ozonated biomass. Recently, combined methods are recomended. For example, ultrasound-
assisted diluted acid hydrolysis of tea processing waste (Germec et al., 2016) and combined
steam-explosion vacuum and dilute-acid spraying of wheat straw (Zoulikha et al., 2015) have been
reported.
Besides pretreatment, enzymatic saccharification (hydrolysis) and fermentation are also the key
processes. The problem of production of butanol from lignocelluloses can result from insufficient
expression of xylanase by solventogenic clostridial strains. Only few strains are capable of express-
ing xylanase. Therefore, it is desirable to identify a new solventogenic Clostridium strains with the
capability of expressing xylanase and utilizing un-pretreated xylan as a substrate for fermentation.
Some Clostridium strains produce xylose isomerase converting xylose into xylulose, which is
a utilizable form of xylose for these strains (Quershi & Ezeji, 2008; Nanda et al., 2014). The
hemicellulotic activity shows C. saccharobutylicum Ox 29 (Berezina et al., 2009). Yan et al. (2016)
described the production of butanol by wild-type Clostridium sp. strain G117 with xylan as the
sole carbon source for fermentation. Strain G117 produced 0.86 g/L butanol and 53.4 mL hydrogen
directly from 60 g/L xylan, with a negligible amount of acetone.
An endo-acting xylanase was isolated from the culture medium of Clostridium sp. strain BOH3
when xylan, glucose, xylose, or sugarcane bagasse hydrolysate (SBH) was used as a carbon source.
Kinetic study of this xylanase revealed that the Michaelis constant (Km ) and the maximal velocity
(Vmax ) are 1.36 mg/ml and 212 mol/minmg protein, respectively (Rajagopalan et al., 2013). In
further studies the authors showed that Clostridium sp. strain BOH3 could utilize 10 g/L xylan for
the efficient production of butanol and hydrogen. After increasing xylan concentration and suple-
menting culture medium with ammonium sulfate and iron (III) chloride, Clostridium strain BOH3
expressed more xylanase and showed higher xylanase activity. As a result, 30 and 50 g/L xylan
were converted giving 12.05 and 14.80 g/L butanol, and 1.78 and 2.65 L/L hydrogen, respectively
(Rajagopalan et al., 2014).
The advantage of butanol producing bacteria is that they can utilize both hexoses and pentoses
hydrolysate sugars as opposed to traditional ethanol-producing yeast strains that cannot use them.
Results obtained by Ezeji & Blaschek (2008) showed that solventogenic clostridia can consume
the sugars present in the dried distillers grains and solubles (DDGS) biomass. The autors tested
five strains: C. beijerinckii BA101, C. acetobutylicum 824, C. beijerinckii P-260, C. butylicum 592
and C. saccharobutylicum 292. In all experiments, the initial sugar level was 60 g/L and included
glucose, cellobiose, mannose, arabinose, galactose and xylose. Except C. beijerinckii BA10, which
preferentially used cellobiose, others strains utilized glucose at higher level (the point at which the
culture was obtained of maximum ABE). In contrast, utilization of xylose, galactose and mannose
was lower.
Although solventogenic Clostridium species are able to use pentose sugars (xylose and arabinose)
forABE production, efficiency of utilization of these carbohydrates tends to decrease in the presence
of preferred sugars as carbon source due to a phenomenon called carbon catabolite repression
(CCR). CCR reduces or prevents the utilization of xylose and arabinose when a preferred carbon
source, such as glucose, is present in the fermentation medium (Tangney et al., 2003; Ren et al.,
2010).
The newly reported Clostridium sp. strain BOH3 is capable of fermenting glucose and xylose
simultaneously. This strain is characterized by its capability to survive under high concentrations
of butanol, direct fermentation of (hemi)cellulosic materials to hydrogen and butanol precursors,
and showed ability to grow in continuous systems (Bramono et al., 2011).
Xin et al. (2014) proved that Clostridium sp. strain BOH3 was capable of fermenting 60 g/L
xylose to 14.9 g/L butanol, which was similar to the 14.5 g/L butanol produced from 60 g/L glu-
cose. Moreover, strain BOH3 consumed glucose and xylose simultaneously. During fermentation
a horticultural waste cellulosic hydrolysate containing either 39.8 g/L glucose and 20.5 g/L xylose
or 58.3 g/L xylose and 5.9 g/L glucose, production of butanol was comparable and amounted to
11.7 and 11.9 g/L, respectively. The high-xylose-utilization capability of strain BOH3 is attributed
to its high xylose-isomerase and xylulokinase activities compared to the low-xylose-utilizing
solventogenic strains, such as Clostridium sp. strain G117.
Recently, ability to direct fermentation of agricultural residues by using solventogenic
Clostridium sp. strain BOH3 has been documented by Rajagopalan et al. (2016). This strain
excreted hydrolyzing enzymes such as cellulase (0.52 U/mL), xylanase (4.10 U/mL) and amylase
(2.05 U/mL). Therefore, it was possible simultaneously saccharification and fermentation. In an
optimized agro-residual medium containing 94.5 g/L rice bran and 36.7 g/L sesame oil cake, strain
BOH3 produced 13.50 g/L butanol and 4.41 L/L hydrogen after 7 days.
Li et al. (2013) significantly enhanced butanol production from cassava starch and cane molasse
by using a continuous co-culture system containing C. beijerinckii and C. tyrobutyricum in free-
cell and immobilized-cell fermentation modes in a fibrous-bed bioreactor. The maximum butanol
production of 6.66 g/L, yield of 0.18 g/g, and productivity of 0.96 g/L h were obtained when
cassava starch was used as the substrate.
In recent years to override intrinsic hierarchy in sugar utilization by the development of strains
enabling the efficient simultaneous fermentation of sugar mixtures into butanol have been attemped.
Yu et al. (2015) to relieve the CCR and enhance xylose utilization by C. tyrobutyricum, co-
overexpressed with aldehyde/alcohol dehydrogenase (adhE2) three genes (xylT, xylA, and xylB)
encoding a xylose proton-symporter, a xylose isomerase and a xylulokinase, respectively, from
C. acetobutylicum ATCC 824. Compared to the strain expressing only adhE2, the engineered
strain had a higher xylose uptake rate and was able to simultaneously consume glucose and xylose
at comparable rates for butanol production. The engineered strain produced more butanol (12.0
vs. 3.2 g/L) with a higher butanol yield (0.12 vs. 0.07 g/g) and productivity (0.17 vs. 0.07 g/L h)
from both glucose and xylose, while host strain consumed little xylose in the fermentation. The
engineered strain was also able to co-utilize glucose and xylose present in soybean hull hydrolysate
for butanol production.
In order to improve the conversion of the cellulose, substrate spectrum of ABE-producing

clostridia can be broadened by the transfer of genes encoding cellulase from other organisms
(Tashiro & Sonomoto, 2010). Others strategies have been proposed using consortium of bacteria
(Zomorrodi & Segr, 2016).
2.2.1 Consolidated bioprocessing (CBP)

The solventogenic bacteria have not been shown ability to directly utilize crystalline cellulose
as a carbon source, although some species of clostridia produce cellulases. Clostridial co-culture
systems containing solventogenic and cellulolytic species, are known as consolidated bioprocessing
(CBP) (Salimi & Mahadevan, 2013; Akinosho et al., 2014). CBP has been known as promising
approach for butanol production since it uses cellulolytic microorganisms that produce cellulase
and microorganisms that perform hydrolysis and fermentation in a single step. CPB reduces the
number of unit operations, and lowers the overall capital cost of the process (Lynd et al., 2005;
Olson et al., 2012).
To cellulosome-forming bacteria are belonged C. thermocellum and C. cellulolyticum (Olson
et al., 2012). C. thermocellum retains many qualities that position it well for use as a CBP organ-
ism, including its fast rate of cellulose digestion from plant biomass and its ability to hydrolyze
both hemicelluloses and cellulose. The distinguishing feature of C. thermocellum, as a platform
for development into a CBP host, is its cellulosome. This multi-enzyme complex consists of
over 20 distinct enzymes (Wertz & Bdu, 2013), housing cellulases, hemicellulases, pectinases,
chitinases, glycosidases, and esterases for the breakdown of lignocellulose (Spinnler et al., 1986;
Zverlov et al., 2005). C. cellulolyticum is the best-understood mesophilic clostridial bacterium,
uses cellulosomes to efficiently degrade the crystalline cellulose and hemicelluloses present in
There are some co-culture systems used for butanol production in the format of CBP are reported
that have been employed:
C. thermocellum and C. saccharoperbutylacetonicum N14 (Nakayama et al., 2011, 2013). This
co-culture system selectively produced up to 7.9 g/L butanol in 9 days with a significant decrease
of hydrogen and acetone production using Avicel cellulose as a carbon source.
C. cellulolyticum and C. acetobutylicum. C. cellulolyticum has a high ability to solublize
crystalline cellulose in pretreated lignocellulosic biomass (Demain et al., 2005), while C. ace-
tobutylicum efficiently ferments sugars derived from cellulose and hemicelluloses (Lee et al.,
2012). Salimi & Mahadevan (2013) have shown that co-culturing these two bacteria allowed to
increase efficiency of cellulose utilization compared to the monoculture of C. cellulolyticum.
The authors found that these two species showed synergism, and that the metabolic activity
of C. acetobutylicum improved the cellulolytic activity of C. cellulolyticum in the co-culture
through exchange of metabolites such as pyruvate. No cellulolytic activity was observed at low
concentration of C. acetobutylicum. The final concentration of butanol detected in the co-culture
batch experiments was up to 0.35 g/L.
C. cellulovorans and C. beijerinckii. Wen et al. (2014) used such co-culture system for butanol
production from alkali-pretreated deshelled corn cobs. In the co-culture a cellulolytic, anaerobic,
butyrate-producing mesophilic C. cellulovorans strain 743B hydrolyzed pre-treated corn cob and
produced butyric acid, while a non-cellulolytic solventogenic C. beijerinckii strain NCIMB 8052,
subsequently consumed generated reducing sugars and butyric acid to produce butanol in one pot
reaction. Under optimized conditions of fermentation, the co-culture produced 2.64 g/L acetone,
8.30 g/L butanol and 0.87 g/L ethanol in less than 80 h, with yield of 0.17 g solvent/g alkali-
pretreated deshelled corn cobs degraded. The growth kinetics and analytical studies showed that
there were mechanisms of cooperation and competition between two strains during co-culturing
process.
Kluyvera and Clostridium. The co-culture system with xylanase producing anaerobic bacteria
Kluyvera strain OM3 and a biofuel producing Clostridium strain BOH3 produced 1.2 g/L butanol
from birch wood xylan. The xylanase of Kluyvera was able to release reducing sugars from birch
wood xylan, and these sugars were further fermented by the solventogenic Clostridium sp.
strain BOH3 to biofuel.This result was comparable to the amount of butanol (1.7 g/L) produced
by the SHF system (separate hydrolysis by the exogenous xylanase application and following
fermentation by Clostridium sp. strain BOH3) (Xin & He, 2013).
Wang et al. (2015b) isolated new microbial consortium N3 and new strain C. celevecrescens
N32 which displayed effective simultaneous saccharification and fermentation of cellulose, and
co-cultured them with C. acetobutylicum ATCC824 under mesophilic conditions. Co-culturing
C. acetobutylicum ATCC824 with the stable consortium N3 resulted in a relatively higher butanol
concentration of 3.73 g/L and higher production yield of 0.145 g/g glucose equivalent. Consi-
derable butanol yield of 2.69 g/L was also acquired by co-culturing C. celevecrescens N32 and
C. acetobutylicum ATCC 824.
2.2.2 Inhibitory effect of hydrolysis by-products on clostridia

During pretreatment of agricultural biomass rich in fiber with weak acids or enzymes, when cel-
lulosic structures were broken down to fermentable sugars, compounds such as salts, furfural,
hydroxymethyl furfural (HMF), acetic, ferulic, glucuronic, -coumaric acids, and phenolic com-
pounds are formed (Ezeji et al., 2007b; Visioli et al., 2014). These compounds have various
inhibitory effects on cell growth, substrate utilization, enzymatic activities and protein, and RNA
synthesis (Liu & Blaschek, 2010; Chandel et al., 2013).
Ezeji et al. (2007b) used corn fiber hydrolysate (CFH) (0.5% v/v sulfuric acid) to produce butanol
by C. beijerinckii BA101. The hydrolysates contained glucose, xylose, arabinose, galactose, and
mannose. The autors sugested that the presence of inhibitors caused poor cell growth and reduced
sugars consumption. When inhibitors were oved by using Ca(OH)2 and Amberlite XAD-resin, total
concentration of residual sugars was 17.0 g/L and 27.6 g/L, respectively, after 96 h of fermentation
that coresponded to efficiency removal of 69.2% and 45.7%, respectively. The ABE production
profiles by C. beijerinckii BA101 using Ca(OH)2 -treated CFH medium over the course of 88 h
showed that the culture produced 7.4 g/L acetone, 7.2 g/L butanol, and 1.0 g/L ethanol resulting in
a total ABE concentration of 15.6 g/L. In treated samples, C. beijerinckii BA101 preferred glucose
as the substrate, although utilized also other sugar present in hydrolysates. This indicated on ability
of C. beijerinckii BA101 to utilize mixed hexose and pentose sugars for ABE production.
The authors also investigated the effect of some of the lignocellulosic hydrolysate inhibitors
associated with C. beijerinckii BA101 growth and acetonebutanolethanol (ABE) production.
When 0.3 g/L r-coumaric and ferulic acids were introduced into the fermentation medium, growth
and ABE production by C. beijerinckii BA101 decreased significantly. Furfural and HMF were
not inhibitory to C. beijerinckii BA101; rather they have stimulatory effect on the growth of
the microorganism and ABE production. Similarly, Lee et al. (2015) found that 57.1, 40.8
and 38.8% of cell growth (OD600 ) of C. beijerinckii was decreased in the presence of 0.5 g/L
p-coumaric acid, ferulic acid and vanillin, respectively. In contrast, syringaldehyde did not inhibit
cell growth and actually increased the OD600 level by 25.9%. All tested phenolic compounds neg-
atively affected butanol production during fermentation. Its production was 98.8, 52.1, 75.9 and
12.1% inhibited by p-coumaric acid, ferulic acid, vanillin and syringaldehyde, respectively.
Wang et al. (2015a) determined the critical operation criteria of butanol fermentation by
C. acetobutylicum via a toxicity evaluation. The inhibitory effect of the inhibitors generated by
acid pretreatment of biomass feedstock on butanol fermentation decreased in the order of formic
acid > oxalic acid > furfural > 5-HMF > Na2 SO4 . C. acetobutylicum had a small tolerance range
for furfural (1.062.6 g/L) and 5-HMF (1.992.3 g/L), while wide range for Na2 SO4 . Results
obtained by authors suggest that all the toxicants should be removed to ensure successful operation.
Thus, removal of accumulated acids in the system is crucial to the long-term operation stability of
butanol production.
Qureshi et al. (2008) showed that fermentation of sulfuric acid treated corn fiber hydrolysate
(SACFH) by C. beijerinckii BA101 inhibited the cell growth and the butanol production (1.7 g/L
ABE), while fermentation of enzyme treated corn fiber hydrolysate (ETCFH) did not reveal any cell
inhibition and resulted in the production of 8.6 g/L ABE and used 24.6 g/L total sugars. Treatment
of SACFH with XAD-4 resin removed some of the inhibitors resulting in the production of 9.3 g/L
ABE which, however, was lower compared with from fermentation of 55 g/L glucose (control
sample) (17.7 g/L ABE). It suggested that some fermentation inhibitors were still present after the
treatment, and inhibitory components should be completely removed from the SACFH prior to
fermentation with C. beijerinckii BA101. Moreover, ABE production from fermentation of 25 g/L
glucose and 25 g/L xylose was 9.9 g/L and 9.6 g/L, respectively, suggesting that the culture was
able to utilize xylose as efficiently as glucose.
Gottumukkala et al. (2013) evaluated the production of butanol by C. sporogenes BE01 using
an hydrolysate obtained with dilute acid pretreatment and enzymatic hydrolysis of rice straw. The
hydrolysate contained 39.02 g/L glucose, 11.35 g/L xylose and 1.71 g/L arabinose. Anionic resin
Seralite SRA400 was used to remove inhibitors (acetic acid and formic acid; furfurals were found
to be absent). Non-detoxified hydrolysate supplemented with yeast extract and calcium carbonate
produced 3.32 g/L butanol at 96 h with a productivity of 0.03 g/L h, while the maximum butanol
production with detoxified hydrolysate was 4.62 g/L with a productivity of 0.05 g/L h.
2.3 Engineering pathways to improve butanol production in solventogenic clostridia

Two principal strategies for improving butanol production in bacteria are being investigated. The
one which we will consider first based on regulatory and metabolic engineering of clostridia
bacteria. Attempts have been made in this area to avoid the production of undesired products, and
to increase both the yield of alcohol and the tolerance of clostridia to butanol. However, butanol
yields and productivities are still unsuitable for industrial applications because the clostridia genome
is still relatively poorly characterized and suitable tools to manipulate their metabolism are lacking
(Atsumi et al., 2008b; Clomburg & Gonzalez, 2010; Chen et al., 2013; Mienda et al., 2015).
The second strategy we will consider is engineering non-producer bacteria such as E. coli,
Pseudomonas putida, Bacillus subtilis, Lactobacillus brevis (Becerra et al., 2015) with hetero-
logous pathways for butanol biosynthesis from natural producers, or with artificially generated
pathways combining enzymes from different genera (Becerra et al., 2015). It is considered espe-
cially advantageous to use synthetic biology and metabolic engineering strategies to introduce the
metabolic pathway for ABE fermentation to E. coli (Atsumi et al., 2008b; Huang et al., 2010).
E. coli do not require anaerobic fermentation, grow fast and are more tolerant to butanol than
clostridia (Atsumi & Liao, 2008; Sakuragi et al., 2011).
The ABE pathway is known as the CoA-dependent pathway, in which hexose sugars are degraded
to pyruvate via the Embden-Meyerhof-Parnas (EMP) pathway (Fig. 7.1a). Some strains of solvent-
producing clostridia also metabolize pentose sugars via the pentose phosphate pathway (Fig. 7.1b).
The acetyl-CoA generated by pyruvate-ferredoxin oxidoreductase (PFOR) can be converted
to either oxidized products (i.e., acetone, acetate, and CO2 ) and reduced products (i.e., butanol,
ethanol, and butyrate). According to some authors, there are two butanol-forming pathways in
C. acetobutylicum, known as the cold and hot channels. In the cold channel, acetate and butyrate
are formed during the acidogenic phase, and then reassimilated to form acetone, ethanol and
butanol. In the hot channel, butanol is formed directly from acetyl-coenzyme A (CoA), with
3-hydroxybutyryl-CoA, crotonyl-CoA, butyryl-CoA and bytyraldehyde as intermediates
(Gheshlaghi et al., 2009; Rabinovitch-Deere, 2013) (Fig 7.1a).
The butanol production in conventional ABE fermentations are usually lower than 13 g/L
(Ramey & Yang, 2004) making butanol production from glucose by ABE fermentation uneco-
nomical. Many researchers have tried to overcome these problems by gene manipulation to knock
out the genes responsible for acetate and butyrate productions (cold channel), and overexpress the
genes for solventogenesis (Fig. 7.2) (Tomas et al., 2003; Sillers et al., 2008; Cooksley et al., 2012;
Jang et al., 2012; Lehmann et al., 2012; Mann et al., 2012).
Harris et al. (2000) showed that C. acetobutylicum strain with inactivation of the butyrate kinase
gene (buk) produced 16.7 g/L butanol, 4.4 g/L acetone and 2.6 g/L ethanol while the strain with
Figure 7.1a. Acetone-butanol-ethanol (ABE) fermentation pathway in Clostridium acetobutylicum with

glucose. Adapted according to Dellomonaco et al. (2010) and Zheng et al. (2015).
combined effect of buk inactivation and overexpression of the alcohol/aldehyde dehydrogenase

(aad) gene produced similar amounts of butanol and acetone but 1.3 times more of ethanol.
Jang et al. (2012) to reinforce the direct butanol-forming flux (hot pathway) simultaneously
inactivated the pta and buk genes in cold pathway, and overexpressed adhE1D485G gene in
C. acetobulylicum. In the engineered strain the ratio of butanol produced through the hot channel
to that produced through the cold channel increased 9.4 times compared to the wild type. Engi-
neered strain produced 18.9 g/L butanol with a yield of 0.71 mol butanol/mol glucose in batch
fermentation. These values were 160% and 245%, respectively, higher than those obtained with
the wild type.
Generaly, the ratio of acetone, butanol and ethanol is approximately 3:6:1 (Zhao et al., 2013;
Janssen et al., 2014; Li et al., 2014). Therefore, a direct way to improve fuel alcohols production is
to avoid acetone formation in the ABE process. This strategy has been pursued by inactivation of the
adc gene, encoding the acetoacetate decarboxylase and necessary for acetone synthesis. Sillers et al.
(2009) expressed the alcohol/aldehyde dehydrogenase (aad) gene from the phosphotransbutyrylase
(ptb) promoter to enhance butanol formation and selectivity, and downregulated acetoacetyl-CoA
Figure 7.1b. Acetone-butanol-ethanol (ABE) fermentation pathway in Clostridium acetobutylicum with

xylose. Adapted according to Dellomonaco et al. (2010) and Zheng et al. (2015).
transferase to minimize acetone production. This led to production of high alcohol (butanol plus
ethanol), overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio.
Inactivating the adc gene to eliminate acetone production, and introduced glutathione biosyn-
thetic capability to improve the robustness of C. acetobutylicum studied Hou et al. (2013). In the
engineered strain acetone production was reduced from 2.64 to 0.15 g/L, whereas butanol produc-
tion was increased from 5.17 to 8.27 g/L. To further improve the alcohol titers, the authors expressed
the hbd, thl,crt, and bcd genes, and amplified the Sol operon to express the adhE and ctfAB genes.
In engineered strain butanol and alcohol production reached 14.86 and 18.11 g/L, respectively, and
the butanol and alcohol yields were 0.336 and 0.409 g/g, respectively.
An increase the butanol ratio to total solvent by disruption acetoacetate decarboxylase gene
(adc) in the industrial strain C. acetobutylicum EA 2018 examined Jiang et al. (2009). The butanol
ratio increased to 80.05%, with acetone production reduced to approximately 0.21 g/L in the adc-
disrupted mutant (2018adc). Futhermore, the regulation of electron flow by the addition of methyl
viologen altered the carbon flux from acetic acid production to butanol production, which resulted
in an increase in the overall yield of butanol from 57 to 70.8%.
Yu et al. (2011) engineered C. tyrobutyricum ATCC 25755 to overexpress aldehyde/alcohol
dehydrogenase (adhE2) from C. acetobutylicum ATCC 824, achieving butanol concentration
of 1.1 g/L. When acetate kinase (ack) and phosphotransbutyrylase (ptb) genes were inactivated,
butanol production from glucose was 10.0 g/L and butanol yield was 27.0% w/w (66% of theoretical
yield).
Production of the mixture of isopropanol, butanol, and ethanol (IBE) instead of acetone, butanol
and ethanol (ABE) is also has been researched. It can be achieved by the conversion of acetone
into isopropanol in ABE fermentation. In this way, IBE without the disruption of pathway can be
Figure 7.2. Improvement ofABE pathway in clostridia toward butanol production. Dash arrows represent mul-
tienzymatic steps; cross arrows represent pathways that have been knocked out; the underlined italicized gene
names represent the pathway that has been overexpressed: thl, thiolase; hbd, 3-hydroxybutyryl-CoA dehydro-
genase; crt, crotonase; bcd/etf, butyryl-CoA dehydrogenase/electron transfer flavoprotein; adhE2, secondary
aldehyde/alcohol dehydrogenase; pta, phosphate acetyltransferase; ack, acetate kinase; adc, acetoacetate
decarboxylase; ptb, phosphate butyryltransferase; buk, butyrate kinase; aad, acetaldehyde dehydrogenase;
EMP, EmbdenMeyerhofParnas.
Explanation of the numbers:
1. C. acetobutyricum ATCC824; buk, aad (according to Harris et al., 2000).
2. C. acetobutyricum ATCC824; buk, pta (according to Jang et al., 2012).
3. C. acetobutyricum ATCC824; adc (according to Hou et al., 2013).
4. C. acetobutyricum EA 2018; adc (according to Jiang et al., 2009).
5. C. tyrobutyricum ATCC 25755; ack, ptb (according to Yu et al., 2011).
produced. The mixed alcohols including isopropanol, butanol and ethanol can be directly used as
a biofuel.
For the isopropanol biosynthesis, an acetoacetyl-CoA transferase transfers the CoA group away
from acetoacetyl-CoA to acetate, forming acetoacetate (Fig. 7.3). Further, acetoacetate is decar-
boxylated to acetone by an acetoacetate decarboxylase. Then, acetone is reduced to isopropanol
Figure 7.3. Adaptation of the clostridia acetone-butanol-ethanol (ABE) fermentation pathway for the
production of isopropanol and butanol (Peralta-Yahya & Keasling, 2010).
by a NADPH-dependent secondary alcohol dehydrogenase (Hanai et al., 2007). The production

of isopropanol from glucose is not redox-balanced. Four moles of NADH is produced, while one
mol of NADPH is consumed per mole of isopropanol. Therefore, an external electron acceptor is
required or a byproduct is served as an electron acceptor (Yan & Liao, 2009).
Bankar et al. (2015) engineered C. acetobutylicum DSM 792 strain to convert acetone into
isopropanol by introducing the secondary alcohol dehydrogenase gene from C. beijerinckii NRRL
B593. During the batch fermentation in controlled bioreactor with medium containing pure glucose,
the maximum total solvent concentration was 18 g/L with 2.51 g/L isopropanol and 10.78 g/L
butanol after 72 h fermentation. Almost 50% acetone was converted into isopropanol with highest
total solvent yield to be 0.39 g/g glucose.
One of the most critical problems in ABE fermentation is solvent toxicity. Clostridial metabolism
is ceased in the presence of 20 g/L or more solvents (Knoshaug & Zhang, 2009). The lipophilic
butanol is more toxic than others. The mechanism of butanol toxicity is related to its hydrophobic-
hydrophilic nature. In clostridia, excess of butanol results in disruption of the phospholipid
component of the cell membrane, and variations in membrane composition and fluidity (Kolek
et al., 2015). As a result membrane destabilization, disruption of membrane-associated functions
including transport processes, glucose uptake, and ATPase activity occur (Lee et al., 2008b).
One of the suggested solution is the use strains which were characterized by greater tolerance
to higher concentrations of butanol. Lee et al. (2005) found that C. beijerinckii BA101 with the
ability of altering glucose uptake system from phosphoenolpyruvate-dependent phosphotransferase
system (PTS) to a non-PTS system (probably energized by the transmembrane proton gradient),
allowed higher butanol tolerance.
The resistance to butanol toxicity can be also realized by engineering native strains to increase
intracellular concentrations of ATP and NADH. Ventura et al. (2013) showed that overexpression
of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes in C. acetobutylicum
ATCC 824 allowed to increase butanol and ethanol concentrations. When fed-batch fermenta-
tion using glucose was carried out, the butanol and total solvent (acetone, butanol and ethanol)
concentrations reached 19.12 and 28.02 g/L, respectively. Tomas et al. (2003) reported that over-
expression of groESL, a class I heat-shock protein, gene in C. acetobutylicum resulted in increase
tolerance of C. acetobutylicum to butanol. The growth of engineered strain was inhibited up
to 85% less by butanol than the control strain. The proposed mechanism was that GroEL and
GroES, protein products of the groESL gene, served to prevent aggregation and assist in pro-
tein folding under heat-shock or stress conditions. Xu et al. (2015) studied the effects of the
cac3319 gene mutation on butanol tolerance and production in C. acetobutylicum. They found
that disrupt cac3919, involving in encoding histidine kinase (HK), could greatly improved butanol
production and tolerance. Compared to control strain ATCC 55025, the cac3319 HK knockout
mutant produced 44.4% more butanol (18.2 vs. 12.6 g/L) with a 90% higher productivity (0.38 vs.
0.20 g/L h).
In addition to using solvent tolerant strains of clostridia, recovery of the solvents during the
fermentation, also referred to as in situ butanol removal, are also applied (Garca et al., 2011; Huang
et al., 2014; Dhamole et al., 2015). Product removal technologies, are suggested and applied in
laboratory and pilot scales (Abdehagh et al., 2014; Huang et al., 2014). Currently, the most applied
techniques are pervaporation (Jitesh et al., 2000; Cai et al., 2013), liquidliquid extraction (Yen &
Wang, 2013), gas stripping (Ezeji et al., 2004; Xue et al., 2012), vacuum fermentation (Mariano
et al., 2012; Qureshi et al., 2014), perstraction (Qureshi & Maddox, 2005) and adsorption (Liu
et al., 2014; Thompson et al., 2014).
2.4 Escherichia coli as host for butanol/isopropanol production

E. coli is a facultative anaerobic, Gram-negative bacteria, capable of heterotrophic growth using
a wide spectrum of organic carbon sources. The glycolysis pathway processes the phosphorylated
sugar into pyruvate, which is accompanied by the release ATP and NADH. Under fermentation
conditions a mixture of succinate, formate, acetate, lactate and ethanol is produced to maintain
redox balance (Clark 1989), with acetate as the main product. Under aerobic conditions, acetyl-CoA
is further processed within the citric acid cycle (TCA cycle) producing succinate (Fig. 7.4).
2.4.1 Butanol
The native pathway for butanol production in C. acetobutylicum reconstructed for the first time in
E. coli Atsumi et al. (2008b). The authors introduced and expressed a set of six genes necessary to
produce of butanol from acetyl-CoA, into two operons. The first operon encoded acetyl-CoA
acetyltransferase (thl) and aldehyde/alcohol dehydrogenase (adhE2) while the second operon
encoded 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), and butyryl-CoA dehy-
drogenase/electron transfer flavoprotein (bcd/etfAB). The engineered E. coli produced 0.014 g/L
butanol from glucose under anaerobic conditions.
Inui et al. (2008) also cloned and expressed butanol producing genes of C. acetobutylicum
ATCC 824 (hbd, crt, bcd/etfAB, alcohol dehydrogenase) in E. coli, in a single vector. In order to
improve the butanol production the authors used two isozymes of alcohol dehydrogenase, adhE1
and adhE2. The E. coli strain with adhE1 produced 0.27 g/L while the strain with adhE2 produced
1.18 g/L butanol during 60 h of cultivation, using glucose as the sole carbon source. This production
corresponded to 7.2% of the theoretical yield.
The effect of polycistronic versus individual expression of key genes in the butanol pathway
on butanol production was studied by Nielsen et al. (2009). The polycistronic version of butanol
pathway the authors constructed in two vectors. The first vector expressed the crt-bcd/etfAB-hbd
operon, while the second vector expressed thl and adhE1. In the individual version of butanol
pathway, genes were expessed individually from dedicated promoters in a total of four vectors.
The expression of polycistronic construct could lead to the production of 0.035 g/L butanol, while
individual expression of the butanol pathway genes improved titres to 0.20 g/L.
Figure 7.4. Fermentative pathways for the production of butanol and isopropanol in engineered Escherichia
coli (adapted according to Clomburg & Gonzales, 2010; Xu & Koffas, 2010; Frster & Gescher, 2014). Dash
arrows represent multienzymatic steps; grey arrows represent reactions under aerobic conditions; cross arrows
represent pathways that have been knocked out. Revelant reactions are represented by the name of the genes cod-
ing for the enzyme (E. coli genes unless otherwise notes in parenthesis as follows: Clostridium acetobutylicum,
Ca; Clostridium beijerinckii, Cb): ppc, phosphoenolpyruvate carboxylase; frdABCD, fumarate reductase;
pfl, puryvate formate lyase; fdhF, formate dehydrogenase; ldhA, lactate dehydrogenase; aceEF-lpdA, pury-
vate dehydrogenase multienzyme complex; adhE, alcohol dehydrogenase; pta, phosphate acetyltransferase;
ackA, acetate kinase; thl, thiolase (Ca); atoB, acetyl-CoA acyltransferase; ctfAB, acetoacetyl-CoA trans-
ferase (Ca); atoAD, acetyl-CoA:acetoacetyl-CoA transferase; adc, acetoacetate decarboxylase (Ca); adh,
secondary alcohol dehydrogenase (Cb); hbd, 3-hydroxybutyryl-CoA dehydrogenase (Ca); crt, crotonase
(Ca); bcd, butyryl-CoA dehydrogenase (Ca); etfAB, electron transfer flavoprotein (Ca); adhE2, secondary
aldehyde/alcohol dehydrogenase (Ca).
Atsumi et al. (2008b) showed that replacement thl form C. acetobutylicum with more specifi-
cally atoB of E. coli allowed an increase in butanol production from 0.014 g/L to about 0.040 g/L.
Nielsen et al. (2009) reported when thl from C. acetobutylicum was replaced with atoB of E. coli
butanol production increased marginally from 0.2 to 0.22 g/L. The authors improved butanol pro-
duction to 0.58 g/L through the expression of formate dehydrogenase (fdh1) from S. cerevisiae
and glyceraldehyde 3-phosphate (gapA) from E. coli. Expression of fdh1 increased the availability
of reducing equivalents through the conversion of formate into CO2 which is coupled with the
generation of NADH, while expression gapA increased glycolytic flux.
To improved butanol production, the host E. coli strain was engineered by deleting the native
patway competing for both carbon flux and reducing power (NADH). Higher butanol production
was accompanied by significantly reducing the amount of lactate, ethanol, and succinate produced.
Astumi et al. (2008b) deleted from wild-type host ldhA, adhE and frdABCD genes that competed
with the butanol pathway for acetyl-CoA and NADH (Fig. 7.4). The resulted strain under semi-
aerobic condition produced nearly twice more butanol (0.14 vs. 0.27 g/L) from glucose, compared to
the native strain. By deleting fnr (an anaerobic regulator repressing the expression of aceEF-lpdA)
to produce additional moles of NADH as reducing power, and inactivating phosphate acetyltrans-
ferase (pta) to eliminate acetate formation, butanol production level increased to 0.37 g/L.
Shen et al. (2011) constructed a modified clostridial butanol pathway by replacing the putative
NADH-independent butyryl-CoA dehydrogenase (bcd) with the NADH-dependent trans-enoyl-
CoA reductase (ter), and coupled irreversible reaction catalyzed by ter with NADH and acetyl-CoA
driving forces. Furthermore, they substituted Clostridium acetacetyl-CoA thiolase (thl) with E. coli
acetyl-CoA acetyltransferase (atoB). In combination with the deletion of three genes (frdABCD,
ldhA, and adhE) involved in mixed-acid fermentation reactions consuming NADH, butanol pro-
duction achieved 15 g/L after 72 h at 88% of the theoretical yield. Next, they performed anaerobic,
pH-controlled fermentation in conjuction with continuous gas stripping, achieving productivity of
0.2 g/L h and titre of 30 g/L (about 70% of the theoretical yield).
Similar approaches of genes replacement and balancing of redox cofactors were employed by
Bond-Watts et al. (2011). To redirect more flux towards butanol formation, the authors replaced
in the butanol biosynthesis pathway the thl by the pdaA from Ralstonia eutropha, and bcd-etfAB
by ter from Treponema denticola. The engineered strain produced 2.95 g/L butanol after 72 h of
fermentation. Further overexpression of E. coli pyruvate dehydrogenase multienzyme complex
(aceEF-lpdA) to provide both NADH and acetyl-CoA for butanol biosynthesis, allowed increase
butanol titre to 4.65 g/L with 28% of the theoretical yield from glucose.
Recently, Saini et al. (2015) proposed an alternative platform for production of butanol with
E. coli, based on the application of butyrate-conversion and butyrate-producing strains in a
co-culture system. A butyrate-conversion strain (BuT-3E) was developed by removal of frdABCD,
ldhA, adhE and pta genes, and recruiting native acetoacetyl-CoA transferase (atoAD) and adhE2
from Clostridium. A butyrate-producing strain (BuT-8L-ato) was equipped with a pathway for
the synthesis of butyrate comprising atoAD and heterologous genes: 3-ketiothiolase (pha), hdb,
crt and ter. During co-culturing the butyrate-conversion strain converted butyrate to butanol with
accompanied production of acetate. Released acetate was re-utilized by the butyrate-producing
strain to synthesize butyrate, which in turn provided the precursor butyrate for butanol production
in strain BuT-3E. At 24 h fermentation butanol titre was 5.5 g/L from glucose. The production yield
accounted for 69% of the theoretical value.
2.4.2 Isopropanol
The engineered fermentative route for isopropanol production in E. coli involves reconstruction the
Clostridium isopropanol biosynthesis pathway by overexpressing three C. acetobutylicum genes
(thl, ctfAB and adc) involved in acetone synsthesis together with the C. beijerinckii secondary
alcohol dehydrogenase (adh) to convert acetone to isopropanol. Hanai et al. (2007) the highest
isopropanol production of 4.9 g/L from glucose, corresponding to 43.5% of the theoretical yield,
obtained for the engineered E. coli strain expressing the combination of thl and adc (C. aceto-
butylicum ATCC 824), atoAD (E. coli), and adh (C. beijerinckii NRRL B593). Unlike Hanai et al.
(2007) which used the polycistronic expression of the thlatoADadc operon, Jojima et al. (2008),
constructed isopropanol pathway in E. coli by expressing the C. acetobutylicum ATCC 824 thl,
ctfAB, adc and the C. beijerinckii NRRL B593 adh genes from a dedicated promoter in a single
vector. After 36 h glucose fed-batch culture the engineered E. coli strain produced 13.6 g/L iso-
propanol, reaching 51% of the theoretical yield. Inokuma et al. (2010) reported that production
of isopropanol could be significantly increased by the use of a gas trapping process for product
revovery from culture broth and allievated product toxicity to E. coli. Fed-batch, pH-controlled
fermentation in SD-8 media supplemented with glucose resulted in 40.1 g/L isopropanol after 60 h
(73.2% of the theoretical yield). By conjucting fed-batch fermentation with the gas-stripping sys-
tem, isopropanol concentration increased to 143 g/L after 240 h fermentation with a molar yield of
67.4% (isopropanol/glucose).
3 NON-FERMENTATIVE ALCOHOL FUELS
Except butanol and isopropanol, longer primary (up to C5) and branched-chain alcohols are not
produced via fermentation (Lamsen & Atsumi, 2012). However, primary higher alcohols can be
synthesized by manipulation of two natural biosynthetic pathways in microorganisms: a keto acid-
based pathway based on amino acid biosynthesis or the fatty acid pathway (Rabinovitch-Deere
et al., 2013). In general, the use of fatty acid metabolism is advantageous for the production of
linear-chain primary alcohols, while that of amino acid metabolism is suitable for branched-chain
alcohols (Choi et al., 2014).
3.1 Production of higher-chain alcohols using the keto acid pathways

The strategy for keto acid-based alcohols production is based on exploiting the amino acid biosyn-
thesis pathways in E. coli to generate 2-ketoacid precursors, which can be used as substrate for the
Ehrlich degradation pathway to alcohols (Connor & Liao, 2009). The Ehrlich degradation pathway
to alcohols consists of two steps. The first is a conversion of 2-keto acids to aldehydes by the expres-
sion of a heterelogous 2-keto acids decarboxylase (kidv, KDC). The second is subsequent reduction
of the aldehydes to alcohol products by an alcohol dehydrogenase (adh2, ADH2) (Rabinovitch-
Deere et al., 2013). Since these two enzymes have broad substrate specificity, the following higher
alcohols it may be produced: propanol, 2-methyl-1-butanol (2MB) and butanol in L-treonine path-
way, and isobutanol and 3-methyl-1-butanol (3MB) from 2-ketoisovalerate (Fig. 7.5). Last two
reactions in the Ehrlich degradation pathway are the non-native once (Atsumi & Liao, 2008).
Atsumi et al. (2008a) reconstructed the Ehrlich pathway in. E. coli to produce higher chain alco-
hols from glucose. The authors coexpressed alcohol dehydrogenase (adh2) with genes encoding
five different 2-ketoacids decarboxylase: pdc6, aro10 and this3 from S. cerevisiae, kivd from
Lactococcus lactis, and pdc from C. acetoburylicum, to determine which combination gave the high-
est alcohol titer. The most active was alcohol dehydrogenase encoded by kivd from L. lactis. This
enzyme showed also the broadest substrate specificity and allowed the highest alcohol production.
Using kivd and adh2, six different 2-ketoacids intermediates were converted to alcohols produced
0.031 g/L propanol, 0.068 g/L 2MB, 0.39 g/L isobutanol, 0.13 g/L 3MB, 0.04 g/L 2-phenylethanol,
and 0.016 g/L butanol.
3.1.1 Propanol and butanol

An increase in the level of 2-ketovalerate in E. coli, which is a precursor of butanol, can be achieved
trought overexpression of leuABCD pathway. Furthermore, overexpessing ilvA lead to increase in
2-ketobutyrate concentration generated from L-threonine.
Atsumi et al. (2008a) by overexpessing ilvA and leuABCD obtained a three times higher produc-
tion of butanol (0.044 g/L) over control strain of E. coli. Next, to improve butanol production, the
authors deleted the ilvD gene that resulted in elimination of 2-keto-3-methylvalerate production.
A cellular control mechanism of enzyme that catalyzes the production of L-threonine is inhibited
when L-threonine has accumulated to a certain level. Therefore, L-threonine has been recognized
as the limiting substrate for butanol and propanol production. After applying of ilvD deletion twice
increase in butanol production was obtained when L-threonine was extremally added.
Shen & Liao (2008) regulated L-threonine synthesis by overexpression thrA, thrB and thrC
genes that relieved the L-threonine feedback. Subsequent, the authors disrupted metA and tdh
genes to eliminate pathways competing with L-threonine biosynthesis. To avoid the diversion of
2-ketobutyrate to amino acids biosynthesis, the genes ilvBN and ilvIH were also knocked out
(Fig. 7.5). Finally, adhE was deleted. As a result, over 1.8 g/L butanol and propanol in near 1:1
ratio after 72 h were produced without the addidion of L-threonine.
Figure 7.5. Non-fermentative pathways for the production of higher alcohols in engineered Escherichia coli (adapted according to Cann & Liao, 2008; Shen & Liao,
2008; Soini et al., 2008; Peralta-Yahya & Keasling, 2010; Zhang et al., 2015). Dash arrows represent multienzymatic steps. Revelant reactions are represented by
the name of the genes coding for the enzyme: ppc, phosphoenolpyruvate carboxylase; aspC, aspartate aminotransferase; thrA, metL, lysC, aspartate kinase/thrA, metL,
homoserine dehydrogenase; asd, aspartate semialdehyde dehydrogenase; thrB, homoserine kinase; thrC, threonine synthase; ilvA, threonine deaminase; tdcB, threonine
dehydratase; cimA, citramalate synthase; leuA, 2-isopropylmalate synthase; leuCD, 2-isopropylmalate isomerase; leuB, 3-isopropylmalate dehydrogenase; ilvGM, ilvIH,
ilvBN, alsS, acetohydroxy acid synthase/acetolactate synthase; ilvC, acetohydroxy acid isomeroreductase; ilvD, dihydroxy acid dehydratase; ilvE, branched-chain amino-acid
aminotransferase; tyrB, leucine aminotransferase; kivd, 2-keto-acids decarboxylase; adh2, alcohol dehydrogenase.
In E. coli 2-ketobutyrate is produced from the deamination of L-threonine, which is formed in six
enzymatic steps from oxaloacetate. A shorter pathway contributing the 2-ketobutyrate formation
was recognized in Leptospira interrogans and Methanocaldococcus jannaschii. The key role in
this pathway plays enzyme citramalate synthase encoded by cimA gene. Atsumi & Liao (2008)
improved production of propanol and butanol by overexpressing mutate cimA from M. jananaschii
and knockouting ilvI, ilvA and tdcB genes. The enginereed strain produced more than 3.5 g/L
propanol and 0.52 g/L butanol after 92 h at 30 C in M9 medium containing 72 g/L glucose and
5 g/L yeast extract.
3.1.2 Isobutanol
As first enginereed E. coli for isobutanol production proposed Atsumi et al. (2008a). The pathway
for isobutanol sythesis was improved in four steps. First, the authors overexpressed the native
ilvIH, ilvC and ilvD to increase concentration of 2-ketoisovalerate. The 2-ketoisovalerate was then
converted to isobutanol by KDC and ADH2 encoded by kivd (L. lactis) and adh2 (S. cerevisiae).
Isobutanol production was 1.7 g/L and was approximately five times higher than control strain. In
second step, the genes coding for proteins involved in by-product formation (adhE, ldhA, frdAB,
fnr and pta) were deleted. As a result isobutanol production increased to 2.2 g/L.
In the third step, the alsS gene from B. subtilis was used to replace ilvIH of E. coli that allowed
increase isobutanol concentration to 3.7 g/L. Lastly, the deletion of pflB gene, encoding pyru-
vate formate lyase, allowed significant increase in isobutanol production. The engineered strain
produced 22 g/L isobutanol between 40 h and 112 h under microaerobic conditions. This level pro-
duction corresponded to the yield of 0.35 g isobutanol/g glucose, which is 86% of the theoretical
maximum.
In later studies, Atsumi et al. (2010a) compared the effect of various alcohol dehydrogenases
(ADH2) (adh2 from S. cerevisiae, adhA from L. lactis and yqhD from E. coli). As host strain
previously engineered strain JCL260 was used (Atsumi et al. 2008a). Overexpession of yqhD
or adhA showed better production of isobutanol than overexpression of adh2. The isobutanol
concentration was over 8 g/L after 24 h.
E. coli is unable to grow at isobutanol concentration above 8 g/L due to toxicity of the product
(Brynildsen & Liao, 2009). Despite this, the ability to produce isobutanol is retained (Atsumi et al.
2008a). However, the final concentration is low (Rutherford et al., 2010). Atsumi et al. (2010b)
used host strain JCL260 to construct strain enables to produced isobutanol in concentration greater
than the toxicity level. The engineered strain SA481 carried five insertions (acrA, gatY, tnaA and
yhb) and one deletion (marCRAB).
At concentration of 6 and 8 g/L isobutanol, the growth of SA481 after 24 h were 13 times and
5 times higher, respectively, than JCL260. However, the improvement of isobutanol tolerance did
not enhance isobutanol production (about 20 g/L). Integrated batch fermentation with air stripping
allowed increase in isobutanol production by E. coli strain JCL260 to about 50 g/L in 72 h (Baez
et al., 2011).
A major barrier in isobutanol production via the amino-acid pathways is NADPH dependency.
NADPH is formed in the phosphate pathway (PPP) or the tricarboxylic acid (TCA) cycle which
function in the presence of oxygen. Under anaerobic conditions, the only available reducing equiv-
alent is NADH, produced through glycolysis (Baez et al., 2011; Lamsen & Atsumi, 2012). Bastian
et al. (2011) overexpressed pntAB encoding a transhydrogenase (E. coli), which catalyzes the
reversible transfer of a hydride ion between NADH and NADP, and constructed NADH-dependent
pathway by engineering a ketol-acid reductoisomerase (enconing by E. coli ilvC) and alcohol dehy-
drogenase (encoding by L. lactis adhA). In this way, under anaerobic conditions, NADH-dependent
strain produced 13.4 g/L isobutanol, reaching 100% the theoretical yield after 24 h.
3.1.3 2-methyl-1-butanol and 3-methyl-1-butanol

2-methyl-1-butanol (2MB) and 3-methyl-1-butanol (3MB) are five carbon alcohols, which in minor
amounts can be produced by S. cerevisiae. Beside some metabolic approaches has been attempted to
increase 2MB production in yeast (Abe & Horikoshi, 2005), production of these 5-carbon alcohols
by engineered E. coli is considered to be a more desirable process (Chen et al., 2013).
Production of 2MB in E. coli harnesses L-isoleucine biosynthesis, sharing the 2-ketobutyrate
with propanol and butanol forming. The first step in L-isoleucine synthesis is formation of 2-keto-
3-methylvalerate (KMV), a immediate precursor of 2MB (Fig. 7.5). KMV synthesis begins with
the condensation of 2-ketobutyrate and puryvate catalyzed by acetohydroxyacid synthase (AHAS).
To improve this step Cann & Liao (2008) tested four different AHAS isozymes: AHAS III encoded
by ilvH (E. coli), AHAS II encoded by ilvGM (E. coli, Salmonella typhimurium and Klebsiella
pneumonia) and AHAS I encoded by ilvBN (E. coli and Corynebacterium glutamicum). Strain with
S. typhimurium AHAS II produced the highest concentration of 2MB. Next, supplying L-threonine,
they tested three different threonine transaminase isozymes encoded by tdcB (E. coli), ilvA (E. coli)
and ilvA (C. glutamicum). Overexpression of C. glutamicum ilvA led to the highest concentration
of 2MB, converting 88% of the supplied L-threonine into 2MB. Further increase in L-threonine
production by overexpression L-threonine pathway (thrABC) along with deletion of metA and tdh
genes, responsible for consuming precursors upstream of 2-ketobutyrate, boosted 2MB production
to 1.25 g/L with a yield of up to 0.17 g 2MB/g glucose (44% of the theoretical value) in 24 h at
30 C under anaerobic conditions.
Production of 3MB can be performed by using the native L-valine (ilvIHDC) and L-leucine
(leuABCD) biosynthesis pathways. The L-valine biosynthesis pathway generates 2-ketoisovalerate,
the precursor for L-valine and isobutanol, which is then converted to 2-keto-4-methylpentanoate by
leuABCD. Next, 2-keto-4-methylpentanoate is reduced to 3MB by KDC and ADH2. Connor & Liao
(2008) to produced 3MB overexpressed ilvIHCD and leuABCD of E. coli together with kivd from
L. lactis and adh2 from S. cerevisiae in butanol high producer JCL260. However, overexpression of
these genes led to low production of 3MB (less than 0.2 g/L), mainly due to the feedback inhibition
of 2-isopropylmalate synthase (IPMS) (encoded by leuA) activity by free L-leucine. To relieve
the feedback inhibition of IPMS, the authors employed a feedback-insensitive mutant of IPMS
encoded by leuAFBR, and inactivated L-leucine synthesis by deleting ilvE (encoded branched-
chain-amino-acid transferase) and tyrB (encoded tyrosine aminotransferase). The final JCL260
strain, expressing the mutated leuA along with alsS (B. substilis), produced 1.28 g/L 3MB in
28 h at glucose concentration of 10 g/L under anaerobic conditions, and minimizing isobutanol
production (less than 0.2 g/L). The overall yield was estimated to be 0.11 g/g, with a maximum
productivity of 0.12 g/L h between 8 and 12 h. In further experiments this recombinant strain was
used by Connor et al. (2010) to enhance 3MB production by random mutagenesis and selection.
The resulting strain produced 9.5 g/L 3MB corresponding to 33% of theoretical maximum after
60 h when a two-phase fermentation strategy with oleyl alcohol was implemented.
Biological production of butanol and its analogues have made significant progress over the past
decade, owing to the advances in metabolic pathway design, strain optimization and exploitation of
novel microorganisms. Through recombination or modification of butanol producing strains, spe-
cific targeted genes will be overexpressed or disrupted for enhancing butanol production, butanol
yield and utilization of substrate. The majority of maximal titres have been produced from engi-
neered E. coli, likely due to the thoroughly studied metabolism, genetic tools and fast growth rate
of the bacterium.
The replacement of sugars lignocellulosic biomass can reduce the cost of the substrate, how-
ever, requires initial pretreatment step to increase polysaccharide accessibility which can result in
formation of compounds that may inhibit bacterial growth and alcohols production. In this term,
development of consolidated bioprocessing (CBP) in which biomass can be broken down efficiently
and subsequently converted into fuel via mixed fermentation using different strains is approach
worth considering in decreasing production time and costs.
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About the Authors
Katarzyna Bukowska, PhD. Eng., received a Masters degree in environmental protection in

2003. In 2008, she received a doctorate in environmental management at the University of Warmia
and Mazury (UWM) in Olsztyn, Poland, specializing in biotechnology in environmental protec-
tion. Since then, she has been employed as an Assistant Professor at the Faculty of Environmental
Sciences, Department of Environmental Biotechnology, UWM Olsztyn. Her scientific interests
concern the following research areas: biogas production, modeling of anaerobic digestion pro-
cesses, and soil remediation. She is the co-author of 15 articles, 9 chapters in monographs and 21
posters or presentations at national and international conferences. She is a member of the Polish and
International Humic Substances Society and European Geosciences Union (Division: Soil System
Sciences, Subdivision: Soil Pollution and Reclamation).
Zygmunt Mariusz Gusiatin, PhD. Eng. was born in 1980. He received a Masters degree in
Environmental Protection in 2003, and a Doctorate in Environmental Management, specializ-
ing in biotechnology in environmental protection from the University of Warmia and Mazury
(UWM) in Olsztyn, Poland, in 2008. Now, he is employed as an Assistant Professor at the Fac-
ulty of Environmental Sciences, Department of Environmental Biotechnology, UWM, Olsztyn.
183
184 About the Authors
His scientific interests concern three research areas: soil remediation, composting and anaerobic
digestion, including digestate management. He has published 30 articles and he is the author or
co-author of 6 monograph chapters, as well as 1 book chapter that was published internationally. He
is a member of the Polish and International Humic Substances Society and European Geosciences
Union (Division: Soil System Sciences, Subdivision: Soil Pollution and Reclamation).
Prof. Ewa Klimiuk works at the University of Warmia and Mazury in Olsztyn, Poland, at the Faculty
of Environmental Sciences, in the Department of Environmental Biotechnology. The research of
Prof. Ewa Klimiuk initially focused on advancement of wastewater treatment, including industrial
wastewater and leachate from municipal landfills. In subsequent years, she dealt with processing
wastewater and waste to produce useful products by technologies such as biogas and composting.
An important part of her research in this area focuses on the microbial production of biopolymers
(polyhydroxyalkanoates) from wastewater and biodiesel by-products. Throughout her entire period
of work she has collaborated with numerous industrial factories and operators. An important stage
of her work was employment at the Lublin University of Technology in the Department of Envi-
ronmental Engineering, Institute of Environmental Engineering, in 20072012.
The achievements of Prof. Klimiuk include 101 original research works published in foreign
and national journals, 2 monographs, and three academic books. Among her many awards and
honours, she received the Badge of Honour of the Ministry of the Environment in 2005, the award
of the President of Olsztyn (Statue of St. James) in the category of science in 2010, and the title of
Honorary Professor of the Lublin University of Technology in 2014.
Assoc. prof. Artur Pawowski works at Lublin University of Technology. Head of Department of
Sustainable Development. He works on issues related to environmental engineering, renewable
sources of energy and multidimensional nature of sustainable development.
About the Authors 185
Author of more than 150 publications, published in English, Polish and Chinese, including the
book Sustainable Development as a Civilizational Revolution. A Multidisciplinary Approach to
the Challenges of the 21st Century (CRC Press, 2011).
Member of European Academy of Science and Arts, Salzburg, Austria, The Committee of Envi-
ronmental Engineering of the Polish Academy of Sciences, Warsaw, Poland, International Academy
of Ecological Safety and Nature Management, Moscow, Russia and International Association for
Environmental Philosophy, Philadelphia, United States.
Editor-in-chief of the scientific journal Problemy Ekorozwoju/ Problems of Sustainable Devel-
opment and member of the editorial board of the Committee of Environmental Engineering
monographs.
Tomasz Pokj, Ph.D., received his Masters degree in environmental protection in 2001 at the
University of Warmia and Mazury in Olsztyn, Poland. In 2006 he defended his Ph.D. thesis Accu-
mulation of polyhydroxyalkanoates with mixed microbial cultures under oxygen and nitrogen
limited conditions at the University of Warmia and Mazury in Olsztyn. Since then, he has been
working in the Department of Environmental Biotechnology, Faculty of Environmental Sciences,
University of Warmia and Mazury in Olsztyn. His research and scientific interests focus on the
synthesis of microbial biopolymers, the production of biogas, the modeling of anaerobic diges-
tion in agricultural biogas plants, and technologies for biofuels. He is the author or co-author of
28 articles, 13 chapters in monographs, 1 book, and 19 posters or presentations at national and
international conferences for industry and academia.

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BIOMASS FOR BIOFUELS

Biomass for Biofuels

Katarzyna Bukowska, Zygmunt Mariusz Gusiatin,

1 Biofuels and sustainable development 3

2 Biomass for fuels classification and composition 15

3 Biomass feedstock for biofuels production 37

4.2 Spent oil and animal fats 52

4 Outlook for advanced biofuels 63

5 Conversion of lignocellulosic biomass into sugars: the effect of the

6 Pretreatment of lignocellulosic biomass 121

3.4 Oxidative delignification 134

7 Fermentative and non-fermentative pathways of butanol and its analogues 155

About the Authors 183

Biofuels and sustainable development

Ewa Klimiuk & Artur Pawowski

1.1 Sustainable development

1.2 Strategies for sustainable development

2 ENVIRONMENTAL ASPECTS OF BIOFUELS PRODUCTION

2.1 Depletion of fossil fuel resources

2.2 Environment pollution

as particulates (soot), monocyclic aromatic hydrocarbons (e.g. benzene) or polycyclic aromatic

Bioethanol Biodiesel Biodiesel Fischer-Tropsch

15% reduction in 10% reduction in carbon 50% reduction in A reduction in nitrogen

2.3 Changing the use of natural space and reducing biodiversity

3 ECONOMIC ASPECTS OF BIOFUELS PRODUCTION

3.1 Cost effectiveness of biofuels production and energy balance

3.2 Energy security

3.3 Loss of government revenue

4 SOCIAL ASPECTS OF BIOFUELS PRODUCTION

4.1 Rural development

4.2 Diversification of production

4.3 Risks associated with the production of biofuels

5 PROSPECTS FOR THE DEVELOPMENT OF THE BIOFUELS MARKET

Biomass for fuels classification and composition

Zygmunt Mariusz Gusiatin & Artur Pawowski

1 DEFINITION AND CLASSIFICATION OF BIOMASS

1.1 Definition of biomass

1.2 Categories and types of biomass

2.1 Criterion of expressing biomass composition

2.2 Biomass composition proximate analysis

Table 2.2. Chemical composition of biomass based on ultimate analysis.

Ultimate analysis (%, dry and ash free)

2.3 Biomass composition ultimate analysis

2.4 Biochemical biomass composition

2.4.1 Characteristic of structural components in biomass

Figure 2.3. Chemical structure of cellulose (Hallac & Ragauskas, 2011).

Figure 2.4. The chemical structure of hemicelluloses in softwood: (a) galactoglucomannan;

Wood Content in Molar

Soft Galacto-glucomannan 58 -D-mannopyranose 3 14 100

2.4.2 Lignin isolation from biomass and its characterization

Table 2.4. The quantities of linkages in lignin from various plants.

Benzyl Phenyl Spiro-

associated with p-hydroxycinnamates (p-coumarate and ferulate), whereas a strong predominance

Biomass feedstock for biofuels production

Katarzyna Bukowska & Artur Pawowski

2 BIOMASS FEEDSTOCK FOR THE FIRST AND NEXT GENERATION

Feedstock Factors affecting sustainability and potential Likelihood of impact

3 BIOMASS FEEDSTOCK FOR THE SECOND AND THIRD GENERATION

3.1 Lignocellulosic biomass

3.1.1 Biomass from short-rotation forestry

Production estimates Growth cycles

* short-rotation coppice (SRC), ** very-short-rotation coppice (VSRC)

Cellulose Xylan Galactan Arabinan Mannan Lignin Extractives Ash