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10/28/2015

MBT Fall2015

MicroalgalBiotechnology:
CultivationandProcessing
HectorDelaHozSiegler,Ph.D.,P.Eng.
DepartmentofChemicalandPetroleumEngineering
UniversityofCalgary
h.siegler@ucalgary.ca

October29th,2015

HectorDelaHozSiegler

Outlineoftodaystalk
I. Introduction to microalgae
What and why
Applications

II. Renewable energy from microalgae


Motivation
Biofuel portfolio
Biodiesel

III. Culturing techniques


Medium requirements
Open ponds and photobioreactors
Phototrophic and heterotrophic

IV. Optimization of heterotrophic cultures


V. Summary

PartI

INTRODUCTIONTOMICROALGAL
BIOTECHNOLOGY
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Microalgae:whatarethey?
Microalgaeareplantlikeunicellularorganisms:apolyphyletic group
ofphotosyntheticeukaryotes.
Sunlight-driven cell factories able to convert carbon dioxide to
potential biofuels, food, and high-value products

ProductsfromMicroalgae

Image source: Rosenberg, J.N., Oyler, G.A., Wilkinson, L., Betenbaugh, M.J. A green light for engineered algae: redirecting metabolism to fuel a biotechnology
revolution ,Current Opinion in Biotechnology, 19 (5), pp. 430-436 (2008)

Applications
Humanand
animalfood

Pharma and
nutraceuticals

CO2 Capture

Microalgae
Biofuels

Wastewater
treatment/
Remediation

Finechemicals:
e.g.antioxidants
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Microalgaeascellfactories
Benefits:
Highlyefficient
Simplenutritionalrequirements
Easilyadaptabletoenvironmentalstresses
Produceandstorehighamountsofoil
Othervaluablebyproducts
Eukaryotic!

Challenges
Lowculturedensity
Slowgrowth:lowproductivity(comparedtobacteriaandyeast)
Highproductioncosts
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SomeCommercialApplications
Species/group

Product

Application areas

Production
facilities

References

Haematococcus
pluvialis /
Chlorophyta

Carotenoids,
astaxanthin

Health food, feed additives


and pharmaceuticals

Open ponds,
PBR

Del Campo et al. (2007)

Odontella aurita
/ Bacillariophyta

Fatty acids

Pharmaceuticals,
cosmetics, baby food

Open ponds

Pulz and Gross (2004)

Isochrysis
galbana /
Chlorophyta

Fatty acids

Animal nutrition

Open ponds,
PBR

Molina Grima et al.


(1994); Pulz and Gross
(2004)

Phaedactylum
tricornutum /
Bacillariophyta

Lipids, fatty
acids

Nutrition, fuel production

Open ponds,
basins, PBR

Yongmanitchai and Ward


(1991); AcienFernandez et al. (2003)

Muriellopsis sp.
/ Chlorophyta

Carotenoids,
Lutein

Health food, food


supplement, feed

Open ponds,
PBR

Blanco et al. (2007); Del


Campo et al. (2007)

Crypthecodinium
cohnii

DHA

Food additive

Fermenters
(heterotrophic)

Carvalho et al.
(2006)

Currently,applicationsofmicroalgalbiotechnologyarelimitedtoniche(small)markets.Though
highvalue!Weexpecttomoveintolargescalemarkets.
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PartII

ALGAEASASOURCEOF
RENEWABLEENERGY
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Energy,economy,andGlobalwarming
100

PrimaryEnergyConsumption(kWyear/person)

Poland
Ghana

Uzbekistan
Canada

US(2002)

10
Russia

South
Korea

France
UK

China
China(2002)

Norway

US

Japan

Brazil

India(2002)

India

0.1

Vanuatu

Chad

0.01
100

1,000

10,000

100,000

GDP($/person/year)

Energyreserves/Energyconsumption

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Climatechangedebate

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Thenaturalcarboncycle

G. A. Olah and colleagues, J.Am.Chem.Soc, 133, 12881-12898, 2011

Carbon CaptureandUtilisation(CCU)

T. Shirvani, X. Yan, O. R. Inderwildi, P. P. Edwards and D. A. King, Energy Environ. Sci., 2011, 4, 37733778

2nd Generation: produced from lignocellulosic


materials, i.e. ethanol from wood chips,
switch grass.
3rd Generation: fuels from microalgae
4th Generation: from crops designed for fuels
in combination with highly efficient microbes.

Landrequiredforsatisfydemand

1st Generation: derived from food-crops, i.e.


ethanol from sugar cane or corn, biodiesel
from canola or soybeans.

Timetorealworldapplication

Biofuels

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Themicroalgalbiofuelsportfolio
Feedstocks

Photosynthesis

CO2

Algal Biomass:

Water

Intermediates

Fuels
Biodiesel

- Oil/Lipids

Green Diesel

Hydrodeoxygenation

Sunlight
Trace elements

- Sugars/Starch

Hydrolysis

- Lignocellulose

Sugars
Bio-oil

Pyrolysis

Excreted products:

Gasoline

Hydrotreating

SynGas

- Hydrogen

Hydrogen

- Alcohols

Alcohols

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BiodieselfromMicroalgae
Biodieselderivedfromoilcropsisapotentialrenewableand
carbonneutralalternativetopetroleumfuels.
Biodieselfromoilcrops,wastecookingoilandanimalfatcannot
realisticallysatisfythedemandfortransportfuels.
Crop

Oil yield
(L/Ha)

Land area needed


(M Ha)

% of existing US
cropping area

Corn

172

1540

846

Soybean

446

594

326

Canola

1190

223

122

Oil Palm

5950

45

24

136900

1.1

58700

4.5

2.5

Microalgae
(70% oil w/w)

Microalgae

Croplandrequirement
bydifferentoilcropsto
replace50%ofall
transportfuelneedsof
theUS.Chisti(2007).
Toooptimistictobetrue!

(30% oil w/w)


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Algaeasasourceofoil
Species

Oil content
(% dw)

Reference

Ankistrodesmus TR-87

28 40

Ben-Amotz and Tornabene (1985)

Botryococcus braunii

25 75

Sheehan et al. (1998); Banerjee et al. (2002); Metzger and Largeau (2005)

Chlorella sp.

28 32

Sheehan et al. (1998), Chisti (2007)

Chlorella protothecoides

15 55

Xu et al. (2006)

Cyclotella DI-35
Dunaliella tertiolecta
Hantzschia DI-160

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36 42
66

Isochrysis sp.

7 33

Nannochloris

20 - 35 (6 - 63)

Sheehan et al. (1998)


Kishimoto et al. (1994); Tsukahara and Sawayama (2005)
Sheehan et al. (1998)
Sheehan et al. (1998); Valenzuela-Espinoza et al. (2002)
Ben-Amotz and Tornabene (1985); Negoro et al. (1991); Sheehan et al. (1998)
Sheehan et al. (1998); Hu et al. (2006)

Nannochloropsis

46 (31 - 68)

Nitzschia TR-114

28 50

Kyle DJ, Gladue RM. WO 91/14427 (Patent)

Phaeodactylum tricornutum

20 31

Sheehan et al. (1998), Chisti (2007)

Scenedesmus TR-84
Stichococcus

45

Sheehan et al. (1998)

33 (9 - 59)

Sheehan et al. (1998)

Tetraselmis suecica

15 32

Thalassiosira pseudonana

(21 - 31)

Sheehan et al. (1998); Zittelli et al. (2006); Chisti (2007)


Brown et al. (1996)

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OilandBiodiesel
Triglycerides:

Glycerol

FattyAcids

BiodieselProduction:

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PolyunsaturatedFattyAcids(PUFA)
Fattyacidswithmultipledoublebonds
EPA: C20:5

DHA: C22:6

C18:3andlongerareessential:mammalscannotsynthesize
C18:3.Needtotakethemfromtheirdiet
Multiplebiologicalfunctionsassignallingmoleculesorbuilding
blocks
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MicroalgaeasaSourceof3PUFA
FishoilhasbeenusedforthecommercialproductionofEPA
andDHA.
Factorsthatlimitfishoilasasourceof3fattyacidsinclude:
taste,odourandstabilityproblems.Highpurificationcost.
Fishobtain3fattyacidsfromtheirdiet.
Severalspeciesofmicroalgaeareprimaryproducersoflong
chainPUFA.
US$1.5billion/yeargeneratedfromtheproductionofDHA
(Pulz andGross,2004).

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PUFAproportionsinMicroalgae(%TFA)
Organisms

ARA (20:4)

EPA (20:5)

DHA (22:6)

Gymnodinium splendens

30

Cricosphaera elongata

28

Isochrysis galbana

15

7.5

Monodus subterraneus

4.7

33

Nannochloropsis sp.

35

Schizochytrium sp.

1.0

2.3

40.9

Chlorella minutissima

5.7

45

Hetermastrix rotundra

28

Chromonas sp.

12.0

6.6

Cryptomonas sp.

16

10

Rhodomonas sp.

8.7

4.6

Asterionella japonica

11

20

Biddulphia sinensis

24

Crypthecodinium cohnii

30

Nitzschia laevis

6.2

19.1

Phaeodactylum Tricornutum

34.5

Skeletonema costatum

29.2

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GeneralProcessDiagram
Solvent
recovery

Culture
Extraction

Crude
Product
Harvesting

debris

Cell disrupter

Dryer
S/L Separator

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PartIII

MICROALGALCULTURING
TECHNIQUES
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Nutritionalrequirements
Dependsonapplication
Foodorhealthoils:foodgradechemicals
Otherwiseindustrialchemicalsorseawater/wastewater

Carbonsource:CO2,sugars,acetate,ethanol
Macronutrients:Nitrogenandphosphorus
Micronutrients:Fe,Mg,Si,S,K
Traces:Ca,Mn,Zn,Co,Se,Cu,Mo
Vitamins:B1,B12,B6,B2
Seawater:Na,K,Mg,Ca,Cl,SO4,HCO3,BO3
Br,F,IO3,Li,Rb,Sr,Ba,Mo,V,Cr,As,Se
NO3,PO4,Fe,Zn,Mn,Cu,Co,Si,Ni

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Multiplewaysofgrowing
Phototrophic

Heterotrophic

Mixotrophic

EnergySource

Applications

FixCO2

Slowgrowth
Noactivityduring
night

CO2 capture

Fast growth
Productiveallday
long

Dontfixgreenhouse
gases
ProduceCO2

Wastewatertreatment
Highvalueproducts
Proteins

FixCO2
Fast growth
Productiveallday
long
Complexand
unknowncell
regulation
CO2 capture
Wastewatertreatment
Highvalueproducts
Proteins
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SolarradiationinCanada

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SolarradiationinAlberta
FortMcMurray:
Edmonton:
MedicineHat:

4181MJ/m2y
4510MJ/m2y
5221MJ/m2y

Munich(GE):
Naples(IT):
KualaLumpur:
Orlando(FL):
Acapulco(MX):
Phoenix(AZ):

4044MJ/m2y
5293MJ/m2y
5622MJ/m2y
5922MJ/m2y
7261MJ/m2y
7621MJ/m2y

Solarradiationdatatakenfrom:U.S.DepartmentofEnergy EnergyPlusWeatherData.
http://apps1.eere.energy.gov/buildings/energyplus/cfm/weather_data.cfm

Culturingtechniques:OpenPonds
Byfar,themostcommon
productionsystem.
Lowinstallationcost
Lagoonsorartificialponds
Highriskofcontamination:
Unwantedalgae
Grazers

Applicationlimitedtofewspecies
(extremophiles).
Unmixedponds:arearangefrom1
200Ha,depth2030cm
Racewaypondsareupto1Ha.
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Culturing:ClosePondsandTanks
Simplerdesignssimilartoopen
ponds,withacover
(greenhouses).
Aimtoreducecontamination
risks.
ControlCO2 looses.
Tanksareusuallymixedby
aeration.
Deeptanksareinefficient.Bad
lighttransmission.
Easytooperate,lowcost.
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Culturing:Photobioreactors
FlatPanelphotobioreactor
ArizonaCenterforAlgalTechnologyandInnovation

TubularPhotobioreactor AlgaeandBiofuelsFacility,South
AustralianResearchandDevelopmentInstitute

FlexibleplasticfilmPhotobioreactor Algenol,Florida

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Culturing:Photobioreactors
Betterculturecontrol
Higherproductivity,andculture
density
Minimalcontaminationrisk
Wellmixed
Excellenttemperaturecontrol
Oxygencontrolisanissue
Highcapitalinvestment
Frequentcleaningrequired
Coolingrequired
32

HeterotrophicProductionofAlgae
Somealgaespeciescangrow
usinganorganiccarbonsource.
Conventionalbioreactorscanbe
used.

33

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Phototrophicvs.Heterotrophic?
Oil content
(%)

Cell conc.
(g/L)

Oil Prod.
(mg/L d)

Ettlia oleoabundans

36 42

2.9

164

Griffiths et al (2009);
Li et al. (2008)

Nannochloropsis sp.

31 68

2.1

204

Rodolfi et al. (2009)

Amphora

40 51

593

Sheehan et al. (1998)

Chlorella sp.

28 32

1.1

139

Hsieh and Wu (2009)

Chlorella vulgaris

Liang et al. (2009)

25 42

1.7

54

Chlorella zofingiensis

25.8

1.9

35

Liu et al. (2010)

Chlorella zofingiensis

51.1

9.6

354

Liu et al. (2010)

Nitzschia laevis

16.5

22.1

914

Wen and Chen (2003)

S. Limacinum (DHA)

17.3

37.9

656

Chi et al. (2009)

A. protothecoides

38.3 53.0

8.4

820

Cheng et al. (2009)

A. protothecoides

50.3 57.8

51.1

3320

Xiong et al. (2008)

Phototrophic

References

Heterotrophic

Specie

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PartIV

MODELBASEDOPTIMIZATIONOF
HETEROTROPHICALGALCULTURES
35

BioprocessOptimization

Continuous/
Realtime

Processconditions
Mediaformulation
Strainselection
Geneticmodification
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TheObjectiveforOptimization
Stress

Oil storing is a metabolic response to stress,


particularly nitrogen deficiency. At nitrogen
deficient conditions, algal cells over-accumulate
lipids.

Nitrogen

As nitrogen is required for protein synthesis, its


deficiency negatively affects growth and cell
functioning. Therefore, conditions that favored oil
accumulation constraint productivity.

The challenge is to maximize biomass production


while keeping a high oil content. It is necessary to
determine the nitrogen supplementation strategy
to achieve this.

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Understandingalgalgrowth
Nitrogen uptake
Cellular growth

Lipid production

38

Analgalgrowthmodel
Cellular growth

Nitrogen uptake

Oil production

39

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Macroscopicbalances

40

Optimization:Problemformulation

Subject to:

41

Simulationresults
Biomassproductivityin
continuouscultures

Lipidproductivityin
continuouscultures
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Experimentalresults

43

Biomassproductivityandgrowthrate

44

Lipidproductivityandproductionrate

45

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Oil Productivity
(g/L h)

Bacteria

Specie
E. coli (gen. modified)

25.4

0.246

Elbahoul et al (2010)

R. opacus PD630

38.4

0.171

Kurosawa et al (2010)

M. ramanniana

67.7

0.17

Hiruta et al (1997)

C. echinulata

26.9

0.07

Kosa et al (2011)

R. toruloides

67.5

0.54

Li et al. (2007)

L. starkeyi

56.0

0.04

Kosa et al. (2011)

C. curvatus

82.7

0.47

Zhang et al. (2011)

Schizochytrium sp.

30

0.096

Ganuza et al (2007)

C. vulgaris

9.7

0.12

Doucha et al. (2011)

A. protothecoides

50.3

0.14

Xiong et al. (2008)

A. protothecoides

49.4

0.43 0.84

Microalgae

Yeasts

Lipid content
(%, w/w)

Molds

Comparativestudy:growthonglucose
References

De la Hoz et al (2012)

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Optimization:closingremarks
Modelbased optimization of heterotrophic microalgal cultures
allowed to reach very high densities, with biomass productivity
greater than 30 g/L d, and as high as 70 g/L d.
High oil content (4060% w/w) can be sustained with a lipid
productivity around 20 g/L d.
High quality monitoring and control is essential to achieve high
productivities.
Better control / sensors = higher productivity.
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Summary
Algaearepromisingorganisms:highlyefficient
Goodsourceofoil:PUFA,biodieselprecursor
Algaecangrowthonsimpleinexpensivemedia
Severalreactortypesandgeometry.Applicationwilllimit
reactorchoice
Severalsuccessfulcommercialapplicationscurrentlyworking.
Alotofresearchisstillneeded!
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