Biochemical Engineering Journal 94 (2015) 9299

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Simultaneous saccharication and fermentation of

xylo-oligosaccharides manufacturing waste residue for l-lactic acid
production by Rhizopus oryzae
Li Zhang a,b , Xin Li a , Qiang Yong a, , Shang-Tian Yang b, , Jia Ouyang a , Shiyuan Yu a
a
b

College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, PR China
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA

a r t i c l e

i n f o

Article history:
Received 12 May 2014
Received in revised form
21 November 2014
Accepted 26 November 2014
Available online 27 November 2014
Keywords:
Cellulose
Fermentation
Filamentous fungi
Lactic acid
Rhizopus oryzae
Xylo-oligosaccharides

a b s t r a c t
High substrate cost and low lactic acid yield are the most pressing concerns in fermentative production
of l-lactic acid by Rhizopus oryzae. In this study, waste residue from corncob after xylo-oligosaccharides
(XOS) manufacturing was used as an alternative abundant, renewable, and inexpensive substrate for
l-lactic acid production. After enzymatic hydrolysis, both glucose and xylose in the hydrolysate were
converted to 34.0 g L1 of l-lactic acid, equivalent to a yield of 0.34 g g1 dry waste residue, by R. oryzae
in separate hydrolysis and fermentation. In contrast, a higher l-lactic acid titer (60.3 g L1 ) and yield
(0.60 g g1 dry waste residue) were achieved in simultaneous saccharication and fermentation (SSF)
with 10% (w/v) substrate loading at 40 C, demonstrating, for the rst time, the feasibility of l-lactic acid
production from XOS manufacturing waste residues. The SSF process for l-lactic acid production from
XOS waste residues was also demonstrated in a 5-L stirred-tank bioreactor, although further optimization
would be necessary.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Lactic acid is a commonly occurring organic acid that can be
produced biologically from renewable carbohydrates, and is valuable due to its wide use in food and related industries [1]. In
addition, a variety of useful chemicals, including plastics, bers,
solvents, and oxygenated chemicals, can be produced from lactic
acid derived from renewable feedstocks by sustainable biotechnological routes [2]. More recently, bio-based l-lactic acid has
attracted increasing attention for its use as a starting material in the
synthesis of poly-lactic acid (PLA) polymers, which are biodegradable and biocompatible with wide applications that conventional
petroleum-based plastics such as polyesters are not suitable or
unfavorable due to environmental concerns [3]. Today, lactic acid
produced in fermentation has become one of the most promising
feedstock monomers in the chemical industry.

Abbreviations: R. oryzae, Rhizopus oryzae; XOS, xylo-oligosaccharides; SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharication
and fermentation.
Corresponding author. Tel.:+86 25 85427587; fax: +86 25 85427587.
Corresponding author. Tel.: +1 614 2926611; fax: +1 614 2923769.
E-mail addresses: swhx@njfu.com.cn (Q. Yong), yang.15@osu.edu (S.-T. Yang).
http://dx.doi.org/10.1016/j.bej.2014.11.020
1369-703X/ 2014 Elsevier B.V. All rights reserved.

Current industrial production of lactic acid uses homolactic acid

bacteria, mainly Lactobacillus spp., cultured in enriched (complex)
media with glucose as substrate, which produce l(+)- or d()-form
of lactic acid with high product yield and productivity, but usually
suffer from high raw material and purication costs [1]. In contrast,
the lamentous fungus Rhizopus oryzae can use relatively inexpensive polysaccharides (e.g., starch) in simple media with minimal
nutrient supplementation and is easy to separate by ltration after
fermentation, and is thus, advantageous for its potential to reduce
lactic acid production cost [48]. It produces optically pure l-lactic
acid, which is the desirable form for food and pharmaceutical applications, and can also use xylose [9], the main sugar component in
hemicellulose, as substrate. Compared to bacteria, it can grow well
at a wider temperature range (up to 40 C) and pH range (from 4 to
9) [5]. Moreover, chitosan present in the fungal mycelia is a highvalue product, and the fungal biomass and byproducts can be used
in animal feeds to improve quality [4].
The current sugar and starch-based feedstock for lactic acid
fermentation accounts for more than 3040% of the total production cost [4]. Recent research on lactic acid production has thus
focused on abundant, low-cost lignocellulosic feedstocks [4,10,11].
More than 20 million tons of corncobs are available annually
in China [12]. Currently, a large amount of this is used to produce xylo-oligosaccharides (XOS) [13,14] from the hemicellulosic

L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 9299

materials, generating large quantities of waste residues, mainly

cellulosic materials, which would cause major environmental, and
economic problems if not properly treated or utilized. Because of
its low cost, high enzymatic digestibility due to low lignin content,
small particle size, and environmental benets of the re-utilization
of this industrial waste, XOS manufacturing waste residues can
be considered as an attractive alternative substrate for l-lactic
acid production. Compared to corncobs and other lignocellulosic
biomass, XOS waste residues with most of xylan removed contain
mainly glucose with a low content of xylose, and thus, would be better for lactic acid fermentation since most microorganisms cannot
use xylose efciently [4].
The goal of this study was to develop an economical fermentation process for l-lactic acid production from XOS waste
residues by R. oryzae. Unlike the conventional separate hydrolysis and fermentation (SHF) process, simultaneous saccharication,
and fermentation (SSF) can synchronize enzymatic hydrolysis and
microbial fermentation in a single step, thus offering various advantages, including increased productivity and reduced processing
time due to reduced product (glucose) inhibition on cellulose
hydrolysis [11,15,16]. In this work, both SSF and SHF were studied and compared for lactic acid production from XOS waste
residues. The hydrolysis and fermentation of XOS waste residues
were studied at different solid loadings and temperatures, and the
results are reported in this paper. To our knowledge, this is the
rst study demonstrating high-titer, high-yield, and cost-effective
l-lactic acid production from lignocellulosic biomass in an SSF
process.

2. Materials and methods

2.1. XOS waste residue
The XOS waste residue derived from alkali-pretreated corncobs
was kindly provided by Jiangsu Kangwei Biologic Co., Ltd. Milled
corncobs was stewed in 12 m3 alkali extraction tank containing
7% (w/v) sodium hydroxide at 8590 C for 1 h. The liquid fraction,
with a high content of hemicellulose was removed by vacuum ltration for further production of XOS; the solid waste residue was
rst soaked in water with a solid/liquid ratio of 1:10 (w/v), and
then neutralized with 72% (w/w) sulfuric acid to adjust the pH to
5.05.5, followed by removing the water with vacuum ltration to
obtain the solid XOS waste residue. The solid fraction was stored
in plastic bags at 4 C until use. Before treatments, the corncobs
contained (%, dry weight basis) 40% cellulose and 31% hemicellulose. After treatments, the solid waste residues contained 65.5%
cellulose and 22.2% hemicellulose or 72.8% glucose, 23.3% xylose,
and 1.9% arabinose.

2.2. Microorganism and cultivation

R. oryzae NLX-M-1 was obtained from Institute of Biochemical Engineering, Nanjing Forestry University, Nanjing, China. The
preculture medium consisted of (g L1 ): 50 glucose, 3 (NH4 )2 SO4 ,
0.75 MgSO4 7H2 O, 0.20 ZnSO4 7H2 O, and 0.30 KH2 PO4 , which was
found to be optimal with glucose as the carbon source. All media
were sterilized by autoclaving at 121 C, 15 psig for 30 min. The
strain was rst cultured on potato-dextrose agar slants at 30 C
for 35 days to generate spores. The preculture used to seed the
fermentation was prepared in 250-mL Erlenmeyer asks, each containing 50 mL preculture medium and 10 g L1 CaCO3 , inoculated
with a spore suspension containing 106 spores mL1 , and incubated
at 30 C for 12 h in a rotary shaker agitated at 170 rpm.

93

2.3. SHF for lactic acid production

Cellic CTec2 (Novozymes), a cellulase complex consisting of
aggressive cellulases, -glucosidases and hemicellulase, was used
for the degradation of cellulose and hemicellulose to fermentable
sugars. Enzymatic hydrolysis trials were performed with the substrate loadings at 5%, 10%, 15%, and 20% (w/v). Unless otherwise
noted, the hydrolysis was carried out in a 250-mL Erlenmeyer ask
with the enzyme dosage of 0.06 g g1 biomass at pH 5.05.5, in a
shaker controlled at 50 C and 150 rpm, for 23 days. The cellulose (glucan) and hemicellulose (xylan) hydrolysis yields (%) were
calculated as the percentages of obtained glucose and xylose in
the hydrolysate to the total glucose and xylose present in the
substrate, respectively. For complete hydrolysis, the theoretical
sugar yields from cellulose and xylan are 1.11 g glucose g1 glucan
and 1.14 g xylose g1 xylan, respectively [17]. All experiments were
duplicated, and the average values are reported. Before use as substrate in fermentation, the enzymatic hydrolysate was centrifuged
and ltered to remove solid residues, and then supplemented with
minerals as follows (g L1 ): 1 (NH4 )2 SO4 , 0.38 MgSO4 7H2 O, 0.10
ZnSO4 7H2 O, 0.15 KH2 PO4 .
The fermentation was then studied in 250-mL Erlenmeyer asks
each containing 100 mL of the enzymatic hydrolysate of XOS waste
residue. CaCO3 was added at 50% of the theoretical amount of glucose derived from the dried material (w/w) to maintain the medium
pH at >6.0 for good cell growth and l-lactic acid production in the
fermentation. After autoclaving at 121 C for 30 min, each ask was
inoculated with the preculture at an inoculation size of 10% (v/v).
Unless otherwise noted, the fermentation was performed at 40 C
in a rotary shaker at 170 rpm for 23 days or until glucose was
depleted or lactic acid production ceased. Unless otherwise noted,
each fermentation condition was studied in duplicate.
2.4. SSF for lactic acid production
The SSF process was studied in 250-mL Erlenmeyer asks with
a 100 mL working volume in a rotary shaker at 170 rpm and 40 C,
unless otherwise noted. The fermentation medium containing the
same inorganic salts as in the SHF medium and XOS waste residue
(5%, 10%, 15%, or 20% w/v), with pH of 5.5, was sterilized by autoclaving at 121 C for 30 min. After cooling, enzymes were added at
the loading rate of 0.06 g g1 biomass, and each ask was then inoculated with the preculture at 10% (v/v). CaCO3 was added at 50% of
the theoretical amount of glucose derived from the dried material
(w/w) after 12 h of fermentation to keep the medium pH at >6.0.
Unless otherwise noted, all batch fermentations were duplicated.
2.5. l-lactic acid production in bioreactor
The SSF process was also studied in a 5-L stirred tank bioreactor (Biostat B, B. Braun) with a rotating brous matrix, made
of a cotton cloth (9 15 0.2 cm) xed on the outer surface of a
perforated stainless steel cup mounted on the impeller shaft, for
cell immobilization [6]. The bioreactor with 3 L of the medium was
sterilized at 121 C for 30 min. After cooling, the bioreactor was
inoculated with 10% (v/v) preculture and operated at 40 C, with
agitation at 200 rpm and aeration at 1.0 vvm. After 12 h, the reactor
pH was maintained at >6.0 by adding CaCO3 solution periodically.
Antifoam 204 from Sigma (0.5 mL per L medium) was added to
prevent foaming during fermentation.
2.6. Analytical methods
The spore concentration was determined by counting the spores
on a haemocytometer under a microscope. Analysis of chemical
composition in XOS waste residues was carried out according to the

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L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 9299

National Renewable Energy Laboratory standard methods for the

determination of structural carbohydrates and lignin in biomass
[18]. For the analysis of fermentation broth samples, 72% (w/w)
sulfuric acid was rst added to neutralize excessive CaCO3 and
acidify calcium lactate. The samples were then heated in boiling water for 5 min to increase the solubility of lactic acid and
deactivate the hydrolytic enzyme and fungus. After centrifugation, the supernatant was analyzed for glucose, xylose, ethanol,
and l-lactic acid with a high performance liquid chromatograph
(HPLC) equipped with an Aminex BioRad HPX-87H column at 45 C,
using 5 mM H2 SO4 as the mobile phase at 0.6 mL min1 . To separate and determine l(+)-lactic acid and d(+)-lactic acid by HPLC,
the SCAS Sumichiral OA-5000 column at 35 C and 1 mM CuSO4
in 5% (v/v) isopropanol at 1.0 mL/min as the mobile phase were
used. The detection was at UV 254 nm. However, no d(+)-lactic acid
was detected, indicating that the R. oryzae strain produced optically
pure l(+)-lactic acid.

3. Results

3.1. SHF of XOS waste residue

3.1.1. Enzymatic hydrolysis of XOS waste residue
Fig. 1 shows the proles of glucose and xylose released from
enzymatic hydrolysis of the waste residue at 5%, 10%, 15%, and 20%
(w/v) substrate loadings. The glucose and xylose titers increased
over time and reached maximum levels in 2460 h, depending on
the solid loading. As expected, more glucose and xylose were produced at higher substrate loadings. For example, increasing the
substrate loading from 5% to 20% also increased the nal glucose
concentrations in the hydrolysates from 31.68 g L1 to 104.97 g L1 .
However, the glucose yield decreased from 87.13% to 72.17% (see
Table 1), probably due to increased solution viscosity, mass transfer limitation, and end-product inhibition [19]. At 5% solid loading,
the enzymatic hydrolysis process was completed in 48 h, with over
30 g L1 glucose and 8 g L1 xylose released from the XOS waste
residue. The conversion yield of cellulose to glucose was 87.1%,
whereas the conversion yield of hemicellulose to xylose was 64.2%,
which could be improved if an endoxylanase with a higher specicity (e.g., Cellic HTec2) was used. No cellobiose accumulation was
observed during the enzymatic hydrolysis process, indicating a
rapid conversion of cellobiose to glucose under the synergism of
cellulase complex. The high enzymatic conversion of XOS waste
residue makes this raw material a promising feedstock for sugarplatform biorenery.

3.1.2. Effect of temperature on l-lactic acid production from

hydrolysates
Usually, a moderate temperature, around 30 C, is required
for fungal cell growth [20], but a higher temperature (>40 C) is
favorable for enzymatic hydrolysis. R. oryzae usually grows at a
temperature between 27 and 35 C [4] and pH 5.06.5 [6,21]. However, it is desirable to perform the fermentation at a temperature
closer to the temperature for enzymatic hydrolysis. Therefore, we
rst studied the effect of temperature on l-lactic acid production
from XOS enzymatic hydrolysates. As shown in Fig. 2, 13.7 g L1 of
l-lactic acid with a yield of 0.274 g g1 waste residue was produced
at 25 C. The l-lactic acid production increased to 15.9 g L1 when
temperature increased to 30 C. However, from 30 C to 40 C, the
l-lactic acid titer and yield did not show any signicant change.
Further increasing the temperature to 45 C resulted in no lactic
acid production, as R. oryzae could not be adapted to grow at the
higher temperature. Thus, 40 C was chosen for the subsequent
fermentation studies.

Fig. 1. Time course proles of sugars released in enzymatic hydrolysis of xylooligosaccharides waste residues at 5%, 10%, 15%, and 20% (w/v) substrate loading.
A: glucose; B: xylose. Data shown are the average with error bar representing the
standard deviation from duplicate runs. Error bar is not visible for some data points
because the standard deviation is smaller than the size of the symbol.

Fig. 2. Effect of temperature on l-lactic acid production from enzymatic

hydrolysates of XOS waste residues by R. oryzae in shake-asks at 5% (w/v) substrate loading. Data shown are the average with error bar representing the standard
deviation from duplicated runs.

L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 9299

95

Table 1
Effects of substrate loading on enzymatic hydrolysis of XOS waste residue.
Substrate loading (%)

Glucose

Xylose
1

Titer (g L
5
10
15
20

31.68
61.32
88.89
104.97

0.00
0.35
0.07
1.12

Yield (%)
87.13
84.32
81.49
72.17

Productivity (g L
0.01
0.49
0.06
0.77

0.66
1.28
1.48
1.75

Titer (g L1 )

0.00
0.01
0.00
0.02

8.10
15.90
23.46
29.38

Productivity (g L1 h1 )

Yield (%)

0.02
0.05
0.39
0.12

64.24
63.06
62.02
58.26

0.16
0.18
1.04
0.23

0.17
0.33
0.39
0.49

0.00
0.00
0.01
0.00

Data shown are the average standard deviation from duplicated runs.

Fig. 3. l-lactic acid fermentation proles of R. oryzae using XOS waste residue hydrolysates as substrate in shake-asks at 40 C and various substrate loadings. A: 5%; B: 10%;
C: 15%; and D: 20% (w/v) loading. Data shown are the average with error bar representing the standard deviation from duplicated runs. Error bar is not visible for some data
points because the standard deviation is smaller than the size of the symbol.

3.1.3. l-lactic acid fermentation of enzymatic hydrolysates

Fig. 3 shows the fermentation proles of R. oryzae grown on XOS
waste residue hydrolysate at various substrate loadings. The fermentation pH was maintained at >6.0 by adding calcium carbonate.
There was not much difference in the pH prole among the different substrate loadings, except for the 20% loading that had a higher
initial pH of 7.5 due to more CaCO3 were added. Nevertheless,
the pH in the range of 5.57.5 should have a minimal effect on the
fermentation [6]. In general, little or no lactic acid was produced
in the rst 12 h, indicating that glucose was mainly used to generate fungal biomass. Thereafter, a rapid consumption of glucose
with fast accumulation of l-lactic acid and ethanol occurred until
all glucose had been consumed, except for the 20% loading, in which
lactic acid production ceased before glucose depletion. During the

fermentation, xylose was also consumed simultaneously with glucose, but at a much slower rate. As can be seen in Fig. 3A, xylose
consumption rate increased signicantly 12 h after glucose depletion, suggesting that xylose uptake by the cells was inhibited or
repressed by glucose [9]. The slower xylose uptake by cells might
also be because xylose transport was much slower than glucose or
was strongly inhibited by glucose. Increasing the substrate loading
from 5% to 15% also increased lactic acid production titer (from
16.5 g L1 to 36.5 g L1 ) and productivity (from 0.34 g L1 h1 to
0.76 g L1 h1 ) (see Table 2). Further increasing the substrate loading to 20% signicantly decreased lactic acid production, which
might be attributed to the limitation in oxygen transfer at the
higher solid loading. The lactic acid yield was 0.330.34 g g1
dry waste residue when the substrate loading was 510%, but

Table 2
Effects of substrate loading on l-lactic acid fermentation by R. oryzae with the enzymatic hydrolysates of XOS waste residues.
Substrate loading (%)

Lactic acid (g L1 )

5
10
15
20

16.53
34.00
36.50
24.44

0.38
0.04
0.15
0.01

Ethanol (g L1 )
5.04
9.50
12.00
15.85

0.45
0.25
0.19
0.71

Data shown are the average standard deviation from duplicated runs.

Lactic acid/ethanol ratio (g g1 )

Lactic acid yield (g g1 )

3.28
3.58
3.04
1.54

0.33
0.34
0.24
0.12

0.01
0.00
0.00
0.00

Lactic acid productivity (g L1 h1 )

0.34
0.71
0.76
0.51

0.01
0.00
0.00
0.00

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L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 9299

decreased signicantly to 0.24 g g1 at 15% loading and 0.12 g g1 at

20% loading. Product (lactic acid) inhibition could adversely affect
microbial accessibility and fungal cell growth, as well as lactic
acid biosynthesis, which might limit lactic acid accumulation to
a higher concentration in the fermentation with higher substrate
loadings. The lower lactic acid production at the higher substrate
loading might also be attributed to the xylose inhibiting lactic
acid production [22]. The fermentation could also be inhibited by
unknown inhibitors produced from the degradation of lignin in the
hydrolysate [23]. The observed diauxic growth with the accelerated
xylose consumption after glucose depletion suggests that xylose
utilization by R. oryzae is regulated by the carbon catabolite repression [24]. The slower xylose uptake by cells might also be because
xylose transport was much slower than glucose or was strongly
inhibited by glucose. Moreover, Maas et al. [25] suggested that more
xylose might be respired to carbon dioxide rather than lactic acid,
and the nutrients were limiting the conversion of xylose.
It is noted that there were signicant amounts of ethanol
co-produced in the fermentation, with the nal titer increased
from 5.0 g L1 at 5% loading to 15.9 g L1 at 20% loading. Ethanol
production suggested insufcient oxygen supply in these shakeask fermentations [26,27]. The lactic acid/ethanol ratio was 3.3
(g g1 ) for 515% substrate loading and 1.5 (g g1 ) at 20% loading. Apparently, 20% loading was more favorable for ethanol
production.
Overall, 10% substrate loading appeared to give the best fermentation results, comparable to previously reported studies on
lactic acid production by R. oryzae from lignocellulosic biomass
in similar SHF processes [12,2225,2730]. Nevertheless, the llactic acid production from enzymatic hydrolysates is a complex
and labor- and time-consuming process. Even the best runs at 10%
loading did not give sufciently high l-lactic acid yield and productivity from the XOS waste residue hydrolysate to be economically
competitive.

Fig. 4. Effect of temperature on l-lactic acid production from XOS waste residues
at 5% (w/v) substrate loading in simultaneous saccharication and fermentation in
shake-asks. Data shown are the average with error bar representing the standard
deviation from duplicated runs.

3.2. SSF of XOS waste residue

3.2.1. Effect of temperature on l-lactic acid production
The SSF process can potentially overcome problems in SHF and
increase l-lactic acid production from XOS waste residues. One
challenge associated with SSF is that the optimal temperatures
and pHs for the saccharication and fermentation processes are
different [31]. It is thus, necessary to determine the optimal temperature for l-lactic acid production in the SSF process. As shown
in Fig. 4, l-lactic acid production increased signicantly in both the
nal titer (from 8.0 g L1 to 25.0 g L1 ) and product yield (from
0.16 g g1 to 0.50 g g1 ) as the temperature increased from 25 C to

Fig. 5. Proles of simultaneous saccharication and fermentation of XOS waste residues by R. oryzae in shake-asks at 40 C and various substrate loadings. A: 5%; B: 10%; C:
15%; and D: 20% (w/v) loading. CaCO3 was added at 12 h, which caused an increase in the pH. Data shown are the average with error bar representing the standard deviation
from duplicated runs. Error bar is not visible for some data points because the standard deviation is smaller than the size of the symbol.

L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 9299

40 C, which might be attributed to the fact that more glucose was

produced from cellulose at the higher temperature in the enzymatic
hydrolysis by cellulase and available for cells to use in the fermentation. However, when the temperature increased to 45 C, no
l-lactic acid was produced, indicating that R. oryzae cells could not
be adapted to survive under this stressful condition. This study conrmed that high-titer lactic acid production can be achieved in a SSF
process at 40 C, a temperature that can improve substrate utilization efciency and reduce production cost by efciently balancing
enzymatic hydrolysis and sugar fermentation processes [20].

3.2.2. l-lactic acid production in SSF process

Fig. 5 shows the fermentation time course data, including glucose, xylose, lactic acid, and ethanol concentration proles, during
the SSF process at different substrate loadings. At 5% substrate loading (Fig. 5A), there was initially a fast accumulation of glucose,
which reached 16 g L1 by 12 h, indicating that the rate of enzymatic hydrolysis was signicantly higher than sugar fermentation
in this period. Thereafter, a rapid decrease in the glucose concentration coupled with a sharp accumulation of l-lactic acid was
observed, indicating that acid production from glucose was faster
than glucose released from cellulose hydrolysis [14]. In fact, the
glucose concentration was kept at near zero since 24 h, suggesting
that all glucose released from cellulose was immediately consumed
in the SSF process. The lactic acid titer reached the maximum
level of 24.9 g L1 at 36 h, achieving a product yield of 0.497 g lactic
acid g1 dry waste residue. Thus, a continuous and simultaneous saccharication and fermentation for l-lactic acid production
from XOS waste residues was realized. Meanwhile, ethanol was
also produced in the SSF, reaching 4.64 g L1 by 24 h. Interestingly,
ethanol production ceased after 24 h, even though lactic acid production continued until 36 h. This was probably because the low
glucose concentration limited cell growth and oxygen uptake by
cells, resulting in a higher oxygen tension that favored lactic acid
biosynthesis. Clearly, the SSF process favored lactic acid production, resulting in a higher lactic acid/ethanol ratio of 5.4 (g g1 ), as
compared to the SHF process.
R. oryzae was also able to ferment pentose for l-lactic acid production [9]. As can be seen in Fig. 5A, the xylose concentration in
the fermentation broth increased to 5 g L1 and remained relatively constant until a signicant decrease was observed after 36 h.
The increased utilization of xylose as carbon source coincided with
the depletion of glucose and cellulose (data not shown). Nevertheless, the utilization of xylose by R. oryzae was not as efcient as
glucose due to catabolite repression and other reasons discussed
earlier [9,22,25].
Since substrate loading plays an important role in the SSF process, we also investigated the fermentation at higher substrate
loadings of 10% (Fig. 5B), 15% (Fig. 5C), and 20% (Fig. 5D). In general, the fermentation proles at 10% loading were similar to those
at 5% loading, but reached a higher lactic acid titer of 60.3 g L1
and yield of 0.60 g g1 dry waste residue with a signicantly higher
productivity (1.0 g L1 h1 vs. 0.69 g L1 h1 ). Further increasing the
substrate loading resulted in a signicant reduction in lactic acid
production, with the nal lactic acid titer dropped to 47.7 g L1 and
yield to 0.32 g g1 waste residue at 15% loading and 33.8 g L1 and

97

0.17 g g1 waste residue at 20% loading, respectively. At the higher

solid loadings of 15% and 20%, sugar consumption seemed to be
greatly reduced, resulting in high concentration levels of glucose
and xylose that were not completely consumed in the fermentation. In fact, xylose consumption was inhibited in the presence of
large amounts of glucose. The poor fermentation performance at
the higher solid loadings can be attributed to increased broth viscosity and reduced oxygen transfer, which not only decreased the
sugar consumption rate but also increased ethanol production, as
indicated by the decreased lactic acid/ethanol ratio at 20% solid
loading (see Table 3). A decrease in oxygen transfer was usually
observed at a higher substrate loading, which was not favorable
for lactic acid production by R. oryzae [6]. It has also been reported
that the fermentation kinetics in the SSF process could be affected
by the reducing sugar concentration in the hydrolysate, as well as
substrate loading, because of increased viscosity and substrate and
product inhibition [11]. In addition, imbalanced osmotic pressure
induced by high sugar concentration was another critical factor that
might have also inhibited cell growth and reduced substrate utilization efciency [32]. Clearly, poor mixing and mass transfer at the
elevated solid loading in shake-asks inhibited the fermentation
with R. oryzae in the SSF process.
Table 3 summarizes and compares the results of SSF at various solid loadings. It is clear that 10% loading was optimal, with
high-titer and high-yield l-lactic acid production from XOS waste
residues in the SSF. Previous studies also found that lactic acid
production from cellulose decreased with increasing the substrate
loading, resulting in a signicant reduction in the product yield and
conversion efciency, even with a prolonged fermentation period
[33,34]. Ruengruglikit and Hang [35] reported that 5% (w/v) was
the optimal loading for corncob as substrate, and a substrate loading below 10% (w/v) was also suggested by Moritz and Duff [36].
Compared to SHF at the same substrate loading of 10%, higher lactic
acid titer (60.3 g L1 vs. 34.0 g L1 ) and yield (0.60 vs. 0.34 g g1 dry
waste residue) were achieved in the SSF process. This can be partially attributed to the fact that lactic acid production became more
favorable than ethanol production in the SSF process, as evidenced
by the much higher lactic acid/ethanol ratio in SSF (10.6 g g1 ) compared to the SHF process (3.6 g g1 ). It is noted that the glucose
inhibition effect on cellulose hydrolysis was alleviated in the SSF
process, which might have also contributed to the higher lactic acid
yield.
3.3. SSF for l-lactic acid production in bioreactor
In order to investigate the scale-up feasibility of the SSF process, fermentation with 10% substrate loading was carried out in
a 5-L stirred tank bioreactor, and the results are shown in Fig. 6.
In general, the fermentation performed well, producing 41.9 g L1
lactic acid with a product yield of 0.42 g g1 dry biomass, which
were lower than those obtained in shake-ask fermentation. The
lower lactic acid production in the stirred-tank bioreactor could be
attributed to the differences in cell morphology, which would affect
mass transfer and lactic acid production [7], and the dissolved oxygen (DO) concentration level. Too much oxygen (high DO) would
promote cell growth and led more substrate into the TCA cycle for

Table 3
Effects of substrate loading on simultaneous saccharication and fermentation for l-lactic acid production from XOS waste residue by R. oryzae.
Substrate loading (%)

Lactic acid (g L1 )

5
10
15
20

24.87
60.29
47.72
33.75

0.10
0.26
0.19
1.01

Ethanol (g L1 )
4.64
5.71
7.28
8.13

0.17
0.32
0.21
1.03

Lactic acid/ethanol ratio (g g1 )

Lactic acid yield (g g1 )

5.36
10.56
6.55
4.15

0.50
0.60
0.32
0.17

Data shown are the average standard deviation from duplicated runs.

0.00
0.00
0.00
0.01

Lactic acid productivity (g L1 h1 )

0.69
1.00
0.66
0.35

0.00
0.00
0.00
0.01

98

L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 9299

Table 4
Comparison of l-lactic acid production from lignocellulosic biomass by R. oryzae.
Strain

Substrate

Titer (g L1 )

Yield (g g1 )

Productivity (g L1 h1 )

References

SHF
R. oryzae HZS6
R. oryzae UMIP 4.77
R. oryzae NRRL 395
R. oryzae NBRC 5378
R. oryzae CBS 112.07
R. oryzae NRRL 395
R. sp. MK-96-1196
R. oryzae NRRL 395
R. oryzae GY 18
R. oryzae NLX-M-1

Corncob
Wheat straw
Waste ofce paper
Wheat straw
Wheat straw
Cassava pulp
Corncob
Wheat bran
Corncob
XOS waste residue

77.2
10
49.1
2
6.8
16.7
26
6

34.0

0.26

0.23
0.24
0.26

0.36
0.34

0.99
0.27
0.51

0.29

0.71

[12]
[22]
[23]
[24]
[25]
[27]
[28]
[29]
[30]
This study

SSF
R. oryzae UMIP 4.77
R. oryzae NBRC 5378
R. sp. MK-96-1196
R. oryzae NRRL 395
R. oryzae NLX-M-1

Paper pulp
Wheat straw
Corncob
Corncob
XOS waste residue

24.1
6
24

60.3

0.23
0.24
0.30
0.60

0.5

1.0

[22]
[24]
[28]
[35]
This study

SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharication and fermentation.

Fig. 6. Proles of simultaneous saccharication and fermentation of XOS waste

residues by R. oryzae at 40 C at 10% (w/v) substrate loading in a 5-L stirred tank
bioreactor. CaCO3 was added at 12 h and periodically thereafter to maintain the pH
above 6.0.

ATP generation and biomass formation, thereby lowering lactic acid

yield. In addition, continuous aeration at a high rate (1 vvm) would
strip away CO2 and ethanol, which could promote more pyruvate
conversion to acetaldehyde and ethanol, thus reducing lactic acid
production. Compared to shake-asks, a relatively high DO and
low pCO2 in the bioreactor with continuous aeration could have
a negative effect on lactic acid production [37]. Further optimization in agitation and aeration rates would be necessary to improve
the fermentation performance. It is noted that the ethanol titer in
the stirred-tank bioreactor was much less than that in asks, only
2.26 g L1 , indicating that some of the produced ethanol was lost as
vapor due to continuous aeration.
4. Discussion
Several studies on l-lactic acid fermentation with lignocellulosic biomass have been reported and are summarized in Table 4
for comparison. For SHF, Park et al. [23] reported the highest lactic acid production of 49.1 g L1 from the enzymatic hydrolysate of
waste ofce paper in a 4-day culture, whereas a moderate lactic acid
yield of 0.36 g g1 corncob was obtained from corncob hydrolysate
[30]. Compared to these SHF studies, comparable lactic acid titer
and yield, but a higher productivity of 0.71 g L1 h1 , were obtained

from XOS waste residues in our study. For SSF, most of the previous studies with R. oryzae produced only up to 24 g L1 of lactic acid
with a moderate yield of 0.230.30 g g1 substrate and a productivity of 0.5 g L1 h1 . On the other hand, our SSF process produced
a high lactic acid titer of 60.3 g L1 with a yield of 0.6 g g1 and
productivity of 1.0 g L1 h1 from XOS waste residues, which were
among the highest ever reported with R. oryzae using lignocellulosic biomass as raw material. It is noted that the lactic acid yield
from XOS waste residues by R. oryzae was comparable to those
obtained from similar lignocellulosic biomass with lactic acid bacteria. Sreenath et al. [15] used Lactobacillus plantarum to produce
lactic acid from alfalfa ber in a SSF process, achieving a yield of
0.61 g g1 dry matter of ber. A coculture of Lactobacillus rhamnosus
and Lactobacillus brevis was used in a SSF process, which produced
lactic acid from corn stover with a yield of 0.70 g g1 cellulose and
hemicellulose [16].
For the same lignocellulosic materials, R. oryzae could produce
more l-lactic acid at a higher nal titer in SSF than in SHF. For example, only 2 g L1 of lactic acid was produced from wheat straw in
SHF, while lactic acid production reached 6 g L1 in SSF [24]. This is
because the inhibition of cellulasecatalyzed reaction by glucose
can be eliminated or greatly alleviated by simultaneously using
glucose in the fermentation, which prevents the accumulation of
glucose generated from the hydrolysis of cellulosic materials in the
SSF process [38]. However, Miura et al. [28] reported that 26 g L1
l-lactic acid was produced from the enzymatic hydrolysate of
corncob, while in the SSF process using a mixed culture of cellulaseproducing Acremonium thermophilus and Rhizopus sp. MK-96-1196
at 35 C, 24 g L1 of l-lactic acid was produced from 100 g L1 of
untreated raw corncob. It is thus, clear that the performance of SSF
will also be dependent on the raw materials used, and each SSF
process will have to be optimized for the conditions used in the
process, including solid loading and agitation and aeration rates.
Further studies including process optimization, lactic acid conversion from xylose and economic analysis will have to be performed
before the process can be commercialized. Better understanding of
the metabolic pathways involved in xylose utilization by R. oryzae is
also needed in order to maximize l-lactic acid production in terms
of nal product titer, yield, and productivity [9].
5. Conclusion
The waste residues from corncobs used in XOS manufacturing
can be used as a low-cost substrate for l-lactic acid production
by R. oryzae. The solid waste residues can be efciently treated

L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 9299

and hydrolyzed with commercial cellulase enzymes to release fermentable sugars, glucose, and xylose, for lactic acid production. A
high cellulose-to-lactic acid conversion and yield can be obtained
in a SSF process with R. oryzae at a relatively high temperature of
40 C. Although lactic acid production in an SSF process has been
demonstrated with starchy materials and lactic acid bacteria [39],
this study is the rst demonstration of SSF for high-titer and highyield l-lactic acid production from lignocellulosic materials by R.
oryzae. The utilization of cheap XOS waste residues for l-lactic acid
production can provide a cost-effective approach for organic acid
production as well as in waste management.
Acknowledgements
This work was supported by the International Advanced
Forestry Technology Introduction Project Funding (Grant No. 20124-18), Natural Science Foundation of Jiangsu Province (Grant No.
BK20131426), Excellent Youth Foundation of Jiangsu Province of
China (BK2012038), the Doctorate Fellowship Foundation of Nanjing Forestry University, Graduate Research Innovation Projects of
Jiangsu Province Ordinary University (CXZZ13 0544), and the Priority Academic Program Development of Jiangsu Higher Education
Institutions.
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