Bioresource Technology 101 (2010) 4959–4964

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Ethanol production from high dry matter corncob using fed-batch

simultaneous saccharification and fermentation after combined pretreatment
Mingjia Zhang a, Fang Wang a, Rongxin Su a,b,*, Wei Qi a,b, Zhimin He a,b
a
State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
b
Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, China

a r t i c l e i n f o a b s t r a c t

Article history: To obtain high concentration of ethanol from cellulose, corncob was pretreated with acid and alkali to
Received 31 August 2009 remove non-cellulose components, and then subjected to simultaneous saccharification and fermenta-
Received in revised form 3 November 2009 tion (SSF). An ethanol concentration as high as 69.2 g/L was achieved with 19% dry matter (DM) using
Accepted 3 November 2009
batch SSF, resulting in an 81.2% overall ethanol yield. A fed-batch process using a high solid concentration
Available online 9 December 2009
was also investigated. Fresh substrate was pretreated with dilute sulfuric acid–sodium hydroxide, and
then added at different amounts during the first 24 h, to yield a final dry matter content of 25% (w/v).
Keywords:
SSF conditions with cellulose loading of 22.8 FPU/g glucan, dry yeast (Saccharomyces cerevisiae) loading
Simultaneous saccharification
and fermentation
of 5 g/L and substrate supplementation every 4 h yielded the highest ethanol concentration of 84.7 g/L
Fed-batch after 96 h. This corresponded to a 79% overall ethanol yield.
Pretreatment Ó 2009 Elsevier Ltd. All rights reserved.
Ethanol
Corncob

1. Introduction Ethanol production from lignocellulose sources such as corncob

The high worldwide demand for energy, the depletion of fossil
fuel reserves, and the concern over global climate change all have
spurred a resurgence of interest in renewable carbohydrate energy
sources (Lynd et al., 2005). Lignocellulose, a waste product from
many agricultural and forestry activities, is a promising carbohy-
drate source for bioconversion to ethanol. Using this abundant
and renewable carbohydrate source in place of fossil fuels is one
of the most effective ways to fight both the energy crisis and envi-
ronmental problems caused by biowaste accumulation and carbon
dioxide emissions (Goldemberg, 2007; Tilman et al., 2006).
In China, nearly 120 million tons of corn (Zea mays L.) is pro-
duced annually from agricultural lands, and results in 20 million
tons of corncob after harvest (Bai et al., 2008). Currently, the hemi-
cellulose component of the corncob has found a use as a source of
high-value health products like xylitol. However, an enormous
quantity of cob residues, rich in cellulose, is still currently dis-
carded as waste. Fully utilizing corncob, or these residues, to pro-
duce biofuels would be a highly attractive option.
* Corresponding author. Address: State Key Laboratory of Chemical Engineering,
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,
China. Tel.: +86 22 27407799; fax: +86 22 27407599.
E-mail address: surx@tju.edu.cn (R. Su).
requires enzymatic hydrolysis of cellulose (by cellulases) to release
sugars (saccharification) that can subsequently be fermented by
yeasts to ethanol. For lignocellulose-based ethanol production to
be economically viable on an industrial scale, the ethanol produced
must be above 4% (v/v) in the fermentation broth (Wingren et al.,
2003). For most types of lignocellulosic materials, this requires
operating at dry mass (DM) concentrations about 15% to achieve
sufficiently high cellulose levels (Jorgensen et al., 2007).
One way to perform this reaction at high dry matter condition is
by a simultaneous saccharification and fermentation (SSF) proce-
dure. Ethanol production by SSF at high dry matter content has
been studied extensively and DM levels of up to 17% have resulted
in production of 52.3 g/L ethanol (Varga et al., 2004). However,
high substrate concentration in the form of fibrous, solid materials
poses two problems: (1) the increased concentrations of inhibitors
such as acetic acid, furfural, and ethanol hamper the performance
of yeast and enzymes and (2) high viscosity results in more power
consumption in the fermentor (Jorgensen et al., 2007) and lowered
mixing and heat transfer efficiency (Georgieva et al., 2008; Jorgen-
sen et al., 2007; Rudolf et al., 2005; Varga et al., 2004). To decrease
these negative effects and to maximize the substrate concentra-
tion, fed-batch SSF might be a better choice.
Another way to obtain ethanol at high concentration is to in-
crease the concentration of the fermentable sugars (hexoses for
Saccharomyces cerevisiae) by improving the availability of cellulose

0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.11.010
4960 M. Zhang et al. / Bioresource Technology 101 (2010) 4959–4964
in the substrate by removing non-cellulosic material by some type
of pretreatment. Due to the robust structure of lignocellulose, a
pretreatment step is essential to disrupt the cell wall macromolec-
ular complexes, to remove hemicellulose and/or lignin, and to in-
crease the surface area accessible to the hydrolytic enzymes
(Mosier et al., 2005; Wyman et al., 2005). Various methods have
been developed to improve biomass fractionation by these pre-
treatment processes, including the use of dilute acid (Saha et al.,
2005), aqueous ammonia (Kim and Lee, 2007), steam explosion
(Ballesteros et al., 2006), wet oxidation (Schmidt and Thomsen,
1998), and hydrothermal treatment (Thomsen et al., 2006). A com-
bination of physical and/or chemical pretreatment methods ap-
pears to be a better approach to both obtain more purified
cellulose content and to enhance digestion. Several combined pre-
treatment technologies have been reported, such as a combination
of sulfuric acid-free EtOH heat treatment followed by mechano-
chemical pulverization, hot-water followed by aqueous ammonia
pretreatment, and H2O2 followed by biological pretreatment (Kim
and Lee, 2006; Teramoto et al., 2008; Yu et al., 2009).
In the present study, corncob was chosen as the raw lignocellu-
losic material. In order to obtain high ethanol concentration, a
combination of acid and alkali pretreatments was used to fraction-
ate the biomass to obtain a substrate with high cellulose content.
This was then used in a fed-batch SSF and the effect of increased
substrate DM on ethanol production was investigated.
2. Methods
2.1. Materials
Corncob was obtained from a local farm (Tianjin, China). It was
premilled and screened, and the fraction between 20 and 80
meshes was used for experiments. All chemicals used were of ana-
lytical grade. Cellulase (GC220, 242 FPU/mL) was purchased from
Genencor International (Palo Alto, CA). Commercial ethanol instant
active dry yeast (S. cerevisiae) was obtained from Angel Yeast Co.,
Ltd., Wuhan, China.
2.2. Pretreatment of corncob
Corncob was pretreated by the different methods described in
Fig. 1.
2.2.1. Soaking in aqueous ammonia (SAA)
Oven-dried corncob was placed in a screw-capped laboratory
bottle (Pyrex glass) and then mixed with 15 wt% aqueous ammonia
at a ratio of 1 g solid per 6 mL liquid. The solid/liquid slurry was
incubated in a water bath at 60 °C for 12 h, with no agitation. After
SAA, the solids were separated by filtering and washed with dis-
tilled water until neutral pH was attained. The solid residue was
dried in a forced-air oven at 105 °C.
Fig. 1. Procedures of different pretreatment methods.
2.2.2. Formic acid/aqueous ammonia pretreatment (FA–AA)
Corncob was treated by mixing with formic acid at a ratio of 1 g
solid per 6 mL liquid and heated at 60 °C for 6 h. After soaking, the
solids were separated by filtering, washed with tap water until
neutral, and then treated by the SAA treatment described above.
2.2.3. Dilute sulfuric acid–sodium hydroxide pretreatment
(H2SO4–NaOH)
Corncob was treated with 2% H2SO4 at a ratio of 1 g solid per
10 mL liquid and heated at 121 °C for 45 min. After treatment,
the solids were separated by filtering, washed with tap water until
neutral and then treated with 2% NaOH at 80 °C at a ratio of 1 g ori-
ginal raw material to 6 mL liquid. The solids were separated by fil-
tering and washed with tap water until neutral. The solid residue
was dried in a forced-air oven at 105 °C.
2.3. SSF-experiments
2.3.1. Batch mode
SSF-experiments were performed in a 6.0 L-reactor (Biostat B.
Braun Biotech International, Melsungen, Germany). The slurry of
pretreated corncob was diluted with water to the desired initial
DM concentration (7.5–19%, w/v). The pH of the medium was con-
trolled at 4.8 during the cultivation by addition of 3 M NaOH. The
reactor containing the diluted slurry was autoclaved at 121 °C for
30 min. The fermentation medium was supplemented with nutri-
ents: (NH4)2HPO4 0.5 g/L, MgSO47H2O 0.025 g/L and yeast extract
1.0 g/L. The experiments were started by adding enzymes (GC220)
30 FPU/g cellulose and prehydrolyzed for 24 h at 50 °C. The tem-
perature was reduced to 37 °C and the dry yeast was added (5 g/
L), converting the saccharification process into a SSF. The time of
yeast addition was referred to as time 0. The experiments were
run for another 96 h. Samples were collected after 0, 1, 2, 4, 8,16,
24, 48, 72, and 96 h, and analyzed for ethanol, sugars, acetic acid
and sugar degradation products. After SSF, the residue solid was
washed, filtered, dried and weighed.
2.3.2. Fed-batch mode
The fed-batch SSF experiments were started with an initial 19%
DM. Another 6% DM was added in 3, 6, and 12 portions during the
first 24 h, giving a final DM concentration of 25%. The fermentation
conditions, enzyme loadings, and nutrient concentrations were the
same as in the batch experiments. Cellulase loading was 22.8 FPU/g
glucan.
2.4. Analysis
The components of corncob before and after pretreatment were
determined according to the standardized methods of the National
Renewable Energy Laboratory (NREL, Golden, CO, USA) (Sluiter
et al., 2004). The glucose, xylose, acetic acid, ethanol content, and
 M. Zhang et al. / Bioresource Technology 101 (2010) 4959–4964 4961

sugar degradation products (HMF and furfural) were determined of corncob increased from 31.0% to 45.7%, concomitant with de-
by high performance liquid chromatography (HPLC) using an creases in lignin content. Acid treatment prior to alkali treatment
Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 65 °C further decreased the non-cellulosic hemicellulose and lignin com-
with 0.005 mol/L H2SO4 as the mobile phase at a flow rate of ponents in the corncob. The FA–AA or H2SO4–NaOH treatments
0.6 mL/min. resulted in substrates with 82.9% and 91.1% cellulose, respectively.
Therefore, the combination of acid and alkali pretreatments were
2.5. Yield calculations effective for the removal of hemicellulose and lignin, resulting in
high cellulose contents in substrate, which was facilitated for the
2.5.1. Hydrolysis yield subsequent SSF operating at high cellulose content to obtain high
The hydrolysis yield of cellulose was calculated by: ethanol concentration. Because corncob was treated by alkali after
acid treatment, no furfural and HMF were detected by HPLC (data
CelluloseS ðgÞ  CelluloseR ðgÞ not shown).
Y cellulose ð%Þ ¼  100
CelluloseS ðgÞ

3.2. Batch SSF using corncob after different pretreatments

2.5.2. Ethanol yield
The ethanol yield (YEthanol) was calculated assuming that 1 g of
glucose present in the liquid would theoretically give 0.511 g of
ethanol and 1 g of cellulose give 1.11 g of glucose. The ethanol
yield (YEthanol) was calculated according to:
Y Ethanol ð%Þ
EthanolProduced ðgÞ  100
¼
CelluloseS ðgÞ  CelluloseR ðgÞ  0:568  GlucoseR ðgÞ  0:511
where CelluloseS (g) is the amount of cellulose in the substrate, Cel-
luloseR is the amount of cellulose in the residual solid after SSF (g),
determined after strong acid hydrolysis, and GlucoseR is the unfer-
mented glucose in the liquor after SSF, determined directly by HPLC
(Varga et al., 2004). The factor of 0.568 is the theoretical conversion
factor for ethanol from cellulose by S. cerevisiae (Varga et al., 2004).
3. Results and discussion
3.1. Components of corncob after different pretreatments
The chemical components of corncob following different pre-
treatments are shown in Fig. 2. After SAA, the cellulose content
100
Untreated
SAA
80
Component content (%)
Table 1 presents the operating conditions and results of the
batch SSF of corncob. Fig. 3 shows the changes in the concentra-
tions of glucose, xylose, and ethanol during the batch SSF under
the four conditions corresponding to the A, B, C, and D modes pre-
sented in Table 1.
Table 1 shows that a combination of acid and alkali pretreat-
ments aided in the final ethanol production by SSF. The SAA pre-
treatment gave a substrate of relatively low cellulose content
that produced only 12.3 g/L ethanol. Pretreatment of corncob with
acid and alkali increased ethanol production to 29.4 g/L for the FA–
AA pretreatment.
The purpose of the pretreatment was to remove lignin and/or
hemicellulose, to disrupt the crystalline structure of cellulose,
and to increase the porosity of the materials, making the materials
more accessible to enzyme attack (Chen et al., 2008; Sun and
Cheng, 2002). Among the pretreatment methods, dilute acid pre-
treatment has been widely studied because it is effective and inex-
pensive. The dilute sulfuric acid pretreatment has been shown to
effectively solubilize and hydrolyze hemicellulose into monomeric
sugars and soluble oligomers, removing it from the cellulose fibers
(Lu et al., 2007). In these experiments, when corncob was treated
with formic acid or dilute sulfuric acid, the hemicellulose content
decreased by 83.2% and 66.8%, respectively.
High ethanol production depends on a high cellulose concentra-
tion. Increases in cellulose content are best affected by removal of
non-cellulose components by pretreatment. The presence of lignin
restricts the access of cellulase to cellulose preventing saccharifica-

FA-AA
tion (Mussatto et al., 2008). Under alkaline conditions, lignin is re-
H2SO4-NaOH
moved from lignocellulose. In this study, acid-treated corncob was
60 subsequently delignified by dilute sodium hydroxide pretreat-
ment, which reduced the lignin content by 81.0%. At the same time,
the cellulose content of the material increased proportionally by
40 53.8%.
The density of the substrate increased after the acid/alkali pre-
treatment but there was no increase in the viscosity of the slurry.
20
Thus, the substrate mixture was easier to stir, even when operated
at high DM concentration. When SSF was carried out at a DM of
0 19%, 62.7 g/L ethanol was obtained, which was almost 2.2-fold
Cellulose Hemicellulose Lignin higher than that obtained with samples that were not pretreated
(Table 1 B and C). A high ethanol concentration (69.2 g/L) was
Fig. 2. Chemical components of corncob after different pretreatments. obtained, corresponding to an 81.2% overall ethanol yield, using

Table 1
SSF conditions and ethanol production parameters for different pretreatment and DM concentrations.

Label Pretreatment DM (%) Enzyme loadings (FPU/g glucan) Ethanol conc. (g/L) YEthanol (%) YCellulose (%)
A SAA 7.5 30 12.3 67.8 93.0
B FA–AA 7.5 30 29.4 89.7 93.2
C FA–AA 19 30 62.7 77.3 90.8
D H2SO4–NaOH 19 30 69.2 81.2 86.0
4962 M. Zhang et al. / Bioresource Technology 101 (2010) 4959–4964

50 50
Glucose a Glucose
Xylose Xylose
b
40 40
Ethanol Ethanol
Concentration (g/L)

Concentration (g/L)
30 30

20 20

10 10

0 0
0 20 40 60 80 100 0 20 40 60 80 100
Time (h) Time (h)

100 100
Glucose Glucose
Xylose c Xylose
d
80 80
Ethanol Ethanol
Concentration (g/L)

60 Concentration (g/L) 60

40 40

20 20

0 0
0 20 40 60 80 100 0 20 40 60 80 100
Time (h) Time (h)

Fig. 3. The concentrations of ethanol (h), glucose (j), xylose (N) during SSF experiments for corn cobs at 7.5% dry matter after different pretreatment methods. (a) SAA
pretreated corncob, (b) FA–AA pretreated corncob, (c) FA–AA pretreated corncob, and (d) H2SO4–NaOH pretreated corncob.

H2SO4–NaOH treated corncob, because strong acid–strong alkali
removed more non-cellulosic material and caused a greater disrup-
tion of biomass structure. Cellulose conversion was decreased
slightly to 86% because of strong inhibition by ethanol and by-
products such as organic acids.
3.3. Fed-batch SSF
In order to increase the final substrate concentration while
avoiding increases in viscosity, fed-batch culture was used in the
SSF process.
Based on the initial DM concentration of 19%, an additional 6%
of substrate was added during the first 24 h. The operating condi-
tions and results are shown in Table 2. Although the conversion of
cellulose decreased slightly to 80.1–82.3%, the final ethanol con-
centrations after 96 h increased to more than 80 g/L in all fed-
batch experiments. No effect of substrate addition time was noted
on the final ethanol concentration, in agreement with previous re-
sults reported for 1.5, 3 and 5 h addition times (Rudolf et al., 2005).
Among the three experiments in the present study, the experiment
‘‘B” was somewhat better than the others (Table 2), with a final
ethanol concentration of 84.7 g/L. This corresponded to an ethanol
yield of 79.6% and a cellulose conversion of 82.3%.
As shown in Figs. 3 and 4, the glucose concentrations during the
batch and fed-batch fermentations decreased rapidly in the first
16 h and did not exceed 1.0 g/L after 24 h. No significant change
in xylose concentrations was found since this pentose is not
metabolized by S. cerevisiae.
A high dry matter content in the SSF process resulted in high
viscosity and uneven slurry distribution in the reactor, so the ac-
tual ethanol yield was far less than the theoretical yield. Use of
the fed-batch mode could alleviate this technical problem by add-
ing fresh substrate only when the viscosity had been decreased
after several hours of reaction. In this study, fed-batch SSF with
25% DM, obtained with acid/alkali pretreated corncob, produced
the highest amount of ethanol, at 84.7 g/L, corresponding to a
79.6% ethanol yield based on the theoretical value. However, the
lower substrate concentration (19% DM) also gave a favorable

Table 2
SSF conditions and ethanol production parameters by fed-batch mode.

Label Pretreatment DM (%) Interval adding time (h) Enzyme loadings (FPU/g glucan) Ethanol conc. (g/L) YEthanol (%) YCellulose (%)
A H2SO4–NaOH 19 + 6 8 22.8 80.2 76.8 80.1
B H2SO4–NaOH 19 + 6 4 22.8 84.7 79.6 82.3
C H2SO4–NaOH 19 + 6 2 22.8 83.3 79.3 81.3
 M. Zhang et al. / Bioresource Technology 101 (2010) 4959–4964 4963

100 paring corncob for ethanol production when added at high concen-
tration to a SSF setup. Pretreatment removed most of the non-
Glucose a cellulosic materials, making cellulose more accessible to enzymes
Xylose
80 that convert it into fermentable sugars. The density of the sub-
Ethanol
Concentration (g/L)

strate, but not its viscosity, increased after pretreatment, which al-
lowed SSF to be performed at high DM concentrations.
60
The fed-batch strategy can alleviate the high viscosity problem
caused by high DM concentration, which is essential for generation
40 of high ethanol amounts. After a prehydrolysis at 50 °C in a fed-
batch process (with a cellulase loading of 22.8 FPU/g cellulose),
the final dry matter in the experiments could be increased up to
20 25%. Ethanol concentrations of up to 84.7 g/L could be produced
from a DM content of 25% by adding fresh substrate every 4 h dur-
ing the first 24 h of SSF. To our knowledge, it is the highest ethanol
0 concentration yet reported and exceeds the technical and eco-
0 20 40 60 80 100 nomic limits of industrial-scale alcohol distillation.
Time (h) Approximately 90% of the ethanol was produced during the first
48 h of the fermentation. From a process-economy aspect, it may
100 be desirable to further shorten this residence time. It seemed that
Glucose a longer residence time was required at higher DM concentrations,
b i.e., the hydrolysis yield was lower. This might be due to some
Xylose
80 inhibitory effects; e.g., an increase in end-product inhibition as
Ethanol
Concentration (g/L)

the sugar uptake rate was lower, but it could also be a reflection
of less efficient mass transport (Sassner et al., 2006).
60

Acknowledgements
40
The authors acknowledge the financial supports received from
the National Natural Science Foundation of China (No. 20976130
20 and 20806057), the Ministry of Science and Technology, P. R. China
(Nos. 2007BAD42B02 and 2006DA62400), and the Ministry of Edu-
0 cation (Nos. IRT0641 and B06006).
0 20 40 60 80 100
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