Bioresource Technology 222 (2016) 431–438

Contents lists available at ScienceDirect

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

Cost-effective simultaneous saccharification and fermentation of L-lactic

acid from bagasse sulfite pulp by Bacillus coagulans CC17
Jie Zhou a,b, Jia Ouyang a,c,⇑, Qianqian Xu a, Zhaojuan Zheng a,b
a
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, China
b
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
c
Key Laboratory of Forest Tree Genetics and Genetic Engineering, Nanjing Forestry University, Nanjing 210037, China

h i g h l i g h t s

SSF demonstrated the remarkable advantage over SHF from BSP by strain CC17.

SSF by strain CC17 could lower about 33.3% fungal cellulase dosage over SHF.

Strain CC17 could convert cellobiose to lactic acid without exogenous b-glucosidase.

110 g/L L-lactic acid was obtained in the fed-batch SSF of BSP by strain CC17.

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

Article history: The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes
Received 26 July 2016 and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic
Received in revised form 27 September Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of
2016
bagasse sulfite pulp (BSP) to produce L-lactic acid. Unexpectedly, SSF by CC17 required approximately
Accepted 29 September 2016
Available online 1 October 2016
33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly,
CC17 can co-ferment cellobiose and xylose without any exogenous b-glucosidase in SSF. Moreover, add-
ing xylanase could increase the concentration of lactic acid produced via SSF. Up to 110 g/L of L-lactic acid
Keywords:
Lactic acid
was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72 g/g cellulose. These results sug-
Bacillus coagulans gest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-
Simultaneous saccharification and efficient fermentation process can be established using this protocol.
fermentation Ó 2016 Elsevier Ltd. All rights reserved.
Bagasse sulfite pulp
Enzyme dosage

1. Introduction interest in lactic acid production (Abdel-Rahman et al., 2013). To

During the last few decades, biological refining chemicals have
drawn global concern due to the environmental impact and energy
stocks (Sanders et al., 2007). Many platform chemicals had been
produced via bioprocesses instead of petrochemical processes,
such as ethanol (Fan et al., 2003), lactic acid (Gao et al., 2011),
fumaric acid (Goldberg et al., 2006), xylonic acid (Toivari et al.,
2012), and other value-added products (Sánchez, 2009). Among
them, lactic acid is an important bio-product due to its high yield
of 0.9 g/g glucose and widespread applications (John et al., 2007).
Specifically, the recent demand for biodegradable and biocompat-
ible poly-lactate polymers (PLA) sharply increased the global
⇑ Corresponding author at: College of Chemical Engineering, Nanjing Forestry
University, Nanjing 210037, China.
E-mail address: hgouyj@njfu.edu.cn (J. Ouyang).
date, the successfully realized commercial production of lactic acid
has involved the utilization of pure sugars or edible crops (Okano
et al., 2010). However, due to shortage of such raw materials, the
high production cost of lactic acid has hindered the larger-scale
application of PLA (Abdel-Rahman et al., 2013; Hu et al., 2015).
Therefore, due to concerns over feedstock costs and the limited
worldwide food availability, lignocellulosic resources have become
attractive and inexpensive raw materials for the production of lac-
tic acid (Neureiter et al., 2004; Zhu et al., 2007).
Although raw materials derived from biomasses are inexpen-
sive, abundant and renewable, several technical barriers to the
use of lignocellulosic materials for lactic acid production still
remain. Due to the complex cell-wall structure of lignocellulose,
lignocellulosic biomasses require pretreatment and saccharifica-
tion for effective lactic acid production (Ouyang et al., 2013a;
Zhang et al., 2014). Undoubtedly, these processes increase the cost

http://dx.doi.org/10.1016/j.biortech.2016.09.119
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
432 J. Zhou et al. / Bioresource Technology 222 (2016) 431–438
of lactic acid production. Moreover, the enzymes involved in enzy-
matic saccharification are generally inhibited by glucose and cel-
lobiose, which leads to a higher cost of the enzymes required for
the process (Nguyen et al., 2013). Thus, to overcome these prob-
lems, much effort has been made to expended an efficient and
cost-effective process for lactic acid production. Compared with
separate hydrolysis and fermentation (SHF), simultaneous saccha-
rification and fermentation (SSF) is regarded as a more promising
method for bioconversion of biomass. During the SSF process, the
fermentable sugars produced by saccharification can be directly
fermented into products in a single vessel, thereby minimizing
the extent of end-product inhibition and decreasing the processing
period (John et al., 2009; Suriyachai et al., 2013; Zhang et al., 2014).
Marques et al. (2008) obtained 72.90 g/L of lactic acid from recy-
cled paper sludge during 168 h of SSF by Lactobacillus rhamnosus
ATCC 7469.
The major difficulty in the SSF process is the mismatch between
the optimal temperatures for saccharification and fermentation.
Most strains, such as Rhizopus oryzae (Huang et al., 2005;
Marques et al., 2008), Lactobacillus delbrueckii subsp. bulgaricus
and Lactobacillus casei (John et al., 2006) perform fermentation at
30–40 °C. However, the optimal temperature for saccharification
is 50 °C. Over the last 15 years, Bacillus coagulans has been demon-
strated to be a suitable microorganism for the efficient conversion
of lignocellulosic biomass into lactic acid. Many reported B. coagu-
lans strains can not only ferment xylose to lactic acid via the pen-
tose phosphate pathway (Patel et al., 2006; Ye et al., 2013), but also
show a robust tolerance of inhibitors (Ouyang et al., 2012). B. coag-
ulans is a thermophilic bacterium with an optimal growth temper-
ature of 50 °C (Budhavaram and Fan, 2009; Ma et al., 2014).
Therefore, this bacterium is an attractive candidate for performing
SSF. B. coagulans strain 36D1 was first applied to the production of
lactic acid from Solka Floc pure cellulose through SSF by Patel et al.,
who obtained a yield rate of approximately 90% (Patel et al., 2005).
However, further studies of this procedure must be conducted
using a more complex lignocellulosic biomass.
Bagasse sulfite pulp (BSP) is produced by the traditional sulfite
pulping process by the pulp and paper industries. Our previous
studies showed that BSP is a potential cellulosic feedstock and that
it has a good hydrolysis performance (Ouyang et al., 2013a). In this
report, we describe a SSF process for lactic acid production from
BSP using the B. coagulans strain CC17. Furthermore, to minimize
the enzyme costs, the enzymatic components and the loading were
optimized. A cost-effective SSF process for lactic acid production
from BSP using the B. coagulans strain CC17 was established.
2. Materials and methods
2.1. Materials, enzymes, and strain
BSP was kindly provided by the Jiangmen Sugar Cane Chemical
Co., Ltd. (Jiangmen, China). The material was washed with distilled
water to remove inhibitors and sulfite groups, filtered and then
stored in sealed plastic bags at 4 °C (Ouyang et al., 2013a).
Cellulase (Celluclast 1.5 L), b-glucosidase (Novozyme 188) and
xylanase (Pentopan Mono BGÒ) were purchased from Sigma-
Aldrich (Germany).
B. coagulans strain CC-17 was deposited in our lab and used in
this study (Zheng et al., 2014).
2.2. Enzymatic hydrolysis of BSP
Enzymatic hydrolysis of BSP was performed in 50 mM citric
acid buffer (pH 4.8) on a rotary shaker set to 50 °C and 150 rpm.
The total working volume was 50 mL in 150-mL Erlenmeyer flasks.
For all of the hydrolysis experiments, the dosages of cellulase (Cel-
luclast 1.5 L) and b-glucosidase (Novozyme 188) were 15 FPIU/g
cellulose and 15 pNPGU/g cellulose, respectively. At specific time
intervals, aliquots were withdrawn and were centrifuged to
remove the insoluble materials. The supernatants were subse-
quently filtered through a 0.22-lm syringe filter and were used
for subsequent analysis. All experiments were performed in dupli-
cate. The cellulose conversion rate was calculated according to the
following equation:
ðCellobiose  1:05 þ GlucoseÞ  0:05  0:9
Cellulose conversion ¼
Cellulose
 100%
where 0.05 is the total volume, 0.9 is the rate of conversion of cel-
lulose to glucose, and 1.05 is the rate of conversion of cellobiose to
glucose.
2.3. Fermentation
2.3.1. Preparation of inoculums
B. coagulans strain CC17, which was maintained in glycerol in
vials at 80 °C, was plated on an agar plate to grow. A loop of cells
collected from a fully populated plate was inoculated into a 150-
mL flask containing 50 mL of the seed culture medium. The seed
culture medium contained the following (g/L): glucose 20, corn
steep powder 2.5, yeast extract 1, NH4Cl 1, MgSO4 0.2, and
CaCO310. The culture was incubated for 14 h at 50 °C and
150 rpm. The initial pH value of the culture was adjusted to 7.2.
The grown culture was used as the seed culture for the fermenta-
tion experiments.
2.3.2. SSF and SHF in batch mode
SSF was conducted in a 250-mL Erlenmeyer flask containing
100 mL of fresh medium. The medium contained different amounts
of BSP, 1.2 g/L corn steep powder, 2.5 g/L yeast extract, 3 g/L
(NH4)2SO4, 0.22 g/L KH2PO4, 0.4 g/L MgSO47H2O, 0.03 g/L FeSO4-
7H2O, and 0.03 g/L MnSO4H2O, and was not sterilized. Before fer-
mentation, various amounts of cellulase, b-glucosidase and
xylanase were added to the broth to obtain the desired enzyme
loadings. CaCO3 (at 1/2 of the cellulose in the BSP, by weight)
and the culture inoculum (10% (v/v)) were added to the SSF med-
ium, the initial pH value of which was approximately 5.6. The pH
value during the fermentation process was controlled by CaCO3
automatically and could be maintained between 5.0 and 5.5.
SHF was conducted in a 150-mL Erlenmeyer flask containing
50 mL of hydrolysate, CaCO3 (at 1/2 of cellulose in the BSP, by
weight), culture inoculum (10% (v/v)). The fermentation basal
medium used for SHF was the same as that used for SSF except
the added enzymes and BSP. The hydrolysate used for SHF was pre-
pared by enzymatically hydrolyzing BSP for 72 h. All the fermenta-
tions were conducted on a rotary shaker at 50 °C and 150 rpm.
Samples were collected periodically to determine the concentra-
tions of L-lactic acid and residual sugars. All experiments were per-
formed in duplicate.
2.3.3. Fed-batch SSF
The fed-batch SSF experiments were conducted with an initial
loading of 30 g/L (with a cellulose content that was the same as
stated below) of BSP and 10% (v/v) of culture inoculum in a total
volume of 100 mL. The feeding time was determined according
to the extent of BSP liquefaction. 3 g cellulose of BSP was added
for 7 times during the first 48 h (at 3, 6, 9, 12, 24, 36, and 48 h),
and the final cellulose concentration was 153.30 g/L. At the begin-
ning of the experiment, 10 FPIU/g cellulose of cellulase, 120 IU/g
hemicellulose of xylanase and 12 g of calcium carbonate were
 J. Zhou et al. / Bioresource Technology 222 (2016) 431–438 433

added to the medium. The fermentation basal medium and the 15 60

conditions were generally identical to those of the batch experi- xylose SHF

Residul sugar concentration (g/L)

SSF

Lactic acid concentration (g/L)

glucose
ments. All experiments were performed in duplicate. 12 cellobiose 50
SHF
SSF
2.4. Analytical methods SHF
40
9

The concentrations of sugars and organic acids in the samples 30

were determined using a high-performance liquid chromatography
system (HPLC, Agilent Technology 1200 series, Germany) equipped
with a Bio-Rad Aminex HPX-87H column (300  7.8 mm) and a
refractive index detector. The mobile phase was 5 mM sulfuric acid
provided at a flow rate of 0.6 mL/min. The column temperature
was maintained at 55 °C. The samples were filtered through
0.22-lm syringe filters prior to injection (Ouyang et al., 2013b;
Zhou et al., 2014).
In the present study, the yield of lactic acid yield from cellulose
was calculated based on the total (initial and added cellulose con-
tent) cellulose concentration in the BSP. The yields of lactic acid
yield from cellulose and hemicellulose were calculated using the
equation shown above.
3. Results and discussion
3.1. Enzymatic hydrolysis and comparison of SHF and SSF by B.
coagulans
The composition of the BSP that was used was found to be
73.84% cellulose, 14.33% hemicellulose and 5.93% lignin by dry
weight, as determined using NREL methods (Sluiter et al., 2006).
To evaluate its hydrolysis performance, enzymatic hydrolysis
experiments were first conducted using various substrate cellulose
loadings and an enzyme cocktail containing 15 FPIU/g cellulose
Celluclast 1.5 L and 15 pNPGU/g cellulose Novozyme 188.
Table 1 summarized the sugar concentrations in BSP after 72 h
of hydrolysis. When the cellulose loading of BSP was less than
100 g/L, the enzymatic hydrolysis yield was greater than 70%, sug-
gesting that BSP was a substrate that was easily degraded through
SSF. When the cellulose loading of BSP was increased to 80 g/L, cel-
lobiose was found to be greatly accumulated. The high level of
accumulated cellobiose indicated that the b-glucosidase concen-
tration in the enzyme cocktail might be insufficient. Enzymatic
hydrolysis was limited by the rate of conversion of cellobiose to
glucose (Andrić et al., 2010). Additionally, the existence of xylose
indicated that the enzyme cocktail might have xylanase activity.
In previous studies, Yoshida also found that pentose could be pro-
duced using Celluclast 1.5 L, demonstrating that Celluclast 1.5 L
has hemicellulose-degradative activity (Yoshida et al., 2008).
SHF and SSF of BSP were conducted at 60 and 80 g/L cellulose
loading with the same enzyme loadings and fermentation condi-
tions. As shown in Table 1, approximately 58.68 g/L and 68.98 g/L
of fermentable sugars (those containing glucose, xylose and cel-
lobiose) were obtained from 60 and 80 g/L cellulose loading of
BSP, respectively, after 72 h of enzymatic hydrolysis. However,
the subsequent fermentation process produced only 32.22 g/L
and 50.54 g/L of lactic acid, respectively (Fig. 1). Residual sugar
analysis showed that glucose was consumed rapidly, whereas
6
20
3
10
SSF
0 0
60 80 60 80
Cellulose content of BSP (g/L)
Fig. 1. Residual sugars and L-lactic acid concentrations after 72 h hydrolysis + 24 h
fermentation with 60 g/L BSP in SHF, 72 h hydrolysis + 48 h fermentaion with 80 g/L
BSP in SHF, and 72 h SSF with both BSP dosage. The enzyme loading of cellulase and
b-glucosidase were 15 FPIU and 15 pNPGU/g cellulose.
xylose and cellobiose clearly accumulated during SHF. These
results demonstrated that the typical carbon catabolite repression
(CCR, glucose repression) occurred during SHF by B. coagulans.
With 60 g/L cellulose loading of BSP, approximately 1.69 g/L of cel-
lobiose and 7.08 g/L of xylose remained after fermentation,
whereas 99.44% of the glucose was utilized by the bacterium in
24 h. In contrast, the SSF experiments demonstrated the remark-
able advantage of SSF over SHF. After 72 h of SSF of 60 g/L cellulose
loading of BSP, only 0.80 g/L of xylose and 0.45 g/L of glucose
remained. This result suggested that almost all of the fermentable
sugars had been converted to lactic acid via SSF in that period. This
phenomenon was also reported by Bertilsson et al. (2009). Com-
pared with the SHF process, SSF led to the reduction of end-
product inhibition by the cellobiose and glucose that were pro-
duced during enzymatic hydrolysis (Kang et al., 2012; Watanabe
et al., 2012). Additionally, in the present study, the slow rate of glu-
cose release during SSF appeared to favor xylose utilization and the
implementation of co-fermentation. Approximately 50.20 g/L lactic
acid was produced during 72 h of SSF at 60 g/L cellulose loading of
BSP. Therefore, SSF not only shortened the processing time by
25.00% but also increased the lactic acid concentration by 43.73%.
SSF at 80 g/L cellulose loading of BSP produced only 55.04 g/L of
lactic acid. Moreover, xylose accumulation was observed at 72 h
of fermentation. The fermentation broth was overly viscous during
the initial stage of fermentation, which was not conductive to the
growth of the strain used. Therefore, the fermentation rate was
limited by this substrate loading and the advantage of SSF could
not be manifested. Considering this problem, 60 g/L was selected
as the optimized substrate cellulose concentration for the follow-
ing SSF experiments.
3.2. Effect of enzyme mixture loading on lactic acid production via SSF
3.2.1. Cellulase
The cost of cellulase is a significant challenge for the commer-
cial conversion of lignocellulosic biomass into renewable chemi-

Table 1
Summary of enzymatic hydrolysis at different substrate cellulose loading.

Cellulose content (g/L) Cellobiose (g/L) Glucose (g/L) Xylose (g/L) Cellulose conversion (%)
40 2.17 ± 0.31 32.36 ± 0.96 6.89 ± 0.23 77.93 ± 2.88
60 3.51 ± 0.02 45.18 ± 0.43 9.99 ± 0.13 73.14 ± 0.68
80 6.31 ± 0.40 58.93 ± 1.08 12.45 ± 0.41 73.76 ± 1.68
100 7.95 ± 0.58 66.41 ± 0.60 14.24 ± 0.21 67.28 ± 1.09

The enzyme loading of Cellulase and b-glucosidase were 15 FPIU and 15 pNPGU/g cellulose.
434 J. Zhou et al. / Bioresource Technology 222 (2016) 431–438

cals (Ou et al., 2009). Two main parameters affect enzyme cost: the
dosage of enzyme needed and the stability of enzymatic activity.
Rodrigues had found that the enzymatic activity of cellulase (Cellu-
clast) reduced in the first 24 h, but it kept stable throughout the
entire process at 50 °C in wheat straw hydrolysis and fermentation
(Rodrigues et al., 2014). Therefore, in this study, only the effect of
the cellulase dosage on lactic acid production was investigated to
determine the optimal cellulase loading and minimize the produc-
tion costs.
SSF was conducted at 60 g/L cellulose loading of BSP, and the
ratio of cellulase and b-glucosidase loading was 1:1 (Table 2). As
shown in Fig. 2A, whatever the cellulase loading was, the lactic acid
concentration increased rapidly during the first 12 h of SSF. How-
ever, thereafter, the lactic acid production rate significantly declined
when the cellulase loading was 5 FPIU/g cellulose, resulting in a low
final lactic acid concentration. When the cellulase loading was
increased from 5 to 10 FPIU/g cellulose, the lactic acid concentration
increased from 34.32 g/L to 45.96 g/L in 72 h of SSF. This result may
be due to the low enzymatic hydrolysis rate at lower cellulase load-
ing limiting lactic acid production. Further increase in the cellulase
loading did not increase the lactic acid concentration. Thus, the opti-
mal cellulase loading for SSF was 10 FPIU/g cellulose. During the SSF
process, the fermentable sugars obtained from enzymatic hydroly-
sis could be immediately fermented to produce chemicals, consider-
ably diminishing their inhibitory effect on the cellulase system,
which contributed to improving the efficiency of hydrolysis. More-
over, the strain which can match the optimum conditions for fungal
cellulase activity is a better choice to lower the loading of cellulase
in SSF than other microbial biocatalyst (Ou et al., 2009). Therefore,
performing SSF using B. coagulans allowed the use of a lower cellu-
lose dosage than that used for SHF. By using the thermophilic B.
coagulans CC17 strain, the cellulase loading for SSF of BSP could be
reduced to 10 FPIU/g cellulose. Compared to 15 FPIU/g cellulose
used in SHF, SSF by strain CC17 could lower about 33.33% of fungal
cellulase dosage.
Fig. 2B–D showed the profiles of fermentable sugar accumula-
tion in the supernatant during the SSF process. In Fig. 2B, glucose
accumulated during the first 6 h of SSF and then was rapidly
consumed. With the increase in cellulase loading, the level of glu-
cose present at 6 h of SSF was increased and the exhaust period of
glucose increased from 12 h to 48 h. As for xylose consumption,
Fig. 2C showed that xylose could be exhausted and the co-
fermentation of glucose and xylose appeared to occur under the
lower cellulase loading condition. However, when the cellulase
loading was greater than 10 FPIU/g cellulose, xylose accumulated,
reaching 8.21 g/L at 72 h of fermentation. Correspondingly, in
Fig. 2B, the glucose content reached 18.42 g/L during the first 6 h
of fermentation. This result indicated that xylose consumption
was also strongly inhibited by CCR even during SSF when the cel-
lulase loading was greater than 10 FPIU/g cellulose. CCR occurs in
many bacterial strains (Görke and Stülke, 2008). In Fig. 2D, cel-
lobiose accumulation was observed only at 6 h of fermentation.
After 6 h of fermentation, the cellobiose concentration remained
low, regardless of the cellulase loading. This result indicated that
cellobiose was consumed without difficulty during the SSF process.
3.2.2. b-Glucosidase
b-Glucosidase, which is an essential enzyme in most cellulose
hydrolytic processes, could increase the hydrolysis yield by reduc-
ing the level of the inhibitor cellobiose. Thus, the effect of b-
glucosidase dosages was investigated in this study. The cellulase
loading was 10 FPIU/g cellulose and the initial cellulose loading
of BSP was 60 g/L (Table 2).
Previous studies found that adding b-glucosidase effectively
improved the outcomes of the SSF process (Han and Chen, 2008;
Krishna et al., 2001). However, as shown in Fig. 3, the maximal lac-
tic acid concentration, approximately 50 g/L, was obtained in this
study when the b-glucosidase loading was only 5 pNPGU/g cellu-
lose. A low level of b-glucosidase loading would help to lower
the total enzyme cost, which is an important factor for economical
lactic acid production. More interestingly, even without the
addition of b-glucosidase, B. coagulans CC17 produced 47.59 g/L
lactic acid of in 72 h of fermentation. This result indicated that
B. coagulans CC17 might have the ability to directly utilize cel-
lobiose to produce lactic acid, as had been previously reported
(Budhavaram and Fan, 2009). Moreover, we found that lactic acid

Table 2
Summarization of fermentation parameters and results in all fermentation experiments.

Experiment Fermentation Cellulose and Cellulase b-glucosidase Xylanase Lactic acid

mode hemicelluloses (g/L) (FPIU/g) (pNPGU/g) (IU/g)
C (g/L) Y1 (g/g) Y2 (g/g)
Comparision of SHF and SSF Batch SHF 60 and 11.64 15 15 0 32.22 0.54 0.45
80 and 15.53 50.54 0.63 0.53
Batch SSF 60 and 11.64 50.20 0.84 0.70
80 and 15.53 55.04 0.69 0.58
Cellulase Effect Batch SSF 60 and 11.64 5 5 0 34.32 0.57 0.48
10 10 45.96 0.77 0.64
15 15 46.58 0.78 0.65
20 20 46.31 0.77 0.65
25 25 47.25 0.79 0.66
b-Glucosidase Effect Batch SSF 60 and 11.64 10 0 0 47.59 0.79 0.66
5 50.24 0.84 0.70
10 47.94 0.80 0.67
15 47.28 0.79 0.66
20 45.70 0.76 0.64
Xylanase Effect Batch SSF 60 and 11.64 10 0 0 49.13 0.82 0.69
80 52.19 0.87 0.73
100 53.95 0.90 0.75
120 55.71 0.93 0.78
140 54.33 0.91 0.76
160 54.04 0.90 0.75
200 54.39 0.91 0.76
Fed-batch Experiment Fed-batch SSF 153.30 and 29.75 10 0 120 110 0.72 0.60

Y1: L-lactic aicd yield to cellulose of BSP.

Y2: L-lactic acid yield to cellulose and hemicellulose of BSP.
 J. Zhou et al. / Bioresource Technology 222 (2016) 431–438 435

50 24
A B

Glucose concentration (g/L)

Lactic aicd concentration (g/L)
40 20

16
30
12
20
8

10
4

0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Time (h) Time (h)
10 6
C D

Cellobiose concentration (g/L)

5
Xylose concentration (g/L)

4
6
3
4
2

2
1

0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Time (h) Time (h)

Fig. 2. Profiles of lactic acid SSF with different cellulase loading. A: lactic acid; B: glucose; C: xylose; D: cellobiose. The substrate cellulose dosage was 60 g/L. The ratio
between cellulase loading and b-glucosidase loading was 1:1. Symbols: square 5 FPIU/g, circle 10 FPIU/g, triangle 15 FPIU/g, inverted triangle 20 FPIU/g, diamond 25 FPIU/g.

60 promising strain for bioconversion because it can utilize the cel-

lobiose, glucose and xylose in raw cellulosic materials for survival
Lactic acid concentration (g/L)

under open conditions. Moreover, B. coagulans CC17 would be

50 expected to co-ferment the cellulose and hemicellulose in a ligno-
cellulosic biomass via SSF.
To study sugar consumption and lactic acid production during
40
the course of SSF in the absence of exogenous b-glucosidase, SSF
was conducted using 60 g/L cellulose of BSP and 10 FPIU/g cellu-
lose of cellulase (Fig. 4). Without the addition of b-glucosidase,
the cellobiose concentration reached a maximal level of approxi-
30
mately 8 g/L during the first 6 h of fermentation, and approxi-
mately 3.63 g/L of glucose and 1.70 g/L of xylose were detected
in the liquid. Subsequently, the obvious co-fermentation of cel-
20
0 5 10 15 20 lobiose, glucose and xylose was observed. The maximal consump-
tion rates of cellobiose, glucose and xylose were 0.39 g/(Lh),
-glucosidase (U/g cellulose)
0.55 g/(Lh) and 0.24 g/(Lh) between 6 h and 12 h of the SSF pro-
Fig. 3. The effect of the b-glucosidase dosages on lactic acid production. The cess, respectively. The changes in the sugar profiles observed dur-
substrate cellulose dosage was 60 g/L. The cellulase loading was 10 FPIU/g cellulose. ing SSF suggest that strain CC17 might have a cellobiose-degrading

concentration was slightly reduced when the b-glucosidase load- 10 50

ing was increased above 10 pNPGU/g cellulose. This result indi-
Lactic acid concentration (g/L)
Cellobiose concentration (g/L)

cated that an excess of b-glucosidase unexpectedly inhibited 8 40

lactic acid production via SSF. As mentioned above, the slow
release of glucose was favor to xylose utilization and implementa- 6 30
tion of co-fermentation. Excessive b-glucosidase promoted the cellobiose
convert of cellobiose from BSP to glucose, which could cause CCR glucose
4 20
of this strain, and the lactic acid production was limited. This is xylose
lactic acid
the first finding of the inhibitory phenomenon of b-glucosidase in
2 10
SSF. Other bacterial species had been reported to utilize pure cel-
lobiose as a carbon source, such as Lactobacillus lactis mutant strain
RM2-24 (Singhvi et al., 2010), Enterococcus mundtii strain QU 25 0 0
0 12 24 36 48 60 72
(Abdel-Rahman et al., 2011) and some engineered Escherichia coli
Time (h)
and Saccharomyces strains (Okano et al., 2010). But only few was
reported to produce lactic acid from cellulose by SSF (Adsul et al., Fig. 4. Time-course of lacic acid SSF without b-glucosidase. The substrate cellulose
2007b). Compared with these strains, B. coagulans CC17 is a dosage was 60 g/L. The cellulase loading was 10 FPIU/g cellulose.
436 J. Zhou et al. / Bioresource Technology 222 (2016) 431–438

pathway that eliminates the need for exogenous b-glucosidase. We 60

also conducted experiments using cellobiose as the sole carbon A

Lactic acid production (g/L)

source. Approximately 41 g/L of lactic acid was produced from
60 g/L of cellobiose. Therefore, the degradation of cellobiose in 55
SSF owed to both the 43.9 pNPGU/mL b-glucosidase present in
the Celluclast 1.5L and the strain’s own cellobiose utilization abil-
ity. Moreover, glucose content was maintained at a low level (<4 g/ 50
L) and xylose was rapidly fermented into lactic acid. Based on the
results of mixed sugars fermentation of glucose, xylose and cel-
lobiose, we hypothesize that the expected intracellular metabolism 45
of cellobiose by strain CC17 may have sufficiently reduced the
extracellular glucose concentration to prevent catabolic repression,
thus allowing xylose fermentation to occur. Therefore, the ability 40
0 80 100 120 140 160 200
of strain CC17 to co-ferment cellobiose and xylose allowed it to
Xylanase loading (IU/g hemicellulose)
simultaneously convert cellulose and xylan to lactic acid in the
absence of exogenous b-glucosidase. B. coagulans CC17 demon- 45 15
strates a distinctive advantage for SSF because it requires less
glucose without xylanase
B
b-glucosidase and can alleviate the inhibition of glucose in mixed 40

Glucose concentration (g/L)

Xylose concentration (g/L)

glucose with xylanase
sugar fermentation. 12
35
3.2.3. Xylanase
30
Due to the association of cellulose and hemicellulose with lig- 9
nin, the enzymatic hydrolysis of native lignocellulosic materials
25
is always a difficult and slow process. In the present study, consid-
ering because BSP contains approximately about 14.33% hemicel- 20 6
lulose, xylanase (Pentopan Mono BGÒ, from Thermomyces xylose without xylanase
xylose with xylanase
lanuginosus) was added to improve the hydrolysis efficiency. As 15
previous study had proved that xylanase has remarkable stability 3
at 50 °C (Damaso et al., 2002), in this study, only the effect of the 0 12 24 36 48
Time (h)
xylanase dosage on lactic acid concentration was investigated.
SSF was conducted at 60 g/L cellulose loading of BSP, and the Fig. 5. The effect of xylanase supplement on lactic acid SSF. The substrate cellulose
cellulase and xylanase loadings were shown in Table 2. As shown dosage was 60 g/L. The cellulase loading was 10 FPIU/g cellulose. A: SSF with
in Fig. 5A, compared to SSF without xylanase added, the lactic acid different dosage of xylanase. B: Corresponding enzymatic hydrolysis experiment
with 0 and 120 IU/g hemicelluloses of xylanase.
concentration was increased by 6.23% and reached 52.19 g/L when
80 IU xylanase/g hemicellulose was added. The maximum lactic
acid concentration reached 55.70 g/L at a xylanase loading of
8 120
120 IU/g hemicellulose. In the corresponding enzymatic hydrolysis
experiment, we found that xylanase supplementation improved
100
both cellulose and xylan hydrolysis (Fig. 5B), which is in good
6
accordance with the result of Hu et al. (2011). Compared with
Residul sugars (g/L)

80

Lactic acid (g/L)

SSF without xylanase supplementation, the xylose and glucose cellobiose
glucose
concentrations were increased by 11.92% and 15.40%, respectively, 4 xylose 60
with the addition of 120 IU xylanase/g hemicellulose. The results lactic acid
suggested that the increase in lactic acid production by SSF due 40
to the addition of xylanase can be partially explained by an 2
improved substrate conversion efficiency. In the investigated range 20
(0–200 IU/g hemicellulose), a saturation phenomenon of xylanase
loading was observed when the xylanase loading was greater than 0 0
0 24 48 72 96 120 144 168 192
120 IU/g hemicelluloses. Therefore, considering the economic ben-
Time (h)
efit, a xylanase loading of 120 IU/g hemicellulose would be the best
choice for SSF of BSP. Fig. 6. Time-course of fed-batch SSF with the final cellulose content of BSP about
153.30 g/L. The fed-batch SSF experiments were carried out with 30 g/L cellulose of
3.3. Lactic acid production by SSF in the fed-batch mode at a high BSP. BSP containing 3 g cellulose was added for 7 times during the first 48 h.
10 FPIU/g cellulose of cellulase, 120 IU/g hemicellulose of xylanase and 12 g calcium
solids loading of BSP carbonate were added into the medium at the initial of the experiment.

BSP is a type of pretreated lignocellulose solid feedstock. A high

substrate loading for SSF resulted in low mass and heat transfer
efficiencies, a low sugar yield, and a low lactic acid yield (data
not shown). Implementing the fed-batch mode could alleviate this
technical problem because fresh substrate is added only when the
viscosity has decreased (Zhang et al., 2010). Therefore, to achieve a
high lactic acid titer and yield, SSF was conducted using the fed-
batch mode, which allowed using a high solids loading while
avoiding increases in viscosity. All of the enzymes and calcium car-
bonate were added at the initial of fermentation. The fed-batch SSF
experiments were conducted using the initial 30 g/L cellulose
loading of BSP. The initial fermentation volume was 100 mL. Dur-
ing the fed-batch SSF process, fresh BSP containing 3 g of cellulose
was added at 3, 6, 9, 12, 24, 36, and 48 h. The final cellulose loading
of BSP was 153.30 g/L. The results of fed-batch SSF were shown in
Fig. 6. The lactic acid yield was shown in Table 2.
As shown in Fig. 6, during fed-batch fermentation, the cel-
lobiose concentration decreased rapidly in the first 24 h and
remained at a constantly low level. The glucose and xylose concen-
trations did not exceed 1.0 g/L throughout 192 h. These results sug-
gested that these sugars were continuously released from the BSP
 J. Zhou et al. / Bioresource Technology 222 (2016) 431–438 437

Table 3
Lignocellulosic materials used for the production of lactic acid by SSF.

Substrate Strain Fermentation Cellulase (FPIU/g) b-glucosidase Lactic acid Fermentation Reference
mode (U/g) Time (h)
C (g/L) Y (g/g)
Waste sugarcan bagasse L. delbrueckii mutant Uc-3 Fed-batch P.janthinellum EU1 (10) 0 67.0 0.83 72 Adsul et al. (2007a,b)
Corn stover L. pentosus ATCC 8041 Fed-batch Spezyme CP (5) 0 74.8 0.65 288 Zhu et al. (2007)
Paper sludge L. rhamnosus ATCC 7469 Batch Celluclast 1.5L (10) 10 72.7 – 168 Marques et al. (2008)
Cellulosic biosludge L. rhamnosus CECT-288 Fed-batch Celluclast 1.5L (12.5) 13 42.0 0.38 48 Romaní et al. (2008)
Acid-pretreatment B. coagulans IPE22 Batch Trichoderma reesei(20) 0 38.4 0.46 90 Zhang et al. (2014)
wheat straw
Corn stover B. coagulans LA204 Fed-batch Cellic CTec2 (30) 0 97.6 0.68 60 Hu et al. (2015)
Empty fruit bunch slurries B. coagulans JI12 Batch Cellic CTec2 (25) 0 80.60 0.49 24 Ye et al. (2014)
Bagasse sulfite pulp B.coagulans CC17 Fed-batch Celluclast 1.5L (10) 0 110 0.72 192 This work

120 IU/g hemicellulose of xylanase was added in this work.

and were subsequently consumed by strain CC17 during the fed-
batch SSF. In this study, fed-batch SSF with a 153.30 g/L cellulose
loading of BSP produced the largest amount of lactic acid, 110 g/
L, corresponding to a 0.72 g/g cellulose yield and a 0.60 g/g cellu-
lose and hemicellulose yield (Table 2). Several previous studies
had reported lactic acid production by SSF from lignocellulosic bio-
mass. In Table 3, compared with the results obtained using the L.
delbrueckii mutant Uc-3 (Adsul et al., 2007a) or L. rhamnosus ATCC
7469 (Marques et al., 2008), B. coagulans CC17 achieved a much
higher lactic acid concentration with the same cellulase loading.
Compared with other B. coagulans strains, such as LA204 (Hu
et al., 2015), JI12 (Ye et al., 2014) and IPE22 (Zhang et al., 2014),
the CC17 strain required a much lower cellulase dosage to produce
high-titer lactic acid. These values meet the requirements for the
economically viable production of lactic acid from lignocellulose
on an industrial scale.
4. Conclusions
In this study, experiments aimed at obtaining high-titer lactic
acid were conducted using Bacillus coagulans strain CC17. The
results showed that the SSF process required 33.33% less cellulase
than that required for the SHF process. We found that B. coagulans
strain CC17 does not require additional b-glucosidase for SSF.
Moreover, adding 120 IU xylanase/g hemicellulose increased the
lactic acid concentration by 13.4%. When fed-batch SSF was con-
ducted under non-sterile conditions, the lactic acid titer and yield
reached 110 g/L and 0.72 g/g cellulose, respectively. Therefore, a
high-titer and cost-effective lactic acid production process by SSF
with strain CC17 has been established in this study.
Acknowledgements
This study was supported by the National Natural Science Foun-
dation of China (51561145015) and the Major Program of the Nat-
ural Science Foundation of Jiangsu Higher Education of China
(16KJA220004). We also kindly acknowledge partial support from
the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).
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