Bioresource Technology 161 (2014) 171–178

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One-pot simultaneous saccharification and fermentation: A preliminary study of a novel configuration for cellulosic ethanol production Jingbo Li, Jianghai Lin, Pengfei Zhou, Kejing Wu, Hongmei Liu, Chunjiang Xiong, Yingxue Gong, Wenjuan Xiao, Zehuan Liu ⇑ Research Center for Molecular Biology, Institutes of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China

h i g h l i g h t s  Delignification was the major effect of the alkali pretreatment at 121 °C for 60 min.  Size reduction enhanced the effectiveness of the mild alkali pretreatment.  One-pot SSF was proposed based on the combined pretreatment.  Laccase increased the ethanol yield by 6.8% for one-pot SSF.  Ethanol yield reached 67.56% for one-pot SSF with laccase supplementation.

a r t i c l e

i n f o

Article history: Received 10 November 2013 Received in revised form 25 February 2014 Accepted 27 February 2014 Available online 12 March 2014 Keywords: One-pot SSF Mild alkali pretreatment Size reduction Enzymatic hydrolysis Bioethanol production

a b s t r a c t Combination of size reduction and mild alkali pretreatment may be a feasible way to produce bioethanol without rinsing and detoxifying the solid substrate. Based on that, a fermentation configuration named one-pot SSF in which pretreatment and fermentation steps were integrated was developed. Additionally, the effect of laccase on fermentation performance was investigated. Delignification was the major effect of the alkali pretreatment at 121 °C for 60 min. The highest glucose and xylose yield, which obtained from the smallest particle at a substrate loading of 2%, was 6.75 and 2.71 g/L, respectively. Laccase improved the fermentation efficiency by 6.8% for one-pot SSF and 5.7% for SSF. Bioethanol from one-pot SSF with laccase supplementation reached 67.56% of the theoretical maximum, whereas that from SSF with laccase supplementation reached 57.27%. One-pot SSF might be a promising configuration to produce bioethanol because of 100% solid recovery, and rinsing water and detoxification elimination. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Pretreatment has been recognized as a must for a long time for saccharification of lignocellulose. A good pretreatment method should: (1) enhance the formation of fermentable sugars or the digestibility of feedstock in subsequent enzymatic hydrolysis; (2) avoid the loss or degradation of carbohydrates; (3) avoid the formation of inhibitors; and (4) be cost-effective (Sun and Cheng, 2002). Acid-based pretreatments including steam explosion, dilute-acid pretreatment, and autohydrolysis at higher severity result in the degradation of hemicellulose and lignin to furfural, 5-HMF and phenolic acids that inhibit enzymatic hydrolysis and yeast fermentation (Cantarella et al., 2004; Klinke et al., 2004; ⇑ Corresponding author. Tel.: +86 20 85222863 801; fax: +86 20 85222863 888. E-mail addresses: [email protected], [email protected] (J. Li), zhliu@jnu. edu.cn (Z. Liu). http://dx.doi.org/10.1016/j.biortech.2014.02.130 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Ximenes et al., 2010). Furthermore, lignocellulosic biomass pretreated by organosolv, phosphoric acid, ionic liquids, dilute acid, steam explosion, lime, alkaline wet oxidation, and fungi or bacteria needs to be washed with water before enzymatic hydrolysis and fermentation, which may result in loss of carbohydrates. Wastewater from pretreated solid effluent also needs to be treated. Detoxification is also needed for most of the above mentioned methods (Chundawat et al., 2010). Pretreated biomass rinsed with water results in the loss of carbohydrates. Meanwhile, the wastewater from this process also needs to be treated. Hence, development of a method that can overcome the above mentioned flaws is meaningful. Size reduction of lignocellulosic biomass can enhance the enzymatic digestibility. Glucose released from both wet oxidized and not wet oxidized wheat straw particles was increased with reduced particle size (Pedersen and Meyer, 2009). Smaller particle size also led to higher glucose yield when microcrystalline cotton

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cellulose was used as substrate (Yeh et al., 2010). However, the maximum conversion rate achieved by physical size reduction was approximately 50%, whereas higher than 70% of conversion rate was reached by using chemical modification regardless of biomass particle size (Vidal et al., 2011). Although size reduction improved the enzymatic digestibility, the efficiency was not high enough. In this case, a combination of size reduction and mild chemical pretreatment method may be of great potential. Compared with sulfuric acid, hydrogen peroxide, and ozone pretreatments, sodium hydroxide pretreatment was the most effective to enhance the enzymatic digestibility of cotton stalks (Silverstein et al., 2007). Yoon et al. (2011) compared the ionic liquid ([EMIM]oAc), acid (H2SO4), and alkali (NaOH) pretreatments for the enzymatic saccharification of sugarcane bagasse (SCB). Their results showed that alkali pretreated SCB obtained the highest reducing sugar yield. Enzymatic convertibility of NaOH pretreated SCB was much higher than that pretreated by H2O2, H2SO4, and steam explosion (Li et al., 2014b). NaOH pretreated corn stover also showed the highest sugar yield (reducing sugar, glucose, and xylose) compared with dilute H2SO4, lime, and NH3/ HCl pretreatments (Chen et al., 2009). Additionally, lowest acetic acid was detected in the enzymatic hydrolysate from NaOH pretreated corn stover. Furfural was not detected in enzymatic hydrolysates from alkali involving pretreatments such as lime, NaOH, and NH3/HCl (Chen et al., 2009). Therefore, NaOH pretreatment may be the competent candidate to be combined with size reduction. The aim of this study was to study the effect of particle size on chemical composition and enzymatic digestibility of NaOH pretreated samples. On this basis, a process called ‘‘one-pot simultaneous saccharification and fermentation (one-pot SSF)’’ was developed to produce ethanol without rinsing the solid and detoxification. 2. Methods 2.1. Preparation of different particle sizes of SCB SCB was kindly provided by the Qianwu sugar refinery plant located in Zhuhai, Guangdong, China. After air-dried, it was stored in woven bags at room temperature until utilization. SCB was grounded by using a hammer mill (FY130, Tianjin Taisitie Instrument Co., Ltd, China) and a jet mill (JM) (NADA Superfine Tech. Co., Ltd, Nanjing, China). Different sizes of samples were obtained by sieving the milled SCB using corresponding mesh sieves. The 400-mesh particle was obtained by using jet milled SCB. The samples were named as Px-Ry which meant that the particles passed x-mesh sieve but were retained by y-mesh sieve. 2.2. Alkali pretreatment Because yeast was sensitive to salt (Moon et al., 2009), a large amount of salt formation from neutralization of the alkali pretreated SCB may depress yeast fermentation. Therefore, low concentration of NaOH was selected. Additionally, alkali pretreated SCB was neutralized by using acid could avoid the loss of carbohydrates and wastewater generation. Each SCB sample was mixed with 0.5% (w/v) NaOH solution at a solid loading of 10%. Thereafter, the mixture was shaken vigorously in order to blend well. Then, it was put into an autoclave within 5 min to suffer the steam cooking. The temperature was kept at 121 °C with a residence time of 1 h. The solid fraction was obtained by filtering through a filter cloth (500-mesh). Hot water (approximately 90 °C) was used to rinse the solid until pH 6–7. All the solids were oven-dried at 85 °C and stored in a sealed plastic bag for enzymatic hydrolysis.

2.3. Enzymatic hydrolysis of the alkali pretreated fractions Enzymatic hydrolysis of the alkali pretreated fractions was conducted at 2% (w/v) of substrate loading in 50 mL 0.1 M citric acid/ citric sodium buffer (pH 4.8), containing 80 lg/mL tetracycline and 60 lg/mL nystatin (dissolved in DMSO). The mixture was incubated at 45 °C in a rotary shaker at 200 rpm overnight to mix well. A blend of Celluclast 1.5 L and Novozym 188 (Novozymes (China) Investment Co. Ltd.) with a loading of 10 FPU/g and 40 pNPGU/g substrate, respectively, was added after acclimation overnight. Samples (1 mL) were taken from the reaction blend at different time intervals. Each sample from the hydrolysate was heated in boiling water for 10 min to inactivate the enzymes. The samples were centrifuged at 6000g for 5 min. The supernatant was filtered through 0.22 lm membrane filter (Sartorius, Germany) for analyzing glucose and xylose by HPLC. 2.4. Microorganism strain, media, and inoculum preparation The Saccharomyces cerevisiae As2.489 strain, referred to as an industrial strain, was used in this work. Yeast extract peptone dextrose (YPD) medium containing 20, 10 and 5 g/L of glucose, yeast extract, and peptone was sterilized by steam autoclaving at 121 °C for 20 min. The strain was grown aerobically at 30 °C in a rotary shaker at 200 rpm in YPD medium for 24 h. Then, 20 mL of activated cells was aseptically transferred to 200 mL of sterile YPD medium in a 500-mL Erlenmeyer flask. The flask was incubated at 30 °C at 200 rpm for 48 h. The cells were harvested by centrifugation in 50-mL sterile centrifuge tubes for 10 min at 5000 rpm at 4 °C using a centrifuge (Anke, Shanghai, China). The cell pellets were washed twice with sterile deionized water. The cells were then resuspended by sterile deionized water and used to initiate fermentation. The entire cell-harvest process was completed within 2 h to ensure the activity of the cells. 2.5. Bioethanol production One-pot SSF and conventional SSF were conducted in 100 mL serum bottles with rubber stoppers. One-pot SSF was conducted with and without laccase supplement. Conventional SSF was performed using rinsed alkali pretreated SCB as control experiment. For one-pot SSF, 5 g of 400-mesh particle was added in 50 mL of 0.5% NaOH and then the blend was steamed at 121 °C for 1 h in an autoclave. After cooling down, 170 ll of H3PO4 was added into the mixture to adjust the pH to approximately 5. Then 2 g/L of yeast extract was supplemented as nitrogen source. For SSF, 5 g of rinsed sample was mixed with 50 mL of water supplemented with 2 g/L of yeast extract, and the pH was also adjusted to approximately 5 by H3PO4. The fermentation media were sterilized in an autoclave at 121 °C for 15 min. Celluclast 1.5 L, Novozym 188, and laccase were filtered through 0.22 lm sterilized membranes (Millipore Corp. Carrigtwohill, Co. Cork, Ireland) before being added to the media. Laccase (derived from Aspergillus oryzae, laccase activity: 1.13 U/mg, cellulase activity: 0.11 FPU/mg, b-glucosidase activity: 0.17 pNPGU/mg, and xylanase activity: 0.24 IU/mg, purchased from Beijing Huamaike Biotechnology Co., Ltd., Beijing, China) treatment was carried out for 12 h at 45 °C at a loading of 5 mg/g substrate before fermentation. A mixture of Celluclast 1.5 L and Novozym 188 with loadings of 15 FPU/g and 60 pNPGU/g substrate, respectively, was added to the media. The yeast cell suspension was added at concentrations of 2 g dry yeast cells/L (DCW/L) to initiate fermentation. Subsequently, the bottles were sealed with rubber stoppers equipped with a water trap, which permitted CO2 removal but without air injection. The bottles were placed on a shaker at 200 rpm at 30 °C. Samples were periodically collected

J. Li et al. / Bioresource Technology 161 (2014) 171–178

under aseptic conditions for determining the concentration of phenolic, glucose, xylose, and ethanol. 2.6. Concept of one-pot SSF Bioethanol production from lignocellulose always includes three steps: (1) biomass pretreatment; (2) enzymatic hydrolysis; (3) fermentation. Based on these three individual steps, three main different process configurations, separate hydrolysis and fermentation (SHF), SSF, and consolidated bioprocessing (CBP), have been applied (Fig. 1A). The CBP process, which combines step 2 and 3 together, employs only one microbial community that can produce cellulases and ferments sugars to ethanol simultaneously. Hydrolysis and fermentation are carried out in the same reactor for SSF and CBP (Fig. 1A). Pretreatment step was not introduced by the proposed fermentation configurations. One-pot SSF in which the

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pretreatment step was combined with SSF was proposed in the present study (Fig. 1B). It merged all three steps together. As shown in Fig. 1C, SCB was pretreated by low concentration of NaOH, and then H3PO4 was added to neutralize the blend, finally SSF was conducted. If this process will be scaled to an industrial level in the future, sterilization of fermentation media could be replaced by the pretreatment at 121 °C. Yeast, sterile nitrogen source and cellulases could be pumped directly to the reactor to produce ethanol (Fig. 1D). The features of one-pot SSF were discussed in the following section. 2.7. Analytical methods Chemical composition of the biomass was analyzed according to the LAP of NREL (Sluiter et al., 2008). Briefly, after a two-step analytical acid hydrolysis procedure, Ba(OH)2 was added to

Fig. 1. Schematic illustration of the one-pot SSF. (A) Main process configurations (MANZANARES, 2010); (B) Configuration of one-pot SSF. (C) Lab-scale one-pot SSF; (D) Industrial scale one-pot SSF; SCB and NaOH solution were mixed and the temperature was hold at 121 °C for 1 h. After cooling, H3PO4 was added, followed by addition of nitrogen source, yeast, and sterile cellulases.

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2.5 mL of acid hydrolysate to adjust the pH to 2 before analysis. Acid soluble lignin was measured using a UV/Vis spectrophotometer (UNICO, UV-2000, Shanghai, China) at 240 nm. Glucose, xylose, and ethanol were determined using a HPLC (Shimadzu, LC-15C) equipped with a refractive index detector (RID-10A). Aminex HPX-87H column with a Cation H+ Cartridge Micro-Guard column was used to separate these components. The column was operated at 55 °C with 5 mM H2SO4 as the mobile phase at the flow rate of 0.6 mL/min. Phenolic content was measured by the Folin–Ciocalteau method as described previously (Li et al., 2013). The total phenolic content was expressed as gallic acid equivalent (GAE, Tianjin Guangfu Fine Chemical Research Institute, Tianjin). 2.8. Calculations and statistical analysis The enzymatic hydrolysis and fermentation efficiencies, ethanol yield, and ethanol productivity were calculated using the following equations.

Enzymatic efficiency for glucoseð%Þ ¼

Glucose concentration ðg=LÞ  100% Solid loading  glucan%  1:11

Enzymatic efficiency for xyloseð%Þ ¼

Xylose concentration ðg=LÞ  100% Solid loading  xylan%  1:14

Fermentation efficiencyð%Þ ¼

Maximum ethanol concentration ðg=LÞ  100% Solid loading  glucan%  1:11  0:51ðg=LÞ

Ethanol yield ¼

Ethanol contentðgÞ Cellulose contentðgÞ

Ethanol productivity ¼

Ethanol concentrationðg=LÞ Corresponding fermentation timeðhÞ

where 1.11 is the coefficient of glucose converted from cellulose, 1.14 is the coefficient of xylose converted from hemicellulose; 0.51 is the coefficient of ethanol converted from glucose. Statistical analysis was carried out by the PASW statistics 18 using one-way ANOVA and Duncan’s multiple range tests. Results were considered statistically significant at a 95% confidence interval (p < 0.05). 3. Results and discussion 3.1. Chemical composition The effect of alkali treatment on the chemical composition of the different particle size fractions is shown in Table 1. Compared

with the untreated one, the cellulose contents in all of the treated ones were significantly increased by approximately 37% by the 0.5% (w/v) NaOH pretreatment at 121 °C for 1 h (p < 0.05). However, particle size had no significant effect on the cellulose content of the alkali pretreated samples. The hemicellulose contents in all the samples were increased to some extent except for P350-R400. As expected, approximately 30% of the lignin was extracted by this specific pretreatment. Acid soluble lignin in alkali pretreated samples was significantly lower than that in untreated (p < 0.05). Approximately 6–11% of the acid soluble lignin was extracted during the pretreatment. Similarly, acid insoluble lignin was also removed by 25–50% during the pretreatment. Correlations between particle size and hemicellulose, acid soluble lignin, and acid insoluble lignin content were not apparent in alkali pretreated samples. The data showed that this kind of pretreatment extracted large part of the lignin and increased the content of cellulose. It seemed that particle size had no significant impact on the chemical composition of the mild alkali pretreated SCB. All of the components including cellulose, hemicellulose, and lignin are solubilised by the alkali pretreatment, but the lignin is solubilised more than the other two components (Silverstein et al., 2007). In the report by Xu et al. (2010), cellulose content in pretreated switchgrass was increased by approximately 40%. Hemicellulose solubilization ranged from 14.36% to 15.49% when 0.5% NaOH was used for pretreatment of Coastal Bermuda grass (Wang et al., 2010). Cellulose content was increased significantly by NaOH pretreatment, but not for hemicellulose in the current study. The similar phenomenon was reported that cellulose content of corn stover which pretreated by 2% of NaOH was increased by 65.6% (Chen et al., 2009). The major effect of alkali pretreatment is the delignification of the biomass (Kim and Holtzapple, 2005). Silverstein et al. (2007) compared the effect of three chemical pretreatment methods on cotton stalks including alkali, acid, and H2O2 pretreatment at 121 °C. The results showed that alkali pretreatment gave rise to 63.65% reduction of lignin, while H2O2 and dilute acid pretreatment led to 29.51% and 24% reduction of lignin. Wang et al. (2010) reported that the lignin reduction was 12.4% and 13.04% when 0.5% NaOH was used for pretreatment of Coastal Bermuda grass. Another study reported that over 30% of lignin was removed from raw switchgrass by 0.5% NaOH at 121 °C for 1 h (Xu et al., 2010). The same as the previous studies, lignin reduction was observed to be the major effect of the NaOH pretreatment in the present study and the reduction rate was ranged from 25% to 50%. 3.2. Enzymatic hydrolysis Enzymatic hydrolysis of the alkali pretreated samples is shown in Fig. 2. Glucose concentration was increased with the decreased particle size, but no significant improvement was observed between P150-R250 and P250-R350 (p > 0.05). JMP400 reached the highest glucose concentration of 6.75 g/L, which was 1.81, 1.4, 1.24, and 1.06-fold of the other samples, respectively. Glucose

Table 1 Chemical composition of the samples. Alkali pretreated samples

Particle size (lm)

Cellulose (%)

Hemicellulose (%)

Acid soluble lignin (%)

Acid insoluble lignin (%)

Untreated 0.5-R50 P100-R150 P150-R250 P250-R350 P350-R400 JMP400

– 5000–270 150–106 106–58 58–43 43–38 638

35.47 ± 0.52a 48.71 ± 0.60b 47.56 ± 1.39b 47.67 ± 0.54b 49.75 ± 0.40b 48.71 ± 0.23b 47.60 ± 1.94b

21.00 ± 0.13a 23.36 ± 0.13b 22.85 ± 0.44b 21.99 ± 0.16c 21.79 ± 0.10a,c 19.58 ± 0.47c 21.39 ± 0.53a,c

4.84 ± 0.04a 4.47 ± 0.04c,d 4.24 ± 0.00e 4.27 ± 0.06b,e 4.51 ± 0.10d 4.35 ± 0.06b,c,e 4.38 ± 0.03b,c

20.62 ± 0.47a 14.89 ± 0.59b 10.50 ± 1.34c 11.51 ± 2.42b,c 13.65 ± 2.55b,c 15.04 ± 1.19b 13.48 ± 1.65b,c

Mean values within the same column with different letters are significantly different at p < 0.05. Mean value ± standard deviation is from two independent experiments.

J. Li et al. / Bioresource Technology 161 (2014) 171–178

Fig. 2. Times course of glucose (A) and xylose (B) concentration of the alkali pretreated samples. Each point in (A) and (B) is a mean value ± standard deviation from two independent experiments. Hydrolysis conditions: 2% substrate, Celluclast 1.5 L 10 FPU and Novozym 188 40 pNPGU/g substrate at 45 °C in a rotary shaker at 200 rpm.

concentration of P350-R400 before 24 h was higher than that of JMP400 but lower after 24 h (Fig. 2A). The initial hydrolysis rates (2 h) of the samples were not significantly different except for 0.5-R50. Thereafter, the hydrolysis rates of the smaller particles were higher than those of the larger ones. At the end of the hydrolysis, JMP400 reached 64.5% of the theoretical maximum whereas 0.5-R50 attained 34.9% of the theoretical maximum. Formation of xylose was also investigated and is shown in Fig. 2B. Xylose concentrations reached maximum at 24 h hydrolysis. The xylose concentration in hydrolysates obtained from 0.5-R50 and JMP400 was the lowest and highest, respectively. This was consistent with the glucose profiles. However, unlike the glucose profiles, the xylose from P350-R400 was significantly lower than that from JMP400 (p < 0.05). Xylose yield from JMP400 was 55.5% of theoretical maximum which was over 24% higher than those from other samples. Smaller particle size was beneficial to the accumulation of glucose and xylose from alkali pretreated SCB. The effect of particle size on enzymatic hydrolysis of lignocellulose is controversial. Some studies reported that no effect of size reduction on cellulose conversion was found in bagasse, rice straw, cardboard, and newspaper (Rivers and Emert, 1988a,b). In contrast,

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some other studies found a reduced particle size resulted in an enhanced cellulose conversion (Pordesimo et al., 2005; Zhu et al., 2009). Harun et al. (2013) reported that the severity of Ammonia Fiber Expansion affected the relationship between particle size and sugar conversion. Positive relationship was presented under low severity conditions, while an opposite relationship was observed under high severity conditions. In this study, enzymatic hydrolysis of NaOH (0.5%) pretreated samples also showed that size reduction benefited glucose and xylose formation (Fig. 2). It was observed in our previous studies that particle size reduction gave rise to an enhanced cellulose conversion of milled and fractionated SCB (data not published). The crystallinity of the hammer and jet milled and fractionated SCB was not affected much but the specific surface area was increased with the size reduction (data not published). Chemical composition of the pretreated samples was not significantly different, indicating that the chemical composition is not the key factor to affect the enzymatic digestibility. It was reported that the increase of surface area could increase the cellulose conversion for substrates produced by different sizereduction processes (Zhu et al., 2009). Therefore, the reduced size and the increased surface area might be the possible reasons for the enhanced enzymatic digestibility. Although the glucose concentration in the enzymatic hydrolysate from untreated JMP400 was 3.5 g/L, it was insufficient to perform fermentation for its low yield (data not published). Therefore, a mild and cost-effective pretreatment, 0.5% NaOH pretreatment at 121 °C for 1 h, was selected. Cotton stalks was pretreated by using 2% of NaOH, H2SO4, and H2O2 at 121 °C for 1 h, the results showed that NaOH pretreatment was the most effective for cellulose conversion (Silverstein et al., 2007). Corn stover was pretreated using 0.15 g/g dry sample of H2SO4 at 106–108 °C for 6 h, 0.4 g/g dry sample of calcium hydroxide at 120 °C for 4 h, 1 g/g dry sample of aqueous ammonia at 26 °C for 24 h, and 0.16 g/g dry sample of sodium hydroxide at 120 °C for 30 min. The results indicated that NaOH pretreated corn stover showed the highest hydrolysis yield (Chen et al., 2009). The enzymatic digestibility of 2% of NaOH pretreated SCB was much higher than that of steam exploded SCB, 2% of H2O2 pretreated SCB, and 2% of H2SO4 pretreated SCB (Li et al., 2014b). It is believed that the intermolecular ester bonds crosslinking hemicellulose and other components such as lignin are destroyed during the alkali pretreatment leads to the enhanced enzymatic hydrolysis (Sun and Cheng, 2002). The glucan and xylan conversion rates of 74.4% and 62.8%, respectively, were obtained from pretreated switchgrass (50 °C, 1.0% NaOH, 12 h) (Xu et al., 2010). The cellulose conversion was 60.8% from NaOH pretreated cotton stalks with a cellulase loading of 40 FPU/g cellulose (121 °C, 2% NaOH, 90 min) (Silverstein et al., 2007). The yield of reducing sugar obtained from alkali pretreated SCB (115 °C, 0.34 M NaOH, 38 min) was 92.8% with a cellulase loading of 30 FPU/g substrate (Yoon et al., 2011). The yield of reducing sugar obtained from steam exploded SCB was 88.95% with a cellulase loading of 10 FPU/g substrate (Li et al., 2013). The enzymatic convertibility of cellulose was 57.4% and 48.9% for wet-oxidized and steam exploded SCB, respectively (Martin et al., 2008). Compared with these studies, the enzymatic hydrolysis efficiency of pretreated JMP400 was acceptable in the present work. Furthermore, a lower NaOH concentration (0.5%) was employed in order to enable the following one-pot SSF. 3.3. Fermentation Conventional SSF was carried out as the control of the one-pot SSF in this work. For all of the fermentations, substrate, cellulase loading and inoculum size were the same. The theoretical ethanol maximum of SSF and one-pot SSF is 26.7 and 20.8 g/L, respectively, based on the cellulose content (Table 1). The value of one-pot SSF is

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calculated from the cellulose content of the untreated sample while that of SSF is from the cellulose content of JMP400. NaCl showed negative influence on yeast growth, ethanol formation, and glucose intake (Moon et al., 2009). Therefore, a lower concentration of NaOH (0.5%), equivalent to 0.73% (w/v) NaCl, was selected to pretreat SCB. High concentration of NaOH pretreatment may transform more lignin into phenolics and inhibit the subsequent one-pot SSF. This was another reason to choose 0.5% NaOH as the pretreatment reagent. The profiles of glucose, ethanol, and xylose during all fermentations are presented in Fig. 3A and B. Laccase could reduce the content of phenolics and thus benefited the fermentability (Jurado et al., 2009). Therefore, laccase was supplemented to SSF and one-pot SSF before fermentation in order to investigate its influence on products. The laccase used in this work showed cellulase and xylanase activity and thus glucose and xylose were detected after laccase treatment for 12 h (Fig. 3A and B). Initial glucose concentration in one-pot SSF with laccase was a little higher than that in SSF with laccase. The ethanol and glucose concentration in the laccase supplemented group was higher during the whole fermentation process (Fig. 3A). This might be because laccase shows auxiliary activity for cellulose and hemicellulose hydrolysis. Ethanol formation stopped increasing much after 72 h of fermentation except for SSF without laccase (Fig. 3A). Glucose concentration in SSFs was lower than that in one-pot SSFs. The fermentation medium of one-pot SSFs contained lignin, hemicellulose, and cellulose degraded components which might depress the utilization of glucose by yeast. SSF with laccase supplementation and one-pot SSF without laccase obtained the highest and lowest ethanol concentration of 15.29 and 12.64 g/L, respectively, corresponding to ethanol yield of 0.32 and 0.36 g/g cellulose, respectively (Table 2). The fermentation efficiency of SSF and one-pot SSF without laccase was 51.57% and 60.73%, respectively, indicating one-pot SSF digested cellulose more efficiently than SSF did. One possible reason may be the cellulase/cellulose loading in one-pot SSF was higher than that in SSF. The positive influence of laccase on xylose release was obvious in all the fermentations. Xylose in one-pot SSFs was more than that in SSFs after 46 h fermentation, while the difference was not obvious before 46 h (Fig. 3B), indicating one-pot SSF could accumulate more xylose. The final xylose concentration in one-pot SSF was 13.55 and 14.25 g/L while that in SSF was 9.79 and 9.74 g/L. If all the xylose converted to ethanol, more ethanol (approximately 6 g/L) would be formed by one-pot SSF. The profile of phenolics was also investigated and is shown in Fig. 3C. The phenolic content in one-pot SSF was approximated 4.5–5-fold more than that in SSF. Laccase had no effect on phenolic content in SSF but enhanced the concentration in one-pot SSF. The main effect of laccase on fermentation was not the detoxification of phenolics but the adding of other enzymatic activities such as cellulase, xylanase, and b-glucosidase. It seemed that phenolic concentration of 1.18 g GAE/L did not depress ethanol formation by yeast, because the ethanol yield and fermentation efficiency of one-pot SSF was higher than that of SSF (Table 2). Our ongoing works showed that the yeast could not grow during one-pot SSF in which 1% of NaOH was used (data not published). It suggests that there is a threshold level of the NaOH concentration when conducting the one-pot SSF. The optimization of NaOH concentration and initial solid loading still needs further exploration. Furthermore, the phenolics could be recovered after the distillation of ethanol and used as antioxidants. The antioxidant activity of pigments from sugarcane juice alcohol wastewater has been studied (Li et al., 2012). As shown in Table 2, the highest fermentation efficiency in the present study was 67.56% from one-pot SSF with laccase. SSF with laccase obtained a fermentation efficiency of 57.27%. As control,

Fig. 3. Time course of the fermentation with S. cerevisiae As2.489: (A) ethanol (open symbols) and glucose (closed symbols); (B) xylose; (C) phenolics. Symbols in (A): s SSF with laccase, r One-pot SSF with laccase, 4 One-pot SSF without laccase, h SSF without laccase. Closed symbols in (A) are the glucose profiles corresponding to the open ones. For one-pot SSF: 10% of untreated JMP400 (dry weight) mixed with 0.5% NaOH, after steam cooking, H3PO4 was added to adjust the pH to 5. Then, 2 g/L yeast extract was supplemented as nitrogen source. For SSF: 10% washed substrate (dry weight) dissolved in distilled water and 2 g/L yeast extract was supplemented as nitrogen source. pH was adjusted to 5 by using H3PO4. Fermentation media were then sterilized. Thereafter, Celluclast 1.5 L 15 FPU/g substrate, Novozym 188 60 pNPGU/g substrate, and 2 g/L S. cerevisiae As2.489 were added to initiate the fermentation.

fermentation efficiency was 82.35% from glucose fermentation. The cellulose conversion rate of one-pot SSF was higher than that of SSF. Laccase enhanced the ethanol concentration, yield and productivity for both one-pot SSF and SSF. It has been reported that 3.36 g/L of ethanol was obtained from pretreated SCB at a substrate loading of 5% (Sasikumar and Viruthagiri, 2010). The fermentation efficiency reached 65% (ethanol yield of 0.28) in a previous study,

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J. Li et al. / Bioresource Technology 161 (2014) 171–178 Table 2 Comparison of various ethanol productions found in the literature and in the current study. Substrate

Etfc

Ety

Etp

FE

Pretreatment

Remarks

Reference

JMP-400 SCB

15.29

0.32

0.45

57.27

0.29

0.33

51.57

14.07

0.40

0.43

67.56

12.64

0.36

0.32

60.73

0.5% NaOH, 121 °C for 1 h

8.40 11.5

0.42b NA

ND NA

82.35c 68

SCB

6.61

0.35

0.19

62.36

SCB

7.19

0.38

0.21

66.89

SCB

3.36

NA

NA

NA

NA

SSF, 10% of substrate loading, 15 FPU/g substrate with laccase SSF, 10% of substrate loading, 15 FPU/g substrate without laccase One-pot SSF, 10% of substrate loading, 15 FPU/g substrate with laccase One-pot SSF, 10% of substrate loading, 15 FPU/g substrate without laccase 2% glucose loading Catalase was added before enzymatic hydrolysis; fermentation was performed using enzymatic hydrolysate without detoxification. 12.9% of substrate loading, 30 mg/g glucan of the optimized enzyme cocktail Substrate was used without rinsing before fermentation, 5% of substrate loading, 10 FPU/g biomass Unwashed substrate was extracted by using ethyl acetate before fermentation, 5% of substrate loading, 10 FPU/g biomass 5% of substrate loading, 15 FPU/g substrate

This study

13.77

0.5% NaOH, 121 °C for 1 h followed by washing with water 0.5% NaOH, 121 °C for 1 h followed by washing with water 0.5% NaOH, 121 °C for 1 h

SCB

NA

0.28

NA

65

Steam explosion with SO2

SCB

25

NA

NA

79.4

Alkali/oxidative

SCB

14.71

NA

NA

68.95

Hydrothermal, 200 °C for 12.5 min

SCB

12.44

NA

NA

50.07

Hydrothermal, 190 °C for 20 min

JMP-400 SCB

Glucose CS

a

Alkaline hydrogen peroxide. Conditions: 24 °C, 0.375 mL of 30% (w/w) H2O2 and 0.5 mL of 5 M NaOH/g biomass

Steam explosion. Conditions: 220 °C, residence time 5 min, LSR: 1:1 Steam explosion. Conditions: 220 °C, residence time 5 min, LSR: 1:1

Substrate was washed before fermentation, cellulose Substrate was washed before fermentation, substrate loading, 20 FPU/g cellulose Substrate was washed before fermentation, substrate loading, 15 FPU/g cellulose Substrate was washed before fermentation, substrate loading, 15 FPU/g cellulose

10 FPU/g 10% of 10% of 10% of

This study This study This study This study Banerjee et al., (2012)

Li et al., (2014a) Li et al., (2014a) Sasikumar and Viruthagiri, (2010) Ewanick and Bura, (2011) Cheng et al., (2008) da Cruz et al., (2012) da Cruz et al., (2012)

a

Glucose fermentation was used as reference experiment. g ethanol/g glucose. c Ethanol/theoretical maximum. NA: not available. ND: not determined. Etfc: Final ethanol concentration (g/L); Ety: ethanol yield (g/g cellulose); Etp: ethanol productivity at the fermentation time of 22 h (g/L h); FE: fermentation efficiency (%); CS: corn stover. b

where SCB was pretreated by steam explosion with SO2 and the pretreated SCB was rinsed before fermentation (Ewanick and Bura, 2011). Alkaline/oxidative pretreated SCB was fermented to ethanol and the final ethanol concentration was 25 g/L corresponding to 79.4% of fermentation efficiency. The alkaline/oxidative pretreated SCB was also rinsed before fermentation and the cellulase loading was 20 FPU/g cellulose (Cheng et al., 2008). Hydrothermal pretreatment was also used for SCB and the fermentation efficiency was 68.95% and 50.07%, respectively, from different pretreatment conditions. Pretreated SCB was still washed with water before fermentation (da Cruz et al., 2012). As previously reported, ethanol yield from steam-exploded SCB without rinsing reached 62.36% and that from solvent extracted steam-exploded SCB reached 66.89% (Li et al., 2014a). The ethanol yield of N-methylmorpholine-N-oxide pretreated SCB was 0.15 (Kuo and Lee, 2009) while that was 0.126–0.141 g ethanol/g bagasse in one-pot SSF without and with laccase. Compared with these results, the fermentation efficiency of one-pot SSF in the present study was comparable. It should be noted that another study reported that alkaline hydrogen peroxide pretreatment could be integrated with enzymatic hydrolysis and fermentation. NaOH and H2O2 loadings were 100 and 112.5 mg/g substrate and lower temperature (24 °C) was employed during pretreatment. Enzymatic hydrolysis was conducted using an optimized enzyme cocktail containing cellulase, b-glucosidase, pectinase, and xylanase and the enzymatic hydrolysate was used as feedstock to produce ethanol. The final ethanol concentration reached 11.5 g/L (fermentation efficiency of 68%) using a glucose- and xylose-utilizing strain of S. cerevisiae (Banerjee et al., 2012). In the current study, an ordinary yeast strain which could not metabolize xylose was used and the final ethanol concentration reached 14.07 g/L (fermentation efficiency of

67.56%, Table 2). NaOH loading was 50 mg/g of milled SCB and a higher temperature (121 °C) was used. The higher temperature could be merged with sterilization process in a realistic scale-up scenario (Fig. 1D). During fermentation, only cellulase and b-glucosidase were loaded. Further optimization focusing on fermenting microorganism, enzyme cocktails, and enzyme loadings may improve the ethanol yield further. Which of the two configurations will be better should consider many factors comprehensively including capital costs, process simplicity, pollutant discharge, compatibility with enzymatic hydrolysis and fermentation at high solid loading to produce high titer of bioethanol etc. These still need to be explored. Based on the above, the features of one-pot SSF are: (1) rinsing water is eliminated; (2) sterilization and pretreatment process can be merged into one; (3) recycle of NaOH or the treatment of the rinsing wastewater is also eliminated; (4) biomass recovery is 100%. However, this unique fermentation configuration needs size reduction of lignocellulose which is thought to be a high energy consumption process and thus to be not economically feasible (Ewanick and Bura, 2010). There may be a compromise between the cost of size reduction and the cost of rinsing water, wastewater treatment, and additional equipment for biomass recovery. Onepot SSF might be a promising option in industrial application to produce bioethanol from biomass.

4. Conclusions A large part of lignin was removed from SCB after 0.5% NaOH pretreatment at 121 °C for 60 min. Chemical composition of the samples resulted from the combined pretreatment was not

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affected much. However, size reduction before NaOH pretreatment enhanced the sugar production from the combined pretreatment. The highest glucose yield which was obtained from the smallest particles reached 64.5% of theoretical maximum. Fermentation efficiency reached 67.56% and 60.73% for one-pot SSF with and without laccase compared to 57.27% and 51.57% for SSF with and without laccase. Further optimization of one-pot SSF may improve the ethanol yield. One-pot SSF might be a new direction for bioethanol production. Acknowledgements This work was supported by the Science and Technology Planning Project of Guangdong Province (No. 2012B020311005), the ‘Fundamental Research Funds for the Central Universities’ (21614333), and the ‘Program for New Century Excellent Talents in University’ (NCET-05-0745). The authors would like to thank Novozymes (China) for their generous gift of Celluclast 1.5 L and Novozym 188. References Banerjee, G., Car, S., Liu, T., Williams, D.L., Meza, S.L., Walton, J.D., Hodge, D.B., 2012. Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation. Biotechnol. Bioeng. 109, 922–931. Cantarella, M., Cantarella, L., Gallifuoco, A., Spera, A., Alfani, F., 2004. Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF. Biotechnol. Prog. 20, 200–206. Chen, M., Zhao, J., Xia, L., 2009. Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility. Biomass Bioenergy 33, 1381–1385. Cheng, K.-K., Zhang, J.-A., Ping, W.-X., Ge, J.-P., Zhou, Y.-J., Ling, H.-Z., Xu, J.-M., 2008. Sugarcane bagasse mild alkaline/oxidative pretreatment for ethanol production by alkaline recycle process. Appl. Biochem. Biotechnol. 151, 43–50. Chundawat, S.P.S., Balan, V., Da Costa, L., Dale, B.E., 2010. Thermochemical pretreatment of lignocellulosic biomass. In: Waldron, Keith (Ed.), Bioalcohol Production. Woodhead Publishing, Cambridge, UK, pp. 24–72. Da Cruz, S.H., Dien, B.S., Nichols, N.N., Saha, B.C., Cotta, M.A., 2012. Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF. J. Ind. Microbiol. Biotechnol. 39, 439–447. Ewanick, S., Bura, R., 2010. Hydrothermal pretreatment of lignocellulosic biomass. In: Waldron, Keith (Ed.), Bioalcohol Production. Woodhead Publishing, Cambridge, UK, pp. 1–23. Ewanick, S., Bura, R., 2011. The effect of biomass moisture content on bioethanol yields from steam pretreated switchgrass and sugarcane bagasse. Bioresour. Technol. 102, 2651–2658. Harun, S., Balan, V., Takriff, M.S., Hassan, O., Jahim, J., Dale, B.E., 2013. Performance of AFEX™ pretreated rice straw as source of fermentable sugars: the influence of particle size. Biotechnol. Biofuels 6, 40. Jurado, M., Prieto, A., Martínez-Alcalá, A., Martínez, A.T., Martínez, M.J., 2009. Laccase detoxification of steam-exploded wheat straw for second generation bioethanol. Bioresour. Technol. 100, 6378–6384. Kim, S., Holtzapple, M.T., 2005. Lime pretreatment and enzymatic hydrolysis of corn stover. Bioresour. Technol. 96, 1994–2006. Klinke, H.B., Thomsen, a.B., Ahring, B.K., 2004. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl. Microbiol. Biotechnol. 66, 10–26.

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