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In-situ corn fiber conversion improves ethanol yield in corn dry-mill process
Industrial Crops & Products 113 (2018) 217–224
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In-situ corn fiber conversion improves ethanol yield in corn dry-mill process a
b
Xiujuan Li , Sitong Chen , He Huang
c,d,⁎⁎
b,⁎
T
, Mingjie Jin
a
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing, 211816, China School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei Street, Nanjing, 210094, China College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, China d Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Corn fiber conversion Dry-mill process Simultaneous saccharification and fermentation (SSF) Cellulase Ethanol
Dry-mill process has been widely applied for ethanol production from corn. However, corn kernel fiber (mostly cellulose) is typically unconverted in the process. This work investigated the effect of corn cellulose conversion on the performance of dry-mill process. Solid loading and pH were optimized first for better design of cellulose conversion in the process. Cellulase was dosed for cellulose conversion during Simultaneous Saccharification and Fermentation (SSF). Addition of cellulase improved ethanol yield by 1.8% compared to traditional SSF without cellulase. Cellulose conversion reached 19.8%. Starch conversion was also enhanced from 98.3% to 99.0% with glycerol yield, which is a major side-product of ethanol fermentation, decreased by 7.3%. Residual starch and residual cellulose resulted from SSF were further studied for their degradability and it was found that 63.2% of residual starch and 14.3% of residual cellulose could be further degraded in the absence of ethanol. This work implies that cellulose conversion is of great benefit to corn ethanol process.
1. Introduction With the development of industrialization and increase of population, global energy demand is increasing dramatically, and the most energy supplies are derived from non-renewable fossil fuel (Kumar and Singh, 2016). With consideration of environmental and social sustainability, renewable energy development has drawn much attention (Zhang et al., 2007). Bioethanol, which can be produced from starchy, sugary and cellulosic materials through fermentation, is considered as the most promising renewable fuel. Starchy crops (e.g. corn) are widely used for bioethanol production due to their availability, ease of conversion, storage capability for a long period and high ethanol yield (Balat, 2011). Currently, most fuel ethanol in United States is produced from corn by dry mill or wet mill process (Bothast and Schlicher, 2005). Although both processes are being employed for ethanol production, 67% of the commercial plants use dry-mill process. Starch comprises 70–72% of the kernel weight on a dry basis, and is readily degraded to fermentable glucose, which can be further fermented to ethanol. However, the biodegradability of starch during enzymatic saccharification is largely determined by its structure that varies with regard to botanical sources and crop hybrids. This variation in structure affects overall starch conversion and thereby ethanol yield. In addition, the amylose and amylopectin ratio also affects
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physicochemical properties of starch and hence affects ethanol yield (Singh and Graeber, 2005; Yangcheng et al., 2013). Recent advances in improving ethanol yield and process profitability include the use of high yield corn varieties (Shi et al., 2013; Zabed et al., 2016), development of non-cooking hydrolysis technique with granular starch hydrolyzing enzyme (GSHE) (Li et al., 2014; Uthumporn et al., 2010; Wang et al., 2005), cell immobilization (Mishra et al., 2016; Najafpour et al., 2004), the use of superior yeast (Favaro et al., 2015; Nakamura et al., 1997; Shigechi et al., 2004), the enhancement of glucose release rate (Khanal et al., 2007) and recovery of high-value co-products (Somavat et al., 2016), etc. Although technologies have been well developed to convert corn starch to ethanol, corn kernel fiber (mostly cellulose), which accounts for around 4 percent of total sugar in corn, is unutilized in the current process. Conversion of corn cellulose is of great interest in the field (Porter et al., 2007). However, most studies focused on conversion of isolated corn fiber (Gáspár et al., 2007; Van Eylen et al., 2011) or DDGS (dried distillers’ grains with solubles) resulted from dry-mill process (Kim et al., 2010; Lau et al., 2008). In-situ conversion of cellulose during dry-mill process is not well studied. Cellulase is a mixture of different cellulytic enzymes that act synergistically on the cellulose molecules to degrade cellulose to glucose (Awafo et al., 2000). The addition of cellulase in the corn ethanol process to in-situ convert
Corresponding author at: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei Street, Nanjing, 210094, China. Corresponding author at: College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, China. E-mail addresses: [email protected] (H. Huang), [email protected] (M. Jin).
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https://doi.org/10.1016/j.indcrop.2018.01.037 Received 11 October 2017; Received in revised form 10 January 2018; Accepted 14 January 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Analytical methods
cellulose could potentially improve ethanol yield as it not only degrades cellulose but may also synergize with glucoamylase to further hydrolyze recalcitrant starch. Cellulase can help disrupt cell wall structure of grain and promote the release of contents including starch and protein, which plays the auxiliary role in improving ethanol yield of starch. Meanwhile, the addition of cellulase may decrease viscosity of fermentation broth and improve its rheological property (Knutsen and Liberatore, 2010), which facilitates saccharification and fermentation. There are two stages in corn ethanol process that cellulase can potentially be dosed, namely liquefaction stage and Simultaneous Saccharification and Fermentation (SSF) stage (performs enzymatic saccharification and fermentation in the same bioreactor). Liquefaction stage uses alpha-amylase and carries out at above 85 °C. This temperature is much higher than most cellulases can tolerate. The other stage cellulases can be dosed is the SSF stage. However, there are several factors need to be investigated. SSF performs at 30 °C, which is lower than the optimal temperature of cellulase (50 °C). Fermentation causes pH decrease rapidly in the first 24 h and results in high ethanol concentration, which both affect cellulase activity. Therefore, it remains unknown how well cellulase can perform during SSF. The main objective of this work was to in-situ convert corn cellulose during dry-mill process and investigate the effect of cellulase addition on SSF performances, including starch conversion, cellulose conversion and ethanol yield. Residual starch and residual cellulose resulted from SSF were further studied for their degradability.
The concentration of maltose, glucose, xylose, organic acids, glycerol and ethanol was analyzed by HPLC with a Biorad Aminex HPX87H column. The analysis conditions were as follows: column temperature (65 °C), mobile phase (5 mM H2SO4, 0.6 mL/min). Total sugar and oligomeric sugar were analyzed according to procedures of NREL-TP-510-42618 and NREL-TP-510-4262, respectively. The starch content was analyzed using NREL analytical procedure NREL-TP-510-42624. Cellulose content was obtained by subtracting starch from total sugar. Ethanol yield was determined based on the theoretical ethanol yield from consumed glucose, which is 0.51 g ethanol/g sugar. Moisture content of biomass was measured by a moisture analyzer (METTLER TOLEDO). The number of yeast cells in the fermentation broth was counted using a hemocytometer (Shanghai Qiujing biochemical reagents Co. Ltd) and microscope (CX31RTSF, OLYMPUS). The cells were stained with methylene blue dye (0.1%) which stains dead cells blue while living cells remain uncolored. The OD value of yeast was determined using ultraviolet and visible spectrophotometer (TU-1810, Beijing Purkinje General Instrument CO. Ltd) at 600 nm. 2.4. Effect of pH and ethanol on cellulase activity Cellulase activity was assayed at different pH (3–5) and different concentrations of ethanol (0–20%) with the modified method of NRELTP-510-42628 using filter paper as substrate. Citric acid-sodium citrate buffer (0.025 M) at different pH and ethanol solution were prepared and sterilized prior to use. Different amount of ethanol was added to the citrate buffer to form an ethanol-buffer mixture with ethanol concentrations of 0, 5, 10, 15, 17.5 and 20% (v/v). With the modified NREL-TP-510-42628 method, 0.5 mL of diluted enzyme was added into 1 mL of citrate-buffer-ethanol mixtures with different pH and ethanol concentration. After 60 min of hydrolysis reaction at 50 °C, 3 mL DNS reagent was added to measure the cellulase activity.
2. Materials and methods 2.1. Materials Corn flour was generously given by China Oil & Foodstuffs Corporation (COFCO), Anhui, China. The glucan and xylan contents of corn were 81.96% and 2.50%, respectively. The starch and cellulose contents of corn were 77.86% and 4.10%, respectively. The alphaamylase, glucoamylase and cellulase used in this study were supplied by Genencor Bio-Products Co. Ltd., Shandong Longda Bio-products Co. Ltd. and Vland Biothech Co. Ltd., respectively. Microorganism and seed culture preparation Saccharomyces cerevisiae was obtained from Angel Yeast Co., Ltd. Seed culture of S. cerevisiae was prepared in YPD medium (10 g/L yeast extract and 20 g/L tryptone) with 100 g/L glucose in a 250 mL Erlenmeyer flask with a working volume of 120 mL. The culture was incubated at 30 °C and 150 rpm under micro-aerophilic conditions with sterile and breathable filtering membrane for 24 h.
2.5. Effect of ethanol concentration on yeast activity The ability of yeast to consume glucose under different concentrations of ethanol was examined in the medium containing 5 g/L glucose and various concentrations of ethanol. The glucose solutions were prepared with citric acid-sodium citrate buffer (0.025 M, pH 4.5) and sterilized prior to use. Ethanol concentration tested ranged from 0 to 20% (v/v). The yeast cells were inoculated at an OD600 of 1.0 (0.44 g/L broth), 3.0 (1.32 g/L broth) and 6.0 (2.64 g/L broth) and cultured at 30 °C, 150 rpm for 48 h. The consumption of glucose was analyzed after incubation.
2.2. Simultaneous saccharification and fermentation (SSF) The corn flour was mixed with deionized water to make slurry at different solid loadings (27%, 30% and 33% w/w). For liquefaction, the pH of the slurry was adjusted to 5.7 using 40% sodium hydroxide solution and 0.048 g of α-amylase was used per 100 g dry corn. The liquefaction was performed in a shake flask at 85 °C for 4 h using water bath with intermittent mixing. The pH of the cooling liquefied mash was adjusted to 4.6 using sulfuric acid for SSF process. SSF was carried out in a 250 mL Erlenmeyer flask with a total mixture weight of 150 g in triplicate at 30 °C, pH 4.6, and 150 rpm. Yeast seed culture was centrifuged at 4000 rpm for 10 min to obtain yeast cell pellet, which was used for inoculation. The initial OD600 for SSF was 1.0, which means 0.44 g yeast cells were used per 1 L fermentation broth. Urea (1.28 g/kg mash) and glucoamylase (0.1 g/100 g dry corn) were added. A rubber stopper with a needle piercing through it was used to cap the flask to maintain largely anaerobic conditions. To monitor the fermentation performance, about 2 mL of samples were drawn at 0, 4, 16, 24, 36, 48, 72, 96 and 120 h and centrifuged at 10,000 rpm for 10 min. The supernatants were immediately sterile filtered through a 0.22 μm filter for HPLC analysis.
2.6. Degradability of residual starch and residual cellulose After SSF at 30% solid loading, the whole broth without any treatments was used as control group to study the degradability of residual starch and residual cellulose. At the same time, to remove ethanol effect, ethanol was evaporated from SSF broth (60 g) with rotary evaporator (RE100-Pro, Scilogex) at 65 °C, 10 min and 0.07 MPa. The broth without ethanol was performed as experimental group. The degradability of residual starch was carried out in a 100 mL Erlenmeyer flask with a total mixture weight of 60 g in triplicates. Alpha-amylase (0.048 g/100 dry corn) was added to make another round liquefaction at 85 °C for 4 h. Glucoamylase (0.1 g/100 g dry corn) was then added to hydrolyze at 30 °C or 60 °C and 150 rpm for 24 h. Residual starch content and released glucose were then analyzed again. The degradability of residual cellulose was tested by adding cellulase (0.6 g/100 dry corn) to the broth for hydrolyze residual cellulose at 50 °C or 30 °C and 250 rpm for 48 h. 218
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Fig. 1. Effect of different solid loading on glucose consumption (a), ethanol yield (b), death rate of yeast (c) and starch conversion (d) during SSF. The death rate of yeast was calculated by dividing viable cells by total cells count. All the experiments were performed in triplicate.
3. Results and discussion
93.6% and 93.3% at 120 h, respectively. There is no significant difference in starch conversion for different solid loadings at 120 h, and so does ethanol yield, but SSF with 33% solid loading was poor in fermentation efficiency with 9.1 g/L glucose unfermented at 120 h. Moreover, SSF at 33% solid loading might have led to higher viscosity, higher osmotic pressure and higher ethanol inhibition, which affected the viability of fermenting yeast (Fig. 1c) and caused poor fermentation efficiency (Kristensen et al., 2009; Nguyen et al., 2014; Wang et al., 2008). Since the SSF at 30% solid loading was fermented completely and further increasing solid loading caused fermentation issues, we subsequently chose to work on 30% solid loading in the future studies. As we all know, pH is a key factor for ethanol fermentation, which has a direct effect on yeast metabolism (Kasemets et al., 2007; Masiero et al., 2014). In addition, the enzyme activities are also affected by pH value. Here, we investigated the effect of different initial pH (4.0, 4.5, 5.0) on 30% solid loading SSF. Surprisingly, no obvious effect of pH on glucose consumption (Fig. 2a), ethanol yield (Fig. 2b), pH change during SSF (Fig. 2c) and death rate of yeast cell (Fig. 2d) was observed. We also investigated the cellulase activities on filter papers at different pH (3–5). As shown in Table 2, the optimum pH for cellulase activity was between 4.6 and 5.0. Considering optimal pH of glucoamylase (4.2–4.6), we selected pH 4.6 as the initial pH of SSF in the subsequent studies.
3.1. Optimization of solid loading and initial pH of SSF Increasing solid loading of SSF increases final ethanol titer, decreases energy consumption and reduces process cost. However, higher solid loading resulted in poor glucose consumption and lower ethanol yield (Jin et al., 2017; Wang et al., 1999). Here we compared the corn ethanol fermentation performances at 27%, 30% and 33% (w/w) solid loadings (Fig. 1). Glucose concentration increased from 0 to 8 h, then rapidly decreased, which indicates that saccharification rate was higher than fermentation rate in the first 8 h and then fermentation rate overtook saccharification rate afterwards. For 27% and 30% solid loadings, glucose was consumed completely within fermentation time of 72 h, and ethanol reached maximums of 111.3 and 127.0 g/L, respectively. For 33% solid loading, there was still 3.7 g/L glucose remaining in the fermentation broth at 72 h and this glucose concentration slightly increased thereafter. This implies that at 33% solid loading saccharification was incomplete in 72 h but the yeast cell viability was already low with a high death rate of 95.0% (Fig. 1c), which caused a considerable amount of glucose unfermented. Byproduct concentrations are shown in Table 1. The main byproduct was glycerol and the glycerol to ethanol ratio was almost identical for all tested solid loadings. For a better understanding of the process at different solid loadings, starch conversions and ethanol yields were determined. The starch conversions at different solid loadings were 97.8%, 98.3% and 98.0% at 120 h, respectively. (Fig. 1d). Correspondingly, the ethanol yields based on the glucose consumption at different solid loading were 93.1%,
3.2. Addition of cellulase significantly improves ethanol yield during SSF Aiming to convert corn cellulose and improve ethanol yield, the addition of cellulase during SSF was investigated at solid loading of
Table 1 Byproducts and ethanol concentrations resulted from SSF at different solid loadings and at different pHs. Experiment with different solid loadings was performed at pH 4.6; Experiment with different initial pHs was performed at solid loading of 30%.
27% solid loading 30% solid loading 33% solid loading Initial pH 4.0 Initial pH 4.5 Initial pH 5.0
Succinic acid (g/L)
Lactic acid (g/L)
Glycerol (g/L)
Acetic acid (g/L)
Ethanol (g/L)
Glycerol /Ethanol
0.51 0.46 0.37 0.45 0.46 0.64
0.48 0.63 0.56 0.52 0.63 0.54
10.08 11.26 12.32 10.75 11.26 11.38
0.21 0.23 0.46 0.57 0.23 0.42
111.33 127.02 137.50 126.52 127.02 126.44
0.09 0.09 0.09 0.08 0.09 0.09
± ± ± ± ± ±
0.01 0.02 0.03 0.01 0.02 0.01
± ± ± ± ± ±
0.01 0.01 0.08 0.02 0.01 0.01
± ± ± ± ± ±
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0.20 0.05 0.21 0.08 0.05 0.13
± ± ± ± ± ±
0.01 0.06 0.01 0.05 0.06 0.05
± ± ± ± ± ±
0.25 0.26 0.21 0.20 0.26 0.23
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Fig. 2. Effect of initial pH on glucose consumption (a), ethanol yield (b), pH change (c) and death rate of yeast (d) during SSF. SSF was carried out at 30% solids loading. The pH of the slurry was adjusted to the predefined value (4.0, 4.5 and 5.0) using 72% sulfuric acid solution before SSF. All the experiments were performed in triplicate.
increased from 98.3% to 99.0%, cellulose conversion reached 19.8% (Fig. 3b). Overall, ethanol yield with the addition of cellulase was improved by 1.8%, which could be a big profit to ethanol plants. For a better understanding of the SSF performances, mass balance comparison between SSF with addition of cellulase and SSF without addition of cellulase were calculated according to the experimental data by shaking flask in our laboratory (Fig. 4). As Fig. 4a shows, 408.7 ± 0.40 kg ethanol was produced from 1 t of corn through SSF with the addition of cellulase at the solid loading of 30%. The ethanol yield for SSF without cellulase addition is 403.5 ± 0.26 kg/tonne corn. The differences between these ethanol yields were statistically significant (P < 0.01). This implies that the improved sugar conversion and decreased glycerol yield resulted in the enhancement of ethanol yield. The experiments’ data had a carbon closure of about 98% (Fig. 4b), which verified the accuracy of our analyses. Considering the cellulase itself used in our study contains 10.40 g/L glucose, we also analyzed the effect of cellulase itself on the improvement of ethanol yield. Based on the SSF with 1 t of corn dry weight and 0.6% cellulase (Fig. 4a), the result showed that 27.68 g ethanol was theoretically
30% and pH 4.6. Cellulase was added simultaneously with glucoamylase at the beginning of SSF. Different dosage of cellulase (0.1%, 0.3%, 0.6%, 0.9% per corn dry weight) was optimized and shown in Supplementary Table S1. With increase of cellulase dosage, ethanol yield increased and glycerol yield decreased until the dosage reached at 0.6%. Higher dosage did not give further increase in ethanol yield. Therefore, we selected 0.6% (w/w) per corn dry weight as the optimal cellulase dosage in SSF. The effect of cellulase addition at the dosage of 0.6% on SSF performances are shown in Fig. 3. Glucose release rate of SSF with the addition of cellulase was faster than the control (without cellulase addition) during initial stage (Fig. 3a). Meanwhile, death rate of yeast was slightly lower (Fig. 3c) and ethanol titer was higher compared to control (Fig. 3a). After 120 h, 129.3 ± 0.30 g/L ethanol was produced with cellulase addition, which is a 1.8% lift compared to the control (127.0 ± 0.26 g/L ethanol). Glycerol production was occurred mostly during the first 24 h of SSF. As Fig. 3c shows, glycerol yield was decreased from 11.3 to 10.5 g/L with the addition of cellulase. With the addition of cellulase in SSF, starch conversion was also slightly
Table 2 Effect of pH and ethanol on cellulase activity. pH
Specific activity (FPU/ml)
5.0 4.6 4.2 3.8 3.4 3.0
116.08 ± 0.64 113.39 ± 0.50 93.78 ± 0.52 72.83 ± 0.54 43.65 ± 1.02 8.62 ± 0.50
a
Specific activity (FPU/ml)
0 5 10 15 17.5 20
114.62 ± 0.50 72.12 ± 1.00 47.36 ± 0.51 33.42 ± 0.50 16.79 ± 0.53 9.40 ± 0.31
b
b
100.00 97.69 80.79 62.75 37.60 7.42
Ethanol (%, v/v)
a
Relative activity (%)
a
Relative activity (%) 100.00 62.92 41.32 29.16 14.65 8.20
Activity assay was performed at 50 °C, and pH 4.8 for 60 min. The cellulase activity at pH 5.0 (116.08 U/mL) and 0% of Ethanol (114.62 U/mL) was defined as 100%, respectively.
220
b
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Fig. 3. Effect of Cellulase addition on SSF performance. a) glucose consumption and ethanol production; b) sugar conversion; c) glycerol production and death rate of yeast; d) residual sugar content. Experiments were performed at 30% (w/w) solid loading. Cellulase at dosage of 0.6% per corn dry basis was added simultaneously with glucoamylase at the beginning of SSF. SSF was performed at 30 °C, pH 4.6, 150 rpm for 120 h. Residual starch and cellulose after SSF without cellulase addition were defined as 100%. All the experiments were performed in triplicate.
insights into further enhancement of sugar conversions. The addition of cellulase for improvement of ethanol yield does not require any additional equipment or control system, the only additional cost would be the cellulase, whose loading could be further reduced by using more advanced cellulase cocktails. The estimated increase in yield may be sufficiently high to carry the additional costs for the addition of cellulases; however, a full techno-economic analysis needs to be performed before any conclusive statements in terms of economic improvement can be made.
produced if the glucose in cellulase solution was converted completely. A very small increase, less than 0.01%, was obtained compared with SSF without the addition of cellulase, which indicated that cellulase solution itself had low impact on the ethanol yield during SSF. To further investigate the effect of cellulase addition on higher solid loading process, we also carried out the SSF at 33% solid loading and 1.2% ethanol lift was observed with cellulase addition (Supplementary Fig. S1). Although the addition of cellulase improved starch and cellulose conversions, there is room for improvement. If the amount of residual starch and residual cellulose after SSF with no cellulase addition was defined as 100%, there were still 59.2% residual starch and 80.1% residual cellulose unconverted with the addition of cellulase (Fig. 3d). Therefore, it would be worthwhile to investigate the influential factors for incomplete degradation of starch and cellulose, which may provide
3.3. High concentration of ethanol is a barrier for further improvement of sugar conversions As discussed above, the incomplete degradation of sugar, especially incomplete cellulose degradation, is observed in SSF even with the Fig. 4. Mass balance comparison between SSF with cellulase addition and SSF without cellulase addition at 30% solid loading. Based on 1 t corn dry weight, carbon balance was calculated without taking xylan into account since xylan/xylose cannot be utilized by this fermenting yeast. Carbon closure was calculated using equation: [(products carbon mass + residual sugar carbon mass)/initial carbon mass] × 100. Initial carbon mass was calculated based on glucan carbon in corn flour. Final carbon mass was calculated based on carbons in ethanol, carbon dioxide, byproducts and residual C6 sugars. All the experiments were performed in triplicate.
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Table 3. SSF at 33% solid loading with the addition of cellulase produced 138.6 g/L ethanol at 72 h, that is about 17.6% (v/v). At that concentration, the death rate of yeast reached 100% after 12 h of incubation (Table 3), which might have caused accumulated glucose unfermented. It indicated that high concentration of ethanol inhibits microorganism growth and viability (Fischer et al., 2008), and further influence glucose consumption and ethanol yield, especially in SSF at 33% solid loading. As discussed above, high concentration of ethanol greatly decreased the activities of yeast and cellulase, which limited the improvement of SSF efficiency through process engineering. Further studies could focus on enhancement of cellulase and yeast tolerances to ethanol.
addition of cellulase. The high ethanol concentration, which was produced during SSF, might be a cause for incomplete sugar conversions. It is well known that ethanol affects the activities of cellulase and yeast. Here, we investigated the cellulase activities on filter papers and yeast activities in the presence of ethanol at different concentrations. In view of actual ethanol concentration during SSF, the effect of ethanol on cellulase activities was studied using ethanol concentration ranging from 0 to 20% (v/v). The specific activity and relative activity of enzyme under different concentrations of ethanol are presented in Table 2. The cellulase activity decreased as the ethanol concentration increased. Ethanol titer during SSF (30% solid loading) with the addition of cellulase reached 125.1 g/L at 48 h, which is about 15.9% (v/v). At that concentration, cellulase activity was already reduced by 71%. When ethanol concentration reached 129.3 g/L at 120 h, that is about 16.4% (v/v), the remaining cellulase activity was about 14.7% to 29.2% of its initial. In addition to ethanol inhibition, pH fluctuation and suboptimal temperature rendered cellulase not working at its full potential. The effect of ethanol on yeast activities was also studied. Ethanol concentration tested ranged from 0 to 20% (v/v). Initial yeast ODs of 1, 3, and 6 were used for study to examine whether ethanol tolerance is related to yeast cell density. As shown in Fig. 5, glucose was completely consumed in 24 h for all tested initial ODs when ethanol concentration was lower than or equal to 10%. When the medium ethanol concentration was 15%, significant inhibition was seen for initial ODs of 1 with 66.9% residual glucose unutilized after 48 h fermentation. However, fermentation with initial OD of 3 still consumed all the glucose in 48 h and fermentation with initial OD of 6 consumed all the glucose in 24 h. It indicated that higher cell density did help in resisting ethanol inhibition. Higher ethanol concentrations (17.5% and 20%) substantially reduced glucose consumption by the yeast for all tested initial ODs with higher OD consumed more glucose. Higher ethanol concentration caused faster cell death (Fig. 5d), which might be the reason for reduced glucose consumption. The death rate of yeast and residual glucose content in the medium with ethanol concentrations of 15%, 17.5% and 20% are shown in
3.4. Residual starch and cellulose in SSF can be further degraded Though ethanol inhibition reduced cellulase activity, it was still unclear if the residual starch and cellulose were degradable by enzymes. Therefore, we investigated the degradability of residual starch and cellulose. The 120 h SSF broth (containing unconverted solids and 129.3 g/L ethanol) with and without ethanol evaporated was used for another round liquefaction using α-amylase and saccharification using glucoamylase (without fermentation). Saccharification was performed at 60 °C first for 24 h. As shown in Fig. 6a, without ethanol evaporated, liquefaction and saccharification released around 4.2 g/L glucose, while removal of ethanol further increased this concentration to 5.7 g/L, which is a 35.7% lift. Starch analysis showed that the residual starch conversions with and without ethanol reached 45.0% and 63.2%, respectively (Fig. 6a). In view of the actual fermentation temperature (30 °C), we also performed the saccharification at 30 °C with comparison to 60 °C. It was found that 30 °C resulted in only 16.0% and 29.6% of residual starch conversions for cases with and without ethanol, respectively. It indicated that SSF temperature did not favor further degradation of residual starch. Considering the starch conversion already reached 99.0% during SSF, residual starch accounted for 1% of the initial total starch. Under SSF conditions (30 °C, with ethanol presence), there was only 16.0% of residual starch further degradable, which Fig. 5. Effect of ethanol concentration on yeast activity. The initial OD of yeast in medium was 1 (a), 3 (b) and 6(c) and the initial glucose concentration was 5 g/L. Death rate of yeast in medium with the initial OD of 6 (d) was measured by hemocytometer using the methylene blue as dye. All the experiments were performed in triplicate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 3 Death rate of yeast and residual glucose in the medium with different concentration of ethanol. Initial OD600 nm of yeast
Time (h)
15% Ethanol (118 g/L) Dr (%)
1
3
6
a b
12 24 48 12 24 48 12 24 48
a
19.12 ± 0.75 80.32 ± 1.02 100.00 ± 0.00 19.42 ± 0.73 64.15 ± 1.00 91.32 ± 1.02 23.43 ± 1.05 93.31 ± 1.02 99.34 ± 0.70
R.glu (%)
b
77.77 ± 0.52 71.01 ± 1.00 66.86 ± 1.00 38.38 ± 1.02 16.45 ± 0.74 4.10 ± 0.21 2.41 ± 0.50 0.39 ± 0.05 0.24 ± 0.05
17.5% Ethanol (138 g/L)
20%Ethanol (158 g/L)
Dr (%)
R.glu (%)
Dr (%)
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 81.23 ± 0.78 100.00 ± 0.00 100.00 + 0.00 79.43 ± 0.76 100.00 ± 0.00 100.00 ± 0.00
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 83.31 ± 0.96 82.15 ± 1.21 82.15 ± 1.21 34.93 ± 0.97 28.59 ± 0.92 28.59 ± 0.92
100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
R.glu (%) ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 98.04 ± 0.64 98.04 ± 0.64 98.04 ± 0.64 82.99 ± 0.51 82.99 ± 0.51 82.99 ± 0.51
Dr: Death rate of yeast in the medium. R.glu: Residual glucose in the medium. The initial glucose concentration was defined as 100%.
cocktail might help in enhancing corn cellulose conversion. Starch conversion with cellulase cocktail might because cellulase disrupted cell wall structure of grain and promoted starch release. It also possible that the cellulase cocktail also contained amylases. In summary, additional sugars (4–9 g/L) could be released under optimum conditions and this sugar stream could potentially further enhance ethanol yield. However, the required optimum conditions do not consistent with current SSF conditions.
means the current process already did very well in converting starch with only 0.16% of the total starch potentially being further converted. With addition of α-amylase and glucoamylase, no residual cellulose was converted (Fig. 6a). The degradability of residual cellulose in 120 h SSF broth was also investigated by addition of 0.6% cellulase at both 50 °C and 30 °C for 48 h. As shown in Fig. 6b, without ethanol evaporated, cellulase hydrolysis released around 5.9 g/L glucose, while removal of ethanol further increased this concentration to 9.3 g/L, which is a 57.6% increase. Starch was also converted by this cellulase cocktail. Starch analysis showed that the residual starch conversions with and without ethanol were 20.2% and 32.4%, while residual cellulose conversions were 8.3% and 14.3%, respectively at 50 °C. Reaction at 30 °C decreased both starch conversion and cellulose conversion, resulting in only 7.5% and 16.3% of residual starch conversions for cases with and without ethanol, and residual cellulose conversions of 2.1% and 6.2%, respectively. This indicates that the residual cellulose was mostly undegradable by current cellulase cocktail. Cellulose pretreatment (Noureddini and Byun, 2010; Porter et al., 2007) or advanced cellulase
4. Conclusion Cellulase addition in SSF at 30% solid loading resulted in 1.8% increase in ethanol yield and 7.3% decrease in glycerol production, with starch and cellulose conversions reaching 99.0% and 19.8%, respectively. High concentration of ethanol inhibited both cellulase and yeast activities. There were 63.2% of residual starch and 14.3% of residual cellulose that were potentially further degraded at optimum temperature in the absence of ethanol. A techno-economic analysis needs to be performed in future study to estimate the economics of this strategy. Fig. 6. Further degradation of residual starch and cellulose. The 120 h SSF broth (containing solids and 129.3 g/L ethanol) with and without ethanol evaporated was investigated for starch further degradation (a) and cellulose further degradation (b). The residual starch was further degraded with amylase and glucoamylase. Saccharification was performed at 60 °C and 30 °C for 24 h. The residual cellulose was further degraded with cellulase at 50 °C and 30 °C for 48 h. The released glucose and xylose were then analyzed again. The residual starch and cellulose content at 0 h were defined as 100%, respectively. All the experiments were performed in triplicate.
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Lau, M.W., Dale, B.E., Balan, V., 2008. Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation. Biotechnol. Bioeng. 99, 529–539. Li, Z., Cai, L., Gu, Z., Shi, Y.C., 2014. Effects of granule swelling on starch saccharification by granular starch hydrolyzing enzyme. J. Agric. Food Chem. 62, 8114–8119. Masiero, S.S., Peretti, A., Trierweiler, L.F., Trierweiler, J.O., 2014. Simultaneous cold hydrolysis and fermentation of fresh sweet potato. Biomass Bioenergy 70, 174–183. Mishra, A., Sharma, A.K., Sharma, S., Bagai, R., Mathur, A.S., Gupta, R.P., Tuli, D.K., 2016. Lignocellulosic ethanol production employing immobilized Saccharomyces cerevisiae in packed bed reactor. Renew. Energy 98, 57–63. Najafpour, G., Younesi, H., Syahidah Ku Ismail, K., 2004. Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Bioresour. Technol. 92, 251–260. Nakamura, Y., Kobayashi, F., Ohnaga, M., Sawada, T., 1997. Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity. Biotechnol. Bioeng. 53, 21–25. Nguyen, C.-N., Le, T.-M., Chu-Ky, S., 2014. Pilot scale simultaneous saccharification and fermentation at very high gravity of cassava flour for ethanol production. Ind. Crop. Prod. 56, 160–165. Noureddini, H., Byun, J., 2010. Dilute-acid pretreatment of distillers’ grains and corn fiber. Bioresour. Technol. 101, 1060–1067. Porter, S.E., Donohoe, B.S., Beery, K.E., Xu, Q., Ding, S.-Y., Vinzant, T.B., Abbas, C.A., Himmel, M.E., 2007. Microscopic analysis of corn fiber using starch- and cellulosespecific molecular probes. Biotechnol. Bioeng. 98, 123–131. Shi, A., Du, Z., Ma, X., Cheng, Y., Min, M., Deng, S., Chen, P., Li, D., Ruan, R., 2013. Production and evaluation of biodiesel and bioethanol from high oil corn using three processing routes. Bioresour. Technol. 128, 100–106. Shigechi, H., Fujita, Y., Koh, J., Ueda, M., Fukuda, H., Kondo, A., 2004. Energy-saving direct ethanol production from low-temperature-cooked corn starch using a cellsurface engineered yeast strain co-displaying glucoamylase and α-amylase. Biochem. Eng. J. 18, 149–153. Singh, V., Graeber, V.J., 2005. Effect of corn hybrid variability and planting location on dry grind ethanol production. Trans. ASAE 48, 709–714. Somavat, P., Li, Q., de Mejia, E.G., Liu, W., Singh, V., 2016. Coproduct yield comparisons of purple, blue and yellow dent corn for various milling processes. Ind. Crop. Prod. 87, 266–272. Uthumporn, U., Zaidul, I.S.M., Karim, A.A., 2010. Hydrolysis of granular starch at subgelatinization temperature using a mixture of amylolytic enzymes. Food Bioprod. Process. 88, 47–54. Van Eylen, D., van Dongen, F., Kabel, M., de Bont, J., 2011. Corn fiber, cobs and stover: enzyme-aided saccharification and co-fermentation after dilute acid pretreatment. Bioresour. Technol. 102, 5995–6004. Wang, S., Ingledew, W.M., Thomas, K.C., Sosulski, K., Sosulski, F.W., 1999. Optimization of fermentation temperature and mash specific gravity for fuel alcohol production. Cereal Chem. 76, 82–86. Wang, P., Singh, V., Xu, L., Johnston, D.B., Rausch, K.D., Tumbleson, M.E., 2005. Comparison of enzymatic (E-mill) and conventional dry-grind corn processes using a granular starch hydrolyzing enzyme. Cereal Chem. 82, 734–738. Wang, D., Bean, S., McLaren, J., Seib, P., Madl, R., Tuinstra, M., Shi, Y., Lenz, M., Wu, X., Zhao, R., 2008. Grain sorghum is a viable feedstock for ethanol production. J. Ind. Microbiol. Biot. 35, 313–320. Yangcheng, H., Jiang, H., Blanco, M., Jane, J.L., 2013. Characterization of normal and waxy corn starch for bioethanol production. J. Agric. Food Chem. 61, 379–386. Zabed, H., Boyce, A.N., Faruq, G., Sahu, J.N., 2016. A comparative evaluation of agronomic performance and kernel composition of normal and high sugary corn genotypes (Zea mays L.) grown for dry-grind ethanol production. Ind. Crop. Prod. 94, 9–19. Zhang, Y.H.P., Ding, S.Y., Mielenz, J.R., Cui, J.B., Elander, R.T., Laser, M., Himmel, M.E., McMillan, J.R., Lynd, L.R., 2007. Fractionating recalcitrant lignocellulose at modest reaction conditions. Biotechnol. Bioeng. 97, 214–223.
All authors have no conflicts of interest. Acknowledgements This work was supported by “National Key R&D Program of China”, Grant No. 2016YFE0105400, “National Natural Science Foundation of China”, Grant No. 21606132, “Natural Science Foundation of Jiangsu Province”, Grant Nos. BK20160823 & BK20170037, and “The Fundamental Research Funds for the Central Universities”, Grant No. 30916011202. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.01.037. References Awafo, V.A., Chahal, D.S., Simpson, B.K., 2000. Evaluation of combination treatments of sodium hydroxide and steam explosion for the production of cellulase-systems by two T. reesei mutants under solid-state fermentation conditions. Bioresour. Technol. 73, 235–245. Balat, M., 2011. Production of bioethanol from lignocellulosic materials via the biochemical pathway: a review. Energy Convers. Manage. 52, 858–875. Bothast, R.J., Schlicher, M.A., 2005. Biotechnological processes for conversion of corn into ethanol. Appl. Microbiol. Biotechnol. 67, 19–25. Favaro, L., Viktor, M.J., Rose, S.H., Viljoen-Bloom, M., van Zyl, W.H., Basaglia, M., Cagnin, L., Casella, S., 2015. Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases. Biotechnol. Bioeng. 112, 1751–1760. Fischer, C.R., Klein-Marcuschamer, D., Stephanopoulos, G., 2008. Selection and optimization of microbial hosts for biofuels production. Metab. Eng. 10, 295–304. Gáspár, M., Kálmán, G., Réczey, K., 2007. Corn fiber as a raw material for hemicellulose and ethanol production. Process Biochem. 42, 1135–1139. Jin, M., Sarks, C., Bals, B.D., Posawatz, N., Gunawan, C., Dale, B.E., Balan, V., 2017. Toward high solids loading process for lignocellulosic biofuel production at a low cost. Biotechnol. Bioeng. 114, 980–989. Kasemets, K., Nisamedtinov, I., Laht, T.M., Abner, K., Paalme, T., 2007. Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study. Antonie Leeuwenhoek 92, 109–128. Khanal, S.K., Montalbo, M., van Leeuwen, J., Srinivasan, G., Grewell, D., 2007. Ultrasound enhanced glucose release from corn in ethanol plants. Biotechnol. Bioeng. 98, 978–985. Kim, Y., Hendrickson, R., Mosier, N.S., Ladisch, M.R., Bals, B., Balan, V., Dale, B.E., Dien, B.S., Cotta, M.A., 2010. Effect of compositional variability of distillers’ grains on cellulosic ethanol production. Bioresour. Technol. 101, 5385–5393. Knutsen, J.S., Liberatore, M.W., 2010. Rheology modification and enzyme kinetics of high solids cellulosic slurries. Energy Fuel 24, 3267–3274. Kristensen, J.B., Felby, C., Jørgensen, H., 2009. Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol. Biofuels 2, 1–11. Kumar, D., Singh, V., 2016. Dry-grind processing using amylase corn and superior yeast to reduce the exogenous enzyme requirements in bioethanol production. Biotechnol. Biofuels 9, 228–239.
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In-situ corn fiber conversion improves ethanol yield in corn dry-mill process a
b
Xiujuan Li , Sitong Chen , He Huang
c,d,⁎⁎
b,⁎
T
, Mingjie Jin
a
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing, 211816, China School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei Street, Nanjing, 210094, China College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, China d Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Corn fiber conversion Dry-mill process Simultaneous saccharification and fermentation (SSF) Cellulase Ethanol
Dry-mill process has been widely applied for ethanol production from corn. However, corn kernel fiber (mostly cellulose) is typically unconverted in the process. This work investigated the effect of corn cellulose conversion on the performance of dry-mill process. Solid loading and pH were optimized first for better design of cellulose conversion in the process. Cellulase was dosed for cellulose conversion during Simultaneous Saccharification and Fermentation (SSF). Addition of cellulase improved ethanol yield by 1.8% compared to traditional SSF without cellulase. Cellulose conversion reached 19.8%. Starch conversion was also enhanced from 98.3% to 99.0% with glycerol yield, which is a major side-product of ethanol fermentation, decreased by 7.3%. Residual starch and residual cellulose resulted from SSF were further studied for their degradability and it was found that 63.2% of residual starch and 14.3% of residual cellulose could be further degraded in the absence of ethanol. This work implies that cellulose conversion is of great benefit to corn ethanol process.
1. Introduction With the development of industrialization and increase of population, global energy demand is increasing dramatically, and the most energy supplies are derived from non-renewable fossil fuel (Kumar and Singh, 2016). With consideration of environmental and social sustainability, renewable energy development has drawn much attention (Zhang et al., 2007). Bioethanol, which can be produced from starchy, sugary and cellulosic materials through fermentation, is considered as the most promising renewable fuel. Starchy crops (e.g. corn) are widely used for bioethanol production due to their availability, ease of conversion, storage capability for a long period and high ethanol yield (Balat, 2011). Currently, most fuel ethanol in United States is produced from corn by dry mill or wet mill process (Bothast and Schlicher, 2005). Although both processes are being employed for ethanol production, 67% of the commercial plants use dry-mill process. Starch comprises 70–72% of the kernel weight on a dry basis, and is readily degraded to fermentable glucose, which can be further fermented to ethanol. However, the biodegradability of starch during enzymatic saccharification is largely determined by its structure that varies with regard to botanical sources and crop hybrids. This variation in structure affects overall starch conversion and thereby ethanol yield. In addition, the amylose and amylopectin ratio also affects
⁎
physicochemical properties of starch and hence affects ethanol yield (Singh and Graeber, 2005; Yangcheng et al., 2013). Recent advances in improving ethanol yield and process profitability include the use of high yield corn varieties (Shi et al., 2013; Zabed et al., 2016), development of non-cooking hydrolysis technique with granular starch hydrolyzing enzyme (GSHE) (Li et al., 2014; Uthumporn et al., 2010; Wang et al., 2005), cell immobilization (Mishra et al., 2016; Najafpour et al., 2004), the use of superior yeast (Favaro et al., 2015; Nakamura et al., 1997; Shigechi et al., 2004), the enhancement of glucose release rate (Khanal et al., 2007) and recovery of high-value co-products (Somavat et al., 2016), etc. Although technologies have been well developed to convert corn starch to ethanol, corn kernel fiber (mostly cellulose), which accounts for around 4 percent of total sugar in corn, is unutilized in the current process. Conversion of corn cellulose is of great interest in the field (Porter et al., 2007). However, most studies focused on conversion of isolated corn fiber (Gáspár et al., 2007; Van Eylen et al., 2011) or DDGS (dried distillers’ grains with solubles) resulted from dry-mill process (Kim et al., 2010; Lau et al., 2008). In-situ conversion of cellulose during dry-mill process is not well studied. Cellulase is a mixture of different cellulytic enzymes that act synergistically on the cellulose molecules to degrade cellulose to glucose (Awafo et al., 2000). The addition of cellulase in the corn ethanol process to in-situ convert
Corresponding author at: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei Street, Nanjing, 210094, China. Corresponding author at: College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, China. E-mail addresses: [email protected] (H. Huang), [email protected] (M. Jin).
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https://doi.org/10.1016/j.indcrop.2018.01.037 Received 11 October 2017; Received in revised form 10 January 2018; Accepted 14 January 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Analytical methods
cellulose could potentially improve ethanol yield as it not only degrades cellulose but may also synergize with glucoamylase to further hydrolyze recalcitrant starch. Cellulase can help disrupt cell wall structure of grain and promote the release of contents including starch and protein, which plays the auxiliary role in improving ethanol yield of starch. Meanwhile, the addition of cellulase may decrease viscosity of fermentation broth and improve its rheological property (Knutsen and Liberatore, 2010), which facilitates saccharification and fermentation. There are two stages in corn ethanol process that cellulase can potentially be dosed, namely liquefaction stage and Simultaneous Saccharification and Fermentation (SSF) stage (performs enzymatic saccharification and fermentation in the same bioreactor). Liquefaction stage uses alpha-amylase and carries out at above 85 °C. This temperature is much higher than most cellulases can tolerate. The other stage cellulases can be dosed is the SSF stage. However, there are several factors need to be investigated. SSF performs at 30 °C, which is lower than the optimal temperature of cellulase (50 °C). Fermentation causes pH decrease rapidly in the first 24 h and results in high ethanol concentration, which both affect cellulase activity. Therefore, it remains unknown how well cellulase can perform during SSF. The main objective of this work was to in-situ convert corn cellulose during dry-mill process and investigate the effect of cellulase addition on SSF performances, including starch conversion, cellulose conversion and ethanol yield. Residual starch and residual cellulose resulted from SSF were further studied for their degradability.
The concentration of maltose, glucose, xylose, organic acids, glycerol and ethanol was analyzed by HPLC with a Biorad Aminex HPX87H column. The analysis conditions were as follows: column temperature (65 °C), mobile phase (5 mM H2SO4, 0.6 mL/min). Total sugar and oligomeric sugar were analyzed according to procedures of NREL-TP-510-42618 and NREL-TP-510-4262, respectively. The starch content was analyzed using NREL analytical procedure NREL-TP-510-42624. Cellulose content was obtained by subtracting starch from total sugar. Ethanol yield was determined based on the theoretical ethanol yield from consumed glucose, which is 0.51 g ethanol/g sugar. Moisture content of biomass was measured by a moisture analyzer (METTLER TOLEDO). The number of yeast cells in the fermentation broth was counted using a hemocytometer (Shanghai Qiujing biochemical reagents Co. Ltd) and microscope (CX31RTSF, OLYMPUS). The cells were stained with methylene blue dye (0.1%) which stains dead cells blue while living cells remain uncolored. The OD value of yeast was determined using ultraviolet and visible spectrophotometer (TU-1810, Beijing Purkinje General Instrument CO. Ltd) at 600 nm. 2.4. Effect of pH and ethanol on cellulase activity Cellulase activity was assayed at different pH (3–5) and different concentrations of ethanol (0–20%) with the modified method of NRELTP-510-42628 using filter paper as substrate. Citric acid-sodium citrate buffer (0.025 M) at different pH and ethanol solution were prepared and sterilized prior to use. Different amount of ethanol was added to the citrate buffer to form an ethanol-buffer mixture with ethanol concentrations of 0, 5, 10, 15, 17.5 and 20% (v/v). With the modified NREL-TP-510-42628 method, 0.5 mL of diluted enzyme was added into 1 mL of citrate-buffer-ethanol mixtures with different pH and ethanol concentration. After 60 min of hydrolysis reaction at 50 °C, 3 mL DNS reagent was added to measure the cellulase activity.
2. Materials and methods 2.1. Materials Corn flour was generously given by China Oil & Foodstuffs Corporation (COFCO), Anhui, China. The glucan and xylan contents of corn were 81.96% and 2.50%, respectively. The starch and cellulose contents of corn were 77.86% and 4.10%, respectively. The alphaamylase, glucoamylase and cellulase used in this study were supplied by Genencor Bio-Products Co. Ltd., Shandong Longda Bio-products Co. Ltd. and Vland Biothech Co. Ltd., respectively. Microorganism and seed culture preparation Saccharomyces cerevisiae was obtained from Angel Yeast Co., Ltd. Seed culture of S. cerevisiae was prepared in YPD medium (10 g/L yeast extract and 20 g/L tryptone) with 100 g/L glucose in a 250 mL Erlenmeyer flask with a working volume of 120 mL. The culture was incubated at 30 °C and 150 rpm under micro-aerophilic conditions with sterile and breathable filtering membrane for 24 h.
2.5. Effect of ethanol concentration on yeast activity The ability of yeast to consume glucose under different concentrations of ethanol was examined in the medium containing 5 g/L glucose and various concentrations of ethanol. The glucose solutions were prepared with citric acid-sodium citrate buffer (0.025 M, pH 4.5) and sterilized prior to use. Ethanol concentration tested ranged from 0 to 20% (v/v). The yeast cells were inoculated at an OD600 of 1.0 (0.44 g/L broth), 3.0 (1.32 g/L broth) and 6.0 (2.64 g/L broth) and cultured at 30 °C, 150 rpm for 48 h. The consumption of glucose was analyzed after incubation.
2.2. Simultaneous saccharification and fermentation (SSF) The corn flour was mixed with deionized water to make slurry at different solid loadings (27%, 30% and 33% w/w). For liquefaction, the pH of the slurry was adjusted to 5.7 using 40% sodium hydroxide solution and 0.048 g of α-amylase was used per 100 g dry corn. The liquefaction was performed in a shake flask at 85 °C for 4 h using water bath with intermittent mixing. The pH of the cooling liquefied mash was adjusted to 4.6 using sulfuric acid for SSF process. SSF was carried out in a 250 mL Erlenmeyer flask with a total mixture weight of 150 g in triplicate at 30 °C, pH 4.6, and 150 rpm. Yeast seed culture was centrifuged at 4000 rpm for 10 min to obtain yeast cell pellet, which was used for inoculation. The initial OD600 for SSF was 1.0, which means 0.44 g yeast cells were used per 1 L fermentation broth. Urea (1.28 g/kg mash) and glucoamylase (0.1 g/100 g dry corn) were added. A rubber stopper with a needle piercing through it was used to cap the flask to maintain largely anaerobic conditions. To monitor the fermentation performance, about 2 mL of samples were drawn at 0, 4, 16, 24, 36, 48, 72, 96 and 120 h and centrifuged at 10,000 rpm for 10 min. The supernatants were immediately sterile filtered through a 0.22 μm filter for HPLC analysis.
2.6. Degradability of residual starch and residual cellulose After SSF at 30% solid loading, the whole broth without any treatments was used as control group to study the degradability of residual starch and residual cellulose. At the same time, to remove ethanol effect, ethanol was evaporated from SSF broth (60 g) with rotary evaporator (RE100-Pro, Scilogex) at 65 °C, 10 min and 0.07 MPa. The broth without ethanol was performed as experimental group. The degradability of residual starch was carried out in a 100 mL Erlenmeyer flask with a total mixture weight of 60 g in triplicates. Alpha-amylase (0.048 g/100 dry corn) was added to make another round liquefaction at 85 °C for 4 h. Glucoamylase (0.1 g/100 g dry corn) was then added to hydrolyze at 30 °C or 60 °C and 150 rpm for 24 h. Residual starch content and released glucose were then analyzed again. The degradability of residual cellulose was tested by adding cellulase (0.6 g/100 dry corn) to the broth for hydrolyze residual cellulose at 50 °C or 30 °C and 250 rpm for 48 h. 218
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Fig. 1. Effect of different solid loading on glucose consumption (a), ethanol yield (b), death rate of yeast (c) and starch conversion (d) during SSF. The death rate of yeast was calculated by dividing viable cells by total cells count. All the experiments were performed in triplicate.
3. Results and discussion
93.6% and 93.3% at 120 h, respectively. There is no significant difference in starch conversion for different solid loadings at 120 h, and so does ethanol yield, but SSF with 33% solid loading was poor in fermentation efficiency with 9.1 g/L glucose unfermented at 120 h. Moreover, SSF at 33% solid loading might have led to higher viscosity, higher osmotic pressure and higher ethanol inhibition, which affected the viability of fermenting yeast (Fig. 1c) and caused poor fermentation efficiency (Kristensen et al., 2009; Nguyen et al., 2014; Wang et al., 2008). Since the SSF at 30% solid loading was fermented completely and further increasing solid loading caused fermentation issues, we subsequently chose to work on 30% solid loading in the future studies. As we all know, pH is a key factor for ethanol fermentation, which has a direct effect on yeast metabolism (Kasemets et al., 2007; Masiero et al., 2014). In addition, the enzyme activities are also affected by pH value. Here, we investigated the effect of different initial pH (4.0, 4.5, 5.0) on 30% solid loading SSF. Surprisingly, no obvious effect of pH on glucose consumption (Fig. 2a), ethanol yield (Fig. 2b), pH change during SSF (Fig. 2c) and death rate of yeast cell (Fig. 2d) was observed. We also investigated the cellulase activities on filter papers at different pH (3–5). As shown in Table 2, the optimum pH for cellulase activity was between 4.6 and 5.0. Considering optimal pH of glucoamylase (4.2–4.6), we selected pH 4.6 as the initial pH of SSF in the subsequent studies.
3.1. Optimization of solid loading and initial pH of SSF Increasing solid loading of SSF increases final ethanol titer, decreases energy consumption and reduces process cost. However, higher solid loading resulted in poor glucose consumption and lower ethanol yield (Jin et al., 2017; Wang et al., 1999). Here we compared the corn ethanol fermentation performances at 27%, 30% and 33% (w/w) solid loadings (Fig. 1). Glucose concentration increased from 0 to 8 h, then rapidly decreased, which indicates that saccharification rate was higher than fermentation rate in the first 8 h and then fermentation rate overtook saccharification rate afterwards. For 27% and 30% solid loadings, glucose was consumed completely within fermentation time of 72 h, and ethanol reached maximums of 111.3 and 127.0 g/L, respectively. For 33% solid loading, there was still 3.7 g/L glucose remaining in the fermentation broth at 72 h and this glucose concentration slightly increased thereafter. This implies that at 33% solid loading saccharification was incomplete in 72 h but the yeast cell viability was already low with a high death rate of 95.0% (Fig. 1c), which caused a considerable amount of glucose unfermented. Byproduct concentrations are shown in Table 1. The main byproduct was glycerol and the glycerol to ethanol ratio was almost identical for all tested solid loadings. For a better understanding of the process at different solid loadings, starch conversions and ethanol yields were determined. The starch conversions at different solid loadings were 97.8%, 98.3% and 98.0% at 120 h, respectively. (Fig. 1d). Correspondingly, the ethanol yields based on the glucose consumption at different solid loading were 93.1%,
3.2. Addition of cellulase significantly improves ethanol yield during SSF Aiming to convert corn cellulose and improve ethanol yield, the addition of cellulase during SSF was investigated at solid loading of
Table 1 Byproducts and ethanol concentrations resulted from SSF at different solid loadings and at different pHs. Experiment with different solid loadings was performed at pH 4.6; Experiment with different initial pHs was performed at solid loading of 30%.
27% solid loading 30% solid loading 33% solid loading Initial pH 4.0 Initial pH 4.5 Initial pH 5.0
Succinic acid (g/L)
Lactic acid (g/L)
Glycerol (g/L)
Acetic acid (g/L)
Ethanol (g/L)
Glycerol /Ethanol
0.51 0.46 0.37 0.45 0.46 0.64
0.48 0.63 0.56 0.52 0.63 0.54
10.08 11.26 12.32 10.75 11.26 11.38
0.21 0.23 0.46 0.57 0.23 0.42
111.33 127.02 137.50 126.52 127.02 126.44
0.09 0.09 0.09 0.08 0.09 0.09
± ± ± ± ± ±
0.01 0.02 0.03 0.01 0.02 0.01
± ± ± ± ± ±
0.01 0.01 0.08 0.02 0.01 0.01
± ± ± ± ± ±
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0.20 0.05 0.21 0.08 0.05 0.13
± ± ± ± ± ±
0.01 0.06 0.01 0.05 0.06 0.05
± ± ± ± ± ±
0.25 0.26 0.21 0.20 0.26 0.23
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Fig. 2. Effect of initial pH on glucose consumption (a), ethanol yield (b), pH change (c) and death rate of yeast (d) during SSF. SSF was carried out at 30% solids loading. The pH of the slurry was adjusted to the predefined value (4.0, 4.5 and 5.0) using 72% sulfuric acid solution before SSF. All the experiments were performed in triplicate.
increased from 98.3% to 99.0%, cellulose conversion reached 19.8% (Fig. 3b). Overall, ethanol yield with the addition of cellulase was improved by 1.8%, which could be a big profit to ethanol plants. For a better understanding of the SSF performances, mass balance comparison between SSF with addition of cellulase and SSF without addition of cellulase were calculated according to the experimental data by shaking flask in our laboratory (Fig. 4). As Fig. 4a shows, 408.7 ± 0.40 kg ethanol was produced from 1 t of corn through SSF with the addition of cellulase at the solid loading of 30%. The ethanol yield for SSF without cellulase addition is 403.5 ± 0.26 kg/tonne corn. The differences between these ethanol yields were statistically significant (P < 0.01). This implies that the improved sugar conversion and decreased glycerol yield resulted in the enhancement of ethanol yield. The experiments’ data had a carbon closure of about 98% (Fig. 4b), which verified the accuracy of our analyses. Considering the cellulase itself used in our study contains 10.40 g/L glucose, we also analyzed the effect of cellulase itself on the improvement of ethanol yield. Based on the SSF with 1 t of corn dry weight and 0.6% cellulase (Fig. 4a), the result showed that 27.68 g ethanol was theoretically
30% and pH 4.6. Cellulase was added simultaneously with glucoamylase at the beginning of SSF. Different dosage of cellulase (0.1%, 0.3%, 0.6%, 0.9% per corn dry weight) was optimized and shown in Supplementary Table S1. With increase of cellulase dosage, ethanol yield increased and glycerol yield decreased until the dosage reached at 0.6%. Higher dosage did not give further increase in ethanol yield. Therefore, we selected 0.6% (w/w) per corn dry weight as the optimal cellulase dosage in SSF. The effect of cellulase addition at the dosage of 0.6% on SSF performances are shown in Fig. 3. Glucose release rate of SSF with the addition of cellulase was faster than the control (without cellulase addition) during initial stage (Fig. 3a). Meanwhile, death rate of yeast was slightly lower (Fig. 3c) and ethanol titer was higher compared to control (Fig. 3a). After 120 h, 129.3 ± 0.30 g/L ethanol was produced with cellulase addition, which is a 1.8% lift compared to the control (127.0 ± 0.26 g/L ethanol). Glycerol production was occurred mostly during the first 24 h of SSF. As Fig. 3c shows, glycerol yield was decreased from 11.3 to 10.5 g/L with the addition of cellulase. With the addition of cellulase in SSF, starch conversion was also slightly
Table 2 Effect of pH and ethanol on cellulase activity. pH
Specific activity (FPU/ml)
5.0 4.6 4.2 3.8 3.4 3.0
116.08 ± 0.64 113.39 ± 0.50 93.78 ± 0.52 72.83 ± 0.54 43.65 ± 1.02 8.62 ± 0.50
a
Specific activity (FPU/ml)
0 5 10 15 17.5 20
114.62 ± 0.50 72.12 ± 1.00 47.36 ± 0.51 33.42 ± 0.50 16.79 ± 0.53 9.40 ± 0.31
b
b
100.00 97.69 80.79 62.75 37.60 7.42
Ethanol (%, v/v)
a
Relative activity (%)
a
Relative activity (%) 100.00 62.92 41.32 29.16 14.65 8.20
Activity assay was performed at 50 °C, and pH 4.8 for 60 min. The cellulase activity at pH 5.0 (116.08 U/mL) and 0% of Ethanol (114.62 U/mL) was defined as 100%, respectively.
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Fig. 3. Effect of Cellulase addition on SSF performance. a) glucose consumption and ethanol production; b) sugar conversion; c) glycerol production and death rate of yeast; d) residual sugar content. Experiments were performed at 30% (w/w) solid loading. Cellulase at dosage of 0.6% per corn dry basis was added simultaneously with glucoamylase at the beginning of SSF. SSF was performed at 30 °C, pH 4.6, 150 rpm for 120 h. Residual starch and cellulose after SSF without cellulase addition were defined as 100%. All the experiments were performed in triplicate.
insights into further enhancement of sugar conversions. The addition of cellulase for improvement of ethanol yield does not require any additional equipment or control system, the only additional cost would be the cellulase, whose loading could be further reduced by using more advanced cellulase cocktails. The estimated increase in yield may be sufficiently high to carry the additional costs for the addition of cellulases; however, a full techno-economic analysis needs to be performed before any conclusive statements in terms of economic improvement can be made.
produced if the glucose in cellulase solution was converted completely. A very small increase, less than 0.01%, was obtained compared with SSF without the addition of cellulase, which indicated that cellulase solution itself had low impact on the ethanol yield during SSF. To further investigate the effect of cellulase addition on higher solid loading process, we also carried out the SSF at 33% solid loading and 1.2% ethanol lift was observed with cellulase addition (Supplementary Fig. S1). Although the addition of cellulase improved starch and cellulose conversions, there is room for improvement. If the amount of residual starch and residual cellulose after SSF with no cellulase addition was defined as 100%, there were still 59.2% residual starch and 80.1% residual cellulose unconverted with the addition of cellulase (Fig. 3d). Therefore, it would be worthwhile to investigate the influential factors for incomplete degradation of starch and cellulose, which may provide
3.3. High concentration of ethanol is a barrier for further improvement of sugar conversions As discussed above, the incomplete degradation of sugar, especially incomplete cellulose degradation, is observed in SSF even with the Fig. 4. Mass balance comparison between SSF with cellulase addition and SSF without cellulase addition at 30% solid loading. Based on 1 t corn dry weight, carbon balance was calculated without taking xylan into account since xylan/xylose cannot be utilized by this fermenting yeast. Carbon closure was calculated using equation: [(products carbon mass + residual sugar carbon mass)/initial carbon mass] × 100. Initial carbon mass was calculated based on glucan carbon in corn flour. Final carbon mass was calculated based on carbons in ethanol, carbon dioxide, byproducts and residual C6 sugars. All the experiments were performed in triplicate.
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Table 3. SSF at 33% solid loading with the addition of cellulase produced 138.6 g/L ethanol at 72 h, that is about 17.6% (v/v). At that concentration, the death rate of yeast reached 100% after 12 h of incubation (Table 3), which might have caused accumulated glucose unfermented. It indicated that high concentration of ethanol inhibits microorganism growth and viability (Fischer et al., 2008), and further influence glucose consumption and ethanol yield, especially in SSF at 33% solid loading. As discussed above, high concentration of ethanol greatly decreased the activities of yeast and cellulase, which limited the improvement of SSF efficiency through process engineering. Further studies could focus on enhancement of cellulase and yeast tolerances to ethanol.
addition of cellulase. The high ethanol concentration, which was produced during SSF, might be a cause for incomplete sugar conversions. It is well known that ethanol affects the activities of cellulase and yeast. Here, we investigated the cellulase activities on filter papers and yeast activities in the presence of ethanol at different concentrations. In view of actual ethanol concentration during SSF, the effect of ethanol on cellulase activities was studied using ethanol concentration ranging from 0 to 20% (v/v). The specific activity and relative activity of enzyme under different concentrations of ethanol are presented in Table 2. The cellulase activity decreased as the ethanol concentration increased. Ethanol titer during SSF (30% solid loading) with the addition of cellulase reached 125.1 g/L at 48 h, which is about 15.9% (v/v). At that concentration, cellulase activity was already reduced by 71%. When ethanol concentration reached 129.3 g/L at 120 h, that is about 16.4% (v/v), the remaining cellulase activity was about 14.7% to 29.2% of its initial. In addition to ethanol inhibition, pH fluctuation and suboptimal temperature rendered cellulase not working at its full potential. The effect of ethanol on yeast activities was also studied. Ethanol concentration tested ranged from 0 to 20% (v/v). Initial yeast ODs of 1, 3, and 6 were used for study to examine whether ethanol tolerance is related to yeast cell density. As shown in Fig. 5, glucose was completely consumed in 24 h for all tested initial ODs when ethanol concentration was lower than or equal to 10%. When the medium ethanol concentration was 15%, significant inhibition was seen for initial ODs of 1 with 66.9% residual glucose unutilized after 48 h fermentation. However, fermentation with initial OD of 3 still consumed all the glucose in 48 h and fermentation with initial OD of 6 consumed all the glucose in 24 h. It indicated that higher cell density did help in resisting ethanol inhibition. Higher ethanol concentrations (17.5% and 20%) substantially reduced glucose consumption by the yeast for all tested initial ODs with higher OD consumed more glucose. Higher ethanol concentration caused faster cell death (Fig. 5d), which might be the reason for reduced glucose consumption. The death rate of yeast and residual glucose content in the medium with ethanol concentrations of 15%, 17.5% and 20% are shown in
3.4. Residual starch and cellulose in SSF can be further degraded Though ethanol inhibition reduced cellulase activity, it was still unclear if the residual starch and cellulose were degradable by enzymes. Therefore, we investigated the degradability of residual starch and cellulose. The 120 h SSF broth (containing unconverted solids and 129.3 g/L ethanol) with and without ethanol evaporated was used for another round liquefaction using α-amylase and saccharification using glucoamylase (without fermentation). Saccharification was performed at 60 °C first for 24 h. As shown in Fig. 6a, without ethanol evaporated, liquefaction and saccharification released around 4.2 g/L glucose, while removal of ethanol further increased this concentration to 5.7 g/L, which is a 35.7% lift. Starch analysis showed that the residual starch conversions with and without ethanol reached 45.0% and 63.2%, respectively (Fig. 6a). In view of the actual fermentation temperature (30 °C), we also performed the saccharification at 30 °C with comparison to 60 °C. It was found that 30 °C resulted in only 16.0% and 29.6% of residual starch conversions for cases with and without ethanol, respectively. It indicated that SSF temperature did not favor further degradation of residual starch. Considering the starch conversion already reached 99.0% during SSF, residual starch accounted for 1% of the initial total starch. Under SSF conditions (30 °C, with ethanol presence), there was only 16.0% of residual starch further degradable, which Fig. 5. Effect of ethanol concentration on yeast activity. The initial OD of yeast in medium was 1 (a), 3 (b) and 6(c) and the initial glucose concentration was 5 g/L. Death rate of yeast in medium with the initial OD of 6 (d) was measured by hemocytometer using the methylene blue as dye. All the experiments were performed in triplicate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 3 Death rate of yeast and residual glucose in the medium with different concentration of ethanol. Initial OD600 nm of yeast
Time (h)
15% Ethanol (118 g/L) Dr (%)
1
3
6
a b
12 24 48 12 24 48 12 24 48
a
19.12 ± 0.75 80.32 ± 1.02 100.00 ± 0.00 19.42 ± 0.73 64.15 ± 1.00 91.32 ± 1.02 23.43 ± 1.05 93.31 ± 1.02 99.34 ± 0.70
R.glu (%)
b
77.77 ± 0.52 71.01 ± 1.00 66.86 ± 1.00 38.38 ± 1.02 16.45 ± 0.74 4.10 ± 0.21 2.41 ± 0.50 0.39 ± 0.05 0.24 ± 0.05
17.5% Ethanol (138 g/L)
20%Ethanol (158 g/L)
Dr (%)
R.glu (%)
Dr (%)
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 81.23 ± 0.78 100.00 ± 0.00 100.00 + 0.00 79.43 ± 0.76 100.00 ± 0.00 100.00 ± 0.00
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 83.31 ± 0.96 82.15 ± 1.21 82.15 ± 1.21 34.93 ± 0.97 28.59 ± 0.92 28.59 ± 0.92
100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
R.glu (%) ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 98.04 ± 0.64 98.04 ± 0.64 98.04 ± 0.64 82.99 ± 0.51 82.99 ± 0.51 82.99 ± 0.51
Dr: Death rate of yeast in the medium. R.glu: Residual glucose in the medium. The initial glucose concentration was defined as 100%.
cocktail might help in enhancing corn cellulose conversion. Starch conversion with cellulase cocktail might because cellulase disrupted cell wall structure of grain and promoted starch release. It also possible that the cellulase cocktail also contained amylases. In summary, additional sugars (4–9 g/L) could be released under optimum conditions and this sugar stream could potentially further enhance ethanol yield. However, the required optimum conditions do not consistent with current SSF conditions.
means the current process already did very well in converting starch with only 0.16% of the total starch potentially being further converted. With addition of α-amylase and glucoamylase, no residual cellulose was converted (Fig. 6a). The degradability of residual cellulose in 120 h SSF broth was also investigated by addition of 0.6% cellulase at both 50 °C and 30 °C for 48 h. As shown in Fig. 6b, without ethanol evaporated, cellulase hydrolysis released around 5.9 g/L glucose, while removal of ethanol further increased this concentration to 9.3 g/L, which is a 57.6% increase. Starch was also converted by this cellulase cocktail. Starch analysis showed that the residual starch conversions with and without ethanol were 20.2% and 32.4%, while residual cellulose conversions were 8.3% and 14.3%, respectively at 50 °C. Reaction at 30 °C decreased both starch conversion and cellulose conversion, resulting in only 7.5% and 16.3% of residual starch conversions for cases with and without ethanol, and residual cellulose conversions of 2.1% and 6.2%, respectively. This indicates that the residual cellulose was mostly undegradable by current cellulase cocktail. Cellulose pretreatment (Noureddini and Byun, 2010; Porter et al., 2007) or advanced cellulase
4. Conclusion Cellulase addition in SSF at 30% solid loading resulted in 1.8% increase in ethanol yield and 7.3% decrease in glycerol production, with starch and cellulose conversions reaching 99.0% and 19.8%, respectively. High concentration of ethanol inhibited both cellulase and yeast activities. There were 63.2% of residual starch and 14.3% of residual cellulose that were potentially further degraded at optimum temperature in the absence of ethanol. A techno-economic analysis needs to be performed in future study to estimate the economics of this strategy. Fig. 6. Further degradation of residual starch and cellulose. The 120 h SSF broth (containing solids and 129.3 g/L ethanol) with and without ethanol evaporated was investigated for starch further degradation (a) and cellulose further degradation (b). The residual starch was further degraded with amylase and glucoamylase. Saccharification was performed at 60 °C and 30 °C for 24 h. The residual cellulose was further degraded with cellulase at 50 °C and 30 °C for 48 h. The released glucose and xylose were then analyzed again. The residual starch and cellulose content at 0 h were defined as 100%, respectively. All the experiments were performed in triplicate.
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Declaration
Lau, M.W., Dale, B.E., Balan, V., 2008. Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation. Biotechnol. Bioeng. 99, 529–539. Li, Z., Cai, L., Gu, Z., Shi, Y.C., 2014. Effects of granule swelling on starch saccharification by granular starch hydrolyzing enzyme. J. Agric. Food Chem. 62, 8114–8119. Masiero, S.S., Peretti, A., Trierweiler, L.F., Trierweiler, J.O., 2014. Simultaneous cold hydrolysis and fermentation of fresh sweet potato. Biomass Bioenergy 70, 174–183. Mishra, A., Sharma, A.K., Sharma, S., Bagai, R., Mathur, A.S., Gupta, R.P., Tuli, D.K., 2016. Lignocellulosic ethanol production employing immobilized Saccharomyces cerevisiae in packed bed reactor. Renew. Energy 98, 57–63. Najafpour, G., Younesi, H., Syahidah Ku Ismail, K., 2004. Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Bioresour. Technol. 92, 251–260. Nakamura, Y., Kobayashi, F., Ohnaga, M., Sawada, T., 1997. Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity. Biotechnol. Bioeng. 53, 21–25. Nguyen, C.-N., Le, T.-M., Chu-Ky, S., 2014. Pilot scale simultaneous saccharification and fermentation at very high gravity of cassava flour for ethanol production. Ind. Crop. Prod. 56, 160–165. Noureddini, H., Byun, J., 2010. Dilute-acid pretreatment of distillers’ grains and corn fiber. Bioresour. Technol. 101, 1060–1067. Porter, S.E., Donohoe, B.S., Beery, K.E., Xu, Q., Ding, S.-Y., Vinzant, T.B., Abbas, C.A., Himmel, M.E., 2007. Microscopic analysis of corn fiber using starch- and cellulosespecific molecular probes. Biotechnol. Bioeng. 98, 123–131. Shi, A., Du, Z., Ma, X., Cheng, Y., Min, M., Deng, S., Chen, P., Li, D., Ruan, R., 2013. Production and evaluation of biodiesel and bioethanol from high oil corn using three processing routes. Bioresour. Technol. 128, 100–106. Shigechi, H., Fujita, Y., Koh, J., Ueda, M., Fukuda, H., Kondo, A., 2004. Energy-saving direct ethanol production from low-temperature-cooked corn starch using a cellsurface engineered yeast strain co-displaying glucoamylase and α-amylase. Biochem. Eng. J. 18, 149–153. Singh, V., Graeber, V.J., 2005. Effect of corn hybrid variability and planting location on dry grind ethanol production. Trans. ASAE 48, 709–714. Somavat, P., Li, Q., de Mejia, E.G., Liu, W., Singh, V., 2016. Coproduct yield comparisons of purple, blue and yellow dent corn for various milling processes. Ind. Crop. Prod. 87, 266–272. Uthumporn, U., Zaidul, I.S.M., Karim, A.A., 2010. Hydrolysis of granular starch at subgelatinization temperature using a mixture of amylolytic enzymes. Food Bioprod. Process. 88, 47–54. Van Eylen, D., van Dongen, F., Kabel, M., de Bont, J., 2011. Corn fiber, cobs and stover: enzyme-aided saccharification and co-fermentation after dilute acid pretreatment. Bioresour. Technol. 102, 5995–6004. Wang, S., Ingledew, W.M., Thomas, K.C., Sosulski, K., Sosulski, F.W., 1999. Optimization of fermentation temperature and mash specific gravity for fuel alcohol production. Cereal Chem. 76, 82–86. Wang, P., Singh, V., Xu, L., Johnston, D.B., Rausch, K.D., Tumbleson, M.E., 2005. Comparison of enzymatic (E-mill) and conventional dry-grind corn processes using a granular starch hydrolyzing enzyme. Cereal Chem. 82, 734–738. Wang, D., Bean, S., McLaren, J., Seib, P., Madl, R., Tuinstra, M., Shi, Y., Lenz, M., Wu, X., Zhao, R., 2008. Grain sorghum is a viable feedstock for ethanol production. J. Ind. Microbiol. Biot. 35, 313–320. Yangcheng, H., Jiang, H., Blanco, M., Jane, J.L., 2013. Characterization of normal and waxy corn starch for bioethanol production. J. Agric. Food Chem. 61, 379–386. Zabed, H., Boyce, A.N., Faruq, G., Sahu, J.N., 2016. A comparative evaluation of agronomic performance and kernel composition of normal and high sugary corn genotypes (Zea mays L.) grown for dry-grind ethanol production. Ind. Crop. Prod. 94, 9–19. Zhang, Y.H.P., Ding, S.Y., Mielenz, J.R., Cui, J.B., Elander, R.T., Laser, M., Himmel, M.E., McMillan, J.R., Lynd, L.R., 2007. Fractionating recalcitrant lignocellulose at modest reaction conditions. Biotechnol. Bioeng. 97, 214–223.
All authors have no conflicts of interest. Acknowledgements This work was supported by “National Key R&D Program of China”, Grant No. 2016YFE0105400, “National Natural Science Foundation of China”, Grant No. 21606132, “Natural Science Foundation of Jiangsu Province”, Grant Nos. BK20160823 & BK20170037, and “The Fundamental Research Funds for the Central Universities”, Grant No. 30916011202. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.01.037. References Awafo, V.A., Chahal, D.S., Simpson, B.K., 2000. Evaluation of combination treatments of sodium hydroxide and steam explosion for the production of cellulase-systems by two T. reesei mutants under solid-state fermentation conditions. Bioresour. Technol. 73, 235–245. Balat, M., 2011. Production of bioethanol from lignocellulosic materials via the biochemical pathway: a review. Energy Convers. Manage. 52, 858–875. Bothast, R.J., Schlicher, M.A., 2005. Biotechnological processes for conversion of corn into ethanol. Appl. Microbiol. Biotechnol. 67, 19–25. Favaro, L., Viktor, M.J., Rose, S.H., Viljoen-Bloom, M., van Zyl, W.H., Basaglia, M., Cagnin, L., Casella, S., 2015. Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases. Biotechnol. Bioeng. 112, 1751–1760. Fischer, C.R., Klein-Marcuschamer, D., Stephanopoulos, G., 2008. Selection and optimization of microbial hosts for biofuels production. Metab. Eng. 10, 295–304. Gáspár, M., Kálmán, G., Réczey, K., 2007. Corn fiber as a raw material for hemicellulose and ethanol production. Process Biochem. 42, 1135–1139. Jin, M., Sarks, C., Bals, B.D., Posawatz, N., Gunawan, C., Dale, B.E., Balan, V., 2017. Toward high solids loading process for lignocellulosic biofuel production at a low cost. Biotechnol. Bioeng. 114, 980–989. Kasemets, K., Nisamedtinov, I., Laht, T.M., Abner, K., Paalme, T., 2007. Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study. Antonie Leeuwenhoek 92, 109–128. Khanal, S.K., Montalbo, M., van Leeuwen, J., Srinivasan, G., Grewell, D., 2007. Ultrasound enhanced glucose release from corn in ethanol plants. Biotechnol. Bioeng. 98, 978–985. Kim, Y., Hendrickson, R., Mosier, N.S., Ladisch, M.R., Bals, B., Balan, V., Dale, B.E., Dien, B.S., Cotta, M.A., 2010. Effect of compositional variability of distillers’ grains on cellulosic ethanol production. Bioresour. Technol. 101, 5385–5393. Knutsen, J.S., Liberatore, M.W., 2010. Rheology modification and enzyme kinetics of high solids cellulosic slurries. Energy Fuel 24, 3267–3274. Kristensen, J.B., Felby, C., Jørgensen, H., 2009. Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol. Biofuels 2, 1–11. Kumar, D., Singh, V., 2016. Dry-grind processing using amylase corn and superior yeast to reduce the exogenous enzyme requirements in bioethanol production. Biotechnol. Biofuels 9, 228–239.
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