Received March 11, 2016; Accepted March 31, 2016; Published April 07, 2016
Citation: Patel H, Divecha J, Shah A (2016) Optimization of Ethanol Production from Enzymatic Hydrolysate of Maize Stover. Adv Recycling Waste Manag 1:106. doi:10.4172/2475-7675.1000106
Copyright: © 2016 Patel H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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For efficient bioethanol production from maize stover, fermentation of glucose and xylose both was attempted using Saccharomyces cerevisiae and Pichia stipitis sequentially from enzymatic hydrolysate of mild alkali treated maize stover. Enzymatic saccharification of mild alkali treated maize stover at high substrate (30%) loading using 13.0 FPU/g commercial cellulase (MAPs 450) and 74.42 U/g crude β-xylosidase (Inhouse produced) after 36 h, yielded 161.32 mg ml-1 reducing sugars. Ethanol production was optimized employing response surface methodology. Under optimized conditions viz. 5% glucose, 14.55% inoculum and Time 35.51 h; 90.65% glucose was utilized and produced 18.93 g l-1 ethanol with 0.53 g l-1 h-1 productivity by Saccharomyces cerevisiae NCIM 3524. Further attempts were made to produce ethanol from xylose present in enzymatic hydrolysate using Pichia stipitis NCIM 3497. However, xylose conversion was not satisfactory as only 71% xylose was utilized.
The progress of bioethanol industries could help to reduce dependence on fossil fuels and moderate global warming . Bioethanol derived from lignocellulose can be a welcome technology due to its sustainability, lower-cost, and significantly larger availability of feedstock reserves as compared to starch-based ethanol [2,3]. Among various types of lignocellulosic biomass, maize stover is observed as one of the encouraging feedstock for the production of cellulosic bioethanol because of its high content of cellulose and hemicellulose . However bioethanol production from lignocellulosic biomass is a challenging process because of its complex chemical structure and organization. Efficient bioconversion of lignocellulosic biomass in to fermentable sugars requires cooperative action of various cellulolytic and hemicellulolytic enzymes at appropriate levels . Moreover, concentration of fermentable sugars should be high enough in enzymatic hydrolysate as at least 4% ethanol concentration is required to develop an economically viable distillation process . Considering economic viability, high gravity ethanol fermentations are attempted at high sugar concentrations .
Another challenging aspect in bioethanol technology is bioconversion of hexoses and pentoses both to ethanol . Unfortunately, there are limited microorganisms that can proficiently convert both of these sugars to ethanol. Saccharomyces cerevisiae and Zymomonas mobilis have been reported for the glucose conversion while Pichia stipitis, Candida shehatae and Pachysolen tannophilus have been reported for the xylose conversion . Various recombinant yeasts and bacteria have been developed during the last few decades that can ferment both the sugars . However, their use for large-scale industrial processes still needs improvements related to the stability of the entire process and to overcome the public concerns for recombinant microorganisms [11,12]. Attempts have been made to utilize both hexoses and pentoses by co-culturing of Saccharomyces cerevisiae and Pichia stipitis . However, co-cultures of these yeasts do not always ensure the efficient xylose conversion which may be because of diauxic behavior of xylose-fermenting yeasts, inhibition by ethanol and oxygen competition between the xylose fermenters and glucose fermenters . Generally microorganisms consume glucose over galactose followed by xylose and arabinose . In co-culture process if fermentation time is not sufficiently long, pentoses remain unutilized in the medium. Hence to overcome the problems of co-culturing, sequential glucose fermentation by Saccharomyces cerevisiae and xylose fermentation by Pichia stipitis can be a logical approach. Such a strategy can help to overcome glucose repression, ethanol inhibition and oxygen competition on xylose fermenting Pichia stipitis. Sequential ethanol fermentation processes using hexose and pentose fermenting yeasts have been reported [15-17].
We have already optimized saccharification of mild alkali treated maize stover using cocktail of in-house produced hemicellulases supplemented with commercial cellulases. This cocktail was effective at high substrate loading and preliminary experiments on ethanol production from enzymatic hydrolysate of maize stover by Saccharomyces cerevisiae 3524, yielded maximum 7.68 g/l ethanol at 3% reducing sugar concentration. In order to generate high concentration of ethanol, the present study was aimed at optimization of ethanol fermentation process using response surface methodology. In addition the research attempted sequential fermentation of hexoses followed by pentoses using Saccharomyces cerevisiae and Pichia stipitis respectively.
Throughout the research work analytical grade chemicals, media and reagents were used and were purchased from Qualigens, Hi-media, Merck, Loba from India. P-nitrophenyl-β-D-xylopyranoside P-nitrophenyl-α-L-arabinofuranoside, P-nitrophenyl-β-D-glucopyranoside and Ethyl ferlulate (Ethyl 4-hydroxy-3-methoxycinnamate) were obtained from Sigma-Aldrich, USA. Maps Enzyme Limited, India provided commercial cellulase MAPs 450.
Maize stover was obtained from local farmer from Panchmahal district, Gujarat. Maize stover was cut to 5 mm length, washed with tap water, air-dried and stored in air tight plastic bag at room temperature.
For production of hemicellulases under solid state fermentation, Aspergillus niger ADH-11 (Accession no KF026012) was used. While for ethanol production standard yeast cultures, Sacchromyces cerevisiae NCIM 3524 and Pichia stipitis NCIM 3497 were procured from NCIM, Pune, India.
Crude β-xylosidase rich hemicellulases was indigenously produced by Aspergillus niger ADH-11 under solid state fermentation using previously optimized conditions . Commercial cellulase was kindly provided by MAPS Enzymes Private Ltd., Gujarat, India. Enzyme doses were used as per necessity in the experiment. The crude β-xylosidase enzyme from Aspergillus nigerADH-11 and commercial cellulase (MAPs 450), were also having other cellulolytic and xylanolytic enzymes (Table 1).
|Enzyme||β-Xylosidase (U ml-1)||α-L-rabinofuranosidase (U ml-1)||Feruloyl esterase (U ml-1)||Xylanase (U ml-1)||Filter paper activity (U ml-1)||Endo-glucanase (U ml-1)||β-Glucosidase (U ml-1)|
ND: Not Detected
Table 1: Composition of enzymes in each source.
β-Xylosidase activity was determined by quantifying p-nitrophenol release from p-nitrophenyl-β-D-xylopyranoside at 50°C. The reaction mixture containing of 2 mM p-nitrophenyl-β-D-xylopyranoside in 50 mM sodium citrate buffer (pH 5.3) was incubated with enzyme at 50°C for 30 min in a total volume of 0.5 ml. The reaction was ended by adding 1 ml of 2 M sodium carbonate. The amount of releasedp-nitrophenol was determined by measuring absorbance at 410 nm. One unit of β-xylosidase activity is defined as amount of enzyme required to release 1 µM of p-nitrophenol per minute under assay condition. α-L-Arabinofuranosidase activity was determined by measuring p-nitrophenol release from p-nitrophenyl-α-L-arabinofuranoside at 50°C. The reaction mixture consisting of 1 mM p-nitrophenyl-α-L-arabinofuranoside in 50 mM sodium citrate buffer (pH 5.3) was incubated with enzyme at 50°C for 10 min in total volume of 0.5 ml. The reaction was terminated by adding 1 ml of 2 M sodium carbonate. The amount of p-nitrophenol released was determined by measuring absorbance at 410 nm. One unit of α-L-arabinofuranosidase activity is defined as amount of enzyme required to release 1 µM of p-nitrophenol per minute under assay condition. Xylanase activity was determined using birch wood xylan solution (1%) as a substrate . The substrate was prepared in 50 mM sodium citrate buffer (pH 5.3). The enzyme reaction was carried out for 10 min at 50°C and was ended by adding 1 ml of DNS reagent. The xylanase activity was determined by quantifying the release of reducing sugar using xylose as standard . One unit of xylanase activity is defined as amount of enzyme required to release 1 µM of xylose per minute under assay condition. According to the IUPAC recommendation, Filter paper activity was measured using Whatmann filter paper no. 1 as a substrate . The reducing sugars, release after 60 min in 50°C, at pH 4.8 (50 mM sodium citrate buffer) was measured as glucose equivalent by DNS method. One unit of filter paper activity is defined as amount of enzyme required to release 1 µM of glucose per minute under assay condition. Endo-glucanase activity was determined by measuring reducing sugarsrelease from carboxymethylcellulose (CMC) at 55°C. The reaction mixture consisting of 2% carboxymethylcellulose in 50 mM sodium citrate buffer (pH 4.8) was incubated with enzyme at 55°C for 30 min in total volume of 1.0 ml. The reaction was terminated by adding 1 ml of DNS reagent. The enzyme activity was determined by measuring the release of reducing sugar using glucose as standard. One unit of endo-glucanase activity is defined as amount of enzyme required to release 1 µM of glucose per minute under assay condition. β-Glucosidase activity was determined by measuring p-nitrophenol release from p-nitrophenyl-β-D-glucopyranoside at 50°C. The reaction mixture consisting of 2 mM p-nitrophenyl-β-D-glucopyranoside in 50 mM sodium citrate buffer (pH 4.8) was incubated with enzyme at 50°C for 30 min in total volume of 0.5 ml. The reaction was terminated by adding 1 ml of 2 M sodium carbonate. The amount of p-nitrophenol released was determined by measuring absorbance at 410 nm. One unit of β-glucosidase activity is defined as amount of enzyme required to release 1 µM of p-nitrophenol per minute under assay condition. Feruloyl esterase activity was determined by measuring ferulic acid release from ethyl ferlulate (Ethyl 4-hydroxy-3-methoxycinnamate) at 50°C. The reaction mixture consists of 2 mM ethyl ferulate in 50 mM sodium citrate buffer (pH 5.3) was incubated with enzyme at 50°C for 30 min in total volume of 1.0 ml. The reaction was terminated by heating in boiling water bath for 10 min. The amount of ferulic acidreleased was determined by HPLC using ferulic acid as standard. One unit of feruloyl esterase activity is defined as amount of enzyme required to release 1 µM of ferulic acid per minute under assay condition.
Maize stover pretreatment
Pretreatment of maize stover was carried out by soaking maize stover in 2% NaOH solution (10% slurry (w/v)) at 30°C for 12 h. The substrate was neutralized with 12 N HCl, filtered with wet muslin cloth and then dried in oven at 80°C. Using Goering and Van Soest  method, composition (cellulose, hemicellulose and lignin) of untreated and pretreated maize stover were determined.
Enzymatic saccharification of pretreated maize stover at high substrate loading using cocktail of crude β-xylosidase and commercial cellulase (MAPs 450)
Maize stover saccharification was carried out using 13 FPU/g commercial cellulase (MAPs 450) supplemented with 74.42 U/g in-house produced β-xylosidase rich crude enzyme from Aspergillus niger ADH-11. Enzymatic sacccharification was carried out in 250 ml plastic bottle containing 30% pretreated maize stover and diluted enzyme in 50 mM sodium citrate buffer (pH 4.8) having 0.1% Tween-80 (v/v) in a total volume of 40 ml. Controls were taken for each reaction in which the active enzyme was substituted with heat inactivated enzyme. The reaction system was fortified with 10 mg % sodium azide. The reaction was performed at 50°C in hybridization oven at 100 rpm. After 36 h, samples were withdrawn and heated in boiling water bath (100°C) for enzyme inactivation. Then filtered using wet muslin cloth by thorough squeezing followed by centrifuged to collect the clear supernatant, which was used for further analysis to estimate reducing sugars by DNS method  and glucose, xylose and arabinose by HPLC method.
Optimization of ethanol production using hexose fermenting Saccharomyces cerevisiae NCIM 3524
Inoculum was prepared using 12 h grown culture (Saccharomyces cerevisiaeNCIM 3524) at 30°C in a medium containing glucose (1%), yeast extract (0.3%), malt extract (0.3%) and peptone (0.5%) having pH 6.5 ± 0.2. The fermentation experiment was carried out in 50 ml screw cap tube at 30 ± 2°C without agitation. The samples were centrifuged for 10 min at 4°C at 15,000 rpm, filtered using 0.2 µ filter and analyzed using HPLC for the presence of sugars and ethanol.
BBD for optimization of ethanol fermentation from hexose sugar: A Box- Behnken factorial design with three factors and three levels was used for optimization of ethanol production. The independent variables were % glucose (X1), % inoculum (X2) and time (X3). Final ethanol yield (g g-1) was the dependent response variable. Selected independent variables were studied at three levels (Table 2) employing BBD with total 15 experimental runs (Table 3). The variables were taken at a central code value considered as zero. The temperature was kept constant at 30°C throughout the experiments runs.
|Variable||Symbols||Coded level of variable|
Table 2: Coded values and experimental range of process variables used in experimental design.
|Run||Sugar concentration (X1)||Inoculum size (X2)||Time (X3)||Ethanol yield (g g-1)|
Table 3: Experimental plan and the results of the Box-Behnken design.
Quadratic models considered as response surface model for predicting the optimal points were expressed according to Eq. (1) and (2).
For statistical calculations the independent variables were coded as:
Where xi is the experimental coded value of the variable; Xo is the middle value of Xi and δXi is the step change.
Ethanol yield (response Y) was explained as a second order response model on three independent variables given by
Where Y is the predicted response variable, βo, βi, βii, βij are fixed regression coefficients of the model, xi, xj (i=1, 2, 3, i ≠ j) represents independent variables in the form of original values.
Interpretation and data analysis
The experimental design results were evaluated and interpreted by MINITAB 16 (PA, USA) statistical software. Optimum fermentation parameters prediction and response contour plot created by the model was also evaluated by the same software. ANOVA was used to establish the significance of the model parameters.
Recovery of ethanol from fermented broth
Ethanol produced by hexose fermenting yeast was recovered using Rota-evaporator under vacuum at 45°C with 120 rpm for 60 min.
Ethanol production using pentose fermenting Pichia stipitis NCIM 3497
Ethanol fermentation using Pichia stipitis NCIM 3497 from fermented broth after distillation was carried out by preparing medium containing 0.3% (w/v) yeast extract, 0.3% (w/v) malt extract, 0.5% (w/v) peptone and adjusting xylose concentration at 1.58% with pH 6.5 ± 0.2. The medium was sterilized at 10 lbs for 20 min. After cooling the medium to 30°C, it was inoculated aseptically with 5.40% (v/v) of 24 h old culture inoculum (Pichia stipitis NCIM 3497 with Absorbance of 0.6 at 660 nm). All the closed screw caps tubes were incubated at 30°C in static condition. The samples were withdrawn after 24 h and analyzed for ethanol production by HPLC and reducing sugars by DNS method .
Ferulic acid was analyzed using high performance liquid chromatography system (Shimadzu, Japan) equipped with photo diode detector (PDA) using a Luna 5u C18 (2) column with dimension of 250 mm × 4.6 mm. Samples were eluted using Water: Acetonitrile : Formic acid (7:2:1) with the flow rate of 0.6 ml min-1. Column was kept at room temperature throughout analysis. Ferulic acid (1 mg ml-1) was used as the standards. Appropriately diluted samples were filtered through 0.2 μ filters to remove any residual debris before injecting in the column. Ferulic acid was quantified using average peak areas of standards.
Monosaccharide sugars were analyzed using high performance liquid chromatography system (Shimadzu, Japan) equipped with a refractive index detector (RID). Sugars were analyzed using a Phenomenex, Rezex ROA-Organic acid H+(8%) column with dimension of 300 mm × 7.8 mm. Samples were eluted using 5 mM H2SO4 with the flow rate of 0.6 ml min-1. Column temperature was kept at 65°C throughout analysis. Glucose, xylose arabinose and ethanol were used as the standards. Appropriately diluted samples were filtered through 0.2 μ filters to remove any residual debris before injecting in the column. Sugars and ethanol were quantified using average peak areas of standards.
Enzymatic hydrolysis of mild alkali treated maize stover using in-house produced hemicellulases supplemented with commercial cellulases was previously optimized in our laboratory at low substrate loading and the cocktail was also found effective at high substrate loading. In order to achieve higher ethanol concentration enzymatic hydrolysis of maize stover was carried out at 30% substrate loading. Effective industrial production of bioethanol from maize stover requires utilization of both hexose and pentose sugars. Hence, the sequential ethanol fermentation was carried out for bioethanol production from enzymatic hydrolysate of maize stover.
Enzymatic saccharification of pretreated maize stover at high substrate loading using cocktail of crude β-xylosidase and commercial cellulase (MAPs 450)
Enzymatic saccharification at high substrate loading potentially offers many advantages. High substrate load have positive effect on economy of the process as it allows use of smaller reactor, produce concentrated sugar syrup and eventually lower distillation cost. The developed cocktail using in-house produced hemicellulases with commercial cellulases was used for saccharification of maize stover at high substrate loading (30%). The result clearly showed that the cocktail was highly effective at high substrate loading and liquefaction of solid residues was observed after 36 h (Figure 1) which contained maximum 161.32 mg ml-1 reducing sugar. This sugar rich hydrolysate mainly contained 67.84 mg ml-1 glucose, 34.62 mg ml-1 xylose and 6.14 mg ml-1 arabinose. Such a concentrated sugar syrup is highly advantageous for commercial ethanol fermentation process as concentration step can be avoided.
Optimization of ethanol production from hexose sugar using RSM
Three variables viz. % glucose, % inoculum and time (h) were selected for response surface optimization and ethanol yield g g-1 was selected as response variable. The results of all the experimental runs (15 run) are shown in Table 3. The experimental results suggest that the variable used in the present study had strong effect on ethanol yield. On the basis of these experimental values, statistical testing was carried out using MINITAB 16. The analysis of the model was tested by Fisher’s ‘F’ and Student’s t-test. Analysis of variance (ANOVA) of ethanol yield showed that the model was significant (P=0.002) as shown in Table 5, mainly due to the square portion of the regression model. A P-value below 0.05 indicates the test parameter are significant.
|% Glucose X % Glucose||-0.006562||0.003106||-2.113||0.088|
|% Inoculum X % Inoculum||-0.001450||0.000497||-2.918||0.033|
|Time X Time||-0.000043||0.000086||-0.503||0.636|
|% Glucose X % Inoculum||0.003250||0.001194||2.723||0.042|
|% Glucose X Time||0.001563||0.000497||3.141||0.026|
|% Inoculum X Time||-0.000208||0.000199||-1.047||0.043|
Table 4: Regression coefficients for ethanol yield g g-1.
|Source||DF||Seq SS||Adj SS||Adj MS||F||P|
Table 5: Analysis of variance for ethanol yield g g-1.
The fitted second-order response surface model as specified by Equation 2 for ethanol yield in coded process variables is as follows:
Where, Y is ethanol yield, and x1, x2 and x3 are uncoded values of % glucose, % inoculum and Time h, respectively. A comparison of the experimentally obtained values with the predicted values indicated that these data are in reasonable agreement as shown in Table 3. The parameter estimates and the corresponding P-value showed that selected variable % glucose and % inoculum had significant square effect (0.024 and 0.052) on ethanol yield (Tables 4 and 5). Also interaction between % glucose and % inoculum, % glucose and time and % inoculum and time had significant square effect (0.042, 0.026 and 0.043) on ethanol yield (Table 4). The R2 value provides a measure of variability in the observed response values that can be explained by the experimental factors and their interactions. Joglekar and May  have suggested that for a good fit of the model, R2 should be at least 80%. Coefficient R2 for reducing sugar yield was observed to be 97.5%.
Application of RSM by Box-Behnken design, predicted that maximum ethanol yield occurred at values of process parameters as 5% glucose, 14.55% inoculum and Time 35.51 h. The model predicted highest (optimum) ethanol yield was 0.364 g g-1 (Figure 2).
Validation of the experimental model
A repeat fermentation for ethanol production from hexoses using Saccharomyces cerevisiaeNCIM 3524 under optimal conditions was carried out for the validation of optimized parameters. The enzymatic hydrolysate was effectively utilized by hexose fermenting yeast and under optimized condition 90.65% glucose was utilized. Ethanol yield was 0.374 g g-1 (18.93 mg ml-1) with 0.53 g l-1 h-1 productivity (Table 6). Before optimization, ethanol yield was only 7.68 mg ml-1 at 3% total reducing sugars which contained only 1.2% glucose. By applying response surface methodology ethanol production was enhanced up to 2.46 fold at high sugar concentration (9% total reducing sugars which contained 5% glucose).
|Initial reducing sugar (g l-1)||90.0|
|Initial glucose (g l-1)||50.0|
|Ethanol (g l-1)||18.93|
|% Reducing sugar consumed||55.55%|
|% Glucose consumed||90.65%|
|Ethanol yield g g-1 glucose||0.374|
|Ethanol yield g g-1 consumed reducing sugar||0.378|
|Productivity (g l-1h-1)||0.53|
Table 6: Ethanol fermentation from enzymatic hydrolysate of maize stover by Saccharomycere cerevisiae under optimized conditions.
In current scenario, cost-competitive ligno-cellulosic biofuel production can be intended by operating the process at high gravity . Li et al. reported maximum 11.8 mg ml-1 ethanol production with 0.110 g l-1 h-1 productivity using Saccharomyces cerevisiae . As compared to this, the productivity of the ethanol from hexose sugar in the present study was higher. A comparison of ethanol production by Saccharomycere cerevisiaefrom various lignocellulosic biomass is shown in Table 7. It can be seen that in the present study ethanol concentration is comparable or even higher as compared to other reports.
|Lignocellulosic Biomass||Fermentation condition||Ethanol concentration (mg/ml)||Ethanol Yield (g/g)||Reference|
|Wheat straw||Reducing sugar 6.5%, inoculum 3.3% at 30°C, 36 h||15.6||0.480|||
|Rice straw||Reducing sugar 6%, inoculum 10% at 28°C, 24 h||12.34||0.093|||
|Sweet sorghum||Sorghum juice, containing 19.9% sucrose and 1.9% (w/w) glucose at 33°C, 24 h||-||0.400|||
|Corn cobs||95.3 g l-1 glucose, 5 g l-1 peptone, 3 g l-1 yeast extract at 30°C, 24 h||45.7||0.480|||
|Maize stover||50 g l-1 glucose (90 g l-1 reducing sugars), 14.55% inoculum at 30°C, 35.51 h||18.93||0.374||Present study|
Table 7: Ethanol yield from various lignocellulosic biomass by Saccharomycere cerevisiae.
Ethanol production byPichia stipitis NCIM 3497
After ethanol recovery, hydrolysate mainly contained xylose, hence ethanol production using Pichia stipitis NCIM 3497 was carried out and only 71% xylose was utilized by yeast with maximum ethanol yield of 0.38 g g-1 (2.23 mg ml-1) with productivity 0.09 g l-1 h-1. Agbogbo and Wenger  reported that Pichia stipitis produced ethanol yield 0.37 g g-1 (10.4 mg ml-1) from enzymatic hydrolysate of corn stover. In the current investigation it was observed that production of ethanol from the remaining pentose sugar was lesser which may be due to existence of fermentative inhibitors produced by the hexose utilizing yeast. Hence, further studies are needed for effective utilization of pentose sugars.
It can be concluded from the present study that enzymatic hydrolysate obtained after saccharification of maize stover at high substrate loading was effectively utilized by Saccharomyces cerevisiae. By statistical optimization of ethanol fermentation process, production was enhanced up to 2.46 fold and ethanol concentration reached to 18.93 mg ml-1. However utilization of pentoses by sequential fermentation was not so effective. Hence, further studies are needed to increase ethanol production from pentoses.
Authors gratefully acknowledge Department of Biotechnology (DBT), New Delhi, India and Gujarat State Biotechnology Mission (GSBTM), Gandhinagar, Gujarat, India for providing the research grants to support this work and also thankful to MAPs Enzyme Limited, India for providing cellulase enzyme.
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