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Enzymatic saccharification of cornstalk by onsite cellulases produced by Trichoderma viride for enhanced biohydrogen production


Correspondence: Guang-Li Cao, Nan-Qi Ren, tel. + 86 28 20 08/86 40 26 95; fax + 86 451 86 28 20 08/86 451 86 40 26 95; e-mails: rnq@hit.edu.cn; glcao1980@yahoo.com.cn


Lignocellulosic biomass, if properly saccharified, could be an ideal feedstock for biohydrogen production. However, the high cellulases cost is the key obstacle to its development. In this work, cost-effective enzyme produced by Trichoderma viride was used to saccharify cornstalk. To obtain high sugar yield, a central composite design of response surface method was used to optimize enzymatic saccharification process. Experimental results showed that the enzymatic saccharification rate reached the highest of 81.2% when pH, temperature, cellulases and substrate concentration were 5, 49.7 °C, 35.7 IU g−1, and 38.5 g L−1, respectively. The cornstalk hydrolysate was subsequently introduced to fermentation by Thermoanaerobacterium thermosaccharolyticum W16, the yield of hydrogen reached the highest level of 90.6 ml H2 g−1 pretreated cornstalk. The present results indicate the potential of using T. thermosaccharolyticum W16 for high yield conversion of cornstalk hydrolysate, which was saccharified by onsite enzyme produced by T. viride.


During last few decades, the excessive consumption of fossil fuels has led to an increased demand in finding alternative energy sources that are environmentally friendly and renewable. Hydrogen has emerged as a promising energy carrier which dose not only satisfy the requirements mentioned above but also has its own advantages such as high calorific value, efficiency, and cleanliness. Among various hydrogen production procedures, anaerobic hydrogen fermentation seems to be favorable owing to its high hydrogen production rate and energy-saving process (Kumar & Das, 2000; Xing et al., 2006; Mohan et al., 2007; Wang et al., 2008; Ren et al., 2009). Nevertheless, the major obstacle to the commercialization of this process is the high production cost. Therefore, the development of an effective method to lower the anaerobic hydrogen production cost is the key issue (Kotay & Das, 2008).

One of the cost-cutting methods is the use of renewable and low-cost feedstock as the substrate for hydrogen production. Lignocellulosic biomass such as agricultural, forestry, and municipal wastes is among the earth's most abundant and sustainable renewable natural resource, which can be used as an excellent bioconversion feedstock (Han et al., 2012). Asian countries possess significant potential for producing biohydrogen from crop residues (Cheng et al., 2011a). In China alone, the annual yield of cornstalk exceeds 0.2 billion dry tons, which is suitable for use as the feedstock for the production of hydrogen (Ohgren et al., 2006; Ezeji et al., 2007; Lv et al., 2008; Cao et al., 2009; Pan et al., 2009). However, cellulosic materials are usually not readily for fermentation because they are made up of a matrix of cellulose and lignin bound by hemicellulose chains (Singh & Bishnoi, 2012a), which are barricade for further use. Currently, one of the effective strategies to produce hydrogen from cellulosic feedstock is the separate hydrolysis and fermentation process (SHF) (Cheng et al., 2011b). This process usually requires appropriate pretreatment of raw materials to remove lignin and hemicellulose from lignocellulose (Cheng & Chang, 2011), followed by the hydrolysis of cellulose and hemicellulose into reducing sugars that could be further fermented. However, during the hydrolysis step, a large amount of expensive commercial enzymes are usually needed, which would further hinder the commercialization of cellulosic hydrogen production (Stork et al., 2009). Consequently, cost-effective and high-efficiency cellulases should be developed. Trichoderma is a well-studied filamentous fungus for cellulases production, the utilization of onsite enzymes produced by the strain for saccharification of lignocellulosic biomass has received extensive attention. Previous studies have shown that there are several factors affecting enzymatic saccharification of cellulose, including substrates, cellulases activity, and reaction conditions (temperature and pH) (Jeya et al., 2009). However, most of them were conducted to estimate the effects of one or two factors. As enzymatic saccharification is subjected to by two and more factors simultaneously, response surface methodology (RSM) was employed in this research to reveal the interactive effects between components and to identify the optimum conditions for reducing sugar production from cornstalk.

In this study, the enzymatic saccharification of alkali-pretreated cornstalk and the optimization of saccharification parameters for maximum reducing sugar production were evaluated first. Then, the cornstalk hydrolysate was fermented by Thermoanaerobacterium thermosaccharolyticum W16 to verify its potential as substrate for hydrogen production.

Materials and methods

Pretreatment of cornstalk

Air-dried cornstalk was obtained from Northeast Agricultural University, Harbin, Heilongjiang province, China. Cornstalk was pretreated with 2% NaOH as described by Ren et al. (2010), except that the reaction temperature was increased to 100 °C. After pretreatment, the solid cellulosic residues were collected by filtration and rinsed thoroughly with water to neutral pH, then dried at 60 °C until constant weight was obtained.

Enzyme extraction and assay

Trichoderma viride CGMCC 3.2876, which was purchased from China General Microbiological Culture Collection Center and maintained on Potato Dextrose Agar (PDA) plates at 4 °C, was used in this study. For cellulases production, 100 mL liquid medium contained (L−1): (NH4)2SO4, 1.4 g; urea, 0.3 g; KH2PO4, 2.0 g; MgSO4·7H2O, 0.3 g; CaCl2, 0.3 g; wheat bran, 20 g; soybean cake powder, 5 g; cellulose power, 8 g; 1 mL trace element solution (Mandels & Reese, 1957), was added in 250 mL conical flask. Each flask was inoculated with 2 × 108 spores of T. viride spore suspension. Then, enzymes production was carried out at 29 °C in a gyratory incubator shaker with a speed of 130 rpm min−1. After 4 day fermentation, the culture medium was harvested by centrifugation at 8000 rpm for 10 min at 4 °C. Clarified supernatant was used as the source of cellulases.

Filter paper activity of cellulase was determined as described by Ghose (1987). Endoglucanase activity was detected using carboxymethyl cellulose as a substrate. The β-glucosidase activity was measured by PNPG assay method (Paquet et al., 1991). Xylanase activity was determined according to Esterbauer et al. (1983). One unit of FPase and xylanase activity was defined as 1 μmol glucose or xylose equivalents released per minute. One unit of β-glucosidase activity was expressed as the amount of enzyme that liberates 1 μmol p-nitro-phenol min−1. Under above conditions, the cellulases activity of crude enzyme from T. viride were 4.2 FPA mL−1, endoglucanase activity of 8.5 IU mL−1, β-glucosidase activity of 5 IU mL−1, and xylanase activity of 53 IU mL−1, respectively.

Optimization of enzymatic saccharification

Independent variables including temperature, pH, cellulases and substrate concentration were optimized for enhancing the reducing sugar yield using a response surface of central composite design (CCD) (Wang et al., 2007). The range and levels of the variables (coded as X1, X2, X3, and X4, respectively) investigated are shown in Table 1. Each of the independent variables was studied at five different levels as per CCD in four variables with a total of 30 experiments with six replications of the center point. All the experiments were carried out at 130 rpm on a rotary shaker for 96 h.

Table 1. Experimental range and levels of the independent process variables to study the saccharification of cornstalk
Independent variableCodeRange and levels
pH X 1 13579
Temperature (°C) X 2 1030507090
Cellulase concentration (IU g−1) X 3 510305065
Substrate concentration (g L−1) X 4 510305065

For predicting the optimal point, a second-order polynomial function was fitted to evaluate the correlativity between independent variables and response in forms of quadratic equation expressed as follows:

display math(1)

the predicted response (Y) was therefore correlated with the set of regression coefficients (b): the intercept (b0), linear (bi), quadratic (bii), and interaction (bij) coefficients; Xi represented the independent variables studied. The response value, sugar yield (Y) was the average of triplicates.

The Design Expert (Vertion, Stat-Ease Inc., Minneapolis, MN, USA) was used for regression and graphical analyses of the obtained data. anova was used to estimate the statistical parameters.

Biohydrogen production from cornstalk hydrolysate

The hydrolyzed slurry obtained at the optimum conditions was boiled to inactivate cellulases. After cooling to the room temperature, the slurry was centrifuged and the reducing sugar concentration of the supernatant was diluted to 10 g L−1 before used as carbon source to produce H2 by T. thermosaccharolyticum W16 isolated by Ren et al. (2008). Hydrogen production was performed as described by Zhao et al. (2012b) in 100 mL serum vials with working volume of 50 mL at 60 °C with an initial pH of 6.5. Cell density, pH, residual carbon substrate concentration, quantity and compositions of produced biogas, and metabolic products were monitored during the course of fermentation. All tests mentioned above were performed in triplicate to determine the reproducibility of the experiments.

Analytical methods

The composition of pretreated and unpretreated cornstalk was measured following the methods described by Sluiter et al. (2006, 2008). Sugar concentration and composition during fermentation were determined using a high-performance liquid chromatography (HPLC) system (LC-10A, Shimadzu Corporation, Kyoto, Japan) (Ren et al., 2010). Cell density in the liquid medium was monitored by measuring turbidity at 600 nm (Zhao et al., 2012a). Hydrogen and the fermentation metabolites were measured following the methods described by Zhao et al. (2012b). The enzyme saccharification rate was calculated as described by Li et al. (2009): enzymatic saccharification (%) = hexose (pentose) released (g) × 0.9 (0.88) × 100/polysaccharides in substrate (g) where 0.9 and 0.88 are the correction coefficient for hydrolysis.

Results and discussion

Saccharification of pretreated cornstalk

As shown in Table 2, the alkaline pretreated cornstalk consisted of 65.2% cellulose, 23.8% hemicellulose, and 7.6% lignin. Comparing with the raw materials, alkaline pretreatment resulted in 78.3% and 48.7% of lignin and hemicellulose removal, respectively, accompanied with cellulose proportion increased by 26.7%. These results indicated that alkaline pretreatment could efficiently fractionate hemicellulose and lignin from cornstalk and allow for the enhancement of enzymatic saccharification (Taherzadeh & Karimi, 2008).

Table 2. Compositions of raw and pretreated corn stalk
Corn stalkComposition (%)aSolid yield (%)bRemoval yield (%)c
  1. a

    Composition was shown as percentage of the solid fraction before and after pretreatment.

  2. b

    Solid yield was shown as percentage of the initial amount of dry matter.

  3. c

    Removal yield was shown as percentage of the amount in the initial material.

Raw material38.525.619.3
NaOH pretreatment65.223.87.5855.26.448.778.3

Preliminary saccharification test showed that when substrate concentration, enzyme concentration, temperature, and initial pH were 35 g L−1, 30 FPA g−1-substrate, 50 °C, and 4.5, respectively, 2% NaOH pretreated cornstalk could release 10.0 g reducing sugar after 72 h of hydrolysis (Fig. 1), the conversion yield reached the maximum of 60%. The results indicated that alkaline pretreated cornstalk could be an eligible substrate for enzyme saccharification.

Figure 1.

Time course of reducing sugar yield from alkaline pretreated cornstalk by Trichoderma viride cellulase.

Optimization of enzymatic saccharification parameters

Optimization of enzymatic hydrolysis parameters was crucial for efficient saccharification, so the influence of key factors were determined by response surface methodology. CCD of response surface analysis was used to optimize the effects of temperature (°C), pH, cellulases concentration (IU g−1), and substrate concentration (g L−1) on enzymatic saccharification of cornstalk to obtain a high total sugar yield for enhanced hydrogen fermentation. Experimental design and results were presented in Table 3. Based on the 4 × 30 CCD analysis, the approximating function of sugar yield obtained by Design-Expert software was given in Eq. (2):

display math(2)
Table 3. The central composite design with four independent variables and the experimental results
RunpHTemperature (°C)Cellulase (IU g−1)Substrate (g L−1)Sugar yield (g L−1)
X 1 X 2 X 3 X 4
13.0070.0020.0050.002.70 ± 0.03
23.0030.0050.0050.007.50 ± 0.05
37.0070.0020.0050.005.06 ± 0.01
45.0010.0035.0035.002.42 ± 0.02
55.0050.0035.0035.0014.66 ± 0.04
67.0030.0050.0050.006.97 ± 0.11
77.0030.0020.0050.006.82 ± 0.09
85.0050.0065.0035.0012.45 ± 0.19
93.0030.0020.0050.005.21 ± 0.07
105.0050.005.0035.007.56 ± 0.02
119.0050.0035.0035.000.92 ± 0.05
125.0050.0035.0035.0014.66 ± 0.05
135.0050.0035.0065.009.73 ± 0.03
145.0090.0035.0035.002.92 ± 0.12
155.0050.0035.0035.0014.66 ± 0.05
165.0050.0035.0035.0014.66 ± 0.05
177.0070.0020.0020.003.91 ± 0.03
183.0030.0050.0020.005.11 ± 0.04
195.0050.0035.005.006.02 ± 0.10
205.0050.0035.0035.0014.66 ± 0.07
213.0030.0020.0020.003.00 ± 0.03
227.0070.0050.0050.007.99 ± 0.05
233.0070.0050.0050.006.99 ± 0.02
245.0050.0035.0035.0014.66 ± 0.12
251.0050.0035.0035.000.08 ± 0.01
263.0070.0050.0020.006.57 ± 0.08
273.0070.0020.0020.002.41 ± 0.02
287.0070.0050.0020.007.55 ± 0.05
297.0030.0020.0020.002.72 ± 0.02
307.0030.0050.0020.003.82 ± 0.03

In the above equation, Y corresponded to response of sugar yield. X1, X2, X3, and X4 were equivalent to independent variables of temperature, pH, cellulases and substrate concentration, respectively.

To validate the statistical results and the model equation, the analysis of variance (anova) was conducted as shown in Table 4. Model fit for sugar yield was highly significant (P < 0.01), whereas the lack of fit was not significant (P > 0.05). Coefficient of determination (R2) was 0.9987, which meant 99.87% of the total variation could be accounted for by the model equation. These findings indicated that the model equation was statistically capable for predicting the effects of temperature, pH, cellulases and substrate concentration on the sugar yield. What's more, the anova analysis showed that the linear and quadratic effect of temperature, pH, cellulases and substrate concentration, and all the interactive effect except interaction of cellulases concentration and substrate concentration on sugar yield were highly significant (P < 0.01), indicating that these terms had great impact on experimental results.

Table 4. Analysis of variance (anova) for the model regression representing sugar yield
SourcesSum of squaresdfMean squareF ValueP-value
Prob > F
  1. Coefficient of determination (R2) = 0.9987.

  2. Adeq precision = 87.980.

X 1 1.6811.6830.76<0.0001
X 2 0.3810.386.970.0185
X 3 38.65138.65706.85<0.0001
X 4 19.42119.42355.13<0.0001
X 1 X 2 2.5012.5045.73<0.0001
X 1 X 3 1.5911.5929.02<0.0001
X 1 X 4 0.7910.7914.380.0018
X 2 X 3 5.5015.50100.59<0.0001
X 2 X 4 5.7215.72104.58<0.0001
X 3 X 4 0.1110.112.070.1709
X 1 2 336.841336.846160.52<0.0001
X 2 2 247.571247.574527.97<0.0001
X 3 2 37.58137.58687.32<0.0001
X 4 2 79.56179.561455.05<0.0001

On the basis of above analysis, the model predicted a maximum sugar yield of 14.92 g L−1 under the conditions of temperature 49.7 °C, pH 5.0, cellulases concentration 35.7 IU g−1, and substrate concentration 38.5 g L−1, respectively.

The response surface plots based on Eq. (2) were shown in Figs 2 and 3. Each response surface showed relative effects of two variables on sugar yield while keeping the other ones constant at middle level. As can be seen from Figs 2 and 3, the response surface of sugar yield showed an obvious peak, indicating that the optimum conditions were inside the design boundary well. Sugar yield increased with temperature, pH, cellulases and substrate concentration increasing from 30 °C, 3, 10 IU g−1, and 10 g L−1 to 50 °C, 5, 35.7 IU g−1, and 38.5 g L−1, respectively, whereas a further increase resulted in reversal of this trend. The results revealed that temperature and pH significantly affected the cellulases activity. Highest sugar yield occurred under the optimum temperature and pH conditions, whereas deviating from the optimum conditions will lead to lower sugar yield due to the deactivation of the enzymes, this conclusion was in good agreement with our previous report by Zhao et al. (2012c). It could also be inferred from this study that sugar yield has positive correlation with cellulases and substrate concentration under the value of 35.7 IU g−1 and 38.5 g L−1, beyond the optimum cellulases and substrate concentration, the soluble sugar yield would be slightly decreased. The phenomenon was the same as described by Guo et al. (2010), which is partly due to inhibition of the enzymatic hydrolysis process by the released glucose (Singh & Bishnoi, 2012a), partly because a probable saturation effect (Pan et al., 2009).

Figure 2.

The response surface plots showing the interaction of temperature and pH, cellulase concentration and pH, substrate concentration and pH on enzymatic saccharification.

Figure 3.

The response surface plots showing the interaction of temperature and cellulases concentration, substrate concentration and temperature, substrate concentration, and cellulases concentration on enzymatic saccharification.

An experiment with the specified conditions optimized above was performed to determine the validity of the predicted values. The sugar yield from the experiments was 14.83 g L−1. The saccharification efficiency reached the highest of 81.2%, which was 21% higher than optimized before (Fig. 1).

The results mentioned above and the validation of the model experiment indicated that sugar yield was strongly dependent on the temperature, pH, cellulases and substrate concentration, and the model was convincible to optimize the experimental parameters and enhance sugar production.

Overall mass balance

An overall mass balance for pretreatment and enzymatic saccharification process of cornstalk was calculated and shown in Fig. 4. This process was carried out under the optimum conditions obtained above. Conversion and saccharification of cornstalk were starting with 100 g dry weight. From the data, a high level of 81.2% of the polysaccharides in pretreated cornstalk was efficiently saccharified to reducing sugars. In addition, 64% of the initial cellulose of corn stalk was converted to glucose. From the overall mass balance, about 57% of theoretical maximum yield of the total initial biomass was converted into monosaccharides, in other words, by going through the alkaline pretreatment and enzymatic saccharification steps, a total of 36.2 g of reducing sugars were obtained from 100 g of cornstalk.

Figure 4.

Mass balance for enzymatic saccharification process of cornstalk.

Trichoderma is well-known for its cellulase production and has been widely used in enzymatic saccharification. Trichoderma reesei, an efficient cellulase producer, was established as an effective species for saccharifying maize straw to yield maximum reducing sugar level of 814 mg g−1-substrate (Chen et al., 2008). Cellulolytic enzymes from T. reesei A1 strain could effectively hydrolyze rice straw, achieving over 70% monosaccharide yield (Vlasenko et al., 1997). These results were lower than the saccharification efficiency of 81.2% obtained under the optimized conditions in this research. The sugar yield obtained in this study is even comparable with that used blends of onsite enzyme produced by A. heteromorphus and T. reesei for the hydrolysis of microwave alkali-pretreated rice straw (Singh & Bishnoi, 2012b) and commercial enzyme from Trichoderma (Celluclast 1.5, Novozyme) (Saha & Cotta, 2008) for saccharification of lime pretreated rice hull. The above all proved that the use of crude enzyme from T. viride for saccharification is both high efficient and low priced.

Hydrogen production from cornstalk hydrolysate

To make fully utilization of substrate, cornstalk hydrolysate was diluted from 14.83 to 10 g L−1. Diluted cornstalk hydrolysate, which mainly contained 7.3 g L−1 of glucose, 2.2 g L−1 of xylose, and 0.56 g L−1 of arabinose, was inoculated with T. thermosaccharolyticum W16 to evaluate its potential for hydrogen production. Figure 5 shows that after 4 h fermentation, H2 quickly evolved and the production rate reached the maximum of 3.1 mmol L−1 h−1 at 20 h of inoculation. Substrate including glucose, xylose, and arabinose all can be consumed, although the organism showed preference for hexose utilization, xylose utilization reached 81% at the end of fermentation. After 36 h cultivation, the cumulative H2 production reached the highest of 2491 mL L−1, equivalent to 90.6 mL H2 g−1 pretreated cornstalk. The H2 content in the biogas was around 52% (data not shown). During H2 fermentation, acetate, butyrate, and ethanol reached the maximum yields of 31.4 mmol L−1, 17.1 mmol L−1, and 10.6 mmol L−1 at 36 h, respectively, followed by small amounts of butanol and pyruvate. Among them, the contents of acetate and butyrate accounted for 70–80% of the total soluble metabolites.

Figure 5.

H2 fermentation by Thermoanaerobacterium thermosaccharolyticum W16 from cornstalk hydrolysate under optimized saccharification conditions.

With respect to hydrogen yield, comparison of this process with others described in literatures is difficult, because the result is greatly depend on the type of feedstock and enzymatic saccharification yields on assay conditions. Comparing our work by using cornstalk hydrolysate with pure substrate (such as glucose) by T. thermosaccharolyticum W16, considerable H2 production efficiency was attained (Ren et al., 2010). This study demonstrated that production of cellulosic biohydrogen from cornstalk was workable by using hydrogen-producing bacteria T. thermosaccharolyticum W16 combined with saccharification of feedstock using crude enzyme produced from T. viride.

This work introduced the crude enzymatic saccharification and its application in cellulose-hydrogen production process from cornstalk. Enzymatic saccharification conditions were investigated using a 24 central composite design to get maximum sugar yield. Experimental results showed that under the optimum conditions, the sugar yield reached the highest of 14.83 g L−1. Subsequently, the maximum hydrogen yield of 111.2 mmol L−1 was obtained from cornstalk hydrolysate by T. thermosaccharolyticum W16, which corresponds to 90.6 mL H2 g−1 pretreated cornstalk. The present results demonstrated that using the crude enzyme from T. viride to saccharify cornstalk under the optimized conditions integrated with hydrogen production is an economical feasible and efficient process of converting lignocellulose to hydrogen.


This research was supported by National Natural Science Foundation of China (No. 51178140, no. 30870037, and no. 31100095), Project 50821002 (National Creative Research Groups), National Science & Technology Pillar Program during the Eleventh Five-Year Plan Period (2008BADC4B01), China Postdoctoral Science Foundation (No. 20110491053), Heilongjiang Postdoctoral Science Foundation (No. LBH-Z11133), the Fundamental Research Funds for the Central Universities (No. HIT. NSRIF. 2011019), and the Open Project of State Key Laboratory of Urban Water Resource and Environment (No. HC201114).