Trichoderma species are widely used for the commercial production of cellulolytic enzymes. In the present investigation, medium components were optimized using a central composite design and response surface methodology to produce endoglucanase (EG) from Trichoderma harzianum KUC1716. From the various medium components tested, cellulose, soy peptone, and thiamine HCl were selected as the optimal carbon, nitrogen, and vitamin sources, respectively. The highest EG (1.97 U/mL) production was obtained with 1.85% cellulose, 0.48% soy peptone, and 0.10% thiamine HCl. EG production in the optimized medium was 2.6 fold higher than in the unoptimized medium. In addition, the crude enzyme preparation from T. harzianum KUC1716 supplemented with β-glucosidase from Schizophyllum commune KUC9397 was used to hydrolyze various types of lignocellulosic materials and showed significant saccharification yields on all lignocellulosic materials, surpassing that of a commercial enzyme cocktail. It was verified that the crude enzyme preparation derived from T. harzianum KUC1716 could replace the commercial enzymes. This highlights the potential of the crude enzymes for use in biomass conversion systems.
Optimization of Endoglucanase Production by Trichoderma harzianum KUC1716 and Enzymatic Hydrolysis of Lignocellulosic Biomass
Hanbyul Lee,a Young Min Lee,a Young Mok Heo,a Hwanhwi Lee,a Joo-Hyun Hong,a Seokyoon Jang,aMihee Min,a Jaejung Lee,b Jun Seok Kim,c Gyu-Hyeok Kim,a and Jae-Jin Kim a*
Trichoderma species are widely used for the commercial production of cellulolytic enzymes. In the present investigation, medium components were optimized using a central composite design and response surface methodology to produce endoglucanase (EG) from Trichoderma harzianumKUC1716. From the various medium components tested, cellulose, soy peptone, and thiamine HCl were selected as the optimal carbon, nitrogen, and vitamin sources, respectively. The highest EG (1.97 U/mL) production was obtained with 1.85% cellulose, 0.48% soy peptone, and 0.10% thiamine HCl. EG production in the optimized medium was 2.6 fold higher than in the unoptimized medium. In addition, the crude enzyme preparation from T. harzianum KUC1716 supplemented with β-glucosidase from Schizophyllum commune KUC9397 was used to hydrolyze various types of lignocellulosic materials and showed significant saccharification yields on all lignocellulosic materials, surpassing that of a commercial enzyme cocktail. It was verified that the crude enzyme preparation derived from T. harzianum KUC1716 could replace the commercial enzymes. This highlights the potential of the crude enzymes for use in biomass conversion systems.
Keywords: Endoglucanase; Optimization; Response surface methodology; Saccharification; Trichoderma harzianum
Contact information: a: Division of Environmental Science & Ecological Engineering, College of Life Science & Biotechnology, Korea University, Seoul, Korea; b: Division of Wood Chemistry and Microbiology, Korea Forest Research Institute, Seoul, Korea; c: Department of Chemical Engineering, Kyonggi University, Suwon, Korea; *Corresponding author: email@example.com
Lignocellulosic materials are abundant, renewable, and sustainable resources worldwide, thus are important sources for various types of industrial processed sugars that can be fermented to ethanol and other organic chemicals (Cardona and Sánchez 2007). Recently, due to the constant fluctuation of oil prices, the significance of biofuel production from lignocellulosic biomass as an alternative energy source has been intensified, and efficient conversion of lignocelluloses to biofuel is gaining interest (Bak et al. 2009).
Cellulose, hemicellulose, and lignin are the three main components of lignocellulosic biomass. Via pretreatment and enzymatic saccharification, cellulose and hemicellulose can be degraded to fermentable sugars, which can be further fermented to ethanol or other products. Among the components, cellulose is the main structural component of lignocellulose, and it is a long chain of glucose molecules. The conversion of cellulose to glucose involves the concerted action of three classes of enzymes: endo-β-1,4-glucanases (EG, EC 18.104.22.168), exo-cellobiohydrolases (CBH, EC 22.214.171.124), and β-glucosidases (BGL, EC 126.96.36.199). EGs randomly hydrolyze internal glycosidic bonds in cellulose to yield reducing or non-reducing new chain ends for CBHs, which subsequently hydrolyze the newly generated cellulose chain ends. By the synergistic action of these enzymes, cellobiose units are released and act as a direct inhibitor of those cellulolytic enzymes. Finally, BGLs hydrolyze cellobiose to glucose and are therefore required to complete the degradation of cellulose.
Trichoderma harzianum is frequently reported as a control agent against fungal pathogens (Arantes and Saddler 2010; Howell 2003). However, recent studies have also revealed the potential of this fungus for cellulase production and industrial applications (Ahmed et al. 2009; Castro et al. 2010). Likewise, T. harzianum has also become a promising system for xylanase production under appropriate conditions (Franco and Ferreira 2004).
In the present study, the medium components, carbon, nitrogen, and vitamin sources, affecting EG production in Trichoderma harzianum KUC1716, were optimized by a central composite design (CCD) using response surface methodology (RSM). A fungal enzyme cocktail from T. harzianumKUC1716 and Schizophyllum commune KUC9397 was utilized to hydrolyze various types of lignocellulosic materials, and its performance was compared to that of a commercial enzyme cocktail (Cellulast 1.5L and Novozyme 188) by evaluating saccharification yields.
Microorganism and Identification
An efficient EG-producing microorganism, T. harzianum KUC1716, was provided by the Korea University Culture Collection (KUC, Seoul, Korea). The microorganism was maintained on potato dextrose agar (Difco, USA). Genomic DNA for identification of the fungus was extracted according to the procedure described by Huh et al. (2011). The part of translation elongation factor (TEF) gene was amplified from isolated genomic DNA with primers EF1-728F (5′-CATCGAGAAGTTCGAGAAGG-3′) and TEF rev (5′-GCCATCCTTGGAGATACCAGC-3′) as described by Carbone and Kohn (1999) and by Samuels et al. (2002). Sequencing of the amplified TEF gene was performed, and the sequence was deposited in GenBank (accession number KR820004).
Inoculum and EG Preparation
T. harzianum KUC1716 was grown on a malt extract agar plate for a week at room temperature. After the cultivation, a spore suspension was obtained by adding 3 mL distilled water to the plate, scraping, and collecting. A total of 106 spores were inoculated into a modified basal medium based on Mandels’ medium for EG production (Juhász et al. 2005). The inoculum was added to 250 mL flasks containing 100 mL of basal medium with various carbon, nitrogen, or vitamin concentrations. After incubation at 27 °C and 150 rpm for a week, the cultures were centrifuged, and the supernatant was used to measure cellulase activity. EG activity was measured using carboxymethyl cellulose (CMC) according to the method of Ghose (1987). One unit per mL of enzyme activity was defined as the amount of enzyme required to release 1 µmol of glucose equivalents from CMC per milliliter of culture medium per minute. All experiments were performed in triplicate.
Effect of Medium Components on EG Production
The effects of carbon sources (e.g., avicel, cellobiose, cellulose, and glucose), nitrogen sources (e.g., casein, corn steep liquor, soy peptone, tryptone, and yeast extract), and vitamin source (e.g., thiamine HCl) on EG production were studied. In addition, carbon source concentrations were varied between 1, 2, and 3% (w/v), nitrogen source concentrations were varied between 0.1, 0.25, 0.5, and 0.75% (w/v), and vitamin concentrations were varied between 0.01, 0.05, 0.1, 0.25, and 0.5%. To determine the optimal medium composition for EG production, the different carbon sources, nitrogen sources, and vitamins were examined sequentially, and the concentration of the best sources were optimized through the CCD.
CCD and Response Surface Analysis
A 20-run CCD using RSM was used to optimize medium components for EG production from T. harzianum KUC1716. Table 1 shows the ranges and levels of the independent variables. The CCD consisted of a 23 full factorial design at a distance of 1.68179 from the origin with 6 central points (Table 2). Based on preliminary experiments, three independent variables that affect EG activity, cellulose (X1, %), soy peptone (X2, %), and thiamine HCl (X3, %), were chosen, and the range for each factor was studied. EG activity (U/mL) served as the dependent output variable. A second-order polynomial equation was fit to the experimental data to predict the optimum point. For three factors, the model equation is,
where Y is the predicted response, b0 is the intercept term, b1, b2, and b3 are linear coefficients, b11, b22, and b33 are squared coefficients, b12, b23, and b13 are interaction terms, and X1, X2, and X3 are the independent variables.
Table 1. Component Ranges and Levels of the Independent Variables
Saccharification was performed in 20 mL bottles in 50 mM sodium acetate buffer (pH 5.0). Four lignocellulosic material samples pre-treated by soaking in aqueous ammonia (SAA) at 30 °C and 70 °C were provided from Kyonggi University, and their chemical composition is presented in Table 4. Substrate concentrations were 1.5% dry mass (w/w). The working volume of enzymatic hydrolysis was eventually 10 mL in the 20 mL bottle. Reactions were performed at 50 °C under shaking at 200 rpm for 60 h according to the National Renewable Energy Laboratory method (Selig et al. 2008). To hydrolyze the biomass, the fungal enzyme cocktail was composed of enzymes from T. harzianumKUC1716 and Schizophyllum commune KUC9397. In a previous study, enzyme preparation obtained by T. harzianum KUC1716 showed low β-glucosidase activity. Consequently, enzyme preparation produced by S. commune KUC9397, absent of filter paper unit but high β-glucosidase activity, was supplemented to improve the hydrolysis yield. For an enzyme cocktail control, Celluclast 1.5 L (Novozymes, Franklinton, NC) and Novozyme 188 (Novozymes A/S, Bagsvaerd, Denmark) were used. The protein content and enzyme activities of the enzyme preparations are summarized in Table 5. After saccharification, enzymatic hydrolysates were boiled for 5 min to inactivate the enzymes and centrifuged at 12,000 rpm for 5 min. Supernatants were filtered with 0.2 μm syringe filters (No. 729022, Macherey-Nagel, Germany). The total reducing sugars were measured by the DNS method (Qi et al. 2004). The saccharification rate (%) was calculated as the ratio of sugar content to initial cellulose and hemicellulose content in the dry substrate. All saccharification experiments were carried out in triplicate.
RESULTS AND DISCUSSION
Effects of Different Nutrient Sources
In the present study, the significance of different carbon, nitrogen, and vitamin sources was investigated to improve the composition of the medium for endoglucanase production using T. harzianum KUC1716. Among the carbon sources, cellulose and Avicel had the strongest inducing effect on the formation of endoglucanase activity, whereas cellobiose and glucose resulted in low levels of endoglucanase activity across their concentration ranges (data not shown). In general, high enzyme activity is detected when organisms are grown on complex substrates as opposed to when they are grown on easily metabolized substrates (Haltrich et al. 1994; Sun and Chen 2008).
Most nitrogen sources in this study had a strong inducing effect on the production of endoglucanase by T. harzianum KUC1716. Enzyme production was greatly affected by soy peptone across its concentration range (data not shown). Although it is common to add peptone to improve cellulase yields (Krishna et al. 2000; Jun et al. 2009), soy peptone is also known as an effective nitrogen source for Trichoderma sp. (Seyis and Aksoz 2005; Rodriguez-Gomez and Hobley 2013).
Little attention has been paid to the role of vitamin source in cellulase production by Trichoderma sp. In a previous study, we reported the effect of thiamine-HCl on cellulase production by Schizophyllum commune, which improved cellulase yield significantly (Lee et al. 2014). As expected, thiamine-HCl showed effective enhancement of endoglucanase production in T. harzianum KUC1716.
The combination of 2% cellulose, 0.5% soy peptone, and 0.1% thiamine HCl was the best for the production of endoglucanase, and interaction effects among the ingredients on endoglucanase production were further assessed.
Statistical Optimization of Endoglucanase Production by RSM
The interaction effects of varying the concentrations of three independent culture medium components (cellulose, soy peptone, and thiamine HCl) on EG production were investigated. The results of the CCD experiments are presented with mean experimental responses in Table 2. The regression equations obtained after an analysis of variance (ANOVA) give the level of EG production as a function of the independent variables tested. The final response equation that represents a suitable model for EG production is given below,
where Y represents EG production, and X1, X2, and X3 are the concentrations of cellulose (%, w/v), soy peptone (%, w/v), and thiamine HCl (%, w/v), respectively.
Table 2. The CCD of RSM and the Mean Experimental Responses of Endoglucanase Production from T. harzianum KUC1716
The model’s coefficient of determination (R2) implies that 87.6% of the sample variation for EG production could be explained by the three independent variables (Table 3). A lower coefficient of variation (CV) indicates higher experimental reliability. The CV of these data is 16.32%, indicating high experiment reliability. The computed F-value (7.87) implies that the model is significant. The P-value (0.0017) was also very low, indicating the model’s significance. In addition, the model’s lack of fit (0.6405) suggests that the obtained experimental data fit well with the model. In this case, five model terms (X1, X2, X12, X22, and X32) were found to be significant for EG production. Cellulose concentration (X1) had an extremely significant effect (P <0.0029) on EG production and was much more influential than the other variables explored in this study.
Response surface curves demonstrate the effect of two independent variables while a third variable remains fixed at zero, and curves for this study are depicted in Fig. 1. The model predicts the optimal values of the most significant three variables to be X1 = -0.15, X2 = -0.09, and X3 = -0.07. Therefore, the optimal concentrations of cellulose, soy peptone, and thiamine HCl are 1.85, 0.48, and 0.10% (w/v), respectively. With this medium composition, the maximum predicted EG production is 1.77 U/mL. The experimentally measured enzyme activity (1.97 U/mL) matches the predicted value and represents a 2.6-fold enhancement in EG activity.
Fig. 1. Statistical optimization of enzyme production using RSM. a. Three dimensional response surface plot for the interaction between cellulose and soy peptone. b. Three dimensional response surface plot for the interaction between cellulose and thiamine HCl. c. Three dimensional response surface plot for the interaction between soy peptone and thiamine HCl
Enzymatic Hydrolysis of Lignocellulosic Materials
Enzymatic hydrolysis of four types of agricultural residue was performed by the enzyme cocktails from T. harzianum KUC1716 and Schizophyllum commune KUC9397. T. harzianum KUC1716 produced large amounts of endoglucanase and exoglucanase but relatively low β-glucosidase activity. To date, no natural microorganism that produces an ideal enzyme preparation for biomass hydrolysis has been discovered (Maeda et al. 2011). Therefore, β-glucosidase from S. commune KUC9397 producing high activity of the enzyme was supplemented to hydrolyze lignocellulosic materials. The enzymatic saccharification yields of SAA-pretreated lignocellulosic materials are shown in Fig. 2. Overall, pretreatments performed at higher temperature were effective for hydrolyzing lignocellulosic materials. The saccharification yields were significantly (p<0.05) higher with higher temperature at pretreatment, perhaps because higher temperature allowed for more efficient pretreatment and better structural preparation of the substrate for enzymatic saccharification. Correlations were also obtained between lignocellulose composition and saccharification yield. The cellulose content had a positive correlation with saccharification yield obtained from the fungal enzyme cocktail (F=26.62, p<0.01, and R2=0.72) and the commercial enzyme cocktail (F=86.53, p<0.01, and R2=0.89). In addition, the lignin content showed a negative correlation with the yield obtained from the fungal enzyme cocktail (F=7.93, p<0.05, and R2=0.44) and the commercial enzyme cocktail (F=9.16, p<0.05, and R2=0.47). It is apparent that the lower lignin ratio improved enzyme accessibility to the polysaccharide chain and resulted in improved saccharification efficiency.
Table 3. ANOVA for the Selected Quadratic Model
In terms of enzyme efficiency, it was apparent that the fungal enzyme cocktail was more efficient at hydrolyzing lignocellulosic materials than the commercial enzyme cocktails. Although this finding could not be explained by the enzyme activities included in the enzyme cocktails, it is likely that accessory enzymes enhance the overall hydrolysis by solubilizing lignocellulose, which hinders access to the cellulose and hemicellulose.
Table 4. Chemical Composition of Pretreated Lignocellulosic Materials
Table 5. Activities of the Enzymes Used for Enzymatic Hydrolysis
Fig. 2. Saccharification yields of lignocellulosic materials over 60 hours. The error bars represent the standard error of triplicate experiments. a. Hydrolysis yield of barley straw. b. Hydrolysis yield of EFB. c. Hydrolysis yield of miscanthus. d. Hydrolysis yield of rice straw. (■, fungal enzyme cocktail; □, commercial enzyme cocktail)
Accessory enzymes such as pectinase might have considerable potential to increase the overall performance of cellulase enzyme mixtures and achieve effective hydrolysis of pretreated lignocellulosic substrates (Hu et al. 2011). It was verified that the crude enzyme preparation derived from T. harzianum KUC1716 and S. commune KUC9397 could replace the commercial enzymes. This highlights the potential of such crude enzymes for use in cellulosic biomass conversion systems.
1. Medium optimization enhanced endoglucanase production 2.6 fold.
2. Crude fungal enzyme from T. harzianum KUC1716 showed hydrolysis potential equal to that of commercial enzymes.
3. This study demonstrated an efficient process to produce endoglucanase by T. harzianum KUC1716 and its potential for biomass hydrolysis for bioethanol applications.
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2013R1A1A2A10011390) and by the Technology Development Program (309016-05) for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea.
Arantes, V., and Saddler, J. N. (2010). “Access to cellulose limits the efficiency of enzymatic hydrolysis: The role of amorphogenesis,” Biotechnol. Biofuels 3(4), 1-11. DOI: 10.1186/1754-6834-3-4
Ahmed, S., Bashir, A., Saleem, H., Saadia, M., and Jamil, A. (2009). “Production and purification of cellulose-degrading enzymes from a filamentous fungus Trichoderma harzianum,” Pak. J. Bot. 41(3), 1411-1419. DOI:
Bak, J. S., Ko, J. K., Han, Y. H., Lee, B. C., Choi, I. G., and Kim, K. H. (2009). “Improved enzymatic hydrolysis yield of rice straw using electron beam irradiation pretreatment,” Bioresour. Technol.100(3), 1285-1290. DOI: 10.1016/j.biortech.2008.09.010
Carbone, I., and Kohn, L. M. (1999). “A method for designing primer sets for speciation studies in filamentous ascomycetes,” Mycologia 91(3), 553-556. DOI: 10.2307/3761358
Cardona, C. A., and Sánchez, Ó. J. (2007). “Fuel ethanol production: Process design trends and integration opportunities,” Bioresour. Technol. 98(12), 2415-2457. DOI: 10.1016/j.biortech.2007.01.002
de Castro, A. M., Ferreira, M. C., da Cruz, J. C., Pedro, K. C. N. R., Carvalho, D. F., Leite, S. G. F., and Pereira, N. (2010). “High-yield endoglucanase production by Trichoderma harzianum IOC-3844 cultivated in pretreated sugarcane mill byproduct,” Enzyme Res. 2010. DOI: 10.4061/2010/854526
Franco, P. F., and Ferreira, H. M. (2004). “Production and characterization of hemicellulase activities from Trichoderma harzianum strain T4,” Biotechnol. Appl. Biochem. 40(3), 255-259. DOI: 10.1042/ba20030161
Ghose, T. K. (1987). “Measurement of cellulase activities,” Pure Appl. Chem. 59(2), 257-268. DOI: 10.1351/pac198759020257
Haltrich, D., Laussamayer, B., Steiner, W., Nidetzky, B., and Kulbe, K. D. (1994). “Cellulolytic and hemicellulolytic enzymes of Sclerotium rolfsii: Optimization of the culture medium and enzymatic hydrolysis of lignocellulosic material,” Bioresour. Technol. 50(1), 43-50. DOI: 10.1016/0960-8524(94)90219-4
Howell, C. R. (2003). “Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts,” Plant Dis. 87(1), 4-10.
Hu, J., Arantes, V., and Saddler, J. N. (2011). “The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: Is it an additive or synergistic effect?” Biotechnol. Biofuels 4(1), 1-14. DOI: 10.1094/pdis.2003.87.1.4
Huh, N., Jang, Y., Lee, J., Kim, G.-H., and Kim, J.-J. (2011). “Phylogenetic analysis of major molds inhabiting woods and their discoloration characteristics. Part 1. Genus Trichoderma,” Holzforschung65, 257-263. DOI: 10.1515/hf.2011.018
Juhász, T., Szengyel, Z., Réczey, K., Siika-Aho, M., and Viikari, L. (2005). “Characterization of cellulases and hemicellulases produced by Trichoderma reesei on various carbon sources,” Process Biochem. 40, 3519-3525. DOI: 10.1016/j.procbio.2005.03.057
Jun, H., Bing, Y., Keying, Z., Xuemei, D., and Daiwen, C. (2009). “Strain improvement of Trichoderma reesei Rut C-30 for increased cellulase production,” Indian J. Microbiol. 49(2), 188-195. DOI: 10.1007/s12088-009-0030-0
Krishna, S. H., Rao, K. S., Babu, J. S., and Reddy, D. S. (2000). “Studies on the production and application of cellulase from Trichoderma reesei QM-9414,” Bioprocess Eng. 22(5), 467-470. DOI: 10.1007/s004490050760
Lee, Y. M., Lee, H., Kim, J. S., Lee, J., Ahn, B. J., Kim, G. H., and Kim, J. J. (2014). “Optimization of medium components for β-glucosidase production in Schizophyllum commune KUC9397 and enzymatic hydrolysis of lignocellulosic biomass,” BioResources 9(3), 4358-4368. DOI: 10.15376/biores.9.3.4358-4368
Maeda, R. N., Serpa, V. I., Rocha, V. A. L., Mesquita, R. A. A., Santa Anna, L. M. M., De Castro, A. M., … and Polikarpov, I. (2011). “Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium funiculosum and Trichoderma harzianum cellulases,” Process Biochem. 46(5), 1196-1201. DOI: 10.1016/j.procbio.2011.01.022
Qi, X. J., Gou, J. X., Han, X. J., and Yan, B. (2004). “Study on measuring reducing sugar by DNS reagent,” J. Cellul. Sci.Technol. 3, 17-20.
Rodriguez-Gomez, D., and Hobley, T. J. (2013). “Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30 ?,” World J. Microbiol. Biotechnol. 29(11), 2157-2165. DOI: 10.1007/s11274-013-1381-6
Samuels, G. J., Dodd, S. L., Gams, W., Castlebury, L. A., and Petrini, O. (2002). “Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus,” Mycologia94(1), 146-170. DOI: 10.2307/3761854
Selig, M., Weiss, N., and Ji, Y. (2008). “Enzymatic saccharification of lignocellulosic biomass,” Technical Report NREL/TP-510-42629. Issue Date 21 Mar 2008
Seyis, I., and Aksoz, N. (2005). “Xylanase production from Trichoderma harzianum 1073 D 3 with alternative carbon and nitrogen sources,” Food Technol. Biotechnol. 43(1), 37-40.
Sun, F., and Chen, H. (2008). “Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment,” Bioresour. Technol. 99(14), 6156-6161. DOI: 10.1016/j.biortech.2007.12.027
Article submitted: May 21, 2015; Peer review completed: August 23, 2015; Revised version received and accepted: September 8, 2015; Published: September 18, 2015.