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Enzymic hydrolysis of rice straw by crude cellulase from Trichoderma reesei
Biolvsotm 'e 7~'c'hnoh~,'y 66 ( 1998 ) 267 - 269 © 1998 Elsevier Science Lid. All righls reserved Primed in G r e a t Britain 0960-8524/98 S - - s e e front mailer ELSEVIER
PI I:S0960-8524(97)00138-7
Short Communication Enzymic Hydrolysis of Rice Straw by Crude Cellulase from Trichoderma reesei Abstract
The cellulase system with a maximum FP cellulase activity of 1"30 lU/ml obtained from fermentation by T. reesei of alkali treated, steamed rice straw was employed at 2 6 F P U g -I substrate for enzymatic saccharification. Production of reducing sugars (as glucose) at 268.5 mg ~ of rice straw treated with 4% sodium hydroxide in combination with 60min of steam pressure (1.05 kg cm 2) was obtained in 48 h of incubation. Analysis of variance applied to the results of the effects of various factors revealed that a substrate concentration of 4% (w/v), a pH of 5"0, a temperature of 50°C, an enzyme concentration of 25 FPUg z substrate and a time of 48h, were optimum for the production of the maximum amount of reducing sugars; 268.5 mgg i of pre-treated rice straw representing a maximum saccharification of 54.3% (cellulose basis). © 1998 Elsevier Science Ltd. All rights reserved Key words: Cellulase, rice straw, Trichoderma reesei, saccharification, enzymatic hydrolysis, pretreatment.
INTRODUCTION Among the easily-available agricultural wastes, rice straw is the most abundant, with 82 million metric tons annually available in India. Rice straw contains approximately 40% cellulose, 18% hemicellulose and 5"5% lignin. Native cellulose is highly resistant to enzymatic hydrolysis. Various chemical and physical pretreatments, e.g. hydrogen peroxide treatment (Takagi, 1987) or steam pretreatment (Brownell & Saddler, 1987), are thus employed to increase the surface area and bulk density and decrease the crystallinity of the cellulose, so as to make it accessible for hydrolysis. The present paper reports on the enzymatic hydrolysis (saccharification) of rice straw using crude cellulase from Trichoderma reesei. The effects of substrate and enzyme concentration, pH and temperature on enzymatic saccharification are reported and assessed.
METHODS Enzyme preparation Crude cellulase from Trichoderma reesei was obtained by its growth on rice straw under solidstate fermentation. A quantity of 20 ml of 25-times concentrated synthetic medium (Chahal & Gray, 1968) was added to 5 g rice straw. These were sterilised, inoculated with 5 mi fungal culture, incubated at 30°C for 10 days, and then mixed well with 100 ml distilled water and shaken for 30 rain, filtered and centrifuged. The clear filtrate was used as crude cellulase which contained 1"30 filter paper (FP) units per ml. General procedure In the experiments where enzyme concentration was held constant, a 10 ml aliquot of the above enzyme preparation (pH 5"0) was added to each 25 ml flask, which contained 2"5 ml of citrate buffer (0"1 M, pH 5"0) and 0"5 g rice straw. The rice straw concentration in the flask was 4%, except in those experiments where the effect of substrate concentration on saccharification was studied. The flasks were then incubated at 50°C for different times. After incubation, the saccharified material was centrifuged at 5000 rpm for 20 min. The reducing sugars in the supernatant were estimated as glucose by the method of Somogyi (1952). Alkali and steam pressure treatment To chemically pretreat the substrate, 10 g of rice straw was immersed in 100ml of 4% sodium hydroxide (w/w) solution, allowed to react at room temperature for 2 days, then subjected to steam pressure at 1-05 kg cm 2 for 60 rain. The straw was neutralised with 1 N HCI, washed thoroughly with distilled water and dried at 60°C. Calculation of conversion
Saccharification (%) (cellulose basis) Amount of reducing sugars (glucose) formed Amount of cellulose present in the substrate x O . 9 x 100
268
Short communication
Statistical analysis
The empirical analysis of the data was done by applying various appropriate tools of statistics. Various regression models were applied according to the nature of the data involved and various hypotheses were tested to examine whether the regression coefficients were significantly different from zero.
RESULTS AND DISCUSSION
T h e alkali and steam pressure treatment of rice straw significantly ( P < 0 - 0 1 ) increased the cellulose content and simultaneously decreased the hemicellulose and lignin contents, which could increase the susceptibility of the remaining solids to enzymatic hydrolysis. T h e saccharification of p r e t r e a t e d rice straw (cellulose content 44.5%) was carried out at pH 3"0-6"0 and a significant ( P < 0 " 0 5 ) increase in the per cent saccharification was observed with an increase in the p H up to 5-0, beyond which a significant ( P < 0 - 0 5 ) decline was observed. T h e decrease in the saccharification as a result of changes in p H to the higher levels is mainly due to the inactivation of cellulases at these p H values.
T h e formation of reducing sugars significantly increased ( P < 0 - 0 5 ) in response to an increase in the t e m p e r a t u r e up to 50°C and maximum production of 268-5 mg of reducing sugar (glucose) equivalents was obtained in 48 h of saccharification. T h e saccharification of p r e t r e a t e d rice straw significantly increased with an increase in the enzyme concentration up to 25 F P U g-~ substrate and an increase in the incubation period up to 48 h, beyond which the increase was not significant (CD = 6-805, P<0"05). A 5-fold increase, from 5 F P U to 25 F P U enzyme units, led to a nearly 2"7-fold increase in the a m o u n t of reducing sugars released after 48 h of hydrolysis. Similarly, the substrate concentration was varied from 2 to 10% (w/v) and its effect on the saccharification of p r e t r e a t e d rice straw was studied. T h e reducing sugars obtained from the hydrolysis of p r e t r e a t e d rice straw were significantly higher (CD = 8.527, P < 0 " 0 5 ) at a 4% concentration of substrate, beyond which a rapid decline (significant at P < 0 " 0 5 ) in the reducing sugars' content was observed. Again, it was found that reducing sugar content increased significantly ( P < 0 " 0 5 ) with hydrolysis time up to 48 h. It is, thus, concluded (Table 1) that a substrate concentration of 4% (w/v), a p H of 5"0, a tempera-
Table 1. Saccharification of alkali treated and steamed rice straw by cellulases of Trichoderma reesei. Estimated equations of the models used, their related statistics and optimum values of different parameters
Parameters
Estimated equations
pH
Log linear: Y= 3-793** Xj0'472"* )(20'213"* R 2 = 0"589, N = 42 X~ = Levels of pH Quadratic: Y= -916934"*+42"81"* X1+2"829"* )(2 -0'424** )(]2-0'023** X 2 - 0"003s XIX2 R 2 = (1"957"*, N = 30 where, X] = Temp. X~ = Quad. term on temp., X~ = Quad. term sacch, time, X]X2 = Interaction (Temp. x Sacch.) Log linear: Y= 2"768** X10"578"* )(20"245** R 2 = 0"898, N = 36 where, X] = cellulase conc. (FPU g ~ substrate) Log linear: Y = 184-680"* - 5-807"* X~ + 1'072* *)(2 R 2 = 0"507, N = 30 where,
Temperature (°C)
Cellulase conc. (FPU g ~ substrate)
Substrate cone. (%, w/v)
X~ = substrate cone. (%, w/v) Time (hi Maximum reducing sugar conc. (mg g ~ substrate) Saccharification (%) (cellulose basis) Y = Amount of reducing sugars (mg/g substrate). )(2 = Saccharification period (hi. b. = Intercept. b j, b2, b3... = regression coefficients. R 2 = Coefficient of determination. N = Number of observations. *Significant at P<0-05. **Significant at P<0.01.
Optimum value 5-0
50°C
25
48 268.5 54.3
Short communication ture of 50°C, an enzyme concentration of 25 F P U g ~ substrate and a time of 4 8 h , were o p t i m u m for the production of the maximum a m o u n t of reducing sugars, 268.5 m g g ~ of pre-treated rice straw; a maximum saccharification of 54"3% (cellulose basis).
REFERENCES Brownell, H. H. & Saddler, J. N. (1987). Steam pretreatment of lignocellulosic material for enhanced enzymatic hydrolysis. Biotech. Bioeng., 29, 228-235. Chahal, D. S. & Gray, W. D. (1968). Growth of selected cellulolytic fungi on wood pulp. In Biodegradation of Materials, Microbial and Allied Aspects, Eds A. H. Walter and J. S. Elphick, pp. 584-593. Elsevier.
269
Somogyi, M. (1952). Notes on sugar determination. J. Biol. Chem., 195, 19-23. Takagi, M. (1987). Pretreatment of lignocellulosic materials with hydrogen peroxide in presence of manganese compounds. Biotech. Bioeng., 29, 165-17(1.
Parminder P. Kaur a*, J. S. Arneja b & Joginder Singh b "Biotechnology Centre, Punjab Agricultural University, Ludhiana - 141004, India 1'Department of Biochemistry, Punjab Agricultural University, Ludhiana - 141004, India (Received 20 April 1997; revised version received 28 August 1997; accepted 16 September 1997) *Author to whom correspondence should be addressed.
PI I:S0960-8524(97)00138-7
Short Communication Enzymic Hydrolysis of Rice Straw by Crude Cellulase from Trichoderma reesei Abstract
The cellulase system with a maximum FP cellulase activity of 1"30 lU/ml obtained from fermentation by T. reesei of alkali treated, steamed rice straw was employed at 2 6 F P U g -I substrate for enzymatic saccharification. Production of reducing sugars (as glucose) at 268.5 mg ~ of rice straw treated with 4% sodium hydroxide in combination with 60min of steam pressure (1.05 kg cm 2) was obtained in 48 h of incubation. Analysis of variance applied to the results of the effects of various factors revealed that a substrate concentration of 4% (w/v), a pH of 5"0, a temperature of 50°C, an enzyme concentration of 25 FPUg z substrate and a time of 48h, were optimum for the production of the maximum amount of reducing sugars; 268.5 mgg i of pre-treated rice straw representing a maximum saccharification of 54.3% (cellulose basis). © 1998 Elsevier Science Ltd. All rights reserved Key words: Cellulase, rice straw, Trichoderma reesei, saccharification, enzymatic hydrolysis, pretreatment.
INTRODUCTION Among the easily-available agricultural wastes, rice straw is the most abundant, with 82 million metric tons annually available in India. Rice straw contains approximately 40% cellulose, 18% hemicellulose and 5"5% lignin. Native cellulose is highly resistant to enzymatic hydrolysis. Various chemical and physical pretreatments, e.g. hydrogen peroxide treatment (Takagi, 1987) or steam pretreatment (Brownell & Saddler, 1987), are thus employed to increase the surface area and bulk density and decrease the crystallinity of the cellulose, so as to make it accessible for hydrolysis. The present paper reports on the enzymatic hydrolysis (saccharification) of rice straw using crude cellulase from Trichoderma reesei. The effects of substrate and enzyme concentration, pH and temperature on enzymatic saccharification are reported and assessed.
METHODS Enzyme preparation Crude cellulase from Trichoderma reesei was obtained by its growth on rice straw under solidstate fermentation. A quantity of 20 ml of 25-times concentrated synthetic medium (Chahal & Gray, 1968) was added to 5 g rice straw. These were sterilised, inoculated with 5 mi fungal culture, incubated at 30°C for 10 days, and then mixed well with 100 ml distilled water and shaken for 30 rain, filtered and centrifuged. The clear filtrate was used as crude cellulase which contained 1"30 filter paper (FP) units per ml. General procedure In the experiments where enzyme concentration was held constant, a 10 ml aliquot of the above enzyme preparation (pH 5"0) was added to each 25 ml flask, which contained 2"5 ml of citrate buffer (0"1 M, pH 5"0) and 0"5 g rice straw. The rice straw concentration in the flask was 4%, except in those experiments where the effect of substrate concentration on saccharification was studied. The flasks were then incubated at 50°C for different times. After incubation, the saccharified material was centrifuged at 5000 rpm for 20 min. The reducing sugars in the supernatant were estimated as glucose by the method of Somogyi (1952). Alkali and steam pressure treatment To chemically pretreat the substrate, 10 g of rice straw was immersed in 100ml of 4% sodium hydroxide (w/w) solution, allowed to react at room temperature for 2 days, then subjected to steam pressure at 1-05 kg cm 2 for 60 rain. The straw was neutralised with 1 N HCI, washed thoroughly with distilled water and dried at 60°C. Calculation of conversion
Saccharification (%) (cellulose basis) Amount of reducing sugars (glucose) formed Amount of cellulose present in the substrate x O . 9 x 100
268
Short communication
Statistical analysis
The empirical analysis of the data was done by applying various appropriate tools of statistics. Various regression models were applied according to the nature of the data involved and various hypotheses were tested to examine whether the regression coefficients were significantly different from zero.
RESULTS AND DISCUSSION
T h e alkali and steam pressure treatment of rice straw significantly ( P < 0 - 0 1 ) increased the cellulose content and simultaneously decreased the hemicellulose and lignin contents, which could increase the susceptibility of the remaining solids to enzymatic hydrolysis. T h e saccharification of p r e t r e a t e d rice straw (cellulose content 44.5%) was carried out at pH 3"0-6"0 and a significant ( P < 0 " 0 5 ) increase in the per cent saccharification was observed with an increase in the p H up to 5-0, beyond which a significant ( P < 0 - 0 5 ) decline was observed. T h e decrease in the saccharification as a result of changes in p H to the higher levels is mainly due to the inactivation of cellulases at these p H values.
T h e formation of reducing sugars significantly increased ( P < 0 - 0 5 ) in response to an increase in the t e m p e r a t u r e up to 50°C and maximum production of 268-5 mg of reducing sugar (glucose) equivalents was obtained in 48 h of saccharification. T h e saccharification of p r e t r e a t e d rice straw significantly increased with an increase in the enzyme concentration up to 25 F P U g-~ substrate and an increase in the incubation period up to 48 h, beyond which the increase was not significant (CD = 6-805, P<0"05). A 5-fold increase, from 5 F P U to 25 F P U enzyme units, led to a nearly 2"7-fold increase in the a m o u n t of reducing sugars released after 48 h of hydrolysis. Similarly, the substrate concentration was varied from 2 to 10% (w/v) and its effect on the saccharification of p r e t r e a t e d rice straw was studied. T h e reducing sugars obtained from the hydrolysis of p r e t r e a t e d rice straw were significantly higher (CD = 8.527, P < 0 " 0 5 ) at a 4% concentration of substrate, beyond which a rapid decline (significant at P < 0 " 0 5 ) in the reducing sugars' content was observed. Again, it was found that reducing sugar content increased significantly ( P < 0 " 0 5 ) with hydrolysis time up to 48 h. It is, thus, concluded (Table 1) that a substrate concentration of 4% (w/v), a p H of 5"0, a tempera-
Table 1. Saccharification of alkali treated and steamed rice straw by cellulases of Trichoderma reesei. Estimated equations of the models used, their related statistics and optimum values of different parameters
Parameters
Estimated equations
pH
Log linear: Y= 3-793** Xj0'472"* )(20'213"* R 2 = 0"589, N = 42 X~ = Levels of pH Quadratic: Y= -916934"*+42"81"* X1+2"829"* )(2 -0'424** )(]2-0'023** X 2 - 0"003s XIX2 R 2 = (1"957"*, N = 30 where, X] = Temp. X~ = Quad. term on temp., X~ = Quad. term sacch, time, X]X2 = Interaction (Temp. x Sacch.) Log linear: Y= 2"768** X10"578"* )(20"245** R 2 = 0"898, N = 36 where, X] = cellulase conc. (FPU g ~ substrate) Log linear: Y = 184-680"* - 5-807"* X~ + 1'072* *)(2 R 2 = 0"507, N = 30 where,
Temperature (°C)
Cellulase conc. (FPU g ~ substrate)
Substrate cone. (%, w/v)
X~ = substrate cone. (%, w/v) Time (hi Maximum reducing sugar conc. (mg g ~ substrate) Saccharification (%) (cellulose basis) Y = Amount of reducing sugars (mg/g substrate). )(2 = Saccharification period (hi. b. = Intercept. b j, b2, b3... = regression coefficients. R 2 = Coefficient of determination. N = Number of observations. *Significant at P<0-05. **Significant at P<0.01.
Optimum value 5-0
50°C
25
48 268.5 54.3
Short communication ture of 50°C, an enzyme concentration of 25 F P U g ~ substrate and a time of 4 8 h , were o p t i m u m for the production of the maximum a m o u n t of reducing sugars, 268.5 m g g ~ of pre-treated rice straw; a maximum saccharification of 54"3% (cellulose basis).
REFERENCES Brownell, H. H. & Saddler, J. N. (1987). Steam pretreatment of lignocellulosic material for enhanced enzymatic hydrolysis. Biotech. Bioeng., 29, 228-235. Chahal, D. S. & Gray, W. D. (1968). Growth of selected cellulolytic fungi on wood pulp. In Biodegradation of Materials, Microbial and Allied Aspects, Eds A. H. Walter and J. S. Elphick, pp. 584-593. Elsevier.
269
Somogyi, M. (1952). Notes on sugar determination. J. Biol. Chem., 195, 19-23. Takagi, M. (1987). Pretreatment of lignocellulosic materials with hydrogen peroxide in presence of manganese compounds. Biotech. Bioeng., 29, 165-17(1.
Parminder P. Kaur a*, J. S. Arneja b & Joginder Singh b "Biotechnology Centre, Punjab Agricultural University, Ludhiana - 141004, India 1'Department of Biochemistry, Punjab Agricultural University, Ludhiana - 141004, India (Received 20 April 1997; revised version received 28 August 1997; accepted 16 September 1997) *Author to whom correspondence should be addressed.