Utility of waste lignocellosics can be tremendously increased if these are first hydrolyzed chemically or enzymatically to glucose which can be subsequently used for making biomass protein, fuel (ethanol) and other fermentation products. Increasing knowledge regarding the mode of action of cellulases and their recent applications has greatly increased the prospects of enzymatic hydrolysis over acid and alkali hydrolysis because of its high saccharification efficiency and avoidance of pollution (Cochet, 1991; Kubicek et al., 1993). However, the cost of production and low yields of cellulases are the major problems in the economics of process and influence their utilization on industrial scale (Macris, 1984).
The efficiency of enzymatic hydrolysis of cellulose to glucose depends upon the source of cellulose, pretreatment methods and mode of fermentation (Gusakov et al., 1987). The present communication reports the studies on the effect of fermentation period, substrate level and inorganic nutrients on the secretion of endoglucanase (a member of cellulase complex) by a filamentous fungus Arachniotus sp. using corn stover as lignocellulosic substrate.
Materials and Methods
Substrate: Corn stover was sun dried, oven dried at 70°C to constant weight and ground to 40 mm mesh size.
Organism and inoculum: Arachniotus sp. procured from Department of Plant Pathology, University of Agriculture, Faisalabad, was maintained on substrate agar slants and inoculum was prepared by the method described by Bajwa et al. (1991).
Culture Cultivation: Duplicate flasks cotnaining different culture media employed for optimization of fermentation period, substrate level and inorganic nutrients were inoculated (5%) aseptically and subjected to continuous shake culture fermentation at pH4 and 30±2°C temperature on orbital shaker (100-120 rpm).
Harvesting: After the stipulated incubation period, the fermented biomass samples were filtered and filtrate was centrifuged at 400 rpm. The supernatant was ultrafiltered and the filtrate thus obtained was subjected to endoglucanase assay by spectrophotometric method described by Wood and Bhat (1988).
Optimization of Culture Conditions
Incubation period: Culture media of corn stover (5%) were fermented for 12, 24, 36 and 48 h to find out the optimum fermentation time.
Substrate level: Four levels of corn stover i.e. 2.5, 5.0, 7.5 and 10 per cent were used in duplicate fermentation media.
Inorganic nutrients: Four different concentrations each of (NH4)2SO4, CaCl2.2H2O, MgSO4.7H2O and KH2PO4, were tested to get the optimum concentrations of the nutrients for maximum endoglucanase production. The experiments were conducted in such a way that condition optimized in one experiment was used in the subsequent studies.
Results and Discussion
The study was undertaken to optimize various culture conditions for the production of endoglucanase by Arachniotus sp. and the results have been discussed as under:
Fermentation Period: It was observed that maximum endoglucanase (0.25 lU mL1) was secreted by the fungus after 24 h of incubation which decreased thereafter and reached a minimum (0.20 IU mL1) after 48 h (Fig. 1). Statistical analysis revealed a highly significant difference (p<0.01) between different time intervals. Santos et al. (1978) used synthetic liquid medium for the growth of P. italicurn and observed maximum endoglucanase activity after 12 hours.
Effect of varying concentrations of inorganic nutrients on endoglucanase production by Arachniotus sp. under optimum conditions*
*Corn stover, 7.5 per cent, pH4 and 30±2°C temperature. 1 - (NH)2S04 (%); 2 = CaCl2.2H2O (%); 3 = MgSO4.7H2O (%) = KH2P04, (%); 5 = Endoglcanase activity after 24 hours (lU/mL/min).
|Fig. 1:||Effect of different incubation periods on endoglucanase production.
|Fig. 2:||Effect different levels of substrate on endoglucanase production.
Maximum endoglucanase activity after 48 hours fermentation of alkali treated wheat straw by P. Janthinellum. The difference in optimum fermentation period may be attributed to difference of substrate and organism.
Substrate Level: Culture media containing 2.5, 5.0, 7.5 and 10 per cent corn stover were fermented for 24 hours and 7.5 per cent substrate level yielded optimum endoglucanase (0.57 IU mL1) activity (Fig. 2). All the substrate levels produced significantly different (p<0.01) endoglucanase except 7.5 and 10 per cent substrate which showed a non significant difference. The results are supported by those of Ortega (1985) who found out 6 per cent CMC as the optimum substrate level for endoglucanase production.
(NH4)2SO4: Four different media containing 0.1, 0.2, 0.3 and 0.4 per cent (NH4)2SO4 were fermented at 7.5 per cent substrate level for 24 h. Results showed optimum production of endoglucanase (0.78 IU mL1) with 0.2 per cent (NH4)2S04 whereas 0.1, 0.3, 0.4 per cent (NH4)2S04 produced 0.63, 0.54 and 0.38 IU mL1 enzyme activities respectively (Table 1). All the treatments showed a highly significant (p<0.01) difference with respect to endoglucanase production as compared to control (with no (NH4)2SO4). The findings are supported by those of (Illanes and Schaffeld (1982) who fermented leached heat pulp as substrate for T. reesei and observed an increase in enzyme activity by the addition of (NH4)2SO4 as nitrogen source. There are 0.2 per cent (NH4)2SO4 as optimum nitrogen source for cellulase production.
CaCI2.2H2O: Addition of 0.01, 0.05, 0.1 and 0.15 per cent CaCl2.2H2O along with optimum concentration of (NH4)2SO4 (0.2%) yielded 0.82, 0.92, 0.74 and 0.69 IU mL1 endoglucanase activity respectively. It was observed that 0.05 per cent CaCI2.2H2O gave higher enyzme yield as compared to other levels tested (Table 1). Statistical analysis revealed a highly significant (p<0.01) effect of CaCI2.2H2O on endoglucanase production. Macris (1984) studied the effect of CaCI2.2H2O on the production of cellulases and observed maximum endoglucanase activity in the presence of 0.03 per cent CaC12.2H2O in glucose and wheat bran medium during fermentation with Alternaria alternata. Mubeen (1997) observed 0.01 per cent CaCI2.2H2O as the optimum level for endoglucanase production from wheat bran by Arachniotous sp.
MgSO4.7H2O: Four different concentrations (0.01, 0.015, 0.02 and 0.025%) of MgSO4.2H2O were added to the fermentation medium of corn stover (7.5%) along with optimum concentrations of (NH4)2SO2 (0.2%) and CaCI2.2H2O (0.05%) and results have been depicted in Table 1. Addition of 0.01 per cent MgSO4.7H2O produced significantly (p<0.05) higher endoglucanase (0.99 lU mL1) than all other levels tested. In general MgSO4.7H2O increased the enzyme production. Results are in line with Kuhad and Singh (1993) who reported 0.05 per cent MgSO4.7H2O as optimum concentration for the production of endoglucanase by P. citrinum using rice husk as substrate. Bahkali (1994) produced maximum endoglucanase in CMC medium containing 0.05 per cent MgSO4.7H2O along with other micro nutrients.
KH2PO4: Effect of 0,1, 0.2, 0.3 and 0.4 per cent KH2PO4 on endoglucanase production was studied under preoptimized culture conditions. Maximum endoglucanase activity (1.13 lU mL1) was recorded in the medium containing 0.2 per cent KH2PO4 and there was a non-significant difference between endoglucanase activities with 0.2 and 0.3 per cent KH2PO4 (Table 1).
Macris et al. (1985) observed 0.2 per cent KH2PO4 as the optimum concentration with cotton fibre and wheat straw as substrates and T. harziamum as organism. Results are in accordance with those of Bahkali (1994) who also reported maximum endoglucanase production with the addition of 0.1 per cent KH2PO4 in cellulose medium fermented with Verticillium tricorpus.