Use of chemostat for enhanced production of β-glucosidase by newly isolated anaerobic cellulolyticClostridium strain RT9

Abstract

A new anaerobic, mesophilic, spore-forming, cellulolyticClostridium strain, RT9, was isolated from bovine rumen fluid. This strain has the ability to hydrolyze cellulose (Sigma cell-100), carboxymethyl-cellulose (CMC), and cellobiose into ethanol, acetate, butyrate, lactate, acetone, H2, and Co2 as the major fermentation products. The bacterium also exhibited endoglucanase, exoglucanase, and β-glucosidase activities in batch culture during growth on chemically defined medium with cellulose as the sole carbon source. Chemostat experiments were conducted under pH, dilution rate, and carbohydrate limitations to study the effect of various parameters on enzymes and product formation. In chemostat with 0.5% cellobiose as the limiting nutrient, maximum β-glucosidase activity and ethanol production occurred at pH values of 5.5 and 6.5, respectively. At low pH, acid accumulation was higher. β-glucosidase production increased up to 17-fold at a low dilution rate, and ethanol raised twofold. Relative activity of β-glucosidase on a percent basis was maximum at pH 6.5 and 45 °C. In a carbon-limited, continuous-culture experiment, when CMC was used as an inducer in cellobiose-growing culture, β-glucosidase activity was higher than the cellobiose-limited chemostat. On the other hand, enzyme declined twofold, and ethanol production increased in glucose limitation.