Optimization of fermentation conditions for cellulase production by Bacillus subtilis CY5 and Bacillus circulans TP3 isolated from fish gut

Abstract

Background. Microbial and fungal cellulases are known to hydrolyse cellulose, which is ingested as plant material by herbivorous/omnivorous fishes. Microbial enzymes have enormous advantage of being produced in large quantities by established fermentation techniques. The present investigation aims to optimize the environmental and nutritional parameters for fermentation to enhance cellulase production by two bacterial strains isolated from fish gastrointestinal tracts. Materials and methods. Two bacterial strains, Bacillus subtilis CY5 and Bacillus circulans TP3, isolated from the gastrointestinal tracts of common carp, Cyprinus carpio L., and Mozambique tilapia, Oreochromis mossambicus (Peters, 1852), respectively were identified as potent cellulase producers. Both strains were cultured in tryptone soya broth for 24 h at 32 ± 2°C, when average viable count of 9.75 · 107 cells · mL-1 culture broth was obtained. This was used as the inoculum for the production medium. The fermentation medium was seeded with 1.0%, 2.0%, 3.0%, 4.0%, and 5.0% inoculum (tryptone soya broth) and incubated in static culture at 40°C to standardize the inoculum size for fermentation. The effect of different production parameters, such as fermentation condition, moisture, pH, temperature, inoculum size, and nitrogen sources on cellulase production by the isolated bacterial strains were studied. Results. Cellulase yield was highest (26 U in B. subtilis and 20.2 U in B. circulans) in solid-state fermentation (SSF). Enzyme production in both the isolates increased in an optimum pH range of 7.0 to 7.5. Minimum cellulase production was observed at 45°C, while maximum production was obtained at 40°C. To standardize the fermentation period for cellulase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 96 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points. Asparagine as the nitrogen source was most effective in B. subtilis CY5, while beef extract proved useful in optimizing enzyme production by B. circulans TP3. Conclusion. The results of this study will help to standardize the requirements for optimum production of cellulase by cellulase-producing fish gut bacteria and might contribute towards better fish feed formulation incorporating plant ingredients, especially in the larval stages when the enzyme system is not efficient.