(613c) Stimulating Cellulase Activity of Microbulbifer Hydrolyticus Using Hot-Water Wood Extract Hydrolisate as Substrate

The advantages often found in the bacterial cellulase production versus fungi cellulase are higher growth rate leading to higher enzyme production, and the production of cellulosomes, providing increased function and synergy. In order to improve enzymatic production it is necessary to optimize the fermentation conditions and substrate utilization for a given microorganism. This study uses Microbulbifer Hydrolitycus IRE-31 (ATCC 700072) to produce cellulase employing hot-water maple sugar wood extract (HWWE) as a substrate. Selective strain medium adaptation of the original M. hydrolyticus IRE31 was performed by gradual increment of HWWE hydrolysate concentration in the marine medium from 2% to 3%, 4%, 5%, 6%, and 10% (v/v). To improve growth the pH level was controlled using 5M NaOH, and 0.02M Ca(OH)₂ .  The effect of 5M NaOH, and 0.02M Ca(OH)₂ on the cellulase activity  of the enzyme present on the broth was measured and compared at different concentrations of HWWE hydrolysate in flask fermentation. The results show higher cellulase activity with 5M NaOH.  In agar the best growth appeared with 0.02M Ca(OH)₂ as base. The addition of 0.5M NaCl in the medium to further the growth of the bacteria reduced the activity of the enzyme in the broth, as the concentration of NaCl was reduced the enzyme activity increased. A comparative study to determine the optimum 0.05M Citrate buffer pH for M. hydrolitycus cellulase activity, measured the activity of the cellulase present in marine broth with HWWE hydrolysate concentrations of 0, 2, 4, 6 and 10% v/v. The citrate buffer pHs ranged from 4.8 to 6.0, with the best cellulase activity observed at pH of 4.8. Tests comparing flask fermentations using different substrates such as xylose, glucose, cellobiose, HWWE hydrolisate and yeast extract, showed higher cellulase activity in the sample broth containing HWWE hydrolysate and no discernible activity in broths containing glucose, yeast extract or xylose. The results indicate a positive correlation with the concentration of HWWE hydrolysate in the marine broth and the cellulase activity. A 1L stirred tank fermentation with 10% v/v  HWWE hydrolysate , set at pH 7.3, 35°C, and 250rpm showed a maximum enzymatic activity in the broth sample of 0.650 μmol/min-ml.