Stimulating Cellulase Activity of Microbulbifer Hydrolyticus Using Hot-Water Wood Extract Hydrolisate as Substrate

Among the cellulase producing microorganisms, bacteria often present the advantage of high growth rate, which leads to high 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 hydrolisate of sugar maple produced during hot-water extraction (hydrolisate) as substrate to stimulate the production of cellulase from Microbulbifer Hydrolitycus IRE-31 (ATCC 700072). Selective strain medium adaptation of the original M. hydrolyticus IRE31 was performed by gradual increment of hydrolisate concentration in the marine medium from 2% to 20% (v/v). To improve growth, the pH level was controlled using 5M sodium hydroxide (NaOH) and 0.02M calcium hydroxide (Ca(OH)₂ ) as bases. The effect of the bases on the bacterial growth and activity of the cellulases present in the broth resulted on higher cellulase activity in flask fermentation with 5M NaOH, while in agar the best growth appeared with 0.02M Ca(OH)₂ as base. A comparative study to determine the optimum 0.05M citrate buffer pH for enzymatic activity of the M. hydrolitycus cellulases present in marine broth supplemented with various hydrolisate concentrations showed best cellulase activity at pH of 4.8. Tests comparing flask fermentations using different substrates such as xylose, glucose, cellobiose, hydrolisate, sodium chloride (NaCl) and yeast extract, exhibited higher cellulase activity in sample broths containing hydrolisate and no discernible activity in broths containing glucose, yeast extract or xylose. The results indicate a positive correlation with the concentration of hydrolisate in the marine broth and the cellulase activity. The addition of 0.25M and 0.20M NaCl in the medium reduced the activity of cellulases in the broth.  Scaling up the study to a1L stirred tank fermentation with 10% v/v hydrolysate, set at pH 7.3, 35°C, and 250rpm displayed a maximum enzymatic activity in the broth sample of 0.650 μmol/min-ml after 10 hours. The evidence from this study supports the inference that the use of hydrolysate as a substrate is promising in the stimulation of the production of cellulase by the marine bacteria.  However, further study is required regarding optimization of the process.