A study on induction of cellulase production by microbulbifer hydrolyticus using hot-water wood extract

This dissertation presents research work that establishes a process using concentrated hot-water wood extract to stimulate the production of cellulases and hemicellulases by Microbulbifer hydrolyticus IRE-31(ATCC 700072), as the first step in the integrated production and utilization of hydrolytic enzymes. M. hydrolyticus potential to hydrolyze cellulose and hemicellulose has been identified in prior studies, but there is little information reported concerning the cellulases and hemicellulases activity performance and productivity of the marine bacteria. This study examines the effectiveness of various substrates on the induction of cellulase production by M. hydrolyticus in shake-flask and scaled--up batch cultivations. Cellobiose, Sugar maple wood powder (WP), concentrated hot-water extract from Paulownia elongata (PWE) and wood extract hydrolysate (WEH) from Sugar maple were used as enzymatic inducers.;Selective strain medium adaptation of the wild type M. hydrolyticus was performed to improve bacterial growth in the media supplemented with WEH and PWE, leading to an increase in sugar maple WEH tolerance from 3% to 20% (v/v) and from 6% to 30% (v/v) PWE in batch cultivations. Batch cultivations with different carbon substrates showed 4 to 8 fold higher cellulase activity in PWE supplemented media. M. hydrolyticus growth is optimal at 0.1 to 0.5 M NaCl, but increased concentrations of NaCl (0.5 M) in the media reduced the cellulase activity of the crude enzymes.;The cellulase and xylanase activity was evident in sugar maple WP, PWE and WEH assays. The cellulolytic enzymes hydrolyzed oligosaccharides present in PWE. The hydrolytic enzymes activity in the PWE media increased the concentrations of xylose and glucose almost simultaneously, in conjunction with that of mannose, arabinose, rhamnose and galactose, demonstrating synergetic action between the cellulase and xylanase of M. hydrolyticus. In contrast to previous results stating that the bacteria does not use mannose and rhamnose, it was discovered that in addition to glucose and xylose, M. hydrolyticus utilizes galactose, mannose, arabinose and rhamnose for growth. Moreover, it was found that M. hydrolyticus almost simultaneously consumed the monosaccharides present. The optimal temperature and pH for cellulase activity for crude cellulase was determined to be at 60°C and 6.5 pH.