Optimization of the Nutritional Requirements of Two Novel Bacterial Cellulose Decomposers Isolated From Lebanese Habitat

Two novel bacterial cellulose decomposers were isolated and identified phenotypically and morphologically as Gram negative short rods none spore formers. Genotypic identification using 16S rRNA was carried out for both isolates.  Bacterial isolate 1 was identified as a member of the species Pseudomonas aeruginosa with 98% similarity thus; it was named Pseudomonas aeruginosa IZ.  Bacterial isolate 2 was identified as a member of the species Serratia marcescens with 98% similarity thus; it was named Serratia marcescens IZ. Physiological, environmental and nutritional factors affecting Psuedomonas aeruginosa IZ and Serratia marcescens IZ cellulase activity were evaluated one factor at a time for optimum production and activity of their cellulase enzyme.  The time course of cellulase activity by the selected bacterial isolates was monitored during 5 days. Maximum enzyme activity by Psuedomonas aeruginosa IZ was obtained at day 2 (0.35 U/ml) whereas maximum enzyme activity by Serratia marcescens IZ was obtained at day 1 (0.2 U/ml).  The effects of Nitrogen source and fermentation technique on cellulase production by the bacterial isolates were studied. It was concluded that fermentation media CM2, which contains corn cobs as a sole source of both carbon and nitrogen, under shaken condition (150 rpm) revealed maximum Psuedomonas aeruginosa IZ cellulase activity after 2 days of incubation (0.71 U/ml) and Serratia marcescens IZ cellulase activity after 1 day of incubation 0.70 (U/ml). Results indicate that the enzymatic activity under these conditions increased 2.02 folds for Psuedomonas aeruginosa IZ, and 3.5 folds Serratia marcescens IZ.  Optimization of the physiological and environmental factors affecting Psuedomonas aeruginosa IZ and Serratia marcescens IZ cellulase activity using Plackett-Burman revealed that optimized factors result in an increase of cellulase activity. Eleven different factors were chosen to perform this optimization process and the main effect of the examined factors affecting cellulase activity by both bacterial isolates was calculated. Data of the present investigation revealed that the rest of the factors studied didn’t affect cellulase enzyme activity or production in a significant manner. By the end of the optimization part, the optimum nutritional conditions for Pseudomonas aeruginosa IZ cellulase production under submerged fermentation using shaken conditions were (g/l): KH₂PO₄ 1.5, K₂HPO₄, 1.79, ZnCl₂, 0.0025, MgSO₄.7H₂O, 0.7, CaCl₂ 0.08 and corn cobs 100. The pH was adjusted to 5.0 and the flask was inoculated with 2% inoculum and incubated at 44˚C for 48 hours under shaken conditions (150 rpm). For Serratia marcescens IZ the optimum nutritional conditions for cellulase production under submerged fermentation using shaken conditions were (g/l): KH₂PO₄ 0.5, K₂HPO₄, 0.5, ZnCl₂, 0.0025, MgSO₄.7H2O, 0.1, CuCl₂ 0.0025, CaCl₂ 0.08 and corn cobs 100. The pH was adjusted to 9.0 and the flask was inoculated with 10% inoculum and incubated at 44˚C for 24 hours under shaken conditions (150 rpm). On the basis of the high productivity of the enzyme at the end of the optimization experiments, the present work envisaged the production of cellulase from Pseudomonas aeruginosa IZ at high scale using corn cobs as a cheap carbon source under submerged fermentation to be exploited commercially for the use in industry.

Keywords: Novel cellulose decomposers, nutritional requirements, optimization