Investigation of pectinase production from pectin and agricultural wastes by Bacillus pumilus (NRLL B-212)

In this study, production of pectinase group enzyms; exo-pectinase, endo-pectinase and pectin lyase by a gram-positive bacterium, Bacillus pumilus was studied in a batch system and the effects of pH, initial concentrations of carbon and nitrogen sources, phosphate and metal ions on enzyme production were investigated. In bioreactor experiments, the effects of mixing speed and air flow rate were examined, oxygen consumption rate and volumetric liquid phase mass transfer coefficients were calculated. Then, the enzyme production was was performed using different agricultural wastes and the enzyme production medium was optimized by response surface methodology.In the first stage of the research, submerged culture fermentation experiments were performed and maximum enzymes production were obtained at pH:8. The effect of initial pectin concentration on enzyme production was examined and 1.0 % pectin was determined as optimum pectin concentration. Ammonium sulfate, yeast extract and peptone were used as nitrogen sources and the highest activity was observed at 0.05% ammonium sulfate. In experiments that examined the effect of metal ions, the highest activity values were obtained in a medium containing 0.02 % NaCl. Phosphate effects were also investigated and maximum exo-pectinase activity was determined as 8.00 U/mL at 0.6 % K2HPO4 + 0.2% KH2PO4 and pectin lyase and endo-pectinase activity were obtained as 27.37 and 8.00 U/mL at 0.3% K2HPO4 + 0.15% KH2PO4 concentrations, respectively.The effects temperature and pH on pectic enzyme activities were tested in the study. For this purpose pH was variated in the range of 5-11, where as temperature was variated from 30oC to 90 oC. The optimum temperatures were found as 40, 50 and 30 oC for pectin lyase, exo and endo pectinase activities, respectively. All the enzymes had maximum activity at pH=10.In the second stage of the study, the effects of stirring and air flow rates on enzyme production were investigated in a 6 L bioreactor and liquid-phase volumetric mass transfer coefficients (kLa) and oxygen consumption rates were also determined. Stirring rate was changed from 200 to 400 rpm, where as air flow rate was changed from 0.1-0.2 vvm. The highest enzyme?s activities were obtained at 300 rpm and 0.1 vvm and were pectin lyase, exo and endo pectinase activities found as 19.5, 10.69 and 102.13 U/mL, respectively.In the last stage of the research, solid state fermentation studies were performed. Agricultural wastes such as wheat bran, sugar beet pulp, sunflower tray, orange peel, banana peel, apple pomace and grape pomace were used and the maximum enzyme activities were determined in the medium containing wheat bran and sugar beet pulp. Furthermore, solid state fermentation parameters were optimized by using response surface method. Solid substrates (wheat bran and sugar beet), ammonium sulfate and yeast extract concentrations were selected as independent variables in the model. The second-order polynomial coefficients were calculated and the results were analyzed using statistical package Design Expert Software. Statistical analysis of the model was carried out using model of variance analysis (ANOVA).Key words:Pectinase, Bacillus pumilus, submerged culture fermentation, solid statefermentation, agricultural wastes, Response Surface Methodolog.