Optimization of Poly-β-Hydroxybutyrate (PHB) Production by an Egyption strain of Rhizobium fabae F44 using Response Surface Methodology

Abstract

The present work is aimed to isolate, purify, identify a newly isolate Rhizobium fabae from different regions in Egypt then, 58 isolates were obtained from the different 9 governorates with 100 % infection plant technique. The 16SrRNA showed a similarity of 99.56 (%) to Rhizobium fabae F44 produce and optimize Poly-β-Hydroxybutyrate polymer (PHB) production by the selected isolate Rhi-zobium fabae F44 using a statistical approach of response surface methodology (RSM). Rhizobium fabae F44 isolate was picked up from Sharkia Governorate in Egypt and then identified by the 16SrRNA gene as Rhizobium fabae. Eleven different variables which affect the production of PHB polymer were screened by Plackett-Burman statistical design approach. Maximization of PHB production was adjusted by the terms of The Face Central Composite Design (FCCD) of RSM and assessed at three coded levels (-1, 0, +1). One way Anova was used to statistically analyze all obtained results in relation to post hoc multiple comparison analysis performed by Tukey's HSD. Appulses of nutritional and physical factors (two carbon sources, two nitrogen sources, mineral salts, pH, temperature, incubation time, inoculum size and agitation rate) were studied using Plack-ett-Burman design method. Out of all tested significant media components, sucrose, yeast extract, agitation rate had the highest significant effects on the response as for PHB production, with confidence level > 98% and were further optimized using FCCD. Predicted Maximum PHB production was observed as (78.51%) which in near the mid-point (0) values (concentrations) of sucrose which reached (25 g/l) and yeast extract of (0.5 g/l) for 48 hrs. at 150 rpm agitation rate. The observed experimental value reached 87.5u/ml was very close result to the predicted one validating the model. So, Response Surface Methodology is an effective statistical approach which can substitute the use of one variable at a time approach due to its adequacy and efficiency to optimize PHB production by Rhizobium fabae F44.