Isolation and Characterization of Exopolysaccharide from Rhizobia Isolates and Optimization Conditions.

Abstract

Bacterial exopolysaccharides are versatile polymeric materials used in modern industrial and agricultural biotechnology. The aim of this thesis was to isolate and characterize exopolysaccharide producing Rhizobia and optimization conditions for exopolysaccharide production. Potentially, 20 different nodulated faba bean were collected, surface sterilized, crushed, serially diluted and inoculated onto yeast extract mannitol agar. Then, seven highly mucoid Rhizobia isolates were selected and grown yeast extract mannitol broth for EPS production. Provisionally, Rhizobia isolates were characterized by biochemical tests. The EPS Produced were precipitated by isopropanol, dried at 500C and concentration of EPS were measured by phenol sulfuric acid methods and finally characterized by FTIR. The quantitative data were analyzed by Minitab V 21.1.0, Origin Pro, 2023, Graph Pad prism V 8 and Design Expert V 13. Four isolates of REPS16, REPS9, REPS5 and REPS11 showed maximum yield of EPS when sucrose supplied as sole carbon sources 223mg/ 100ml, 215 mg/100ml, 201mg/100ml and 200 mg/100ml while, myo-inositol gave minimum EPS yield 33mg/100ml-10mg/100ml whilst, nitrogen sources indicate220mg- 100mg /100ml in yeast extract. The factors of sucrose, glucose, yeast extract, beef extracts peptone, temperature and pH were found to have a profound impact on EPS production (p<0.0001). As per 2-D and 3-D graph judging from these experimental results, the preliminary optimization and optimized media for producing EPSs was 1.5 folds increase in sucrose, glucose and yeast extract, beef extract, peptone at 280C, pH 7, 96hrs and 3.5mg/100ml NaCl. As per FTIR indicated that, all of the extracted EPS from Rhizobia isolates were contained exopolysaccharide functional groups of – CH-, C=O, C-O-C, -COOR, -OH, ester, aldehydes, ketone groups and some traces of amines and sulphonates components of carbohydrates moieties. Overall, this study's findings showed that it may be possible to isolate potential Rhizobia that produce exopolysaccharide from the faba bean's root nodule, and enables industrial exopolysaccharide production and maximizing yield as whole.