Optimization Of Conditions For Exopolysaccharide Producing Rhizobia From Root Nodule Of Field Pea (Pisum Sativum

Abstract

Exopolysaccharides (EPS) are long-chain biopolymer with branched, recurring unit of sugars and non-carbohydrate constituents connected to exterior of cells. The objective of this thesis was to isolate and characterize Rhizobia that produce exopolysaccharides and optimize the conditions for its production. Pea plants with nodules were collected. Five highly mucoid Rhizobia isolates were selected and grown on yeast extract mannitol broth for EPS production. The Rhizobia isolates were characterized by morphological and biochemical tests. The EPS Produced were extracted by isopropanol, dried at 600C and total sugars were estimated by phenol sulfuric acid methods and finally characterized by FTIR. The quantitative data collected in this investigation was analyzed by Origin Program,2024, and Design Expert V 13.All rhizobia samples exhibit circular colony shapes, white translucent color, entire margins, raised elevation, mucoidal consistency, and are classified as Gram-negative. All isolates tested positive for the indole, catalase, starch hydrolase, and urease tests, while all showed negative results for the oxidase test. From the different carbon sources, sucrose yielded the highest EPS (213 mg/100 ml), From nitrogen sources tested, yeast extract Yielded (205 mg/100 ml.Temperature 28°C resulted in the highest EPS production(218mg/100ml,pH 7 resulted in the highest EPS production at (210 mg/100 ml),96 hours of incubation was optimal for EPS production across all isolates(222 mg/100 ml) .Ps17 achieved the highest EPS production of 222 mg/100 ml after 96 hours, 2.5%Nacl resulted in the highest EPS production(220mg/100ml). The interaction between pH 7 and 28°C yielded the highest EPS production of 500 mg/100 ml. All isolates exhibited similar FT-IR spectra, showing key peaks for hydroxyl groups (3000 cm-1), CH stretching (2970 cm-1), and carbohydrate ring vibrations (1000-1100 cm-1). Sucrose and yeast extract are the most effective carbon and nitrogen sources respectively for EPS production, with optimum results at 28°C, pH 7, and a 96-hour incubation period. Ps17 demonstrated the highest EPS yield of 222 mg/100 ml, and a combination of 8 mg/100 ml sucrose and PH7 achieved up to500 mg/100 ml, with the best overall production at 500 mg/100 ml observed with pH 7 and 28°C.For maximal EPS production, sucrose and yeast extract, an incubation temperature of 28°C, and pH of 7 and optimize conditions based on the interaction between pH and temperature to achieve up to 500 mg/100 m