Determination of production of poly-beta-hydroxybutyrate (PHB) of some rhizobium bacteria, discrimination of strains by total cell protein profiles obtained by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isolation of ...

IV DETERMINATION OF PRODUCTION OFPOLY-p- HYDROXYBUTYRATE (PHB) OF SOME Rhizobium BACTERIA, DISCRIMINATION OF STRAINS BY TOTAL CELL PROTEIN PROFILES OBTAINED BY SODIUM DODECYL SULPHATE POLYACRYLAMTOE GEL ELECTROPHORESIS (PAGE) AND ISOLATION OF PLASMH) DNA (Ph.D.Thesis) Nazime MERCAN GAZİ UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY June 2002 ABSTRACT In the research, a total of 31 of Rhizobium strains (R melihti, R japonicum, R phaseoli, R leguminosarum, R viciae, Rhizobium sp.) were used. Determination of carbon source utilization showed that these strains could use glucose, dextrose and mannitole as monosaccharides while they showed differences in the utilization of trehalose, xylose, raffinose, maltose, galactose, sucrose, lactose and pyruvic acid-sodyum salt. Generally, Rhizobium strains did not grow at low pH, but R. viciae F69, Fill, R phaseoli Fa28 and Fa41, R cicer Nl, N2, N4, N6 strains were able to grow at pH 4,5 and these strains were characterized to be acid tolerant. Total cell protein profiles of bacteria were analysed using SDS- PAGE and molecular weights of proteins were found between 11-215 kD. Based on the protein profiles, it was possible to distinguish all of the strains. According to the SDS-PAGE analysis, it has been found strains belonging to the same species showed similarities in their protein patterns and differences from other species. PHB production by Rhizobium bacteria was determined by a spectrophotometry method. A qualitative method using Nile Blue A reagent T, «EiMET-M VMM 'lrt!i^SY0Mwas used for the detection of PHB in the cells. The color of the colonies under 254 nm-ultraviolet ligth at 254 nm ranged from pink to orange depending on the amount of PHB in the cells. It was found that the PHB contents of the strains the cell dry weight were between 5,37-57,09%. The effects of different carbon and nitrogen sources on the production of PHB by some of the strains were examined. For this purpose the strains R viciae Fill (57,09%), R meliloti Yll (39,25%) and B. japonicum S irat Fab (34,43%) which had been shownto have the highest PHB production were used. The maximum yield of PHB îor R, meliloti Yll and R japonicum S irat Fab strains were 83,75% and 34,31% of the cell dry weight respectively when they were grown in glucose medium as the carbon source. R viciae Fill produced a high level of PHB (41,22%) in the mannitol medium. The percentage yield of PHB by the strains (except Fill) was not found high in the media containing nitrogen sources. While the highest PHB accumulation was obtained with the strain R meliloti Yll (26,23%) and B. japonicum S irat Fab (26,78%) grown in the medium containing protease peptone, R viciae Fill was produced 48,13% of PHB in medium containing asparagine. The PHB concentration of the cells decreased with decreasing pH of the cultures. Amoung the strains showing the highest production of PHB R meliloti Yll was selected for mutation studies. Acriflavine, 5-bromourasiI, nitrosoguanidine as chemical mutations and UV irridiation at 254 nm as physical mutation were applied on the selected strain. The PHB production by parent and mutant strains were determined periodically and continued one year interval. Compared with the parent strains the percentage were determined in the first period of measurement The amount of PHB in some mutant strains increased in the second period measurement. In addition, the effects of physical and chemical mutations on the production of PHB by the strains were different. Ability of strains in the production of PHB by R meliloti Yll, B. japonicum S irat Fab and R viciae Fill grown in different concentration of beet molasses were studied. While R meliloti Yll strain produced maximum of PHB asVI 56,31% in medium containing 0,5% beet molasses, R viciae Fill and R japonicum S irat Fab strains in beet molasses medium showed production of PHB at lower percentage. A lower yield of PHB for R viciae Fill was observed with increasing viscosity of the medium. The agar diffusion method was used to study the general inhibitory effect of Rhizobium strains on some test bacteria. None of the strains were shown to have any inhibitory affect on test bacteria. Plasmid DNA profiles of Rhizobium strains and their molecular weight were determined. According to the results, only 13 strains carried plasmid, while the did not carry. Molecular weights of plasmids ranged between 13,5-16,74 kb. Science Code Key Words Page number Advisor : 401.02.00 : Rhizobium, isolation, utizilation of carbon sources, bioplastic (PHB), different carbon and nitrogen sources, beet molasses, antimicrobial activity, protein profile, plasmid DNA :175 : Prof. Dr. Yavuz BEYATLI