The Influence of Agitation Rates, pH and Calcium Carbonate on L-lysine Production by Bacillus subtilis using Agricultural Products as Carbon and Nitrogen Sources

L-Lysine is an essential amino acid that is required in the diet of humans and animals. It is utilized in human medicine, cosmetics and pharmaceutical industry. ’The influence of agitation rates, pH and calcium carbonate on L-lysine production by Bacillus subtilis using agricultural products as carbon and nitrogen sources was studied. The L-lysine-producing bacteria had already been isolated from Nigerian soil. They were purified and Identified as B. subtilis PR13 and B. subtilis PR9, using cultural, biochemical and molecular characteristics. Optimization of some parameters which included agitation rates, pH values and CaCO3 concentrations, on L-lysine production by the Bacillus species was carried out. The L-lysine was produced in 250 ml flasks containing fermentation media (FM1 and FM2). The findings revealed that, enhanced L-lysine yield of 2.10 and 1.33 mg/ml was observed at agitation rate of 180 rpm for B. subtilis PR13 and PR9 respectively. There was a positive correlation between agitation rates and L- lysine production by B. subtilis PR13 and PR9 (r = 0.96 and 0.83 respectively). The pH of 7.5, stimulated optimum L- lysine yield of 2.27 mg/ml for PR13 and 1.38 mg/ml for PR9. There was a positive correlation between pH values and L-lysine production by B. subtilis PR13 and PR 9 (r = 0.63 and 0.50 respectively). The supplementation of 40g/l of CaCO3, enhanced optimum L-lysine yield of 2.18 mg/ml for B. subtilis PR 13 and 1.30 mg/ml for B. subtilis PR9. There was a positive correlation between varying concentrations of calcium carbonate and L-lysine production by the B. subtilis PR13 (r =0.35), while negative correlation was observed for B. subtilis PR 9 (r = -0.10). The results obtained in the study illustrated that the optimization of process parameters could increase the L-lysine yield from agricultural products by B. subtilis PR13 and B. subtilis PR9.

Optimization of Nutritional Parameters for the Production of L-Methionine from Newly Isolated Bacillus cereus Strain.

Aims: This study evaluated nutritional parameters for optimum methionine production Study Design: Methionine was assayed using the colorimetric method of Greenstein and Wintz (1961). Place and Duration of Study: Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria, between April 2010 and November 2011. Methodology: Bacillus cereus RS 16 was previously isolated from a soil ecovar in Owerri, Nigera. It was maintained on nutrient agar (Oxoid) slants at 4ºC. Bacillus cereus 16 was confirmed by 16Sr RNA conducted at Macrogen Incoporated Korea. Methionine production was carried out in a submerged medium. A 10% (v/v) seed culture was used to inoculate a 100ml Erlenmeyer flask containing 30 ml of fermentation medium in a rotatory shaking incubator at 170 rpm and 30ºC. Growth and methionine accumulation was determined from the broth. Nutritional parameters were studied to determine optimum methionine production Results: Glucose and ammonium chloride were the best carbon and nitrogen source for Lmethionine production. Maximum methionine (4.55mg/ml) were obtained with 80.0g glucose, 20.0g ammonium chloride, 20.0g calcium carbonate, 0.1.g DL-ornithine monohydrate, 0.1g peptone, 0.5g potassium dihydrogen phosphate, 0.5g potassium hydrogen phosphate, 0.01g magnesium sulphate heptahydrate, 0.001g manganese sulphate tetrahydrate, 0.001g ferrous sulphate heptahydrate at 72h fermentation period. There was a remarkable increase of methionine level to 4.55 mg/ml after optimization compared to methionine level of 1.92 mg/ml before optimization Conclusion: This present investigation has determined optimum parameters for maximum production of L- methionine by the newly isolated mesophilic bacterium Bacillus cereus strain RS 16. This information has enabled formulation of media composition for maximum methionine production by this organism.

Improved Cultural Conditions for Methionine Accumulation in Submerged Cultivation of Bacillus cereus S8.

Aims: To improve the cultural conditions for enhanced methionine production by Bacillus cereus S8 Study design: Study of the fermentation process in shake flask culture. Place and Duration of Study: Department of Applied Microbiology and Brewing, Nnamdi Azikwe University, Awka, Nigeria between 2011 to 2012. Methodology: The effects of medium/fermenter volume ratio, carbon and nitrogen sources, growth stimulators, vitamins and amino acid on methionine accumulation in the broth culture of Bacillus cereus S8 were investigated. The time course for methionine production was also studied. Results: A 20% medium/fermenter volume ratio improved methionine yield. Glucose and ammonium sulphate at 6.0 and 1.0% respectively stimulated methionine accumulation by Bacillus cereus S8. Yeast extract, peptone, DL-leucine and all vitamins studied enhanced methionine production. A methionine yield of 3.23mg/ml was produced after 96h fermentation and at a pH of 6.90. Conclusion: Improving the cultural conditions of Bacillus cereus S8 in submerged medium stimulated methionine increase.

A New Approach to Screening for Methionine-Producing Bacteria.

Aim: To find a method of screening for active Methionine-producing organisms. Study Design: Examination of cross-section of soil. Place and Duration of Study: Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Nigeria between April 2010 and August 2011. Methodology: Bacterial isolates (200) from soil were screened for Methionine producers on solid agar medium seeded with Methionine auxotroph, Escherichia coli. The agar plates were observed for halo growth of the E. coli which indicates Methionine production by the isolate. Methionine production in submerged medium by the isolates was investigated. Results: A total of 24 bacterial isolates were recovered as Methionine producers. Six of the active isolates used for submerged fermentation accumulated Methionine in a range of 0.46 – 1.40mg/ml. A close relationship was established between the nature of the halo growths of E. coli auxotroph on solid agar and the Methionine yields of the active bacterial isolates in submerged medium. Conclusion: It is a new and fast approach to screening for active Methionine producers.