LYSINE ACCUMULATION IN THE BROTH CULTURE OF Bacillus Species ISOLATED FROM SOIL USING CARBOHYDRATES AND SEED MEALS

AUTHOR: OKPALLA, JUDE

DEPARTMENT: APPLIED MICROBIOLOGY AND BREWING

AFFILIATION: NNAMDI AZIKIWE UNIVERSITY, AWKA

Lysine production by bacterial organisms capable of utilizing carbohydrates and seed meals as carbon and nitrogen sources was investigated. Bacterial isolates (460) from different soil samples in Awka town were screened for lysine production on solid agar medium seeded with lysineauxotroph, Escherichia coli. The agar plates were observed for halo growth of the E.coli, which indicates lysine production by the isolate. A total of 37 bacterial isolates were observed to be lysine producers. They were further cultured in submerged medium and 3 of the isolates identified as Bacillus subtilis PR 13, Bacillus subtilis PR9 and Bacillus pumilus SS16, were chosen as the best lysine producers. The maximum growth andlysine production were obtained at 20ml medium volume and 10% inoculum size for B. subtilis PR13 and B. subtilis SS16, and 25ml medium volume and 8% inoculum size for B. subtilis PR9. The enzyme hydrolysis of the agricultural carbon sources showed that maize produced the highest reducing sugar of 5.2mg/ml. Lysine was maximally produced at 180rpm, pH 7.5, 4% CaCO3, 6% (w/v) millet, 4% (w/v) soyabean meal, for B. subtilis PR13; and 180rpm, pH 7.5, 4% CaCO3, 6% (w/v) sorghum, 4% defatted peanut meal, for B. subtilis PR9. For B.pumilus SS16, lysine was enhanced at 200rpm, pH 7.5, 4% CaCO3, 6% (w/v) sorghum and 2% (w/v) defatted soyabean meal. CuSO4 at 5􀟤g/ml stimulated the highest lysineconcentration of 3.18􀟤g/ml in B. subtilis PR13. Lysine accumulation was enhanced by 0.005g/l of MgSO4 in B. subtilis PR13 and 0.005g/l of FeSO4 in B. subtilis PR9 and B. pumilus SS16. Optimum lysine yield was observed at 1􀟤g/ml of biotin, 0.04units/ml penicillin and 0.1􀟤g/ml stearic acid for B. subtilis PR13, while 10􀟤g/ml nicotinic acid, 0.04 units/ml penicillin and 0.2%(v/v) linoleic acid stimulated lysine production in B.subtilis PR9. For B.pumilus SS16, lysine accumulation was improved with 1.0 g/ml of riboflavin, 0.05􀟤g/ml vancomycin and 0.2% (v/v) linoleic acid. Glycine at 0.01%(w/v) enhanced lysine production in all the Bacillus strains except B.subtilis PR9, while yeast extract at 0.1%(w/v) improved growth and lysineyield in all the Bacillus strains except B.subtilis PR13. Effect of fermentation time on lysine production showed that maximum lysine accumulation was obtained after 96h for B. subtilis PR13 and B subtilis PR9 and 72h for B. pumilus SS16. The UV irradiated mutants PRMU – 8 and PRMU – 14 derived from B.subtilis PR13; MUPR – 4 obtained from B.subtilis PR9 and SSMU – 4 and SSMU – 9 obtained from B. pumilus SS16 produced higher yields of lysine than the parent strains. All the S-amino-ethyl-cysteine (AEC) resistant mutants with the exception of ART – 7 produced higherlysine levels than the parent strains.

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