Optimization of Fermentation Conditions of Bacillus thuringiensis EC1 for Enhanced Methionine Production


Bacillus thuringiensis EC1, isolated from the fermented oil bean seed, Pentachletra macrophila Benthan, produced a methionine yield of 1.89 mg/ml. The influence of cultural conditions on methionine accumulation by B. thuringiensis EC1 showed that a 20% medium/fermenter volume ratio and a 5% inoculum size increased methionine yield. The carbon of choice was maltose and at 8% level stimulated methionine production. Among the nitrogen sources studied, ammonium sulphate was found to be the best and at 1% concentration produced a methionine yield of 2.56 mg/ml. All growth-promoting substances and their mixtures enhanced methionine accumulation by B. thuringiensis EC1. The effect of Vitamins on methionine production showed that riboflavin and thiamine HCl at 1.0 μg/ml yielded 2.49 mg/ml and 2.80 mg/ml methionine respectively. The influence of bivalent metals on methionine accumulation indicated that Zn2+ at all concentration stimulated methionine production. Mg2+ and Ba2+ at 0.1 μg/ml and 10.0 μg/ml respectively improved methionine yield. Optimizing the cultural conditions of B. thuringiensis EC1 in submerged medium gave a methionine yield of 3.18 mg/ml.

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Anakwenze, V. , Ezemba, C. and Ekwealor, I. (2014) Optimization of Fermentation Conditions of Bacillus thuringiensis EC1 for Enhanced Methionine Production. Advances in Microbiology, 4, 344-352. doi: 10.4236/aim.2014.47041.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Mueller, J.H. (1922) A New Sulphur-Containing Amino Acid Isolated from Casein. Proceedings of the Society of Experimental Biology and Medicine, 19, 161.
[2] Noftsger, S., St-Pierre, N.R. and Sylvester, J.T. (2005) Determination of Rumen Degradability and Ruminal Effects of Three Sources of Methionine in Lactating Cows. Journal of Dairy Science, 88, 223-237.
[3] Kumar, D. and Gomes, K. (2005) Methionine Production by Fermentation. Biotechnology Advances, 23, 41-61.
[4] Pham, C.B., Galvez, C.F. and Padolina, W.G. (1992) Methionine Fermentation by Batch Fermentation from Various Carbohydrates. ASEAN Food Journal, 7, 34-37.
[5] Umerie, S.C., Ekwealor, I.A. and Nawabo, I.O. (2000) Lysine Production from Various Carbohydrates and Seed Meals. Bioresource Technology, 75, 249-252.
[6] Okamoto, K. and Ikeda, M. (2000) Development of Industrially Stable Process for L-Threonine Fermentation by an L-Methionine Auxotrophic Mutant of Escherichia coli. Journal of Bioscience and Bioengineering, 89, 87-89.
[7] Hermann, T. (2003) Industrial Production of Amino Acids by Coryneform bacteria. Journal of Biotechnology, 104, 155-172.
[8] Kase, H. and Nakayama, K. (1975) L-Methionine Production by Methionine Analog-Resistant Mutants of Corynebacterium glutamicum. Agricultural and Biological Chemistry, 39, 153-160.
[9] Nakayama, K., Araki, K. and Kase, H. (1978) Microbial Production of Essential Amino Acid with Corynebacterium glutamicum Mutants. Advances in Experimental Medicine and Biology, 105, 649-661.
[10] Dike, K.S. and Ekwealor, I.A. (2012) Studies on Process and Physical Parameters for the Production of L Methionine from Newly Isolated Bacillus cereus Strains. Asian Journal of Biological Science, 5, 96-104.
[11] Ozulu, U.S., Nwanah, O.U., Ekwealor, C.C., Dike, S.K., Nwikpo, C.L. and Ekwealor, I.A. (2012) A New Approach to Screening for Methionine-Producing Bacteria. British Microbiology Research Journal, 2, 36-39.
[12] Greenstein, J.P. and Wintz, M. (1961) Methionine. Chemistry of the Amino Acid. John Wiley & Sons, Hoboken, 2125-2155.
[13] Miller, G.L. (1959) Use of Dinitrosalicyclic Acid Reagent for Detection of Reducing Sugars. Analytical Chemistry, 31, 427-431.
[14] Mondal, S., Das, Y.B. and Chatterjee, S.P. (1996) Methionine Production by Microorganisms. Folia Microbiologica, 41, 465-472.
[15] Mondal, S. and Chatterjee, S.P. (1994) Enhancement of Methionine Production by Methionine Analogue Resistant Mutants of Brevibacterium heali. Acta Biotechnologica, 14, 199-204.
[16] Khongsay, N., Laopaiboon, L., Jaisil, P. and Laopaiboon, P. (2012) Optimization of Agitation and Aeration for Very High Gravity Ethanol Fermentation from Sweet Sorghum Juice by Saccharomyces cerevisiae Using an Orthogonal Array Design. Energy, 5, 561-576.
[17] Bisht, S.D., Yadav, S.K. and Darmwal, N.S. (2012) Enhanced Production of Extracellular Alkaline Lipase by an Improved Strain of Pseudomonas aeruginosa MTCC 10,055. American Journal of Applied Science, 9, 158-167.
[18] Shah, A.H., Hameed, A., Ahmad, S. and Majid, K.G. (2002) Optimization of Culture Conditions for L-Lysine Fermentation by Corynebacterium glutamicum. Journal of Biological Science, 2, 151-156.
[19] Rahman, R.N., Geok, P. L., Basri, M. and Salleh, A.B. (2005) Physical Factors Affecting the Production of Organic Solvent-Tolerant Protease by Pseudomonas aeruginosa Strain K. Bioresource Technology, 96, 429-436.
[20] Banik, A.K. and Majumdar, S.K. (1974) Studies on Methionine Fermentation. Part I. Selection of Mutants of Micrococcus glutamicus and Optimum Conditions for Methionine Production. Indian Journal of Experimental Biology, 12, 363-365.
[21] Camila, R.S., Andréia, B.D. and Meire, L.L.M. (2007) Effect of the Culture Conditions on The Production of an Extracellular Protease by Thermophilic Bacillus sp. and Some Properties of the Enzymatic Activity. Brazilian Journal of Microbiology, 38, 253-258.
[22] Anike, N. and Okafor, N. (2008) Secretion of Methionine by Microorganisms Associated with Cassava Fermentation. African Journal of Food, Agriculture, Nutrition and Development, 8, 77-90.
[23] Carlos, E.T. and Meire, L.L.M. (2000) Culture Conditions for the Production of Thermostable Amylase by Bacillus sp. Brazilian Journal of Microbiology, 31, 298-302.
[24] Morinaga, Y., Tani, Y. and Yamada, H. (1984) Homocysteine Trans-Methylation in Methanol Utilizing Bacteria and Its Application to L-Methionine Production. Agricultural and Biological Chemistry, 48, 143-148.
[25] Phadatare, S.U., Deshpande, V.V. and Srinivasan, M.C. (1993) High Activity Alkaline Protease from Conidiobolus caronatu (NCL 86.8.20): Enzyme Production and Compatibility with Commercial Detergents. Enzyme Microbial Technology, 15, 72-76.
[26] Ekwealor, I.A. and Obeta, J.A.N. (2005) Studies on Lysine Production by Bacillus megaterium. African Journal of Biotechnology, 4, 633-638. http://dx.doi.org/10.5897/AJB2005.000-3115
[27] Tani, Y., Lim, W.J. and Yang, H.C. (1988) Isolation of L-Methionine Enriched Mutants of a Methylotrophic Yeast, Candida boidinii No 2201. Journal of Fermentation Technology, 66, 153-158.
[28] Sen, S.K. and Chatterjee, S.P. (1989) Influence of B-Vitamins and Trace Elements on Lysine Production by Micrococcus varians 2fa. Acta Biotechnologica, 9, 63-67.
[29] Ekwealor, I.A. and Obeta, J.A.N. (2007) Effect of Vitamins and Bivalent Metals on Lysine Yield in Bacillus megaterium. African Journal of Biotechnology, 6, 1348-1351.
[30] Hughes, M.N. and Poole, P.K. (1989) Metals and Microorganisms. Chapman and Hall, Boca Raton, 1-37.
[31] Sigel, I. (1983) Metal Ions in Biological Systems: Zinc and Its Role, Biology and Nutrition. Marcel Dekker Inc., New York.
[32] El-Sayed, A.S.A. (2009) L-Methioninase Production by Aspergillus flavipes under Solid-State Fermentation. Journal of Basic Microbiology, 49, 331-341.
[33] Saxena, R. and Singh, R. (2011) Amylase Production by Solid-State Fermentation of Agro-Industrial Wastes Using Bacillus sp. Brazilian Journal of Microbiology, 42, 1334-1342.
[34] Murgov, I.D. and Zaitseva, Z.M. (1973) Optimization of the Composition of Nutrient Medium for the Biosynthesis of L-Lysine by Brevibacterium flavum 178 Using Mathematical Planning of the Experiment. Applied Microbiology, 9, 845-851.

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