Enhancing Production of Amino Acids from Bacillus spp. Using Batch and Fed-batch Fermentation Strategies

Abstract

Aims: Production of amino acids from black strap sugar cane molasses by Bacillus sp. R22EG1 strain and Bacillus sp. R20EG2 using batch and fed-batch (pulsed and continuous feeding) cultures was investigated to achieve the maximum concentration of free amino acids. Place and Duration of Study: Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt, between December 2011 and March 2012. Methodology: Two Bacillus strains namely R22EG1 and R20EG2, were used as amino acid produces. The amino acid producing bacteria were grown in the bioreactor with batch and fed batch cultivation. The fed-batch fermentations were performed in two strategies using pulsed and continuous feeding of black strap sugar cane molasses. In the first strategy (fed-batch by pulsed feeding), the amount of black strap sugar cane molasses (50 ml.L-1) was added to the fermentation vessel. Two, three and four additions of this amount of black strap sugar cane molasses were also added during the first 12 and 48 h of cultivation periods. In the second strategy, the black strap sugar cane molasses was fed continuously during the first 12, 18 and 24 h of cultivation periods at rates of 4.17, 2.78 and 2.08 ml.L-1.h-1, respectively (fed-batch by continuous feeding). The cell dry weight, amino acid concentration and residual sugar were determined as well as the growth and production parameters were calculated. Results: The biological activity of Bacillus sp. R22EG1 and Bacillus sp. R20EG2 strains duringproduction of amino acids on 5% black strap sugar cane molasses medium for 72 h at 30°C was investigated in bioreactor as a batch and fed-batch cultures. In batch culture, the highest figures of amino acid concentration (2.30 and 2.83 g.L-1), yield (9.52 and 11.71%), and conversion coefficient (11.09 and 13.44%) were recorded after 72 h and 60 h of fermentation periods by Bacillus sp. R22EG1 and R20EG2 strains, respectively, whereas the maximum productivity, approximately, 0.044 and 0.059 g.L-1.h-1 were observed after 12 h and 24 -36 h for Bacillus sp. R22EG1 and R20EG2 strains, respectively. Two feeding strategies (pulsed and continuous) were studied during production of amino acids using fed-batch culture. The highest cell dry weight, amino acid concentration and yield were recorded after three pulsed molasses addition during the first 12 h of fermentation periods with specific addition rate of 0.204 ml.L-1.h-1 (0.099 g.L-1.h-1 sugar) for the tested strains. The continuous feeding rate of 4.17 ml.L -1.h-1 (2.01 g.L-1.h-1 sugar) was more favorable than pulsed feeding during 12 h for amino acid production in fed-batch culture, as it increased the amino acid concentration by Bacillus sp. R22EG1 and R20EG2 strains, approximately, (1.53 & 1.42) fold than pulsed feeding and about (1.64 & 1.59) fold than that produced in batch bioreactor technique, after a 48 h fermentation period. The highest content of free amino acid species in culture supernatants was glutamic acid produced by both strains. Conclusion: The maximum production of amino acids from continuous fed–batch by Bacillus sp. R22EG1 strain and Bacillus sp. R20EG2 was 3.76 and 4.49 g.L-1 with continuous feeding at 4.17 ml.L-1.h-1 (2.01 g.L-1.h-1 sugar) at 48 h, respectively. These results were 1.53 & 1.42 fold higher than pulsed feeding and 1.64 & 1.59 fold higher than batch fermentation by Bacillus sp. R22EG1 and R20EG2 strains, respectively. The highest content species of free amino acids was glutamic acid using a Bichrom 30 amino acid analyzer