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

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 during production 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 a nd 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