Theromphilic bacterial cultures of Bacillus species capable of producing antibiotic bacitracin were isolated from local habitats and were screened for the production of bacitracin. Culture No.39 gave the maximum yield of bacitgracin (37.2.1.U/mL) whereas rest of the cultures produced less quantity of bacitracin. It was identified as Bacillus licheniformis. The culture Bacillus l icheniformis 39 was mutated by both UV irradiation and NYG (N-methyl-N-nitro-nitrosoguanidine). The mutatedstrain was designated as Bacillus l.icheriformis M-39.
In submerged fermentation, 20 hours old vegetative inoculuim (5%v/v) gave the maximu yield of bacitracin when the culture was grown in medium containing L-glutamic acid, L-alanine, citric acid and inorganic salts. It was found that amino acids L-glutamic acid, L-alanine, the divalent metal ions (Mg, Fe and Mn) sodium dihydrogen phosphate were essential for bacitracin production whereas Na2So4 and CaCl2 were deleted from the medium as they decreased the bacitracin yield.
Carbohydrates in the form of sugars and starches were employed for the production of bacitracin. Among sugars glucose gave the maximum yield of anitibiotic and among starches, soluble starch was found best suited for bacitracin production. Glucose also proved as interfering carbon source so it gave maximum yield at 0.5% (w/v) concentration agter its complete consumption. Soluble starch was also used at slowly utilized carbon source in addition to glucose to fulfill the carbon needs of cultures after the depletion of glucose. Catablite repression of glucose was also studied in addition to the effect of various organic acids on the production of bacitracin and citric acid gave the maximum yield.
Organic and inorganic nitrogen sources were tried to enhance the yield of bactracin but they were found ineffective in the presence amino acids of the fermentation medium. Effect of bacitracin constituent amino acid, their stereoisomerism, and some other amino acids were studied on bacitracin production. L-leucine increased the bacitracin yield upto 72% on 1.0 g/l addition. Bacitracin yield was further increased to 15% when a combination of five other amino amino acids was used with L-leucine.
Inhibition of antibiotic bacitracin on its own production was studied. The inhibition increased upto 140 l.U/mL addition bacitracin in fermentation medium at the start of fermentation. The inhibition was due to the formation of chelating complex with divalent metal ions. The inhibitory effect of bacitracin was maximum with 6.0 mM concentration of Mn2+.
To economize the feasibility of the process agricultural by-products such as corn steep-liquor, soybean, corn, sunflower and cotton seed meals were tried in place of amino acids. The results obtained by using soybean meal were encouraging. The optima medium for the submerged fermentation of bacitracin contained soybean meal, glucose , starch, citric acid and mineral salts produced bacitracin 94.3 l.U.mL at the flask level.
The environmental factors such as incubation temperature, initial pH, aeration and agitation rates were also optimized. The optimum bacitgracin yield (156.4 l.U/mL) was obtained in 30 L fermenter with working volume 15 L at 37 C with initial pH of 7.0. The agitation and aeration rates were 250 rpm and 1.0 L/L/min respectively.
The production of bacitracin was also carried out by solid state fermentation by using soybean meal as solid substrate. The other agricultural by-products were also tried but did not give better yield than soybean meal. Carbohydrate and nitrogen sources were also found ineffective to increase the antibiotic yield.
Isolation of bacitracin from the fermented broth was carried out by its precipitation as metal salt by divalent metal ions. Zn2 was found the most suited for this purpose and it gave the maximum percentage recovery of 67.4% and antibiotic activity of 6.9 l.U/mL in the optimized conditions of temperature, pH and carrier quantity.