Study On Microbial Agent Of Bacillus Subtilis For Animal Manure Composting

Microbial agent for composting mainly refers to a class of microbial preparation which are used to composting process of manure, plant residues and some life wastes. Bacillus subtilis is a kind of aerobic spore-forming bacterium, and is widespread in nature. Because B. subtilis can produce protease, amylase, cellulase and hemicellulase, it is one of the main microbial species in bacteria agent for animal manure composting. The number of spores and viable cells number are important parameters of bacteria agent. Production cost is also an important issue that is concerned for microbial agents manufacturing enterprises. In this work, spore-forming conditions of B.subtilis DK36 were investigated, and the fermentation process was optimized. On this basis, production technology of the microbial agent was studied. In order to reduce production cost, dephenolizing cottonseed protein as a nitrogen source was used to cultivate B.subtilis DK36. The main results are as follows:1) Study on spore-forming conditions of B.subtilis DK36. When glucose, sucrose, fructose, soluble starch, corn flour and wheat bran were used as carbon source, the cells grew well. Glucose and sucrose as carbon source did not make the cell produce spores. When wheat bran was used as a carbon source, sporulation was the most, followed by corn flour and soluble starch. Peptone, yeast extract, soy peptone, soybean powder, potassium nitrate, sodium nitrate, ammonium nitrate made the cells form a lot of spores, and soytone was the best among them. Urea inhibited cell growth and sporulation. Adding Ca2+, Mg2+, Na+, Fe2+and Mn2+could promote formation of spores, and the best one was manganese sulfate. Cu2+ had inhibitory effect on cell growth and sporulation. The optimal spore-forming medium was: bran 15.0 g/L, soy peptone 5.0 g/L, manganese sulfate 0.1 g/L, calcium carbonate 3.0 g/L. The optimal spore fermentation conditions were: culture temperature 30℃, rotation speed 200 r/min, inoculation of 2%, initial pH 7.0. Under this fermentation condition, a spore number of 3.6×109 cfu/mL was obtained.2) Cultivation of B.subtilis DK36 using dephenolizing cottonseed protein. When cottonseed protein was used as nitrogen source, the bacteria cells grew well. Glucose, sucrose, corn flour and wheat bran could promote the cell growth. But glucose and sucrose made the bacteria cell not form spores, and corn flour and wheat bran could make produce a great deal spores. Adding Ca2+, Mn2+, Mg2+and K+ had a significant role in promoting sporulation. And, it was found that potassium had a synergism effect with magnesium, manganese and calcium. It was also found that dephenolizing cottonseed protein as a nitrogen source cause sporulation slowly. The best medium was: corn flour 30.0 g/L, cottonseed protein 30.0 g/L, magnesium sulfate 2.0 g/L and potassium dihydrogen phosphate 4.5 g/L. The optimum fermentation conditions were: culture temperature 30℃, rotation speed 200 r/min, inoculation 8%, and the number of spores and spores rate were 3.5×109cfu/mL and 98.7% respectively.3) Preparation of microbial agent of B.subtilis DK36. Although the spores could withstand high temperatures, but as drying temperature increased, the viable cells in microbial agent decreased significantly. When the broth was pH7.0, the agents had the highest number of viable cells. Supporter types had a significant influence on the quality of microbial agent. When wheat bran, diatomaceous earth and activated carbon were used as supporters, their viable cell numbers were similar. Second flour and corn had poor effect on microbial agent. Addition of glucose, starch and dextrin had hardly the impact on the viable cell numbers. The optimal preparation process was: the ratio of fermentation and the supporter 1.5: 1, drying temperature 70℃, broth pH 7.0, wheat bran as supporter, moisture content 3.3%, and theviable cell number of the agent was 26 million/g.