Acetoin Fermentation Conditions Of Three Strains Of Bacillus

This experiment measured the performance of Bacillus subtilis B3,Bacillus amyloliquefaciens PEBA20,Bacillus amyloliquefaciens NTBA three strains of Bacillus fermented acetoin,and from the bacteria The growth,the pH of the fermentation medium,and the relationship between the two fermentation metabolites 2,3-butanediol and tetramethylpyrazine and acetoin were studied.The research results are as follows:1.Parallel fermentation experiments were carried out with three strains of Bacillus,and the OD₆₀₀00 value and the yield of acetoin in the fermentation process were examined.The OD₆₀₀00 value of B.subtilis B3 was higher than that of B.amyloliquefaciens PEBA20 and B.amyloliquefaciens NTBA.The yield of B.subtilis B3 acetoin was higher than that of the other two strains of Bacillus,and the maximum yield was 12.59 g/L.It is considered that B.subtilis B3 is a strain having superior performance in fermentation to produce acetoin.2.Mutagenesis of Bacillus subtilis B3 was carried out by atmospheric pressure room temperature plasma（ARTP）mutameter to determine the acetoin-producing properties and self-genetic stability of the mutant strains.A strain of acetoin was compared with the starting strain B3.The high mutant strain G4 had a maximum yield of 14.44 g/L for acetoin,which was 14.7%higher than that of the original strain B3.3.During the fermentation and culture of the three strains of Bacillus,the yield change of acetoin was significantly related to the change of OD₆₀₀00 value.When the bacteria are exponentially increasing,the yield of acetoin is low.When the growth of the cells exceeds the exponential growth period,the yield of acetoin begins to increase greatly until the yield of acetoin.After reaching the maximum value,the OD₆₀₀00 value of the bacterial cell volume began to show a downward trend at the same time.In the test fermentation system,the spore rate of the three strains of Bacillus was always low and the change range was small,which was not related to the yield change of acetoin.4.Three strains of Bacillus were cultured in YPG fermentation medium at pH 7.0.The pH range of the whole fermentation process fluctuated between 6.2 and 6.8,with little change.B.subtilis B3 was tested in three initial pH（5.5,6.5,7.0）mediums.It was found that the initial OD₆₀₀00 value was the highest under the initial pH of 7.0,and the maximum yield of acetoin was also the highest.However,in the fermentation culture with an initial pH of 5.5,the yield of acetoin was the smallest,and the amount of OD₆₀₀00 was the smallest.It can be seen that the acid-base performance of the culture system greatly affects the metabolism of Bacillus acetoin.5.During the fermentation process of the three strains of Bacillus,the similar changes in the yield dynamics of acetoin,tetramethylpyrazine and 2,3-butanediol were all changed first and then decreased,and then the steady state was maintained.The difference is that they have inconsistent time points when they produce three substances to reach maximum yield.The time to maximum yield of B.subtilis B3 producing acetoin,tetramethylpyrazine and2,3-butanediol was 120 h,96 h and 72 h,respectively.The time to maximum yield of B.amyloliquefaciens PEBA20 was 144 h,120 h,and 72 h,respectively.The time to reach maximum yield of B.amyloliquefaciens NTBA was 144 h,144 h,and 96 h.In comparison,it was found that the three strains of Bacillus produced 2,3-butanediol to reach the maximum yield first,and the maximum yield of tetramethylpyrazine was reached before the acetoin or at the same time as the acetoin.6.During the fermentation of B.subtilis B3,glucose was added continuously,so that the maximum yield of acetoin,tetramethylpyrazine and 2,3-butanediol was increased by 4%,3.3%and 2.5%respectively,compared with the starting strain B3.but the increase is not high,and the time point at which the fermentation reaches the maximum yield has not changed.7.In the initial stage of B.subtilis B3 fermentation,the maximum yield of acetoin,tetramethylpyrazine,2,3-butanediol can be increased by adding 3 g/L of 2,3-butanediol to the medium.Compared with the starting strain B3,it increased by 13%,10.2%,and 9.5%,respectively,but the time point at which the fermentation reached the maximum yield did not change.It indicates that the addition of 2,3-butanediol at the initial stage of fermentation can greatly increase the yield of acetoin and increase the yield of tetramethylpyrazine and 2,3-butanediol.