Artificial Consortium That Produces Riboflavin Improves The Production Of Acetoin By Paenibacillus Polymyxa

The introduction of NADH/NAD⁺regeneration system can regulate the distribution of acetoin and 2,3-butanediol.Studies have shown that coculture fermentation can utilize the excess NADH produced by one of the strains to form a NADH regeneration system to increase the yield of butanol.Therefore,a reasonable coculture system could promote the accumulation of acetoin.In this work,we studied the effects of exogenous vitamin B₂ addition and coculture system on acetoin and2,3-butanediol production of Paenibacillus polymyxa CJX518,so as to explore the effective fermentation methods to enhance the production of acetoin.At the same time,through the study of the downstream application of acetoin,we investigated the production of tetramethylpyrazine utilizing the acetoin precursor by microbial fermentation to solve the problem of separation and purification of acetoin.The main findings are as follows:1)Exogenous addition of vitamin B₂ can significantly improve the titer and yield of acetoin.Experiments showed that the VB₂ addition of 5mg/L and 10mg/L at 0h could promote the conversion of 2,3-butanediol to acetoin,and the addition of 5mg/L vitamin B2 at 0,12,and 24 h could also promote the titer and yield of acetoin.There was no obvious difference between the two groups.The titer of acetoin was increased by 122.2%,and the yield of acetoin increased to 0.40 g·g^-1 glucose from 0.16 g·g^-1glucose.Exogenous addition of vitamin B₂ could affect the dissolved oxygen and pH in the fermentation broth,and through the intracellular coenzyme analysis,vitamin B₂could also affect the intracellular NADH/NAD⁺ratios.The enzyme activity analysis showed that vitamin B₂ could increase the activity of NADH dehydrogenase and2,3-butanediol dehydrogenase in Paenibacillus polymyxa CJX518,and promote the conversion of 2,3-butanediol to acetoin and then enhance acetoin titer.2)Coculture of Paenibacillus polymyxa CJX518 and Escherichia coli LS02T producing VB₂ could promote the titer of acetoin.The results showed that the titer of acetoin increased by 74.2%when Escherichia coli LS02T was firstly cultured for 4h and then Paenibacillus polymyxa CJX518 was inoculated with the ratio of E.coli:P.polymyxa(0.1:0.2 OD₆₀₀).After the optimization of fermentation conditions,the acetoin titer increased by 76.7%under batch fermentation,and the accumulation of acetoin was 57.24 g/L,the productivity was 0.298 g/?L·h?and the yield of acetoin was 0.355 g·g^-1 glucose under fed-batch fermentation.3)According to the peak time of fermented samples and tetramethylpyrazine standard both in 13.96 min and the molecular weight of 136.1,it was proved that microbial fermentation could also produce tetramethylpyrazine in Paenibacillus polymyxa CJX518.The results showed that the fermentation temperature and inorganic ammonium salt species and concentrations could affect the production of tetramethylpyrazine.Meanwhile,the coculture of Paenibacillus polymyxa CJX518and Escherichia coli LS02T could enhance tetramethylpyrazine titer than pure culture.The above studies showed that coculture of Paenibacillus polymyxa and Escherichia coli had great potential,which provided a new strategy for improving the production capacity of acetoin and promoting its industrial application.