Regulation Of Hydrogen Production From Fermentation Of Cotton Stalk Hydrolysate By Enterobacter Cloacae WL1318 And Construction Of FhlA Gene Prokaryotic Expression Vector

With the constant consumption of fossil energy and the outbreak of energy crisis,the development of green and renewable energy has become a major initiative of the people’s livelihood in this century.As a new energy carrier,hydrogen gas is regarded as one of the most ideal and clean fuels on earth currently.Compared to traditional hydrogen production methods,microbial fermentative hydrogen production can utilize multiple industrial and agricultural wastes,such as straw cellulose material,industrial effluents and sludge as fermentative substrate for biological hydrogen production,which presents more economic feasibility in the production mode.This research developed toward a hydrogen-producing bacterium Enterobacter cloacae WL1318obtained in the group’s previous research work,the factors influencing the bacterial hydrogen production from fermentation of cotton stalk hydrolysate was studied,and the optimum fermentation conditions for hydrogen production was obtained.Regulation of the fermentation process based on the main influencing factor of hydrogen production was further developed.On the basis,metabolic flux analysis was developed toward the hydrogen production process by E.cloacae WL1318 from fermentation of cotton stalk hydrolysate,the metabolic flux distribution in the biohydrogen synthesis and competitive metabolic branch was illustrated,and the main regulation node was also stated.Meanwhile,the formate hydrogen lyase transcription activator fhlA gene affecting the biohydrogen synthesis was cloned,and the prokaryotic expression vector was constructed.（1）The initial sugar concentration,fermentation temperature and initial pH value exhibited significant effects on biohydrogen synthesis,reducing sugar utilization and bacterial growth in the hydrogen production process of E.cloacae WL1318 from fermentation of cotton stalk hydrolysate.When the initial sugar concentration was 40 g/L,the fermentation temperature was 37℃,and the initial pH value was 7.5 the highest daily hydrogen yield and cumulative hydrogen yield were obtained,and the relatively high glucose and xylose utilization,bacterial growth were also acquired,which hence determined as the optimum fermentative hydrogen production condition.The alternative pH regulation treatments avoided the sharp dropping of the p H value of fermentative broth,which was beneficial to biohydrogen synthesis and bacterial growth,while affected the glucose and xylose utilization in cotton stalk hydrolysate insignificantly.The hydrogen production potentials（P）in the alternative pH regulation treatments were all higher than that in the no regulation treatment,the daily hydrogen yield enhanced about 1.5 times than that in the no regulation treatment;and the cumulative hydrogen yield enhanced 15%and 30%of that in the no regulation treatment in the treatments of 24 h regulating the pH value and 48 h regulating the pH value respectively.The alternative pH regulation exhibited great influence on the bacterial growth of E.cloacae from fermentation of cotton stalk hydrolysate,which made the peak value of viable cells obtain just after the regulation time point,and all the bacterial growth OD₆₀₀s higher than the no regulation treatment after the regulation time point.（2）During the fermentation period of hydrogen production by E.cloacae WL1318 from fermentation of cotton stalk hydrolysate,,the absolute and relative metabolic fluxes of glucose,xylose and xylulose decreased gradually,indicating that glucose and xylose were effectively metabolized and promoted the synthesis and fermentation of the cell material.After 24 h of fermentation,pyruvate decarboxylase activity and pyruvate metabolic flux were higher,and then decreased slowly,indicating that pyruvate was rapidly decarboxylated and provided carbon skeleton for other metabolic branches.Citric acid in the fermentation 24⁴8 h has a higher metabolic flux,indicating that the metabolic flow of pyruvate nodules flow to the tricarboxylic acid cycle.Lactic acid synthesis branch in the fermentation of 48⁹6 h exhibited competition for biological hydrogen synthesis of metabolic flux distribution,lactate metabolic flux and lactate dehydrogenase activity were higher.The activity of acetaldehyde dehydrogenase was higher at 48h and 96 h after fermentation,and the metabolic flux of ethanol was increased at 120 h after fermentation,which indicated that the metabolic branch of acetic acid and ethanol synthesis played a more important competition in biological hydrogen synthesis.（3）Taking E.cloacae WL1318 genomic DNA as template,according to the GeneBank of Enterobacter cloacae and relatives fhlA gene sequences design primers,FF1 and FR1 for up and down primers,amplification fhlA genes,purpose fragment rubber cutting after recovery,with FF2and FR2 as upstream and downstream primers,fhlA gene cloning,and connect to the prokaryotic expression vector of pET-28a.FhlA gene nucleotide sequence length is 2060 bp and 1212 bp completed ORF,encoded 403 amino acids,by bioinformatics analysisthe.Protein found by Blastp than build a evolutionary tree is the formate-hydrogenase lyase system activating transcription factor,formic acid and hydrogen cracking of intracellular proteins.Through double enzyme digestion and sequencing verification,it was proved that it was successfully connected to prokaryotic expression vector pET-28a-fhlA,laying an experimental and theoretical foundation for the subsequent construction of recombinant strains.