| Fermentative hydrogen production from organic wastewater is a promising approach which can effectively remove organic matters and simultaneously achieve energy recovery.H2-ethanol co-production is one of the main fermentation pathways in hydrogen-producing bacteria.Ethanoligenens harbinense is the representative specie among the H2-ethanol co-producing microorganisms and the dominant bacterial group in ethanol-type hydrogen-producing reactors.However,the molecular regulatory mechanism of H2-ethanol co-production in E.harbinense has not been well understood.In order to overcome the bottleneck,large-scale quantitative proteomic and acetylproteomic strategies were employed in this study,which allowed in-depth analysis of the metabolic regulatory mechanism at molecular level.E.harbinense YUAN-3 was selected as the target strain since it is the representative strain of E.harbinense.This study aims to provide technical support and theoretical proof for the targeted regulation of fermentative hydrogen production industry.Acetic acid is another liquid end-product of E.harbinense besides ethanol.It is also one of the major factors that inhibit fermentation process.Molecular mechanism of acetic acid induced-fermentation inhibition was investigated in strain YUAN-3.Firstly,exogenous acetic acid of 10 m M,20 m M and 30 m M was added to PYG medium based on p H variation.The hydrogen production rate of strain YUAN-3 significantly decreased and the fermentation period was significantly expanded in the stress of exogenous acetic acid.However,the yield of each end-product was not significantly changed.Secondly,protein sample preparation method for proteomic analysis was optimized,results indicated that the combination of homogenization and phenol extraction method was more applicable to E.harbinense.Thirdly,through quantitative proteomic approach,78,121 and 216 proteins were differentially expressed after strain YUAN-3 was cultured in the medium supplemented with exogenous acetic acid of 10 m M,20 m M and 30 m M.Bioinformatics analysis revealed that acetic acid had greater impact on basic proteins,low molecular weight proteins and cytoplasmic proteins of strain YUAN-3.The expression levels of proteins involved in growth,carbon and phosphate uptake were down-regulated,and this is the main reason why biogas production rate of strain YUAN-3 significantly decreased in acetic acid stress.Dihydropyrimidinase,dihydropyrimidine dehydrogenase and ?-alanine synthase which involved in ?-alanine and pyrimidine metabolism,and proteins including thioredoxin,peroxiredoxin which involved in oxidative stress response play important roles in defending acetic acid inducedintracellular acidification.Molecular mechanism of metabolite profile change in E.harbinense YUAN-3 was studied.Results showed that ethanol accumulation altered the distribution of end-product yields.Ethanol yield of strain YUAN-3 increased by 15.1%,30.1%,and 27.4% in 50 m M,100 m M,and 200 m M ethanol stress,respectively.Sharp decreasing of H2 yield from 1888.6 ± 45.8 m L·L-1 to 837 ± 64.7 m L·L-1 and acetic acid yield from 1767.7 ± 45 mg·L-1 to 160.6 ± 44.7 mg·L-1 were observed in strain YUAN-3 with increasing exogenous ethanol(0 m M to 200 m M).However,CO2 yield was not significantly changed during ethanol accumulation.Quantitative proteomic analysis indicated that 263 proteins of strain YUAN-3 were differentially expressed in 50 m M,100 m M,and 200 m M of exogenous ethanol.Bioinformatics analysis showed that ethanol had greater impact on acidic proteins,high molecular weight proteins and cytoplasmic proteins of strain YUAN-3.The upregulation of bifunctional acetaldehyde-Co A/alcohol dehydrogenase(ADHE)was the main reason why ethanol production was enhanced while H2 and acetic acid yields declined in strain YUAN-3 during ethanol accumulation.Meanwhile,ethanol accumulation significantly changed the expression level of proteins related to the glycolysis,and proteins involved in carbon and nitrogen metabolic regulation,this is another reason why ethanol induced metabolic change in strain YUAN-3.Moreover,desulfoferrodoxin,glutathione peroxidase and proteins involved in histidine biosynthesis play important roles in ethanol tolerance of strain YUAN-3.Mechanism of metabolic enhancement by supplementing E.harbinense YUAN-3 with L-cysteine was analyzed.Experimental results revealed that biogas production rate,end-products yield and dry cell weight of strain YUAN-3 increased correspondingly by adding L-cysteine to PYG medium within the concentration of 0-2 m M.Further quantitative proteomic and acetylproteomic analysis revealed that L-cysteine mainly regulated the metabolic activity of strain YUAN-3 through the following ways.Firstly,the expression level of proteins participated in L-cysteine and L-methionine metabolism were significantly changed by adding L-cysteine to PYG medium,which inferred that increasing L-cysteine within the concentration of 0-2 m M could reduce the accumulation of homocysteine.Therefore,the metabolic activity of strain YUAN-3 was enhanced.Secondly,proteins involved in energy production,growth and enzyme activity regulation were acetylated,which further influenced the metabolic activity of strain YUAN-3.Thirdly,the acetylation level of proteome decreased by adding L-cysteine to PYG medium,which subsequently reduced acetyl Co A consumption.This is a reason why Lcysteine enhances the metabolic activity of strain YUAN-3.Fourthly,proteins related to glycolysis,ethanol and hydrogen production pathway of strain YUAN-3 were tightly regulated by acetylation.In addition,the data showed that 28% proteins of E.harbinense genome were acetylated,which confirmed that an abundant of acetylated proteins existed in E.harbinense and play important roles in metabolic regulation. |
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