Screening And Identification Of Low-temperature Hydrogen-producing Bacteria And Its Metabolomics Study

Fermentation hydrogen production technology has the advantages of mild reaction conditions,wide range of raw materials,low consumption,and no pollution.It is a very promising technology,and it has been widely concerned by researchers.At present,the main problems of the fermentation hydrogen production technology are(1)the hydrogen production of the strain is not high;(2)When hydrogen-producing bacteria are fermented in cold areas,the cost of hydrogen production becomes higher due to the higher fermentation temperature.In order to solve problems,this paper screened an anaerobic hydrogen-producing strain with high efficiency at low temperature,optimized its growth conditions through intermittent experiments and carried out metabolomics research on the anaerobic hydrogen-producing bacteria,so as to understand the physiological and biochemical characteristics of hydrogen-producing bacteria.And it is of great significance to increase the hydrogen production of hydrogen-producing bacteria,laying a foundation for the subsequent industrial production of hydrogen-producing bacteria.In order to solve the problem of high cost of hydrogen production due to high fermentation temperature,this study used Hungate anaerobic rolling tube technology to separate and screen two plants with good yield from the activated sludge in the continuous stirred tank reactor at low temperature.The anaerobic hydrogen-producing bacteria with hydrogen ability were named XY-18 and XY-50 respectively;under normal temperature conditions,an anaerobic hydrogen-producing bacteria with high efficiency was screened and named XY-72.Through intermittent experiments to compare the gas production and hydrogen production of hydrogen-producing bacteria XY-18 and hydrogen-producing bacteria XY-50 under low temperature conditions,the experimental results show that the hydrogen production and gas production of hydrogen-producing bacteria XY-18 are higher than hydrogen-producing bacteria XY-50;and then comparing the hydrogen-producing ability of hydrogen-producing bacteria XY-18 and hydrogen-producing bacteria XY-72 under low temperature conditions,the results show that hydrogen-producing bacteria XY-18 has good hydrogen production performance at low temperature,and its hydrogen production The amount is 30%higher than that of hydrogen-producing bacteria XY-72.In order to identify the species from which hydrogen-producing bacteria were obtained,the 16S r DNA gene sequence analysis in this study found that the gene sequence homology of hydrogen-producing bacteria XY-18 and Clostridium acidisoli strain CK74 reached 99.58%.Scanning electron microscopy and other methods confirmed that the hydrogen-producing bacteria XY-18 was a strain of the genus Clostridium,and was named Clostridium sp.XY-18.In this experiment,the effects of various factors on the growth of Clostridium sp.XY-18 under low temperature conditions were carried out by batch experiments.The experimental results showed that the optimal inoculation amount of Clostridium sp.XY-18 was 1%;the optimal concentration of L-cysteine for hydrogen production of Clostridium sp.XY-18 was 1.0 g·L^-1;the optimal concentration of glucose was 20 g·L^-1;the optimum growth and hydrogen production temperature of Clostridium sp.XY-18was 20?.Clostridium sp.XY-18 obtained a maximum specific hydrogen production rate of 1.2 mol H₂/mol glucose under optimized culture conditions.In this study,metabolomics was used to analyze the metabolite changes of Clostridium sp.XY-18 at 10?and 20?.The results showed that a total of 134metabolites were screened at 10?and 20?,of which 4 metabolites were significantly down-regulated,namely cellobiose,uric acid,galactonic acid,erucic acid,and 130metabolites were significantly down-regulated.Up-regulated,respectively,are 3-hydroxybutyric acid,L-proline,L-threonine,6-phosphogluconic acid,glutathione,etc.The main enriched metabolic pathways of differential metabolites are amino acid metabolism,carbon metabolism,phosphotransferase system,pentose phosphate pathway,bacterial chemotaxis and other pathways,and these metabolic pathways are significant.