Hydrogen Production Performance And Metabolomic Study Of Acid-tolerant Strain Clostridium. Sp H-20

Combining environmental governance with the process of obtaining clean energy is the main strategy to deal with environmental pollution and the energy crisis.The dark fermentation biological hydrogen production technology can use the widely distributed high-carbon water organic waste as the raw material for bio-H₂ production and achieve a win-win situation of energy recovery and environmental protection.It is one of the most valuable secondary energy production technologies today.At present,the bottleneck of the industrial application of dark fermentation hydrogen production technology is the low yield of the fermentation system.The product inhibition effect is the biggest reason for this bottleneck.The application of aciduric bacteria with high-efficiency hydrogen-producing capability may provide a new approach for solving this problem.On the other hand,the research on the acid-resistant mechanism of acid-tolerant and efficient hydrogen-producing strains can provide theoretical support for the directional regulation of the hydrogen-producing fermentation process,and can also develop the stable operation of the dark-fermentation hydrogen-producing system.Using the Hungate anaerobic rolling tube separation method,the activated sludge in the anaerobic reactor was selected as the screening object.After several rounds of separation operations,we finally screened out the strain H-20.H₂-producing bacteria H-20 has been identified as G⁺,and exists in two forms,single cell and cell chain in LM-1 medium.The 16S r DNA sequence similarity between this strain and Clostridium acidisoli CK74^T strain is 99.42%,and it is named Clostridium sp H-20.The p H of the fermentation broth is crucial to the hydrogen production process.In order to clarify the hydrogen production performance of acid-resistant strain,various initial p H was set in this experiment to explore the acid resistance of H-20 strain.The results demonstrated that within the initial p H range of 4.5-7.0,the maximum accumulative H₂ production of H-20 strain was 3202±50 m L/L culture;when the initial p H dropped to 4.5,the accumulative hydrogen production could still reach 2806±20m L/L culture,which is much higher than that of the normal hydrogen-producing bacteria H-35 in the control group,showing better acid-tolerance hydrogen-producing ability,and possess high research value.The stable acid-base environment is conducive to the bio-H₂ production of the strain,and the industrial field usually chooses to add buffer substances to make the fermentation process continue.The citric acid-sodium citrate buffer system is safe,non-toxic,and has great buffering capability.Therefore,in this experiment,citric acid-sodium citrate was adopted to prepare the fermentation broth with buffering capacity.By changing the p H of the buffer system,the effect of this buffer system on the hydrogen production of H-20 strain was studied.The results show that this buffer system can shorten the lag period of H-20 strain by two hours when the p H is 4.5,and increase the biomass of the fermentation system by about 15%on average.The untargeted metabolomic analysis strategy of GC-MS was used to explore the effect of different degrees of acid stress on the metabolism of H-20 strain.The results of bioinformatics analysis illustrated that 92 differential metabolites were screened in the H-1/H-0 comparison group,of which 61 metabolites were up-regulated and 31 were down-regulated,and these differential metabolites were significantly enriched to 11.There are 106 differential metabolites in the H-2/H-0 comparison group,including 16differential metabolites up-regulated,90 differential metabolites down-regulated,and18 pathways of these 106 differential metabolites are significantly enriched;In the H-3/H-0 comparison group,111 differential metabolites were screened,of which 16metabolites were up-regulated and 95 were down-regulated.These 111 differential metabolites were significantly enriched in 23 metabolic pathways.The significantly enriched pathways in the above three control groups are mainly related to the metabolism of carbohydrates,purines and amino acids,and involve some signal transduction systems.Therefore,it is speculated that hydrogen-producing bacteria H-20 may rely on mechanisms related to the metabolism of these substances to resist the acid stress in the environment.