Mechanism Of The Hydrogen Production Promotion Of Clostridium From Rice Paddy Soil By Ferrihydrite With Low Concentration

Hydrogen can not only be used by microorganisms as an electronic donor in paddy soil systems,but also as a regulator to promote the growth of rice.But the existing research on hydrogen-producing microorganisms in rice fields is still relatively scarce.In this study,a hydrogen-producing strain BY-1 was screened from paddy soil by enrichment culture,and was preliminarily identified as Clostridium based on 16S r RNA data.Subsequently,the whole genome sequencing technology was used to conduct in-depth analysis of the hydrogen-producing metabolic pathways of the strains.Finally,starting from the in situ factors affecting the hydrogen production of Clostridium in the paddy rice environment,the effect of low amount of ferrihydrite on the hydrogen metabolism of strain BY-1 was explored,and the mechanism of ferrihydrite promoting hydrogen production was further explored.The main results are as follows:First,hydrogen-producing strain BY-1 was isolated from the collected paddy soil by enrichment culture.And then according to the of strain BY-1 the morphology and the identification results of 16S r RNA,BY-1 was preliminarily determined to be a Clostridium microorganism.Analysis of hydrogen-producing metabolism of BY-1 using glucose as a metabolic substrate,the experimental results showed that the strain had hydrogen-producing capacity,and it's hydrogen production efficiency was 1.12 mol H₂/mol glucose.After 60 h of dark culture,the main by-products of strain BY-1 in the process of hydrogen-producing metabolism were butyric acid,lactic acid and acetic acid,and the final yields were 0.43 mmol,0.40 mmol and 0.175 mmol,respectively.Add different concentrations of ferrihydrite(50,100and 150 mg·L^-1),the reduction efficiency of ferrihydrite after 60 h can be close to 80%of the added amount,which indicates that the strain has a strong iron reduction ability.Second,BY-1 was sequenced whole genome by a combination of second and third generation sequencing techniques,with a total gene number of 4894 and a total length of5130817 bp.Based on gene annotation results,this strain contains genes Hya B and Hyb C encoding the large subunit structure of[Ni-Fe]hydrogenase.It is also contains genes hyd A,which encodes the structure of small subunits in the[Ni-Fe]hydrogenase.Subsequently,the hydrogen-producing metabolic pathway of BY-1 was analyzed according to the gene annotation results.And the results showed that the strain had all the genes required for the metabolism of lactic acid,acetic acid,ethanol and acetic acid.BY-1 can use two potential extracellular electron transport modes(nanowires and secretory electron shuttles)to achieve fe(III)reduction.Third,this study focused on low concentration(0–50 mg·L^-1 concentration range)ferrihydrite.At first,we studied the effect of ferrihydrite at different concentrations on the hydrogen production,the enzymatic activity and the biomass of strain BY-1.Meanwhile,we monitored the changes of main metabolites,glucose content and Fe(II)content in the fermentation broth.The results showed that in the low amount range,ferrihydrite has no obvious p H buffering effect on the dark fermentation system,but can effectively promote the production of hydrogen.The optimum addition amount of ferrihydrite was 15 mg·L^-1.Under this condition,hydrogen production increased by 85.34%compared with the blank control group.The utilization rate of glucose increased from 77.66%of the blank control group to91.40%.The maximum biomass and hydrogen-producing enzyme activity of Clostridium were 1.82 times and 1.80 times that of the blank control group,respectively.The production of acetic acid and butyric acid of Clostridium increased,while the production of lactic acid decreased.Compared with the blank group,the production of acetic acid and butyric acid was increased by 71.42%and 28.98%,respectively,and the production of lactic acid was decreased by 12.55%.The Fe(II)content in the culture system increased with the reaction time,and the highest concentration was 6.36 mg·L^-1,but the total electron consumption for iron reduction only accounted for 0.12%of the electron consumption for hydrogen production.In conclusion,low concentration ferrihydrite can still significantly promote the hydrogen production of Clostridium without obvious p H buffering effect,and the potential mechanism of promoting hydrogen-producing of Clostridium are as follows.First,promote the growth of Clostridium and the utilization of glucose.Second,increase the enzymatic activity of hydrogenase.Third,regulate its metabolism in favor of hydrogen-producing.In summary,this study not only deepens the understanding of hydrogen-producing microorganisms in paddy soil,but also explores the influence of low amounts of ferrihydrite on the hydrogen metabolism of Clostridium from the in situ influencing factors that affect Clostridium hydrogen production.The study found that low amounts of ferrihydrite can significantly promote Clostridium hydrogen production without obvious p H buffering effect,which provides a theoretical basis for understanding the impact of hydrometalite on hydrogen-producing microorganisms in the natural environment.