Generation Mechanisms Of Phosphine By Microorganisms In Anaerobic Digestion Process

Phosphine has the characteristics of diversity and extensiveness in nature.These characteristics determined that phosphine played an essential role in the biogeochemical phosphorus cycle.Besides,phosphine was regarded as a biological signal gas to explore unknown life in the universe.Therefore,it was of great significance to explore the origin of phosphine.The non-biological source of phosphine had been clearly understood.However,there were many controversies about the biological source of phosphine.So far,the mechanism of microbial production phosphine is still a mystery.In this study,through the enrichment of phosphine-producing microbial communities in the process of anaerobic digestion,the relationship between metabolites and phosphine was analyzed,and the characteristics of phosphine released by microbial anaerobic digestion were clarified.The critical steps of anaerobic digestion to produce phosphine were revealed.The mechanism of microbial phosphine production was explained by analyzing the substrate dehydrogenation,extracellular polymers,metabolic pathways,and phosphorus metabolism-related genes.The research results provided a scientific basis for further improving the biogeochemical phosphorus cycle,provided evidence support for phosphine as a biological signal exploration life in the universe,and provided a theoretical reference for the study of phosphorus recycling.The main results were as follows:(1)The characteristics of phosphine released by anaerobic digestion as follows:the first stage,the hydrolysis of organic matter indirectly promoted the production of phosphine;the second stage,the process of producing hydrogen and acetic acid released phosphine,and the third stage of methane production inhibited the production of phosphine.The anaerobic digestion was initiated to produce phosphine by designing the culture medium.In this process,the production of phosphine was effectively promoted by inhibiting methane production.The maximum concentration of phosphine was 110 mg m^-3,which was much higher than that of the control group 20?g m^-3.The analysis of metabolites showed that butyric acid production indirectly promotes the production of phosphine.In contrast,the simultaneous production of hydrogen and acetic acid directly promotes the formation of phosphine,and the p H range beneficial to the production of phosphine is 6.0-7.0.Molecular biology studies have shown that Azotobacter produces hydrogen,and Ruminococcaceae produces acid to promote the production of phosphine.(2)The effect of carbon influence was studied using tricarboxylic acid(TCA)cycle L-malic acid and citric acid as carbon sources,and the effect of substrate dehydrogenation on the production of phosphine was analyzed.Studies have found that L-malic acid as a carbon source was quickly absorbed and utilized by microorganisms.The primary metabolite was acetic acid.The maximum concentration of acetic acid was 673 mg L^-1,much higher than the maximum concentration of lactic acid,31 mg L^-1.The average concentration of phosphine was 210 mg m^-3.On the contrary,microorganisms used citric acid with low efficiency,and the primary metabolite was lactic acid.The maximum concentration of lactic acid was 150.18 mg L^-1,higher than the maximum concentration of acetic acid 35.31 mg L^-1,and the average concentration of phosphine was 105 mg m^-3.When NADH was added to the medium,the yield of phosphine was increased by 17%compared to the control group.NADH was given antioxidant properties and provided reducing power[H]to promote the production of phosphine by microorganisms.The reducing power produced by the substrate dehydrogenation process was the key to the formation of phosphine.The use of L-malic acid to produce acetic acid by microorganisms was the process of forming reducing power,which promoted the production of phosphine.Dehydrogenase had a linear relationship with the production of phosphine.However,the metabolism of citric acid to produce lactic acid consumed reducing power,which was not conducive to the production of phosphine,and dehydrogenase did not correlate with phosphine.(3)The functional bacteria with high phosphine production were screened and identified.The isolated phosphine-producing strain is named Pseudescherichia sp.SFM4,which was closely related to Pseudescherichia vulneris(97%similarity).SFM4 belongs to the Proteobacteria phylum,Gammaproteobacteria class,Enterobacteriales order,Enterobacteriaceae family,and Escherichia genus.The best initial conditions for SFM4 to produce phosphine was p H 7.47,with nitrogen,carbon,and phosphorus loadings of 6.17,300,and 10 mg L^-1,respectively.The influence of these factors on the production of phosphine was ranked:p H>nitrogen load>phosphorus load>carbon load.As phosphine concentration increased,the concentration of OD600 and DNA increased,and the fluorescence intensity corresponding to the byproducts of soluble microbial metabolism increased accordingly.It showed that microorganisms produced a large amount of phosphine in the logarithmic growth phase,and metabolic pathway analysis also showed that gene duplication and translation account for the highest abundance in the process of phosphine production.(4)The mechanism of microbial phosphine production was that inorganic phosphorus enters the cell through the phosphorus transport system to synthesize organophosphates with C-P bond.The organophosphates received the reducing power[H]from pyruvate regeneration and hydrogen and generated phosphine under phosphatase and phosphate reductase.The selected functional strain SFM4 was used as the research object to analyze the mechanism of microbial phosphine production.The study found that the process of catabolism of macromolecule glucose by SFM4 to produce pyruvate did not produce phosphine,while the pyruvic acid regeneration process produced phosphine through the TCA cycle with the maximum concentration of phosphine 78 mg m^-3.The maximum amount of hydrogen produced was 2.5 mg L^-1.SFM4 produced phosphine in the process of microbial aggregation.It was necessary to reach a specific density and stability to produce phosphine.Phosphine could be a signal molecule released by microbial quorum sensing.Microbes responded to phosphine stimulation.The protein concentration increased,while polysaccharides decreased in the T-EPS,and volatile solids(VS)concentration increased.EPS analysis showed that phosphine was produced inside the cell rather than extracellular.Exogenous hydrogen was used as an electron donor to help the formation of phosphine,and atomic hydrogen[H]as a carrier for electron transfer was essential for the formation of phosphine.When atomic hydrogen was quenched by 20 mmol L^-1 tert-butanol,the production of phosphine was suppressed entirely.The determination of the functional genes of phosphorus metabolism showed that the elemental phosphorus that SFM4 produces phosphine comes from the decomposition of intracellular organophosphates by phosphatase,especially organophosphates containing C-P bonds.(5)Construction of anaerobic digestion-microbial electrolysis cell(AD-MEC)to enhance phosphine production.When the cathode potential changed from 0 to-0.9 V,the maximum current density at-0.9 V was-0.03 A m^-2 with the maximum phosphine 192 mg m^-3.The cathode potential has a positive effect on the formation of phosphine,and gaseous phosphine leads to a deficit of phosphorus in the reaction system,realizing 24%of phosphorus resources.In conclusion,microbial production of hydrogen and acetic acid is a key step in the release of phosphine in anaerobic digestion.Hydrogen and NADH formed by acid production provide reducing power[H]to promote the production of phosphine;in addition,microorganisms decompose organophosphorus in intracellular to provide phosphorus for phosphine production.The research results provide a basis for a deep understanding of the mechanism of microbial phosphine production and provide technical support for the resource utilization of phosphine.