Enhancemenet And Mechanism Of Phenol Anaerobic Biotransformation Based On The Interspecies Electron Transfer

Phenol is a toxic pollutant,which widely exists in industrial wastewater such as coal chemical and pharmaceutical plants,and it causes great harm to the ecological environment and human health.Anaerobic biotechnology is an important method for treating phenol wastewater.It has the advantages of low economic cost and recyclable biogas.However,it also facesinhibition ofacetotrophic methanogenic activity and low efficiency of microbial electron transfer due to the slow hydrolysis rateof phenol.This thesis is oriented to the problems in the anaerobic biotransformation process of phenol and based on the interspecies electron transfer mechanism of anaerobic microorganisms.It explores a variety of methods to enhancetheelectron transfer and promote phenol anaerobic biotransformation,such as magnetite（Fe₃O₄）coupling zero-valent iron（ZVI）,Fe₃O₄coupling exogenous hydrogen（H₂）,and granular activated carbon（GAC）coupling exogenous hydrogen.The thesis alsoanalyzesthe influencesof the methods onthe interspecies electron transfer performance of microorganisms,the metabolic activity of the microorganismsand the structure of anaerobic microbial communities.Finally,the thesisutilizesmetagenomics to analyze the mechanism of GAC coupled with exogenous hydrogen to enhance interspecies electron transfer and promote phenol anaerobic biotransformationat the molecular and gene level.The details are as follows:Firstly,Fe₃O₄coupled with ZVI was added to the anaerobic digestion of of phenol to enhance the anaerobic biotransformation.The results showed that Fe₃O₄coupled with ZVI had a significant synergistic effect on anaerobicdegradation and methanogenesis of phenol.The phenol degradation rate and methane production of Fe₃O₄/ZVI-added group wereincreased by 8.8–23.1%and 11.9–31.6%,respectively compared with Fe₃O₄-added group,and enhanced by5.9–17.1%and 4.4–18.3%,respectively compared with ZVI-added group.By analysis,Fe₃O₄coupled with ZVI had a significant synergistic effect on anaerobicacetotrophic methanogenesis.The synergistic effects mainly included two aspects:（1）Fe₃O₄strengthened the growth of acetate oxidizing bacteria and promoted the oxidation of acetate to H₂ and CO₂.ZVI created the condition of microhydrogen which promoted the enrichment of hydrogenotrophic methanogens and accelerated the electron transfer in biotransformation process of hydrogen to methane.That is,Fe₃O₄-coupled ZVI strengthened the methanogenesis pathway of the syntrophic acetate oxidizing and hydrogenotrophic methanogenesis（SAO+HM）.（2）Fe₃O₄ coupled with ZVI enhanced the electron transfer efficiency between acetate oxidizing bacteria and hydrogenotrophic methanogens,which played a vital role on the synergistic effect of Fe₃O₄and ZVI on the anaerobic digestion of phenol.Secondly,to construct the dominant methanogenesis pathway of the syntrophic acetate oxidizing and hydrogenotrophic methanogenesis（SAO+HM）,Fe₃O₄ coupled with exogenous hydrogen（H₂）was added to the anaerobic digestion of phenol.The results indicated that Fe₃O₄ could significantly strengthen the anaerobic phenol degradation,and H₂ played a vital role in the methane conversion rate of phenol.However,Fe₃O₄ coupled with exogenous hydrogen had no synergistic promotion effect on the anaerobic digestion of phenol.Fe₃O₄/H₂ could obviously reduce the conductivity and extracellular protein of sludge,and further indicated that DIET produced by Fe₃O₄and IET produced by exogenous hydrogen interfered with each other in the process of extracellular electron transport,so as to reduce the efficiency of extracellular electron transport.The results of 16Sr RNA showed that the coupling of Fe₃O₄ with H₂increased significantly the relative abundance of the phenol oxidizing bacteria Syntrophus and Syntrophorhabdu and the acetate oxidizing bacteria Clostridium＿sensu＿stricto＿12.The hydrogenotrophic methanogen Methanobacterium（3.72%）became the dominant methanogenwith the presence of H₂.Fe₃O₄ coupled with H₂madethe dominantacetoclastic methanogenesis pathway（AM）convert to the dominant methanogenesis pathway of the syntrophic acetate oxidizing and hydrogenotrophic methanogenesis（SAO+HM）in anaerobic digestion of phenol.Thirdly,in order to improve the transfer efficiency of extracellular electrons,GAC coupled with H₂was added to the anaerobic digestion of phenol.The results indicated that GAC coupled with H₂ could significantly increase the conversion rate of phenol to methane.When GAC coupled with exogenous hydrogen was added to the anaerobic digestion of phenol,the hydrogenotrophic methanogenic activity of the sludge was 9.3times of the acetotrophic methanogenic activity,and the activity of the electron transport system of sludge and the secretion of extracellular polymers were both achieved a synergistic increase,indicating that the interspecies electron transfer efficiency between the syntrophic bacteria and hydrogenotrophic methanogens was enhanced.The results of 16Sr RNA indicated that H₂ enhanced the enrichment of Cryptanaerobacter,a phenol degrading bacteria,and accelerated the biotransformation of phenol to benzoate.At the same time,H₂ increased the relative abundance of hydrogenotrophic methanogens（HM）and syntrophicacetate oxidizing bacteria（SAO bacteria）,and reduced the relative abundance of acetotrophic methanogens.In the anaerobic digestion of phenol,GAC coupled with H₂ promoted the dominant SAO+HM pathway and improved the electron transfer efficiency between syntrophic bacteria and hydrogenotrophic methanogens.Fourthly,the metagenomics was utilized to analyze the mechanism of GAC coupled with H₂for enhancing the phenol anaerobic biotransformation.The results showed that GAC coupled with exogenous hydrogen can promote the syntrophic bacteria（Syntrophorhabdus,Syntrophaceae,Syntrophorhabdaceae）,the electroactive bacteria（Geobacter,Geobacteraceae）As well as the enrichment of methanogens（Methanothrix,Methanobacterium）,GAC provides a good enrichment medium for functional flora.The relative abundances of functional proteins in the process of hydrogenotrophic methanogenesis and functional proteins in the process of interspecies electron transfer on the GAC surface were significantly increased.At the same time,the functional genes of the hydrogenotrophic methanogenesis pathway and the functional genes controlling bacterial pili growth were significantly increased.From the molecular level and gene level,it was proved that GAC couplled with exogenous hydrogen can enhance the electron transfer efficiency and the hydrogenotrophic methanogenesis pathway in the phenol anaerobic system.By analyzing the correlation between functional bacteria,functional proteins and functional genes,a dominant hydrogenotrophic methanogenesis pathway was constructed and the direct interspecies electron transfer（DIET）between syntrophic bacteria and hydrogenotrophic methanogens via extracellular pili was generated on the surface of GAC.That is the intrinsic reason for the enhanced anaerobic biotransformation of phenol by GAC/exogenous hydrogen.