Research Of Extracellular Electron Transfer In Psychrophilic Electroactive Biofilm Based On Omics

The extracellular electron transfer efficiency between the anode biofilm and the electrode interface is one of the important factors affecting the performance of bioelectrochemical system(BES).As an important ecological factor,temperature affects the metabolism of electroactive bacteria(EAB)and the maturation of anode biofilm.At present,the regulation mechanism of EET between psychrophilic electroactive biofilm and electrode interface needs to be further studied.Therefore,the relationship between the QS signal molecules and the EET is explored.Based on the metatranscriptomics sequencing,the functional microbes and genes responding to temperature change were identified and analyzed.Combined with metagenomics,single cell genomics and metaproteomics methods,EAB and functional proteins responding to EET stress at low temperature were identified,and the metabolic pathway of psychrophilic EAB was reconstructed.The relationship between the QS signal molecules and EET was explored.It was found that there were rich types of signal molecules in the start-up period,but with the extension of operation cycle,only C6-HSL,3-oxo-C8-HSL and 3-oxoC12-HSL were detected.The QS system was affected by temperature.The secretion of 3-oxo-C8-HSL was affected by low temperature,while the concentration of C10-HSL was related to temperature.C8-HSL and 3-oxo-C14-HSL were substrate dependent.Only C6-HSL always existed in different BES operation cycles under different conditions,which indicated that C6-HSL was mainly involved in basic metabolic activities of microorganisms.It was found that las-QS system was not directly involved in the regulation of EET,and rhl-QS played a key role in the EET base on the Pseudomonas aeruginosa pure-cultured system.While,PQS(Pseudomonas quinolones signal)system inhibited the EET efficiency.Among them,C4-HSL could restore the current output of the gene deficient strain to the level of the normal strain,while PQS signal molecule has no significant contribution to the current output.Compared with QS,anode biomass had no significant effect on EET.The response of anodic biofilm community to temperature shifts was investigated.It was found that the power output was limited by low temperature disturbance,which led to power overshoot of biofilm.Low temperature disturbance affected the diversity of microbial community,resulting in the decrease of operational taxonomic units(OTUs),Shannon index and Simpson index.However,low temperature had no obvious effect on the phylogeny of dominant EAB,and Geobacter always dominated the community.Under low temperature,the abundance of genes related to membrane transport decreased,while the abundance of genes related to translation increased.Many up-regulated genes participated in the cold shock response including cold shock protein(csp A),which indicated that the biofilm community adapted to the low temperature disturbance.In the process of low temperature disturbance,Acinetobacter was a highly responsive species to temperature changes.More differentially expressed genes(DEGs)were up-regulated in the initial cooling stage.With further cooling down,the down regulated genes were dominant in the system.239 DEGs were continuously up-regulated with decreasing temperature,while 19 DEGs were continuously down-regulated.The up-regulated DEGs,which were sensitive to temperature under low temperature disturbance,included fli C,pil C,pil Q and pil U genes related to the synthesis of type IV pili,indicating that the biofilm still had the ability to synthesize nanowires and maintained EET activity at low temperature.At low temperature,most of the ATP generated in oxidative phosphorylation was used to maintain basic metabolic activities and protein synthesis for cold adaption,resulting in less electrons participating in EET.Many genes with unknown functions were potentially important for the community to adapt to low temperature and realize EET.The response of biofilm to EET rate shifts under low temperature was analyzed.It was found that the mature and stable electrochemical active biofilm could be enriched in reactor at 4 ℃.Using saturated calomel electrode as reference electrode,high current appeared at-300 m V,but decreased at relatively high potential(0 m V).The psychrophilic biofilm community was dominated by Geobacter,and the short-term change of EET rate did not obviously affect the community structure.The main metabolic processes of anodic biofilm community at low temperature were amino acid metabolism,carbohydrate metabolism and energy metabolism.16 of differentially expressed proteins(DEPs)sensitive to anodic potential mainly came from Geobacter.The expression of respiratory metabolism related functional proteins(phosphorylation,propionic acid metabolism purine,ribonucleoside triphosphate binding and TCA cycle)indicated that the basic metabolism of biofilm remained active at low temperature.The optimal potential promoted the up regulation of respiratory metabolism related DEPs,and some oxidative metabolism reactions were inhibited under suboptimal conditions.While,some amino acid metabolism affected the EET of biofilm.At high EET rate,more DEPs interacted with each other actively.The proteins involved in energy metabolism were similar under optimal potential and suboptimal potential,while some interacting DEPs involved in nucleic acid or amino acid metabolism were different.The draft genome of psychrophilic EAB was reconstructed and its metabolic activity was analyzed.The identification of cytochrome c and pilus related functional genes and proteins showed the activity of EET,indicating that EET of psychrophilic EAB at low temperature can be realized mainly through the directly transfer mechanism of nanowires and cytochrome c.In this study,the interaction mechanism between psychrophilic electroactive biofilm and electrode were revealed,which laid a theoretical basis for the optimal regulation of BES.