Enrichment And Isolation Of Exoelecrogens From Environment And Studis On Electricity Generation Mechanism

With the rapid development of petroleum industry,the soil and aquatic system polluted by leakage of petroleum transportation seriously affects human living environment.As a new type of bioremediation technology,MFCs（Microbial fuel cells）can generate clean electricity while removing organic contaminants in soil and present a good prospect of ecological restoration.In MFCs system,facultative anaerobic exoelectrogens such as Shewanella sp.is more conveniently operated than strictly anaerobic exoelectrogens Geobacter sp.,and can be used in biodegradation,heavy metal recovery and carbon cycle promotion in sewage,showing a good application prospect.However,the electrochemical activity of Shewanella sp.in terms of its power output and stability is much lower than that of the anaerobic model exoelectrogen Geobacter sp.,therefore,it is necessary to screen more efficient facultative anaerobic exoelectrogens from environment.In this study,different exoelectrogens were screened out from oil-contaminated soils and sewage,and the taxonomy and biochemical activity of which were studied.The main research contents and conclusions are as follows:（1）The petroleum-contaminated soils was severed as inoculum to enrich and screen exoelectrogens by three-electrode system and MFC device,respectively.The organic matter degradation in contaminated soil in MFC anodic chamber and the characteristics of electrochemical active biofilm formed on the electrode were also analyzed meanwhile.The experimental results show that when the petroleum contaminated soil served as inoculum and substrate to enrich exoelectrogens,the biofilms initially enriched on working electrode surface with the highest current density of 45μA cm^-2 in the three-electrode system.However,by using the mixed bacteria after inoculated with ferric citrate medium as an inoculum in three-electrode system,the maximum output current density of biofilm formed on electrode was approximately 580μA cm^-2,which has a significantly higher electricity generation ability than that of the initial biofilms enrichment state.At the same time,the petroleum-contaminated soil can be directly degraded as substrate to a certain extent in MFC device,and the maximum output power density is about 220 m W m^-2.Ultimately,two exoelectrogens were isolated from the enriched mixed bacteria on the electrode,named strain OS-1 and OS-2,respectively.By compared with NCBI database,the strain OS-1 has the highest homology with Geobacter sulfurreducens PCA（99.56%）,and the strain OS-2 has the highest homology with Ochrobactrum ciceri Ca-34^T（99.78%）.（2）The sewage was served as inoculum to screen and enrich facultative anaerobic exoelectrogens by using a three-electrode system.A facultative anaerobic exoelectrogen LZ-1 was isolated and the related biological and electrochemical characters were studied.The results showed that:1.The strain LZ-1 has the highest homology with Citrobacter freundii ATCC 8090（99.78%）,and can use various substrates for its growth such as sucrose,glucose,sodium citrate,sodium acetate and sodium pyruvate etc..2.When sodium acetate was served as substrate,the current density produced by strain LZ-1 biofilm can reach to 880μA cm^-2,which is significantly higher than that of anaerobic G. sulfurreducens PCA(650μA cm^-2).3.The biomass of strain LZ-1 and G.sulfurreducens PCA were analyzed,and it was found that the facultative anaerobic strain LZ-1 exhibit average biomass amount(461.8?g cm^-2),which was approximately 190?g cm^-2 higher than G.sulfurreducens PCA(271.8?g cm^-2).4.XRD analysis confirmed that the strain LZ-1 has strong ferric iron reduction ability that can reduce ferric citrate to Fe CO₃.5.In order to reveal the facultative anaerobic exoelectrogen electron transfer mechanism,the specific primers were designed and varied through PCR program.6.In the dual-chamber MFC,the maximum output voltage of biofilm formed by strain LZ-1 was about 0.65 V, and the maximum output power density(1233 m W m^-2)was higher than that of G. sulfurreducens PCA(832 m W m^-2),which also shows a stable electrochemical activity at the same condition.（3）The strain LZ-1 biofilm enrichment and electrochemical activity changes were real-time monitored by spectroelectrochemistry technology for the first time,and the relationship between cytochrome c redox state changes in cell membrane and the related applied potentials were further analyzed.The results showed that when the biofilm produced current density lower than 150μA cm^-2,the cytochrome c could be completely oxidized at potentials higher than-0.15 V（vs.SCE）or could be totally reduced at potentials lower than-0.60 V with or without substrate in the solution.When the biofilm produced current density higher than 450μA cm^-2,the cytochrome c could be completely oxidized at potentials higher than-0.15 V（vs.SCE）without substrate in solution.However,if there was substrate in the solution,the cytochrome c could not be completely oxidized even at a high potential of 0.30 V（vs.SCE）and the reduced cytochrome c still accounts for about 68%of the overall.（4）The three-electrode system and dual-chamber MFC device were used to study the growth of strain LZ-1 under different pH conditions and the electrochemical performance of biofilm enrichment.The results showed that when the biofilm current density reached to 400μA cm^-2and there was no substrate in the solution,the cytochrome c in LZ-1 biofilm could be completely oxidized at an external potential of 0.30 V and totally reduced at -0.60 V under pH 6.0-8.0 conditions.Moreover,by using MFC system,the strain LZ-1 biofilm formed on anode shows a high power density output under slightly alkaline condition of pH 8.0(the maximum output was 1385 m W m^-2).On the other hand,the influence of oxygen on electricity generation of the facultative anaerobic strain LZ-1 biofilm was investigated.It was concluded that when oxygen exsit,the strain LZ-1 easily transfers electrons to oxygen and limit its electricity production ability.In addition,after a long period of 100 h aeration,the strain LZ-1 biofilm showed a certain degree of oxygen tolerance and its electricity production capacity did not have a significant effect.