Study On Degradation And Synchronous Electricity Production Of Aromatic Pollutants By Pseudomonas Putida In Microbial Fuel Cell

Aromatic compounds exist widely in the environment.Because of their long-distance migration,difficulty in natural degradation,and easy bioaccumulation,aromatic compounds can exist for a long time in the ecological environment.The existence of aromatic pollutants in air,soil and water will cause potential harm to human health,and have strong carcinogenicity,teratogenicity and mutagenicity under some specific conditions.At present,the treatment methods for aromatic pollutants mainly include physical methods,chemical methods and biological methods.Among them,biological methods are widely used to treat aromatic pollutants due to their advantages of no secondary pollution and environmental friendliness.Microbial fuel cell(MFC)is a kind of device which combines traditional pollutant degradation with electrochemical technology and uses microorganisms to directly convert the chemical energy of pollutants into electric energy.The extracellular electron transport of microorganisms is the basis of the operation and application of MFCs.It has been reported that extracellular electron transport is mainly in three ways:direct contact,intermediate-mediated and nanowires,but the mechanism of extracellular electron transfer(EET)is not thorough enough.Pseudomonas putida can efficiently metabolize a variety of aromatic compounds,which is one of the best choices to remove aromatic pollutants from the environment.The P.putida B6-2 used in this paper was obtained from the oil-contaminated soil by Professor Xu Ping’s research group.And P.putida B6-2 can degrade a variety of aromatic compounds efficiently,diversified and deeply from the oil-contaminated soil,and has great potential for bioremediation of the compound polluted environment.In this work,P.putida B6-2 was used as anode microorganism to construct MFC to study its degradation of many kinds of aromatic pollutants and synchronous electricity generation.After that,in order to deeply understand the electricity production mechanism of P.putida B6-2,the MFC was characterized by electrochemistry,scanning electron microscopy(SEM)and atomic force microscopy.On this basis,through transcriptomics data analysis combined with molecular biology methods to find key genes related to electricity production and verify their functions.The main contents and results of this paper are as follows:(1)Simultaneous electricity generation and degradation of different aromatic pollutants by P.putida B6-2 in MFCs.The MFC constructed with P.putida B6-2 as the anode microorganisms to investigated the degradation and simultaneous electricity generation of a variety of aromatic mode pollutants by P.putida B6-2,such as biphenyl(BP),dibenzothiophene(DBT),and carbazole(CA).The experimental results show that P.putida B6-2 in MFC has a strong degradation ability to BP,DBT,and CA.When using BP as the sole substrate,P.putida B6-2 can completely degrade 462 mg L-1 BP within 200 h;and when the three mixtures(BP(462 mg L-1),DBT(55 mg L-1)and CA(50 mg L-1))are used as substrates,P.putida B6-2 can still degrade the three substances completely within 200 h.In addition,the MFCs constructed with P.putida B6-2 as anode microorganisms all have good electricity production performance.The power generation trend of the constructed MFCs is similar in the power generation cycle.The power generation cycle is about 1000 h.At the initial stage,the MFC constructed with BP as the substrate has a shorter start-up period(50 h)than the other three MFCs.During the stable period of electricity production,the peak electricity production of MFC constructed reached 473 mV with BP,DBT and CA as the substrates,followed by MFC with BP as the substrate(446 mV),and when BP and CA as the substrates the peak power generation is only 334 mV.Similar to the power generation cycle,the maximum power density of MFCs is ranked as follows:MFC with BP,DBT and CA as substrates(642 mW m-2)>MFC with BP as a substrate(550 mW m-2)>MFC with BP and DBT as substrates(484 mW m-2)>MFC with BP and CA as substrates(427 mW m-2).In summary,the MFC constructed with P.putida B6-2 can not only efficiently degrade a variety of aromatic compounds,but also realize energy recovery.(2)A preliminary study on the mechanism of EET in MFC anode was carried out.Firstly,the electrochemical activities of anodic biofilm and anodic solution were studied by electrochemical methods respectively.At the end of electricity generation,the response current of the anode biofilm did not show an obvious redox peak,indicating that P.putida B6-2 in MFC may not use redox protein for direct EET,while the chemical results of anolyte showed a pair of obvious redox peaks(-0.1 V and 0.2 V),which indicates that there are redox substances in the anolyte,and it is speculated that P.putida B6-2 in MFC may use an electron mediator to carry out indirect EET.But this pair of redox peaks can only be observed at the end of power generation.Therefore,the anodic biofilm in the stable phase was studied by SEM.SEM results show that P.B6-2 formed dense nanowire-like extracellular appendages in the anode biofilm during the stable period of electricity production.After that,the formation process of the anode biofilm on the anode material was systematically characterized by SEM,combined with the electrochemical analysis results and the entire electricity generation process analysis,it is speculated that the nanowires produced by P.putida B6-2 may be related to electricity generation.Combined with the results of electrochemical analysis,it was inferred that the existence of nanowires may be related to the electricity generation process.Finally,the conductivity of the nanowires generated by P.putida B6-2 was characterized using a conductive atomic force microscope.However,due to the destruction of the nanowires during sample processing,the generation of P.putida B6-2 could not be observed under the conductive atomic force microscope.Of nanowires,so it is impossible to directly measure its electrical conductivity.(3)The transcriptome of P.putida B6-2 was sequenced and the differential gene that may be related to extracellular electron transport were knocked out for functional exploration.P.putida B6-2 was used as the experimental strain and BP as the substrate,which was cultured under shaking flask and MFC respectively.When the MFC was in a stable discharge and the biofilm grew well and the shake flask culture was in logarithmic growth phase,P.putida B6-2 cells were collected from anodic biofilm and shake flask culture medium respectively,and the total RNA,of bacteria under two different conditions were extracted and sequenced by prokaryotic transcriptome.Based on the analysis of the results obtained,it is inferred that the extracellular appendage of nanowires of P.putida B6-2 is related to the flagellum gene fliC.After that,the flagellum gene fliC was knocked out by homologous recombination technology,and the knockout strain was obtained after screening and verification.The knockout strains and wild-type strains were cultured under the condition of MFC,and their degradation,electricity production capacity and nanowire growth were compared to judge the function of flagellum gene fliC.Finally,the complementary plasmid was constructed according to the fliC gene,and the complementary plasmid was transformed into the knockout strain to obtain the complementary strain,screened and verified,and the changes of parameters such as the electricity production performance of the complementary strain under hypoxia stress were observed,compared with the knockout strain and wild-type strain,which further confirmed that the nanowires extracellular appendage of P.putida B6-2 was related to flagella gene fliC under hypoxia stress.To sum up,P.putida B6-2 can not only achieve the degradation of a variety of aromatic pollutants in MFCs,but also achieve synchronous power generation to achieve energy recovery.In addition,by means of electrochemical analysis and scan electron microscopic characterization,it was found for the first time that P.putida B6-2 may realize EET through nanowires.Finally,transcriptome analysis and gene knockout methods were used to determine that flagellar gene fliC may be related to the extracellular appendage of nanowires of P.putida B6-2.