Extracellular Electron Transfer Mediated By Cysteine Of The Gram-positive Bacterium Lysinibacillus Varians GY32

Bacterial extracellular electron transfer(EET)is the process by which bacteria transfer electrons generated by intracellular metabolism to extracellular electron receptors.It is an important driving force for material metabolism,element cycling,energy conversion and other processes in natural environment.The bioelectrochemical technology based on microbial EET has shown great application potential in pollutant degradation,environmental remediation,healthcare,etc.The current research on EET is mainly based on Gram-negative bacteria(such as Geobacter and Shewanella),while there is little understanding of EET mechanisms of Gram-positive bacteria,which limits the cognition and application of the microbial electron transfer mechanisms.Due to the difference between the cell walls of Gram-positive bacteria and negative bacteria,the EET mechanisms are very different.At present,the EET of Gram-positive bacteria has been found mainly in the following two ways:(1)EET based on electron mediators;(2)EET based on c-type cytochromes,and there is little understanding of other EET mechanisms.Lysinibacillus varians GY32 is a Gram-positive bacterium isolated from e-waste-contaminated river sediment.The length of a single cell can exceed 1 mm,and the electrode can be used as an electron acceptor for EET.This study mainly focused on the types of metabolic substrates,gene expression characteristics,and electron transfer mechanisms of strain GY32 in the EET process.The main results and conclusions were as follows:(1)Strain GY32 could use a variety of small-molecule organic acids and amino acids that are ubiquitous in the environment as electron donors,and electrode and ferric iron as extracellular electron acceptors for EET.The electrode respiration characteristics of strain GY32 were studied by microbial fuel cells(MFC).It was found that strain GY32 could utilize formic acid,acetic acid and 8 amino acids(histidine,glycine,serine,glutamic acid,glutamine,leucine acid,isoleucine,asparagine)as the substrates for electrode respiration.In the electricity generation process of MFC,the longfilament GY32 cells were interwoven into a unique centimeter-scale conductive cell network around the electrode.In addition,the strain GY32 could use acetic acid for ferric iron reduction.The broad-spectrum electron donor source and special long-filament cell structure of strain GY32 indicate that it may perform an EET function in the ecological environment and make an important impact on surrounding microbial communities and the transformation of minerals and organic matter.(2)Strain GY32 synthesized a small quantity of c-type cytochromes,possessed nanowire appendages,and secreted electron mediators under the condition of electrode respiration.It had a variety of potential EET pathways,among which the indirect electron transfer based on electron mediators was the main one.Although there were 6 c-type cytochrome genes in the genome of strain GY32 and their expression levels increased under the condition of MFC electrode respiration,these c-type cytochromes were not detected in the cellular outer layer by many methods such as visible spectroscopy,Raman spectroscopy,and c-type cytochrome localization analysis.It is speculated that c-type cytochromes are difficult to form a complete transmembrane EET pathway,and other substances are needed to assist in completing the EET process of GY32.The electrochemical analysis of the substances in the electricity generation process of GY32 showed that the effect of GY32 culture supernatant accounted for 80% of the current output,but that flavin electron mediators could not promote the electricity generation of GY32,indicating that other substances mainly mediated its EET process.In addition,GY32 could synthesize a variety of linear extracellular appendages with different morphologies,including nanowires with conductive property.The fimbriae gene analysis showed that the protein sequence contained up to 13.8% aromatic amino acids,suggesting that it may be the nanowire with the most conductive potential.(3)Combined omics analysis and bioelectrochemical test showed that strain GY32 could secrete Cys as an electron mediator for EET,revealing a novel bacterial EET pathway mediated by Cys.By comparing the transcriptome and proteome of strain GY32 under the two conditions of aerobic culture and electrode respiration,it was found that the expression levels of heme-related genes were significantly up-regulated under the condition of electrode respiration,including two c-type cytochromes,suggesting that these c-type cytochromes participated in the EET process of GY32.In addition,under the condition of electrode respiration,the expression levels of the genes in flavin synthesis pathway were down-regulated,which was consistent with the experiment result that flavin substances were not involved in the EET of GY32.Interestingly,during the electrode respiration process,the expression levels of Cys synthesis-related proteins were significantly up-regulated.Further metabolite analysis and electrochemical analysis showed that GY32 could secrete and use Cys as an electron mediator for electrode respiration during the electricity generation process.(4)Strain GY32 could significantly promote the electrical activity of Shewanella decolorationis S12 and the sediment microbial community.By co-culture with Shewanella decolorationis S12,it was found that GY32 could increase current density of the MFC and improve coulombic efficiency of the system by 1.5 times.By constructing the mutant of S12 to compare the electrochemical activity,it was found that Cys may be the key substance to improve the electrochemical performance of the co-culture system.The output current of the sediment microbial fuel cell(SMFC)constructed from contaminated sediment could be effectively increased by adding GY32.The abundance of GY32 gradually increased toward the electrode.This study revealed the EET process and related mechanisms of the Gram-positive bacterium L.varians GY32 with the electrode and other electroactive bacteria mediated mainly by the secretion of Cys.The results expand the knowledge of EET pathways of electroactive microorganisms,and provide an important reference for comprehensive understanding of the EET network of environmental bacteria and its action mechanisms.