Role Of Microbial Extracellular Electron Transfer Mediated By Extracellular Polymeric Substances In The Reduction Of Typical Pollutants

Extracellular electron transfer(EET)is essential to information and energy exchange of microbial cells with the external environment,and plays a pivotal role in microbial dissimilatory metal reduction,conversion of organic pollutants,and sustainable bioenergy applications.Although many key details remain unresolved,EET is known to proceed via two general strategies:contact-dependent direct transfer through multiheme cytochromes or nanowires,and indirect transfer through dissolved redox-active electron-shuttling mediators that are produced by bacteria or naturally present substances.Most microbial cells are enveloped by extracellular polymeric substance(EPS).EPS play a key role in cell surface attachment and microhabitat formation to offer resistance to desiccation,antibiotics,high salinity,and extreme temperature and p H conditions.EPS is an important interface medium for material and energy exchange of microbial cells with the external environments or with other cells.Hence,it is essential to identify the functional components in EPS(exfoliated from bacteria and in intact cells)responsible for extracellular electron transfer,and elucidated the underlying mechanisms for the microbial extracellular reduction,which provides valuable insight on the possible impact of microbial EPS on biogeochemical cycling and the detoxification of high-oxidation-state pollutants in the environment.The role of EPS in redox transformation of high-oxidation-state pollutants by bacteria is not well understood.Here,nitroaromatic compounds(NACs),arsenate and selenate are chosen acting as probing pollutants,and the tested bacterial strains are Gram-positive Bacillus subtilis(B.subtilis)and Gram-negative Escherichia coli DH5?(E.coli).Using batch reaction experiments and an array of chemical,electrochemical,and biological analyses integrated with spectroscopic analyses,we determined the functional components in EPS exfoliated from bacteria and in intact cells responsible for the reduction of probing pollutants,and further elucidated the underlying mechanisms for the nonenzymatic extracellular reductive reaction.For the reduction of different probing pollutants,EPS act as different roles,such as reducing agent,electron shuttles and permeability barrier.The main findings achieved are summarized as follows:(1)The functional components in EPS exfoliated from bacteria and in intact cells responsible for the reduction of NACs were identified and further elucidated the underlying mechanisms for the nonenzymatic extracellular reductive reaction.The results of batch experiments showed that a nitroaromatic compound(1,3-dinitrobenzene)can be readily reduced to 3-hydroxylaminonitrobenzene and 3-nitroaniline in aqueous suspension of common bacteria(E.coli or B.subtilis)or in aqueous dissolved EPS extracted from the bacteria.The loss ratio of 1,3-dinitrobenzene by E.coli was unaffected after knocking out the nfs A gene encoding nitroreductase,but was suppressed by removing EPS attached to cells.In contrast,the loss ratio was enhanced by adding aqueous dissolved EPS to E.coli or B.subtilis suspension.The residual 1,3-dinitrobenzene and products formed after reduction were only presented outside the bacterial cells.Thus,bacterial reduction of 1,3-dinitrobenzene was mediated by nonenzymatic extracellular reduction.This was further corroborated by the observation that the stoichiometric demand of electrons in 1,3-dinitrobenzene reduction was nearly equal to the quantity of electrons donated by bacterial cells in the electrochemical cell experiment.Inhibition on the reduction of 1,3-dinitrobenzene by chemical probes combined with fluorescence detection demonstrated that reducing sugars in EPS might act as electron donors,while cytochromes and some low-molecular weight molecules(flavins and quinones)were involved as electron transfer mediators.(2)Linear relationships were observed between the reduction kinetics and the one-electron reduction potentials for a series of substituted dinitrobenzenes in the presence of bacterial cells or dissolved EPS.Their close linear regression slope values suggest that the extracellular matrix and the exfoliated EPS utilized the same reducing agents(likely hydroquinones and reduced flavins)as terminal electron donors to reduce NACs.In addition,the difference of apparent kinetic isotope effect for 1,3-DNB reduction between B.subtilis cells and B.subtilis EPS could be due to the obviously more enriched and diversified electron transfer mediators in the extracellular matrix of living cells.These results reveal a previously unrecognized mechanism for nonenzymatic extracellular reduction of NACs by common bacteria.(3)The role of EPS in bacterial reduction of arsenate was confirmed in this research.The results show that arsenate can be readily reduced to arsenite on cell surfaces of common bacteria(E.coli or B.subtilis)or in aqueous dissolved EPS extracted from different microorganisms(E.coli,B.subtilis,P.chrysosporium,D.gigas,and a natural biofilm)in the absence of exogenous electron donors.The efficiency of arsenate reduction by E.coli after 7-h incubation was only moderately reduced after knocking out the arsenic resistance genes(ars B and ars C).Most of the reduced arsenite was present outside the bacterial cells,including for the E.coli blocked mutant lacking ars B and ars C.Thus,extracellular processes dominated arsenate reduction.Arsenate reduction was facilitated by removing EPS attached to E.coli or B.subtilis,which was attributed to enhanced access to reduced extracellular cytochromes.This highlights the role of EPS as a permeability barrier to arsenate reduction.Fourier-transform infrared(FTIR)combined with other chemical analyses implicated some low-molecular weight(<3 k Da)molecules as electron donors(reducing saccharides)and electron transfer mediators(quinones)in arsenate reduction by dissolved EPS alone.These results indicate that EPS act as both reducing agent and permeability barrier for access to reduced biomolecules in bacterial reduction of arsenate.(4)In the absence of exogenous electron donors,selenate(Se O₄^2-)was readily reduced to Se⁰ nanoparticles(Se NPs)in aqueous suspension of Gram-negative bacteria(Escherichia coli),Gram-positive bacteria(Bacillus subtilis)or dissolved EPS extracted from Bacillus subtilis.The majority of Se NPs was formed outside cells and immobilized by EPS.Selenate reduction was facilitated by adding dissolved EPS and was weakened by removing EPS attached to bacterial cells.This highlights the role of EPS as reducing agent to selenate reduction.Batch experiments implicate some low-molecular weight(<3 k Da)molecules dominanted selenate reduction by dissolved EPS,indicating the crucial role of EPS in the reduction of selenate.The results highlight the possible impact of microbial EPS on biogeochemical cycling and the detoxification of selenium in the environment.