Disruption of the Putative Cell Surface Polysaccharide Biosynthesis Gene SO3177 in Shewanella oneidensis MR-1 Enhances Adhesion to Electrodes and Current Generation in Microbial Fuel Cells

Abstract

A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an increased number of colonies exhibiting rough morphology. One such isolate, strain 4A, generated 50% more current in an MFC than wild-type MR-1. Determination of the transposon insertion site in strain 4A followed by deletion and complementation experiments revealed that the SO3177 gene, encoding a putative formyltransferase and situated in a cell surface polysaccharide biosynthesis gene cluster, was responsible for the increased current. Transmission electron microscopy showed that a layered structure at the cell surface, stainable with ruthenium red, was impaired in the SO3177 mutant (⌬SO3177), confirming that SO3177 is involved in the biosynthesis of cell surface polysaccharides. Compared to the wild type, ⌬SO3177 cells preferentially attached to graphite felt anodes in MFCs, while physicochemical analyses revealed that the cell surface of ⌬SO3177 was more hydrophobic. These results demonstrate that cell surface polysaccharides affect not only the cell adhesion to graphite anodes but also the current generation in MFCs.Dissimilatory metal-reducing bacteria (DMRB) conserve energy for growth by coupling the oxidation of organic compounds to the reduction of metal compounds (29). DMRB are of great interest not only for their importance in the biogeochemical cycling of metals (25) but also for their utility in biotechnological processes, such as microbial fuel cells (MFCs) (24,40). In recent years, the ability of many DMRB, including members of the genera Shewanella (5,12,20,31), Geobacter (2), Aeromonas (34), Desulfobulbus (19), and Phodoferax (9), to generate current in MFCs has been described.Among DMRB, Shewanella oneidensis MR-1 is one of the most extensively studied due to its metabolic versatility (28), annotated genome sequence (17), and genetic accessibility. In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to an anode without an exogenously added mediator (20), it has become a model organism for the study of microbial current generation in MFCs. Extensive studies have been performed to understand the mechanisms of extracellular electron transfer (EET) to solid materials, such as MFC anodes and metal oxides, in strain MR-1. Multiple mechanisms, including direct EET through the physical contact of bacterial cells via outer membrane (OM) cytochromes (42) and conductive nanowires (16) and mediated EET via self-produced electron shuttles such as quinones and flavins (27,30,39,41), have been identified.Although OM cytochromes and electron shuttles have been identified to play central roles in EET, it is reasonable to speculate that this complex catabolic process is also influenced by other (extra)cellular components. To date, only limited studies hav...

Disruption of Putrescine Biosynthesis in Shewanella oneidensis Enhances Biofilm Cohesiveness and Performance in Cr(VI) Immobilization

“…Comparative analyses of the abilities of several different Shewanella strains to adhere to hematite (␣-Fe 2 O 3 ) revealed that strains with more hydrophobic and electronegative cell surfaces exhibited better adhesion (42). In addition, Shewanella cells with a more hydrophobic and electronegative surface have been shown to enhance not only cell adhesion to graphite electrodes but also cell-cell adhesion as evidenced by strong autoaggregation (25). The hyperadherent phenotype of CP2-1-S1 suggests that its cell surface physicochemical properties, such as charge and hydrophobicity, might have been altered.…”

“…Previous studies demonstrated that cell surface charges and hydrophobicity play an important role in cell adhesion to biotic and abiotic surfaces (25,41,42). Hydrophobic interactions have been shown to be an important factor controlling the adhesion of Shewanella cells to amorphous Fe(III) oxide (41).…”

“…Transposon mutagenesis of S. oneidensis MR-1 was carried out through filter mating with Escherichia coli harboring TnM using the helper strain RK600 according to previously described protocols (25)(26)(27). LB medium was used for bacterial growth, and when necessary, the medium was supplemented with 25 g/ml of kanamycin, 15 g/ml of tetracycline, 10 g/ml of gentamicin, and/or 6 g/ml of chloramphenicol.…”

Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

“…The suspension was centrifuged and the cells were then washed with DM before use in electrochemical experiments. Mutant strains of MR-1 lacking complete genes for MtrC and SO3177 were previously constructed by allele replacement using a two-step homologous recombination method (28,37). The growth procedures for the two mutant strains were the same as described for the WT MR-1 strain.…”

Spectroelectrochemical Investigation on Biological Electron Transfer Associated with Anode Performance in Microbial Fuel Cells

“…To confirm the involvement of heme molecules in the OM c-Cyts proteins of MR-1 cells in the observed EET reaction between bacteria and electrodes, we examined mutant strains with inframe deletions of the genes encoding MtrA, MtrB, MtrC, OmcA, and CymA. Each mutant was constructed by allele replacement using a two-step homologous recombination method, as previously reported (Saltikov & Newman 2003;Kouzuma et al, 2010).…”