Project description:Microtoming Coupled with Microarray Analysis to Evaluate Potential Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in Anode Biofilms Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in A Current Producing Biofilm Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. In order to further compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, mature anode biofilms were treated to stop turnover over of mRNA and then encased in resin which was sectioned into 100 nm shavings with a diamond knife and pooled into inner (0-20 µm from anode surface) and outer (30-60 µm) fractions. Whole genome DNA microarray analysis of RNA extracted from the shavings revealed that, at a 2-fold lower threshold, there were 146 genes that had significant (p<0.05), differences in transcript abundance between the inner and outer portions of the biofilm. Only 1 gene, GSU0093, a hypothetical ABC transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting that cells in the outer biofilm had lower metabolic rates. However, the differences in transcript abundance were relatively low (<3-fold) and the outer biofilm did not have significantly lower expression of the genes for TCA cycle enzymes which previous studies have demonstrated are sensitive indicators of changes in rates of metabolism in G. sulfurreducens. There also was no significant difference in the transcript levels for outer-surface cell components thought to be important in electron transfer in anode biofilms. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of the metabolic staining and gene expression studies suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms.

Project description:Microtoming Coupled with Microarray Analysis to Evaluate Potential Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in Anode Biofilms Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in A Current Producing Biofilm Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. In order to further compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, mature anode biofilms were treated to stop turnover over of mRNA and then encased in resin which was sectioned into 100 nm shavings with a diamond knife and pooled into inner (0-20 µm from anode surface) and outer (30-60 µm) fractions. Whole genome DNA microarray analysis of RNA extracted from the shavings revealed that, at a 2-fold lower threshold, there were 146 genes that had significant (p<0.05), differences in transcript abundance between the inner and outer portions of the biofilm. Only 1 gene, GSU0093, a hypothetical ABC transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting that cells in the outer biofilm had lower metabolic rates. However, the differences in transcript abundance were relatively low (<3-fold) and the outer biofilm did not have significantly lower expression of the genes for TCA cycle enzymes which previous studies have demonstrated are sensitive indicators of changes in rates of metabolism in G. sulfurreducens. There also was no significant difference in the transcript levels for outer-surface cell components thought to be important in electron transfer in anode biofilms. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of the metabolic staining and gene expression studies suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms. Three biological replicates were hybridized in triplicate on a coustom affimetrix tilling array using prokaryotic protocol (p69Affy, p75 Adobe) for labeling, hybridization and scanning.

Project description:G. sulfurreducens can generate electricity from the oxidation of organic compounds. This is because it can take electrons from organic compounds and ship them out to the outer surface of the cell where they can then be deposited on various insoluble electron acceptors including electrodes. Cells attatched to the surface of an electrode oxidize acetate and and deposit the electrons derived from acetate onto the surface of the electrode after which they can travel through an electrical circuit, producing a current. Microbial fuel cells powered by acetate oxidation by Geobacter species are called Geobatteries. In this experiment we compared gene expression in a biofilm of the wild type strain growing on the surface of an electrode within a current-producing Geobattery to gene expression in a wild type biofilm that is not producing current, but is growing on the surface of an electrode. In both cases, the cells were growing in a flow-through two chambered H-cell Geobattery setup. This consists of two glass chambers, an anoxic anode chamber containing G. sulfurreducens, a graphite electrode, a reference electrode and growth medium and an oxic chamber containing the counter electrode. The two chambers are connected by a cation selective membrane and a wire connected to a potentionstat. A potentiostat is an instrument which maintains the redox potential of the anode at a fixed value relative to a reference electrode. Media continuously flowed through the anoxic anode chamber at a dilution rate of 0.15. In the experimental condition, the Geobattery was operational. The circuit was closed and G. sulfurreducens attached to the electrode generated current as it oxidized acetate. The redox potential at the anode was poised at 300 mV by the potentiostat. In the control condition, everything was the same, except that the medium in the anode chamber contained fumarate as electron acceptor, and the anode was not hooked up to the potentiostat i.e. the circuit was open. This prevented the anode from serving as an electron acceptor. Nevertheless a thick G. sulfurreducens biofilm grew on the surface of the electrode. The control and experimental geobatteries were harvested when current in the operational/experimental Geobatteries reached 10 mA. Keywords: two condition comparison

Project description:The conductive pili of Geobacter sulfurreducens are essential for optimal extracellular electron transfer to Fe(III) and long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene for PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, slow rates of Fe(III) reduction were detected after extended (> 30 days) incubation in the presence of Fe(III) oxide. After seven consecutive transfers the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, proteomic, and gene deletion studies indicated that this adaptation was associated with greater production of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every three days, the wild-type strain out-competed the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron-shuttling producing Fe(III) reducers in most soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current; consistent with the concept that long-range electron transport through G. sulfurreducens biofilms cannot be achieved without PilA-pili. An eight-chip study using total RNA recovered from four separate cultures of Geobacter sulfurreducens JS-1 (experimental condition) or Geobacter sulfurreducens KN400 (control condition) grown with acetate (10mM)-Fe(III) oxide (100 mmol l-1) exponential growth. Each chip measures the expression level of 3,328 genes from Geobacter sulfurreducens KN400 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

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Geobacter sulfurreducens

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