Spatially Resolved Confocal Resonant Raman Microscopic Analysis of Anode‐Grown Geobacter sulfurreducens Biofilms

Abstract

When grown on the surface of an anode electrode, Geobacter sulfurreducens forms a multi-cell thick biofilm in which all cells appear to couple the oxidation of acetate with electron transport to the anode, which serves as the terminal metabolic electron acceptor. Just how electrons are transported through such a biofilm from cells to the underlying anode surface over distances that can exceed 20 microns remains unresolved. Current evidence suggests it may occur by electron hopping through a proposed network of redox cofactors composed of immobile outer membrane and/or extracellular multi-heme c-type cytochromes. In the present work, we perform a spatially resolved confocal resonant Raman (CRR) microscopic analysis to investigate anode-grown Geobacter biofilms. The results confirm the presence of an intra-biofilm redox gradient whereby the probability that a heme is in the reduced state increases with increasing distance from the anode surface. Such a gradient is required to drive electron transport toward the anode surface by electron hopping via cytochromes. The results also indicate that at open circuit, when electrons are expected to accumulate in redox cofactors involved in electron transport due to the inability of the anode to accept electrons, nearly all c-type cytochrome hemes detected in the biofilm are oxidized. The same outcome occurs when a comparable potential to that measured at open circuit (-0.30 V vs. SHE) is applied to the anode, whereas nearly all hemes are reduced when an exceedingly negative potential (-0.50 V vs. SHE) is applied to the anode. These results suggest that nearly all c-type cytochrome hemes detected in the biofilm can be electrochemically accessed by the electrode, but most have oxidation potentials too negative to transport electrons originating from acetate metabolism. The results also reveal a lateral heterogeneity (x-y dimensions) in the type of c-type cytochromes within the biofilm that may affect electron transport to the electrode.

A virus-based nanoplasmonic structure as a surface-enhanced Raman biosensor

“…The optimum conditions were found to be 1 μl of a NC solution of $ 0.2 OD at 535 nm. Raman spectra were collected with a Renishaw in via Raman microscope (Hoffman Estates, IL) with 100 Â objective and laser light 785 nm (with pinhole) (Lebedev et al 2014b). The excitation energy was kept below 0.06 mW.…”

Electron Transport through Early Exponential‐Phase Anode‐Grown Geobacter sulfurreducens Biofilms

“…4 G. sulfurreducens cells possess an abundance of multi‐heme c ‐type cytochromes ( c ‐Cyt) on their outer membrane, in the extracellular polymeric substance (EPS), and along certain protein filaments (pili) contained in the EPS 1g. In the case of stationary phase G. sulfurreducens biofilms (full‐grown biofilms that have achieved a maximum thickness and current), a substantial body of published data supports the hypothesis that EET occurs by redox conduction (also referred to as incoherent multistep hopping),5 in which c ‐Cyt hemes act as electron‐transport cofactors 2c. 4, 5d, 6 Redox conduction occurs through a sequence of discrete electron‐transfer reactions between neighboring reduced and oxidized cofactors in a bucket brigade manner, requiring a redox gradient (spatial variation in cofactor oxidation state—portion of cofactors in oxidized vs. reduced state) to drive electron transport in a given direction 5a.…”

Potential regulates metabolism and extracellular respiration of electroactive Geobacter biofilm

“…The electrochemical activity of G. sulfurreducens is promoted by forming biofilms and aggregates, where the extracellular electron transfer proteins in matrix may be shared by nearby cells as common electron relays (Reguera, Pollina, Nicoll, & Lovley, ; Rollefson, Stephen, Tien, & Bond, ; Virdis, Millo, Donose, & Batstone, ; Yates et al, ; Yates et al, ). The common electron transfer network provides a convenient mechanism to coordinate global microbial metabolism in biofilms (Allan et al, ; Lebedev, Strycharz‐Glaven, & Tender, ; Schrott, Ordonez, Robuschi, & Busalmen, ). As a result, G. sulfurreducens biofilms can be acclimated by conditions to produce microbial current of several times higher on electrodes and sequestrate more uranium than planktonic cells (Cologgi, Speers, Bullard, Kelly, & Reguera, ; Jiang et al, ).…”

“…Electrochemical potential is a typical variable affecting the current production and redox state of cyt c in G. sulfurreducens biofilms (Lebedev et al, ; Robuschi et al, ; Torres et al, ; Wei, Liang, Cao, & Huang, ). Current production by G. sulfurreducens was found to vary with the potential between −0.4 and +0.8 V versus standard hydrogen electrode (SHE) and the maximum current was reached at +0.2 V (Li et al, ; Rimboud, Pocaznoi, Erable, & Bergel, ; Wagner, Call, & Logan, ; Zhu, Yates, & Logan, ).…”