scholarly journals Evidence of streamlined extracellular electron transfer pathway from biofilm structure, metabolic stratification, and long-range electron transfer parameters.

2021 ◽  
Author(s):  
Fernanda Jiménez Otero ◽  
Grayson L. Chadwick ◽  
Matthew D. Yates ◽  
Rebecca L. Mickol ◽  
Scott H. Saunders ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Transfer ◽  
Current Density ◽  
Geobacter Sulfurreducens ◽  
Wild Type ◽  
Electron Diffusion ◽  
Electrochemical Systems ◽  
Anabolic Activity ◽  
Current Production ◽  
Transfer Parameters

A strain of Geobacter sulfurreducens , an organism capable of respiring solid extracellular substrates, lacking four out of five outer membrane cytochrome complexes (strain extABCD + ) grows faster and produces greater current density compared to wild type grown under identical conditions. To understand cellular and biofilm modifications altered in extABCD + responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild type and extABCD + biofilms spanning 5 μm gaps. Our results reveal that extABCD + biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to wild type (based on increased rates of 15 N incorporation). We also observed an increased rate of electron transfer through extABCD + vs. wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multi-heme cytochromes streamlines electron transfer to electrodes. The combination of imaging, physiological and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than wild type. Importance Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4x higher current compared to wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

scholarly journals Unexpected Genomic Features of High Current Density-Producing Geobacter sulfurreducens strain YM18

2021 ◽  
Author(s):  
Takashi Fujikawa ◽  
Yoshitoshi Ogura ◽  
Koki Ishigami ◽  
Yoshihiro Kawano ◽  
Miyuki Nagamine ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Current Density ◽  
Iron Reduction ◽  
High Current Density ◽  
Model Organism ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
High Current ◽  
Current Production ◽  
Current Densities

Abstract Geobacter sulfurreducens produces high current densities and it has been used as a model organism for extracellular electron transfer studies. Nine G. sulfurreducens strains were isolated from biofilms formed on an anode poised at –0.2 V (vs. SHE) in a bioelectrochemical system in which river sediment was used as an inoculum. The maximum current density of an isolate, strain YM18 (9.29 A/m2), was higher than that of the strains PCA (5.72 A/m2), the type strain of G. sulfurreducens, and comparable to strain KN400 (8.38 A/m2), which is another high current producing strain of G. sulfurreducens. Genomic comparison of strains PCA, KN400, and YM18 revealed that omcB, xapD, spc, and ompJ, which are known to be important genes for iron reduction and current production in PCA, were not present in YM18. In the PCA and KN400 genomes, two and one region (s) encoding CRISPR/Cas systems were identified, respectively, but they were missing in the YM18 genome. These results indicate that there is genetic variation in the key components involved in extracellular electron transfer among G. sulfurreducens strains.

scholarly journals Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III) oxide, Mn(IV) oxide, or electrode reduction by Geobacter sulfurreducens

2017 ◽  
Author(s):  
Fernanda Jiménez Otero ◽  
Chi Ho Chan ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Outer Membrane ◽  
Electron Acceptor ◽  
Single Gene ◽  
Gene Clusters ◽  
Geobacter Sulfurreducens ◽  
Transcriptional Analysis ◽  
Growth Defect ◽  
Redox Potentials ◽  
Wild Type

AbstractAt least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and −0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an ΔomcBC background, such as extEFG, were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens, depending on the available extracellular electron acceptor.

scholarly journals Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III)-oxide, Mn(IV)-oxide, or electrode reduction by Geobacter sulfurreducens .

2018 ◽  
Author(s):  
Fernanda Jiménez Otero ◽  
Chi Ho Chan ◽  
Daniel R Bond
Keyword(s):  
Electron Transfer ◽  
Outer Membrane ◽  
Electron Acceptor ◽  
Single Gene ◽  
Gene Clusters ◽  
Geobacter Sulfurreducens ◽  
Transcriptional Analysis ◽  
Growth Defect ◽  
Redox Potentials ◽  
Wild Type

At least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c -type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and -0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an Δ omcBC background, such as extEFG , were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the Δ extABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens , depending on the available extracellular electron acceptor.

scholarly journals Biofilm and Nanowire Production Leads to Increased Current in Geobacter sulfurreducens Fuel Cells

2006 ◽  
Vol 72 (11) ◽  
pp. 7345-7348 ◽  
Author(s):  
Gemma Reguera ◽  
Kelly P. Nevin ◽  
Julie S. Nicoll ◽  
Sean F. Covalla ◽  
Trevor L. Woodard ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Energy Efficient ◽  
Geobacter Sulfurreducens ◽  
Electricity Production ◽  
Anode Surface ◽  
Efficient Manner ◽  
Electrically Conductive ◽  
Genetic Studies ◽  
Electronic Network ◽  
Current Production

ABSTRACT Geobacter sulfurreducens developed highly structured, multilayer biofilms on the anode surface of a microbial fuel cell converting acetate to electricity. Cells at a distance from the anode remained viable, and there was no decrease in the efficiency of current production as the thickness of the biofilm increased. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili. These pili may represent an electronic network permeating the biofilm that can promote long-range electrical transfer in an energy-efficient manner, increasing electricity production more than 10-fold.

scholarly journals Identification of Different Putative Outer Membrane Electron Conduits Necessary for Fe(III) Citrate, Fe(III) Oxide, Mn(IV) Oxide, or Electrode Reduction byGeobacter sulfurreducens

2018 ◽  
Vol 200 (19) ◽  
Author(s):  
Fernanda Jiménez Otero ◽  
Chi Ho Chan ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Outer Membrane ◽  
Electron Acceptor ◽  
Gene Clusters ◽  
Geobacter Sulfurreducens ◽  
Transcriptional Analysis ◽  
Metal Reduction ◽  
Wild Type ◽  
Redox Proteins ◽  
Content Type

ABSTRACTAt least five gene clusters in theGeobacter sulfurreducensgenome encode putative “electron conduits” implicated in electron transfer across the outer membrane, each containing a periplasmic multihemec-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single-gene-cluster deletions and all possible multiple-deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III) and Mn(IV) oxides, and graphite electrodes poised at +0.24 V and −0.1 V versus the standard hydrogen electrode (SHE). Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously describedomcBCcluster caused defects, but deletion of additional components in an ΔomcBCbackground, such asextEFG, were needed to produce defects greater than 50% compared to findings with the wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCDmutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III) oxide, Mn(IV) oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than the wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth conditions showed all of theseextclusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing onlyextABCDdetected no significant changes in expression of genes encoding known redox proteins or pilus components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth ofG. sulfurreducens, depending on the available extracellular electron acceptor.IMPORTANCEGram-negative metal-reducing bacteria utilize electron conduits, chains of redox proteins spanning the outer membrane, to transfer electrons to the extracellular surface. Only one pathway for electron transfer across the outer membrane ofGeobacter sulfurreducenshas been linked to Fe(III) reduction. However,G. sulfurreducensis able to respire a wide array of extracellular substrates. Here we present the first combinatorial genetic analysis of five different electron conduits via creation of new markerless deletion strains and complementation vectors. Multiple conduit gene clusters appear to have overlapping roles, including two that have never been linked to metal reduction. Another recently described cluster (ExtABCD) was the only electron conduit essential during electrode reduction, a substrate of special importance to biotechnological applications of this organism.

scholarly journals The electrically conductive pili of Geobacter soli

2020 ◽  
Author(s):  
Shiyan Zhuo ◽  
Guiqin Yang ◽  
Li Zhuang
Keyword(s):  
Electron Transfer ◽  
Outer Surface ◽  
Electronic Materials ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Oxide Reduction ◽  
Electrically Conductive ◽  
Current Production ◽  
Current Densities ◽  
Pilin Gene

AbstractElectrically conductive pili (e-pili) enable electron transport over multiple cell lengths to extracellular environments and play an important role in extracellular electron transfer (EET) of Geobacter species. To date, the studies of e-pili have mainly focused on Geobacter sulfurreducens and the closely related Geobacter metallireducens because of their developed genetic manipulation systems. We investigated the role of G. soli pili in EET by directly deleting the pilin gene, pilA, which is predicted to encode e-pili. Deletion of pilA, prevented the production of pili, resulting in poor Fe(III) oxide reduction and low current production, implying that G. soli pili is required for EET. To further evaluate the conductivity of G. soli pili compared with G. sulfurreducens pili, the pilA of G. soli was heterologously expressed in G. sulfurreducens, yielding the G. sulfurreducens strain GSP. This strain produced abundant pili with similar conductivity to the control strain that expressed native G. sulfurreducens pili, consistent with G. soli as determined by direct measurement, which suggested that G. soli pili is electrically conductive. Surprisingly, strain GSP was deficient in Fe(III) oxide reduction and current production due to the impaired content of outer-surface c-type cytochromes. These results demonstrated that heterologous pili of G. sulfurreducens severely reduces the content of outer-surface c-type cytochromes and consequently eliminates the capacity for EET, which strongly suggests an attention should be paid to the content of c-type cytochromes when employing G. sulfurreducens to heterologously express pili from other microorganisms.IMPORTANCEThe studies of electrically conductive pili (e-pili) of Geobacter species are of interest because of its application prospects in electronic materials. e-Pili are considered a substitution for electronic materials due to its renewability, biodegradability and robustness. Continued exploration of additional e-pili of Geobacter soli will improve the understanding of their biological role in extracellular electron transfer and expand the range of available electronic materials. Heterologously expressing the pilin genes from phylogenetically diverse microorganisms has been proposed as an emerging approach to screen potential e-pili according to high current densities. However, our results indicated that a Geobacter sulfurreducens strain heterologously expressing a pilin gene produced low current densities that resulted from a lack of content of c-type cytochromes, which were likely to possess e-pili. These results provide referential significance to yield e-pili from diverse microorganisms.

scholarly journals A Geobacter sulfurreducens Strain Expressing Pseudomonas aeruginosa Type IV Pili Localizes OmcS on Pili but Is Deficient in Fe(III) Oxide Reduction and Current Production

2013 ◽  
Vol 80 (3) ◽  
pp. 1219-1224 ◽  
Author(s):  
Xing Liu ◽  
Pier-Luc Tremblay ◽  
Nikhil S. Malvankar ◽  
Kelly P. Nevin ◽  
Derek R. Lovley ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Structural Features ◽  
Geobacter Sulfurreducens ◽  
Control Strain ◽  
Oxide Reduction ◽  
Content Type ◽  
Type Iv ◽  
Current Production ◽  
Pilin Protein

ABSTRACTThe conductive pili ofGeobacterspecies play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili ofGeobacter sulfurreducenshave a metal-like conductivity, independent of the presence ofc-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain ofG. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene ofPseudomonas aeruginosaPAO1. Strain PA expressed and properly assembledP. aeruginosaPilA subunits into pili and exhibited a profile of outer surfacec-type cytochromes similar to that of a control strain expressing theG. sulfurreducensPilA. Surprisingly, the strain PA pili were decorated with thec-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of theG. sulfurreducensPilA that are necessary for conductivity.

scholarly journals Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens

2021 ◽  
Author(s):  
Xinying Liu ◽  
David Jeffrey Fraser Walker ◽  
Stephen Nonnenmann ◽  
Dezhi Sun ◽  
Derek R. Lovley
Keyword(s):  
Electron Transfer ◽  
Electron Transport ◽  
Long Range ◽  
Geobacter Sulfurreducens ◽  
Metal Corrosion ◽  
Extracellular Electron Transfer ◽  
Wild Type ◽  
Electrically Conductive ◽  
In The Wild ◽  
Pilin Gene

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c-type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90 % of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in E. coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c-type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90 % filaments with a 3 nm diameter, but greatly reduced conductance, further indicating that the 3 nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c-type cytochromes, including OmcS, was deleted yielded only 3 nm diameter filaments with the same conductance as in the wild-type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer.

scholarly journals Interspecies Electron Transfer via Hydrogen and Formate Rather than Direct Electrical Connections in Cocultures of Pelobacter carbinolicus and Geobacter sulfurreducens

2012 ◽  
Vol 78 (21) ◽  
pp. 7645-7651 ◽  
Author(s):  
Amelia-Elena Rotaru ◽  
Pravin M. Shrestha ◽  
Fanghua Liu ◽  
Toshiyuki Ueki ◽  
Kelly Nevin ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Formate Dehydrogenase ◽  
Geobacter Sulfurreducens ◽  
Close Relative ◽  
Wild Type ◽  
Geobacter Metallireducens ◽  
Content Type ◽  
Interspecies Electron Transfer ◽  
Essential Components ◽  
Electrical Connections

ABSTRACTDirect interspecies electron transfer (DIET) is an alternative to interspecies H2/formate transfer as a mechanism for microbial species to cooperatively exchange electrons during syntrophic metabolism. To understand what specific properties contribute to DIET, studies were conducted withPelobacter carbinolicus, a close relative ofGeobacter metallireducens, which is capable of DIET.P. carbinolicusgrew in coculture withGeobacter sulfurreducenswith ethanol as the electron donor and fumarate as the electron acceptor, conditions under whichG. sulfurreducensformed direct electrical connections withG. metallireducens. In contrast to the cell aggregation associated with DIET,P. carbinolicusandG. sulfurreducensdid not aggregate. Attempts to initiate cocultures with a genetically modified strain ofG. sulfurreducensincapable of both H2and formate utilization were unsuccessful, whereas cocultures readily grew with mutant strains capable of formate but not H2uptake or vice versa. The hydrogenase mutant ofG. sulfurreducenscompensated, in cocultures, with significantly increased formate dehydrogenase gene expression. In contrast, the transcript abundance of a hydrogenase gene was comparable in cocultures with that for the formate dehydrogenase mutant ofG. sulfurreducensor the wild type, suggesting that H2was the primary electron carrier in the wild-type cocultures. Cocultures were also initiated with strains ofG. sulfurreducensthat could not produce pili or OmcS, two essential components for DIET. The finding thatP. carbinolicusexchanged electrons withG. sulfurreducensvia interspecies transfer of H2/formate rather than DIET demonstrates that not all microorganisms that can grow syntrophically are capable of DIET and that closely related microorganisms may use significantly different strategies for interspecies electron exchange.

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