Biocathode extracellular electron transfer (EET) may be exploited for biotechnology applications, including microbially mediated O 2 reduction in microbial fuel cells and microbial electrosynthesis. However, biocathode mechanistic studies needed to improve or engineer functionality have been limited to a few select species that form sparse, homogeneous biofilms characterized by little or no growth. Attempts to cultivate isolates from biocathode environmental enrichments often fail due to a lack of some advantage provided by life in a consortium, highlighting the need to study and understand biocathode consortia in situ. Here, we present metagenomic and metaproteomic characterization of a previously described biocathode biofilm (؉310 mV versus a standard hydrogen electrode [SHE]) enriched from seawater, reducing O 2 , and presumably fixing CO 2 for biomass generation. Metagenomics identified 16 distinct cluster genomes, 15 of which could be assigned at the family or genus level and whose abundance was roughly divided between Alpha-and Gammaproteobacteria. A total of 644 proteins were identified from shotgun metaproteomics and have been deposited in the the ProteomeXchange with identifier PXD001045. Cluster genomes were used to assign the taxonomic identities of 599 proteins, with Marinobacter, Chromatiaceae, and Labrenzia the most represented. RubisCO and phosphoribulokinase, along with 9 other Calvin-Benson-Bassham cycle proteins, were identified from Chromatiaceae. In addition, proteins similar to those predicted for iron oxidation pathways of known iron-oxidizing bacteria were observed for Chromatiaceae. These findings represent the first description of putative EET and CO 2 fixation mechanisms for a selfregenerating, self-sustaining multispecies biocathode, providing potential targets for functional engineering, as well as new insights into biocathode EET pathways using proteomics. Bioelectrochemical systems (BES) use microorganisms as catalysts to drive complex electrochemical reactions, such as electricity generation by microbial fuel cells (MFCs) (1), wastewater treatment (2), and microbial electrosynthesis (3-6), that would not be possible without living cells. The term "biocathode" refers to a biofilm, constituted of a single organism or microbial consortium, that has formed on the cathode of a BES and consumes electrons (e Ϫ ). Cathodes hold great potential as a stable electron source to drive microbial metabolism (7); however, little is known about the underlying extracellular electron transfer (EET) pathways that could be exploited for biocathode functional engineering. Although biocathode EET has been demonstrated for a variety of microorganisms, including acetogens (5) and a methanogenic archaeon (6), studies aimed at identifying EET conduits from the electrode to cells have mostly been confined to the model organisms Geobacter (8) and Shewanella (9), due to the massive effort put forth to understand how these iron-reducing bacteria are able to catalyze EET at bioanodes (10-12). The ability of iron-...

Here, we report on the development of a genetic system for Marinobacter sp. strain CP1, previously isolated from the Biocathode MCL community and shown to oxidize iron and grow as a cathodic biofilm. Sequence analysis of the small and large subunits of the 16S rRNA gene of CP1, as well as comparison of select conserved proteins, indicate that it is most closely related to Marinobacter adhaerens HP15 and Marinobacter sp. ES.042. In silico DNA–DNA hybridization using the genome-to-genome distance calculator (GGDC) predicts CP1 to be a new species of Marinobacter described here as Marinobacter atlanticus. CP1 is competent for transformation with plasmid DNA using conjugation with Escherichia coli donor strain WM3064 and constitutive expression of green fluorescent protein (GFP) is stable in the absence of antibiotic selection. Targeted double deletion mutagenesis of homologs for the M. aquaeoli fatty acyl-CoA reductase (acrB) and fatty aldehyde reductase (farA) genes resulted in a loss of production of wax esters; however, single deletion mutants for either gene resulted in an increase in total wax esters recovered. Genetic tools presented here for CP1 will enable further exploration of wax ester synthesis for biotechnological applications, as well as furthering our efforts to understand the role of CP1 within the Biocathode MCL community.

Here we show that long-distance extracellular electron transport occurs in a cathodic biofilm capable of CO2 fixation and O2 respiration.

Bacteria that directly use electrodes as metabolic electron donors (biocathodes) have been proposed for applications ranging from microbial electrosynthesis to advanced bioelectronics for cellular communication with machines. However, just as we understand very little about oxidation of analogous natural insoluble electron donors, such as iron oxide, the organisms and extracellular electron transfer (EET) pathways underlying the electrode-cell direct electron transfer processes are almost completely unknown. Biocathodes are a stable biofilm cultivation platform to interrogate both the rate and mechanism of EET using electrochemistry and to study the electroautotrophic organisms that catalyze these reactions. Here we provide new evidence supporting the hypothesis that the uncultured bacterium “Candidatus Tenderia electrophaga” directly couples extracellular electron transfer to CO2 fixation. Our results provide insight into developing biocathode technology, such as microbial electrosynthesis, as well as advancing our understanding of chemolithoautotrophy.