Abstract De.fer.ri.vi.bri.o.na'ce.ae. N.L. masc. n. Deferrivibrio type genus of the family; L. fem. pl. n. suff. ‐ aceae , ending to denote a family; N.L. fem. pl. n. Deferrivibrionaceae , the Deferrivibrio family. Chrysiogenota / Deferribacteres / Deferribacterales / Deferrivibrionaceae The Deferrivibrionaceae family belongs to the Deferribacterales order within the Chrysiogenota phylum and accommodates a single genus Deferrivibrio . Only two representatives have been described, Deferrivibrio essentukiensis Es71‐Z0220 T and Deferrivibrio metallireducens V6Fe1 T , which are genetically, but not phenotypically, very similar. The family Deferrivibrionaceae includes anaerobic chemoorgano‐ or chemolithotrophic bacteria that inhabit aquifers, hot vents of marine sediments, and petroleum reservoirs. They are moderate thermophiles, neutrophiles, and halotolerant microorganisms that use organic acids, peptides, or molecular hydrogen as energy sources, and Fe(III) compounds in insoluble form, Mn(IV), nitrate, or elemental sulfur as electron acceptors. Some members of this family ferment organic acids and peptides and perform autotrophic carbon fixation, probably via the reversed oxidative TCA cycle. DNA G + C content (%) : 34.04–34.8 (genome sequence). Type genus : Deferrivibrio Zavarzina et al. 2022, VL211.

ABSTRACT Microbially induced corrosion (MIC), caused by iron-cycling microorganisms that directly uptake electrons from metallic iron, is a serious economic and environmental problem. Iron corrosion is inhibited at pH above 9.0 in the presence of carbonate by the formation of a passivating film, but the possibility of direct oxidation of metallic iron by anaerobic alkaliphiles has not been thoroughly investigated. This bioinduced process may pose a serious environmental hazard under anaerobic alkaline conditions of underground radioactive waste disposal in metal containers with bentonite clays. We used Geoalkalibacter ferrihydriticus , an anaerobic iron-cycling bacterium capable of both dissimilatory iron reduction and anaerobic iron oxidation, as a model organism to investigate the microbial ability to utilize Fe 0 from steel wire as an electron donor under anaerobic autotrophic conditions at pH 9.5. During bacterial growth, corrosion of the steel wire was induced and accompanied by intense H 2 production and precipitation of a solid phase. Mössbauer spectroscopy revealed that green rust with siderite admixture was the major mineral formed during Fe oxidation. Protons appeared to be the only thermodynamically favorable electron acceptor for G. ferrihydriticus . Their reduction could lead to hydrogen production. Genomic analysis supported the proposal of such a metabolic mode for the organism. Thus, we have shown that MIC can be realized under anaerobic alkaline conditions by iron-cycling microorganisms in the absence of organic substrates. Microbial hydrogen production may facilitate the further development of authigenic microflora, which could further increase corrosion in radioactive waste repositories and reduce the barrier properties of bentonite clays. IMPORTANCE Microbially induced corrosion (MIC) is a problem with significant economic damage. MIC processes occurring under anaerobic conditions at neutral pH have been actively studied over the last decades. Meanwhile, MIC processes under anaerobic alkaline conditions remain very poorly understood, although they represent a serious environmental problem, as such conditions are characteristic of the geological disposal of nuclear waste stored in metal containers isolated by clays. Our studies of the corrosion of steel by the anaerobic iron-cycling bacterium Geoalkalibacter ferrihydriticus at pH 9.5 in the absence of any organic matter have shown that this process is possible and can be accompanied by the active release of hydrogen. The formation of this gas can trigger the development of an authigenic anaerobic microflora that uses it as an electron donor and can negatively affect the insulating properties of the clay barrier through microbial metabolic activity.

Analysis of the anaerobic microbial communities from the therapeutic muds of Tambukan Lake performed by molecular and cultivation approaches revealed their high taxonomic diversity. Microorganisms involved in transformations of oxidized and reduced sulfur compounds represented 40–70

Polyextremophilic microbial communities of Baikal Rift Zone hot springs have been studied fragmentarily, and these studies have typically focused on either phototrophic microbial mats or on the whole microbial community from one or a few sites. In our work, we conducted the first large-scale screening of microbial communities from seven hot spring groups in the Baikal Rift Zone, using metabarcoding of the V3-V4 regions of the 16S rRNA gene. Analysis of alpha and beta diversity, as well as co-occurrence network analysis, revealed that the microbial diversity of the studied springs is highly dependent on temperature values. This approach allowed classifying microbial communities into four distinct groups, characterized by significantly different taxa representing the key functional roles of primary producers, heterotrophic consumers, and terminal destructors of organic matter. Sulfate-reducing bacteria constituted a major metabolic group driving the final stage of organic matter mineralization. Moreover, the presence of alkalithermophilic dissimilatory iron reducers, whose existence was debatable, was proved in the studied samples by cultural methods. The phylotypes that gained an advantage on selective media with synthesized ferrihydrite and hydrogen or acetate added as an electron donor belonged to the genus Parvivirga of the order Anaerosomatales and several unknown representatives of the phylum Bacillota.

A study of iron-reducing prokaryotes that use the poorly crystalline mineral ferrihydrite as an electron acceptor revealed their widespread occurrence on the planet. However, the ability of iron reducers to develop under polyextreme conditions (at elevated values of pH and temperature) has not been previously demonstrated. To confirm the existence of alkalothermophilic iron reducers, we studied the water and sediment samples from the Goryachinsk nitrogen-rich thermal water basin. These springs are associated with young tectonic fractures in the Baikal rift zone; their water is characterized by low salinity, alkaline pH (8.8‒9.2) and temperature of 53‒55°C. Enrichment cultures reducing synthesized ferrihydrite were obtained by adding this mineral to the water and sediment samples from the Goryachinsk thermal springs, followed by anaerobic incubation at 50 and 55°C. The 16S rRNA gene profiling of the water and sediment samples and of the primary enrichment cultures revealed high taxonomic diversity, almost exclusively represented by bacterial taxa. Subsequent transfers with ferrihydrite and organic acids or hydrogen as electron donors resulted in stable enrichment cultures of organo- and lithotrophic alkalothermophilic iron reducers. Members of the genus Parvivirga, one of the first cultured representatives of the OPB41 group (now the order Anaerosomatales, phylum Actinomycetota), constituted 30 to 50

The microbiome of deep continental aquifers is considered the most slowly evolving part of the biosphere. The Yessentukskoye Mineral Water Basin (YMWB), located in the pre-Caucasus region, contains three closely spaced but distinct aquifers, the Upper Cretaceous, the Lower Cretaceous, and the Upper Jurassic, which represent unique objects for subsurface biosphere research due to gas-hydrogeochemical and thermal anomalies of the area. We analyzed the geological and hydrogeochemical parameters of the three aquifers and a recharge area of the YMWB and investigated their microbial communities using metagenomic and cultivation-based approaches within a long-term survey. Correlation analysis of the obtained data revealed stable and highly stratified microbial communities inhabiting four distinct ecosystems. Their structure and the metabolic traits of their prokaryotic populations were similar to those presumed to have dominated the Earth’s biosphere during several critical periods of its evolutionary history, that is, the Early Archean, the period of banded iron formations accumulation, and the Great Oxidation Event. Among the YMWB strata, the Upper Jurassic aquifer, supersaturated with CO2, influenced by magmatic activity, and highly enriched with thermophilic autotrophic hydrogenotrophic acetogens, turned out to be the first described modern ecosystem based on the primary production by a process predicted to support the Last Universal Common Ancestor (LUCA). The characterization of the YMWB microbial communities reveals a contemporary model environment of the early stages of Earth’s development and thus contributes to the understanding of the evolutionary traits in microbial populations that may have played a critical role in the formation of the modern biosphere.IMPORTANCEContinental subsurface environments are estimated to harbor up to one-fifth of the planet’s total biomass, representing the most stable and slowly evolving part of the biosphere. Among the deep subsurface inhabitants, the microbial communities of drinking mineral waters remain the least studied. Our interdisciplinary study of the Yessentukskoye Mineral Water Basin shows how hydrochemical and hydrodynamic factors shape different subsurface ecosystems, whose microbial populations influence the composition of mineral waters. A comprehensive analysis reveals the similarity of these ecosystems to those predicted for the early Earth. The deepest of the studied aquifers is the first described modern ecosystem with the most probable primary producer performing hydrogenotrophic acetogenesis. Thus, our results contribute to the understanding of the genesis of modern drinking water resources and expand the knowledge of the evolutionary traits that may have played a critical role in the formation of the Earth’s biosphere.

Abstract Ge.o.al.ka.li.bac.te.ra.ce'ae. N.L. masc. n. Geoalkalibacter , type genus of the family; L. fem. pl. n. suff. ‐ aceae , ending to denote a family; N.L. fem. pl. n. Geoalkalibacteraceae , the family of the genus Geoalkalibacter . Desulfobacterota_F / Desulfuromonadia / Desulfuromonadales / Geoalkalibacteraceae The family is a member of the order Desulfuromonadales in the phylum Desulfobacterota_F and consists of a single genus Geoalkalibacter . The family Geoalkalibacteraceae includes strictly anaerobic chemolitho‐ or chemoorganotrophic bacteria that inhabit soda lakes or petroleum reservoirs. They are alkalitolerant or alkaliphiles, halotolerant or moderate halophiles using molecular hydrogen or a wide range of organic compounds as energy sources and either soluble or insoluble Fe(III), Mn(IV), or sulfur compounds as electron acceptors. Members of this family are electroactive microorganisms and can also use Fe(II)‐containing minerals or cathodes as electron donors. DNA G + C content (mol%) : 57.05–60.57 (genome analysis). Type genus : Geoalkalibacter Zavarzina et al. 2006, VL115 emend. Greene et al. 2009.

A study of the metabolic group of iron-reducing prokaryotes that use the weakly crystalline mineral ferrihydrite as an electron acceptor has revealed its widespread distribution on Earth. However, the ability of iron reducers to develop in polyextreme conditions – at elevated pH values and temperatures – has not yet been demonstrated. To prove the existence of alkalithermophilic iron reducers, we studied water and sediment samples from the Goryachinsk nitrogen springs. These springs are confined to young tectonic faults of the Baikal rift zone, and their waters are characterized by low mineralization, high pH (8.8‒9.2) and temperature (53‒55°C). By adding synthesized ferrihydrite to water and bottom sediment samples from the Goryachinsk springs and incubating them under anaerobic conditions at temperatures of 50 and 55°C, enrichment cultures were obtained that reduce this mineral. Profiling of water and sediment samples and primary enrichment cultures by the 16S rRNA gene revealed their high phylogenetic diversity, represented almost exclusively by bacterial taxa. Further transfers with ferrihydrite and organic acids or hydrogen added as an electron donor allowed us to obtain stable enrichment cultures of organo- and lithotrophic alkalithermophilic iron reducers. From 30 to 50% of their total representation were representatives of the genus Parvivirga , one of the first cultivated representatives of the OPB41 group (now the order Anaerosomatales) of the phylum Actinomycetota.

The Yessentuki mineral water deposit (YMWD) is a well-known source of balneologically valuable drinking mineral water, but it has rarely been investigated in terms of the microbes inhabiting it. In this work, we have studied the microbial communities of the continuously operating production well 9, penetrating the Lower Cretaceous aquifer of the YMWD, and characterized, in detail, two novel representatives of class Ignavibacteria (Bacteroidota). One representative of the so-called XYB12-FULL-38-5 group within the family Melioribacteraceae has been isolated in pure culture, designated strain 09-Me, and physiologically characterized. It is a facultatively anaerobic thermotolerant microorganism capable of fermentation and respiration on simple and complex sugars (lichenan, xanthan gum, glucomannan, curdlan, pachyman). In addition to oxygen, ferric iron, arsenate, and elemental sulfur were also used as electron acceptors. Phylogenomic and physiological analyses reveal this novel isolate to represent a novel genus and species for which the name Stygiobacter electus gen. nov., sp. nov. is proposed. The second representative of the family Melioribacteraceae described here belonged to the so-called DSXH01 group, which comprises the dominant group (up to 28%) of the microbial community of well 9 water. The organism was characterized through the analysis of its genome, assembled from metagenome of well 9 (Ess09-04 MAG). Genes encoding enzymes of carbohydrate utilization and genes responsible for aerobic and anaerobic respiration have been identified in the genomes of both bacteria. The investigation of the environmental distribution of Stygiobacter genus-related bacteria and representatives of the lineage DSXH01 has shown that they all are typical inhabitants of the subsurface biosphere, and are often found in bioreactors. These data significantly expand our knowledge on the microbes of subsurface water basins and pave the way for future studies of the novel members of Ignavibacteria class.

— Analysis of the composition of microbial communities of the Yessentukskoe mineral water deposit (YMWD) by molecular and classical microbiological methods revealed the presence of numerous uncultured bacteria of high taxonomic rank in the studied water-bearing horizons. Selective media with ferrihydrite (5Fe 2 O 3 ·9Н 2 O), magnetite (Fe 3 O 4 ), and siderite (FeCO 3 ) were used to obtain enrichment and pure cultures of iron-reducing microorganisms belonging to novel taxa in the phylum Actinobacterota (previously the uncultured order OPB41), order Deferribacterales , and the family Melioribacteraceae , as well as those related to the phylum Synergistota . Anaerobic iron-oxidizing bacteria belonged to the genera Ciceribacter and Bradyrhizobium. The results obtained expand our knowledge on biodiversity of the deep subsurface biosphere and reveal unexpected new metabolic features of previously characterized bacterial taxa.

The anaerobic oxidation of fatty acids and alcohols occurs near the thermodynamic limit of life. This process is driven by syntrophic bacteria that oxidize fatty acids and/or alcohols, their syntrophic partners that consume the products of this oxidation, and the pathways for interspecies electron exchange via these products or direct interspecies electron transfer (DIET). Due to the interdependence of syntrophic microorganisms on each other’s metabolic activity, their isolation in pure cultures is almost impossible. Thus, little is known about their physiology, and the only available way to fill in the knowledge gap on these organisms is genomic and metabolic analysis of syntrophic cultures. Here we report the results of genome sequencing and analysis of an obligately syntrophic alkaliphilic bacterium ‘Candidatus Contubernalis alkaliaceticus’. The genomic data suggest that acetate oxidation is carried out by the Wood–Ljungdahl pathway, while a bimodular respiratory system involving an Rnf complex and a Na+-dependent ATP synthase is used for energy conservation. The predicted genomic ability of ‘Ca. C. alkaliaceticus’ to outperform interspecies electron transfer both indirectly, via H2 or formate, and directly, via pili-like appendages of its syntrophic partner or conductive mineral particles, was experimentally demonstrated. This is the first indication of DIET in the class Dethiobacteria.

Haloalkaliphilic microorganisms are double extremophiles functioning optimally at high salinity and pH. Their typical habitats are soda lakes, geologically ancient yet widespread ecosystems supposed to harbor relict microbial communities. We compared metabolic features and their determinants in two strains of the natronophilic species Dethiobacter alkaliphilus, the only cultured representative of the class “Dethiobacteria” (Bacillota). The strains of D. alkaliphilus were previously isolated from geographically remote Mongolian and Kenyan soda lakes. The type strain AHT1T was described as a facultative chemolithoautotrophic sulfidogen reducing or disproportionating sulfur or thiosulfate, while strain Z-1002 was isolated as a chemolithoautotrophic iron reducer. Here, we uncovered the iron reducing ability of strain AHT1T and the ability of strain Z-1002 for thiosulfate reduction and anaerobic Fe(II) oxidation. Key catabolic processes sustaining the growth of both D. alkaliphilus strains appeared to fit the geochemical settings of two contrasting natural alkaline environments, sulfur-enriched soda lakes and iron-enriched serpentinites. This hypothesis was supported by a meta-analysis of Dethiobacterial genomes and by the enrichment of a novel phylotype from a subsurface alkaline aquifer under Fe(III)-reducing conditions. Genome analysis revealed multiheme c-type cytochromes to be the most probable determinants of iron and sulfur redox transformations in D. alkaliphilus. Phylogeny reconstruction showed that all the respiratory processes in this organism are likely provided by evolutionarily related early forms of unconventional octaheme tetrathionate and sulfite reductases and their structural analogs, OmhA/OcwA Fe(III)-reductases. Several phylogenetically related determinants of anaerobic Fe(II) oxidation were identified in the Z-1002 genome, and the oxidation process was experimentally demonstrated. Proteomic profiling revealed two distinct sets of multiheme cytochromes upregulated in iron(III)- or thiosulfate-respiring cells and the cytochromes peculiar for Fe(II) oxidizing cells. We suggest that maintaining high variation in multiheme cytochromes is an effective adaptive strategy to occupy geochemically contrasting alkaline environments. We propose that sulfur-enriched soda lakes could be secondary habitats for D. alkaliphilus compared to Fe-rich serpentinites, and that the ongoing evolution of Dethiobacterales could retrace the evolutionary path that may have occurred in prokaryotes at a turning point in the biosphere’s history, when the intensification of the sulfur cycle outweighed the global significance of the iron cycle.

Two heterotrophic bacteroidetes strains were isolated as satellites from autotrophic enrichments inoculated with samples from hypersaline soda lakes in southwestern Siberia. Strain Z-1702T is an obligate anaerobic fermentative saccharolytic bacterium from an iron-reducing enrichment culture, while Ca. Cyclonatronum proteinivorum OmegaT is an obligate aerobic proteolytic microorganism from a cyanobacterial enrichment. Cells of isolated bacteria are characterized by highly variable morphology. Both strains are chloride-independent moderate salt-tolerant obligate alkaliphiles and mesophiles. Strain Z-1702T ferments glucose, maltose, fructose, mannose, sorbose, galactose, cellobiose, N-acetyl-glucosamine and alpha-glucans, including starch, glycogen, dextrin, and pullulan. Strain OmegaT is strictly proteolytic utilizing a range of proteins and peptones. The main polar lipid fatty acid in both strains is iso-C15:0, while other major components are various C16 and C17 isomers. According to pairwise sequence alignments using BLAST Gracilimonas was the nearest cultured relative to both strains (<90% of 16S rRNA gene sequence identity). Phylogenetic analysis placed strain Z-1702T and strain OmegaT as two different genera in a deep-branching clade of the new family level within the order Balneolales with genus. Based on physiological characteristics and phylogenetic position of strain Z-1702T it was proposed to represent a novel genus and species Natronogracilivirga saccharolityca gen. nov., sp. nov. (= DSMZ 109061T =JCM 32930T =VKM B 3262T). Furthermore, phylogenetic and phenotypic parameters of N. saccharolityca and C. proteinivorum gen. nov., sp. nov., strain OmegaT (=JCM 31662T, =UNIQEM U979T), make it possible to include them into a new family with a proposed designation Cyclonatronaceae fam. nov..

The physicochemical properties of siderites (FeCO 3 ) of different origin have attracted attention due to challenges in the diagnostics with the aim of evaluating the involvement of bacteria in the formation of iron-rich sedimentary deposits. A comparative study of siderites of chemical and bacterial origin was performed by Mӧssbauer spectroscopy, X-ray diffraction, and scanning electron microscopy for the purpose of determining the possible characteristics, which will allow the identification of biogenic siderite. It was found that all the characterized siderites have significant differences in the crystal morphology associated with the physicochemical conditions of their formation. Siderites of bacterial origin are characterized by a smaller crystal size compared to siderites of hydrothermal or sedimentary origin. The inhomogeneity of the nearest environment of the iron atoms, which was found for siderites of bacterial origin and which is manifested in the Mӧssbauer spectra as a larger width of the quadrupole shift distribution, can be used as a diagnostic indication of biogenic siderites.

The Yessentukskoye deposit of Caucasian mineral waters contains balneologically valuable drinking mineral water, which is extracted from the Upper Cretaceous 1 km subsurface aquifer and is almost unexplored by microbiologists. We have sampled this water via continuously operating production wells, characterized the phylogenetic diversity of its microbial community, and obtained enrichments of thermophilic iron reducers from the source aquifer. From the enrichments, a novel anaerobic thermophilic bacterium, reducing Fe(III) in the mineral ferrihydrite with acetate as the electron donor, was isolated into a pure culture. The novel isolate, designated as strain Es71-Z0220 T belonging to Deferribacterales order, is thermophilic, neutrophilic, halotolerant, motile vibrio. It utilizes synthesized ferrihydrite, fumarate, nitrate or elemental sulfur as the electron acceptors with organic acids as the electron donors. The strain is incapable of soluble Fe(III) complexes reduction and fermentative growth. The draft genome assembly of strain Es71-Z0220 T resulted in 65 contigs with a total size of ca. 2.3 Mb. On the basis of whole-genome phylogenetic reconstruction and physiological characterization, the novel isolate was considered to represent a novel family, genus and species for which the name Deferrivibrio essentukiensis gen. nov., sp. nov. is proposed. Genome analysis revealed key determinants of anaerobic respiration and carbon substrate utilization pathways in the organism with peculiarities related to putative Fe(III)-reducing electron transfer chain. Considering the revealed metabolic features of Deferrivibrio essentukiensis , the involvement of the organism in its subsurface environment in biogeochemical by carbon cycling by coupling the organic matter oxidation with Fe(III) minerals reduction is discussed.

The continental subsurface harbors microbial populations highly enriched in uncultured taxa. OPB41 is an uncultured order-level phylogenetic lineage within the actinobacterial class Coriobacteriia . OPB41 bacteria have a wide geographical distribution, but the physiology and metabolic traits of this cosmopolitan group remain elusive. From two contrasting subsurface environments, a terrestrial mud volcano and a deep subsurface aquifer, located in the central part of Eurasia, within the Caucasus petroleum region, we have isolated two pure cultures of anaerobic actinobacteria belonging to OPB41. The cells of both strains are small non-motile rods forming numerous pili-like appendages. Strain M08DHB T is mesophilic, while strain Es71-Z0120 T is a true thermophile having a broad temperature range for growth (25–77°C). Strain M08DHB T anaerobically reduces sulfur compounds and utilizes an aromatic compound 3,4-dihydroxybenzoic acid. Strain Es71-Z0120 T is an obligate dissimilatory Fe(III) reducer that is unable to utilize aromatic compounds. Both isolates grow lithotrophically and consume molecular hydrogen or formate using either thiosulfate, elemental sulfur, or Fe(III) as an electron acceptor. Genomes of the strains encode the putative reductive glycine pathway for autotrophic CO 2 fixation, Ni-Fe hydrogenases, putative thiosulfate/polysulfide reductases, and multiheme c -type cytochromes presumably involved in dissimilatory Fe(III) reduction. We propose to assign the isolated strains to the novel taxa of the species–order levels and describe strain M08DHB T as Anaerosoma tenue gen. nov., sp. nov., and strain Es71-Z0120 T as Parvivirga hydrogeniphila gen. nov., sp. nov., being members of Anaerosomatales ord. nov. This work expands the knowledge of the diversity, metabolic functions, and ecological role of the phylum Actinomycetota .

Caucasian Mineral Waters is a unique territory, where various types of mineral waters with overall daily flow over 16 000 m 3 are concentrated in a relatively small area. The Yessentukskoye deposit is characterized by high diversity of water types, of which Yessentuki nos. 17 and 4 are produced in the greatest amounts. Biogeochemical activity of microorganisms inhabiting the subsurface hydrosphere is one of the proposed mechanisms responsible for the genesis of these waters. The influence of microbial communities on the quality of balneological water resources is presently quite poorly studied. This is the first report on characterization of two communities inhabiting the water-bearing rocks and mineral waters of the Yessentukskoye deposit. The 16S rRNA gene profiling of these communities revealed predominance of uncultured archaea of the phylum Hadarchaeota (36.6%) in the Yessentuki no. 17 water retrieved from the well 46 and of several new classes of uncultured actinobacteria (29.4%) in the Yessentuki no. 4 water retrieved from the well 49-E. Significant differences were revealed in the structure of microbial communities inhabiting the water-bearing horizons of these two wells having different hydrochemical characteristics. Enrichment and pure cultures of the microorganisms belonging to the less abundant taxa were obtained. Analysis of metadata on genomic properties of prokaryotes of the dominant taxa, revealed in this work, indicates their ability to grow chemoautotrophically and thus, their potential involvement in redox transformations of the water-bearing rocks and the gas component of mineral waters.

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacteriumCarboxydothermus ferrireducensthat drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) byC. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutivec-type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.