Climate change is rapidly transforming Arctic landscapes where increasing soil temperatures speed up permafrost thaw. This exposes large carbon stocks to microbial decomposition, possibly worsening climate change by releasing more greenhouse gases. Understanding how microbes break down soil carbon, especially under the anaerobic conditions of thawing permafrost, is important to determine future changes. Here, we studied the microbial community dynamics and soil carbon decomposition potential in permafrost and active layer soils under anaerobic laboratory conditions that simulated an Arctic summer thaw. The microbial and viral compositions in the samples were analyzed based on metagenomes, metagenome-assembled genomes, and metagenomic viral contigs (mVCs). Following the thawing of permafrost, there was a notable shift in microbial community structure, with fermentative Firmicutes and Bacteroidota taking over from Actinobacteria and Proteobacteria over the 60-day incubation period. The increase in iron and sulfate-reducing microbes had a significant role in limiting methane production from thawed permafrost, underscoring the competition within microbial communities. We explored the growth strategies of microbial communities and found that slow growth was the major strategy in both the active layer and permafrost. Our findings challenge the assumption that fast-growing microbes mainly respond to environmental changes like permafrost thaw. Instead, they indicate a common strategy of slow growth among microbial communities, likely due to the thermodynamic constraints of soil substrates and electron acceptors, and the need for microbes to adjust to post-thaw conditions. The mVCs harbored a wide range of auxiliary metabolic genes that may support cell protection from ice formation in virus-infected cells.
OBJECTIVE: As the Arctic warms, thawing permafrost unlocks carbon, potentially accelerating climate change by releasing greenhouse gases. Our research delves into the underlying biogeochemical processes likely mediated by the soil microbial community in response to the wet and anaerobic conditions, akin to an Arctic summer thaw. We observed a significant shift in the microbial community post-thaw, with fermentative bacteria like Firmicutes and Bacteroidota taking over and switching to different fermentation pathways. The dominance of iron and sulfate-reducing bacteria likely constrained methane production in the thawing permafrost. Slow-growing microbes outweighed fast-growing ones, even after thaw, upending the expectation that rapid microbial responses to dominate after permafrost thaws. This research highlights the nuanced and complex interactions within Arctic soil microbial communities and underscores the challenges in predicting microbial response to environmental change.

The mobility and bioavailability of phosphate in paddy soils are closely coupled to redox-driven Fe-mineral dynamics. However, the role of phosphate during Fe-mineral dissolution and transformations in soils remains unclear. Here, we investigated the transformations of ferrihydrite and lepidocrocite and the effects of phosphate pre-adsorbed to ferrihydrite during a 16-week field incubation in a flooded sandy rice paddy soil in Thailand. For the deployment of the synthetic Fe-minerals in the soil, the minerals were contained in mesh bags either in pure form or after mixing with soil material. In the latter case, the Fe-minerals were labeled with 57Fe to allow the tracing of minerals in the soil matrix with 57Fe Mössbauer spectroscopy. Porewater geochemical conditions were monitored, and changes in the Fe-mineral composition were analyzed using 57Fe Mössbauer spectroscopy and/or X-ray diffraction analysis. Reductive dissolution of ferrihydrite and lepidocrocite played a minor role in the pure mineral mesh bags, while in the 57Fe-mineral-soil mixes more than half of the minerals was dissolved. The pure ferrihydrite was transformed largely to goethite (82-85%), while ferrihydrite mixed with soil only resulted in 32% of all remaining 57Fe present as goethite after 16 weeks. In contrast, lepidocrocite was only transformed to 12% goethite when not mixed with soil, but 31% of all remaining 57Fe was found in goethite when it was mixed with soil. Adsorbed phosphate strongly hindered ferrihydrite transformation to other minerals, regardless of whether it was mixed with soil. Our results clearly demonstrate the influence of the complex soil matrix on Fe-mineral transformations in soils under field conditions and how phosphate can impact Fe oxyhydroxide dynamics under Fe reducing soil conditions.

Biological nitrogen fixation (BNF) by methanotrophic bacteria has been shown to play an important role in maintaining fertility. However, this process is still limited to aerobic methane oxidation with sufficient oxygen. It has remained unknown whether and how methanotrophic BNF proceeds in hypoxic environments. Herein, we incubated paddy soils with a ferrihydrite-containing mineral salt medium to enrich methanotrophic bacteria in the presence of methane (20%, v/v) under oxygen constraints (0.27%, v/v). The resulting microcosms showed that ferrihydrite-dependent aerobic methane oxidation significantly contributed (81%) to total BNF, increasing the 15N fixation rate by 13-fold from 0.02 to 0.28 μmol 15N2 (g dry weight soil) -1 d-1. BNF was reduced by 97% when ferrihydrite was omitted, demonstrating the involvement of ferrihydrite in methanotrophic BNF. DNA stable-isotope probing indicated that Methylocystis, Methylophilaceae, and Methylomicrobium were the dominant methanotrophs/methylotrophs that assimilated labeled isotopes (13C or 15N) into biomass. Metagenomic binning combined with electrochemical analysis suggested that Methylocystis and Methylophilaceae had the potential to perform methane-induced BNF and likely utilized riboflavin and c-type cytochromes as electron carriers for ferrihydrite reduction. It was concluded that ferrihydrite mediated methanotrophic BNF by methanotrophs/methylotrophs solely or in conjunction with iron-reducing bacteria. Overall, this study revealed a previously overlooked yet pronounced coupling of iron-dependent aerobic methane oxidation to BNF and improves our understanding of methanotrophic BNF in hypoxic zones.

Some thermophilic bacteria from deep-sea hydrothermal vents grow by dissimilatory iron reduction, but our understanding of their biogenic mineral transformations is nascent. Mineral transformations catalyzed by the thermophilic iron-reducing bacterium Desulfovulcanus ferrireducens during growth at 55°C were examined using synthetic nanophase ferrihydrite, akaganeite, and lepidocrocite separately as terminal electron acceptors. Spectral analyses using visible-near infrared (VNIR), Fourier-transform infrared attenuated total reflectance (FTIR-ATR), and Mössbauer spectroscopies were complemented with x-ray diffraction (XRD) and transmission electron microscopy (TEM) using selected area electron diffraction (SAED) and energy dispersive X-ray (EDX) analyses. The most extensive biogenic mineral transformation occurred with ferrihydrite, which produced a magnetic, visibly dark mineral with spectral features matching cation-deficient magnetite. Desulfovulcanus ferrireducens also grew on akaganeite and lepidocrocite and produced non-magnetic, visibly dark minerals that were poorly soluble in the oxalate solution. Bioreduced mineral products from akaganeite and lepidocrocite reduction were almost entirely absorbed in the VNIR spectroscopy in contrast to both parent minerals and the abiotic controls. However, FTIR-ATR and Mössbauer spectra and XRD analyses of both biogenic minerals were almost identical to the parent and control minerals. The TEM of these biogenic minerals showed the presence of poorly crystalline iron nanospheres (50-200 nm in diameter) of unknown mineralogy that were likely coating the larger parent minerals and were absent from the controls. The study demonstrated that thermophilic bacteria transform different types of Fe(III) (oxyhydr)oxide minerals for growth with varying mineral products. These mineral products are likely formed through dissolution-reprecipitation reactions but are not easily predictable through chemical equilibrium reactions alone.

In methane (CH4) generating sediments, methane oxidation coupled with iron reduction was suggested to be catalyzed by archaea and bacterial methanotrophs of the order Methylococcales. However, the co-existence of these aerobic and anaerobic microbes, the link between the processes, and the oxygen requirement for the bacterial methanotrophs have remained unclear. Here, we show how stimulation of aerobic methane oxidation at an energetically low experimental environment influences net iron reduction, accompanied by distinct microbial community changes and lipid biomarker patterns. We performed incubation experiments (between 30 and 120 days long) with methane generating lake sediments amended with 13C-labeled methane, following the additions of hematite and different oxygen levels in nitrogen headspace, and monitored methane turnover by 13C-DIC measurements. Increasing oxygen exposure (up to 1%) promoted aerobic methanotrophy, considerable net iron reduction, and the increase of microbes, such as Methylomonas, Geobacter, and Desulfuromonas, with the latter two being likely candidates for iron recycling. Amendments of 13C-labeled methanol as a potential substrate for the methanotrophs under hypoxia instead of methane indicate that this substrate primarily fuels methylotrophic methanogenesis, identified by high methane concentrations, strongly positive δ13CDIC values, and archaeal lipid stable isotope data. In contrast, the inhibition of methanogenesis by 2-bromoethanesulfonate (BES) led to increased methanol turnover, as suggested by similar 13C enrichment in DIC and high amounts of newly produced bacterial fatty acids, probably derived from heterotrophic bacteria. Our experiments show a complex link between aerobic methanotrophy and iron reduction, which indicates iron recycling as a survival mechanism for microbes under hypoxia.

Microbial dissimilatory iron reduction is a fundamental respiratory process that began early in evolution and is performed in diverse habitats including aquatic anoxic sediments. In many of these sediments microbial iron reduction is not only observed in its classical upper zone, but also in the methane production zone, where low-reactive iron oxide minerals are present. Previous studies in aquatic sediments have shown the potential role of the archaeal methanogen Methanosarcinales in this reduction process, and their use of methanophenazines was suggested as an advantage in reducing iron over other iron-reducing bacteria. Here we tested the capability of the methanogenic archaeon Methanosarcina barkeri to reduce three naturally abundant iron oxides in the methanogenic zone: the low-reactive iron minerals hematite and magnetite, and the high-reactive amorphous iron oxide. We also examined the potential role of their methanophenazines in promoting the reduction. Pure cultures were grown close to natural conditions existing in the methanogenic zone (under nitrogen atmosphere, N2:CO2, 80:20), in the presence of these iron oxides and different electron shuttles. Iron reduction by M. barkeri was observed in all iron oxide types within 10 days. The reduction during that time was most notable for amorphous iron, then magnetite, and finally hematite. Importantly, the reduction of iron inhibited archaeal methane production. When hematite was added inside cryogenic vials, thereby preventing direct contact with M. barkeri, no iron reduction was observed, and methanogenesis was not inhibited. This suggests a potential role of methanophenazines, which are strongly associated with the membrane, in transferring electrons from the cell to the minerals. Indeed, adding dissolved phenazines as electron shuttles to the media with iron oxides increased iron reduction and inhibited methanogenesis almost completely. When M. barkeri was incubated with hematite and the phenazines together, there was a change in the amounts (but not the type) of specific metabolites, indicating a difference in the ratio of metabolic pathways. Taken together, the results show the potential role of methanogens in reducing naturally abundant iron minerals in methanogenic sediments under natural energy and substrate limitations and shed new insights into the coupling of microbial iron reduction and the important greenhouse gas methane.

Iron minerals in soils and sediments play important roles in many biogeochemical processes and therefore influence the cycling of major and trace elements and the fate of pollutants in the environment. However, the kinetics and pathways of Fe mineral recrystallization and transformation processes under environmentally relevant conditions are still elusive. Here, we present a novel approach enabling us to follow the transformations of Fe minerals added to soils or sediments in close spatial association with complex solid matrices including other minerals, organic matter, and microorganisms. Minerals enriched with the stable isotope 57Fe are mixed with soil or sediment, and changes in Fe speciation are subsequently studied by 57Fe Mössbauer spectroscopy, which exclusively detects 57Fe. In this study, 57Fe-labeled ferrihydrite was synthesized, mixed with four soils differing in chemical and physical properties, and incubated for 12+ weeks under anoxic conditions. Our results reveal that the formation of crystalline Fe(III)(oxyhydr)oxides such as lepidocrocite and goethite was strongly suppressed, and instead formation of a green rust-like phase was observed in all soils. These results contrast those from Fe(II)-catalyzed ferrihydrite transformation experiments, where formation of lepidocrocite, goethite, and/or magnetite often occurs. The presented approach allows control over the composition and crystallinity of the initial Fe mineral, and it can be easily adapted to other experimental setups or Fe minerals. It thus offers great potential for future investigations of Fe mineral transformations in situ under environmentally relevant conditions, in both the laboratory and the field.

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple ΔppcABCDE ΔpgcA mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. IMPORTANCE Many inner and outer membrane cytochromes used by Geobacter for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that Geobacter periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of Geobacter electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.

Geobacter sulfurreducens is a ubiquitous iron-reducing bacterium in soils, and in engineered systems, it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work, we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C, H, O, N, and ash content) showed high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 ± 0.2 μg/g dry weight) and lipids (32 ± 0.5% dry weight/dry weight) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. IMPORTANCE Geobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively \"breathing\" metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal and, as a ubiquitous iron reducer in soils around the world, G. sulfurreducens has an impact on the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organism\'s composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria-these observations are important for future microbiologists and biotechnologists working with the organism.

Uncultured bacteria of the candidate phylum MBNT15, distantly related to Desulfobacterota, have been identified in a broad range of mostly organic-rich aquatic environments. We assembled a near-complete genome of a member of MBNT15 from a boreal peatland metagenome and used genomic data to analyze the metabolic pathways of this bacterium and its ecological role. This bacterium, designated SHF-111, was predicted to be rod shaped, it lacks flagellar machinery but twitching motility is encoded. Genome-based phylogenetic analysis supported the phylum-level classification of the MBNT15 lineage. Genome annotation and metabolic reconstruction revealed the presence of the Embden-Meyerhof, Entner-Doudoroff and pentose phosphate pathways, as well as the complete tricarboxylic acid (TCA) cycle, and suggested a facultatively anaerobic chemoheterotrophic lifestyle with the ability to ferment peptides, amino acids, fatty acids and simple sugars, and completely oxidize these substrates through aerobic and anaerobic respiration. The SHF-111 genome encodes multiple multiheme c-type cytochromes that probably enable dissimilatory iron reduction. Consistently, the relative abundance of MBNT15 in peatlands positively correlated with iron concentration. Apparently, in the wetland ecosystem, MBNT15 representatives play the role of scavengers, carrying out the complete mineralization of low molecular weight organic substances formed as a result of microbial degradation of complex polymeric substrates. Comparative genome analysis of the MBNT15 phylum revealed that vast majority of its members are capable of aerobic respiration and dissimilatory iron reduction and some species also can reduce sulfur and nitrogen compounds, but not sulfate. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Deferrimicrobium borealis, within a candidate phylum Deferrimicrobiota.