Setting restorative goals with a regional outlook: Mine-rehabilitation outcomes influence landscape connectivity.

Mining is increasing worldwide and is typically associated with highly negative environmental impacts, such as habitat loss and fragmentation. To counteract these effects and improve restoration practices, decisions, such as setting mine rehabilitation goals, could incorporate the assessment and restoration of landscape connectivity into their regional and local-scale planning. The overarching goal of this work was to assess landscape connectivity for flora and fauna and explore mine-rehabilitation scenarios that can be used as a tool for prioritising biodiversity outcomes. Our study area comprised the Fitzroy Basin (Queensland, Australia), where the disturbance footprints from mining cover a cumulative area of 121,239 ha. We considered two scenarios: rehabilitation to agriculture and restoration to native ecosystems. To compare these scenarios, we created differential maps, which highlighted that restoring to native ecosystems represented connectivity gains over agricultural rehabilitation goals. These maps revealed three ways to prioritise rehabilitation outcomes, giving priority to mines that: 1) presented medium to high connectivity values, 2) covered a large area of influence (contributing to connectivity on a larger scale, regardless of current flow values), and 3) showed a gain of important paths/corridors. We explored four case studies and found that three benefited from restoration outcomes, while the fourth did not benefit by either scenario. Our methods can be used for decision-making in restoration ecology and conservation, including mine rehabilitation priorities and goals, as well as for evaluating connectivity gains or losses.

The role of plants in ironstone evolution: iron and aluminium cycling in the rhizosphere.

The Carajás plateaus in Brazil host endemic epilithic vegetation ("campo rupestre") on top of ironstone duricrusts, known as canga. This capping rock is primarily composed of iron(III) oxide minerals and forms a physically resistant horizon. Field observations reveal an intimate interaction between canga's surface and two native sedges (Rhynchospora barbata and Bulbostylis cangae). These observations suggest that certain plants contribute to the biogeochemical cycling of iron. Iron dissolution features at the root-rock interface were characterised using synchrotron-based techniques, Raman spectroscopy and scanning electron microscopy. These microscale characterisations indicate that iron is preferentially leached in the rhizosphere, enriching the comparatively insoluble aluminium around root channels. Oxalic acid and other exudates were detected in active root channels, signifying ligand-controlled iron oxide dissolution, likely driven by the plants' requirements for goethite-associated nutrients such as phosphorus. The excess iron not uptaken by the plant can reprecipitate in and around roots, line root channels and cement detrital fragments in the soil crust at the base of the plants. The reprecipitation of iron is significant as it provides a continuously forming cement, which makes canga horizons a 'self-healing' cover and contributes to them being the world's most stable continuously exposed land surfaces. Aluminium hydroxide precipitates ("gibbsite cutans") were also detected, coating some of the root cavities, often in alternating layers with goethite. This alternating pattern may correspond with oscillating oxygen concentrations in the rhizosphere. Microbial lineages known to contain iron-reducing bacteria were identified in the sedge rhizospheric microbiome and likely contribute to the reductive dissolution of iron(III) oxides within canga. Drying or percolation of oxygenated water to these anaerobic niches have led to iron mineralisation of biofilms, detected in many root channels. This study sheds light on plants' direct and indirect involvement in canga evolution, with possible implications for revegetation and surface restoration of iron mine sites.

Anthropedogenesis in coal mine overburden; the need for a comprehensive, fundamental biogeochemical approach.

The primary solid waste product of surface coal mining is overburden (also known as spoil), the material that is moved to access the underlying coal seams. Once removed, this material is generally stored in large (>100 m in height) piles until it is re-contoured in preparation for post-mining rehabilitation, often remaining there for decades. Under ideal conditions, these new landforms would have at least 30 cm veneer of topsoil applied as a growth medium for plants. However, topsoil deficit conditions are common in coal mines, so the forced use of overburden that possess poor chemical, biological, and physical properties as the growth medium hinders plant establishment. To reach the characteristics of a functional soil that can support plants, it is necessary to dramatically improve spoil quality, i.e., accelerate pedogenesis, as an integral part of the rehabilitation process. For decades, overburden rehabilitation has often taken the traditional approach of using the agricultural practice of fertilizer application or focusing primarily on the types of plants used to stabilize these young landforms. In contrast, success improved when the rehabilitation procedures employed a more holistic approach to establishing self-sustaining plant-soil ecosystems. Here we identify constraints that impede spoil-to-soil transformation, discuss the global treatment options practiced post-mining for coal spoils, and describe how a comprehensive biogeochemical approach can be implemented in spoil reclamation processes going forward. Integrating the revitalization of soil organisms, reclamation of soil chemistry, soil structure, and landform into rehabilitation procedures can accelerate the transformation of coal spoils to functional soils. We believe that a shift in the nature of the question from "what chemicals and seeds should we add to coal spoil during site rehabilitation?" to something fundamentally more specific: "How do we induce pedogenic functions in coal spoils to develop fertile soils?" is needed.

Integrating microalgae production into mine closure plans.

Examples of successful mine closure and acceptable regional transitioning of mining areas are scarce. The recent changes to the environmental, social and governance (ESG) obligations of mining companies should help to ensure that water and land resources as well as post-mining employment opportunities are considered as a part of mine closure. Integrating microalgae production into mine closure plans is a potential opportunity for mining companies to improve many ESG outcomes. Mine sites with sufficient suitable land and water resources in high solar radiation geographies may be able to economically grow microalgae to capture atmospheric CO2, re-purpose saline mine waters, treat acidic and near-neutral pH metalliferous waters as well as produce soil ameliorants (biofertiliser, biostimulants and/or biochar) to improve mine rehabilitation outcomes. Microalgae production facilities may also provide an alternative industry and employment opportunities to help transition regional mining towns that have become reliant on mining activities. The potential economic, environmental and social benefits of using mine-influenced water for microalgae production may offer an opportunity to successfully close and transition some mining landscapes.

Preservation of Terrestrial Microorganisms and Organics Within Alteration Products of Chondritic Meteorites from the Nullarbor Plain, Australia.

Meteorites that fall to Earth quickly become contaminated with terrestrial microorganisms. These meteorites are out of chemical equilibrium in the environments where they fall, and equilibration promotes formation of low-temperature alteration minerals that can entomb contaminant microorganisms and thus preserve them as microfossils. Given the well-understood chemistry of meteorites and their recent discovery on Mars by rovers, a similarly weathered meteorite on Mars could preserve organic and fossil evidence of a putative past biosphere at the martian surface. Here, we used several techniques to assess the potential of alteration minerals to preserve microfossils and biogenic organics in terrestrially weathered ordinary chondrites from the Nullarbor Plain, Australia. We used acid etching of ordinary chondrites to reveal entombed fungal hyphae, modern biofilms, and diatoms within alteration minerals. We employed synchrotron X-ray fluorescence microscopy of alteration mineral veins to map the distribution of redox-sensitive elements of relevance to chemolithotrophic organisms, such as Mn-cycling bacteria. We assessed the biogenicity of fungal hyphae within alteration veins using a combination of Fourier-transform infrared spectroscopy and pyrolysis gas chromatography-mass spectrometry, which showed that alteration minerals sequester and preserve organic molecules at various levels of decomposition. Our combined analyses results show that fossil microorganisms and the organic molecules they produce are preserved within calcite-gypsum admixtures in meteorites. Furthermore, the distributions of redox-sensitive elements (e.g., Mn) within alteration minerals are localized, which qualitatively suggests that climatically or microbially facilitated element mobilization occurred during the meteorite's residency on Earth. If returned as part of a sample suite from the martian surface, ordinary chondrites could preserve similar, recognizable evidence of putative past life and/or environmental change.

Recoding of stop codons expands the metabolic potential of two novel Asgardarchaeota lineages.

Asgardarchaeota have been proposed as the closest living relatives to eukaryotes, and a total of 72 metagenome-assembled genomes (MAGs) representing six primary lineages in this archaeal phylum have thus far been described. These organisms are predicted to be fermentative heterotrophs contributing to carbon cycling in sediment ecosystems. Here, we double the genomic catalogue of Asgardarchaeota by obtaining 71 MAGs from a range of habitats around the globe, including the deep subsurface, brackish shallow lakes, and geothermal spring sediments. Phylogenomic inferences followed by taxonomic rank normalisation confirmed previously established Asgardarchaeota classes and revealed four additional lineages, two of which were consistently recovered as monophyletic classes. We therefore propose the names Candidatus Sifarchaeia class nov. and Ca. Jordarchaeia class nov., derived from the gods Sif and Jord in Norse mythology. Metabolic inference suggests that both classes represent hetero-organotrophic acetogens, which also have the ability to utilise methyl groups such as methylated amines, with acetate as the probable end product in remnants of a methanogen-derived core metabolism. This inferred mode of energy conservation is predicted to be enhanced by genetic code expansions, i.e., stop codon recoding, allowing the incorporation of the rare 21st and 22nd amino acids selenocysteine (Sec) and pyrrolysine (Pyl). We found Sec recoding in Jordarchaeia and all other Asgardarchaeota classes, which likely benefit from increased catalytic activities of Sec-containing enzymes. Pyl recoding, on the other hand, is restricted to Sifarchaeia in the Asgardarchaeota, making it the first reported non-methanogenic archaeal lineage with an inferred complete Pyl machinery, likely providing members of this class with an efficient mechanism for methylamine utilisation. Furthermore, we identified enzymes for the biosynthesis of ester-type lipids, characteristic of bacteria and eukaryotes, in both newly described classes, supporting the hypothesis that mixed ether-ester lipids are a shared feature among Asgardarchaeota.

Accelerating microbial iron cycling promotes re-cementation of surface crusts in iron ore regions.

Accelerating microbial iron cycling is an innovative environmentally responsible strategy for mine remediation. In the present study, we extend the application of microbial iron cycling in environmental remediation, to include biocementation for the aggregation and stabilization of mine wastes. Microbial iron reduction was promoted monthly for 10 months in crushed canga (a by-product from iron ore mining, dominated by crystalline iron oxides) in 1 m3 containers. Ferrous iron concentrations reached 445 ppm in treatments and diverse lineages of the candidate phyla radiation dominated pore waters, implicating them in fermentation and/or metal cycling in this system. After a 6-month evaporation period, iron-rich cements had formed between grains and 20-cm aggregates were recoverable from treatments throughout the 1-m depth profile, while material from untreated and water-only controls remained unconsolidated. Canga-adapted plants seeded into one of the treatments germinated and grew well. Therefore, application of this geobiotechnology offers promise for stabilization of mine wastes, as well as re-formation of surface crusts that underpin unique and threatened plant ecosystems in iron ore regions.

Biocement stabilization of an experimental-scale artificial slope and the reformation of iron-rich crusts.

Novel biotechnologies are required to remediate iron ore mines and address the increasing number of tailings (mine waste) dam collapses worldwide. In this study, we aimed to accelerate iron reduction and oxidation to stabilize an artificial slope. An open-air bioreactor was inoculated with a mixed consortium of microorganisms capable of reducing iron. Fluid from the bioreactor was allowed to overflow onto the artificial slope. Carbon sources from the bioreactor fluid promoted the growth of a surface biofilm within the artificial slope, which naturally aggregated the crushed grains. The biofilms provided an organic framework for the nucleation of iron oxide minerals. Iron-rich biocements stabilized the artificial slope and were significantly more resistant to physical deformation compared with the control experiment. These biotechnologies highlight the potential to develop strategies for mine remediation and waste stabilization by accelerating the biogeochemical cycling of iron.

A widely distributed hydrogenase oxidises atmospheric H2 during bacterial growth.

Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H2) using group 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to recycle H2 produced by nitrogenase. However, given this hydrogenase is also present in various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in bacterial metabolism. Here we investigated the role of this enzyme in three species from different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated during exponential growth compared to stationary phase, in contrast to the profile of the persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays confirmed that all three strains aerobically respire H2 to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H2 supported mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, we used phylogenomic analyses to show that this hydrogenase is widely distributed and is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric model of the physiological role of atmospheric H2 oxidation and extend this process to two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have broader relevance for understanding how bacteria conserve energy in different environments and control the biogeochemical cycling of atmospheric trace gases.

Characterisation of iron oxide encrusted microbial fossils.

Robust methods for the characterisation of microbial biosignatures in geological matrices is critical for developing mineralogical biosignatures. Studying microbial fossils is fundamental for our understanding of the role microorganisms have played in elemental cycling in modern and ancient environments on Earth and potentially Mars. Here, we aim to understand what promotes the fossilisation of microorganisms after the initial stages of biomineralisation, committing bacteriomorphic structures to the geological record within iron-rich environments. Mineral encrusted cell envelope structures were routinely identified within a goethite-rich vein that cross-cut the saprolite (iron ore) of a weathered banded iron formation (BIF) system in the Quadrilátero Ferrífero, Brazil. The preservation of potential organic and mineralogical biosignatures associated with these fossils was characterised using the following high-resolution analytical techniques: scanning and transmission electron microscopy, focused ion beam scanning electron microscopy, nanoscale secondary ion mass spectrometry, synchrotron-based Fourier transform infrared spectroscopy and Raman spectroscopy. Electron microscopy demonstrated that mineral nucleation associated with a range of cell envelope structures typically followed the extant cell templates. These biologically-influenced iron-rich minerals are microcrystalline with minimal secondary growth. In contrast, intracellular mineralisation formed larger minerals that grew inward from the cell membrane to infill intracellular voids after cell death. A three dimensional reconstruction of encrusted cell envelopes in a fossilised biofilm suggests that microorganisms may be able to replicate, during the initial stages of mineralisation. Carbon and nitrogen signatures are preserved associated with the cell envelope structures; however, there were no conclusive mineralogical biosignatures associated with the mineralised cell envelopes highlighting the classical importance of morphology and elemental biosignatures in determining the biogenicity of bacteriomorphic structures.

Biogeochemical cycling of iron oxides in the rhizosphere of plants grown on ferruginous duricrust (canga).

Goethite-cemented duricrusts, also known as canga, commonly occur as a capping rock protecting underlying iron ore deposits. The processes that govern canga formation are still unclear but include recurrent partial dissolution and recrystallisation of goethite through biogeochemical cycling of iron, hypothesised to be catalysed by plants and bacteria. In the present study, the effect of plant exudates on mobilisation of iron in canga was examined using model plants grown on crushed canga in RHIZOtest devices, which separate roots from substrate by a semi-permeable membrane. Moderate plant-induced acidification of the canga was detected, however the primary driver of mineral dissolution was the synergistic effect of reductive and ligand-promoted dissolution, identified by an increase in organic acids concentration and the presence of low concentrations of free ferrous iron. Whilst organic acids exudation lasted, iron cations were stabilised in solution; once the organic acids were degraded by microorganisms, the free cations precipitated as iron oxy-hydroxides. Mineralogical analysis and high-resolution microscopy confirmed our hypothesis that plants that grow in this iron-rich substrate contribute to iron dissolution indirectly (e.g., during phosphate solubilisation), and that the resulting surplus iron not taken up by the plants is redeposited, promoting the cementation of the residual minerals. Understanding the contribution of plants to the iron cycling in canga is crucial when formulating post-mining rehabilitation strategies for iron ore sites.

Eukaryotic Colonization of Micrometer-Scale Cracks in Rocks: A "Microfluidics" Experiment Using Naturally Weathered Meteorites from the Nullarbor Plain, Australia.

The advent of microfluidics has revolutionized the way we understand how microorganisms propagate through microporous spaces. Here, we apply this understanding to the study of how endolithic environmental microorganisms colonize the interiors of sterile rock. The substrates used for our study are stony meteorites from the Nullarbor Plain, Australia; a semiarid limestone karst that provides an ideal setting for preserving meteorites. Periodic flooding of the Nullarbor provides a mechanism by which microorganisms and exogenous nutrients may infiltrate meteorites. Our laboratory experiments show that environmental microorganisms reach depths greater than 400 µm by propagating through existing brecciation, passing through cracks no wider than the diameter of a resident cell (i.e., ∼5 µm). Our observations are consistent with the propagation of these eukaryotic cells via growth and cell division rather than motility. The morphology of the microorganisms changed as a result of propagation through micrometer-scale cracks, as has been observed previously for bacteria on microfluidic chips. It has been suggested that meteorites could have served as preferred habitats for microorganisms on ancient Mars. Based on our results, the depths reached by terrestrial microorganisms within meteorites would be sufficient to mitigate against the harmful effects of ionizing radiation, such as UV light, in Earth's deserts and potentially on Mars, if similar processes of microbial colonization had once been active there. Thus, meteorites landing in ancient lakes on Mars, that later dried out, could have been some of the last inhabited locations on the surface, serving as refugia before the planet's surface became inhospitable. Finally, our observations suggest that terrestrial microorganisms can colonize very fine cracks within meteorites (and potentially spaceships and rovers) on unexpectedly short timescales, with important implications for both recognition of extraterrestrial life in returned geological samples and planetary protection.

Biogeochemical cycling of iron: Implications for biocementation and slope stabilisation.

Microbial biofilms growing in iron-rich seeps surrounding Lake Violão, Carajás, Brazil serve as a superb natural system to study the role of iron cycling in producing secondary iron cements. These seeps flow across iron duricrusts (referred to as canga in Brazil) into hydraulically restricted lakes in northern Brazil. Canga caps all of the iron ore deposits in Brazil, protecting them from being destroyed by erosion in this active weathering environment. Biofilm samples collected from these seeps demonstrated heightened biogeochemical iron cycling, contributing to the relatively rapid, seasonal formation of iron-rich cements. The seeps support iron-oxidising lineages including Sideroxydans, Gallionella, and an Azoarcus species revealed by 16S rRNA gene sequencing. In contrast, a low relative abundance of putative iron reducers; for example, Geobacter species (<5% of total sequences in any sample), corresponds to carbon limitation in this canga-associated ecosystem. This carbon limitation is likely to restrict anoxic niches to within biofilms. Examination of a canga rock sample collected from the edge of Lake Violão revealed an array of well- to poorly-preserved microbial fossils in secondary iron cements. These heterogeneous cements preserved bacterial cell envelopes and possibly extracellular polymeric substances within the microfossil iron-rich cements (termed biocements). Bacterial iron reduction initiates the sequence, and intuitively is the rate-limiting step in this broadly aerobic environment. The organic framework of the active- and paleo-biofilms appears to provide a scaffold for the formation of some cements within canga and likely expedites cement formation. The accelerated development of these resilient iron-rich biocements in the lake edge environment compared with surroundings duricrust-associated environments may provide insights into new approaches to remediate mined land, aiding to stabilise slopes, reduce erosion, restore functional hydrogeology and provide a substrate akin to natural canga for revegetation using endemic canga plant species, which have adapted to grow on iron-rich substrates.

Goethite Reduction by a Neutrophilic Member of the Alphaproteobacterial Genus Telmatospirillum.

In tropical iron ore regions, biologically mediated reduction of crystalline iron oxides drives ongoing iron cycling that contributes to the stability of surface duricrusts. This represents a biotechnological opportunity with respect to post-mining rehabilitation attempts, requiring re-formation of these duricrusts. However, cultivated dissimilatory iron reducing bacteria typically reduce crystalline iron oxides quite poorly. A glucose-fermenting microbial consortium capable of reducing at least 27 mmol/L goethite was enriched from an iron duricrust region. Metagenome analysis led to the recovery of a metagenome assembled genome (MAG) of an iron reducer belonging to the alphaproteobacterial genus Telmatospirillum. This is the first report of iron reduction within the Telmatospirillum and the first reported genome of an iron-reducing, neutrophilic member of the Alphaproteobacteria. The Telmatospirillum MAG encodes putative metal transfer reductases (MtrA, MtrB) and a novel, multi-heme outer membrane cytochrome for extracellular electron transfer. In the presence of goethite, short chain fatty acid production shifted significantly in favor of acetate rather than propionate, indicating goethite is a hydrogen sink in the culture. Therefore, the presence of fermentative bacteria likely promotes iron reduction via hydrogen production. Stimulating microbial fermentation has potential to drive reduction of crystalline iron oxides, the rate limiting step for iron duricrust re-formation.

Microbial Diversity in Actively Forming Iron Oxides from Weathered Banded Iron Formation Systems.

The surface crust that caps highly weathered banded iron formations (BIFs) supports a unique ecosystem that is a post-mining restoration priority in iron ore areas. Geochemical evidence indicates that biological processes drive the dissolution of iron oxide minerals and contribute to the ongoing evolution of this duricrust. However, limited information is available on present-day biogeochemical processes in these systems, particularly those that contribute to the precipitation of iron oxides and, thus, the cementation and stabilization of duricrusts. Freshly formed iron precipitates in water bodies perched on cangas in Karijini National Park, Western Australia, were sampled for microscopic and molecular analyses to understand currently active microbial contributions to iron precipitation in these areas. Microscopy revealed sheaths and stalks associated with iron-oxidizing bacteria. The iron-oxidizing lineages Sphaerotilus, Sideroxydans, and Pedomicrobium were identified in various samples and Leptothrix was common in four out of five samples. The iron-reducing bacteria Anaeromyxobacter dehalogens and Geobacter lovleyi were identified in the same four samples, with various heterotrophs and diverse cyanobacteria. Given this arid, deeply weathered environment, the driver of contemporary iron cycling in Karijini National Park appears to be iron-reducing bacteria, which may exist in anaerobic niches through associations with aerobic heterotrophs. Overall oxidizing conditions and Leptothrix iron-oxidizers contribute to net iron oxide precipitation in our sampes, rather than a closed biogeochemical cycle, which would result in net iron oxide dissolution as has been suggested for canga caves in Brazil. Enhancements in microbial iron oxide dissolution and subsequent reprecipitation have potential as a surface-crust-ecosystem remediation strategy at mine sites.

Microbial Populations of Stony Meteorites: Substrate Controls on First Colonizers.

Finding fresh, sterilized rocks provides ecologists with a clean slate to test ideas about first colonization and the evolution of soils de novo. Lava has been used previously in first colonizer studies due to the sterilizing heat required for its formation. However, fresh lava typically falls upon older volcanic successions of similar chemistry and modal mineral abundance. Given enough time, this results in the development of similar microbial communities in the newly erupted lava due to a lack of contrast between the new and old substrates. Meteorites, which are sterile when they fall to Earth, provide such contrast because their reduced and mafic chemistry commonly differs to the surfaces on which they land; thus allowing investigation of how community membership and structure respond to this new substrate over time. We conducted 16S rRNA gene analysis on meteorites and soil from the Nullarbor Plain, Australia. We found that the meteorites have low species richness and evenness compared to soil sampled from directly beneath each meteorite. Despite the meteorites being found kilometers apart, the community structure of each meteorite bore more similarity to those of other meteorites (of similar composition) than to the community structure of the soil on which it resided. Meteorites were dominated by sequences that affiliated with the Actinobacteria with the major Operational Taxonomic Unit (OTU) classified as Rubrobacter radiotolerans. Proteobacteria and Bacteroidetes were the next most abundant phyla. The soils were also dominated by Actinobacteria but to a lesser extent than the meteorites. We also found OTUs affiliated with iron/sulfur cycling organisms Geobacter spp. and Desulfovibrio spp. This is an important finding as meteorites contain abundant metal and sulfur for use as energy sources. These ecological findings demonstrate that the structure of the microbial community in these meteorites is controlled by the substrate, and will not reach homeostasis with the Nullarbor community, even after ca. 35,000 years. Our findings show that meteorites provide a unique, sterile substrate with which to test ideas relating to first-colonizers. Although meteorites are colonized by microorganisms, the microbial population is unlikely to match the community of the surrounding soil on which they fall.

The Proteome and Lipidome of Thermococcus kodakarensis across the Stationary Phase.

The majority of cells in nature probably exist in a stationary-phase-like state, due to nutrient limitation in most environments. Studies on bacteria and yeast reveal morphological and physiological changes throughout the stationary phase, which lead to an increased ability to survive prolonged nutrient limitation. However, there is little information on archaeal stationary phase responses. We investigated protein- and lipid-level changes in Thermococcus kodakarensis with extended time in the stationary phase. Adaptations to time in stationary phase included increased proportion of membrane lipids with a tetraether backbone, synthesis of proteins that ensure translational fidelity, specific regulation of ABC transporters (upregulation of some, downregulation of others), and upregulation of proteins involved in coenzyme production. Given that the biological mechanism of tetraether synthesis is unknown, we also considered whether any of the protein-level changes in T. kodakarensis might shed light on the production of tetraether lipids across the same period. A putative carbon-nitrogen hydrolase, a TldE (a protease in Escherichia coli) homologue, and a membrane bound hydrogenase complex subunit were candidates for possible involvement in tetraether-related reactions, while upregulation of adenosylcobalamin synthesis proteins might lend support to a possible radical mechanism as a trigger for tetraether synthesis.

Hydrogenotrophic culture enrichment reveals rumen Lachnospiraceae and Ruminococcaceae acetogens and hydrogen-responsive Bacteroidetes from pasture-fed cattle.

Molecular information suggests that there is a broad diversity of acetogens in the rumen, distinct from any currently isolated acetogens. We combined molecular analysis with enrichment culture techniques to investigate this diversity further. Methane-inhibited, hydrogenotrophic enrichment cultures produced acetate as the dominant end product. Acetyl-CoA synthase gene analysis revealed putative acetogens in the cultures affiliated with the Lachnospiraceae and Ruminococcaceae as has been found in other rumen studies. No formyltetrahydrofolate synthetase genes affiliating with acetogens or with 'homoacetogen similarity' scores >90% were identified. To further investigate the hydrogenotrophic populations in these cultures and link functional gene information with 16S rRNA gene identity, cultures were subcultured quickly, twice, through medium without exogenous hydrogen, followed by incubation without exogenous hydrogen. Comparison of cultures lacking hydrogen and their parent cultures revealed novel Lachnospiraceae and Ruminococcaceae that diminished in the absence of hydrogen, supporting the hypothesis that they were likely the predominant acetogens in the enrichments. Interestingly, a range of Bacteroidetes rrs sequences that demonstrated <86% identity to any named isolate also diminished in cultures lacking hydrogen. Acetogens or sulphate reducers from the Bacteroidetes have not been reported previously; therefore this observation requires further investigation.

Methanothermobacter thermautotrophicus modulates its membrane lipids in response to hydrogen and nutrient availability.

Methanothermobacter thermautotrophicus strain ΔH is a model hydrogenotrophic methanogen, for which extensive biochemical information, including the complete genome sequence, is available. Nevertheless, at the cell membrane lipid level, little is known about the responses of this archaeon to environmental stimuli. In this study, the lipid composition of M. thermautotrophicus was characterized to verify how this archaeon modulates its cell membrane components during growth phases and in response to hydrogen depletion and nutrient limitation (potassium and phosphate). As opposed to the higher abundance of phospholipids in the stationary phase of control experiments, cell membranes under nutrient, and energy stress were dominated by glycolipids that likely provided a more effective barrier against ion leakage. We also identified particular lipid regulatory mechanisms in M. thermautotrophicus, which included the accumulation of polyprenols under hydrogen-limited conditions and an increased content of sodiated adducts of lipids in nutrient-limited cells. These findings suggest that M. thermautotrophicus intensely modulates its cell membrane lipid composition to cope with energy and nutrient availability in dynamic environments.