Microbial methane cycling in sediments of Arctic thermokarst lagoons.

Thermokarst lagoons represent the transition state from a freshwater lacustrine to a marine environment, and receive little attention regarding their role for greenhouse gas production and release in Arctic permafrost landscapes. We studied the fate of methane (CH4 ) in sediments of a thermokarst lagoon in comparison to two thermokarst lakes on the Bykovsky Peninsula in northeastern Siberia through the analysis of sediment CH4 concentrations and isotopic signature, methane-cycling microbial taxa, sediment geochemistry, lipid biomarkers, and network analysis. We assessed how differences in geochemistry between thermokarst lakes and thermokarst lagoons, caused by the infiltration of sulfate-rich marine water, altered the microbial methane-cycling community. Anaerobic sulfate-reducing ANME-2a/2b methanotrophs dominated the sulfate-rich sediments of the lagoon despite its known seasonal alternation between brackish and freshwater inflow and low sulfate concentrations compared to the usual marine ANME habitat. Non-competitive methylotrophic methanogens dominated the methanogenic community of the lakes and the lagoon, independent of differences in porewater chemistry and depth. This potentially contributed to the high CH4 concentrations observed in all sulfate-poor sediments. CH4 concentrations in the freshwater-influenced sediments averaged 1.34 ± 0.98 µmol g-1 , with highly depleted δ13 C-CH4 values ranging from -89‰ to -70‰. In contrast, the sulfate-affected upper 300 cm of the lagoon exhibited low average CH4 concentrations of 0.011 ± 0.005 µmol g-1 with comparatively enriched δ13 C-CH4 values of -54‰ to -37‰ pointing to substantial methane oxidation. Our study shows that lagoon formation specifically supports methane oxidizers and methane oxidation through changes in pore water chemistry, especially sulfate, while methanogens are similar to lake conditions.

Metabolic potential of microbial communities from ferruginous sediments.

Ferruginous (Fe-rich, SO4 -poor) conditions are generally restricted to freshwater sediments on Earth today, but were likely widespread during the Archean and Proterozoic Eons. Lake Towuti, Indonesia, is a large ferruginous lake that likely hosts geochemical processes analogous to those that operated in the ferruginous Archean ocean. The metabolic potential of microbial communities and related biogeochemical cycling under such conditions remain largely unknown. We combined geochemical measurements (pore water chemistry, sulfate reduction rates) with metagenomics to link metabolic potential with geochemical processes in the upper 50 cm of sediment. Microbial diversity and quantities of genes for dissimilatory sulfate reduction (dsrAB) and methanogenesis (mcrA) decrease with increasing depth, as do rates of potential sulfate reduction. The presence of taxa affiliated with known iron- and sulfate-reducers implies potential use of ferric iron and sulfate as electron acceptors. Pore-water concentrations of acetate imply active production through fermentation. Fermentation likely provides substrates for respiration with iron and sulfate as electron donors and for methanogens that were detected throughout the core. The presence of ANME-1 16S and mcrA genes suggests potential for anaerobic methane oxidation. Overall our data suggest that microbial community metabolism in anoxic ferruginous sediments support coupled Fe, S and C biogeochemical cycling.

Global distribution of microbial abundance and biomass in subseafloor sediment.

The global geographic distribution of subseafloor sedimentary microbes and the cause(s) of that distribution are largely unexplored. Here, we show that total microbial cell abundance in subseafloor sediment varies between sites by ca. five orders of magnitude. This variation is strongly correlated with mean sedimentation rate and distance from land. Based on these correlations, we estimate global subseafloor sedimentary microbial abundance to be 2.9⋅10(29) cells [corresponding to 4.1 petagram (Pg) C and ∼0.6% of Earth's total living biomass]. This estimate of subseafloor sedimentary microbial abundance is roughly equal to previous estimates of total microbial abundance in seawater and total microbial abundance in soil. It is much lower than previous estimates of subseafloor sedimentary microbial abundance. In consequence, we estimate Earth's total number of microbes and total living biomass to be, respectively, 50-78% and 10-45% lower than previous estimates.

Microbial assemblages in Arctic coastal thermokarst lakes and lagoons.

Several studies have investigated changes in microbial community composition in thawing permafrost landscapes, but microbial assemblages in the transient ecosystems of the Arctic coastline remain poorly understood. Thermokarst lakes, abrupt permafrost thaw features, are widespread along the pan-Arctic coast and transform into thermokarst lagoons upon coastal erosion and sea-level rise. This study looks at the effect of marine water inundation (imposing a sulfate-rich, saline environment on top of former thermokarst lake sediments) on microbial community composition and the processes potentially driving microbial community assembly. In the uppermost lagoon sediment influenced from marine water inflow, the microbial structures were significantly different from those deeper in the lagoon sediment and from those of the lakes. In addition, they became more similar along depth compared with lake communities. At the same time, the diversity of core microbial consortia community decreased compared with the lake sediments. This work provides initial observational evidence that Arctic thermokarst lake to lagoon transitions do not only substantially alter microbial communities but also that this transition has a larger effect than permafrost thaw and lake formation history.

Microbial anabolic and catabolic utilization of hydrocarbons in deep subseafloor sediments of Guaymas Basin.

Guaymas Basin, located off the Gulf of California, is a hydrothermally active marginal basin. Due to steep geothermal gradients and localized heating by sill intrusions, microbial substrates like short-chain fatty acids and hydrocarbons are abiotically produced from sedimentary organic matter at comparatively shallow depths. We analyzed the effect of hydrocarbons on uptake of hydrocarbons by microorganisms via nanoscale secondary ion mass spectrometry (NanoSIMS) and microbial sulfate reduction rates (SRR), using samples from two drill sites sampled by IODP Exp. 385 (U1545C and U1546D). These sites are in close proximity of each other (ca. 1 km) and have very similar sedimentology. Site U1546D experienced the intrusion of a sill that has since then thermally equilibrated with the surrounding sediment. Both sites currently have an identical geothermal gradient, despite their different thermal history. The localized heating by the sill led to thermal cracking of sedimentary organic matter and formation of potentially bioavailable organic substrates. There were low levels of hydrocarbon and nitrogen uptake in some samples from both sites, mostly in surficial samples. Hydrocarbon and methane additions stimulated SRR in near-seafloor samples from Site U1545C, while samples from Site U1546D reacted positively only on methane. Our data indicate the potential of microorganisms to metabolize hydrocarbons even in the deep subsurface of Guaymas Basin.

Microbial impact on initial soil formation in arid and semiarid environments under simulated climate change.

The microbiota is attributed to be important for initial soil formation under extreme climate conditions, but experimental evidence for its relevance is scarce. To fill this gap, we investigated the impact of in situ microbial communities and their interrelationship with biocrust and plants compared to abiotic controls on soil formation in initial arid and semiarid soils. Additionally, we assessed the response of bacterial communities to climate change. Topsoil and subsoil samples from arid and semiarid sites in the Chilean Coastal Cordillera were incubated for 16 weeks under diurnal temperature and moisture variations to simulate humid climate conditions as part of a climate change scenario. Our findings indicate that microorganism-plant interaction intensified aggregate formation and stabilized soil structure, facilitating initial soil formation. Interestingly, microorganisms alone or in conjunction with biocrust showed no discernible patterns compared to abiotic controls, potentially due to water-masking effects. Arid soils displayed reduced bacterial diversity and developed a new community structure dominated by Proteobacteria, Actinobacteriota, and Planctomycetota, while semiarid soils maintained a consistently dominant community of Acidobacteriota and Proteobacteria. This highlighted a sensitive and specialized bacterial community in arid soils, while semiarid soils exhibited a more complex and stable community. We conclude that microorganism-plant interaction has measurable impacts on initial soil formation in arid and semiarid regions on short time scales under climate change. Additionally, we propose that soil and climate legacies are decisive for the present soil microbial community structure and interactions, future soil development, and microbial responses.

An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting.

Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ~50% of cells were recovered from deep samples (100-365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 10(4)-10(8) cells cm(-3). We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.

Liquid scintillation counting at the limit of detection in biogeosciences.

Liquid scintillation is widely used to quantify the activity of radioisotopes. We present an overview of the technique and its application to biogeosciences, particularly for turnover rate measurements. Microbial communities and their metabolism are notoriously difficult to analyze in low energy environments as biomass is exceedingly sparse and turnover rates low. Highly sensitive methods, such as liquid scintillation counting, are required to investigate low metabolic rates and conclusively differentiate them from the background noise of the respective analyzer. We conducted a series of experiments to explore the effects of luminescence, measurement time and temperature on scintillation measurements. Luminescence, the spontaneous emission of photons, disproportionally affects samples within the first few hours after sample preparation and can be minimized by following simple guidelines. Short measurement times will negatively affect liquid scintillation analysis or if background noise makes up a significant proportion of the detected events. Measurement temperature affected liquid scintillation analysis only when the temperature during the measurement reached approximately 30°C or higher, i.e. the liquid scintillation analyzer was placed in an environment without temperature control, but not in cases where chemicals were stored at elevated temperatures prior to measurement. Basic understanding on the functionality of a liquid scintillation analyzer and simple precautions prior to the measurement can significantly lower the minimum detection limit and therefore allow for determination of low turnover rates previously lost in the background noise.

A modified isooctane-based DNA extraction method from crude oil.

Microbes from oil reservoirs shape petroleum composition through processes such as biodegradation or souring. Such processes are considered economically detrimental and might pose health and safety hazards. It is therefore crucial to understand the composition of a reservoir's microbial community and its metabolic capabilities. However, such analyses are hindered by difficulties in extracting DNA from such complex fluids as crude oil. Here, we present a novel DNA extraction method from oils with a wide American Petroleum Institute (API) gravity (density) range. We investigated the ability to extract cells from oils with different solvents and surfactants, the latter both nonionic and ionic. Furthermore, we evaluated three DNA extraction methods. Overall, the best DNA yields and the highest number of 16S rRNA reads were achieved with isooctane as a solvent, followed by an ionic surfactant treatment using sodium dodecyl sulfate and DNA extraction using the PowerSoil Pro Kit (Qiagen). The final method was then applied to various oils from oil reservoirs collected in aseptic conditions. Despite the expected low cell density of 101-103 cells/ml, the new method yielded reliable results, with average 16S rRNA sequencing reads in the order of 41431 (±8860) per sample. Thermophilic, halophilic, and anaerobic taxa, which are most likely to be indigenous to the oil reservoir, were found in all samples. API gravity and DNA yield, despite the sufficient DNA obtained, did not show a correlation.

Clay-associated microbial communities and their relevance for a nuclear waste repository in the Opalinus Clay rock formation.

Microorganisms are known to be natural agents of biocorrosion and mineral transformation, thereby potentially affecting the safety of deep geological repositories used for high-level nuclear waste storage. To better understand how resident microbial communities of the deep terrestrial biosphere may act on mineralogical and geochemical characteristics of insulating clays, we analyzed their structure and potential metabolic functions, as well as site-specific mineralogy and element composition from the dedicated Mont Terri underground research laboratory, Switzerland. We found that the Opalinus Clay formation is mainly colonized by Alphaproteobacteria, Firmicutes, and Bacteroidota, which are known for corrosive biofilm formation. Potential iron-reducing bacteria were predominant in comparison to methanogenic archaea and sulfate-reducing bacteria. Despite microbial communities in Opalinus Clay being in majority homogenous, site-specific mineralogy and geochemistry conditions have selected for subcommunities that display metabolic potential for mineral dissolution and transformation. Our findings indicate that the presence of a potentially low-active mineral-associated microbial community must be further studied to prevent effects on the repository's integrity over the long term.

Enrichment of rare methanogenic Archaea shows their important ecological role in natural high-CO2 terrestrial subsurface environments.

Introduction: Long-term stability of underground CO2 storage is partially affected by microbial activity but our knowledge of these effects is limited, mainly due to a lack of sites. A consistently high flux of mantle-derived CO2 makes the Eger Rift in the Czech Republic a natural analogue to underground CO2 storage. The Eger Rift is a seismically active region and H2 is produced abiotically during earthquakes, providing energy to indigenous microbial communities. Methods: To investigate the response of a microbial ecosystem to high levels of CO2 and H2, we enriched microorganisms from samples from a 239.5 m long drill core from the Eger Rift. Microbial abundance, diversity and community structure were assessed using qPCR and 16S rRNA gene sequencing. Enrichment cultures were set up with minimal mineral media and H2/CO2 headspace to simulate a seismically active period with elevated H2. Results and discussion: Methane headspace concentrations in the enrichments indicated that active methanogens were almost exclusively restricted to enrichment cultures from Miocene lacustrine deposits (50-60 m), for which we observed the most significant growth. Taxonomic assessment showed microbial communities in these enrichments to be less diverse than those with little or no growth. Active enrichments were especially abundant in methanogens of the taxa Methanobacterium and Methanosphaerula. Concurrent to the emergence of methanogenic archaea, we also observed sulfate reducers with the metabolic ability to utilize H2 and CO2, specifically the genus Desulfosporosinus, which were able to outcompete methanogens in several enrichments. Low microbial abundance and a diverse non-CO2 driven microbial community, similar to that in drill core samples, also reflect the inactivity in these cultures. Significant growth of sulfate reducing and methanogenic microbial taxa, which make up only a small fraction of the total microbial community, emphasize the need to account for rare biosphere taxa when assessing the metabolic potential of microbial subsurface populations. The observation that CO2 and H2-utilizing microorganisms could only be enriched from a narrow depth interval suggests that factors such as sediment heterogeneity may also be important. This study provides new insight on subsurface microbes under the influence of high CO2 concentrations, similar to those found in CCS sites.

Subseafloor sedimentary life in the South Pacific Gyre.

The low-productivity South Pacific Gyre (SPG) is Earth's largest oceanic province. Its sediment accumulates extraordinarily slowly (0.1-1 m per million years). This sediment contains a living community that is characterized by very low biomass and very low metabolic activity. At every depth in cored SPG sediment, mean cell abundances are 3 to 4 orders of magnitude lower than at the same depths in all previously explored subseafloor communities. The net rate of respiration by the subseafloor sedimentary community at each SPG site is 1 to 3 orders of magnitude lower than the rates at previously explored sites. Because of the low respiration rates and the thinness of the sediment, interstitial waters are oxic throughout the sediment column in most of this region. Consequently, the sedimentary community of the SPG is predominantly aerobic, unlike previously explored subseafloor communities. Generation of H(2) by radiolysis of water is a significant electron-donor source for this community. The per-cell respiration rates of this community are about 2 orders of magnitude higher (in oxidation/reduction equivalents) than in previously explored anaerobic subseafloor communities. Respiration rates and cell concentrations in subseafloor sediment throughout almost half of the world ocean may approach those in SPG sediment.

Biological Sulfate Reduction in Deep Subseafloor Sediment of Guaymas Basin.

Sulfate reduction is the quantitatively most important process to degrade organic matter in anoxic marine sediment and has been studied intensively in a variety of settings. Guaymas Basin, a young marginal ocean basin, offers the unique opportunity to study sulfate reduction in an environment characterized by organic-rich sediment, high sedimentation rates, and high geothermal gradients (100-958°C km-1). We measured sulfate reduction rates (SRR) in samples taken during the International Ocean Discovery Program (IODP) Expedition 385 using incubation experiments with radiolabeled 35SO4 2- carried out at in situ pressure and temperature. The highest SRR (387 nmol cm-3 d-1) was recorded in near-surface sediments from Site U1548C, which had the steepest geothermal gradient (958°C km-1). At this site, SRR were generally over an order of magnitude higher than at similar depths at other sites (e.g., 387-157 nmol cm-3 d-1 at 1.9 mbsf from Site U1548C vs. 46-1.0 nmol cm-3 d-1 at 2.1 mbsf from Site U1552B). Site U1546D is characterized by a sill intrusion, but it had already reached thermal equilibrium and SRR were in the same range as nearby Site U1545C, which is minimally affected by sills. The wide temperature range observed at each drill site suggests major shifts in microbial community composition with very different temperature optima but awaits confirmation by molecular biological analyses. At the transition between the mesophilic and thermophilic range around 40°C-60°C, sulfate-reducing activity appears to be decreased, particularly in more oligotrophic settings, but shows a slight recovery at higher temperatures.

Metagenome-Assembled Genome of a Putative Methanogenic Methanosarcina sp. Strain Enriched from Terrestrial High-CO2 Subsurface Sediments.

A metagenome-assembled genome (MAG), named Methanosarcina sp. strain ERenArc_MAG2, was obtained from a 3-month-old H2/CO2 atmosphere enrichment culture, originally inoculated with 60-m deep drill core sediment collected from the tectonic Eger Rift terrestrial subsurface. Annotation of the recovered draft genome revealed putative archaeal methanogenesis genes in the deep biosphere.

Circular Metagenome-Assembled Genome of Methanobacterium sp. Strain ERen5, a Putative Methanogenic, H2-Utilizing Terrestrial Subsurface Archaeon.

A circular, single-contig Methanobacterium sp. metagenome-assembled genome (MAG) was recovered from high-CO2 enrichments inoculated with drill core material from the tectonic Eger Rift terrestrial subsurface. Annotation of the recovered MAG highlighted putative methanogenesis genes, providing valuable information on archaeal activity in the deep biosphere.

Expanding the phylogenetic distribution of cytochrome b-containing methanogenic archaea sheds light on the evolution of methanogenesis.

Methane produced by methanogenic archaea has an important influence on Earth's changing climate. Methanogenic archaea are phylogenetically diverse and widespread in anoxic environments. These microorganisms can be divided into two subgroups based on whether or not they use b-type cytochromes for energy conservation. Methanogens with b-type cytochromes have a wider substrate range and higher growth yields than those without them. To date, methanogens with b-type cytochromes were found exclusively in the phylum "Ca. Halobacteriota" (formerly part of the phylum Euryarchaeota). Here, we present the discovery of metagenome-assembled genomes harboring methyl-coenzyme M reductase genes reconstructed from mesophilic anoxic sediments, together with the previously reported thermophilic "Ca. Methylarchaeum tengchongensis", representing a novel archaeal order, designated the "Ca. Methylarchaeales", of the phylum Thermoproteota (formerly the TACK superphylum). These microorganisms contain genes required for methyl-reducing methanogenesis and the Wood-Ljundahl pathway. Importantly, the genus "Ca. Methanotowutia" of the "Ca. Methylarchaeales" encode a cytochrome b-containing heterodisulfide reductase (HdrDE) and methanophenazine-reducing hydrogenase complex that have similar gene arrangements to those found in methanogenic Methanosarcinales. Our results indicate that members of the "Ca. Methylarchaeales" are methanogens with cytochromes and can conserve energy via membrane-bound electron transport chains. Phylogenetic and amalgamated likelihood estimation analyses indicate that methanogens with cytochrome b-containing electron transfer complexes likely evolved before diversification of Thermoproteota or "Ca. Halobacteriota" in the early Archean Eon. Surveys of public sequence databases suggest that members of the lineage are globally distributed in anoxic sediments and may be important players in the methane cycle.

Detection and quantification of microbial cells in subsurface sediments.

Quantification of total cell abundance is one of the most fundamental parameters in the exploration of subsurface life. Despite all recent advances in molecular techniques, this parameter is usually determined by fluorescence microscopy. In order to obtain reliable and reproducible results, it is important not just to focus on the actual cell enumeration but also to consider the entire chain of processing. Starting with the retrieval of the sample, over subsampling and sample processing to the final step of fluorescence microscopy, there are many potential sources of contamination that have to be assessed and, if possible, avoided. Because some degree of sample contamination will always occur, it is necessary to employ some form of contamination control. Different tracers are available, each one with its specific advantages and drawbacks. In many cases, the problems arise not after the sample has arrived in a well-equipped laboratory with highly trained personnel, but much earlier at the drill site or in a field camp. In this review, I discuss the different aspects of cell enumeration in subsurface sediment, evaluating every step in the long process chain.

Prokaryotic cells of the deep sub-seafloor biosphere identified as living bacteria.

Chemical analyses of the pore waters from hundreds of deep ocean sediment cores have over decades provided evidence for ongoing processes that require biological catalysis by prokaryotes. This sub-seafloor activity of microorganisms may influence the surface Earth by changing the chemistry of the ocean and by triggering the emission of methane, with consequences for the marine carbon cycle and even the global climate. Despite the fact that only about 1% of the total marine primary production of organic carbon is available for deep-sea microorganisms, sub-seafloor sediments harbour over half of all prokaryotic cells on Earth. This estimation has been calculated from numerous microscopic cell counts in sediment cores of the Ocean Drilling Program. Because these counts cannot differentiate between dead and alive cells, the population size of living microorganisms is unknown. Here, using ribosomal RNA as a target for the technique known as catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH), we provide direct quantification of live cells as defined by the presence of ribosomes. We show that a large fraction of the sub-seafloor prokaryotes is alive, even in very old (16 million yr) and deep (> 400 m) sediments. All detectable living cells belong to the Bacteria and have turnover times of 0.25-22 yr, comparable to surface sediments.

Deep sub-seafloor prokaryotes stimulated at interfaces over geological time.

The sub-seafloor biosphere is the largest prokaryotic habitat on Earth but also a habitat with the lowest metabolic rates. Modelled activity rates are very low, indicating that most prokaryotes may be inactive or have extraordinarily slow metabolism. Here we present results from two Pacific Ocean sites, margin and open ocean, both of which have deep, subsurface stimulation of prokaryotic processes associated with geochemical and/or sedimentary interfaces. At 90 m depth in the margin site, stimulation was such that prokaryote numbers were higher (about 13-fold) and activity rates higher than or similar to near-surface values. Analysis of high-molecular-mass DNA confirmed the presence of viable prokaryotes and showed changes in biodiversity with depth that were coupled to geochemistry, including a marked community change at the 90-m interface. At the open ocean site, increases in numbers of prokaryotes at depth were more restricted but also corresponded to increased activity; however, this time they were associated with repeating layers of diatom-rich sediments (about 9 Myr old). These results show that deep sedimentary prokaryotes can have high activity, have changing diversity associated with interfaces and are active over geological timescales.

Organic matter mineralization in modern and ancient ferruginous sediments.

Deposition of ferruginous sediment was widespread during the Archaean and Proterozoic Eons, playing an important role in global biogeochemical cycling. Knowledge of organic matter mineralization in such sediment, however, remains mostly conceptual, as modern ferruginous analogs are largely unstudied. Here we show that in sediment of ferruginous Lake Towuti, Indonesia, methanogenesis dominates organic matter mineralization despite highly abundant reactive ferric iron phases like goethite that persist throughout the sediment. Ferric iron can thus be buried over geologic timescales even in the presence of labile organic carbon. Coexistence of ferric iron with millimolar concentrations of methane further demonstrates lack of iron-dependent methane oxidation. With negligible methane oxidation, methane diffuses from the sediment into overlying waters where it can be oxidized with oxygen or escape to the atmosphere. In low-oxygen ferruginous Archaean and Proterozoic oceans, therefore, sedimentary methane production was likely favored with strong potential to influence Earth's early climate.