Methanogenic Sediments Research Articles

Stand age-related effects of mangrove on archaeal methanogenesis in sediments: Community assembly and co-occurrence patterns

Mangrove sediment is a key source of methane emissions; however, archaea community structure dynamics and methanogenesis activities during long-term mangrove restoration remain unclear. In this study, microcosm incubations revealed a substantial reduction in microbial-mediated methane production potential from mangrove sediments with increasing stand age; methane production rates decreased from 0.42 ng g−1 d−1 in 6-year-old stands to 0.23 ng g−1 d−1 in 64-year-old stands. High-throughput sequencing revealed a reduction in community diversity because of specific microorganism colonization and species loss, notably a decline in the relative abundance of Bathyarchaeia in sediments of 64-year-old stands. In addition, mangrove sediments, especially those in older stands (20- and 64-year-old), had more complex and stable co-occurrence microbial networks than mudflats. Furthermore, archaea community assembly in older stands was dominated by stochastic processes wherein dispersal limitation was prominent, and that in younger stands (6- and 12-year-old) was driven by deterministic processes. The proportion of dispersal limitation of Bathyarchaeia and traditional methanogens in sediment decreased with an increase in stand age. Quantitative polymerase chain reaction analysis confirmed a decrease in Bathyarchaeia (from 3.50 to 0.54 copies g−1) and mcrA gene (from 3.83 to 0.25 copies g−1) abundance in mangrove sediments with an increase in stand age. These findings demonstrate the critical role of Bathyarchaeia in methanogenesis; the decline in microbial interactions and abundance, and the reduced proportion of dispersal limitation of Bathyarchaeia and traditional methanogens collectively contributed to the mitigation of microbial-mediated methane production potential in older mangrove stands.

Survival strategies of aerobic methanotrophs under hypoxia in methanogenic lake sediments

BackgroundMicrobial methane oxidation, methanotrophy, plays a crucial role in mitigating the release of the potent greenhouse gas methane from aquatic systems. While aerobic methanotrophy is a well-established process in oxygen-rich environments, emerging evidence suggests their activity in hypoxic conditions. However, the adaptability of these methanotrophs to such environments has remained poorly understood. Here, we explored the genetic adaptability of aerobic methanotrophs to hypoxia in the methanogenic sediments of Lake Kinneret (LK). These LK methanogenic sediments, situated below the oxidic and sulfidic zones, were previously characterized by methane oxidation coupled with iron reduction via the involvement of aerobic methanotrophs.ResultsIn order to explore the adaptation of the methanotrophs to hypoxia, we conducted two experiments using LK sediments as inoculum: (i) an aerobic "classical" methanotrophic enrichment with ambient air employing DNA stable isotope probing (DNA-SIP) and (ii) hypoxic methanotrophic enrichment with repeated spiking of 1% oxygen. Analysis of 16S rRNA gene amplicons revealed the enrichment of Methylococcales methanotrophs, being up to a third of the enriched community. Methylobacter, Methylogaea, and Methylomonas were prominent in the aerobic experiment, while hypoxic conditions enriched primarily Methylomonas. Using metagenomics sequencing of DNA extracted from these experiments, we curated five Methylococcales metagenome-assembled genomes (MAGs) and evaluated the genetic basis for their survival in hypoxic environments. A comparative analysis with an additional 62 Methylococcales genomes from various environments highlighted several core genetic adaptations to hypoxia found in most examined Methylococcales genomes, including high-affinity cytochrome oxidases, oxygen-binding proteins, fermentation-based methane oxidation, motility, and glycogen use. We also found that some Methylococcales, including LK Methylococcales, may denitrify, while metals and humic substances may also serve as electron acceptors alternative to oxygen. Outer membrane multi-heme cytochromes and riboflavin were identified as potential mediators for the utilization of metals and humic material. These diverse mechanisms suggest the ability of methanotrophs to thrive in ecological niches previously thought inhospitable for their growth.ConclusionsOur study sheds light on the ability of enriched Methylococcales methanotrophs from methanogenic LK sediments to survive under hypoxia. Genomic analysis revealed a spectrum of genetic capabilities, potentially enabling these methanotrophs to function. The identified mechanisms, such as those enabling the use of alternative electron acceptors, expand our understanding of methanotroph resilience in diverse ecological settings. These findings contribute to the broader knowledge of microbial methane oxidation and have implications for understanding and potential contribution methanotrophs may have in mitigating methane emissions in various environmental conditions.

Influence of sedimentary deposition on the microbial assembly process in Arctic Holocene marine sediments.

The sea-level rise during the Holocene (11-0 ky BP) and its resulting sedimentation and biogeochemical processes may control microbial life in Arctic sediments. To gain further insight into this interaction, we investigated a sediment core (up to 10.7 m below the seafloor) from the Chuckchi Shelf of the western Arctic Ocean using metabarcoding-based sequencing and qPCR to characterize archaeal and bacterial 16S rRNA gene composition and abundance, respectively. We found that Arctic Holocene sediments harbor local microbial communities, reflecting geochemical and paleoclimate separations. The composition of bacterial communities was more diverse than that of archaeal communities, and specifically distinct at the boundary layer of the sulfate-methane transition zone. Enriched cyanobacterial sequences in the Arctic middle Holocene (8-7 ky BP) methanogenic sediments remarkably suggest past cyanobacterial blooms. Bacterial communities were phylogenetically influenced by interactions between dispersal limitation and environmental selection governing community assembly under past oceanographic changes. The relative influence of stochastic and deterministic processes on the bacterial assemblage was primarily determined by dispersal limitation. We have summarized our findings in a conceptual model that revealed how changes in paleoclimate phases cause shifts in ecological succession and the assembly process. In this ecological model, dispersal limitation is an important driving force for progressive succession for bacterial community assembly processes on a geological timescale in the western Arctic Ocean. This enabled a better understanding of the ecological processes that drive the assembly of communities in Holocene sedimentary habitats affected by sea-level rise, such as in the shallow western Arctic shelves.

Hafnium isotope evidence for enhanced weatherability at high southern latitudes during Oceanic Anoxic Event 2

The Oceanic Anoxic Event 2 (OAE 2; ca. ∼94 Ma) represents one of the most extreme carbon cycle perturbations of the Phanerozoic, which coincided with major environmental and climate reorganization in both terrestrial and marine realms. Chemical weathering of continental silicate rocks is thought to have played a crucial role during OAE 2, through enhanced release of bio-essential nutrients to the ocean, promoting high rates of marine primary production and organic carbon burial, but also due to its effect on atmospheric CO2 drawdown, which altogether possibly drove the OAE 2 termination. Yet, the evolution of continental chemical weathering during OAE 2 remains poorly defined, especially in high-latitude regions. In this study, we present a combined hafnium-neodymium isotope investigation of the clay-size detrital fraction (△εHfclay) of late Cenomanian to early Turonian sediments from the southwest Australian margin, at a site (International Ocean Discovery Program U1516) located in the southern high latitudes (∼62°S) during the late Cretaceous. The reliability of △εHfclay as a proxy for continental chemical weathering in ancient anoxic marine sediments was assessed by analyzing a suite of samples retrieved from methanogenic sediments experiencing marine silicate weathering at ocean margins, suggesting negligible effect of reverse weathering on hafnium-neodymium isotope compositions. At Site U1516, the early stage of OAE 2 was characterized by relatively low △εHfclay values (−5.9 ± 2), typical of reduced chemical weathering in nearby continental regions. At the onset of the most prominent carbon isotope excursion, an abrupt decrease in △εHfclay points towards accelerated export of poorly weathered sediments resulting from the abrupt reactivation of river systems in southwest Australia. This period was followed by a pronounced △εHfclay shift towards positive values, indicative of intensifying chemical weathering conditions during the OAE 2 interval showing the highest δ13C anomaly. Based on these results, we posit that enhanced hydrological cycle, most likely caused by a southward shift of the westerlies, led to a large increase in weatherability at southern high latitudes during peak OAE 2 warmth. This finding provides empirical support for the potential role played by high-latitude weathering systems in driving the termination of OAE 2, via weathering-driven consumption of atmospheric CO2 and accelerated riverine fluxes of nutrients leading to enhanced organic carbon burial in marine sediments.

Using thermodynamics to obtain geochemical information from genomes.

Thermodynamic characterization of the relative stabilities of chemical compounds is a pillar of conceptual models in various fields of geosciences. Analogous models applied to genomes can yield new information about the relationship between genomes and their geochemical environments. In this perspective article, we present a chemical and thermodynamic analysis of prokaryotic lineages that have been the target of previous phylogenomic studies of evolutionary adaptation to varying redox conditions. The thermodynamic model development begins by quantifying the effects of hydrogen activity (aH2 ) and temperature on the relative stabilities of organic compounds with different carbon oxidation state. When applied to proteins instead of metabolites, the same techniques can be used to identify combinations of aH2 and temperature at which reference proteomes for Class I or Class II methanogens are relatively stable. The calculated aH2 values are compatible with reported measurements for habitats of methanogens ranging from highly reducing submarine hydrothermal systems to less reducing environments including methanogenic sediments. In contrast to the transition between the two classes of methanogenic archaea, that between basal and terrestrial groups of Thaumarchaeota (denoting the origin of ammonia-oxidizing archaea) occurs at a less-reducing redox boundary. These examples reveal the consequences of energy minimization driving evolution and show how geochemical calculations involving biomolecules can be used to quantify and better understand the coevolution of the geosphere and biosphere.

Cultivation and biogeochemical analyses reveal insights into methanogenesis in deep subseafloor sediment at a biogenic gas hydrate site

Gas hydrates deposited in subseafloor sediments are considered to primarily consist of biogenic methane. However, little evidence for the occurrence of living methanogens in subseafloor sediments has been provided. This study investigated viable methanogen diversity, population, physiology and potential activity in hydrate-bearing sediments (1–307 m below the seafloor) from the eastern Nankai Trough. Radiotracer experiments, the quantification of coenzyme F430 and molecular sequencing analysis indicated the occurrence of potential methanogenic activity and living methanogens in the sediments and the predominance of hydrogenotrophic methanogens followed by methylotrophic methanogens. Ten isolates and nine representative culture clones of hydrogenotrophic, methylotrophic and acetoclastic methanogens were obtained from the batch incubation of sediments and accounted for 0.5–76% of the total methanogenic sequences directly recovered from each sediment. The hydrogenotrophic methanogen isolates of Methanocalculus and Methanoculleus that dominated the sediment methanogen communities produced methane at temperatures from 4 to 55 °C, with an abrupt decline in the methane production rate at temperatures above 40 °C, which is consistent with the depth profiles of potential methanogenic activity in the Nankai Trough sediments in this and previous studies. Our results reveal the previously overlooked phylogenetic and metabolic diversity of living methanogens, including methylotrophic methanogenesis.

Controls on volatile fatty acid concentrations in marine sediments (Baltic Sea)

Abstract Volatile fatty acids (VFA) are key intermediates in the degradation pathway of organic matter (OM) in marine sediments. They are products of fermentation and are utilized as substrates in the terminal steps of microbial OM mineralization, such as sulfate reduction and methanogenesis. We studied the distribution of formate, acetate, propionate and butyrate concentrations in the pore water throughout 100-m deep sedimentary sequences in the Baltic Sea. The sediments included both sulfate and methane zones and were retrieved during the Integrated Ocean Drilling Program Expedition 347. VFA concentrations were consistently low throughout the sulfate and methane zones, ranging from few hundred nanomolar to about hundred micromolar, thus pointing to efficient microbial utilization in the terminal OM mineralization steps in both zones. Concentrations of formate followed a different depth trend than the other three VFAs suggesting a different mechanistical control of the formate turnover. Calculations of the Gibbs energy reveal that acetoclastic methanogenesis is exergonic (up to −30 kJ (mol CH4)−1) throughout the sulfate zone and suggest that methanogens can potentially coexist with sulfate reducers in the sulfate zone while sharing acetate as a substrate. In the deeper methane-rich sediments, the energy yield of acetoclastic methanogenesis is generally very low (less negative than −10 kJ (mol CH4)−1). The uniformly low acetate concentrations in the methane zone suggest its active microbial turnover but the pathway from acetate to methane still remains unclear. Interestingly, acetoclastic sulfate reduction still yields at least −20 kJ (mol sulfate)−1 in the methane zone and, thus, sulfate reducers may utilize acetate as electron donor in the methane zone, which is in agreement with the earlier findings of sulfate reducers in methanogenic sediments.

Trophic strategy of diverse methanogens across a river-to-sea gradient.

Methanogens are an important biogenic source of methane, especially in estuarine waters across a river-to-sea gradient. However, the diversity and trophic strategy of methanogens in this gradient are not clear. In this study, the diversity and trophic strategy of methanogens in sediments across the Yellow River (YR) to the Bohai Sea (BS) gradient were investigated by high-throughput sequencing based on the 16S rRNA gene. The results showed that the diversity of methanogens in sediments varied from multitrophic communities in YR samples to specific methylotrophic communities in BS samples. The methanogenic community in YR samples was dominated by Methanosarcina, while that of BS samples was dominated by methylotrophic Methanococcoides. The distinct methanogens suggested that the methanogenic community of BS sediments did not originate from YR sediment input. High-throughput sequencing of the mcrA gene revealed that active Methanococcoides dominated in the BS enrichment cultures with trimethylamine as the substrate, and methylotrophic Methanolobus dominated in the YR enrichment cultures, as detected to a limited amount in in situ sediment samples. Methanosarcina were also detected in this gradient sample. Furthermore, the same species of Methanosarcina mazei, which was widely distributed, was isolated from the area across a river-to-sea gradient by the culture-dependent method. In summary, our results showed that a distribution of diverse methanogens across a river-to-sea gradient may shed light on adaption strategies and survival mechanisms in methanogens.

CO2 conversion to methane and biomass in obligate methylotrophic methanogens in marine sediments

Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13C-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60–86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways (~40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Although methane formation from methanol is the primary pathway of methanogenesis, the observed high DIC incorporation into lipids is likely linked to CO2-dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.

Kinetics of organic carbon mineralization and methane formation in marine sediments (Aarhus Bay, Denmark)

Abstract Sediments were sampled at nine stations on a transect across a 7–10 m thick Holocene mud layer in Aarhus Bay, Denmark, to investigate the linkages between CH4 dynamics and the rate and depth distribution of organic matter degradation. High-resolution sulfate reduction rates determined by tracer experiments (35S-SRR) decreased by several orders of magnitude down through the mud layer. The rates showed a power law dependency on sediment age: SRR (nmol cm−3 d−1) = 106.18 × Age−2.17. The rate data were used to independently quantify enhanced SO42− transport by bioirrigation. Field data (SO42–, TCO2, T13CO2, NH4+ and CH4 concentrations) could be simulated with a reaction-transport model using the derived bioirrigation rates and assuming that the power law was continuous into the methanogenic sediments below the sulfate-methane transition zone (SMTZ). The model predicted an increase in anaerobic organic carbon mineralization rates across the transect from 2410 to 3540 nmol C cm−2 d−1 caused by an increase in the sediment accumulation rate. Although methanogenesis accounted for only ∼1% of carbon mineralization, a large relative increase in methanogenesis along the transect led to a considerable shallowing of the SMTZ from 428 to 257 cm. Methane gas bubbles appeared once a threshold in the sedimentation accumulation rate was surpassed. The 35S-measured SRR data indicated active sulfate reduction throughout the SO42− zone whereas quasi-linear SO42− gradients over the same zone indicated insignificant sulfate reduction. This apparent inconsistency, observed at all stations, was reconciled by considering the transport of SO42− into the sediment by bioirrigation, which accounted for 94 ± 2% of the total SO42− flux across the sediment-water interface. The SRR determined from the quasi-linear SO42− gradients were two orders of magnitude lower than measured rates. We conclude that models solely based on SO42− concentration gradients will not capture high SRRs at the top of the sulfate reduction zone if they do not properly account for (i) SO42− influx by bioirrigation, and/or (ii) the continuity of organic matter reactivity with sediment depth or age.

Conductive Particles Enable Syntrophic Acetate Oxidation between Geobacter and Methanosarcina from Coastal Sediments

ABSTRACTCoastal sediments are rich in conductive particles, possibly affecting microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate-oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure, and their survival depends on successful partnership. Here, we demonstrate that conductive particles enable the partnership between SAO bacteria (i.e., Geobacter spp.) and methanogens (Methanosarcina spp.) from the coastal sediments of the Bothnian Bay of the Baltic Sea. Baltic methanogenic sediments were rich in conductive minerals, had an apparent isotopic fractionation characteristic of CO2-reductive methanogenesis, and were inhabited by Geobacter and Methanosarcina. As long as conductive particles were delivered, Geobacter and Methanosarcina persisted, whereas exclusion of conductive particles led to the extinction of Geobacter. Baltic Geobacter did not establish a direct electric contact with Methanosarcina, necessitating conductive particles as electrical conduits. Within SAO consortia, Geobacter was an efficient [13C]acetate utilizer, accounting for 82% of the assimilation and 27% of the breakdown of acetate. Geobacter benefits from the association with the methanogen, because in the absence of an electron acceptor it can use Methanosarcina as a terminal electron sink. Consequently, inhibition of methanogenesis constrained the SAO activity of Geobacter as well. A potential benefit for Methanosarcina partnering with Geobacter is that together they competitively exclude acetoclastic methanogens like Methanothrix from an environment rich in conductive particles. Conductive particle-mediated SAO could explain the abundance of acetate oxidizers like Geobacter in the methanogenic zone of sediments where no electron acceptors other than CO2 are available.

Spatial distribution of dissolved methane and its source in the western Arctic Ocean

Recent Arctic warming and decreasing sea-ice can promote the release of methane (CH4), a greenhouse gas, from the Arctic Ocean, thereby providing a strong climate feedback. However, the dynamics of dissolved CH4 in the Arctic Ocean remain uncertain, especially in western areas. This report describes the horizontal and vertical distributions of concentration and stable carbon isotope ratio (δ13C value) of CH4 in the western Arctic Ocean. Surface layer samples used for this study were supersaturated with CH4 in comparison to the atmosphere. Especially high CH4 concentrations (up to 10.3 nmol kg−1) were observed at stations in the continental shelf area. At the bottom layer of the shelf stations, the CH4 concentration was higher (up to 55.9 nmol kg−1). Its δ13C value was lower (down to − 63.8‰) than in the surface layer, which suggests that CH4 in the shelf water is produced mainly by methanogens in sediment. At deeper stations in the Canada Basin (seafloor > 300 m depth), the maxima of CH4 concentration were detected at depths of 10–50 m and 100–200 m, although δ13C values were lowest at 50 m depth. The shallower CH4 maximum coincided with the DO maximum, suggesting CH4 production by plankton activity or sinking particles. The deeper CH4 maximum corresponded to the nutrient maximum, suggesting horizontal advection of shelf water from the coastal shelf area. From the results, we were able to confirm that the dynamics of dissolved CH4 in the western Arctic Ocean in summer 2012 varied with area and depth.

Depth Distribution and Assembly of Sulfate-Reducing Microbial Communities in Marine Sediments of Aarhus Bay

Most sulfate-reducing microorganisms (SRMs) present in subsurface marine sediments belong to uncultured groups only distantly related to known SRMs, and it remains unclear how changing geochemical zones and sediment depth influence their community structure. We mapped the community composition and abundance of SRMs by amplicon sequencing and quantifying the dsrB gene, which encodes dissimilatory sulfite reductase subunit beta, in sediment samples covering different vertical geochemical zones ranging from the surface sediment to the deep sulfate-depleted subsurface at four locations in Aarhus Bay, Denmark. SRMs were present in all geochemical zones, including sulfate-depleted methanogenic sediment. The biggest shift in SRM community composition and abundance occurred across the transition from bioturbated surface sediments to nonbioturbated sediments below, where redox fluctuations and the input of fresh organic matter due to macrofaunal activity are absent. SRM abundance correlated with sulfate reduction rates determined for the same sediments. Sulfate availability showed a weaker correlation with SRM abundances and no significant correlation with the composition of the SRM community. The overall SRM species diversity decreased with depth, yet we identified a subset of highly abundant community members that persists across all vertical geochemical zones of all stations. We conclude that subsurface SRM communities assemble by the persistence of members of the surface community and that the transition from the bioturbated surface sediment to the unmixed sediment below is a main site of assembly of the subsurface SRM community.IMPORTANCE Sulfate-reducing microorganisms (SRMs) are key players in the marine carbon and sulfur cycles, especially in coastal sediments, yet little is understood about the environmental factors controlling their depth distribution. Our results suggest that macrofaunal activity is a key driver of SRM abundance and community structure in marine sediments and that a small subset of SRM species of high relative abundance in the subsurface SRM community persists from the sulfate-rich surface sediment to sulfate-depleted methanogenic subsurface sediment. More generally, we conclude that SRM communities inhabiting the subsurface seabed assemble by the selective survival of members of the surface community.

Carbon isotope biogeochemistry of acetate in sub-seafloor sediments in the Sea of Okhotsk near Sakhalin Island, Russia

Biogeochemical processes involving acetate in sub-seafl oor sediments were investigated by examining the stable carbon isotopic relationships of acetate and other relevant carbon-bearing materials (i.e., total organic carbon (TOC) and ΣCO2) in fi ve piston cores retrieved from the Sea of Okhotsk off Sakhalin Island, Russia. The cores were classifi ed into two types on the basis of SO42– content: (I) those with sulfate-reducing sediments in which the depletion of pore-water SO42– with increasing depth was slight, and (II) those with methanogenic sediments in which SO42– concentrations were less than 2 mM more than 3 m below the seafl oor. Acetate was detected in all cores. The acetate content in methanogenic sediments (2.6–23.0 μM) was relatively higher than in the sulfate-reducing sediment (2.8–8.8 μM). In the sulfate-reducing sediments, the depth profi les of δ13Cacetate approximately parallel those of δ13CTOC, with the δ13Cacetate values (–39‰ to –33‰) depleted by about 12‰ relative to δ13CTOC (–24.5‰ to –22.3‰). These approximately parallel depth profi les suggest that the principal acetate production process in the sulfate-reducing sediments is fermentation of dissolved organic compounds. The fermentation products, however, tend to be similar or slightly enriched in 13C compared to their substrates. Therefore, the 13C depletion of acetate relative to TOC in the sulfate reduction zone suggests that some portion of the acetate was synthesized by acetogenesis in which the synthesized acetate is depleted in 13C compared with its precursor. Given the large contribution of land-derived organic matter in the studied sediments, organoautotrophic acetogenesis using the lignin-derived syringate monomer, which originates from land plants, is likely. In the methanogenic sediments, the δ13Cacetate values in sediments throughout the cores (–39‰ to –25‰) were depleted compared to δ13CTOC (–25.5‰ to –21.4‰). This suggests some acetogenic contribution to the total acetate production. The depth profi les of δ13Cacetate in methanogenic sediments did not parallel those of either δ13CTOC or δ13CΣCO2, probably because of the mixed isotopic effect from some production and consumption processes; namely fermentation, acetogenesis, and acetoclastic methanogenesis. * Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan ** Research Institute for Global Change, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237-0061, Japan *** Institute of Low Temperature Science, Hokkaido University, N19W8 Kita-ku, Sapporo, Hokkaido 060-0819, Japan **** Present address: Graduate School of Environmental Studies, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 4648601, Japan a Corresponding author. Tel: +81358414250. Fax: +81358418791. e-mail: ijiri@eps.s.u-tokyo.ac.jp Present address: Department of Earth and Planetary Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Microbial Sulfate Reduction Potential in Coal-Bearing Sediments Down to ~2.5 km below the Seafloor off Shimokita Peninsula, Japan.

Sulfate reduction is the predominant anaerobic microbial process of organic matter mineralization in marine sediments, with recent studies revealing that sulfate reduction not only occurs in sulfate-rich sediments, but even extends to deeper, methanogenic sediments at very low background concentrations of sulfate. Using samples retrieved off the Shimokita Peninsula, Japan, during the Integrated Ocean Drilling Program (IODP) Expedition 337, we measured potential sulfate reduction rates by slurry incubations with 35S-labeled sulfate in deep methanogenic sediments between 1276.75 and 2456.75 meters below the seafloor. Potential sulfate reduction rates were generally extremely low (mostly below 0.1 pmol cm−3 d−1) but showed elevated values (up to 1.8 pmol cm−3 d−1) in a coal-bearing interval (Unit III). A measured increase in hydrogenase activity in the coal-bearing horizons coincided with this local increase in potential sulfate reduction rates. This paired enzymatic response suggests that hydrogen is a potentially important electron donor for sulfate reduction in the deep coalbed biosphere. By contrast, no stimulation of sulfate reduction rates was observed in treatments where methane was added as an electron donor. In the deep coalbeds, small amounts of sulfate might be provided by a cryptic sulfur cycle. The isotopically very heavy pyrites (δ34S = +43‰) found in this horizon is consistent with its formation via microbial sulfate reduction that has been continuously utilizing a small, increasingly 34S-enriched sulfate reservoir over geologic time scales. Although our results do not represent in-situ activity, and the sulfate reducers might only have persisted in a dormant, spore-like state, our findings show that organisms capable of sulfate reduction have survived in deep methanogenic sediments over more than 20 Ma. This highlights the ability of sulfate-reducers to persist over geological timespans even in sulfate-depleted environments. Our study moreover represents the deepest evidence of a potential for sulfate reduction in marine sediments to date.

Culture Independent Genomic Comparisons Reveal Environmental Adaptations for Altiarchaeales.

The recently proposed candidatus order Altiarchaeales remains an uncultured archaeal lineage composed of genetically diverse, globally widespread organisms frequently observed in anoxic subsurface environments. In spite of 15 years of studies on the psychrophilic biofilm-producing Candidatus Altiarchaeum hamiconexum and its close relatives, very little is known about the phylogenetic and functional diversity of the widespread free-living marine members of this taxon. From methanogenic sediments in the White Oak River Estuary, NC, USA, we sequenced a single cell amplified genome (SAG), WOR_SM1_SCG, and used it to identify and refine two high-quality genomes from metagenomes, WOR_SM1_79 and WOR_SM1_86-2, from the same site. These three genomic reconstructions form a monophyletic group, which also includes three previously published genomes from metagenomes from terrestrial springs and a SAG from Sakinaw Lake in a group previously designated as pMC2A384. A synapomorphic mutation in the Altiarchaeales tRNA synthetase β subunit, pheT, caused the protein to be encoded as two subunits at non-adjacent loci. Consistent with the terrestrial spring clades, our estuarine genomes contained a near-complete autotrophic metabolism, H2 or CO as potential electron donors, a reductive acetyl-CoA pathway for carbon fixation, and methylotroph-like NADP(H)-dependent dehydrogenase. Phylogenies based on 16S rRNA genes and concatenated conserved proteins identified two distinct sub-clades of Altiarchaeales, Alti-1 populated by organisms from actively flowing springs, and Alti-2 which was more widespread, diverse, and not associated with visible mats. The core Alti-1 genome suggested Alti-1 is adapted for the stream environment with lipopolysaccharide production capacity and extracellular hami structures. The core Alti-2 genome suggested members of this clade are free-living with distinct mechanisms for energy maintenance, motility, osmoregulation, and sulfur redox reactions. These data suggested that the hamus structures found in Candidatus Altiarchaeum hamiconexum are not present outside of stream-adapted Altiarchaeales. Homologs to a Na+ transporter and membrane bound coenzyme A disulfide reductase that were unique to the brackish sediment Alti-2 genomes, could indicate adaptations to the estuarine, sulfur-rich environment.

Methanogenesis at extremely haloalkaline conditions in the soda lakes of Kulunda Steppe (Altai, Russia)

Microbial methanogenesis at extreme conditions of saline alkaline soda lakes has, so far, been poorly investigated. Despite the obvious domination of sulfidogenesis as the therminal anaerobic process in the hypersaline soda lakes of Kulunda Steppe (Altai, southwestern Siberia), high concentrations of methane were detected in the anaerobic sediments. Potential activity measurements with different substrates gave results significantly deviating from what is commonly found in hypersaline habitats with neutral pH. In particular, not only a non-competitive methylotrophic pathway was active, but also lithotrophic and, in some cases, even acetate-dependent methanogenesis was found to be present in hypersaline soda lake sediments. All three pathways were functioning exclusively within the alkaline pH range between 8 and 10.5, while the salt concentration was the key factor influencing the activity. Methylotrophic and, to a lesser extent, lithotrophic methanogenesis were active up to soda-saturating conditions (4 M total Na(+)). Acetate-dependent methanogenesis was observed at salinities below 3 M total Na(+). Detection of methanogens in sediments using the mcrA gene as a functional marker demonstrated domination of methylotrophic genera Methanolobus and Methanosalsum and lithotrophic Methanocalculus. In a few cases, acetoclastic Methanosaeta was detected, as well as two deep lineage methanogens. Cultivation results corresponded well to the mcrA-based observations. Enrichments for natronophilic methylotrophic methanogens resulted in isolation of Methanolobus strains at moderate salinity, while at salt concentrations above 2 M Na(+) a novel member of the genus Methanosalsum was dominating. Enrichments with H2 or formate invariably resulted in domination of close relatives of Methanocalculus natronophilus. Enrichments with acetate at low salt concentration yielded two acetoclastic alkaliphilic Methanosaeta cultures, while at salinity above 1 M Na(+) syntrophic associations were apparently responsible for the observed acetate conversion to methane. Overall, the results indicated the presence of functionally structured and active methanogenic populations in Siberian hypersaline soda lakes.

Isotopic compositions of archaeal membrane lipids in methanogenic sediments

Isotopic compositions of archaeal membrane lipids in methanogenic sediments

Iron-mediated anaerobic oxidation of methane in brackish coastal sediments.

Methane is a powerful greenhouse gas and its biological conversion in marine sediments, largely controlled by anaerobic oxidation of methane (AOM), is a crucial part of the global carbon cycle. However, little is known about the role of iron oxides as an oxidant for AOM. Here we provide the first field evidence for iron-dependent AOM in brackish coastal surface sediments and show that methane produced in Bothnian Sea sediments is oxidized in distinct zones of iron- and sulfate-dependent AOM. At our study site, anthropogenic eutrophication over recent decades has led to an upward migration of the sulfate/methane transition zone in the sediment. Abundant iron oxides and high dissolved ferrous iron indicate iron reduction in the methanogenic sediments below the newly established sulfate/methane transition. Laboratory incubation studies of these sediments strongly suggest that the in situ microbial community is capable of linking methane oxidation to iron oxide reduction. Eutrophication of coastal environments may therefore create geochemical conditions favorable for iron-mediated AOM and thus increase the relevance of iron-dependent methane oxidation in the future. Besides its role in mitigating methane emissions, iron-dependent AOM strongly impacts sedimentary iron cycling and related biogeochemical processes through the reduction of large quantities of iron oxides.

Real-time monitoring of subsurface microbial metabolism with graphite electrodes.

Monitoring in situ microbial activity in anoxic submerged soils and aquatic sediments can be labor intensive and technically difficult, especially in dynamic environments in which a record of changes in microbial activity over time is desired. Microbial fuel cell concepts have previously been adapted to detect changes in the availability of relatively high concentrations of organic compounds in waste water but, in most soils and sediments, rates of microbial activity are not linked to the concentrations of labile substrates, but rather to the turnover rates of the substrate pools with steady state concentrations in the nM–μM range. In order to determine whether levels of current produced at a graphite anode would correspond to the rates of microbial metabolism in anoxic sediments, small graphite anodes were inserted in sediment cores and connected to graphite brush cathodes in the overlying water. Currents produced were compared with the rates of [2-14C]-acetate metabolism. There was a direct correlation between current production and the rate that [2-14C]-acetate was metabolized to 14CO2 and 14CH4 in sediments in which Fe(III) reduction, sulfate reduction, or methane production was the predominant terminal electron-accepting process. At comparable acetate turnover rates, currents were higher in the sediments in which sulfate-reduction or Fe(III) reduction predominated than in methanogenic sediments. This was attributed to reduced products (Fe(II), sulfide) produced at distance from the anode contributing to current production in addition to the current that was produced from microbial oxidation of organic substrates with electron transfer to the anode surface in all three sediment types. The results demonstrate that inexpensive graphite electrodes may provide a simple strategy for real-time monitoring of microbial activity in a diversity of anoxic soils and sediments.