Growth of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a High-Pressure Membrane Capsule Bioreactor

Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-μm-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the Desulfuromonadales and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.     

Growth and Population Dynamics of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a Continuous-Flow Bioreactor

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes (μ_ANME-2 = 0.167 · week⁻¹), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (μ_ANME-1 = 0.218 · week⁻¹). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 · week⁻¹, and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.     

Distribution of Methanogenic and Sulfate-Reducing Bacteria in Near-Shore Marine Sediments

The distribution of methanogenic and sulfate-reducing bacteria was examined in sediments from three sites off the coast of eastern Connecticut and five sites in Long Island Sound. Both bacterial groups were detected at all sites. Three distributional patterns were observed: (i) four sites exhibited methanogenic and sulfate-reducing populations which were restricted to the upper 10 to 20 cm, with a predominance of sulfate reducers; (ii) three sites in western Long Island Sound exhibited a methanogenic population most abundant in sediments deeper than those occupied by sulfate reducers; (iii) at one site that was influenced by fresh groundwater, methanogens and sulfate reducers were numerous within the same depths; however, the number of sulfate reducers varied vertically and temporally with sulfate concentrations. It was concluded that the distributions of abundant methanogenic and sulfate-reducing bacteria were mutually exclusive. Methanogenic enrichments yielded all genera of methanogens except Methanosarcina, with the methanobacteria predominating.     

Community Size and Metabolic Rates of Psychrophilic Sulfate-Reducing Bacteria in Arctic Marine Sediments

The numbers of sulfate reducers in two Arctic sediments with in situ temperatures of 2.6 and −1.7°C were determined. Most-probable-number counts were higher at 10°C than at 20°C, indicating the predominance of a psychrophilic community. Mean specific sulfate reduction rates of 19 isolated psychrophiles were compared to corresponding rates of 9 marine, mesophilic sulfate-reducing bacteria. The results indicate that, as a physiological adaptation to the permanently cold Arctic environment, psychrophilic sulfate reducers have considerably higher specific metabolic rates than their mesophilic counterparts at similarly low temperatures.     

Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments

Differences in methylmercury (CH₃Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (Desulfovibrio desulfuricans, Desulfobulbus propionicus, Desulfococcus multivorans, Desulfobacter sp. strain BG-8, and Desulfobacterium sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family Desulfobacteriaceae) than in pure cultures of members of groups that are not able to use acetate (e.g., the family Desulfovibrionaceae). Little or no Hg methylation was observed in cultures of Desulfobacterium or Desulfovibrio strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the Desulfobacterium and Desulfobacter groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family Desulfobacteriaceae have a greater potential to methylate Hg than members of the family Desulfovibrionaceae have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH₃Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH₃Hg formation and for developing remediation strategies for Hg-contaminated sediments.     

Cultivation of Autotrophic Ammonia-Oxidizing Archaea from Marine Sediments in Coculture with Sulfur-Oxidizing Bacteria

The role of ammonia-oxidizing archaea (AOA) in nitrogen cycling in marine sediments remains poorly characterized. In this study, we enriched and characterized AOA from marine sediments. Group I.1a crenarchaea closely related to those identified in marine sediments and “Candidatus Nitrosopumilus maritimus” (99.1 and 94.9% 16S rRNA and amoA gene sequence identities to the latter, respectively) were substantially enriched by coculture with sulfur-oxidizing bacteria (SOB). The selective enrichment of AOA over ammonia-oxidizing bacteria (AOB) is likely due to the reduced oxygen levels caused by the rapid initial growth of SOB. After biweekly transfers for ca. 20 months, archaeal cells became the dominant prokaryotes (>80%), based on quantitative PCR and fluorescence in situ hybridization analysis. The increase of archaeal 16S rRNA gene copy numbers was coincident with the amount of ammonia oxidized, and expression of the archaeal amoA gene was observed during ammonia oxidation. Bacterial amoA genes were not detected in the enrichment culture. The affinities of these AOA to oxygen and ammonia were substantially higher than those of AOB. [¹³C]bicarbonate incorporation and the presence and activation of genes of the 3-hydroxypropionate/4-hydroxybutyrate cycle indicated autotrophy during ammonia oxidation. In the enrichment culture, ammonium was oxidized to nitrite by the AOA and subsequently to nitrate by Nitrospina-like bacteria. Our experiments suggest that AOA may be important nitrifiers in low-oxygen environments, such as oxygen-minimum zones and marine sediments.Archaea have long been known as extremophiles, since most cultivated archaeal strains were cultivated from extreme environments, such as acidic, hot, and high-salt environments. The view of archaea as extremophiles (i.e., acidophiles, thermophiles, and halophiles) has radically changed by the application of molecular technologies, including PCR in environmental microbiology. Using Archaea-specific PCR primers, novel archaeal 16S rRNA gene sequences were discovered in seawater (23, 27). Following these discoveries, an ever-increasing and unexpectedly high variety of archaeal 16S rRNA gene sequences has been reported from diverse “nonextreme” environments (67). This indicates that archaea are, like bacteria, ubiquitous in the biosphere rather than exclusively inhabiting specific extreme niches. Archaea are abundant in water columns of some oceanic provinces (33, 36) and deep-subsea floor sediments (11, 12, 48). Despite the increasing number of reports of the diversity and abundance of these nonextreme archaea by molecular ecological studies, their physiology and ecological roles have remained enigmatic.Oxidation of ammonia, a trait long thought to be exclusive to the domain Bacteria (13), was recently suggested to be a trait of archaea of the crenarchaeal groups I.1a and I.1b, based on a metagenome analysis (79) and supported by the discovery of archaeal amoA-like genes in environmental shotgun sequencing studies of Sargasso Sea water (80) and genomic analysis of “Candidatus Cenarchaeum symbiosum,” a symbiont of a marine sponge (30). Molecular ecological studies indicated that these ammonia-oxidizing archaea (AOA) are often predominant over ammonia-oxidizing bacteria (AOB) in ocean waters (9, 53, 87), soils (17, 47), and marine sediments (61). Critical evidence for autotrophic archaeal ammonia oxidation was obtained by the characterization of the first cultivated mesophilic crenarchaeon (group I.1a), “Candidatus Nitrosopumilus maritimus SCM1,” from an aquarium (38), and a related archaeon from North Sea water (87) and subsequently by enrichment of thermophilic AOA (22, 31). Whole-genome-based phylogenetic studies recently indicated that the nonthermophilic crenarchaea, including the AOA, likely form a phylum separate from the Crenarchaeota and Euryarchaeota phyla (15, 16, 72). This proposed new phylum was called Thaumarchaeota (15).Microorganisms in marine sediments contribute significantly to global biogeochemical cycles because of their abundance (85). Nitrification is essential to the nitrogen cycle in marine sediments and may be metabolically coupled with denitrification and anaerobic ammonium oxidation, resulting in the removal of nitrogen as molecular nitrogen and the generation of greenhouse gases, such as nitrous oxide (19, 75). Compared with studies on archaeal nitrification in the marine water column, only limited information on archaeal nitrification in marine sediments is available so far. Archaeal amoA genes have been retrieved from marine and coastal sediments (8, 26, 61), and the potentially important role of AOA in nitrification has been suggested based on the abundance of archaeal amoA genes relative to that of bacterial amoA genes in surface marine sediments from Donghae (South Korea) (61). Cultivation of AOA, although difficult (38), remains essential to estimating the metabolic potential of archaea in environments such as soils (47) and marine sediments (61). Here, we report the successful enrichment of AOA of crenarchaeal group I.1a from marine sediments by employing a coculture with sulfur-oxidizing bacteria (SOB) which was maintained for ca. 20 months with biweekly transfers. In this way, we were able to characterize AOA from marine sediments, providing a clue for the role of AOA in the nitrogen cycle of marine sediments.     

Community Structure, Cellular rRNA Content, and Activity of Sulfate-Reducing Bacteria in Marine Arctic Sediments

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell⁻¹ day⁻¹), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.     

Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sediment

Substrate-electron acceptor combinations and specific metabolic inhibitors were applied to anoxic saltmarsh sediment spiked with mercuric ions (Hg²⁺) in an effort to identify, by a direct approach, the microorganisms responsible for the synthesis of hazardous monomethylmercury. 2-Bromoethane sulfonate (30 mM), a specific inhibitor of methanogens, increased monomethylmercury synthesis, whereas sodium molybdate (20 mM), a specific inhibitor of sulfate reducers, decreased Hg²⁺ methylation by more than 95%. Anaerobic enrichment and isolation procedures yielded a Desulfovibrio desulfuricans culture that vigorously methylated Hg²⁺ in culture solution and also in samples of presterilized sediment. The Hg²⁺ methylation activity of sulfate reducers is fully expressed only when sulfate is limiting and fermentable organic substrates are available. To date, sulfate reducers have not been suspected of Hg²⁺ methylation. Identification of these bacteria as the principal methylators of Hg²⁺ in anoxic sediments raises questions about the environmental relevance of previous pure culture-based methylation work.     

Metabolism of Trimethylamine, Choline, and Glycine Betaine by Sulfate-Reducing and Methanogenic Bacteria in Marine Sediments

The response of methanogenesis and sulfate reduction to trimethylamine, choline, and glycine betaine was examined in surface sediments from the intertidal region of Lowes Cove, Maine. Addition of these substrates markedly stimulated methanogenesis in the presence of active sulfate reduction, whereas addition of other substrates, including glucose, acetate, and glycine, had no effect on methane production. Sulfate reduction was stimulated simultaneously with methanogenesis by the various quaternary amines and all other substrates examined. Incubation of exogenous trimethylamine, choline, or glycine betaine with either bromoethane sulfonic acid or sodium molybdate was used to establish pathways of degradation of the substrates. Methanogenesis dominated the metabolism of trimethylamine, although limited nonmethanogenic activity, perhaps by sulfate-reducing bacteria, was observed. Acetate was oxidized primarily by sulfate reducers. Both choline and glycine betaine were fermented stoichiometrically to acetate and trimethylamine; apparently, neither substrate could be utilized directly by methanogens or sulfate reducers, and the activities of fermenters, methanogens, and sulfate reducers were all required to effect complete mineralization. These observations support the hypothesis that the presence of quaternary amines can mediate the coexistence of sulfate reduction and methanogenesis in marine surface sediments; they also implicate methanogens in the nitrogen cycle of marine sediments containing quaternary amines.     

Identification of Aerobic Methane-Oxidizing Bacteria in Coastal Sediments of the Crimean Peninsula

Microbiology - Methane oxidation rates and diversity of methane-oxidizing microorganisms in the upper sediment layers of the Crimean Peninsula coastal regions (Black Sea) were investigated....     

An Anaerobic Methane-Oxidizing Community of ANME-1b Archaea in Hypersaline Gulf of Mexico Sediments

Sediments overlying a brine pool methane seep in the Gulf of Mexico (Green Canyon 205) were analyzed using molecular and geochemical approaches to identify geochemical controls on microbial community composition and stratification. 16S rRNA gene and rRNA clone libraries, as well as mcrA gene clone libraries, showed that the archaeal community consists predominantly of ANME-1b methane oxidizers; no archaea of other ANME subgroups were found with general and group-specific PCR primers. The ANME-1b community was found in the sulfate-methane interface, where undersaturated methane concentrations of ca. 100 to 250 μM coexist with sulfate concentrations around 10 mM. Clone libraries of dsrAB genes and bacterial 16S rRNA genes show diversified sulfate-reducing communities within and above the sulfate-methane interface. Their phylogenetic profiles and occurrence patterns are not linked to ANME-1b populations, indicating that electron donors other than methane, perhaps petroleum-derived hydrocarbons, drive sulfate reduction. The archaeal component of anaerobic oxidation of methane is comprised of an active population of mainly ANME-1b in this hypersaline sediment.     

Diversity of Sulfate-Reducing Bacteria in Oxic and Anoxic Regions of a Microbial Mat Characterized by Comparative Analysis of Dissimilatory Sulfite Reductase Genes

Sequence analysis of genes encoding dissimilatory sulfite reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the Desulfonema-like organisms within the δ-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating Desulfonema-like organisms with oxic habitats.     

Electron Donors Utilized by Sulfate-Reducing Bacteria in Eutrophic Lake Sediments

Mineralization rates of ¹⁴C-labeled substrates were determined in the presence and absence of Na₂MoO₄, an inhibitor of sulfate reduction, in the profundal sediments of a shallow eutrophic lake. Sulfate reduction was inhibited by Na₂MoO₄ at all concentrations tested (0.2 to 200 mM), whereas methane production was inhibited at Na₂MoO₄ concentrations greater than 20 mM. Initial mineralization rates of glucose were unaffected by Na₂MoO₄; however, Na₂MoO₄ decreased the mineralization rates of lactate (58%), propionate (52%), an amino acid mixture (85%), and acetate (14%). These decreases in the rates of mineralization were attributed to inhibition of sulfate reduction. Hydrogen stimulated the reduction of ³⁵SO₄²⁻ 2.5- to 2.8-fold, demonstrating potential hydrogen oxidation by sulfate-reducing bacteria. These results indicate that sulfate reducers utilize an array of substrates as electron donors and are of potential significance to the in situ mineralization of lactate, propionate, and free amino acids in these sediments.     

Seasonal Rates of Methane Oxidation in Anoxic Marine Sediments

Methane concentrations and rates of methane oxidation were measured in intact sediment cores from an inshore marine sediment at Jutland, Denmark. The rates of methane oxidation, determined by the appearance of ¹⁴CO₂ from injected ¹⁴CH₄, varied with sediment depth and season. Most methane oxidation was anoxic, but oxygen may have contributed to methane oxidation at the sediment surface. Cumulative rates (0- to 12-cm depth) for methane oxidation at Kysing Fjord were 3.34, 3.48, 8.60, and 17.04 μmol m⁻² day⁻¹ for April (4°C), May (13°C), July (17°C), and August (21°C), respectively. If all of the methane was oxidized by sulfate, it would account for only 0.01 to 0.06% of the sulfate reduction. The data indicate that methane was produced, in addition to being oxidized, in the 0- to 18-cm sediment stratum.     

Comparative biochemistry of Archaea and Bacteria.

This review compares exemplary molecular and metabolic features of Archaea and Bacteria in terms of phylogenetic aspects. The results of the comparison confirm the coherence of the Archaea as postulated by Woese. Archaea and Bacteria share many basic features of their genetic machinery and their central metabolism. Similarities and distinctions allow projections regarding the nature of the common ancestor and the process of lineage diversification.     

Demethylation of Dimethylsulfoniopropionate and Production of Thiols in Anoxic Marine Sediments

Dimethylsulfoniopropionate (DMSP) is a natural product of algae and aquatic plants, particularly those from saline environments. We investigated whether DMSP could serve as a precursor of thiols in anoxic coastal marine sediments. The addition of 10 or 60 μM DMSP to anoxic sediment slurries caused the concentrations of 3-mercaptopropionate (3-MPA) and methanethiol (MSH) to increase. Antibiotics prevented the appearance of these thiols, indicating biological formation. Dimethyl sulfide (DMS) and acrylate also accumulated after the addition of DMSP, but these compounds were rapidly metabolized by microbes and did not reach high levels. Acrylate and DMS were probably generated by the enzymatic cleavage of DMSP. MSH arose from the microbial metabolism of DMS, since the direct addition of DMS greatly increased MSH production. Additions of 3-methiolpropionate gave rise to 3-MPA at rates similar to those with DMSP, suggesting that sequential demethylation of DMSP leads to 3-MPA formation. Only small amounts of MSH were liberated from 3-methiolpropionate, indicating that demethiolation was not a major transformation for 3-methiolpropionate. We conclude that DMSP was degraded in anoxic sediments by two different pathways. One involved the well-known enzymatic cleavage to acrylate and DMS, with DMS subsequently serving as a precursor of MSH. In the other pathway, successive demethylations of the sulfur atom proceeded via 3-methiolpropionate to 3-MPA.     

Effect of Salinity on Mercury-Methylating Activity of Sulfate-Reducing Bacteria in Estuarine Sediments

The biomethylation of mercury was measured in anoxic estuarine sediments that ranged in salinity from 0.03 to 2.4% with or without added molybdate, an inhibitor of sulfate reducers. Mercury methylation was inhibited by molybdate by more than 95%, regardless of sediment salinity. In the absence of inhibitor, high-salinity sediments methylated mercury at only 40% of the level observed in low-salinity sediments. In response to molybdate inhibition of sulfate reducers, methanogenesis increased up to 258% in high-salinity sediments but only up to 25% in low-salinity sediments. In contrast to an earlier low-salinity isolate, a Desulfovibrio desulfuricans strain from high-salinity sediment required 0.5 M sodium for optimal growth and mercury methylation activity. The formation of negatively charged mercuric chloride complexes at high salinity did not noticeably interfere with the methylation process. Results of these studies demonstrate that sulfate reducers are responsible for mercury methylation in anoxic estuarine sediments, regardless of the prevailing salinity.     

Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.