The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

Dynamic modeling of anaerobic methane oxidation coupled to sulfate reduction: role of elemental sulfur as intermediate.

The process dynamics of anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR), and the potential role of elemental sulfur as intermediate are presented in this paper. Thermodynamic screening and experimental evidence from the literature conclude that a prominent model to describe AOM-SR is based on the concept that anaerobic methane oxidation proceeds through the production of the intermediate elemental sulfur. Two microbial groups are involved in the process: (a) anaerobic methanotrophs (ANME-2) and (b) Desulfosarcina/Desulfococcus sulfur reducers cluster (DSS). In this work, a dynamic model was developed to explore the interactions between biotic and abiotic processes to simulate the microbial activity, the chemical composition and speciation of the liquid phase, and the gas phase composition in the reactor headspace. The model includes the microbial kinetics for the symbiotic growth of ANME-2 and DSS, mass transfer phenomena between the gas and liquid phase for methane, hydrogen sulfide, and carbon dioxide and acid-base reactions for bicarbonate, sulfide, and ammonium. A data set from batch experiments, running for 250 days in artificial seawater inoculated with sediment from Marine Lake Grevelingen (The Netherlands) was used to calibrate the model. The inherent characteristics of AOM-SR make the identification of the kinetic parameters difficult due to the high correlation between them. However, by meaningfully selecting a set of kinetic parameters, the model simulates successfully the experimental data for sulfate reduction and sulfide production. The model can be considered as the basic structure for simulating continuous flow three-phase engineered systems based on AOM-SR.

Anaerobic methane oxidation coupled to sulfate reduction in a biotrickling filter: Reactor performance and microbial community analysis.

The aim of this work was to evaluate the performance of a biotrickling filter (BTF) packed with polyurethane foam and pall rings for the enrichment of microorganisms mediating anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) by activity tests and microbial community analysis. A BTF was inoculated with microorganisms from a known AOM active deep sea sediment collected at a depth of 528 m below the sea level (Alpha Mound, Gulf of Cadiz). The microbial community analysis was performed by catalyzed reporter deposition - fluorescence in situ hybridization (CARD-FISH) and 16S rRNA sequence analysis. The AOM occurrence and rates in the BTF were assessed by performing batch activity assays using 13C-labelled methane (13CH4). After an estimated start-up time of ∼20 days, AOM rates of ∼0.3 mmol l-1 day-1 were observed in the BTF, values almost 20 times higher than previously reported in a polyurethane foam packed BTF. The microbial community consisted mainly of anaerobic methanotrophs (ANME-2, 22% of the total number of cells) and sulfate reducing bacteria (SRB, 47% of the total number of cells). This study showed that the BTF is a suitable reactor configuration for the enrichment of microbial communities involved in AOM coupled to SR at ambient pressure and temperature with a relatively short start-up time.

Effect of pressure and temperature on anaerobic methanotrophic activities of a highly enriched ANME-2a community.

This study investigated the effect of temperature and methane partial pressure on the anaerobic methane-oxidizing and sulfate-reducing (AOM-SR) activities by a highly enriched ANME-2a community. The ANME-2a-enriched biomass was incubated at different pressures, i.e., 2, 10, 20, and 30 MPa at 15 °C for 80 days. The response of the microbial community with temperature was investigated in incubations at 4, 15, and 25 °C at 10 MPa. Among all tested conditions, the incubation at 10 MPa pressure and 15 °C showed the highest AOM-SR activity of the studied ANME-2a phylotype, whereas activity at 2 MPa pressure and 15 °C was almost comparative to the response at 10 MPa pressure. The finding of the most favorable conditions for AOM-SR activity by the studied AOM-SR community comparable to the in situ pressure and temperature (15 °C at 10 MPa) suggests that the studied ANME-2a phylotype was well adapted to the conditions similar to its origin. The microbial community analysis showed that the bacterial community composition shifted upon changing the incubation temperature and pressure.

Enrichment of sulfate reducing anaerobic methane oxidizing community dominated by ANME-1 from Ginsburg Mud Volcano (Gulf of Cadiz) sediment in a biotrickling filter.

This study was performed to enrich anaerobic methane-oxidizing archaea (ANME) present in sediment from the Ginsburg Mud Volcano (Gulf of Cadiz) in a polyurethane foam packed biotrickling filter (BTF). The BTF was operated at 20 (±2) °C, ambient pressure with continuous supply of methane for 248 days. Sulfate reduction with simultaneous sulfide production (accumulating ∼7 mM) after 200 days of BTF operation evidenced anaerobic oxidation of methane (AOM) coupled to sulfate reduction. High-throughput sequence analysis of 16S rRNA genes showed that after 248 days of BTF operation, the ANME clades enriched to more than 50% of the archaeal sequences, including ANME-1b (40.3%) and ANME-2 (10.0%). Enrichment of the AOM community was beneficial to Desulfobacteraceae, which increased from 0.2% to 1.8%. Both the inoculum and the BTF enrichment contained large populations of anaerobic sulfur oxidizing bacteria, suggesting extensive sulfur cycling in the BTF.

Enrichment of ANME-2 dominated anaerobic methanotrophy from cold seep sediment in an external ultrafiltration membrane bioreactor.

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a microbially mediated unique natural phenomenon with an ecological relevance in the global carbon balance and potential application in biotechnology. This study aimed to enrich an AOM performing microbial community with the main focus on anaerobic methanotrophic archaea (ANME) present in sediments from the Ginsburg mud volcano (Gulf of Cadiz), a known site for AOM, in a membrane bioreactor (MBR) for 726 days at 22 (± 3)°C and at ambient pressure. The MBR was equipped with a cylindrical external ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m/min. Sulfide production with simultaneous sulfate reduction was in equimolar ratio between days 480 and 585 of MBR operation, whereas methane consumption was in oscillating trend. At the end of the MBR operation (day 726), the enriched biomass was incubated with 13C labeled methane, 13C labeled inorganic carbon was produced and the AOM rate based on 13C-inorganic carbon was 1.2 µmol/(gdw d). Microbial analysis of the enriched biomass at 400 and 726 days of MBR operation showed that ANME-2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The major relevance of this study is the enrichment of an AOM consortium in a MBR system which can assist to explore the ecophysiology of ANME and provides an opportunity to explore the potential application of AOM.

Anaerobic Methane-Oxidizing Microbial Community in a Coastal Marine Sediment: Anaerobic Methanotrophy Dominated by ANME-3.

The microbial community inhabiting the shallow sulfate-methane transition zone in coastal sediments from marine Lake Grevelingen (The Netherlands) was characterized, and the ability of the microorganisms to carry out anaerobic oxidation of methane coupled to sulfate reduction was assessed in activity tests. In vitro activity tests of the sediment with methane and sulfate demonstrated sulfide production coupled to the simultaneous consumption of sulfate and methane at approximately equimolar ratios over a period of 150 days. The maximum sulfate reduction rate was 5 µmol sulfate per gram dry weight per day during the incubation period. Diverse archaeal and bacterial clades were retrieved from the sediment with the majority of them clustered with Euryarchaeota, Thaumarcheota, Bacteroidetes, and Proteobacteria. The 16S rRNA gene sequence analysis showed that the sediment from marine Lake Grevelingen contained anaerobic methanotrophic Archaea (ANME) and methanogens as archaeal clades with a role in the methane cycling. ANME at the studied site mainly belong to the ANME-3 clade. This study provides one of the few reports for the presence of ANME-3 in a shallow coastal sediment. Sulfate-reducing bacteria from Desulfobulbus clades were found among the sulfate reducers, however, with very low relative abundance. Desulfobulbus has previously been commonly found associated with ANME, whereas in our study, ANME-3 and Desulfobulbus were not observed simultaneously in clusters, suggesting the possibility of independent AOM by ANME-3.

Anaerobic oxidation of methane coupled to thiosulfate reduction in a biotrickling filter.

Microorganisms from an anaerobic methane oxidizing sediment were enriched with methane gas as the substrate in a biotrickling filter (BTF) using thiosulfate as electron acceptor for 213days. Thiosulfate disproportionation to sulfate and sulfide were the dominating sulfur conversion process in the BTF and the sulfide production rate was 0.5mmoll-1day-1. A specific group of sulfate reducing bacteria (SRB), belonging to the Desulforsarcina/Desulfococcus group, was enriched in the BTF. The BTF biomass showed maximum sulfate reduction rate (0.38mmoll-1day-1) with methane as sole electron donor, measured in the absence of thiosulfate in the BTF. Therefore, a BTF fed with thiosulfate as electron acceptor can be used to enrich SRB of the DSS group and activate the inoculum for anaerobic oxidation of methane coupled to sulfate reduction.

Local conditions structure unique archaeal communities in the anoxic sediments of meromictic Lake Kivu.

Meromictic Lake Kivu is renowned for its enormous quantity of methane dissolved in the hypolimnion. The methane is primarily of biological origin, and its concentration has been increasing in the past half-century. Insight into the origin of methane production in Lake Kivu has become relevant with the recent commercial extraction of methane from the hypolimnion. This study provides the first culture-independent approach to identifying the archaeal communities present in Lake Kivu sediments at the sediment-water interface. Terminal restriction fragment length polymorphism analysis suggests considerable heterogeneity in the archaeal community composition at varying sample locations. This diversity reflects changes in the geochemical conditions in the sediment and the overlying water, which are an effect of local groundwater inflows. A more in-depth look at the archaeal community composition by clone library analysis revealed diverse phylogenies of Euryarchaeota and Crenarachaeota. Many of the sequences in the clone libraries belonged to globally distributed archaeal clades such as the rice cluster V and Lake Dagow sediment environmental clusters. Several of the determined clades were previously thought to be rare among freshwater sediment Archaea (e.g., sequences related to the SAGMEG-1 clade). Surprisingly, there was no observed relation of clones to known hydrogentrophic methanogens and less than 2 % of clones were related to acetoclastic methanogens. The local variability, diversity, and novelty of the archaeal community structure in Lake Kivu should be considered when making assumptions on the biogeochemical functioning of its sediments.