Aerobic Methanotrophic Communities in the Bottom Sediments of Lake Baikal

The results of the first systematical investigation into the aerobic methanotrophic communities inhabiting the bottom sediments of Lake Baikal have been reported. Use of the radioisotopic method revealed methane consumption in 12 10- to 50-cm-long sediment cores. The maximum methane consumption rates (495–737 µl/(dm³ day) were recorded in sediments in the regions of hydrothermal vents and oil and gas occurrence. Methane consumption was most active in the surface layers of the sediments (0–4 cm); it decreased with the sediment depth and became negligible or absent at depths below 20 cm. The number of methanotrophic bacteria usually ranged from 100 to 1000 cells/cm³ of sediment and reached 1 million cells/cm³ in the regions of oil and gas occurrence. The seventeen enrichment cultures obtained were represented mainly by morphotype II methanotrophs. Phylogenetic analysis of the enrichment cultures in terms of the amino acid sequence of the α subunit of the membrane-bound methane monooxygenase (MMO) revealed the predominance of methanotrophs of the genus Methylocystis. The results obtained suggest the presence of an active aerobic methanotrophic community in Lake Baikal.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 562–571.Original Russian Text Copyright © 2005 by Gainutdinova, Eshinimaev, Tsyrenzhapova, Dagurova, Suzina, Khmelenina, Namsaraev, Trotsenko.     

Aerobic methanotrophs from the coastal thermal springs of Lake Baikal

The number, activity, and diversity of aerobic methanotrophic bacteria in the sediments of three coastal thermal springs of Lake Baikal were analyzed. The average number of methanotrophs was 10³–10⁴ cells per 1 cm³ of sediment. The highest number of methanotrophs (10⁸ cells/cm³ of silt) and the highest potential rate of methane uptake [7.7 nmol CH₄/(cm³ day)] were revealed in sediments from the Sukhaya thermal spring. The methods of molecular ecology (DGGE, FISH, analysis of pmoA gene fragments) showed the predominance in most enrichment cultures of methanotrophs of type II, i.e., of the genera Methylocystis and Methylosinus. In only one enrichment culture (from the Sukhaya thermal spring), a type I methanotroph was revealed; its similarity to Methylococcus capsulatus Bath did not exceed 80%. These results demonstrate a widespread occurrence and high activity of the aerobic methanotrophic community in the coastal thermal springs of Lake Baikal.     

Bacterial processes of the methane cycle in the bottom sediments of Baikal lake

The activity of methanogenic and methanotrophic bacteria was evaluated in bottom sediments of Lake Baikal. Methane concentration in Baikal bottom sediments varied from 0.0053 to 81.7 ml/dm3. Bacterial methane was produced at rates of 0.0004-534.7 microliters CH4/(dm3 day) and oxidized at rates of 0.005-1180 microliters CH4/(dm3 day). Peak methane production and oxidation were observed in Frolikha Bay near a methane vent. Methane was emitted into water at rates of 49.2-4340 microliters CH4/(m2 day). Rates of bacterial methane oxidation in near-bottom water layers ranged from 0.002 to 1.78 microliters/(1 day). Methanogens and methanotrophs were found to play an important role in the carbon cycle through all layers of sediments, particularly in the areas of methane vent and gas-hydrate occurrence.     

pmoA-based analysis of methanotrophs in a littoral lake sediment reveals a diverse and stable community in a dynamic environment

Diversity and community structure of aerobic methane-oxidizing bacteria in the littoral sediment of Lake Constance was investigated by cloning analysis and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the pmoA gene. Phylogenetic analysis revealed a high diversity of type I and type II methanotrophs in the oxygenated uppermost centimeter of the sediment. T-RFLP profiles indicated a high similarity between the active methanotrophic community in the oxic layer and the inactive community in an anoxic sediment layer at a 10-cm depth. There were also no major changes in community structure between littoral sediment cores sampled in summer and winter. By contrast, the fingerprint patterns showed substantial differences between the methanotrophic communities of littoral and profundal sediments.     

pmoA-Based Analysis of Methanotrophs in a Littoral Lake Sediment Reveals a Diverse and Stable Community in a Dynamic Environment

Diversity and community structure of aerobic methane-oxidizing bacteria in the littoral sediment of Lake Constance was investigated by cloning analysis and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the pmoA gene. Phylogenetic analysis revealed a high diversity of type I and type II methanotrophs in the oxygenated uppermost centimeter of the sediment. T-RFLP profiles indicated a high similarity between the active methanotrophic community in the oxic layer and the inactive community in an anoxic sediment layer at a 10-cm depth. There were also no major changes in community structure between littoral sediment cores sampled in summer and winter. By contrast, the fingerprint patterns showed substantial differences between the methanotrophic communities of littoral and profundal sediments.     

Microbial communities of the discharge zone of oil- and gas-bearing fluids in low-mineral Lake Baikal

At the site of natural ingress of oil, microbial diversity in the Central Baikal bottom sediments differing in the chemical composition of pore waters was studied by molecular biological techniques. The sediments saturated with oil and methane were found to contain members of 10 bacterial and 2 archaeal phyla. The oxidized sediment layer contained methanotrophic bacteria belonging to the Alphaproteobacteria, which had a specific structure of the pmoA gene and clustered together with uncultured methanotrophs from cold ecosystems. The upper sediment layer also contained oil-oxidizing bacteria and the alkB genes most closely related to those of Rhodococcus. The microbial community of reduced sediments exhibited lower diversity and was represented mostly by the organisms involved in hydrocarbon biodegradation.     

Methane oxidation in landfill cover soil

Methane oxidation in the cover soil of the Khmet'evo municipal landfill in Moscow oblast was investigated. Methane emission from the experimental parcel of the site was highly inhomogeneous. At a depth of 45-60 cm, the pore gas mainly consisted of CH4 (60-70%) and CO2 (30-40%). In the upper horizons of the cover soil, the concentration of these gases sharply decreased. Techniques for estimation of the methane-oxidizing activity in the cover soil of the landfill were tested. The rate of methane oxidation in the soil, the factor limiting methane emission from the surface of the site, correlated with the cell number of culturable methanotrophic bacteria. The method of indirect immunofluorescence revealed ten known species of methanotrophic bacteria in enrichment cultures obtained from samples of the cover soil. Our results also indicate the presence of unknown psychrotolerant methanotrophs that are active at the low temperatures characteristic of Moscow oblast.     

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.     

Methane formation and oxidation in the meromictic oligotrophic Lake Gek-Gel (Azerbaijan)

The production and oxidation of methane and diversity of culturable aerobic methanotrophic bacteria in the water column and upper sediments of the meromictic oligotrophic Lake Gek-Gel (Azerbaijan) were studied by radioisotope, molecular, and microbiological techniques. The rate of methane oxidation was low in the aerobic mixolimnion, increased in the chemocline, and peaked at the depth where oxygen was detected in the water column. Aerobic methanotrophic bacteria of type II belonging to the genus Methylocystis were identified in enrichment cultures obtained from the chemocline. Methane oxidation in the anaerobic water of the monimolimnion was much more intense than in the aerobic zone. However, below 29–30 m methane concentration increased and reached 68 μM at the bottom. The highest rate of methane oxidation under anaerobic conditions was revealed in the upper layer of bottom sediments. The rate of methane oxidation significantly exceeding that of methane production suggests a deep source of methane in this lake.     

Microbial communities from Arctic marine sediments respond slowly to methane addition during ex situ enrichments

Anaerobic methanotrophic archaea (ANME) consume methane in marine sediments, limiting its release to the water column, but their responses to changes in methane and sulfate supplies remain poorly constrained. To address how methane exposure may affect microbial communities and methane- and sulfur-cycling gene abundances in Arctic marine sediments, we collected sediments from offshore Svalbard that represent geochemical horizons where anaerobic methanotrophy is expected to be active, previously active, and long-inactive based on reaction-transport biogeochemical modelling of porewater sulfate profiles. Sediment slurries were incubated at in situ temperature and pressure with different added methane concentrations. Sediments from an active area of seepage began to reduce sulfate in a methane-dependent manner within months, preceding increased relative abundances of anaerobic methanotrophs ANME-1 within communities. In previously active and long-inactive sediments, sulfur-cycling Deltaproteobacteria became more dominant after 30 days, though these communities showed no evidence of methanotrophy after nearly 8 months of enrichment. Overall, enrichment conditions, but not methane, broadly altered microbial community structure across different enrichment times and sediment types. These results suggest that active ANME populations may require years to develop, and consequently microbial community composition may affect methanotrophic responses to potential large-scale seafloor methane releases in ways that provide insight for future modelling studies.     

Comparison of Aerobic Methanotrophic Communities in Littoral and Profundal Sediments of Lake Constance by a Molecular Approach

Analysis of pmoA and 16S rRNA gene clone libraries of methanotrophic bacteria in Lake Constance revealed an overall dominance of type I methanotrophs in both littoral and profundal sediments. The sediments exhibited minor differences in their methanotrophic community structures. Type X methanotrophs made up a significant part of the clone libraries only in the profundal sediment and were also found only there as a prominent peak by T-RFLP analyses.     

Anaerobic Methane Oxidation in Enrichment Cultures from Deep Sediments of a Mud Volcano Peschanka (South Baikal)

Under laboratory conditions, the microbial communities of bottom sediments of a mud volcano Peschanka (Lake Baikal) were found to carry out anaerobic methane oxidation (AOM). After 16 days of anaerobic cultivation of the enrichment cultures, methane content in the gas phase decreased, and microbial consortia were established. The content of carbon, nitrogen, and oxygen determined by energy dispersive X-ray spectroscopy (EDS) was higher than in the nearby sediment particles. The presence of bacteria of the NC10 phylum and archaea of the ANME-2d cluster was established by fluorescent in situ hybridization (FISH).     

Activity and diversity of methanotrophic bacteria at methane seeps in eastern Lake Constance sediments

The activity and community structure of aerobic methanotrophic communities were investigated at methane seeps (pockmarks) in the littoral and profundal zones of an oligotrophic freshwater lake (Lake Constance, Germany). Measurements of potential methane oxidation rates showed that sediments inside littoral pockmarks are hot spots of methane oxidation. Potential methane oxidation rates at littoral pockmark sites exceeded the rates of the surrounding sediment by 2 orders of magnitude. Terminal restriction fragment length polymorphism (T-RFLP) analysis of the pmoA gene revealed major differences in the methanotrophic community composition between littoral pockmarks and the surrounding sediments. Clone library analysis confirmed that one distinct Methylobacter-related group dominates the community at littoral pockmarks. In profundal sediments, the differences between pockmarks and surrounding sediments were found to be less pronounced.     

Shifts in identity and activity of methanotrophs in arctic lake sediments in response to temperature changes

Methane (CH(4)) flux to the atmosphere is mitigated via microbial CH(4) oxidation in sediments and water. As arctic temperatures increase, understanding the effects of temperature on the activity and identity of methanotrophs in arctic lake sediments is important to predicting future CH(4) emissions. We used DNA-based stable-isotope probing (SIP), quantitative PCR (Q-PCR), and pyrosequencing analyses to identify and characterize methanotrophic communities active at a range of temperatures (4°C, 10°C, and 21°C) in sediments (to a depth of 25 cm) sampled from Lake Qalluuraq on the North Slope of Alaska. CH(4) oxidation activity was measured in microcosm incubations containing sediments at all temperatures, with the highest CH(4) oxidation potential of 37.5 μmol g(-1) day(-1) in the uppermost (depth, 0 to 1 cm) sediment at 21°C after 2 to 5 days of incubation. Q-PCR of pmoA and of the 16S rRNA genes of type I and type II methanotrophs, and pyrosequencing of 16S rRNA genes in (13)C-labeled DNA obtained by SIP demonstrated that the type I methanotrophs Methylobacter, Methylomonas, and Methylosoma dominated carbon acquisition from CH(4) in the sediments. The identity and relative abundance of active methanotrophs differed with the incubation temperature. Methylotrophs were also abundant in the microbial community that derived carbon from CH(4), especially in the deeper sediments (depth, 15 to 20 cm) at low temperatures (4°C and 10°C), and showed a good linear relationship (R = 0.82) with the relative abundances of methanotrophs in pyrosequencing reads. This study describes for the first time how methanotrophic communities in arctic lake sediments respond to temperature variations.     

Methane oxidation by methanotrophs and its effects on the phosphate flux over the sediment-water interface in a eutrophic lake

The effect of methane oxidation in aerobic sediment on oxygen consumption and phosphate flux was investigated in diffusion chambers. The diffusion chambers consisted of two compartments separated by a Teflon membrane. In the upper chamber a thin sediment layer was present and the lower chamber was continuously flushed with gas. The hydrophobic membrane allowed for diffusion of gases from the lower chamber through the sediment layer toward the headspace of the upper chamber. In experiments with a methane oxidation rate of 9.8 mmol m^–2 day^–1, the oxygen consumption rate increased by a factor of two compared with controls without methane oxidation (8.6 vs 17.7 mmol m^–2 day^–1). Methane oxidation significantly decreased oxygen penetration depth (2.5–4.0 vs 1.0–2.0 mm). However, despite the shrinkage of the oxidized microlayer, no differences were found in phosphate flux across the sediment water interface. Batch experiments with standard additions of methane revealed that the growth of methanotrophic bacteria contributes to the phosphate uptake of aerobic sediment. From the batch experiments a molar ratio of carbon to phosphate of 45 mol:mol was calculated for the growth of methanotrophs. Results suggest that a decrease in chemical phosphate adsorption caused by a decrease in the oxygen penetration depth could be compensated for entirely by the growth of methanotrophic bacteria. Send offprint requests to: A.J.C. Sinke     

Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity

This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the α-subunit of particulate methane monooxygenase ( pmoA ) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum . Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.     

Abundance and Activity of Methanotrophic Bacteria in Littoral and Profundal Sediments of Lake Constance (Germany)

The abundances and activities of aerobic methane-oxidizing bacteria (MOB) were compared in depth profiles of littoral and profundal sediments of Lake Constance, Germany. Abundances were determined by quantitative PCR (qPCR) targeting the pmoA gene and by fluorescence in situ hybridization (FISH), and data were compared to methane oxidation rates calculated from high-resolution concentration profiles. qPCR using type I MOB-specific pmoA primers indicated that type I MOB represented a major proportion in both sediments at all depths. FISH indicated that in both sediments, type I MOB outnumbered type II MOB at least fourfold. Results obtained with both techniques indicated that in the littoral sediment, the highest numbers of methanotrophs were found at a depth of 2 to 3 cm, corresponding to the zone of highest methane oxidation activity, although no oxygen could be detected in this zone. In the profundal sediment, highest methane oxidation activities were found at a depth of 1 to 2 cm, while MOB abundance decreased gradually with sediment depth. In both sediments, MOB were also present at high numbers in deeper sediment layers where no methane oxidation activity could be observed.     

Interactions between Methane Oxidation and Nitrification in Coastal Sediments

Galveston Bay sediments exhibit substantial spatial and seasonal variability in rates of nitrification and aerobic methane oxidation. We examined the biogeochemical and microbiological controls on these processes using aerobic enrichment slurries. Potential aerobic methane and ammonia oxidation rates from unamended control slurries were compared to rates in slurries amended with methane, ammonium, or methane + ammonium. Bacterial community composition was monitored using denaturing gradient gel electrophoresis (DGGE) analysis of PCR amplified ribosomal and functional gene DNA. Potential methane and ammonia oxidation rates increased over time in sediments amended with methane and ammonium, respectively. The highest potential methane oxidation rates occurred in treatments receiving both ammonium and methane suggesting that methanotrophs in the enrichment cultures were nitrogen limited. The highest ammonia oxidation rates occurred in treatments amended with ammonium only. Treatments receiving both ammonium and methane exhibited ammonia oxidation rates and porewater ammonium concentrations similar to those measured in the unamended control suggesting that methanotrophs may have inhibited ammonia oxidation by sequestering available ammonia. Sequence analysis revealed a decrease in general bacterial community diversity over time and a shift in ammonia-oxidizing bacterial composition corresponding with methane availability. However, methanotroph community composition similarities between treatments with different relative methane oxidation rates suggest that changes in physiological activity, as well as shifts in community composition, contributed to the observed patterns in potential rates.     

Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments

Methane (CH₄) emitted from high-latitude lakes accounts for 2–6% of the global atmospheric CH₄ budget. Methanotrophs in lake sediments and water columns mitigate the amount of CH₄ that enters the atmosphere, yet their identity and activity in arctic and subarctic lakes are poorly understood. We used stable isotope probing (SIP), quantitative PCR (Q-PCR), pyrosequencing and enrichment cultures to determine the identity and diversity of active aerobic methanotrophs in the water columns and sediments (0–25 cm) from an arctic tundra lake (Lake Qalluuraq) on the north slope of Alaska and a subarctic taiga lake (Lake Killarney) in Alaska''s interior. The water column CH₄ oxidation potential for these shallow (∼2 m deep) lakes was greatest in hypoxic bottom water from the subarctic lake. The type II methanotroph, Methylocystis, was prevalent in enrichment cultures of planktonic methanotrophs from the water columns. In the sediments, type I methanotrophs (Methylobacter, Methylosoma and Methylomonas) at the sediment-water interface (0–1 cm) were most active in assimilating CH₄, whereas the type I methanotroph Methylobacter and/or type II methanotroph Methylocystis contributed substantially to carbon acquisition in the deeper (15–20 cm) sediments. In addition to methanotrophs, an unexpectedly high abundance of methylotrophs also actively utilized CH₄-derived carbon. This study provides new insight into the identity and activity of methanotrophs in the sediments and water from high-latitude lakes.     

Bacterial Processes of the Methane Cycle in Bottom Sediments of Lake Baikal

The activity of methanogenic and methanotrophic bacteria was evaluated in bottom sediments of Lake Baikal. Methane concentration in Baikal bottom sediments varied from 0.0053 to 81.7 ml/dm³. Bacterial methane was produced at rates of 0.0004–534.7 l CH₄/(dm³ day) and oxidized at rates of 0.005–1180 l CH₄/(dm³ day). Peak methane production and oxidation were observed in Frolikha Bay near a methane vent. Methane was emitted into water at rates of 49.2–4340 l CH₄/(m² day). Rates of bacterial methane oxidation in near-bottom water layers ranged from 0.002 to 1.78 l/(l day). Methanogens and methanotrophs were found to play an important role in the carbon cycle through all layers of sediments, particularly in the areas of methane vent and gas-hydrate occurrence.