Different Temperature Optima for Methane Formation When Enrichments from Acid Peat Are Supplemented with Acetate or Hydrogen

Laboratory studies of methane formation in peat samples from an acid subarctic mire in Sweden indicated the presence of a low-temperature-adapted methanogenic flora. Enrichment culture studies with ethanol, acetate, hydrogen, or a combination of these as substrate for methane formation provided evidence for the existence of two different methanogenic populations in the peat: one, unaffected by hydrogen and using acetate, with a temperature optimum at 20°C; the other, oxidizing hydrogen, with a temperature optimum at ca. 28°C.     

Anaerobic Methane Oxidation: Occurrence and Ecology

Anoxic sediments and digested sewage sludge anaerobically oxidized methane to carbon dioxide while producing methane. This strictly anaerobic process showed a temperature optimum between 25 and 37°C, indicating an active microbial participation in this reaction. Methane oxidation in these anaerobic habitats was inhibited by oxygen. The rate of the oxidation followed the rate of methane production. The observed anoxic methane oxidation in Lake Mendota and digested sewage sludge was more sensitive to 2-bromoethanesulfonic acid than the simultaneous methane formation. Sulfate diminished methane formation as well as methane oxidation. However, in the presence of iron and sulfate the ratio of methane oxidized to methane formed increased markedly. Manganese dioxide and higher partial pressures of methane also stimulated the oxidation. The rate of methane oxidation in untreated samples was approximately 2% of the CH₄ production rate in Lake Mendota sediments and 8% of that in digested sludge. This percentage could be increased up to 90% in sludge in the presence of 10 mM ferrous sulfate and at a partial pressure of methane of 20 atm (2,027 kPa).     

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.Subject terms: Biogeochemistry, Biodiversity, Environmental microbiology     

Evidence for the Existence of Psychrophilic Methanogenic Communities in Anoxic Sediments of Deep Lakes

In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5°C), slurries were first incubated at temperatures between 4 and 60°C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6°C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15°C, with maximal rates in slurries kept at 6°C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6°C after preincubation at 30°C.     

Methanogenesis from Methylated Amines in a Hypersaline Algal Mat

Methane ebullition and high rates of methane production were observed in sediments of a hypersaline pond (180‰) which contained sulfate in excess of 100 mM. The highest rates of methane production were observed in surface sediments associated with an algal mat dominated by a Spirulina sp. The mat contained a methylated amine, glycine betaine (GBT), at levels which accounted for up to 20% of the total mat nitrogen. GBT was apparently the source of trimethylamine (TMA), which was also present in the sediment at relatively high concentrations. Patterns of substrate metabolism by the methanogenic populations in sediment slurries suggested that TMA was a major methane precursor. Neither exogenous hydrogen nor acetate stimulated methanogenesis, while addition of a variety of amines including TMA, trimethylamine oxide, GBT, and choline resulted in substantial increases with yields of >70%. The temperature optimum for methanogenesis in this system was 45 to 55°C, which coincided with the observed sediment temperature. Patterns and rates of methane production in this and other hypersaline algal mats may be determined by a complex interaction between salinity, the use of methylated amines for osmoregulation by algae, and the formation of TMA by fermentation.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Methane Production in Minnesota Peatlands

Rates of methane production in Minnesota peats were studied. Surface (10- to 25-cm) peats produced an average of 228 nmol of CH₄ per g (dry weight) per h at 25°C and ambient pH. Methanogenesis rates generally decreased with depth in ombrotrophic peats, but on occasion were observed to rise within deeper layers of certain fen peats. Methane production was temperature dependent, increasing with increasing temperature (4 to 30°C), except in peats from deeper layers. Maximal methanogenesis from these deeper regions occurred at 12°C. Methane production rates were also pH dependent. Two peats with pHs of 3.8 and 4.3 had an optimum rate of methane production at pH 6.0. The addition to peat of glucose and H₂-CO₂ stimulated methanogenesis, whereas the addition of acetate inhibited methanogenesis. Cysteine-sulfide, nitrogen-phosphorus-trace metals, and vitamins-yeast extract affected methane production very little. Various gases were found to be trapped or dissolved (or both) within peatland waters. Dissolved methane increased linearly to a depth of 210 cm. The accumulation of metabolic end products produced within peat bogs appears to be an important mechanism limiting carbon turnover in peatland environments.     

Isolation and Characterization of Methanomicrobium paynteri sp. nov., a Mesophilic Methanogen Isolated from Marine Sediments

A new mesophilic methanogenic bacterial species isolated from marine sediments collected in the Cayman Islands is described. Cells are small rods occuring singly without filaments, are not motile, and do not possess flagella. Colonies are semitransparent and off-white in color. After 2 weeks of incubation at 37°C colonies are 1 to 2 mm in size, circular, and have entire edges. Only hydrogen-carbon dioxide is a substrate for growth and methane formation. Cells can tolerate a variety of organic secondary buffers (bicarbonate-CO₂ being the primary buffer). Cells do not require yeast extract or Trypticase, but do require acetate, for growth. The optimum growth temperature is 40°C. The optimum sodium concentration is 0.15 M. The optimum pH for growth is 7.0. The minimum generation time is 4.8 h. The DNA base composition is 44.9 mol% guanine plus cytosine. The name Methanomicrobium paynteri is proposed in honor of M. J. B. Paynter. The type strain is G-2000 (=ATCC 33997, =DSM 2545).     

Effect of Temperature and Retention Time on Methane Production from Beef Cattle Waste

The effect of temperature and retention time on the rate of methane production from waste of beef cattle fed a finishing diet was investigated by using continuously mixed 3-liter working volume anaerobic fermentors. The temperatures ranged from 30 to 65°C with 5°C increments between fermentors. The fermentors were fed once per day with 6% volatile solids (organic matter). Retention time for each temperature was varied from 18 to 2.5 days. After 3-volume turnovers, samples were obtained on 4 consecutive days. The highest methane production rate (liters/liter of fermentor per day) and methane yield at that rate (liters/gram of volatile solids) were 1.27 and 0.19 at 9 days and 30°C, 1.60 and 0.16 at 6 days and 35°C, 2.28 and 0.23 at 6 days and 40°C, 2.42 and 0.24 at 6 days and 45°C, 2.83 and 0.14 at 3 days and 50°C, 2.75 and 0.14 at 3 days and 55°C, 3.18 and 0.14 at 2.5 days and 60°C, and 1.69 and 0.17 at 6 days and 65°C. Volatile solids degradation at these retention times and temperatures was between 46 and 54%. The concentrations of volatile acids in the 30 to 55°C fermentors were generally below 2,000 mg/liter, with the exception of the 3-day retention time. The 60 and 65°C fermentors were usually above this level for all retention times. These studies indicate potential rates of methane production from the fermentation of untreated waste of beef cattle fed high-grain finishing diets. This information should serve as preliminary guidelines for various kinetic analyses and aid in economic evaluations of the potential feasibility of fermenting beef cattle waste to methane.     

Temperature Adaptations in the Terminal Processes of Anaerobic Decomposition of Yellowstone National Park and Icelandic Hot Spring Microbial Mats

The optimum temperatures for methanogenesis in microbial mats of four neutral to alkaline, low-sulfate hot springs in Yellowstone National Park were between 50 and 60°C, which was 13 to 23°C lower than the upper temperature for mat development. Significant methanogenesis at 65°C was only observed in one of the springs. Methane production in samples collected at a 51 or 62°C site in Octopus Spring was increased by incubation at higher temperatures and was maximal at 70°C. Strains of Methanobacterium thermoautotrophicum were isolated from 50, 55, 60, and 65°C sites in Octopus Spring at the temperatures of the collection sites. The optimum temperature for growth and methanogenesis of each isolate was 65°C. Similar results were found for the potential rate of sulfate reduction in an Icelandic hot spring microbial mat in which sulfate reduction dominated methane production as a terminal process in anaerobic decomposition. The potential rate of sulfate reduction along the thermal gradient of the mat was greatest at 50°C, but incubation at 60°C of the samples obtained at 50°C increased the rate. Adaptation to different mat temperatures, common among various microorganisms and processes in the mats, did not appear to occur in the processes and microorganisms which terminate the anaerobic food chain. Other factors must explain why the maximal rates of these processes are restricted to moderate temperatures of the mat ecosystem.     

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25°C), stoppered bottles incubated and sampled at 37 to 85°C showed more methane upon analysis than bottles incubated at 25°C: values at 50, 63, and 85°C were 109, 126, and 125%, respectively, of the 25°C value. All variation between 4 and 50°C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63°C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.     

Methanogenesis in Big Soda Lake, Nevada: an Alkaline, Moderately Hypersaline Desert Lake

Incubated sediment slurries from Big Soda Lake, Nevada, produced significant levels of CH₄, and production was inhibited by 2-bromoethanesulfonic acid and by autoclaving. Methane production was stimulated by methanol, trimethylamine, and, to a lesser extent, methionine. Surprisingly, hydrogen, acetate, and formate amendments provided only slight or no stimulation of methanogenesis. Methane production by sediment slurries had a pH optimum of 9.7. A methanol-grown enrichment culture containing a small, epifluorescent coccus as the predominant organism was recovered from sediments. The enrichment grew best when FeS or autoclaved sediment particles were included in the media, had a pH optimum of 9.7, and produced ¹⁴CH₄ from ¹⁴CH₃OH. The methane formed by methanolgrown enrichment cultures was depleted in ¹³C by 72 to 77‰ relative to the methanol.     

High Pressure Enhances the Growth Rate of the Thermophilic Archaebacterium Methanococcus thermolithotrophicus without Extending Its Temperature Range

Temperature and hydrostatic pressure are essential in determining the assemblage of species in their specific biotopes. To evaluate the effect of high pressure on the range of viability of thermophiles, the pressure and temperature dependence of the growth of the methanogenic archaebacterium Methanococcus thermolithotrophicus was investigated. High pressure up to 50 MPa enhanced the growth rate without extending the temperature range of viability. The optimum temperature remained unaltered (65°C). Beyond 50 MPa, cell lysis predominated over cell proliferation. Destabilization was also observed at temperatures below and above the optimum growth temperature (<60°C, ≥70°C) and at low substrate concentrations.     

Effects of Incubation Temperature on Growth and Production of Exopolysaccharides by an Antarctic Sea Ice Bacterium Grown in Batch Culture

The sea ice microbial community plays a key role in the productivity of the Southern Ocean. Exopolysaccharide (EPS) is a major component of the exopolymer secreted by many marine bacteria to enhance survival and is abundant in sea ice brine channels, but little is known about its function there. This study investigated the effects of temperature on EPS production in batch culture by CAM025, a marine bacterium isolated from sea ice sampled from the Southern Ocean. Previous studies have shown that CAM025 is a member of the genus Pseudoalteromonas and therefore belongs to a group found to be abundant in sea ice by culture-dependent and -independent techniques. Batch cultures were grown at −2°C, 10°C, and 20°C, and cell number, optical density, pH, glucose concentration, and viscosity were monitored. The yield of EPS at −2°C and 10°C was 30 times higher than at 20°C, which is the optimum growth temperature for many psychrotolerant strains. EPS may have a cryoprotective role in brine channels of sea ice, where extremes of high salinity and low temperature impose pressures on microbial growth and survival. The EPS produced at −2°C and 10°C had a higher uronic acid content than that produced at 20°C. The availability of iron as a trace metal is of critical importance in the Southern Ocean, where it is known to limit primary production. EPS from strain CAM025 is polyanionic and may bind dissolved cations such at trace metals, and therefore the presence of bacterial EPS in the Antarctic marine environment may have important ecological implications.     

Kinetics of chlorinated ethylene dehalogenation under methanogenic conditions

Kinetics were determined for methanogenic activity and chlorinated ethylene dehalogenation by a methanol-enriched, anaerobic sediment consortium. The culture reductively dechlorinated perchloroethylene (PCE) to trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), vinylchloride (VC), and ethylene and ethane. The absence : of methanol or the addition of 2-bromoethanesulfonic. acid in the presence of methanol suppressed both methanogenic activity and dechlorination. In contrast, acetate production continued in the presence of 2-bromoethanesulfonic acid. These results suggest that dechlorination was strongly linked to methane formation and not to acetate production. A kinetic model, developed to describe both methanogenesis and dechlorination, successfully predicted experimentally measured concentrations of biomass, methane, substrate, and chlorinated ethylenes. The average maximum specific dehalogenation rates for PCE, TCE, 1,1-DCE, and VC were 0.9 +/- 0.6, 0.4 +/- 0.1, 12 +/- 0.1, and 2.5 +/- 1.7 mumol contaminant/ g. DW/day, respectively. This pattern for dechlorination rates is distinctly different than that reported for transition metal cofactors, where rates drop by approximately one order of magnitude as each successive chlorine is removed. The experimental results and kinetic analysis suggest that it will be impractical to targeting methanol consuming methanogenic organisms for in situ ground-water restoration. (c) 1995 John Wiley & Sons, Inc.     

Evidence for para Dechlorination of Polychlorobiphenyls by Methanogenic Bacteria

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.     

Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 °C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA- and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.     

Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.     

Effect of Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH₄ production rate, a decrease in the steady-state H₂ partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), “novel Euryarchaeota” (23%), and Methanosarcinacaeae (7%). Further incubation at 30°C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15°C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Kinetics of Homoacetic Fermentation of Lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converted lactate to acetate at mesophilic temperatures (30 to 42°C) and at pHs between 6.6 and 9.6. The production of acetate was found to be growth associated. Approximately 0.96 g of acetic acid and 0.066 g of cells were formed from each gram of lactic acid consumed at 37°C. The concentration of the substrate (lactate) had little or no effect on the growth rate; however, the fermentation was inhibited by acetic acid. The bacterium grew at an optimal pH of 7.6 and an optimal temperature of 37°C. Small amounts of bicarbonate were stimulatory to bacterial growth. Bacterial growth was enhanced, however, by the use of higher concentrations of bicarbonate in the media, only because higher buffer capacities were obtained and proper medium pH could be maintained for growth. Based on its ability to convert lactate to acetate, this homoacetic bacterium may be important in the anaerobic methanogenic process when lactate is a major intermediary metabolite.