Thermophilic methanogenesis in a hot-spring algal-bacterial mat (71 to 30 degrees C).

Algal-bacterial mats which grow in the effluent channels of alkaline hot springs provided an environment suitable for studying natural thermophilic methane producing bacteria. Methane was rapidly produced in cores taken from the meat and appeared to be an end product of decomposition of the algal-bacterial organic matter. Formaldehyde prevented production of methane. Initial methanogenic rate was lower and methanogenesis became exponential when samples were permitted to cool before laboratory incubation. Methanogenesis occurred and methanogenic bacteria were present over a range of 68 to 30 degrees C, with optimum methanogenesis near 45 degrees C. The temperature distribution of methanogenesis in the mat is discussed relative to published results on standing crop, primary production, and decomposition in the thermal gradient. The depth distribution of methanogenesis was similar to that of freshwater sediments, with a zone of intense methanogenesis near the mat surface. Methanogenesis in deeper mat layers was very low or undetectable despite large numbers of viable methanogenic bacteria and could not be stimulated by addition of anoxic source water, sulfide, or a macronutrient solution.     

Terminal Processes in the Anaerobic Degradation of an Algal-Bacterial Mat in a High-Sulfate Hot Spring

The algal-bacterial mat of a high-sulfate hot spring (Bath Lake) provided an environment in which to compare terminal processes involved in anaerobic decomposition. Sulfate reduction was found to dominate methane production, as indicated by comparison of initial electron flow through the two processes, rapid conversion of [2-¹⁴C]acetate to ¹⁴CO₂ and not to ¹⁴CH₄, and the lack of rapid reduction of NaH¹⁴CO₃ to ¹⁴CH₄. Sulfate reduction was the dominant process at all depth intervals, but a marked decrease of sulfate reduction and sulfate-reducing bacteria was observed with depth. Concurrent methanogenesis was indicated by the presence of viable methanogenic bacteria and very low but detectable rates of methane production. A marked increased in methane production was observed after sulfate depletion despite high concentrations of sulfide (>1.25 mM), indicating that methanogenesis was not inhibited by sulfide in the natural environment. Although a sulfate minimum and sulfide maximum occurred in the region of maximal sulfate reduction, the absence of sulfate depletion in interstitial water suggests that methanogenesis is always severely limited in Bath Lake sediments. Low initial methanogenesis was not due to anaerobic methane oxidation.     

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

The short-term effects of temperature on methanogenesis from acetate or CO(2) in a thermophilic (58 degrees C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with C-labeled methane precursors (CH(3)COO or CO(2)). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60 degrees C and was completely inhibited at 65 degrees C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58 degrees C and did not grow or produce methane at 65 degrees C. An accidental shift of digestor temperature from 58 to 64 degrees 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 CH(3)COO was optimal at 65 degrees C and completely inhibited at 75 degrees C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70 degrees C. Methanogenesis from CO(2) in the sludge was optimal at 65 degrees C and still proceeded at 75 degrees C. A CO(2)-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75 degrees C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65 degrees C produced more methane than sludge incubated at 60 degrees C, and no acetate accumulated at 65 degrees C. Methanogenesis was severely inhibited in sludge incubated at 70 degrees C, but since neither acetate nor H(2) 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.     

Microbiology of Methanogenesis in Thermal, Volcanic Environments

Microbial methanogenesis was examined in thermal waters, muds, and decomposing algal-bacterial mats associated with volcanic activity in Yellowstone National Park. Radioactive tracer studies with [¹⁴C]glucose, acetate, or carbonate and enrichment culture techniques demonstrated that methanogenesis occurred at temperatures near 70°C but below 80°C and correlated with hydrogen production from either geothermal processes or microbial fermentation. Three Methanobacterium thermoautotrophicum strains (YT1, YTA, and YTC) isolated from diverse volcanic habitats differed from the neotype sewage strain ΔH in deoxyribonucleic acid guanosine-plus-cytosine content and immunological properties. Microbial methanogenesis was characterized in more detail at a 65°C site in the Octopus Spring algal-bacterial mat ecosystem. Here methanogenesis was active, was associated with anaerobic microbial decomposition of biomass, occurred concomitantly with detectable microbial hydrogen formation, and displayed a temperature activity optimum near 65°C. Enumeration studies estimated more than 10⁹ chemoorganotrophic hydrolytic bacteria and 10⁶ chemolithotrophic methanogenic bacteria per g (dry weight) of algal-bacterial mat. Enumeration, enrichment, and isolation studies revealed that the microbial population was predominantly rod shaped and asporogenous. A prevalent chemoorganotrophic organism in the mat that was isolated from an end dilution tube was a taxonomically undescribed gram-negative obligate anaerobe (strain HTB2), whereas a prevalent chemolithotrophic methanogen isolated from an end dilution tube was identified as M. thermoautotrophicum (strain YTB). Taxonomically recognizable obligate anaerobes that were isolated from glucose and xylose enrichment cultures included Thermoanaerobium brockii strain HTB and Clostridium thermohydrosulfuricum strain 39E. The nutritional properties, growth temperature optima, growth rates, and fermentation products of thermophilic bacterial strains 39E, HTB2, and YTB were determined.     

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.     

Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California

Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.     

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.     

Inhibition Experiments on Anaerobic Methane Oxidation

Anaerobic methane oxidation is a general process important in controlling fluxes of methane from anoxic marine sediments. The responsible organism has not been isolated, and little is known about the electron acceptors and substrates involved in the process. Laboratory evidence indicates that sulfate reducers and methanogens are able to oxidize small quantities of methane. Field evidence suggests anaerobic methane oxidation may be linked to sulfate reduction. Experiments with specific inhibitors for sulfate reduction (molybdate), methanogenesis (2-bromoethanesulfonic acid), and acetate utilization (fluoroacetate) were performed on marine sediments from the zone of methane oxidation to determine whether sulfate-reducing bacteria or methanogenic bacteria are responsible for methane oxidation. The inhibition experiment results suggest that methane oxidation in anoxic marine sediments is not directly mediated by sulfate-reducing bacteria or methanogenic bacteria. Our results are consistent with two possibilities: anaerobic methane oxidation may be mediated by an unknown organism or a consortium involving an unknown methane oxidizer and sulfate-reducing bacteria.     

Blockage of methanogenesis in marine sediments by the nitrification inhibitor 2-chloro-6-(trichloromethyl) pyridine (Nitrapyrin or N-Serve)

Nitrapyrin or N-Serve [2-chloro-6-(trichloromethyl)pyridine] blocked methanogenesis associated with slurries of marine sediments. Both nitrapyrin and chloroform, an established inhibitor of methanogenic bacteria, were effective at micromolar concentrations. Chemical hydrolysis of nitrapyrin resulted in the release of three molar equivalents of chloride ions and the loss of its ability to inhibit methane production. Thus, the potency of nitrapyrin in blocking methanogenesis seemed to depend upon its trichloromethyl moiety; this conclusion was supported in experiments with other substituted pyridine compounds.     

Methanogenic capabilities of ANME‐archaea deduced from 13C‐labelling approaches

Anaerobic methanotrophic archaea (ANME) are ubiquitous in marine sediments where sulfate dependent anaerobic oxidation of methane (AOM) occurs. Despite considerable progress in the understanding of AOM, physiological details are still widely unresolved. We investigated two distinct microbial mat samples from the Black Sea that were dominated by either ANME‐1 or ANME‐2. The ¹³C lipid stable isotope probing (SIP) method using labelled substances, namely methane, bicarbonate, acetate, and methanol, was applied, and the substrate‐dependent methanogenic capabilities were tested. Our data provide strong evidence for a versatile physiology of both, ANME‐1 and ANME‐2. Considerable methane production rates (MPRs) from CO₂‐reduction were observed, particularly from ANME‐2 dominated samples and in the presence of methane, which supports the hypothesis of a co‐occurrence of methanotrophy and methanogenesis in the AOM systems (AOM/MPR up to 2:1). The experiments also revealed strong methylotrophic capabilities through ¹³C‐assimilation from labelled methanol, which was independent of the presence of methane. Additionally, high MPRs from methanol were detected in both of the mat samples. As demonstrated by the ¹³C‐uptake into lipids, ANME‐1 was found to thrive also under methane free conditions. Finally, C₃₅‐isoprenoid hydrocarbons were identified as new lipid biomarkers for ANME‐1, most likely functioning as a hydrogen sink during methanogenesis.     

Bacterial methanogenesis in holocene sediments of the Baltic sea

Microbial methanogenesis was proved geochemically, based on the abundance of methanogenic bacteria and methane production rates in experiments with radioactive carbon. The results are compared with direct measurements of methane concentrations in mud samples taken with a hermetic sampler. The migration of methane formed in sediments occurs during filtration of porewater rather than at the expense of gas diffusion.     

Effect of ammonia on the methanogenic activity of methylaminotrophic methane producing Archaea enriched biofilm

Ammonia is a metabolic product in the decomposition of protein wastes, and has a recognized inhibitory effect on methanogenesis; this effect has been slightly quantified on methanogenic biofilms and particularly those populated by methanogenic Archaea which produce ammonia as a catabolic product from methylated amines. This paper presents studies on the effect of ammonia on maximum methanogenic activity of anaerobic biofilms enriched by methylaminotrophic methane producing Archaea (mMPA). The effect of unionized free ammonia on the specific maximum methanogenic activity of a mMPA enriched biofilm was studied, using 250 mL flasks containing ceramic rings colonized by 30 day-old experimental biofilm and adding 48.8 (control system), 73.8, 98.8, 148.8, 248.8, 448.8 and 848.8 mg NH(3)-N/L. The systems were maintained for ten days at a pH of 7.5 and temperature of 37 degrees C. The results showed that at 848.8 mg NH(3)-N/L, biofilm methane production required 36 h adaptation period, prior to entering into maximum production phase. The highest maximum methanogenic activity reached a value of 2.337+/-0.213 g COD methane/g VSS *day when 48.8 mg NH(3)-N/L was added, and inhibition was clearly observed in those systems above 148.8 mg NH(3)-N/L, producing under 1.658+/-0.185 g COD methane/g VSS *day. The lowest methanogenic activity reached was 0.639+/-0.162 g COD methane/g VSS *day at the system added with 848.8 mg NH(3)-N/L. When applying the Luong and non-competitive inhibition models, the best fit was obtained with the non-competitive model, which predicted 50% inhibition of methanogenic activity at 365.288 mg NH(3)-N/L.     

Methanogenic symbionts of anaerobic ciliates and their contribution to methanogenesis in an anoxic rice field soil

Methanogenesis in rice field soils starts soon after flooding while potentially competing processes like reduction of sulphate and iron take place. Early methanogenesis is mainly driven by hydrogen, while later in the season acetate tends to become more important. Anaerobic ciliates are abundant during this period, and their endosymbionts use hydrogen produced by the ciliates to reduce carbon dioxide to methane. These endosymbiotic methanogens are protected from the competition for substrates with other bacteria that may control methanogenesis outside the protozoan cells. Thus, we focussed on early methanogenesis and on the potential contribution from ciliates and their endosymbionts. Only ciliates of the genus Metopus were found to harbour methanogens, as identified by the F(420)-fluorescence of the endosymbionts. We followed the population dynamics of the ciliates with time, and calculated the ratio of symbiotic methane production to overall methanogenesis. Symbiotic methane production was calculated from the species-specific numbers of methanogenic endosymbionts times the cell-specific methane production of the symbionts. According to this calculation, the symbionts' contribution to overall methane production was only 6.4% at the beginning and decreased with time. In a second experiment, colchicine and cycloheximide were used to inhibit all eukaryotes, comparing the remaining methane production rate to a control without inhibitors. In the inhibition experiment, the contribution from symbionts decreased from 40% to 6% during the first days after flooding, and dropped to near zero within 2 weeks. However, nearly all methane produced from H(2)/CO(2) could be attributed to the ciliates' symbionts between days 5 and 10 after flooding. Both experiments showed that the contribution of methanogenic symbionts to overall methane production is a transient phenomenon, restricted to the first 2 weeks.     

Temperature limitation of methanogenesis in aquatic sediments.

Microbial methanogenesis was examined in sediments collected from Lake Mendota, Wisconsin, at water depths of 5, 10, and 18 m. The rate of sediment methanogenesis was shown to vary with respect to sediment site and depth, sampling date, in situ temperature, and number of methanogens. Increased numbers of methanogenic bacteria and rates of methanogenesis correlated with increased sediment temperature during seasonal change. The greatest methanogenic activity was observed for 18-m sediments throughout the sampling year. As compared with shallower sediments, 18-m sediment was removed from oxygenation effects and contained higher amounts of ammonia, carbonate, and methanogenic bacteria, and the population density of methanogens fluctuated less during seasonal change. Rates of methanogenesis in 18-m sediment cores decreased with increasing sediment depth. The optimum temperature, 35 to 42 C, for sediment methanogenesis was considerably higher than the maximum observed in situ temperature of 23 C. The conversion of H2 and [14C]carbonate to [14C]methane displayed the same temperature optimum when these substrates were added to sediments. The predominant methanogenic population had simple nutritional requirements and were metabolically active at 4 to 45 C. Hydrogen oxidizers were the major nutritional type of sediment methanogens; formate and methanol fermentors were present, but acetate fermentors were not observed. Methanobacterium species were most abundant in sediments although Methanosarcina, Methanococcus, and Methanospirillum species were observed in enrichment cultures. A chemolithotropic species of Methanosarcina and Methanobacterium was isolated in pure culture that displayed temperature optima above 30 C and had simple nutritional requirements.     

Inhibitory effects of nitrogen oxides on a mixed methanogenic culture

The effect of nitrate, nitrite, nitric oxide (NO), and nitrous oxide on a mixed, mesophilic (35 degrees C) methanogenic culture was investigated. Short-term inhibition assays were conducted at a concentration range of 10-350 mg N/L nitrate, 17-500 mg N/L nitrite, 0.02-0.8 mg N/L aqueous NO, and 19-191 mg N/L aqueous nitrous oxide. Simultaneous methane production and N-oxide reduction was observed in 10 and 30 mg N/L nitrate and 0.02 mg N/L aqueous NO-amended cultures. However, addition of N-oxide resulted in immediate cessation of methanogenesis in all other cultures. Methanogenesis completely recovered subsequent to the complete reduction of N-oxides to nitrogen gas in all N-oxide-amended cultures, with the exception of the 500 mg N/L nitrite- and 0.8 mg N/L aqueous NO-amended cultures. Partial recovery of methanogenesis was observed in the 500 mg N/L nitrite-amended culture in contrast to complete inhibition of methanogenesis in the 0.8 mg N/L aqueous NO-amended culture. Accumulation of volatile fatty acids was observed in both cultures at the end of the incubation period. Among all N-oxides, NO exerted the most and nitrate exerted the least inhibitory effect on the fermentative/methanogenic consortia. The effect of multiple additions of nitrate (300 mg N/L) on the same methanogenic culture was also investigated. Long-term exposure of the methanogenic culture to nitrate resulted in an increase of N-oxide reduction rates and decrease of methane production rates, which was attributed to changes in the microbial community structure due to nitrate addition.     

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.     

Methanogens and sulfate-reducing bacteria in oil sands fine tailings waste

In the past decade, the large tailings pond (Mildred Lake Settling Basin) on the Syncrude Canada Ltd. lease near Fort McMurray, Alta., has gone methanogenic. Currently, about 60%-80% of the flux of gas across the surface of the tailings pond is methane. As well as adding to greenhouse gas emissions, the production of methane in the fine tailings zone of this and other settling basins may affect the performance of these settling basins and impact reclamation options. Enumeration studies found methanogens (10(5)-10(6) MPN/g) within the fine tailings zone of various oil sands waste settling basins. SRB were also present (10(4)-10(5) MPN/g) with elevated numbers when sulfate was available. The methanogenic population was robust, and sample storage up to 9 months at 4 degrees C did not cause the MPN values to change. Nor was the ability of the consortium to produce methane delayed or less efficient after storage. Under laboratory conditions, fine tailings samples released 0.10-0.25 mL CH4 (at STP)/mL fine tailings. The addition of sulfate inhibited methanogenesis by stimulating bacterial competition.     

Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.     

Dynamics of the methanogenic archaeal community during plant residue decomposition in an anoxic rice field soil

Incorporation of plant residues strongly enhances the methane production and emission from flooded rice fields. Temperature and residue type are important factors that regulate residue decomposition and CH(4) production. However, the response of the methanogenic archaeal community to these factors in rice field soil is not well understood. In the present experiment, the structure of the archaeal community was determined during the decomposition of rice root and straw residues in anoxic rice field soil incubated at three temperatures (15 degrees C, 30 degrees C, and 45 degrees C). More CH(4) was produced in the straw treatment than root treatment. Increasing the temperature from 15 degrees C to 45 degrees C enhanced CH(4) production. Terminal restriction fragment length polymorphism analyses in combination with cloning and sequencing of 16S rRNA genes showed that Methanosarcinaceae developed early in the incubations, whereas Methanosaetaceae became more abundant in the later stages. Methanosarcinaceae and Methanosaetaceae seemed to be better adapted at 15 degrees C and 30 degrees C, respectively, while the thermophilic Methanobacteriales and rice cluster I methanogens were significantly enhanced at 45 degrees C. Straw residues promoted the growth of Methanosarcinaceae, whereas the root residues favored Methanosaetaceae. In conclusion, our study revealed a highly dynamic structure of the methanogenic archaeal community during plant residue decomposition. The in situ concentration of acetate (and possibly of H(2)) seems to be the key factor that regulates the shift of methanogenic community.     

Methanogenesis in rumen ciliate cultures ofEntodinium caudatum andEpidinium ecaudatum after long-term cultivation in a chemically defined medium

The methanogenic activity in the presence of Entodinium caudatum and Epidinium ecaudatum was well preserved after long-term cultivation. Microscopic observation revealed that methane production in the presence of E. caudatum was probably caused by their intracellular methanogenic activity, while methane production in the presence of E. ecaudatum f caudatum et ecaudatum could be attributed to both the methanogenic bacterial fraction of their external surface and their intracellular activity. Methane production per protozoan cell of E. caudatum and E. ecaudatum was 2.1 nmol per cell per d and 6.0 nmol per cell per d, respectively. E. caudatum was responsible for almost the entire methane production in the culture. The activity of free methanogens constituted approximately 50% of the total methane production in the E. ecaudatum culture. Decrease of digestibility of substrates and differences in the fermentation end products accompanied the inhibition of methanogenesis in both cultures by penicillin G, streptomycin, chloramphenicol, 2-bromoethanesulfonate, and pyromellitic diimide. E. caudatum appeared to be more sensitive than E. ecaudatum to the compounds tested. Hydrogen recoveries based on both volatile fatty acids and methane production suggested that the methanogenic population appeared not to be fully able to consume hydrogen produced in the protozoan cultures. The culture conditions tested were found to be suitable for experiments on the relationship between rumen ciliates and rumen bacteria.