Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil

Temperature is an important factor controlling CH(4) production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37 degrees C or in a temperature gradient block covering the same temperature range at intervals of 1 degrees C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH(4) production rates increased with temperature, with an apparent activation energy of 61 kJ mol(-1). Steady-state partial pressures of the methanogenic precursor H(2) also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H(2) plus CO(2)-dependent methanogenesis was kept at -20 to -25 kJ mol of CH(4)(-1) over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 microM. Simultaneously, the relative contribution of H(2) as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to (14)CH(4), so that the carbon and electron flow to CH(4) was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Activity, structure and dynamics of the methanogenic archaeal community in a flooded Italian rice field

Methane production was studied in an Italian rice field over two consecutive years (1998, 1999) by measuring the rates of total and acetate-dependent methanogenesis in soil and root samples. Population dynamics of methanogens were followed by terminal restriction fragment length polymorphism and real-time PCR targeting archaeal SSU rRNA genes. Rates of total and acetate-dependent methanogenesis in soil increased during the season, reached a maximum at about 70-80 days after flooding and then decreased again. In contrast, the size of the archaeal community remained relatively constant. Therefore, the seasonal changes in the methanogenic processes were probably not caused by changes in the size of the methanogenic community but in its activity. During the 1998/1999 winter period, a slight decrease in archaeal cell numbers was found. In both years, the dominant groups were methanogens affiliated with Rice cluster I, Methanosaetaceae, Methanosarcinaceae and Methanobacteriaceae. Correspondence analysis showed, however, that the archaeal community structure was different in 1998 and 1999. Methanogens with potential acetoclastic activity made up a larger fraction of the total archaeal community in 1999 (32-53%) than in 1998 (20-32%). Furthermore, the frequency of Methanosaetaceae relative to Methanosarcinaceae was significantly higher in 1999 than in 1998. This difference could be explained by the much lower soil acetate concentrations in 1999, to which Methanosaetaceae are physiologically better adapted than Methanosarcinaceae. Over the season, however, the composition of the archaeal community remained relatively constant and thus did not reflect the observed seasonal change in CH(4) production activity. The analysis of rice root samples in 1999 showed that the archaeal community structure on the roots was similar to that in soil but with acetoclastic methanogens being relatively less common. This observation is in agreement with domination of CH(4) production by H(2)/CO(2)-dependent methanogenesis on roots. Our study provided a link between size, structure and function of the methanogenic community in an Italian rice field.     

Effect of Inhibition of Acetoclastic Methanogenesis on Growth of Archaeal Populations in an Anoxic Model Environment

Methyl fluoride is frequently used to specifically inhibit acetoclastic methanogenesis, thus allowing determination of the relative contribution of acetate versus H₂/CO₂ to total CH₄ production in natural environments. However, the effect of the inhibitor on growth of the target archaeal population has not yet been studied. Therefore, we incubated rice roots as an environmental model system under anoxic conditions in the presence and absence of CH₃F, measured the activity and Gibbs free energy (ΔG) of CH₄ production, and determined the abundance of individual archaeal populations by using a combination of quantitative (real-time) PCR and analysis of terminal restriction fragment length polymorphism targeting the 16S rRNA gene. It was shown that CH₃F specifically inhibited not only acetoclastic methanogenic activity but also the proliferation of Methanosarcina spp, which were the prevalent acetoclastic methanogens in our environmental model system. Therefore, inhibition experiments with CH₃F seem to be a suitable method for quantifying acetoclastic CH₄ production. It is furthermore shown that the growth and final population size of methanogens were consistent with energetic conditions that at least covered the maintenance requirements of the population.     

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.     

Methane production potential and methanogenic archaea community dynamics along the Spartina alterniflora invasion chronosequence in a coastal salt marsh

Invasion by the exotic species Spartina alterniflora, which has high net primary productivity and superior reproductive capacity compared with native plants, has led to rapid organic carbon accumulation and increased methane (CH₄) emission in the coastal salt marsh of China. To elucidate the mechanisms underlying this effect, the methanogen community structure and CH₄ production potential as well as soil organic carbon (SOC), dissolved organic carbon, dissolved organic acids, methylated amines, aboveground biomass, and litter mass were measured during the invasion chronosequence (0–16 years). The CH₄ production potential in the S. alterniflora marsh (range, 2.94–3.95 μg kg^?1 day^?1) was significantly higher than that in the bare tidal mudflat. CH₄ production potential correlated significantly with SOC, acetate, and trimethylamine concentrations in the 0–20 cm soil layer. The abundance of methanogenic archaea also correlated significantly with SOC, and the dominant species clearly varied with S. alterniflora-driven SOC accumulation. The acetotrophic Methanosaetaceae family members comprised a substantial proportion of the methanogenic archaea in the bare tidal mudflat while Methanosarcinaceae family members utilized methylated amines as substrates in the S. alterniflora marsh. Ordination analysis indicated that trimethylamine concentration was the primary factor inducing the shift in the methanogenic archaea composition, and regressive analysis indicated that the facultative family Methanosarcinaceae increased linearly with trimethylamine concentration in the increasingly sulfate-rich salt marsh. Our results indicate that increased CH₄ production during the S. alterniflora invasion chronosequence was due to increased levels of the non-competitive substrate trimethylamine and a shift in the methanogenic archaea community.     

Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10⁷ per g of dry soil for the H₂-CO₂-utilizing methanogens and of up to 10⁶ for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H₂-CO₂, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.     

Functional and structural response of the methanogenic microbial community in rice field soil to temperature change

The microbial community in anoxic rice field soil produces CH₄ over a wide temperature range up to 55°C. However, at temperatures higher than about 40°C, the methanogenic path changes from CH₄ production by hydrogenotrophic plus acetoclastic methanogenesis to exclusively hydrogenotrophic methanogenesis and simultaneously, the methanogenic community consisting of Methanosarcinaceae, Methanoseataceae, Methanomicrobiales, Methanobacteriales and Rice Cluster I (RC‐1) changes to almost complete dominance of RC‐1. We studied changes in structure and function of the methanogenic community with temperature to see whether microbial members of the community were lost or their function impaired by exposure to high temperature. We characterized the function of the community by the path of CH₄ production measuring δ¹³C in CH₄ and CO₂ and calculating the apparent fractionation factor (α_app) and the structure of the community by analysis of the terminal restriction fragment length polymorphism (T‐RFLP) of the microbial 16S rRNA genes. Shift of the temperature from 45°C to 35°C resulted in a corresponding shift of function and structure, especially when some 35°C soil was added to the 45°C soil. The bacterial community (T‐RFLP patterns), which was much more diverse than the archaeal community, changed in a similar manner upon temperature shift. Incubation of a mixture of 35°C and 50°C pre‐incubated methanogenic rice field soil at different temperatures resulted in functionally and structurally well‐defined communities. Although function changed from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a rather narrow temperature range of 42–46°C, each of these temperatures also resulted in only one characteristic function and structure. Our study showed that temperature conditions defined structure and function of the methanogenic microbial community.     

Methanogenic Pathway and Archaeal Community Structure in the Sediment of Eutrophic Lake Dagow: Effect of Temperature

Methanogenic degradation of organic matter is an important microbial process in lake sediments. Temperature may affect not only the rate but also the pathway of CH₄ production by changing the activity and the abundance of individual microorganisms. Therefore, we studied the function and structure of a methanogenic community in anoxic sediment of Lake Dagow, a eutrophic lake in north-eastern Germany. Incubation of sediment samples (in situ 7.5°C) at increasing temperatures (4, 10, 15, 25, 30°C) resulted in increasing production rates of CH₄ and CO₂ and in increasing steady-state concentrations of H₂. Thermodynamic conditions for H₂/CO₂ -dependent methanogenesis were only exergonic at 25 and 30°C. Inhibition of methanogenesis with chloroform resulted in the accumulation of methanogenic precursors, i.e., acetate, propionate, and isobutyrate. Mass balance calculations indicated that less CH₄ was formed via H₂ at 4°C than at 30°C. Conversion of ¹⁴CO₂ to ¹⁴CH₄ also showed that H₂/CO₂ -dependent methanogenesis contributed less to total CH₄ production at 4°C than at 30°C. [2–¹⁴ C]Acetate turnover rates at 4°C accounted for a higher percentage of total CH₄ production than at 30°C. Collectively, these results showed a higher contribution of H₂-dependent methanogenesis and a lower contribution of acetate-dependent methanogenesis at high versus low temperature. The archaeal community was characterized by cloning, sequencing, and phylogenetic analysis of the 16S rRNA genes retrieved from the sediment. Sequences were affiliated with Methanosaetaceae, Methanomicrobiaceae, and three deeply branching euryarchaeotal clusters, i.e., group III, Rice cluster V, and a novel euryarchaeotal cluster, the LDS cluster. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that 16S rRNA genes affiliated to Methanosaetaceae (20–30%), Methanomicrobiaceae (35–55%), and group III (10–25%) contributed most to the archaeal community. Incubation of the sediment at different temperatures (4–30°C) did not result in a systematic change of the archaeal community composition, indicating that change of temperature primarily affected the activity rather than the structure of the methanogenic community.     

Unexpected competitiveness of Methanosaeta populations at elevated acetate concentrations in methanogenic treatment of animal wastewater

Acetoclastic methanogenesis is a key metabolic process in anaerobic digestion, a technology with broad applications in biogas production and waste treatment. Acetoclastic methanogenesis is known to be performed by two archaeal genera, Methanosaeta and Methanosarcina. The conventional model posits that Methanosaeta populations are more competitive at low acetate levels (<1 mM) than Methanosarcina and vice versa at higher acetate concentrations. While this model is supported by an extensive body of studies, reports of inconsistency have grown that Methanosaeta were observed to outnumber Methanosarcina at elevated acetate levels. In this study, monitoring of anaerobic digesters treating animal wastewater unexpectedly identified Methanosaeta as the dominant acetoclastic methanogen population at both low and high acetate levels during organic overloading. The surprising competitiveness of Methanosaeta at elevated acetate was further supported by the enrichment of Methanosaeta with high concentrations of acetate (20 mM). The dominance of Methanosaeta in the methanogen community could be reproduced in anaerobic digesters with the direct addition of acetate to above 20 mM, again supporting the competitiveness of Methanosaeta over Methanosarcina at elevated acetate levels. This study for the first time systematically demonstrated that the dominance of Methanosaeta populations in anaerobic digestion could be linked to the competitiveness of Methanosaeta at elevated acetate concentrations. Given the importance of acetoclastic methanogenesis in biological methane production, findings from this study could have major implications for developing strategies for more effective control of methanogenic treatment processes.

     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Community analysis of methanogenic archaea within a riparian flooding gradient

Anoxic soils in river floodplains (or riparian soils) are a source of methane emission. However, little is known about the ecology and community structure of archaeal methanogenic microbes, which are a crucial component of methane flux in those habitats. We studied the archaeal community in the vertical profile of four different sites along the River Waal in the Netherlands. These sites differ in their annual flooding regime ranging from never or seldom to permanently flooded. The archaeal community structure has been characterized by terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of the archaeal SSU rRNA gene and the mcrA gene. The latter gene codes for the alpha-subunit of methyl-coenzyme M reductase. Additionally, the potential methanogenic activity was determined by incubation of soil slurries under anoxic conditions. The community composition differed only slightly with the depth of the soil (0-20 cm). However, the diversity of archaeal SSU rRNA genes increased with the frequency of flooding. Terminal restriction fragment length polymorphism analysis of mcrA gene amplicons confirmed the results concerning methanogenic archaea. In the never and rarely flooded soils, crenarchaeotal sequences were the dominant group. In the frequently and permanently flooded soils, Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae and the uncultured Rice Clusters IV and VI (Crenarchaeota) were detectable independently from duration of anoxic conditions. Methanosaetaceae, on the other hand, were only found in the permanently and frequently flooded soils under conditions where concentrations of acetate were < 30 microM. The results indicate that methanogens as well as other archaea occupy characteristic niches according to the flooding conditions in the field. Methanosaetaceae, in particular, seem to be adapted (or proliferate at) to low acetate concentrations.     

Effect of Substrate Concentration on Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosarcina barkeri and M. acetivorans and in Rice Field Soil

Methanosarcina is the only acetate-consuming genus of methanogenic archaea other than Methanosaeta and thus is important in methanogenic environments for the formation of the greenhouse gases methane and carbon dioxide. However, little is known about isotopic discrimination during acetoclastic CH₄ production. Therefore, we studied two species of the Methanosarcinaceae family, Methanosarcina barkeri and Methanosarcina acetivorans, and a methanogenic rice field soil amended with acetate. The values of the isotope enrichment factor (ɛ) associated with consumption of total acetate (ɛ_ac), consumption of acetate-methyl (ɛ_ac-methyl) and production of CH₄ (ɛ_CH4) were an ɛ_ac of −30.5‰, an ɛ_ac-methyl of −25.6‰, and an ɛ_CH4 of −27.4‰ for M. barkeri and an ɛ_ac of −35.3‰, an ɛ_ac-methyl of −24.8‰, and an ɛ_CH4 of −23.8‰ for M. acetivorans. Terminal restriction fragment length polymorphism of archaeal 16S rRNA genes indicated that acetoclastic methanogenic populations in rice field soil were dominated by Methanosarcina spp. Isotope fractionation determined during acetoclastic methanogenesis in rice field soil resulted in an ɛ_ac of −18.7‰, an ɛ_ac-methyl of −16.9‰, and an ɛ_CH4 of −20.8‰. However, in rice field soil as well as in the pure cultures, values of ɛ_ac and ɛ_ac-methyl decreased as acetate concentrations decreased, eventually approaching zero. Thus, isotope fractionation of acetate carbon was apparently affected by substrate concentration. The ɛ values determined in pure cultures were consistent with those in rice field soil if the concentration of acetate was taken into account.Methane (CH₄) is the most abundant reduced gas in the earth''s atmosphere and is an important greenhouse gas with a high global-warming potential (7). It is presently a matter of discussion whether the contribution of CH₄ to the greenhouse effect will increase in the future (3, 23). This has made it necessary and more urgent to understand natural processes that lead to the production of CH₄.Methanogenesis, the microbial formation of CH₄, is the final step in the degradation of organic matter in anoxic environments like natural wetlands, lake sediments, and flooded rice fields. The most important precursors for the production of CH₄ are acetate (equation 1) and CO₂ (equation 2) with the following reactions (8): (1) (2)Acetate is the most important substrate since it contributes more than 67% to microbial methanogenesis during anoxic degradation of polysaccharides. In methanogenic environments only two genera of archaea, Methanosaeta and Methanosarcina, are capable of using acetate (2). While Methanosaeta can be considered a specialist that uses only acetate, Methanosarcina can use a wide range of substrates besides acetate, for example, H₂/CO₂, methanol, methylamines, and methylated sulfides. Among methanogens, Methanosarcinaceae also display the largest environmental distribution. They can be found in freshwater sediments and soil, marine habitats, landfills, and animal gastrointestinal tracts (46).Additionally, differences between Methanosarcina and Methanosaeta were found for isotope fractionation of stable carbon. The fractionation factor (α) or, equivalently, the enrichment factor (ɛ) during acetoclastic methanogenesis in Methanosarcina barkeri strains typically ranges from an α of 1.021 to 1.027 or an ɛ of −27‰ to −21‰ (14, 27, 48), whereas isotope fractionation in Methanosaeta spp. is weaker, i.e., an α of 1.007 (ɛ = −7‰) for Methanosaeta thermophila (43) and an α of 1.010 (ɛ = −10‰) for Methanosaeta concilii (34). It is suggested that the two archaeal genera differ in isotope fractionation due to differences in their biochemical activation of acetate to acetyl-coenzyme A (acetyl-CoA) (34). However, isotopic data for acetoclastic methanogens are rare. For instance, all data for Methanosarcina refer to only one species, namely M. barkeri.Hence, in this study we investigated whether differences in carbon isotope fractionation within the genus Methanosarcina occur. Therefore, we determined isotope ratios of stable carbon in cultures of the acetoclastic species M. barkeri and Methanosarcina acetivorans. Second, we were interested if these data, obtained from pure cultures, could also be applied to understand natural environments. For that reason, we determined isotope fractionation during acetoclastic methanogenesis in the model system rice field soil. Furthermore, we discuss the effect of substrate concentration on carbon isotope fractionation and the importance of monitoring isotope fractionation during the course of acetate consumption.     

Methane emission from feather moss stands

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH₄. However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH₄ turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH₄ cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH₄ emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH₄ h^?1 gdw^?1. Temperature and water content positively influenced CH₄ emission. Methanogenic potentials determined under N₂ atmosphere in darkness ranged between 22 and 157 pmol CH₄ h^?1 gdw^?1. Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH₄ was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 10⁴ and 10⁵ copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH₄ was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH₄ produced was depleted in ¹³C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3–62 mM). The δ¹³C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH₄ by hydrogenotrophic methanogenesis and causing net emission of CH₄ under ambient conditions, albeit at low rates.     

Methanogenic diversity and activity in municipal solid waste landfill leachates

Archaeal microbial communities present in municipal solid waste landfill leachates were characterized using a 16S rDNA approach. Phylogenetic affiliations of 239 partial length 16S rDNA sequences were determined. Sequences belonging to the order Methanosarcinales were dominant in the clone library and 65% of the clones belonged to the strictly acetoclastic methanogenic family Methanosaetaceae. Sequences affiliated to the metabolically versatile family Methanosarcinaceae represented 18% of the retrieved sequences. Members of the hydrogenotrophic order Methanomicrobiales were also recovered in limited numbers, especially sequences affiliated to the genera Methanoculleus and Methanofollis. Eleven euryarchaeal and thirteen crenarchaeal sequences (i.e. 10%) were distantly related to any hitherto cultivated microorganisms, showing that archaeal diversity within the investigated samples was limited. Lab-scale incubations were performed with leachates mixed with several methanogenic precursors (acetate, hydrogen, formate, methanol, methylamine). Microbial populations were followed using group specific 16S rRNA targeted fluorescent oligonucleotidic probes. During the incubations with acetate, acetoclastic methanogenesis was rapidly induced and led to the dominance of archaea hybridizing with probe MS1414 which indicates their affiliation to the family Methanosarcinaceae. Hydrogen and formate addition induced an important acetate synthesis resulting from the onset of homoacetogenic metabolism. In these incubations, species belonging to the family Methanosarcinaceae (hybridizing with probe MS1414) and the order Methanomicrobiales (hybridizing with probe EURY496) were dominant. Homoacetogenesis was also recorded for incubations with methanol and methylamines. In the methanol experiment, acetoclastic methanogenesis took place and archaea hybridizing with probe MS821 (specific for Methanosarcina spp.) were observed to be the dominant population. These results confirm that acetoclastic methanogenesis performed by the members of the order Methanosarcinales is predominant over the hydrogenotrophic and methylotrophic pathways in landfill leachates.     

Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Changing Feeding Regimes To Demonstrate Flexible Biogas Production: Effects on Process Performance,Microbial Community Structure,and Methanogenesis Pathways

Flexible biogas production that adapts biogas output to energy demand can be regulated by changing feeding regimes. In this study, the effect of changes in feeding intervals on process performance, microbial community structure, and the methanogenesis pathway was investigated. Three different feeding regimes (once daily, every second day, and every 2 h) at the same organic loading rate were studied in continuously stirred tank reactors treating distiller''s dried grains with solubles. A larger amount of biogas was produced after feeding in the reactors fed less frequently (once per day and every second day), whereas the amount remained constant in the reactor fed more frequently (every 2 h), indicating the suitability of the former for the flexible production of biogas. Compared to the conventional more frequent feeding regimes, a methane yield that was up to 14% higher and an improved stability of the process against organic overloading were achieved by employing less frequent feeding regimes. The community structures of bacteria and methanogenic archaea were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA and mcrA genes, respectively. The results showed that the composition of the bacterial community varied under the different feeding regimes, and the observed T-RFLP patterns were best explained by the differences in the total ammonia nitrogen concentrations, H₂ levels, and pH values. However, the methanogenic community remained stable under all feeding regimes, with the dominance of the Methanosarcina genus followed by that of the Methanobacterium genus. Stable isotope analysis showed that the average amount of methane produced during each feeding event by acetoclastic and hydrogenotrophic methanogenesis was not influenced by the three different feeding regimes.     

Substrate limitation of sediment methane flux,methane oxidation and use of stable isotopes for assessing methanogenesis pathways in a small arctic lake

Among predicted impacts of climate change in the Arctic are greater thaw depth and shifts in vegetation patterns and hydrology that are likely to increase organic carbon and nutrient loading to lakes. We measured substrate limitation of sediment methane (CH₄) flux, examined pathways of methanogenesis, and potential CH₄ oxidation using stable isotope labeled acetate in intact sediment cores from arctic lake GTH 112 (68°40′20″N, 149°14′57″W). We hypothesized that the acetoclastic pathway would dominate methanogenesis, reflecting dissolved organic carbon supply from the surrounding landscape, and that sediment CH₄ flux would be stimulated by addition of acetate. Experiments demonstrated acetate limitation of sediment CH₄ flux with short-term CH₄ flux response to availability of acetate, high rates of CH₄ oxidation, and strong dominance of the acetoclastic over the hydrogenotrophic methanogenic pathway. The experiments also indicated that isotopic fractionation effects during isotope enrichment experiments are large during methanogenesis and can alter the methanogenic pathways being investigated. Under oxic conditions, CH₄ oxidation at the sediment–water interface or in the water column is likely to account for much of diffusive CH₄ flux, but under anoxic hypolimnetic conditions and increased substrate availability, conditions that are likely to occur with climate change, sediment CH₄ flux will likely increase, with oxidation utilizing a smaller portion of sediment CH₄ production.     

Phylogenetic characterization of methanogenic assemblages in eutrophic and oligotrophic areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1

Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µ_max = 0.050–0.055 h^–1) and a maximum value of the specific acetate consumption rate (q _infs ^supps ) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K _m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. Offprint requests to: R. Moletta