Peter Claus

Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions

The prototypical representatives of the Euryarchaeota—the methanogens—are oxygen sensitive and are thought to occur only in highly reduced, anoxic environments. However, we found methanogens of the genera Methanosarcina and Methanocella to be present in many types of upland soils (including dryland soils) sampled globally. These methanogens could be readily activated by incubating the soils as slurry under anoxic conditions, as seen by rapid methane production within a few weeks, without any additional carbon source. Analysis of the archaeal 16S ribosomal RNA gene community profile in the incubated samples through terminal restriction fragment length polymorphism and quantification through quantitative PCR indicated dominance of Methanosarcina, whose gene copy numbers also correlated with methane production rates. Analysis of the δ13C of the methane further supported this, as the dominant methanogenic pathway was in most cases aceticlastic, which Methanocella cannot perform. Sequences of the key methanogenic enzyme methyl coenzyme M reductase retrieved from the soil samples before incubation confirmed that Methanosarcina and Methanocella are the dominant methanogens, though some sequences of Methanobrevibacter and Methanobacterium were also detected. The global occurrence of only two active methanogenic archaea supports the hypothesis that these are autochthonous members of the upland soil biome and are well adapted to their environment.

Pathway of CH4 formation in anoxic rice field soil and rice roots determined by 13C-stable isotope fractionation.

In anoxic rice fields methane is produced by either reduction of CO2 or cleavage of acetate. We measured the delta 13C-values of CH4 and CO2, acetate and organic carbon during time course experiments with anoxic methanogenic soil and root samples and used these values to calculate the fractions of CH4 (and acetate) produced from CO2 reduction. Comparison with radiotracer and/or inhibitor studies constrained the kinetic fractionation factors used for calculation. The fractionation factors for the conversion of CO2 to CH4 and of acetate to CH4 were on the order of alpha = 1.07 (epsilon = -70%) and epsilon > or = - 20%, respectively. The pathway of CH4 production changed with time of anoxic incubation. Anoxic slurries of rice field soil first produced CH4 predominantly (>50%) from CO2, then predominantly (>80%) from acetate and finally (after about one month) according to the theoretically expected ratio (33% CO2 and 67% acetate). Anoxic rice roots, on the other hand, initially produced CH4 exclusively from CO2, followed by contribution of acetate of about 40-60%. Rice roots also produced acetate that partially originated (< or = 1 30%) from reduction of CO2 as determined by calculation of isotopic fractionation using fractionation factors from the literature. The results demonstrate that there is quite some variability in pathways of CH4 production, and also indicate that isotopic fractionation factors may be different in different habitats and change with time.

Temporal change of 13C-isotope signatures and methanogenic pathways in rice field soil incubated anoxically at different temperatures

Production of CH4 and CO2 was quantified in anoxically incubated soil samples taken from an Italian rice field. The rates increased with temperature between 10 and 37°C. The δ13C of the accumulated CO2, CH4 and acetate changed with time in a systematic way. The data were used in mass balance equations to constrain isotopic fractionation factors and pathways of CH4 production. The calculations were further constrained by the determination of 14CH4 production from 14CO2 at steady state. At 50°C, CH4 was exclusively produced from CO2, indicating a fractionation factor of αCO2/CH4 = 1.073. Between 10 and 37°C, the results showed a temporal change in the methanogenic pathway. A relatively high (40–60%) CO2-derived fraction of CH4 production in the beginning was followed by a phase in which contribution of CO2-derived CH4 decreased to low (<15%) values, and ultimately by the steady state phase in which values increased to <40% (the theoretically expected value). The rate of change from one phase to the next increased with temperature. Incubation temperature had a strong effect on the overall fractionation of 13C during the formation and consumption of acetate, with stronger fractionation at low than at high temperature. The results further showed that, especially at low temperatures, fractionation occurred during acetate turnover and acetoclastic methanogenesis, despite the fact that steady-state conditions caused (apparent) substrate-limitation.

Phosphate inhibits acetotrophic methanogenesis on rice roots.

ABSTRACT The contribution of acetate- and H2/CO2-dependent methanogenesis to total CH4 production was determined in excised washed rice roots by radiolabeling, methyl fluoride inhibition, and stable carbon isotope fractionation. Addition of ≥20 mM phosphate inhibited methanogenesis, which then was exclusively from H2/CO2. Otherwise, acetate contributed about 50 to 60% of the total methanogenesis, demonstrating that phosphate specifically inhibited acetotrophic methanogens on rice roots.

Carbon isotope fractionation during acetoclastic methanogenesis by Methanosaeta concilii in culture and a lake sediment.

ABSTRACT The isotope enrichment factors (ε) in Methanosaeta concilii and in a lake sediment, where acetate was consumed only by Methanosaeta spp., were clearly less negative than the ε usually observed for Methanosarcina spp. The fraction of methane produced from acetate in the sediment, as determined by using stable isotope signatures, was 10 to 15% lower when the appropriate ε of Methanosaeta spp. was used.

Response of the methanogenic microbial communities in Amazonian oxbow lake sediments to desiccation stress

Methanogenic microbial communities in soil and sediment function only when the environment is inundated and anoxic. In contrast to submerged soils, desiccation of lake sediments happens only rarely. However, some predictions suggest that extreme events of drying will become more common in the Amazon region, and this will promote an increase in sediments drying and exposure. We asked whether and how such methanogenic communities can withstand desiccation stress. Therefore, we determined the rates and pathways of CH(4) production (analysis of CH(4) and δ(13) C of CH(4), CO(2) and acetate), the copy numbers of bacterial and archaeal 16S rRNA genes and mcrA genes (quantitative PCR), and the community composition of Archaea and Bacteria (T-RFLP and pyrosequencing) in oxbow lake sediments of rivers in the Brazilian Amazon region. The rivers were of white water, black water and clear water type. The measurements were done with sediment in fresh state and after drying and rewetting. After desiccation and rewetting the composition of both, the archaeal and bacterial community changed. Since lake sediments from white water rivers exhibited only negligible methanogenic activity, probably because of relatively high iron and low organic matter content, they were not further analysed. The other sediments produced CH(4), with hydrogenotrophic methanogenesis usually accounting for > 50% of total activity. After desiccation and rewetting, archaeal and bacterial gene copy numbers decreased. The bacterial community showed a remarkable increase of Clostridiales from about 10% to > 30% of all Bacteria, partially caused by proliferation of specific taxa as the numbers of OTU shared with fresh sediment decreased from about 9% to 3%. Among the Archaea, desiccation specifically enhanced the relative abundance of either Methanocellales (black water) and/or Methanosarcinaceae (clear water). Despite the changes in gene copy numbers and composition of the microbial community, rates of CH(4) production even increased after desiccation-rewetting, demonstrating that the function of the methanogenic microbial community had not been impaired. This result indicates that the increase in extreme events of drying may increase methane production in flooded sediments.

Structure and function of the methanogenic microbial communities in Uruguayan soils shifted between pasture and irrigated rice fields

Irrigated rice fields in Uruguay are temporarily established on soils used as cattle pastures. Typically, 4 years of cattle pasture are alternated with 2 years of irrigated rice cultivation. Thus, oxic upland conditions are rotated with seasonally anoxic wetland conditions. Only the latter conditions are suitable for the production of CH4 from anaerobic degradation of organic matter. We studied soil from a permanent pasture as well as soils from different years of the pasture-rice rotation hypothesizing that activity and structure of the bacterial and archaeal communities involved in production of CH4 change systematically with the duration of either oxic or anoxic conditions. Soil samples were taken from drained fields, air-dried and used for the experiments. Indeed, methanogenic archaeal gene copy numbers (16S rRNA, mcrA) were lower in soil from the permanent pasture than from the pasture-rice alternation fields, but within the latter, there was no significant difference. Methane production started to accumulate after 16 days and 7 days of anoxic incubation in soil from the permanent pasture and the pasture-rice alternation fields respectively. Then, CH4 production rates were slightly higher in the soils used for pasture than for rice production. Analysis of δ(13) C in CH4, CO2 and acetate in the presence and absence of methyl fluoride, an inhibitor of aceticlastic methanogenesis, indicated that CH4 was mainly (58-75%) produced from acetate, except in the permanent pasture soil (42%). Terminal restriction fragment length polymorphism (T-RFLP) of archaeal 16S rRNA genes showed no difference among the soils from the pasture-rice alternation fields with Methanocellaceae and Methanosarcinaceae as the main groups of methanogens, but in the permanent pasture soil, Methanocellaceae were relatively less abundant. T-RFLP analysis of bacterial 16S rRNA genes allowed the distinction of permanent pasture and fields from the pasture-rice rotation, but nevertheless with a high similarity. Pyrosequencing of bacterial 16S rRNA genes generally revealed Firmicutes as the dominant bacterial phylum, followed by Proteobacteria, Acidobacteria and Actinobacteria. We conclude that a stable methanogenic microbial community established once pastures have been turned into management by pasture-rice alternation despite the fact that 2 years of wetland conditions were followed by 4 years of upland conditions that were not suitable for CH4 production.

Methane production potentials, pathways, and communities of methanogens in vertical sediment profiles of river Sitka

Biological methanogenesis is linked to permanent water logged systems, e.g., rice field soils or lake sediments. In these systems the methanogenic community as well as the pathway of methane formation are well-described. By contrast, the methanogenic potential of river sediments is so far not well-investigated. Therefore, we analyzed (a) the methanogenic potential (incubation experiments), (b) the pathway of methane production (stable carbon isotopes and inhibitor studies), and (c) the methanogenic community composition (terminal restriction length polymorphism of mcrA) in depth profiles of sediment cores of River Sitka, Czech Republic. We found two depth-related distinct maxima for the methanogenic potentials (a) The pathway of methane production was dominated by hydrogenotrophic methanogenesis (b) The methanogenic community composition was similar in all depth layers (c) The main TRFs were representative for Methanosarcina, Methanosaeta, Methanobacterium, and Methanomicrobium species. The isotopic signals of acetate indicated a relative high contribution of chemolithotrophic acetogenesis to the acetate pool.

Distance-Decay and Taxa-Area Relationships for Bacteria, Archaea and Methanogenic Archaea in a Tropical Lake Sediment

The study of of the distribution of microorganisms through space (and time) allows evaluation of biogeographic patterns, like the species-area index (z). Due to their high dispersal ability, high reproduction rates and low rates of extinction microorganisms tend to be widely distributed, and they are thought to be virtually cosmopolitan and selected primarily by environmental factors. Recent studies have shown that, despite these characteristics, microorganisms may behave like larger organisms and exhibit geographical distribution. In this study, we searched patterns of spatial diversity distribution of bacteria and archaea in a contiguous environment. We collected 26 samples of a lake sediment, distributed in a nested grid, with distances between samples ranging from 0.01 m to 1000 m. The samples were analyzed using T-RFLP (Terminal restriction fragment length polymorphism) targeting mcrA (coding for a subunit of methyl-coenzyme M reductase) and the genes of Archaeal and Bacterial 16S rRNA. From the qualitative and quantitative results (relative abundance of operational taxonomic units) we calculated the similarity index for each pair to evaluate the taxa-area and distance decay relationship slopes by linear regression. All results were significant, with mcrA genes showing the highest slope, followed by Archaeal and Bacterial 16S rRNA genes. We showed that the microorganisms of a methanogenic community, that is active in a contiguous environment, display spatial distribution and a taxa-area relationship.

Structure and function of methanogenic microbial communities in sediments of Amazonian lakes with different water types

Tropical lake sediments are a significant source for the greenhouse gas methane. We studied function (pathway, rate) and structure (abundance, taxonomic composition) of the microbial communities (Bacteria, Archaea) leading to methane formation together with the main physicochemical characteristics in the sediments of four clear water, six white water and three black water lakes of the Amazon River system. Concentrations of sulfate and ferric iron, pH and δ13 C of organic carbon were usually higher, while concentrations of carbon, nitrogen and rates of CH4 production were generally lower in white water versus clear water or black water sediments. Copy numbers of bacterial and especially archaeal ribosomal RNA genes also tended to be relatively lower in white water sediments. Hydrogenotrophic methanogenesis contributed 58 ± 16% to total CH4 production in all systems. Network analysis identified six communities, of which four were comprised mostly of bacteria found in all sediment types, while two were mostly in clear water sediment. Terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing showed that the compositions of the communities differed between the different sediment systems, statistically related to the particular physicochemical conditions and to CH4 production rates. Among the archaea, clear water, white water, and black water sediments contained relatively more Methanomicrobiales, Methanosarcinaceae and Methanocellales, respectively, while Methanosaetaceae were common in all systems. Proteobacteria, Deltaproteobacteria (Myxococcales, Syntrophobacterales, sulfate reducers) in particular, Acidobacteria and Firmicutes were the most abundant bacterial phyla in all sediment systems. Among the other important bacterial phyla, clear water sediments contained relatively more Alphaproteobacteria and Planctomycetes, whereas white water sediments contained relatively more Betaproteobacteria, Firmicutes, Actinobacteria, and Chloroflexi than the respective other sediment systems. The data showed communities of bacteria common to all sediment types, but also revealed microbial groups that were significantly different between the sediment types, which also differed in physicochemical conditions. Our study showed that function of the microbial communities may be understood on the basis of their structures, which in turn are determined by environmental heterogeneity.