A. N. Nozhevnikova

Shift from Acetoclastic to H2-Dependent Methanogenesis in a West Siberian Peat Bog at Low pH Values and Isolation of an Acidophilic Methanobacterium Strain

ABSTRACT Methane production and archaeal community composition were studied in samples from an acidic peat bog incubated at different temperatures and pH values. H2-dependent methanogenesis increased strongly at the lowest pH, 3.8, and Methanobacteriaceae became important except for Methanomicrobiaceae and Methanosarcinaceae. An acidophilic and psychrotolerant Methanobacterium sp. was isolated using H2-plus-CO2-supplemented medium at pH 4.5.

Isolation and characterization of new strains of methanogens from cold terrestrial habitats

Five strains of methanogenic archaea (MT, MS, MM, MSP, ZB) were isolated from permanently and periodically cold terrestrial habitats. Physiological and morphological studies, as well as phylogenetic analyses of the new isolates were performed. Based on sequences of the 16S rRNA and methyl-coenzyme M reductase a-subunit (mcrA) genes all new isolates are closely related to known mesophilic and psychrotolerant methanogens. Both, phylogenetic analyses and phenotypic properties allow to classify strains MT, MS, and MM as members of the genus Methanosarcina. Strain MT is a new ecotype of Methanosarcina mazei, whereas strains MM and MS are very similar to each other and can be assigned to the recently described psychrotolerant species Methanosarcina lacustris. The hydrogenotrophic strain MSP is a new ecotype of the genus Methanocorpusculum. The obligately methylotrophic strain ZB is closely related to Methanomethylovorans hollandica and can be classified as new ecotype of this species. All new isolates, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. In spite of the ability of all isolates to grow at temperatures as low as 1-5 degrees C, all of them have their growth optima in the range of moderate temperatures (25-35 degrees C). Thus, they can be regarded as psychrotolerant organisms. Psychrotolerant methanogens are thought to play an important role in methane production in both, habitats under seasonal temperature variations or from permanently cold areas.

Methanogenesis at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H2/CO2 as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H2/CO2-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.

Methanogenic degradation of organic matter by anaerobic bacteria at low temperature

Abstract Microbial decomposition of organic matter in a cold climatic zone is important since it is in the zone of boreal forests and marshes that the atmospheric content of the greenhouse gases, such as CO 2 and CH 4 , undergoes the greatest changes. Decomposition of various organic substrates by a psychrophilic methanogenic community studied over the year at three temperatures: 28°C (control), 15°C, and 6°C. Substrate dynamics and product concentrations explained the trophic interactions in the microbial community. The high rate of acetogenesis, observed at low temperature, did not correlate with the relatively low rate of the acetoclastic methanogenesis.

Organic removal and microbiological features of UASB-reactor under various organic loading rates

Abstract The performance of a laboratory upflow anaerobic sludge blanket reactor (UASB-reactor) fed synthetic wastewater was investigated under organic loading rates (OLRs) ranging from 3·4 to 44·9 g COD/l·day. The distribution of substrates, intermediate byproducts, pH, VSS, specific sludge activities (acidogenic, acetoclastic, lithotrophic) and the number of different groups of microorganisms at different heights in the reactor were monitored. The conditions for active granular biomass formation were found for the OLRs investigated. When using high-flow UASB-reactors, attention should be paid to the efficient retention of the most active biomass circulating in the sludge blanket zone. The numbers of methanogens were one to two orders of magnitude higher in the lower part of the reactor (sludge bed zone) than in the upper part (sludge blanket zone). Electron micrographs of the granules showed that the predominant microbial biomass was Methanothrix. However, increasing the OLR led to a substantial increase of Methanosarcina in the granules. Significant amounts of other bacteria were found distributed within the granules between Methanothrix filaments.

Evaluation of kinetic coefficients using integrated Monod and Haldane models for low-temperature acetoclastic methanogenesis

The integrated Monod and Haldane models were used to evaluate the kinetic coefficients and their standard deviations using the methane accumulation curves of low-temperature acetoclastic methanogenesis. The linear and exponential approximations and the limitations of their applicability were deduced from the integrated models. The samples of lake sediments and biomass taken from a low-temperature upflow anaerobic sludge blanket (UASB) reactor were used as inoculum in batch assays for acetate methanation. In comparison, the Monod and Haldane models were applied to evaluate the kinetic coefficients for mesophilic acetoclastic methanogenesis accomplished by the pure culture of Methanosarcina barkeri strain MS. The Monod and Haldane models and their approximations were fitted by using non-linear regression. For the wide range of initial acetateconcentrations (4.2-84 mM: 5-100 mM) applied to the UASB biomass at 11 and 22 degrees C and for the lake sediment samples at 6 and 15 degrees C, a better fit was obtained with the Haldane models and their exponential approximations, respectively. For the lake sediments the values of inhibition coefficients decreased at decreasing temperatures. At the highest temperature of 30 degrees C no difference was found between the Haldane and Monod models and the simpler Monod model should be preferred. The values of the maximum growth rate of biomass were highest at 30 degrees C (lake sediment) and 22 degrees C (the UASB biomass) being in a range presented in the literature for mesophilic acetoclastic methanogenesis.

Methanosarcina lacustris sp. nov., a new psychrotolerant methanogenic archaeon from anoxic lake sediments.

A new psychrotolerant methanogenic archaeon strain ZS was isolated from anoxic lake sediments (Switzerland). The cells of the organism were non-motile cocci, 1.5-3.5 microm in diameter. The cells aggregated and formed pseudoparenchyma. The cell wall was Gram-positive. The organism utilized methanol, mono-, di-, trimethylamine and H2/CO2 with methane production. The temperature range for growth was 1-35 degrees C with an optimum at 25 degrees C. The DNA G+C content of the organism was 43.4. mol%. Analysis of the 16S rRNA gene sequence showed that strain ZS was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (95.6-97.6% of sequence similarity). The level of DNA-DNA hybridization of strain ZS with Methanosarcina barkeri and Methanosarcina mazei was 15 and 31%, respectively. Based on the results of physiological and phylogenetic studies strain ZS can be assigned to a new species of the genus Methanasarcina. The name Methanosarcina lacustris sp. nov. is proposed. The type strain is ZS (= DSM 13486T, VKM B-2268).

New species of psychrophilic acetogens: Acetobacterium bakii sp. nov., A. paludosum sp. nov., A. fimetarium sp. nov.

Three strains of new acetogenic bacteria were isolated from several low temperature environments. Cells were gram-positive, oval-shaped flagellated rods. The organisms fermented H2/CO2, CO, formate, lactate, and several sugars to acetate. Strains Z-4391 and Z-4092 grew in the temperature range from 1 to 30°C with an optimum at 20°C; strain Z-4290 grew in the range from 1 to 35°C with an optimum at 30°C. The DNA G+C content of strains Z-4391, Z-4092, and Z-4290 was 42.1, 41.7, and 45.8 mol% respectively.

Acetogenesis at Low Temperature

Most habitats on earth have a low average annual temperature, and more than 80% of the biosphere is permanently cold. In general, the study of mesophilic processes have dominated the interests of biologists. However, though poorly characterized, microbial processes in cold climatic zones may play important roles in the biological cycles and global ecology.

Modelling low-temperature methane production from cattle manure by an acclimated microbial community

The modified model of anaerobic digestion suggested earlier was used to describe a low temperature methanogenesis during a batch cattle-manure digestion by an acclimated microbial community investigated by Kotsyurbenko et al. (1993). The model showed that the pH inhibition of acetoclastic methanogens and propionate-consuming syntrophs is significant in explanation of the long-term constant values of acetate and propionate concentrations in spite of an appreciable rate of methanogenesis. A low rate of acetate production during acidogenesis as well as propionate and butyrate degradation at 6°C is compensated by a low rate of acetate consumption during acetoclastic methanogenesis. The system dynamics was described well by two alternative microbial pathways with or without participation of homoacetogenic bacteria. The corresponding kinetic coefficients were evaluated. The independent experimental data during a low-temperature cattle manure digestion without inoculation were used for the model verification. It was shown that a dominance of homoacetogenic bacteria over hydrogen-consuming methanogens needs to be considered to describe the hydrogen, methane, VFA and pH time-profiles.