Methanomethylovorans thermophila sp. nov., a thermophilic, methylotrophic methanogen from an anaerobic reactor fed with methanol.

A novel thermophilic, obligately methylotrophic, methanogenic archaeon, strain L2FAW(T), was isolated from a thermophilic laboratory-scale upflow anaerobic sludge blanket reactor fed with methanol as the carbon and energy source. Cells of strain L2FAW(T) were non-motile, irregular cocci, 0.7-1.5 mum in diameter and usually occurred singly (sometimes forming clusters of two or four cells). The cells stained Gram-negative and lysed immediately in 0.1 % (w/v) SDS. Growth was inhibited by chloramphenicol and tetracycline, but not by penicillin, bacitracin, spectinomycin, vancomycin or kanamycin. Methanol and mono-, di- and trimethylamine were used as substrates, but H2/CO2, formate, acetate, propanol, dimethyl sulfide and methanethiol were not. The temperature range for growth was 42-58 degrees C, with an optimum at 50 degrees C. The fastest growth was observed at a salinity below 100 mM NaCl; no growth occurred above 300 mM NaCl. The optimal pH for growth was 6.5; growth was observed from pH 5 to pH 7.5. The G+C content of the genomic DNA was 37.6 mol%. Analysis of the 16S rRNA gene sequence and the partial methyl-CoM reductase gene sequence revealed that the organism was phylogenetically closely related to Methanomethylovorans hollandica DMS1T (98 % similarity for the 16S rRNA gene sequence and 91 % similarity for the methyl-CoM reductase gene sequence). The DNA-DNA relatedness between L2FAW(T) and Methanomethylovorans hollandica DMS1T was 46 %. On the basis of these results, strain L2FAW(T) (=DSM 17232T=ATCC BAA-1173T) represents the type strain of a novel species, for which the name Methanomethylovorans thermophila sp. nov. is proposed.

Carbon monoxide conversion by thermophilic sulfate-reducing bacteria in pure culture and in co-culture with Carboxydothermus hydrogenoformans.

Biological sulfate (SO(4)) reduction with carbon monoxide (CO) as electron donor was investigated. Four thermophilic SO(4)-reducing bacteria, Desulfotomaculum thermoacetoxidans (DSM 5813), Thermodesulfovibrio yellowstonii (ATCC 51303), Desulfotomaculum kuznetsovii (DSM 6115; VKM B-1805), and Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum (DSM 14055), were studied in pure culture and in co-culture with the thermophilic carboxydotrophic bacterium Carboxydothermus hydrogenoformans (DSM 6008). D. thermoacetoxidans and T. yellowstonii were extremely sensitive to CO: their growth on pyruvate was completely inhibited at CO concentrations above 2% in the gas phase. D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were less sensitive to CO. In pure culture, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were able to grow on CO as the only electron donor and, in particular in the presence of hydrogen/carbon dioxide, at CO concentrations as high as 50-70%. The latter SO(4) reducers coupled CO oxidation to SO(4) reduction, but a large part of the CO was converted to acetate. In co-culture with C. hydrogenoformans, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum could even grow with 100% CO (P(CO) = 120 kPa).

Effect of carbon monoxide, hydrogen and sulfate on thermophilic (55 degrees C) hydrogenogenic carbon monoxide conversion in two anaerobic bioreactor sludges.

The conversion routes of carbon monoxide (CO) at 55 degrees C by full-scale grown anaerobic sludges treating paper mill and distillery wastewater were elucidated. Inhibition experiments with 2-bromoethanesulfonate (BES) and vancomycin showed that CO conversion was performed by a hydrogenogenic population and that its products, i.e. hydrogen and CO2, were subsequently used by methanogens, homo-acetogens or sulfate reducers depending on the sludge source and inhibitors supplied. Direct methanogenic CO conversion occurred only at low CO concentrations [partial pressure of CO (PCO) <0.5 bar (1 bar=10(5) Pa)] with the paper mill sludge. The presence of hydrogen decreased the CO conversion rates, but did not prevent the depletion of CO to undetectable levels (<400 ppm). Both sludges showed interesting potential for hydrogen production from CO, especially since after 30 min exposure to 95 degrees C, the production of CH4 at 55 degrees C was negligible. The paper mill sludge was capable of sulfate reduction with hydrogen, tolerating and using high CO concentrations (PCO>1.6 bar), indicating that CO-rich synthesis gas can be used efficiently as an electron donor for biological sulfate reduction.

Temperature characteristics of methanogenic archaea and acetogenic bacteria isolated from cold environments.

In most terrestrial ecosystems of boreal and northern climate zones degradation of organic matter with methane production occurs at low temperature. Two psychrophilic methanogenic archaea and four acetogenic bacteria were described until now. Recently, we isolated 12 new strains of methanogenic archaea and 3 strains of acetogenic bacteria from different natural and man-made cold environments including tundra permafrost wet land, sediments of deep lakes, silt of sludge disposal pond, pig and cattle manure digested at 6 degrees C, and an anaerobic EGSB-reactor operated at 9 degrees C. The temperature characteristics of microorganisms isolated from cold environments are discussed. All isolates are able to grow below 10 degrees C, most of them grow at such low temperature as 1 degrees C. The upper temperature limit for most growing at low temperature acetogens is 30 degrees C, and the temperature optima is 20 degrees C and below. Most isolated methanogens have temperature optima around 25 degrees C, and upper temperature limits at 30-40 degrees C. Whether microorganisms able to grow at low temperature are classified as mesophiles, psychrophiles, or psychrotrophs (psychrotolerants) is an issue of this article. We propose to modify the basic temperature definition of anaerobic microorganisms growing at low temperature.

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).

[Growth characteristics of haploid and diploid Hansenula polymorpha yeasts on methanol in continuous culture]. / Kharakteristika rosta gaploidnykh i diploidnykh drozhzhei Hansenula polymorpha na metanole v usloviiakh nepreryvnogo kul'tivirovaniia.

The rate of growth and the yield of biomass of a Hunsenula polymorpha homozygous diploid strain ML-3 X ML-3 were nearly identical with those of the parent haploid culture ML-3. The two cultures were eliminated from the fermenter at D = 0.20 to 0.21 h-1. In contrast, a heterozygous diploid strain ML-3 X VKM 1397 could grow at D = 0.23 to 0.25 h-1 and its biomass yield reached 38% while the yields of the haploid and homozygous diploid strains were 35 to 36%. The pH of the medium had the same effect on the three cultures: a change in the pH from 4.0 to 3.5 and then to 3.0 did not influence the yield of biomass; a further pH drop to 2.5 made the cells be eliminated from the fermenter.

[Continuous cultivation of Hansenula polymorpha and Candida krusei on mixtures of methyl and ethyl alcohols]. / Nepreryvnoe kul'tivirovanie Hansenula polymorpha i Candida krusei na smesi metilovogo i étilovogo spirtov.

The object of this work was to study whether it would be possible to cultivate together ther the methanol-assimilating yeast Hansenula polymorpha M-2 requiring biotin and thiamine for its growth and the vitamin-prototrophic yeast Candida krusei E-10 actively growing in a medium with ethanol, without vitamin addition to the medium. The growth of the mixed culture in a medium containing ethanol and methanol depended on the pH of the medium; the biomass yield was maximal at pH 3.5 which was the suboptimal value for H. polymorpha M-2. The biomass yield decreased when the relative content of methanol in the mixed substrate was increased. At the optimal pH 3.5 and the optimal methanol-to-ethanol ratio (1:1), the productivity exceeded 2.0 g/litre per hour and the dilution rate D reached 0.17 h-1, the maximal value for H. polymorpha M-2 at this pH. The cultural broth obtained upon the growth of C. krusei E-10 on ethanol as a pure culture could not be used for the cultivation of H. polymorpha M-2 on methanol unless the vitamins were added.