Microbiology and biochemistry of the enhanced biological phosphate removal process

Eight oligonucleotides which are complementary to conserved tracts of 16S rRNA from phylogenetically defined groups of methanogens were designed and characterized for use as hybridization probes for studies in environmental and determinative microbiology. The target-group specificity and temperature of dissociation for each probe were characterized. In general, the probes were very specific for the target methanogens and did not hybridize to the rRNAs of nontarget methanogens. Together, the eight probes circumscribe methanogens now represented in pure culture (with the exception of members of the family Methanothermaceae). Three probes are order specific; two identify members of the order Methanobacteriales, and one is specific for the order Methanococcales. The fourth probe encompasses three families belonging to the order Methanomicrobiales, the third order within the current classification. The fifth probe is specific for the remaining family within this order (Methanosarcinaceae). Three additional probes encompass different genera within the Methanosarcinaceae.

Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens

Biological methanogenesis plays a major role in the carbon cycle on Earth. Methanogenesis is the terminal step in carbon flow in many anaerobic habitats, including marine and freshwater sediments, marshes and swamps, flooded soils, bogs, geothermal habitats, and animal gastrointestinal tracts. CH4 escaping from anaerobic habitats can serve as a carbon and energy source for aerobic methanotrophic bacteria, and can escape to the atmosphere, where it is a major participant in atmospheric chemical reactions and is an important greenhouse gas.

Physiological Ecology of Methanogens

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.

Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval.

Taxonomic, Phylogenetic, and Ecological Diversity of Methanogenic Archaea

A novel thermophilic acetotrophic Methanosaeta strain was isolated from a thermophilic anaerobic digest or by using acetate enrichment and serial dilution in the presence of vancomycin and neomycin. This isolate, designated Methanosaeta sp. strain PT, resembled Methanosaeta sp. strain CALS-1 morphologically; however, it occasionally formed filaments longer than 100 μm and exhibited autofluorescence. The content of coenzyme F420 was much higher than that of Methanosaeta reference strains, and coenzyme F420 with four glutamyl residues on the side chain was the predominant component. Furthermore, a comparative analysis of the antigenic fingerprint of strain PT with the fingerprints of reference organisms showed that this isolate was not related antigenically to the reference methanogens, including Methanosaeta sp. (“Methanothrix” sp.) strain CALS-1 and Methanosaeta concilii (“Methanothrix soehngenii”) Opfikon. Strain PT formed visible colonies in a deep agar medium when high cell concentrations were present. However, transfer of a colony into liquid medium resulted in no growth. Strain PT could utilize only acetate as a sole carbon and energy source. The optimum temperature and optimum pH for methanogenesis were near 55°C and 6.7, respectively. The specific methane formation rate μCH
4 under optimum conditions was 0.47 day−1, and the doubling time was 1.49 days. The DNA base composition was 52.7 mol% guanine plus cytosine.

Isolation and Characterization of a Novel Thermophilic Methanosaeta Strain

Methanethiol, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide were efficiently removed from contaminated air by Thiobacillus thioparus TK-m and oxidized to sulfate stoichiometrically. More than 99.99% of dimethyl sulfide was removed when the load was less than 4.0 g of dimethyl sulfide per g (dry cell weight) per day.

Removal of methanethiol, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide from contaminated air by Thiobacillus thioparus TK-m

Three thermophilic Methanothrix (“Methanosaeta”) strains, strains PT T (= DSM 6194T) (T = type strain), CALS-1 (= DSM 3870), and Z-517 (= DSM 4774), were characterized chemotaxonomically and compared with five mesophilic strains, Methanothrix soehngenii (“Methanosaeta concilii”) GP6 (= DSM 3671), Opfikon (= DSM 2139), FE (= DSM 3013), UA, and PM. These methanogens were exclusively acetotrophic and had a characteristic sheathed structure. The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine. The thermophilic strains often had phase-refractive gas vesicles inside their cells. Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Methanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups. The difference between the thermophiles and mesophiles was also verified by comparing partial 16S rRNA sequences (ca. 30 base differences in ca. 540 bases). On the basis of our results, we propose the name Methanothrix thermophila for the three thermophilic strains. The type strain of M. thermophila is strain PT (= DSM 6194). We also propose that the name Methanothrix thermoacetophila (“Methanosaeta thermoacetophila”), which was given to strain Z-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate.

Characterization of three thermophilic strains of Methanothrix ("Methanosaeta") thermophila sp. nov. and rejection of Methanothrix ("Methanosaeta") thermoacetophila.

Removal of dimethyl sulfide, methyl mercaptan, and hydrogen sulfide by immobilized Thiobacillus thioparus TK-m

A new thermophilic sulfate-reducing bacterium, strain TSB, that was spore-forming, rod-shaped, slightly motile and gram-positive, was isolated from a butyrate-containing enrichment culture inoculated with sludge of a thermophilic methane fermentation reactor. This isolate could oxidize benzoate completely. Strain TSB also oxidized some fatty acids and alcohols. SOinf4sup2-, SOinf3sup2-, S2Oinf3sup2-and NOinf3sup-were utilized as electron acceptors. With pyruvate or lactate the isolate grew without an external electron acceptor and produced acetate. The optimum temperature for growth was 62°C. The G+C content of DNA was 52.8 mol%. This isolate is described as a new species, Desulfotomaculum thermobenzoicum.

Eiichi Mikami

Papers

Isolation and Characterization of a Novel Thermophilic Methanosaeta Strain

Removal of methanethiol, dimethyl sulfide, dimethyl disulfide, and hydrogen sulfide from contaminated air by Thiobacillus thioparus TK-m

Characterization of three thermophilic strains of Methanothrix ("Methanosaeta") thermophila sp. nov. and rejection of Methanothrix ("Methanosaeta") thermoacetophila.

Removal of dimethyl sulfide, methyl mercaptan, and hydrogen sulfide by immobilized Thiobacillus thioparus TK-m

Isolation and characterization of a thermophilic benzoate-degrading, sulfate-reducing bacterium, Desulfotomaculum thermobenzoicum sp. nov.