A. Yu. Kallistova

Enumeration of Methanotrophic Bacteria in the Cover Soil of an Aged Municipal Landfill

The enumeration of methanotrophic bacteria in the cover soil of an aged municipal landfill was carried out using (1) fluorescent in situ hybridization (FISH) with horseradish peroxidase-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition-FISH (CARD-FISH), and (2) most probable number (MPN) method. The number of methanotrophs was determined in cover soil samples collected during April–November 2003 from a point with low CH4 emission. The number of types I and II methanotrophs obtained by CARD-FISH varied from 15 ± 2 to 56 ± 7 × 108 cells g−1 absolute dry mass (adm) of soil and methanotrophs of type I dominated over type II. The average number of methanotrophs throughout the cover soil profile was highest during May–September when the cover soil temperature was above 13°C. Methanotrophs accounted for about 50% of the total bacterial population in the deepest cover soil layer owing to higher availability of substrate (CH4). A lower number of methanotrophs (7 × 102 to 17 × 105 cells g−1 adm of soil) was determined by the MPN method compared to the CARD-FISH counts, thus confirming previous results that the MPN method is limited to the estimation of the culturable species that can be grown under the incubation conditions used. The number of culturable methanotrophs correlated with the methane-oxidizing activity measured in laboratory assays. In comparison to the incubation-based measurements, the number of methanotrophs determined by CARD-FISH better reflected the actual characteristics of the environment, such as release and uptake of CH4, temperature, and moisture, and availability of substrates.

[Candidatus "Jettenia moscovienalis" sp. nov., a New Species of Bacteria Carrying out Anaerobic Ammonium Oxidation].

A new species of bacteria oxidizing ammonium with nitrite under anoxic conditions was isolated from the activated sludge of a semi-industrial bioreactor treating digested sludge of the Kuryanovo wastewater treatment plant (Moscow, Russia). Physiological, morphological, and molecular genetic characterization of the isolate was carried out. The cells were ovoid (∼0.5 × 0.8 μm), with the intracellular membrane structures characteristic of anammox bacteria (anammoxosome and paryphoplasm); unlike other anammox bacteria, it possessed extensive intracellular membrane structures located in layers parallel to the cytoplasmic membrane, but never close to the anammoxosome. The cells formed aggregates 5–28 μm in diameter and readily attached to solid surfaces. The cells were morphologically labile, easily plasmolyzed, and lost their content. Doubling time was 28 days, μmax = 0.025 day−1; optimal temperature and pH for growth were 20–45°C and 8.0, respectively. Phylogenetic analysis of the 16S rRNA gene sequences suggested its classification as a new species of the candidate genus Jettenia (order Planctomycetales). The name Candidatus “Jettenia moscovienalis” sp. nov. was proposed for the new bacterium.

Sulfate reduction and methanogenesis in the Shira and Shunet meromictic lakes (Khakasia, Russia)

The biogeochemical and molecular biological study of the chemocline and sediments of saline meromictic lakes Shira and Shunet (Khakasia, Russia) was performed. A marked increase in the rates of sulfate reduction and methanogenesis was revealed at the medium depths of the chemocline. The rates of these processes in the bottom sediments decreased with depth. The numbers of the members of domains Bacteria, Archaea, and of sulfate-reducing bacteria (SRB) were determined by fluorescence in situ hybridization with rRNA specific oligonucleotide probes labeled with horseradish peroxidase and subsequent tyramide signal amplification. In the chemocline, both the total microbial numbers and those of Bacteria were shown to increase with depth. The archaea and SRB were present in almost equal numbers. In the lake sediments, a drastic decrease in microbial numbers with depth was revealed. SRB were found to prevail in the upper sediment layer and archaea in the lower one. This finding correlated with the measured rates of sulfate reduction and methanogenesis.

Microbial diversity of methanogenic communities in the systems for anaerobic treatment of organic waste

Methane production via anaerobic degradation of organic-contaminated wastewater, semiliquid, or solid municipal waste of complex composition by methanogenic microbial communities is a multistage process involving at least four groups of microorganisms. These are hydrolytic bacteria (polysaccharolytic, proteolytic, and lipolytic), fermentative bacteria, acetogenic bacteria (syntrophic, proton-reducing), and methanogenic archaea; complex trophic interactions exist between these groups. The review provides information concerning the diversity of the major microbial groups identified in the systems for wastewater and concentrated waste treatment, solid-phase anaerobic fermentation, and landfills for disposal of municipal solid waste, and also specifies the sources of isolation of the type strains. The research demonstrates that both new microorganisms and those previously isolated from natural habitats may be found in waste treatment systems. High microbial diversity in the systems for organic waste treatment provides for stable methanogenesis under fluctuating environmental conditions.

Methane formation and oxidation in the meromictic oligotrophic Lake Gek-Gel (Azerbaijan).

The production and oxidation of methane and diversity of culturable aerobic methanotrophic bacteria in the water column and upper sediments of the meromictic oligotrophic Lake Gek-Gel (Azerbaijan) were studied by radioisotope, molecular, and microbiological techniques. The rate of methane oxidation was low in the aerobic mixolimnion, increased in the chemocline, and peaked at the depth where oxygen was detected in the water column. Aerobic methanotrophic bacteria of type II belonging to the genus Methylocystis were identified in enrichment cultures obtained from the chemocline. Methane oxidation in the anaerobic water of the monimolimnion was much more intense than in the aerobic zone. However, below 29–30 m methane concentration increased and reached 68 μM at the bottom. The highest rate of methane oxidation under anaerobic conditions was revealed in the upper layer of bottom sediments. The rate of methane oxidation significantly exceeding that of methane production suggests a deep source of methane in this lake.

Microbial composition of the activated sludge of Moscow wastewater treatment plants

The contribution of the major technologically important microbial groups (ammonium- and nitrite-oxidizing, phosphate-accumulating, foam-inducing, and anammox bacteria, as well as planctomycetes and methanogenic archaea) was characterized for the aeration tanks of the Moscow wastewater treatment facilities. FISH investigation revealed that aerobic sludge were eubacterial communities; the metabolically active archaea contributed insignificantly. Stage II nitrifying microorganisms and planctomycetes were significant constituents of the bacterial component of activated sludges, with Nitrobacter spp. being the dominant nitrifiers. No metabolically active anammox bacteria were revealed in the sludge from aeration tanks. The sludge from the aeration tanks using different wastewater treatment technologies were found to have differing characteristics. Abundance of the nitrifying and phosphate-accumulating bacteria in the sludge generally correlated with microbial activity in microcosms and with efficiency of nitrogen and phosphorus removal from wastewater. The highest microbial numbers and activity were found in the sludge of the tanks operating according to the technologies developed in the universities of Hannover and Cape Town. The activated sludge from the Novokur’yanovo facilities, where abundant growth of filamentous bacteria resulted in foam formation, exhibited the lowest activity. The group of foaming bacteria included Gordonia spp. and Acinetobacter spp utilizing petroleum and motor oils, Sphaerotilus spp. utilizing unsaturated fatty acids, and Candidatus ‘Microthrix parvicella’. Thus, the data on abundance and composition of metabolically active microorganisms obtained by FISH may be used for the technological control of wastewater treatment.

Role of anammox bacteria in removal of nitrogen compounds from wastewater

The review deals with the unique microbial group responsible for anaerobic ammonium oxidation with nitrite (anammox), and with the role of this process in development of the biotechnology for removal of nitrogen compounds from wastewater. The history of the study of this process is briefly related. Up-to date knowledge on the intracellular organization, energy metabolism, growth stoichiometry, and physiology of anammox bacteria is described, and the main methods for cultivation of these microorganisms are characterized. Special attention is paid to the problems associated with practical application of anammox bacteria, which result from their extremely slow growth, the absence of pure cultures, and the interaction with other microbial groups.

[Intensification of Microbial Decomposition of Organic Fraction of Municipal Waste: Laboratory and Field Experiments].

Methods of intensifying the anaerobic microbial decomposition of the organic fraction of municipal solid waste (MSW) on an MSW landfill and in anaerobic reactors were studied. It was discovered that it is preferable for the initiation and stabilization of the process of anaerobic digestion of organic waste in laboratory bioreactors at 20 and 50°C to use a mixture of activated suspension of soil from the anaerobic zone of the landfill and digested sewage sludge. Stimulation of methanogenesis was shown in field conditions when digested sewage sludge was added directly into the upper layer of anaerobic zone of the landfill. The investigation of methane production during fermentation of concentrated food waste with a mixture of excessive activated sludge in the laboratory under thermophilic conditions (50°C) has shown that the main problem at the first stage of the process was the acidification of the digested mixture due to the accumulation of volatile fatty acids. It was shown that for stable operation of the bioreactor under thermophilic conditions the amount of inoculum added during the start up should be no less than 30-50%—based on the total volatile suspended solids. A sharp decrease in the digestion temperature from 50 to 20°C did not cause methanogenesis termination, since the thermophilically fermented biomass contained both thermophilic and mesophilic methanogens.

Culturable psychrotolerant methanotrophic bacteria in landfill cover soil

Methanotrophs closely related to psychrotolerant members of the genera Methylobacter and Methylocella were identified in cultures enriched at 10°C from landfill cover soil samples collected in the period from April to November. Mesophilic methanotrophs of the genera Methylobacter and Methylosinus were found in cultures enriched at 20°C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40°C from a sample collected in May (the temperature of the cover soil was 11.5–12.5°C). In addition to methanotrophs, methylobacteria of the genera Methylotenera and Methylovorus and members of the genera Verrucomicrobium, Pseudomonas, Pseudoxanthomonas, Dokdonella, Candidatus Protochlamydia, and Thiorhodospira were also identified in the enrichment cultures. A methanotroph closely related to the psychrotolerant species Methylobacter tundripaludum (98% sequence identity of 16S rRNA genes with the type strain SV96T) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15°C, into the landfill cover soil resulted in a decrease in methane emission from the landfill surface in autumn (October, November). The inoculum used was demonstrated to contain methanotrophs closely related to Methylobacter tundripaludum SV96.

Methane formation and oxidation by prokaryotes

The review deals with systematization and generalization of new information concerning the phylogenetic and functional diversity of prokaryotes involved in the methane cycle. Methane is mostly produced by methanogenic archaea, which are responsible for the terminal stage of organic matter decomposition in a number of anoxic ecotopes. Although phylogeny, physiology, and biochemistry of methanogens have been extensively studied, important discoveries were made recently. Thus, members of deep phylogenetic lineages within the Euryarchaeota phylum (Methanomassiliicoccales, “Candidatus Methanofastidiosa,” “Methanonatronarchaeia”) and even outside it (“Ca. Verstraetearchaeota” and “Ca. Bathyarchaeota”) were reported to carry out methyl-reducing methanogenesis. Moreover, evidence was obtained on aerobic methane production by marine heterotrophic bacteria, which demethylate polysaccharide esters of methylphosphonic acid. Methanotrophic microorganisms oxidize methane both aerobically and anaerobically, decreasing significantly the release of this greenhouse gas into the atmosphere. In the presence of oxygen methane is oxidized by methanotrophic members of Alpha- and Gammaproteobacteria, as well as by Verrucomicrobia. Methanotrophic gammaproteobacteria have been recently revealed in hypoxic and even anoxic environments, where they probably oxidize methane either in a trophic consortium with oxygenic phototrophs and/or methylotrophs or using electron acceptors other than oxygen. Anaerobic methane oxidation has been known for a long time. Sulfat- and nitrate-dependent anaerobic methane oxidation carried out by the ANME archaea via reverse methanogenesis are the best studied processes. While metal-dependent anaerobic methane oxidation is considered possible, the mechanisms and agents responsible for this process have not been reliably identified. Intracellular oxygen production during nitrite-dependent anaerobic methane oxidation was shown for bacteria “Ca. Methylomirabilis oxyfera.” These findings stimulate interest in the processes and microorganisms of the methane cycle.