[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 degrees 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 degrees 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 volatile suspended solids. A sharp decrease in the digestion temperature from 50 to 20 degrees C did not cause methanogenesis termination, since the thermophilically fermented biomass contained both thermophilic and mesophilic methanogens.

[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 x 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, µ(max) = 0.025 day(-1); optimal temperature and pH for growth were 20-45 degrees 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.

[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 degrees C from the same cover soil samples. A thermotolerant methanotroph related to Methylocaldum gracile was identified in the culture enriched at 40 degrees C from a sample collected in May (the temperature of the cover soil was 11.5-12.5 degrees 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 r-RNA genes with the type strain SV96(T)) was isolated in pure culture. The introduction of a mixture of the methanotrophic enrichments, grown at 15 degrees 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.

[Microbial composition of the activated sludges of the 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 sludges were eubacterial communities; the metabolically active archaea contributed insignificantly. Stage II nitrifying microorganisms and planctomycetes were significant constituents of the bacterial component of activated sludge, with Nitrobacter spp. being the dominant nitrifier. 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 differ in characteristics. Abundance of the nitrifying and phosphate-accumulating bacteria in the sludges 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 sludges of the tanks operating according to the technologies developed in the universities of Hanover 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.

[Lipid composition of activated sludge in a pilot plant for anaerobic ammonium oxidation].

The lipid composition of the microbial community inhabiting activated sludge in a pilot reactor for the anaerobic oxidation of ammonium (anammox) at the Kur'yanovo Treatment Plant (Moscow) has been studied. The fatty acid composition is mostly based on common fatty acids C14-C18 (95%) with both normal and isomeric structures. The biomass of activated sludge was found to contain lipids with the so-called ladderane substances (ladder alcohols and fatty acids) that are common for anammox bacteria: C20-[3]-ladderane and C20-[5]-ladderane alcohols and C18-and C20-[3]-ladderane and C18- and C20-[5]-ladderane acids. In addition, the native extract contained both simple and compound ethers of the above-mentioned substances with residues of phosphocholine, phosphoethanolamine, and phosphoglycerine. The spectra of the electron impact and tandem mass spectrometry of certain substances have been obtained and published for the first time.

Comparison of different thickening methods for active biomass recycle for anaerobic digestion of wastewater sludge.

The effect of returning solids to the digester, after one of three thickening processes, on volatile solids reduction (VSR) and gas production was investigated. Three different thickening methods were compared: centrifugation, flotation and gravitational sedimentation. The amount and activity of retained biomass in thickened recycled sludge affected the efficiency of digestion. Semi-continuous laboratory digesters were used to study the influence of thickening processes on thermophilic sludge digestion efficiency. Centrifugation was the most effective method used and caused an increase of VSR from 43% (control) up to 70% and gas generation from 0.40 to 0.44 L g(-1) VS. Flotation and gravitational sedimentation ways of thickening appeared to be less effective if compared with centrifugation. These methods increased VSR only by up to 65 and 51%, respectively and showed no significant increase of gas production. The dewatering capacity of digested sludge, as measured by its specific resistance to filtration, was essentially better for the sludge digested in the reactors with centrifugated and settled recycle. The VS concentration of recycle (g L(-1)), as reflecting the amount of retained biomass, appeared to be one of the most important factors influencing the efficiency of sludge digestion in the recycling technology.

Anaerobic thermophilic digestion of sewage sludge with a thickened sludge recycle.

The process of anaerobic thermophilic digestion of municipal wastewater sludge with a recycled part of thickened digested sludge, was studied in semi-continuous laboratory digesters. This modified recycling process resulted in increased solids retention time (SRT) with the same hydraulic retention time (HRT) as compared with traditional digestion without recycling. Increased SRT without increasing of HRT resulted in the enhancement of volatile substance reduction by up to 68% in the reactor with the recycling process compared with 34% in a control conventional reactor. Biogas production was intensified from 0.3 L/g of influent volatile solids (VS) in the control reactor up to 0.35 L/g VS. In addition, the recycling process improved the dewatering properties of digested sludge.

Aerobic biological treatment of thermophilically digested sludge.

Aerobic biological treatment of digested sludge was studied in a continuously operated laboratory set-up. An aerated reactor was filled with thermophilically digested sludge from the Moscow wastewater treatment plant and inoculated with special activated sludge. It was then operated at the chemostat mode at different flow rates. Processes of nitrification and denitrification, as well as dephosphatation, occurred simultaneously during biological aerobic treatment of thermophilically digested sludge. Under optimal conditions, organic matter degradation was 9.6%, the concentrations of ammonium nitrogen and phosphate decreased by 89 and 83%, respectively, while COD decreased by 12%. Dewaterability of digested sludge improved significantly. The processes were found to depend on hydraulic retention time, oxygen regime, and temperature. The optimal conditions were as follows: hydraulic retention time 3-4 days, temperature 30-35 degrees C, dissolved oxygen levels 0.2-0.5 mg/L at continuous aeration or 0.7-1 mg/L at intermittent aeration. Based on these findings, we propose a new combined technology of wastewater sludge treatment. The technology combines two stages: anaerobic digestion followed by aerobic biological treatment of digested sludge. The proposed technology makes it possible to degrade the sludge with conversion of approximately 45% volatile suspended solids to biogas, to improve nitrogen and phosphorus removal in reject water from sludge treatment units, and to achieve removal of malodorous substances after 8-9 days of anaerobic-aerobic sludge treatment.

[Anoxygenic phototrophic bacteria of the high-altitude meromictic Lake Gek-Gel, Azerbaijan].

The anoxygenic phototrophic bacterial community of the high-altitude meromictic Lake Gek-Gel (Azerbaijan) was investigated in September 2003. The highest concentration of bacteriochlorophyll e (48 microg/l) was detected at a depth of 30 m; the peak of bacteriochlorophyll a (4.5 microg/l) occurred at 29 m. Phylogenetic analysis revealed that brown-colored green sulfur bacteria Chlorobium phaeobacteroides predominated in the lake. Nonsulfur purple bacteria phylogenetically close to Blastochloris sulfoviridis were found in insignificant amounts; these organisms have not been previously reported in Lake Gek-Gel.

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 CH(4) emission. The number of types I and II methanotrophs obtained by CARD-FISH varied from 15 +/- 2 to 56 +/- 7 x 10(8) 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 degrees C. Methanotrophs accounted for about 50% of the total bacterial population in the deepest cover soil layer owing to higher availability of substrate (CH(4)). A lower number of methanotrophs (7 x 10(2) to 17 x 10(5) 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 CH(4), temperature, and moisture, and availability of substrates.

[Sulfate reduction and methanogenesis in the Shira and Shunet meromictic lakes (Khakass Republic, Russia)].

The biogeochemical and molecular biological study of the chemocline and sediments of saline meromictic lakes Shira and Shunet (Khakass Republic, 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 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 correlates with the measured rates of sulfate reduction and methanogenesis.

[Methane oxidation in landfill cover soil].

Methane oxidation in the cover soil of the Khmet'evo municipal landfill in Moscow oblast was investigated. Methane emission from the experimental parcel of the site was highly inhomogeneous. At a depth of 45-60 cm, the pore gas mainly consisted of CH4 (60-70%) and CO2 (30-40%). In the upper horizons of the cover soil, the concentration of these gases sharply decreased. Techniques for estimation of the methane-oxidizing activity in the cover soil of the landfill were tested. The rate of methane oxidation in the soil, the factor limiting methane emission from the surface of the site, correlated with the cell number of culturable methanotrophic bacteria. The method of indirect immunofluorescence revealed ten known species of methanotrophic bacteria in enrichment cultures obtained from samples of the cover soil. Our results also indicate the presence of unknown psychrotolerant methanotrophs that are active at the low temperatures characteristic of Moscow oblast.

Aerobic and anaerobic microbial degradation of poly-beta-hydroxybutyrate produced by Azotobacter chroococcum.

Food industry wastewater served as a carbon source for the synthesis of poly-beta-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5 degrees C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11 degrees C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3-7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.

Production and oxidation of methane at low temperature by the microbial population of municipal sludge checks situated in north-east Europe.

Methanogenic and methane-oxidizing activities of the microbial population of sewage sludge checks (Moscow and Syktyvkar regions) were studied at temperatures ranging from 5 to 25 degrees C. The number of methanogens in silt samples reached 10(10) cells/ml. A temperature decrease from 25 to 5 degrees C led to a sharp decrease of methanogenesis in the silt samples. Nevertheless, methanogenesis was still significant even at 5 degrees C. Different organic substrates, including polymeric and aromatic compounds, were degraded with methane production at 6 degrees C. At depths of 20-40 cm the number of methanotrophic bacteria reached 10(11) cells/ml. Methane oxidative activity of the microbial populations in the silt was less sensitive to the 25 degrees to 5 degrees C temperature decrease. Ten methanotrophic species, able to grow at 6 degrees C, were enriched from the Syktyvkar sludge lagoon and identified by indirect immunofluorescence. Enrichments obtained from the Syktuvkar region (62 N) contained more species of methane oxidizing bacteria able to grow at low temperature then methanogenic enrichments obtained from Moscow region (56 N).

[Growth of mesophilic methanotrophs at low temperatures]. / Rost mezofil'nykh metanotrofov pri nizkikh temperaturakh.

The optimal growth of mesophilic methanotrophic bacteria (collection strains of the genera Methylocystis, Methylomonas, Methylosinus, and Methylobacter) occurred within temperature ranges of 31-34 degrees C and 23-25 degrees C. None of the strains studied were able to grow at 1.5 or 4 degrees C. Representatives of six methanotrophic species (strains Mcs. echinoides 2, Mm. methanica 12, Mb. bovis 89, Mcs. pyriformis 14, Mb. chroococcum 90, and Mb. vinelandii 87) could grow at 10 degrees C (with a low specific growth rate). The results obtained suggest that some mesophilic methane-oxidizing bacteria display psychrotolerant (psychrotrophic) but not psychrophilic properties. In general, the Rosso model, which describes bacterial growth rate as a function of temperature, fits well the experimental data, although, for most methanotrophs, with symmetrical approximations for optimal temperature.