A. G. Dorofeev

A novel bacterium carrying out anaerobic ammonium oxidation in a reactor for biological treatment of the filtrate of wastewater fermented sludge

A new genus and species of bacteria capable of ammonium oxidation under anaerobic conditions in the presence of nitrite is described. The enrichment culture was obtained from the Moscow River silt by sequential cultivation in reactors with selective conditions for anaerobic ammonium oxidation. Bacterial cells were coccoid, ∼0.4 × 0.7 μm, with the intracellular membrane structures typical of bacteria capable of anaerobic ammonium oxidation (anammoxosome and paryphoplasm). The cells formed aggregates 5–25 μm in diameter (10 μm on average). They were readily adhered to solid surfaces. The cells were morphologically labile: they easily lost their content and changed their morphology during fixation for electron microscopy. The organism was capable of ammonium oxidation with nitrite. The semisaturation constants Ks for nitrite and ammonium were 0.38 mg N-NO2/L and 0.41 mg N-NH4/L, respectively. The maximal nitrite concentrations for growth were 90 and 75 mg N-NO2/L for single and continuous application, respectively. The doubling time was 32 days, μmax = 0.022 day−1, the optimal temperature and pH were 20°C and 7.8–8.3, respectively. According to the results of 16S rRNA gene sequencing, the bacterium was assigned to a new genus and species within the phylum Planctomycetes. The proposed name for the new bacterium is Candidatus Anammoximicrobium moscowii gen. nov., sp. nov. (a microorganism carrying out anaerobic ammonium oxidation, isolated in the Moscow region).

[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.

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.

[Nonlinearity in the growth of bacterial colonies: conditions and causes].

The universally recognized kinetic model of colony growth, introduced by Pirt, predicts a linear increase of colony size. The linearity follows from the assumption that the colony expands through the growth of only such cells that are located immediately behind the moving colony front, in the so-called peripheral zone of constant width and density. In this work, Pirts model was tested on two bacteria—Alcaligenes sp. and Pseudomonas fluorescens—having markedly distinct cultural properties and grown on an agarized medium with pyruvate. The colony size dynamics was followed for different densities of the inoculum, ranging from a single cell to a microdroplet of bacterial suspension (105–106 cells), and for different depths of the agar layer, determining the amount of available substrate. A linear growth mode was observed only with P. fluorescens and only in the case of growth from a microdroplet. When originating from a single cell, colonies of both organisms displayed nonlinear growth with a distinct peak of Kr (the rate of colony radius increase) occurring after 2–3 days of growth. The growth of P. fluorescens colonies showed virtually no dependence on the depth of the agarized medium, whereas the rate of colony size increase of Alcaligenes sp. turned out to be directly related to the medium layer thickness. The departure from linearity is consistently explained by a new kinetic scheme stipulating a possible contribution to the colony growth not only of peripheral cells but also (much more distinct in Alcaligenes) of cells at the colony center. The colony growth dynamics is determined not only by the concentration of the limiting substrate but also by the amount of autoinhibitor, the synthesis of which is governed by the age of cells. The distinctions of growth from a single cell and microdroplet could also originate as a result of dissociation into the R- and S-forms and competition between the corresponding subpopulations for oxygen and the common substrate.

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.

Modeling of anammox process with the biowin software suite

Mathematical modeling of the biotechnology for the removal of ammonium nitrogen from wastewater based on the anammox process was performed with the specialized BioWin software suite (EnviroSim Associates Ltd., Canada). Nitrogen removal by means of the transformation of ammonium nitrogen to molecular nitrogen was conducted in a continuous stirred bioreactor carrying both suspended and immobilized activated sludge. Both basic values of the kinetic and stoichiometric coefficients are incorporated in the BioWin software, and those that changed based on the results of the experimental studies were used for the calculations. The optimal temperature and dissolved oxygen concentration revealed by mathematical modeling were 35°C and 0.14 mg/L. The results obtained from calculations were similar to those obtained in the experiments. The calculated and experimental concentrations of ammonium, nitrites, and nitrates in the treated water were similar and comprised 10.7 and 11.7% of the initial concentration entering the bioreactor, respectively. The selected mathematical model possessed a high predictive ability for the calculation of biotechnologies based on the anammox process

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]-lad-derane 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.

Growth of the thermophilic bacterium Geobacillus uralicus as a function of temperature and pH: An SCM-based kinetic analysis

The synthetic chemostat model (SCM), originally developed to describe nonstationary growth under widely varying concentrations of the limiting substrate, was modified to account for the effects of nontrophic factors such as temperature and pH. The bacterium Geobacillus uralicus, isolated from an ultradeep well (4680 m), was grown at temperatures ranging from 40 to 75°C and at pH varying from 5 to 9. The biomass kinetics was reasonably well described by the SCM, including the phase of growth deceleration observed in the first hours after a change in the cultivation temperature. At an early stage of batch growth in a neutral or alkalescent medium, bacterial cells showed reversible attachment to the glass surface of the fermentation vessel. The temperature dependence of the maximum specific growth rate (μm) was fitted using the equation μm = A exp (λT )/{1 + expB[1 – C/(T + 273)]}, where A, λ, B, and C are constants. The maximum specific growth rate of 2.7 h–1 (generation time, 15.4 min) was attained on a complex nutrient medium (peptone and yeast extract) at 66.5°C and pH 7.5. On a synthetic mineral medium with glucose, the specific growth rate declined to 1.2 h–1, and the optimal temperature for growth decreased to 62.3°C.

Development of the First Russian Anammox-Based Technology for Nitrogen Removal from Wastewater

A pilot single-stage setup with the reactor volume of 20 m3 was constructed for ammonium removal from the filtrate of thermophilically digested sludge. The setup was operated at temperatures of 20–37 ℃, dissolved oxygen concentrations of 0.1–0.7 mg/L, pH of 5.7–8.5, hydraulic retention time of 12–36 h, and filtrate output of up to 30 m3/day. The efficiency of nitrogen removal was 75–90%, nitrogen load was 0.9–1.1 kg N/(m3·day), and the specific volumetric nitrogen removal capacity of the reactor reached 0.8–1.0 kg N/(m3·day). The sludge retained activity at low pH (5.7) and enhanced nitrite concentration (up to 250 mg/L). A correlation was established between conductivity reduction of the treated liquid and nitrogen removal efficiency, and the formula for calculation of ammonium concentration using the conductivity was proposed.

Approaches to cultivation of “nonculturable” bacteria: Cyclic cultures

The work deals with more efficient procedures for the isolation and cultivation of “nonculturable” microorganisms (NM) from environmental sources. The techniques for NM cultivation in situ and under laboratory conditions are discussed. A new approach is considered, viz., cultivation under cyclically varying conditions with the cycle duration comparable to the duration of the cell cycle. Cyclic cultivation implies sequential changes of several cultivation phases with different growth conditions. An established sequence of growth phases provides for the competitiveness of the target microorganisms and for accumulation of their biomass. Cultivation of phosphate-accumulating bacteria, nonculturable microorganisms which have not been previously isolated in pure culture, in an SBR reactor is discussed as an example of cyclic cultures.