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Formation of a community of phosphate-accumulating microorganisms in a laboratory sequential batch reactor (SBR) ensuring alternated aerobic and anaerobic conditions during periodic removal and addition of the medium were investigated. The bioreactor removed 50% phosphorus from the incoming medium after 22 days from the start-up. Microscopy and X-ray microassay revealed the of cells of diverse morphology that contained phosphorus-enriched granules. High-throughput sequencing of the 16S rRNA gene fragments carried out on days 0, 8, 15, and 22 showed changes in the community composition and its decreasing diversity. On day 22, approximately twofold increase of the relative abundances of Bacteroidetes (up to 43% of the 16S rRNA gene sequences) and Proteobacteria of the classes alpha (up to 15%) and beta (up to 27%) was observed. While at the onset of the reactor operation, typical PAOs related to “Candidatus Accumulibacter” (class Betaproteobacteria) constituted 0.2% of the community, they were not detected on day 22. The most likely PAO candidates were beta-proteobacteria of the genus Dechloromonas, the share of which increased from 0.7 to 11% by the time of the highest phosphorus removal from the inflowing medium. The relative abundance of heterotrophs of the genus Zoogloea (family Rhodocyclaceae) increased from 0.1 to 11.5%.

Background: The biological activities of dark Chinese teas are largely due to their microbial post-fermentation. Herbal teas are traditional Russian beverages that hold special value, owing to their taste and useful medicinal properties. However, no data are available in the literature on using microbial post-fermentation for enhancing their biological activity. The goal of this work was to demonstrate that the fungus Eurotium isolated from Chinese black teas can be used for the post-fermentation of herbal teas produced from bay willow and apple leaves.Methods: Eurotium cristatum was isolated from brick Chinese tea Fujan and identified using conventional methods of microbiology and molecular biology. Low molecular weight metabolites (phenols, amines, sugars, and amino acids) were determined by HPLC. E. cristatum was grown in association with the bacterium Bacillus amyloliquefaciens.Results: It was revealed to exhibit valuable biosynthetic features, such as a lack of mycotoxins, zero antimicrobial activity, and the capacity to synthesize neuroactive amines. B. amyloliquefaciens displayed a wide spectrum of antibiotic (antimicrobial and antifungal) activities that anifested themselves even with antibiotic-resistant bacteria). While growing on green unfermented tea (Camellia sp.) E. cristatum produced and modified neuroactive amines, such as dopamine, serotonin, and epinephrine. The fungus efficiently grew during the post-fermentation of herbal teas from both bay willow and apple leaves. Even though Camellia leaves substantially differed from bay willow and apple leaves in terms of phenol content, the growth of E. cristatum on Camellia was also sufficiently good. This suggests that the growth of Eurotium fungi is not influenced by the phenolic compounds. The data obtained on the composition of phenolic compounds, carbohydrates, and amino acids in the fermented plants and raw material provide evidence that the growth of the fungus proceeds depends on the hydrolysis of high molecular weight phenols and cell biopolymers in the fermented material.Conclusion: Thus, the ability of E. cristatum to grow on plant leaves of significantly different biochemical composition provides foundations for new technologies aiming to obtain post-fermented herbal teas with high biological activity that are enriched in low molecular weight compounds including biogenic amines.

The stages of formation and development of activated sludge biofilm carrying out the anammox process in a laboratory sequencing batch reactor (SBR) with complete biomass retention on the carrier were investigated using light, epifluorescence, and electron microscopy. Light microscopy revealed biofilm formation on the carrier to occur within one week. Rod-shaped and filamentous microorganisms were the first to attach on the carrier and acted as a skeleton for biofilm formation, thus playing an important role in colonization of the carrier. Epifluorescence microscopy revealed physiologically active anammox bacteria of the genera Candidatus “Brocadia” and Ca. “Jettenia” in the biofilms. Selective autofluorescence of the colonies of anammox bacteria Ca. “Jettenia” was observed. This autofluorescence was probably caused by specific proteins in the extracellular polymeric substances (EPS), their synthesis and/or amount depending on the colony age and the physiological state of the cells. Structural organization of the colonies of anammox bacteria was investigated by electron microscopy. High-throughput sequencing of the 16S rRNA gene fragments revealed the presence of sequences affiliated with members of the phyla Chloroflexi, Bacteroidetes, Planctomycetes, and Proteobacteria in the biofilms. Apart from anammox bacteria, anaerobic and facultatively anaerobic organotrophs, stage I nitrifiers, denitrifiers, and sulfate reducers were detected. Members of the genus Ca. “Brocadia” were predominant among the anammox bacteria, probably due to better adhesion of their cells to the carrier or to their competitive advantage over Ca. “Jettenia” in the presence of organic acids (acetate and formate) in the medium.

Persisters (P) are the cells surviving, but not reproducing in the presence of lethal doses of antibiotics (AB). These are the cells of a scarce (0.001–1.0%) subpopulation, emerging due to a phenotypic transition, which may be stochastic or induced by stress impacts. Growing in fresh medium, P reproduce the structure of the parent population, which is considered the cause of: (a) recurrent infections; (b) inefficiency of antibiotic treatment; and (c) development of antibiotic resistance, as was discussed in numerous articles and reviews. The goal of the present work was to investigate P formation by bacteria contaminating human skin and their properties, which have not been studied previously. Two approaches were used to measure the frequency of occurrence was determined for P in the populations of stationary-phase cultures of Staphylococcus capitis Р205-1 (UNIQEM, Russia), which is sensitive to polymyxin, Na sulfacyl, and co-trimoxazole, and Staphylococcus epidermidis 14990 АТСС, which is resistant to these antibiotics: (1) traditional, by application of lethal AB doses and (2) by using a lysis solution (LS), which killed the ordinary vegetative cells and selected the persister cells resistant to AB and LS (PAB and PLS, respectively). This is the first report on applicability of the method involving the lysis solution for determination of P abundance in strains with multidrug AB resistance (for the S. epidermidis strain). Unlike most bacterial strains retrieved from collections , the share of P in stationary-phase cultures of staphylococci inhabiting human skin was quite high: ~10% for both S. epidermidis PLS and S. capitis. Heterogeneity in resistance to the lysis solution was revealed in the PAB obtained by the standard procedure: PLS share in S. capitis was ~1% of PAB abundance. These PLS were resistant to AB cross-action. The differences in PLS abundance in the studied strains (10% for S. epidermidis and below 1.0% for S. capitis) correlated with their AB resistance. Long-term incubation (4 months) under standard conditions resulted in the cycle of staphylococci development ending by emergence of dormant forms (DF); their share for both strains was ~0.1% of the number of viable cells in the stationary-phase cultures. The DF of staphylococci had all characteristics of dormant cells: emerged in the cycle of culture development; preserved viability under nongrowth conditions; had no experimentally detectable metabolism (absence of respiration); had specific ultrastructural organization; and were heat-resistant (200‒600 times higher than the vegetative cells at 90°C for 15 min). The correlation between PLS abundance in the cultures of staphylococci and the share of heat-resistant DF confirms our earlier suggestion on persisters as DF percursors.

High efficiency of a combined preparation including synergistic polymyxin B and 4-hexylresorcinol was shown for treatment of experimental sepsis caused by an antibiotic-resistant highly virulent hypermucoid Klebsiella pneumoniae strain KPM9Pmr in mice. Complex therapy with polymyxin B (1 mg/kg) and 4-hexylresorcinol (30 mg/kg) led to cure in 80%; in 20% of these mice, no bacterial cells were found. After treatment with polymyxin B alone, only 50% animals survived and all of them contained bacterial cells. Comparative analysis of the results of monotherapy and combined treatment indicates that 4-hexylresorcinol not only increases the efficiency of antibiotic, but also minimizes persistence of the infection agent and therefore, the risk of development of antibiotic resistance.

A new approach is described to increasing the numbers of viable cells in long-term stored populations (cultures) of the hydrocarbon-oxidizing bacterium Acinetobacter junii, which involves application of humic substances (HSs). HSs are produced due to condensation and oxidation of phenolic compounds, including alkylresorcinols, factors of intercellular microbial communication with stress-potentiating and antioxidant activity. HS addition to the stationary-phase bacterial culture was shown to result in 10- to 15-fold increase in cell viability for bacterial preparations stored for 1 to 4 months under provocative conditions (free air supply, growth medium, and temperatures of 20–25°C). Analysis of death curves for experimental and control A. junii cultures revealed two phases, with the initial stage of rapid death (0.167 log CFU/mL day), usual for autolysis of the regular stationary cells, and the stage of slower death (log 0.015 CFU/mL day), typical of persister cells. This was previously shown to be the feature determining this type of phenotypic heterogeneity in the populations of opportunistic bacteria. The concentration dependence of HSs effect on persisters formation was determined. While treatment of A. junii stationary cultures with hydrogen peroxide (0.03–0.3%) as a stress factor also resulted in enhanced persisters formation (fourfold), unlike the HS-treated variants they survived for not more than 1.5 to 2 months. Plating of the control and experimental variants (with HSs or H2O2) after long-term storage (2–4 months) revealed their phase variation spectrum to change due to replacement of the dominant colony morphology phenotype by the minor ones (40% and more), which is one of the features of the dormant cystlike bacterial forms. The ratios of the variants in the control and experiments (with HSs) were different. Comparison of the chemical composition of HS preparations indicated that the most oxidized hydrophilic HSs affected formation of persister cells, while less oxidized humates with higher antioxidant activity were responsible for the viability prolongation. Thus, the effect of HSs on bacterial populations includes induction of persister cell formation (their increased number), prolongation of the colony-forming capacity in the cells surviving under provocative storage conditions, and alteration of the phase variant spectrum of the stored populations. Formation of stress-induced persister cells developing into mature dormant forms supporting survival of the population (species) was established for saprotrophic bacteria (in the case of A. junii). In practical terms, HS-induced formation of persister cells may be recommended for development of diverse bacterial preparations.

Addition of humic compounds (9 preparations) to the cultures of hydrocarbon-oxidizing bacteria was shown to result in up to 15-fold increase in the number of viable cells in the cultures stored in the growth media with access to air at 20‒25°C. The most oxidized humates exhibited the highest activity in induction of persister formation, which subsequently matured to dormant forms, while unoxidized humates with antioxidant activity supported viability persistence.

—Biotechnologies involving phosphate-accumulating organisms, which collect inorganic phosphates from the medium as polyphosphates during cyclic growth under aerobic and anaerobic conditions, are presently applied for phosphorus removal from wastewater. Betaproteobacteria of the candidate genus ‘Candidatus Accumulibacter’ carry out phosphate accumulation in most systems for wastewater treatment. However, no member of ‘Ca. Accumulibacter’ has been isolated in pure culture. Metagenomic analysis of the microbial consortium formed in a laboratory setup for phosphate removal from wastewater and removing up to 80% phosphorus from the medium was carried out. Members of the phyla Proteobacteria (82.5%), Bacteroidetes (10.5%), and Chloroflexi (1.6%) predominated in the community. Among the proteobacteria, Betaproteobacteria were revealed, among which ‘Ca. Accumulibacter’ predominated, as well as Gammaproteobacteria (26.8%), which mainly belonged to the family ‘Ca. Competibacteraceae.’ Metagenomic data were used to obtain the genome of the dominant phosphate-accumulating bacterium, which belonged to a new ‘Ca. Accumulibacter’ species. The studied community was promising both for further basic research on metabolism of phosphate-accumulating organisms and for improvement of existing biotechnologies for phosphorus removal from wastewater.

—Approaches to stabilizing the cells of lactic acid organisms (LAO) investigated in the present work resulted in maintaining the viable cell titer at 107–108 cells/mL, the value recommended for the LAO titer in foodstuffs, for 12 months. Natural LAO associations selected for long-term preservation of LAO cell viability exhibited high efficiency. Lactic acid bacteria Lactobacillus plantarum and yeasts Candida parapsilosis were isolated from the association isolated from marketed curdled milk and selected for 6 months. The death rate of these LAO in binary culture (50% of each component) was several times lower than in monocultures. Adding other approaches for cell stabilization in a binary LAO culture (anoxic conditions and stabilizing antioxidant and biopolymer supplements) resulted in a 5- to 6-fold increase in the number of viable cells after 12-month storage. The algorithm for combined application of several approaches to prolongation of cell viability in the cultures of lactic acid microorganisms during long-term storage (up to 12 months) was not reported previously. It may be recommended for LAO cell stabilization both in fermented milk products and in leaven cultures. This is the first report of C. parapsilosis as the yeast component of a stable association with lactic acid bacteria.

The review deals with specific bacterial physiological groups developing under periodically varying ambient conditions and possessing a cyclic type of metabolism (CTM). CTM is characterized by unbalanced two-phase growth occurring during the period less than the generation time. During the first phase, the carbon and/or energy source are accumulated, while during the second phase biomass biosynthesis occurs using the accumulated substrate. Known CTM organisms of the groups of phosphate-accumulating and glycogen-accumulating bacteria are described, and their major biochemical models are discussed. Development of microorganisms with obligate CTM is probably impossible or significantly suppressed under stable ambient conditions, which may be the reason why the major CTM organisms can not be cultured using traditional batch and continuous cultivation, while highly enriched cultures may be obtained by cyclic cultivation. Ecological aspects of CTM are discussed, and the possible environments for CTM microorganisms in natural ecosystems are determined.

Binary biofilms containing gram-positive bacteria Micrococcus thailandicus HB or Kocuria rhizophila strain 4A-2G and gram-negative bacteria Escherichia coli strain K-12 or strain ET12567 pRAG56 were obtained. Strain E. coli ET12567 contained a plasmid encoding aminoglycoside phosphotransferase, the enzyme which inactivates kanamycin. A new kinetic approach was developed for selective detection of metabolically active gram-positive bacteria in binary biofilms with E. coli, based on the differences in reduction rate of the electron acceptor, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was found that the studied E. coli strains did not exert a protective effect upon the action of antibiotics vancomycin and oxacillin on gram-positive bacteria in the binary biofilms under formation. However, a protective effect was found in the case of preformed binary biofilms: the growth of gram-positive bacteria in binary biofilms was observed at the concentrations of antibiotics oxacillin and kanamycin, which almost completely suppressed growth in the mono-species biofilms of these gram-positive bacteria. The presence of a protective effect was confirmed by several methods (dynamics of MTT reduction, densitometric evaluation of biofilm staining, measurement of the optical density of MTT extracts, and CFU enumeration).

Compaction and biocrystallization of the nucleoid are presently considered as a necessary and important stage in the transformation of the cell ultrastructure during change of microbial cultures strategies from growth to survival. Nucleoid biocrystallization in the stationary phase cells is achieved due to structural regularity of the DNA complexes with the histone-like Dps protein. Our experiments with Escherichia coli mutants, overproducers of the Dps protein, confirmed nucleoid biocrystallization in the late stationary phase cells. Since nucleoid biocrystallization was revealed in E. сoli cells without Dps overproduction at late stages of starvation, it is constitutive in the cycle of development of microbial populations. The present work concentrated on detection of the nucleoid biocrystalline structure in (1) long-starved (21 day in the chemostat mode) bacterial cells (genera Arthrobacter and Pseudomonas), (2) dormant ametabolic (anabiotic) cells of such prokaryotes as archaea and non-spore-forming bacteria, (3) endospores of bacilli, (4) streptomycete exospores, and (5) in the cells surviving in permafrost for (2‒3 Ma). The topics discussed include nucleoid biocrystallization as a necessary stage of maturation of the dormant microbial cells providing for survival and preservation of the species, dynamics of nucleoid biocrystallization during maturation of the dormant cells, and its possible role for the preservation of genetic information in the case of autolysis of most of the cells in a developing culture.

The preservation of activity of extracellular enzymes in soil is presently associated with their immobilization on organic or inorganic carriers. Enzyme immobilization results, however, in a significant decrease in enzymatic activity. In the present work, the mechanism responsible for promotion of the catalytic activity was revealed, as well as the favorable effect of low-molecular alkylhydrozybenzenes of the class of alkylresorcinols, which are common in soil organic matter, on stability of immobilized enzymes (exemplified by amylases) by their post-translational modification. Optimal conditions (enzyme to sorbent ratio, pH optimum, CaCl2 concentration, and sorption time) for amylase sorption on a biological sorbent (yeast cell walls) were determined and decreased activity of the immobilized enzyme compared to its dissolved state was confirmed. Alkylresorcinols (C7AHB) at concentrations of 1.6 to 80 mM were found to cause an increase of amylase activity both in the case of already sorbed enzymes (by 30%) and in the case of a free dissolved enzyme with its subsequent immobilization (by 50–60%). In both cases, the optimal C7AHB concentration was 16 mM. Amylase stability was determined for C7AHB-modified and unmodified enzymes immobilized on the biological sorbent after two cycles of freezing (–20°C) and thawing (4°C). Inverse dependence was revealed between increasing stability of C7AHB-modified enzymes and an increase in their activity, as well as higher stability of immobilized modified amylases than of the dissolved modified enzyme. Investigation of the effect of C7HOB-modification in the preservation of activity in immobilized amylases after four freeze–thaw cycles revealed: (1) better preservation of activity by the modified immobilized enzymes compared to immobilized ones; (2) differences in the dynamics of activity loss within compared pairs, with activity of immobilized amylases decreasing after the second cycle to a lower level (42%) than activity of the modified immobilized enzymes after the fourth cycle (48%). These results demonstrate that in the preservation of activity of extracellular enzymes in soil both stabilization mechanisms are of importance: immobilization on organic carriers and modification of the enzyme conformation by low-molecular compounds with the functions of chemical chaperones.

Dynamics of the composition of the microbial community was studied during start-up of a single-stage completely mixed constant flow laboratory setup for ammonium removal by the nitritation/anammox process from the filtrate of digested sludge of the Kuryanovo wastewater treatment plant (KWTP), Moscow. To decrease the period of the start-up, the setup was initially inoculated with two types of activated sludge (nitrifying sludge from a KWTP aeration tank and sludge from a sequencing batch reactor enriched with anammox bacteria). The start-up and adjustment stage was therefore decreased to 35–40 days, and nitrogen removal efficiency reached 80% after 120 days of the setup operation. Taxonomic analysis of the composition of the microbial community was carried out by pyrosequencing of the 16S rRNA fragments obtained using the universal and planctomycetes-specific primers. In the course of adaptation of activated sludge to increasing nitrogen load, microbial community of the setup became less diverse and more specialized. The contribution of anammox bacteria of the family Brocadiaceae, closely related to Candidatus “Brocadia caroliniensis,” increased gradually. Members of the order Nitrosomonadales were involved in ammonium oxidation to nitrite. While nitrite-oxidizing bacteria of the genus Nitrospira were also detected, their share decreased with accumulation of the activated sludge. The contribution of other bacteria varied as well: the shares of the phyla Ignavibacteria, Chloroflexi, and Acidobacteria increased significantly (up to 13, 12, and 10%, respectively of the total number of reads), while relative abundance of the Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Synergistetes, Aminicenantes, Thermotogae, and Cloacimonetes decreased. Thus, application of pyrosequencing made it possible to monitor succession of the bacterial community involved in nitrogen removal by nitritation/anammox process.

Effect of human inherent immunity factors of, a gene-encoded antibacterial peptide indolicidin (Ind) and a cytokine interleukin 1 (IL1) on formation of antibiotic-tolerant persister cells surviving in the presence of ciprofloxacin (Cpf, 100 μg/mL) and ampicillin (Amp, 100 μg/mL) in submerged bacterial cultures (Staphylococcus aureus FGA 209P, Escherichia coli K12, and Pseudomonas aeruginosa PAO1) was studied. While Ind in physiological concentrations (0.3 and 3.0 μg/mL) introduced to the lag- or exponential-phase cultures of test organisms exhibited no reliable effect on population growth, the number of persisters increased at 3.0 μg/mL. Bactericidal Ind concentrations (9 μg/mL) suppressed S. aureus growth (-0.1% of surviving cells) with subsequent recovery due to development of the more antibiotic-tolerant white variant. Treatment with Cpf after Ind addition resulted in mutual potentiation of their antimicrobial activity, with the number of S. aureus persisters 2 to 3 orders of magnitude lower than in the case of the antibiotic alone. IL1, another immunity factor, when introduced (0.1-1 ng/mL) to the exponentially growing S. aureus culture (but not to the lag phase culture) had a temporary growth-static effect, with the number of persisters surviving Cpf treatment (100 μg/mL) increasing by 1 to 2 orders of magnitude. Electron microscopy revealed significant alterations in the outer cell envelope layer of surviving S. aureus cells, which should be associated with their changed antigenic properties. Thus, the factors of human inherent immunity have a dose-dependent effect on the growth of bacterial populations. In combination with antibiotics, they exhibit synergism of antimicrobial action (indolicidin) and minimize (indolicidin) or increase (interleukin 1) the frequency of formation of persister cells responsible for survival of a population subjected to an antibiotic attack.

Regulation of streptomycete growth and development by distant interactions of physical nature was shown using a vial-in-vial experimental setup, providing physical isolation of the inducer and detector cultures. Some effects of distant interaction were observed with Streptomyces netropsis proliferating submerged culture as an inducer and a surface culture of the same strain as a detector. The growth rate of the detector strain doubled. Spore germination was stimulated, as was indicated by a 30% increase in the number of colony-forming units (CRU) in the detector culture plated as a spore suspension. The phase variation spectrum also changed, with an increased share of the colonies of the morphotype predominant under the standard conditions.

Effect of sublethal doses of physical and chemical stressors (heat shock for 2 h at 45 degrees C and addition of C12-alkylhydroxybenzene, a microbial alarmone) on development of resistance to the subsequent lethal antibiotic attack and the role of the time interval between these treatments were studied on a submerged batch culture of Escherichia coli 12. The interval sufficient for the development of stress response provides for development of temporary adaptive resistance to the antibiotic attack, resulting in increased number of surviving persister cells. The interval below the time required for the stress response potentiates cell death and results in a decreased number of persisters. Heterogeneity of the fractions (10(-4) to 10(-2)% of the intial CFU number) surviving lethal doses of an antibiotic (a mpicillin or ciprofloxacin) was found. Apart from a low number of antibiotic-resistant cells (up to 0.005% of surviving cells), the fractions contained antibiotic-tolerant forms, such as temporarily resistant metabolically adapted cells, long-term persisters, and the cells of slowly growing SCV variants with small colonies (d ≤ 1 mm). Persisters are hypothesized to act as precursors for cystlike dormant cells (CLC), in which the cell differentiation stage is completed and the processes of cell ametabolism (transition to the anabiotic state) are still incomplete.

Efficiency of MALDI mass spectrometry for differentiation between phenotypic phase variants (in colony morphology and virulence/avirulence) was investigated for saprotrophic and opportunistically pathogenic bacteria of five genera (Acinetobacter, Arthrobacter, Rhodococcus, Corynebacterium, and Escherichia). Analysis of MALDI spectra (on the SA and HCCA matrices) included (1) determination of similarity of the protein spectra as a percentage of the common protein peaks to the total amount of proteins, which reflects the phylogenetic relationships of the objects and has been recommended for identification of closely related species; (2) comparison of intensities of the common peaks; and (3) the presence of specific peaks as determinative characteristics of the variants. Under the standard analytical conditions, the similarity between the MALDI profiles was shown to increase in the row: genus–species–strain–variant. Assessment of intensities of the common peaks was most applicable for differentiation between phase variants, especially in the case of high similarity of their profiles. Phase variants (A. oxydans strain K14) with similar colony morphotypes (S, R, M, and Sm) grown on different media (LB agar, TSA, and TGYg) exhibited differences in their protein profiles reflecting the differences in their physiological characteristics. This finding is in agreement with our previous results on screening of the R. opacus with similar colony morphology and different substrate specificity in decomposition of chlorinated phenols. Analysis of MALDI spectra is probably the only efficient method for detection of such variants.

The paper presents results of study of anoxic ammonia oxidation at low temperature conducted at JSC Mosvodokanal, Moscow, Russia. The study was carried out in two reactors, 5 l each, operated at the temperature of 5–10 °С. Both reactors were inoculated with the soil, collected from waste water sludge landfill, that presumably, contained low-temperature adapted nitrifying bacteria. Reactor No. 1 contained nitrifying bacteria only. Reactor No. 2 was further inoculated by anammox bacteria. Filtrate from digested sludge belt thickeners was added into the reactors to achieve a final N-NH4 concentration of 70–95 mg/l. The reactors were operated as sequencing batch reactors. After 90 days of incubation maximal nitrification rate in reactor No. 1 was 1.4 mg N-NH4/g VSS*h, and in reactor No. 2–1.0 mg N-NH4/g VSS*h. Estimated doubling time of nitrifying bacteria was 45 days. Total mineral nitrogen removal in the 1st reactor was 20% (via process of heterotrophic denitrification), and in the 2nd – 60% (via both the processes of heterotrophic and autotrophic nitrate reduction). Through the process of autotrophic denitrification (anammox), two times as much nitrogen was removed, compared to the heterotrophic denitrification process. Anammox process rate was 0.4 mg N-NH4/g VSS*h.

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.

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.

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.

Effect of the antibiotics kanamycin and ampicillin on growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M67AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.

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 x 0.7 mm, 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, μ(max) = 0.022 day(-1), the optimal temperature and pH were 20 degrees C and 7.8-8.3, respectively. According to the 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 microorganisms carrying out anaerobia ammonium oxidation, isolated in the Moscow region).

Aim. The creation of genetically engineered fusion protein SPA-BAPmut and its application as a secondary immunoreagent in immunoassays. Methods. Gene cloning, PCR, electrophoresis, DNA sequencing, bacteria cells culturing, protein expression and purification, ELISA, Western-blotting were used. Results. The DNA sequences encoding Staphylococcus aureus protein A (SPA) and bacterial alkaline phosphatase with enhanced catalytic activity (BAPmut) were used for construction of gene encoding fusion protein SPA-BAPmut that was expressed in the high-productive Escherichia coli system and obtained in a soluble form. Cultivation conditions to provide a high-level expression of SPA-BAPmut (> 1 g/l) were determined. The target protein was obtained with purity more than 95 % using IMAX method. SPA-BAPmut is thermostable, and both parts of fusion protein (SPA and BAPmut) retain their IgG binding and alkaline phosphatase activity for a long time. SPA-BAPmut was used as a substitute of secondary antibodies in immunoassays. As little as 5 ng of the antigen could be detected in Western blotting and 1 g/ml of IgG in ELISA. Conclusions. The possibility of using SPA-BAPmut as universal secondary immunoreagent for different types of immunoassays was shown. Мета. Створення генно-інженерного злитого білка SPA-BAPmut та його застосування як вторинного імунореагенту в імунологічних тестах. Методи. Клонування генів, ПЛР, секвенування ДНК, культивування бактерій, електрофорез, синтез і очищення білків, ELISA, вестерн-блот. Результати. З використанням послідовновностей ДНК, що кодують білок А Staphylococcus aureus (SPA) і бактерійну лужну фосфатазу з покращеними каталітичними властивостями (BAPmut), сконструйовано ген злитого білка SPA-BAPmut та забезпечено його препаративне отримання у розчинній формі внаслідок синтезу в клітинах Escherichia coli. Визначено умови ферментації, за яких вихід SPA-ВAPmut становить приблизно 1 г/л культури E. coli. Із застосуванням методу металоафінної хроматографії одержано цільовий білок з чистотою понад 95 %. SPA-ВAPmut термостабільний, а обидва його компоненти (SPA і ВAPmut) зберігають імуноглобулінзв’язувальну і фосфатазну активність тривалий час. SPA-BAPmut дозволяє виявляти щонайменше 5 нг антигену та 1 мкг/мл антитіл. Висновки. Показано можливість використання SPA-ВAPmut як універсального вторинного імунореагенту в імунохімічних тестах. Цель. Создание генно-инженерного слитого белка SPA-BAPmut и eго использование как вторичного иммунореагента в иммунологических тестах. Методы. Клонирование генов, ПЛР, секвенирование ДНК, культивирование бактерий, электрофорез, биосинтез и очистка белков, ELISA, вестерн-блот. Результаты. С использованием последовательностей ДНК, кодирующих белок А Staphylo- coccus aureus (SPA) и бактериальную щелочную фосфатазу с улучшенными каталитическими свойствами (BAPmut), сконструирован ген слитого белка SPA-BAPmut и обеспечено его препаративное получение в растворимой форме вследствие синтеза в клетках Escherichia coli. Определены условия ферментации, при которых выход SPA-ВAPmut составляет около 1 г/л культуры E. coli. С применением метода металлоаффинной хроматографии целевой белок получен с чистотой более 95 %. SPA-ВAPmut термостабилен, а оба его компонента (SPA и ВAPmut) сохраняют иммуноглобулинсвязывающую и фосфатазную активность на протяжении длительного времени. SPA-BAPmut позволяет детектировать 5 нг антигена и 1 мкг/мл антител. Выводы. Показана возможность применения SPA-ВAPmut как универсального вторичного иммунореагента в иммунохимических тестах.

Evaluation of effectiveness of Mexidol in optimization of hypolipidemic therapy in ischemic stroke and diabetes mellitus patients.Authors analyzed the indicators of lipid status: total cholesterol, low-density lipoproteins, high density lipoproteins, triglycerides and concentration of platelet factor-4, β-tromboglobulin, von Willebrand factor in 68 patients with acute ischemic stroke and diabetes mellitus. Authors investigate the dynamics of these parameters (1(st), 21(st), 3-d and 6(th) month after onset stroke) depending on timing and dose of Mexidol.Long time therapy of Mexidol may optimize of hypolipidemic therapy in ischemic stroke and diabetes mellitus patients.Цель исследования - определение эффективности терапии мексидолом для оптимизации вторичной профилактики сосудистых событий посредством воздействия на показатели сосудисто-тромбоцитарного гемостаза и липидного спектра крови у пациентов, перенесших ишемический инсульт (ИИ), на фоне сахарного диабета 2-го типа (СД2). Материал и методы. Проводили исследование содержания общего холестерина, холестерина липопротеидов низкой плотности, холестерина липопротеидов высокой плотности, триглицеридов в сыворотке крови; определяли концентрации 4-пластиночного фактора (4ПФ), β-тромбоглобулина (βТГ) и фактора Виллебранда у 68 пациентов с ИИ и СД2. Проведен динамический контроль исследуемых показателей (1-е, 21-е сутки, 3-й и 6-й месяцы от начала ИИ) на фоне терапии мексидолом. Результаты и заключение. Продолжительная терапия мексидолом предоставляет дополнительные возможности для нормализации липидного спектра крови и оптимизации вторичной профилактики сосудистых событий у пациентов с ИИ и СД2.

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.

The adaptogenic effect of the chemical analogues of alkylhydroxybenzenes (AHBs), bacterial extracellular autoregulators (the individual compound C7-AHB and its technical preparation Sidovit), was demonstrated for two pseudomonad species, Pseudomonas aeruginosa and P. fluorescens. The protective effect of AHBs resulted in increased growth rate and biomass accumulation in bacteria grown under suboptimal conditions within the species tolerance range. The adaptogenic effect of AHBs (10–50 μmg/l) was more pronounced under more unfavorable growth conditions. In the case of P. fluorescens, the individual compound C7-AHB increased the biomass yield by 30% under alkaline conditions (pH 9.5), when the growth rate decreased by 80–90% compared to the optimum (pH 5.5–7.5). The Sidovit preparation, containing a mixture of natural AHBs with C7-AHB as the main component, increased the growth rate of P. aeruginosa by 40–60% at nonoptimal temperatures (45 and 10°C) or under enhanced salinity (1% NaCl). The action of AHBs as regulators of the rpoS and SOS response stress regulons was demonstrated to be among the mechanisms of their adaptogenic effect, as was demonstrated with the relevant reporter genes in the model strains E. coli C600 thi, thr, leuΔ(pro-lac) with the osmE-lacZ and umuD-lacZ hybrid operons, respectively. AHBs are technologically and economically acceptable as adaptogenic supplements for bacterial preparations used in soil bioremediation and oil spillage removal under conditions unfavorable for microbial growth, including increased salinity, extreme pH, and fluctuating sub- or supraoptimal temperatures.

Alkylhydroxybenzenes (AHB) were found to control the activation of protective functions of microorganisms by inducing stress gene expression and increasing the frequency of the intrapopulation phase transitions which are responsible for the phenotypic variability of bacteria. We established the dependence of the regulatory effects of AHB on their structure (alkyl radical length) and concentration. A reversion assay using the tryptophan auxotrophic strain Bacillus subtilis trpA5 B 1733 indicated a relationship between the reversion frequency that was 40–120 times higher than the background value and phase transition’s intensity (with R → S transition rates up to 87% in contrast to 2% in the control experiment) induced by specific doses (5–100 mg/ml) of long-chain AHB such as C12-AHB acting for a short time (1 h) on vegetative (dividing or stationary-phase) B. subtilis cells. Using four test strains constructed from Escherichia coli C600 thi, thr, leuΔ(pro-lac) with transcriptional or translational vectors containing the hybrid umuD-lacZ or osmE-lacZ operons, we demonstrated that AHB perform the regulatory functions involved in controlling the SOS response gene expression and the general rpoS -dependent stationary-phase regulon, respectively. The dose-dependent effect of long-chain AHB (within the 50–100 µg/ml range) resulting in a two- to threefold increase in the stress gene expression, similar to the effect of natural stress factors such as UV irradiation and starvation, provides evidence that AHB function as alarmones (danger signals). From the fact that the osm E gene is upregulated by 35–70 µg/ml C12-AHB (its regulation level is increased up to twofold), it follows that C12-AHB controls rpoS-dependent regulation and the transition to the stationary phase. The effect of the short-chain homologue C7-AHB substantially differs from that of C12-AHB. C7-AHB in concentrations of 10–100µg/ml causes a significant decrease in osmE and umuD expression. A 30-min preincubation of cells with 10–100µg/ml C7-AHB protected them from UV irradiation, as was observed as a 3.6-fold decrease in umuD expression. Comparative analysis of the marker gene’s expression values in the strains with the transcriptional and translational vectors demonstrates that AHB nonspecifically activate stress regulons at the transcription level.

The changes in the state of Bacillus subtilis spores that occur during germination were analyzed using dynamic phase microscopy (DPM). DPM is based on monitoring and analyzing the interference image of a specimen in a coherent laser beam. The optical path difference (the phase thickness of the specimen, PT) depends on the geometrical height of the specimen and its refractive index. We demonstrated that the maximum PT value is a convenient criterion of the physiological state of the organism involved: PT is ≥ 80 nm, ∼40–50 nm, and ≤ 20 in dormant, developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40°C results in a reversible twofold decrease (from 80 to 40 nm) in their PT under conditions that do not promote the development of the bacteria; this decrease is irreversible under growth-promoting conditions; (ii) the PT values of germinating spores oscillate with a considerable fluctuation amplitude (up to 7 nm), in contrast to the limited fluctuation amplitude (within 1 nm) in dormant spores; (iii) activated spores were heterogenous with respect to the PT pattern: a majority of the spores exhibited a usual spatial profile (with a maximum thickness in the center), whereas a minor fraction of them were characterized by an erythrocyte-like profile with a concave center; this implies that the central zone of the spore was more rapidly hydrated (with a decrease in refractive index) than the peripheral zone.

Examples are considered of the involvement of low-molecular-weight autoregulators in the development of resistance of proliferating microbial cultures to unfavorable environmental impacts of various intensity, including impacts programmed to occur in the developmental cycle (“new medium stress,” starvation stress) and nonprogrammed impacts. It was shown that extracellular adaptation factors control the reversible adhesion of cells in submerged cultures and the processes of cell reactivation in the poststress period and are involved in the stabilization of cellular biopolymers (proteins and DNA) and subcellular structures (membranes); the adaptogens of the phenolic type also act as efficient scavengers of reactive oxygen species. The protective effect of the adaptogenic autoregulators is manifested in the increase of resistance of microbial cells to stressors of various nature and in the preservation of the cell proliferative ability.

Information about the functions of extracellular autoregulators, which adapt microorganisms to the stresses “scheduled” in the development cycle of microbial cultures (stresses of new medium, starvation, or space exhaustion (high cell density)) is summarized in the review. In a number of bacteria and yeasts, derivatives of alkylhydroxybenzenes (AHB), particularly of the class of alkyl resorcinols, act as autoregulators with adaptogenic functions. The chemical structure of AHB determines their amphiphility; capacity for physical and chemical interaction with membrane lipids, proteins, and DNA; properties as natural modifiers of biological membranes and enzymes; and the expression of antioxidant activity. Increase of AHB concentration up to the critical level (10−5-10−4 M) results in cessation of cell division and in transition of the microbial culture to the stationary phase; further increase to 10−4-10−3 M induces a transition of some of the cells of a post-stationary culture to the anabiotic state with the formation of cystlike resting cells (CRC), even in non-spore-forming bacteria. AHB participate in the regulation of the phenotypic variability of bacteria. The dynamics of extra-and intracellular concentrations of AHB in growing microbial cultures and the polymodality of their effect determine the adaptogenic functions of AHB as autoinhibitors of culture growth, autoinducers of anabiosis, and autoinhibitors of germination of resting forms. Manifestation of any given function depends on the concentration of AHB, the physiological state of the recipient cells, and on environmental factors. The species nonspecificity of AHB effects points to their significant role in the regulation of the development and functioning of microbial communities.

The population composition of polycultures of Pseudomonas aeruginosa dissociants (R + M and R + S + M) developing on media with various contents and ratios of nitrogen and phosphorus has been studied. Irrespective of its proportion (10 to 90%) in the inoculum, the R variant accounted for 65 to 84% of the whole population of linear-phase and stationary-phase binary cultures of R and M dissociants, which differ in terms of energy metabolism and nutritional requirements. After prolonged cultivation, the population in the binary culture contained only R cells (100%), which are characterized by minimum requirements with respect to the main biogenic elements. These data agree with the predictive data of model studies and can be attributed to regulation of the population composition of bacterial cultures by trophic factors. It was established that the proportion of M cells, which are distinguished by maximum nutrient requirements and enhanced stability, increased during two developmental stages of the Ps. aeruginosa polycultures (R + M and R + S + M): the lag phase and the decay stage. This result cannot be due to the influence of trophic factors and presumably results from changes in the levels of autoregulatory factors (anabiosis autoinducers) involved in stress resistance and plausibly in the adaptive interconversion of dissociants upon transfer to a new medium (during the lag phase) and under starvation conditions (at the onset of the decay phase).

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.

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2-3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h-1, the fraction of proteins in cells being almost the same (60-70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3-5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.

This work tests 6 hypothetical models simulating the growth, respiration, and adhesion of cells to the walls of the cultivation flask. All the models postulate the synthesis of antiadhesins (AAs), i.e., extracellular metabolites decreasing the degree of cell adhesion. The models have the following distinguishing features: (model 1) the blocking of sorption centers on the glass walls by antiadhesins (the competitive inhibition of adhesion); (model 2) the noncompetitive inhibition of adhesion; (model 3) the accelerated release of bound cells; (model 4) a combination of models 1 and 3; (model 5) a combination of models 1 and 3 with a delay; (model 6) a combined action of two AAs, one of which, AA1, inhibits cell adhesion, and the other, AA2 (its synthesis is induced when the concentration of AA1 reaches a threshold level), stimulates the detachment of bound cells. Model 6 fits the relevant experimental data best. The delay effect is relatively small. The sigmoid character of the curve showing cell adhesion as a function of the antiadhesin concentration implies the existence of a strong cooperative effect in the adhesion inhibition. The models proposed satisfactorily simulate the growth, respiration, and adhesion of cells and AA synthesis in a batch bacterial culture grown either in a fresh nutrient medium or in the medium supplemented with the filtrate of a mature culture of the same species.

One of the two putative groups of antiadhesins was identified in Pseudomonas fluorescensby the method of gas chromatography–mass spectrometry. A mixture of high-molecular unbranched hydrocarbons (HC) with a chain length from 21 to 33 carbon atoms reduced cell adhesion to a glass surface. These HC accumulated in the culture liquid to a total concentration of 10–15 μg/l; the concentrations of individual HC ranged from 0.1 to 3.0 μg/l. After the addition of individual HC to the bacterial culture, the number of cells attached to the glass surface decreased. This decrease in cell adhesion was due to the enhanced aggregation of the bacterial cells, which promoted mechanical (hydrodynamic) cell detachment from the surface.

Exchange of information between bacteria via physical signals, referred to as “distant interactions” (DI), is the subject of this review. All cases of DI reported to date are discussed, as well as the history of these studies and the place of DI in bacterial communication. Bacterial DI are a particular case of DI occurring in nature (in plants, animals, and fungi). Along with the chemical signals of intracellular communications, DI play a significant role in the life of microorganisms, especially during critical and transitional periods.

The effect of the gaseous metabolites of onePseudomonas fluorescens culture on the attachment of cells of anotherP. fluorescens culture to glass was studied. Gaseous metabolites increased the number of unattached cells by 10–30% and the mean residence time of cells attached to glass by 100%. These effects were presumably due to the yet unidentified compound, which we called volatile antiadhesin. This compound could be adsorbed by activated charcoal and HAYESEP-Q adsorbent.

The effects of distant interactions (LRI) and culture air on the adhesion ofPseudomonas fluorescens cells were studied. OneP. fluorescens culture was found to diminish the adhesion of cells of another, glassscreened,P. fluorescens culture by 30% (in the absence o air exchange between cultures). This effect was interpreted to be due to penetrating LRI. Under the combined action ofLRI and culture air (the latter alone reduced cell adhesion by only several percent), the amount of unattached cells increased 2-to 30-fold (on the average, by a factor of nine). Such a great reduction of cell adhesion indicated the synergistic action ofLRI and culture air.

Some properties of the adhesion-modifying factors of Pseudomonas fluorescens are described. Adhesin, which promotes the adhesion of P. fluorescens cells, is a hydrophobic compound of a protein nature with a molecular mass of more than 10 kDa located either at the cell surface or in the medium. Antiadhesin, which suppresses the adhesion of P. fluorescens cells, is a thermolabile hydrophobic compound of a nonprotein nature with a molecular mass of less than 3 kDa. Heating makes antiadhesin hydrophilic. The role of adhesin and antiadhesin in the adhesion and adaptation of P. fluorescens cells is discussed.

Two factors affecting the adhesionof Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, antiadhesin (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.

AP. fluorescens NCIMB 9046 culture resuscitated from the lyophilized state on LB agar and stored in a mineral medium with glucose for 15 days comprised 90% of the S variant and 10% of the R variant. The R variant was more adhesive to glass and grew faster than the S variant.

The biological effects of the peptide and protein fractions of human brain extract are examined using anEscherichia coli culture as a simple and adequate test system. Both fractions bind superoxide radical and N-ethylmaleimide, the protein fraction being more active than the peptide fraction. In addition, the protein fraction inhibits the growth of the bacteria.

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.

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day−1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day−1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.