[Composition of the oil-slime microbial community determined by analysis of the 16S rRNA gene].

Analysis of the 16S rRNA genes of the cultured microorganisms of industrial oil-slime revealed predominance (-85-90%) of the Gammaproteobacteria in the community of aerobic heterotrophs and specific oil-slime degraders. Relation of the isolated strains with members of the genera Pseudomonas, Stenotrophomonas, and Enterobacter was established. Analysis of the same gene in the total DNA from the oil-slime revealed greater microbial diversity (-20 operative taxonomic units determined by T-RFLP) than in the cultured part of the community, which included -12 different colony types. Three major restriction fragments were found, with their total area -50%. These results demonstrated the low morphological and phylogenetic diversity of the oil-slime bacterial community.

Participation of oxygen in the bacterial transformation of 2,4,6-trinitrotoluene.

The exposure of Bacillus cereus ZS18 cell suspensions to 2,4,6-trinitrotoluene (TNT) in the absence of other oxidizable substrates increases oxygen uptake, exceeding the basal level of respiration of the bacterium 1.5- and 2-fold with 50 and 100 mg/liter of TNT, respectively. The interaction of both living and to less extent dead bacterial cells with TNT results in the accumulation of superoxide anion (O2*-) in the extracellular medium, which was revealed by the EPR spectroscopy. The accumulation of O2*- decreased by 50-70% in the presence of Cu,Zn-superoxide dismutase of animal origin. In the presence of living bacterial cells, the level of TNT decreased progressively, yielding hydroxylaminodinitrotoluenes together with O2*-. In the presence of heat-killed cells, a moderate decrease in TNT was observed, and the appearance of O2*- was not accompanied by the production of any detectable TNT metabolites. Chelating agents inhibited the transformation of TNT and decreased the formation of O2*-. The demonstrated generation of O2*- during the interaction of TNT with K4[Fe(CN)6] together with the observed effects of chelating agents suggest the participation of iron in the one-electron reduction of TNT and the functioning of an extracellular redox cycle with the involvement of molecular oxygen.

[Hydride-mediated reduction of 2,4,6-trinitrotoluene by yeasts as the way to its deep degradation].

Broad screening of microorganisms from natural and anthropogenic ecological niches has revealed strains Candida sp. AN-L15 and Geotrichum sp. AN-Z4 which transform 2,4,6-trinitrotoluene (TNT) via alternative pathways (with the domination of hydride ion-mediated reduction of the aromatic ring) and produce relatively high amounts of nitrites. According to the spectrophotometry data, the hydride attack of TNT by Candida sp. AN-L15 and Geotrichum sp. AN-Z4 grown at pH 5.0-8.0 leads to the mono- and dihydride complexes of TNT (H(-)-TNT and 2H(-)-TNT, respectively) and to protonated forms of the latter. Analysis by HPLC, GC-mass spectrometry, and ion chromatography revealed the products of deep conversion of TNT. The growth of the yeast strains in a weakly acidic medium with TNT (440 microM) is accompanied by formation of 2,4-dinitrotoluene (2,4-DNT, up to 18.2 microM). Together with accumulation of nitrites (up to 76.0 microM, depending on pH of the medium), these findings demonstrate the capacity of both strains for TNT denitration. Formation of 2,4-DNT reflects the realization of one of the possible mechanisms of TNT ortho-nitro group elimination and switching over to the pathways of metabolism of dinitrotoluenes, which are much more easily biodegradable than TNT. Simultaneously with the dominating TNT hydride attack, the mechanism of 4- and 6-electron reduction of the nitro group also functions in Candida sp. AN-L15 and Geotrichum sp. AN-Z4. Realization of the studied mechanisms of TNT transformation under growth of Candida sp. AN-L15 on n-alkane is important for bioremediation in the cases of combined pollution by oil products and explosives.

[Modeling of lactic acid fermentation of leguminous plant juices].

Lactic acid fermentation of leguminous plant juices was modeled to provide a comparative efficiency assessment of the previously selected strains of lactic acid bacteria as potential components of starter cultures. Juices of the legumes fodder galega, red clover, and alfalfa were subjected to lactic acid fermentation in 27 variants of experiment. Local strains (Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, and Lactobacillus sp. RS 4) and the collection strain Lactobacillus plantarum BS 933 appeared the most efficient (with reference to the rate and degree of acidogenesis, ratio of lactic and acetic acids, and dynamics of microflora) in fermenting fodder galega juice; Lactobacillus sp. RS 1, Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, Lactobacillus sp. RS 4, and L. plantarum BS 933 were the most efficient for red clover juice. Correction of alfalfa juice fermentation using the tested lactic acid bacterial strains appeared inefficient, which is explainable by its increased protein content and a low level of the acids produced during fermentation.

[Fermentation of a high-protein plant biomass by introduction of lactic acid bacteria].

Lactic acid bacteria displaying increased ability to produce lactic acid, medium proteolytic activity, and tolerance to osmotic stress were isolated under selective conditions from phyllosphere and rhizosphere of registered and raised cultivars of legumes. Lactic fermentation of poorly ensilable leguminous plants (red clover and Caucasian goat's rue) was performed by introduction of rifampin-resistant homofermenting representatives of the genus Lactobacillus (selected according to a set of technologically important characteristics). The results demonstrate that introduction of active local strains of lactobacteria, as well as the collection strain Lactobacillus plantarum BS 933, enhances activation of ensiling and increases the quality of fodder, as assessed according to the standard criteria (a decrease in pH of the medium, the ratio of lactic acid to fatty acid homologues, and the composition of silage microflora).

[Initial stages of 2,4,6-trinitrotoluene transformation by microorganisms]. / Nachal'nye étapy transformatsii 2,4,6-trinitrotoluola mikroorganizmami.

Screening of a wide range of microorganisms (32 strains) isolated from various anthropogenic and natural environments and of a number of collection strains showed that the early stages of 2,4,6-trinitrotoluene (TNT) transformation by the majority of the strains studied resulted in the formation of hydroxylaminodinitrotoluenes (HADNTs). The levels of HADNTs were in a number of cases comparable to the initial TNT level. The alternative reductive attack at TNT through the reduction of the aromatic ring was not characteristic of most of the prokaryotes studied. The susceptibility to the toxic effect of TNT was different for gram-positive and gram-negative bacteria.

[Distribution and physiology of microorganisms in petrochemical oily sludge of plant]. / Osobennosti raspredeleniia i fiziologicheskogo sostoianiia mikroorganizmov nefteshlama--otkhoda neftekhimicheskogo proizvodstva.

The occurrence, vertical distribution, and the physiological state of microorganisms in a petrochemical oily sludge deposit were studied. The total number and the number of viable microbial cells at depths of 0.2 and 3 m were about 10 and 10(8) cells/g dry wt. sludge. Most microbial cells taken from the middle (1 m deep) and the bottom (3 m deep) sludge horizons showed a delayed colony-forming ability, which suggested that the cells occurred in a hypometabolic state. The relative number of microaerobic denitrifying microorganisms steeply increased with depth. The amount of microorganisms tolerant to 3, 5, and 10% NaCl and capable of growing at 7 and 40 degrees C varied from 10(2) to 10(8) CFU/g dry wt. sludge. Petrochemical oily sludge was found to maintain the growth of heterotrophs, among which the degraders of oily sludge and ten different individual polycyclic aromatic hydrocarbons were detected. The occurrence of highly adaptable microorganisms with an adequate metabolic potential in the petrochemical oily sludge deposit implies that its bioremediation is possible without introducing special microorganisms.

Models of 2,4,6-trinitrotoluene (TNT) initial conversion by yeasts.

The original models of the initial steps of 2,4,6-trinitrotoluene (TNT) conversion by yeasts are presented. Saccharomyces sp. ZS-A1 reduced nitro groups of TNT producing isomeric monohydroxylaminodinitrotoluenes (HADNT) as the key initial metabolites (molar ratio HADNT/TNT was up to 0.81), whereas aminodinitrotoluenes (ADNT) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Conversely, Candida sp. AN-L13 transformed TNT almost quantitatively into H-TNT, thus realizing the alternative attack, consisting of the TNT aromatic ring reduction. The third type of conversion, revealed in Candida sp. AN-L14, is the combination of both above mechanisms and produces an equimolar mix of HADNT and H-TNT. In the toxicity tests with Paramecium caudatum, the supernatant of Saccharomyces sp. ZS-A1, which converts TNT into HADNT, was most toxic while the supernatant of Candida sp. AN-L13 (TNT --> H-TNT) was least toxic. The microorganisms converting TNT quantitatively to the reactive metabolites can be useful for their immobilization through the detoxifying interaction with the soil components such as humic compounds.

[Alternative pathways of the initial transformation of 2,4,6-trinitrotoluene by yeasts]. / Al'ternativnye puti nachal'noi transformatsii 2,4,6-trinitrotoluola drozhzhami.

A new model for the initial transformation of 2,4,6-trinitrotoluene (TNT) by facultatively anaerobic and aerobic yeasts is presented. The model is based on the data that Saccharomyces sp. ZS-A1 was able to reduce the nitrogroups of TNT with the formation of 2- and 4-hydroxyaminodinitrotoluenes (2-HADNT and 4-HADNT) as the major early TNT metabolites (the molar HADNT/TNT ratio reached 0.81), whereas aminodinitrotoluenes (ADNTs) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Candida sp. AN-L13 almost completely transformed TNT into H-TNT through the reduction of the aromatic ring. Candida sp. AN-L14 transformed TNT through a combination of the two mechanisms described. Aeration stimulated the production of HADNT from TNT, whereas yeast incubation under stationary conditions promoted the formation of HADNT. The transformation of TNT into HADNT led to a tenfold increase in the acute toxicity of the TNT preparation with respect to Paramecium caudatum, whereas the increase in the toxicity was about twofold in the case of the alternative attack at the aromatic ring.

Biotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteria.

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp. , and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO2 from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO2 from the nitroaromatic ring.

[Microbiological aspects of fermenting chicken manure at various temperatures]. / Mikrobiologicheskie aspekty fermentatsii kurinogo pometa pri razlichnykh temperaturakh.

Mesophilic and thermophilic groups of methanogenic bacteria were found in untreated fowl manure. The thermophilic anaerobic fermentation of fowl manure leads to the development of both individual methanogens utilizing only some of the methane precursors, and syntrophic associations that are also able to utilize complex substrates. The selection of methanogenic bacteria during fowl manure anaerobic fermentation results in accumulation of thermophilic forms utilizing acetate and propionate.

Microbial bioconversion of pollutants.

Microorganisms totally detoxicate xenobiotics of various chemical structures, which are serious and, in some cases, very hazardous pollutants. At present, the efforts of a number of researchers promoted the establishment in this country of a collection of microorganisms able to degrade volatile toxic pollutants--toluene, isomeric xylenes, styrene, alpha-methylstyrene, crotonaldehyde; widely distributed xenobiotics chlorobenzoic acids; isomeric aryldicarboxylic acids; and ecologically hazardous pollutants such as aromatic nitrocompounds. The active strains-destructors are mainly representatives of the genera Pseudomonas and Rhodococcus. Research into their physiological characteristics, key enzymes, pathways of xenobiotics degradation, genetic mechanisms determining the degradation of these foreign compounds, and behaviour of the strains in a real environment made it possible to develop the theoretical principles of using these microbial cultures to purify real industrial wastes and remediate polluted areas of soil and water. Improvement of the methods of immobilizing the active xenobiotics-degrading strains on cheap and efficient carriers made it possible to significantly intensify the cleanup process of industrial wastes and eliminate a number of problems during the development of the biotechnologies for industrial waste cleanup. Successfully operated at present are the biotechnologies of the local cleanup of waste waters of terephthalate production, microbial purification of industrial waste waters in nylon-66 production from hexamethylenediamine, purification of coke production wastes from phenols, waste waters of polyisocyanate production from aromatic amines, local purification of waste waters in synthetic rubber production from alpha-methylstyrene, acetaldehyde production wastes from crotonaldehyde and mercury. Microbial strains constructed by gene engineering methods for the cleanup of contaminated soils from dicofol and 3-chlorobenzoate were successfully applied (Golovleva et al. 1988).

[Transformation of 2,4,6-trinitrotoluene during oxygen and nitrate respiration in Pseudomonas fluorescens]. / Prevrashchenie 2,4,6-trinitrotoluola v usloviiakh kislorodnogo i nitratnogo dykhaniia Pseudomonas fluorescens.

A study of the metabolic pathway and the rate of 2,4,6-trinitrotoluene (TNT) transformation depending on the nature of the electron acceptor in the electron transport chain of Pseudomonas fluorescens B-3468 revealed that the first reaction of nitroreduction of TNT resulting in formation of 2-amino-4,6-dinitrotoluene (2A) and 4-amino-2,6-dinitrotoluene (4A) became more active in case of nitrate respiration as compared to oxygen respiration; a TNT decrease was 100 and 66%, respectively. The same tendency but much more pronounced was observed at the next stage of nitroreduction that lead to 2,4-diamino-6-nitrotoluene (2,4DA). On the contrary, aerobic conditions are more preferable for the subsequent destruction of 2,4DA. Thus monoamino derivatives, 2A and 4A, predominated under anaerobic conditions, whereas 2,4DA under anaerobic ones (85 and 69% of the total nitrogen-containing metabolites), respectively. Phloroglucinol and pyrogallol accumulated in the culture liquid when the bacteria were grown on a medium containing 2,4DA as a sole source of nitrogen. Their role as intermediates was proved by the results obtained by studying oxidative activity of the cells grown in the presence of 2,4DA and phloroglucinol.

[Possibilities for the deep bacterial destruction of 2,4,6-trinitrotoluene]. / Izuchenie vozmozhnosti glubokoi bakterial'noi destruktsii 2,4,6-trinitrotoluola.

The possibility of 2,4,6-trinitrotoluene (TNT) deep destruction (the aromatic cycle fission inclusive) by Pseudomonas fluorescens B-3468 is reported for the first time. The formation of nitrogen-free metabolites, viz. phloroglucinol and pyrogallol, is preceded by the NAD(P)H-dependent deamination of 2,4-diamino-6-nitrotoluene (2,4-DA), a TNT intermediate. 30% of 2,4-DA nitrogen in released as ammonium under the action of an induced cell-free extract in the presence of the preferential co-factor NADH (together with FAD). The elevated pyrogallol-decomposing activity in cells grown on 2,4-DA, phloroglucinol and pyrogallol as well as the induction of the pyrocatechase activity in cells grown on the above substrates, together with the earlier reported accumulation of phloroglucinol and pyrogallol upon 2,4-DA utilization, indicated that the enzyme might be involved in the TNT cycle cleavage. The participation of NADH-dependent glutamate dehydrogenase in 2,4-DA nitrogen utilization is supported by experimental evidence.

[Yeasts utilizing the surface-active substance Laurox-9]. / O drozhzhakh, utiliziruiushchikh poverkhnostno-aktivnoe veshchestvo lauroks-9.

Yeasts capable of utilizing the non-ionogenic surfactant Laurox-9 were identified as Cryptococcus humicolus (Daszewska) Golubev and Rhodotorula mucilaginosa (Jöerg) Harrison. IR spectrometry used for the quantitative assay of Laurox-9 showed that R. mucilaginosa utilized it as a sole carbon source. The first step of Laurox-9 metabolism was its hydrolytic cleavage yielding lauric acid and polyethyleneglycol. Optimal conditions were found for the electron-cytochemical localization of hydrolase involved in the primary degradation of Laurox-9 by yeasts. Its localization of the exocellular components was established.

[Regulation of terephthalate catabolism in Rhodococcus rubropertinctus]. / Reguliatsiia katabolizma tereftalata u Rhodococcus rubropertinctus.

The regulation of terephthalate catabolism was studied in Rhodococcus rubropertinctus which decomposed this synthetic monomer. The pathway (a) of terephthalate (TP) catabolism is as follows: TP----benzoate----4-hydroxybenzoate----protocatechuate----pyrocatechol-- --cycle ortho-cleavage. The following results were obtained when studying why two other catabolic pathways were realized if benzoate and 4-hydroxybenzoate were taken as a sole carbon source, namely, (b) benzoate----pyrocatechol----cycle cleavage and (c) 4-hydroxybenzoate----protocatechuate----cycle cleavage. TP seemed to cause the divergence of pathways (a) and (b) by repressing the system of benzoate oxidation to pyrocatechol. In pathway (c), benzoate repressed the synthesis of enzymes which catalysed protocatechuate oxidation. Pathway (b) was switched over to (a) when the strain was grown in a medium containing TP and benzoate at a benzoate concentration above 5 mM. Here, the concentration of benzoate (first exogenous and later formed from TP) played a key role. R. rubropertinctus growth in a medium with TP and glucose had diauxic characteristics.

[Final stages of the preliminary metabolism of 2,4,6-trinitrotoluene in Pseudomonas fluorescens]. / Zakliuchitel'nye étapy podgotovitel'nogo obmena 2,4,6-trinitrotoluola u Pseudomonas fluorescens.

The work was aimed at studying the transformation of 2,4-diamino-6-nitrotoluene (2,4-DA), an intermediate product in 2,4,6-trinitrotoluene catabolism by microorganisms. The results allow one to propose the following scheme for the terminal steps of TNT preparatory metabolism: 2,4-DA----[phloroglucinol carboxylic acid]----phloroglucinol----pyrogallol----ring cleavage.

[Characteristics of plasmid pBS271 controlling epsilon-caprolactam degradation by bacteria in the genus Pseudomonas]. / Kharakteristika plazmidy pBS271, kontroliruiushchei degradatsiiu épsilon-kaprolaktama bakteriiami roda Pseudomonas.

Conjugative plasmids control the ability of five Pseudomonas strains isolated from the rectifiers of chemical plants to grow on epsilon-caprolactam as a sole carbon and nitrogen source. All the plasmids have a high molecular mass of their DNA (ca. 300 MDa) and control epsilon-caprolactam degradation at least to succinate. One of the plasmids (pBS271) belongs to the incompatibility group P-2 and suppresses the growth of a broad spectrum of temperate and virulent P. aeruginosa bacteriophages as well as that of some P. putida bacteriophages.

[Bacterial destruction of terephthalic acid and its isomers]. / Bakterial'naia destruktsiia tereftralevoi kisloty i ee izomerov.

A strain of Mycobacterium lacticolum destructing the isomeric aryldicarbonic acids: teraphthalic, isophthalic and phthalic (in decreasing degree) has been selected from bacterial community of terephthalate containing industrial sewage biotreatment. The dominating role of M. lacticolum in microbial community increases during the continuous-flow cultivation under condition of terephthalate concentration increasing. Minor members of microbial community are not able to oxidize terephthalate and utilize it in compound culture, but the community's capacity to attack high concentrations of this substance is much lesser than in case of M. lacticolum.