[Microbial degradation of glyphosate herbicides (review)].

This review analyzes the issues associated with biodegradation of glyphosate (N-(phosphonomethyl)glycine), one of the most widespread herbicides. Glyphosate can accumulate in natural environments and can be toxic not only for plants but also for animals and bacteria. Microbial transformation and mineralization ofglyphosate, as the only means of its rapid degradation, are discussed in detail. The different pathways of glyphosate catabolism employed by the known destructing bacteria representing different taxonomic groups are described. The potential existence of alternative glyphosate degradation pathways, apart from those mediated by C-P lyase and glyphosate oxidoreductase, is considered. Since the problem of purifying glyphosate-contaminated soils and water bodies is a topical issue, the possibilities of applying glyphosate-degrading bacteria for their bioremediation are discussed.

New approaches to identification and activity estimation of glyphosate degradation enzymes.

We propose a new set of approaches, which allow identifying the primary enzymes of glyphosate (N-phosphonomethyl-glycine) attack, measuring their activities, and quantitative analysis of glyphosate degradation in vivo and in vitro. Using the developed approach we show that glyphosate degradation can follow different pathways depending on physiological characteristics of metabolizing strains: in Ochrobactrum anthropi GPK3 the initial cleavage reaction is catalyzed by glyphosate-oxidoreductase with the formation of aminomethylphosphonic acid and glyoxylate, whereas Achromobacter sp. MPS12 utilize C-P lyase, forming sarcosine. The proposed methodology has several advantages as compared to others described in the literature.

[Sorption and microbial degradation of glyphosphate in soil suspensions].

Sorption and microbial destruction of glyphosphate, the active agent of the herbicide Groundbio, in suspensions of sod-podzolic and gray forest soils has been studied. According to the values of the adsorptive capacity (3560 and 8200 mg/kg, respectively) and the Freundlich constants (Kf, 15.6 and 18.7, respectively), these soils had a relatively high sorption capacity as related to the herbicide. Sorbed glyphosphate is represented by extractable and bound (inextractable) fractions. After long-term incubation of sterile suspensions, the ratio of these fractions reached 2 : 1 for sod-podzolic soil and 1 : 1 for gray forest soil. Inoculation of a native suspension of sod-podzolic soil with cells of a selected degrader strain Ochrobactum anthropi GPK 3 resulted in a 25.4% decrease in the total glyphosphate content (dissolved and extractable), whereas in a noninoculated suspension, the loss did not exceed 5.5%. The potential for the use of a selected bacterial strain for intensification of the glyphosphate destruction processes in soil systems is demonstrated for the first time.

[Thiomorpholine transformation by the fungus Bjerkandera adusta].

A screening of lignin-degrading basidial fungi that can grow in the presence of thiomorpholine derivatives (the mixture of 1,4-perhydrothiazines) has been performed. Strain Bjerkandera adusta VKM F-3477 was shown to have the maximal rate of growth in the presence of these compounds, and its capacity for thiomorpholine degradation was studied. The methods of quantitative analysis of thiomorpholine and its degradation products on the basis of thin layer chromatography and high-performance liquid chromatography were developed. It was shown that the B. adusta strain did not utilize thiomorpholine as a carbon source but transformed it into thiomorpholine sulfoxide that accumulated in the medium. Mn peroxidase produced by B. adusta in the course of thiomorpholine transformation is not directly involved in its oxidation.

[Microbial degradation of organophosphonates by soil bacteria].

Bacteria that can utilize glyphosate (GP) or methylphosphonic acid (MPA) as a sole phosphorus source have been isolated from soil samples polluted with organophosphonates (OP). No matter which of these compounds was predominant in the native habitat of the strains, all of them utilized methylphosphonate. Some of the strains isolated from GP-polluted soil could utilize both phosphorus sources. Strains growing on glyphosate only were not isolated. The isolates retained high destructive activity after long-term storage of cells in lyophilized state, freezing to -20 degrees C, and maintenance on various media under mineral oil. When phosphorus-starved cells (with 2% phosphorus) were used as inoculum, the efficiency of OP biodegradation significantly increased (1.5-fold).

[Microbial degradation of mustard gas reaction masses: isolation and selection of degradative microorganisms, analysis of organic components of reaction masses and their biodegradation]. / Mikrobiologicheskaia degradatsiia reaktsionnykh mass iprita: vydelenie i selektsiia mikroorganizmov-destruktorov, analiz organicheskikh komponentov reaktsionnykh mass i ikh biodestruktsiia.

Bacterial strains growing in medium with mustard gas reaction masses (RM) as carbon sources were obtained. Growth cessation in the above medium was caused by the exhaustion of bioutilizable substrates, first of all monoethanolamine (MEA) and ethyleneglycol (EG), rather than by the accumulation of toxic metabolites in the culture liquid or in the cells. The main RM components, 1,4-perhydrothiazines (PHT), formed in the course of chemical detoxication of mustard gas, were identified and analyzed. The predominant component of PHT mixture was N-(2-hydroxyethyl)-2-methyl-1,4-perhydrothiazine hydrochloride. Concentrations of all the PHT decreased by 50% in the course of culture growth; their destruction was a result of microbial metabolism.

[Thiodiglycol metabolism in Alcaligenes xylosoxydans subsp. denitrificans]. / Metabolism tioglikolia kul'turoi Alcaligenes xylosoxydans subsp. denitrificans.

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C-S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO4(2-) ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.

[The bioutilization of thiodiglycol (a breakdown product of mustard gas): isolation of degraders and investigation of degradation conditions]. / Bioutilizatsiia tiodiglikolia--protukta detoksikatsii iprita: vydelenie shtammov-destruktorov, izuchenie uslovii protsessa biodegradatsii.

The Alcaligenes xylosoxydans subsp. denitrificans strain TD1 capable of degrading thiodiglycol (TDG), a breakdown product of mustard gas, was isolated from soil contaminated with breakdown products of this chemical warfare agent. The selected stable variant of TD1 (strain TD2) can grow on TDG with a lag phase of 4-8 h and a specific growth rate of 0.04-0.045 h-1. Optimal conditions for the biodegradation of TDG (pH, the concentration of TDG in the medium, and its relative content with respect to the bacterial biomass) were determined. TDG was found to be degraded with the formation of diglycolsulfoxide and thiodiglycolic acid. The data obtained can be used to develop approaches to the bioremediation of mustard gas-contaminated soils.

[Utilization of detoxification products of yperite-lewisite mixtures]. / Utilizatsiia produktov detoksikatsii ipritno-liuizitnoi smesi.

An approach to developing an ecologically safe technology for disposal of yperite-lewisite mixtures has been developed. The technology includes three sequential stages: (1) detoxification by alkaline hydrolysis (resulting in the loss of toxic properties); (2) electrochemical treatment of detoxification products (electrocoagulation, to eliminate arsenic salts, and electrochemical oxidation, to convert all organic components into bioutilizable matter); and (3) degradation of the compounds obtained at the two preceding stages by a bred microbial association.

[Biosynthesis of protein and reserve substances by yeasts growing on different substrates with limited sources of mineral nutrition]. / Biosintez belka i rezervnykh veshchestv u drozhzhei, rastushchikh na raznykh substratakh v usloviiakh limitirovaniia istochnikami mineral'nogo pitaniia.

The content of protein and reserve compounds was studied in yeast cells growing on glucose, hexadecane and ethanol in a complete medium and in media limited with a source of nitrogen, phosphorus or sulfur. It was shown that the proportion between these components characterises the direction of biosynthetic processes in the cell depending on cultivation conditions. This is strongly shifted towards protein synthesis in the period of exponential growth when constructive processes are active. When the growth is limited with the sources of mineral nutrition, the content of protein drops down abruptly while that of the reserve compounds, lipids and carbohydrates, rises. Glycogen prevails in cells grown on glucose whereas lipids are the major component of cells cultivated on hexadecane. Nitrogen, phosphorus and sulfur deficiency causes quantitative and qualitative changes in the composition of free and bound amino acids in yeast biomass. The degree of these changes depends on the nature of the growth--limiting factor.

[Conditions and dynamics of citrate, isocitrate and polyol excretion in yeast growth on glucose]. / Usloviia i dinamika ékskretsii tsitrata, izotsitrata i poliolov pri roste drozhzhei na gliukoze.

The dynamics of metabolite excretions by Candida guilliermondii, Candida zeylanoides and Torulopsis candida was studied during their batch cultivation in media with glucose excess and low concentrations of N, P or S sources; the latter were the growth limiting factors and determined the transition to phase of retardation and the stationary phase of growth. Growth limitation with the above components in the presence of a carbon source results in the following: (a) the growth becomes linear and the phase of retardation falls into two periods differing in the absolute growth rate which initially is relatively high but then decreases; (b) protein content drastically decreases while lipid content and in particular, of glycogen increases, i. e. the direction of biosynthetic processes changes; (c) intensive excretion of citrate and isocitrate begins and continues (including the stationary phase) until the carbon source is exhausted. In contrast to citrate and isocitrate, polyols are excreted throughout all growth phases (including the exponential one) and strictly periodically, i. e. these metabolites are repeatedly excreted and re-utilized. The fact that polyols are excreted during the exponential growth phase proves that their overproduction apparently is not connected with the limitation of the cultural growth.

[Metabolite excretion by yeasts of the genus Candida in media lacking sources of N, P, S, or Mg and having different carbon sources]. / Ekskretsiia metabolitov Drozhzhami roda Candida pri defitsite istochnikov N, P, S ili Mg v sredakh s razlichnymi istochnikami ugleroda.

The purpose of this work was to study the effect produced by limiting the growth of yeasts belonging to the genus Candida on the excretion of cell metabolites. Changes in the composition and quantitative ratios between substances excreted depending on the carbon source (glucose, ethanol, hexadecane) and factors limiting growth of the cultures (N, P, S or Mg sources) were examined. It has been shown that (a) limitation of the yeast growth results in the majority of cases (but not always) in the excretion of macroquantities of these or other metabolites; (b) many microorganisms differ substantially from one another in their capacity to excrete metabolites; (c) the total number of metabolites found in the medium in macroquantities in different variants of the experiment is relatively small, and all of them belong to these or other intermediates of the oxidative metabolism of the carbon source (the acids of the citric acid cycle etc.) or are involved in the process of gluconeogenesis (polyols); (d) the composition and quantitative proportions of the excreted metabolites depend on the organism, carbon source and growth limiting factor.

[Effect of cultivation conditions on growth of Candida lipolytica yeasts and alpha-ketoacid biosynthesis in the presence of thiamine deficiency]. / Vliianie uslovii kul'tivirovaniia na rost drozhzhei Candida lipolytica i biosintez alpha-ketokislot v usloviiakh defitsita tiamina.

The effect of pH and aeration on the growth of Candida lipolytica and the biosynthesis of alpha-keto acids on acetate and glucose was studied in batch cultures at thiamine deficiency. If the initial thamine concentration was the same, then, irrespective of the carbon source, the yeast biomass in a medium saturated with oxygen by 5--10% was 1.5--2.0 times higher than in a medium with 60--90% [O2]. The rate of alpha-keto acid biosynthesis, on the contrary, decreased in the conditions of low aeration. The biomass increased and the rate of acid production decreased when pH was changed from 6.0 to 8.0 in a medium with acetate. However, at all studied values of pH and [O2] in a medium with either acetate or glucose, the growth at the deceleration phase was of a linear character, and was accompanied with the accumulation of alpha-keto acids in the cultural broth. The rate of acid production was maximal when the specific growth rate decreased to 0.04--0.06 hr-1. The presence of a linear phase in the conditions of thiamine deficiency suggests that here the growth of yeasts is determined by the constant activity of one of the thiamine dependent enzymes per unit volume of the cultural broth. However, the value of this activity seems to change depending on the cultivation conditions which, in turn, causes changes in the rates of biosynthetic processes.

[Kinetics of Candida lipolytica yeast growth and biosynthesis of alpha-keto acids with thiamine deficiency in media with different carbon sources]. / Kinetika rosta drozhzhei Candida lipolytica i biosintez alpha-ketokislot pridefitsite tiamina v sredakh s razlichnymi istochnikami ugleroda.

The growth kinetics of Candida lipolytica on glucose, acetate and hexadecane was studied in batch cultures at thiamine deficiency. The growth at the deceleration phase is of a linear character. The transition from the exponential phase to the linear one is accompanied with the accumulation of alpha-keto acids in the cultural broth, which is also observed in the stationary phase. The rate of acid production in the linear phase increases as the specific growth rate decreases, and reaches the maximum value in media with different carbon sources at mu = 0.01--0.06 h-1. Apparently, the deceleration of growth is due to a decrease in the activity of a thiamine-dependent enzyme (pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase or transketolase) which is a limiting point of biosynthetic processes. Here, a linear growth is determined by the constant activity of this enzyme per unit volume of the cultural broth which, in turn, depends on the constant concentration of the coenzyme, thiamine diphosphate, in the same volume.

[Respiratory cyanide resistance in Candida lipolytica and the supersynthesis of citric acids]. / Tsianidrezistentnost' dykhaniia Candida lipolytica i sverkhsintez limonnykh kislot.

The interrelationship between the cyanide resistance of respiratio and the process of citric acid synthesis was studied with various strains of Candida lipolytica. When the synthesis of citric acids was intensive, the respiration of cells was almost entirely inhibited by cyanide. The respiration became cyanide resistant only when the biosynthesis of citric acids was over. Induction of the cyanide resistance of respiration with antimycin A stopped the biosynthesis of citric acids. The cyanide resistance of respiration in yeast cultures incapable of considerable production of citric acids was displayed when the growth ceased. There was a strong direct correlation between the rate of citric acid synthesis and the degree of inhibition of the respiration by cyanide. It was concluded therefore that the process of citric acid overproduction could reset the reduction equivalents, thus making unnecessary the cyanide resistant pathway of electron transport.

[Growth kinetics of thiamine requiring Candida lipolytica during thiamine limitation: the existence of a linear growth phase]. / Wachstumskinetik einer thiaminbedürftigen Candida lipolytica bei Thiaminlimitation: Auftreten einer linearen Wachstumsphase.

The growth kinetics of batch cultures of the thiamine requiring Candida lipolytica 695 was investigated. It was established that a phase of logarithmic growth is followed by a linear growth phase. The time length of the linear phase and the relative increasing of the biomass during the linear phase are nearly constant and independend of the concentration of thiamine. The existence of the linear phase is discussed in connection with a constant activity of one of the two 2-ketoacid dehydrogenases as a bottle neck enzyme which needs thiamine pyrophosphate as coenzyme. Critical intracellular thiamine concentrations were calculated necessary for transition from the logarithmic to the linear phase and from the logarithmic to the stationary growth phase. Without the existence of the second critical thiamine concentration the linear growth would continue infinitely.

[Oxidation of aliphatic alcohols and acids by yeasts capable and incapable of growth on n-alkanes].

Yeast cultures, capable and incapable fo growth on hydrocarbons, were tested for their ability to oxidize aliphatic alcohols and acids, intermediate products of n-alkane degradation. The cultures belonging to 7 genera and 13 species did not grow on liquid paraffins and tetradecane. However, they grew on tetradecanol and oxidized aliphatic alcohols and fatty acids, the pattern of oxidation of the substrates being the same as in the case of alkane-oxidizing yeast cultures grown on glucose. None of 45 yeast strains (12 genera and 36 species) which did not grow on paraffin was able to oxidize tetradecane. As follows from this and earlier evidence (Lozinov et al., 1976a), the only reaction typical of yeasts capable of growth on n-alkanes is the oxigenase reaction of their oxidation to alcohols.

[The activity of citrate glyoxylate and pentosephosphate cycle enzymes during yeast growth on hexadecane and glucose]. / Aktivnost' fermentov tsitratnogo, glioksilatnogo i pentozofosfatnogo tsiklov pri roste drozhzei na geksadekane i na gliukoze

The activity of key enzymes of the citrate (CC), glyoxylate (GC), and pentose phosphate cycles (PPC) was determined in the cells of Candida lipolytica and Candida tropicalis growing on hexadecane and glucose. The activity of isocitrate lyase and malate synthase (enzymes of GC) was very high in hexadecane grown cells but was almost absent in "glucose" grown. The activity of citrate synthase and aconitase was 2-4 times and 1.5-2 times, respectively, higher in the cells, grown on n-alkane. The activity of citrate synthase of "hexadecane" and "glucose" yeasts was higher than the activity of other enzymes of CC (aconitase and isocitrate dehydrogenase), especially in the "hexadecane" cells. The activity of the key enzymes of PPC was almost the same in the yeast cells, grown on "hexadecane" and "glucose". Possible factors causing differences in the activities of the enzymes of GC and CC are discussed. Yeast organisms incapable to grow on n-alkanes were found to be able to grow on acetate (43 strains belonging to 35 yeast species have been studied) and to have a high activity of isocitrate lyase. Therefore, the absence of growth of many yeast strains on n-alkanes is not caused by the absence of the activity of enzymes of GC in these strains.