I. T. Ermakova

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 of glyphosate, 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.

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°C, and maintenance on various media under mineral oil. When phosphorusstarved cells (with 2% phosphorus) were used as inoculum, the efficiency of OP biodegradation significantly increased (1.5-fold).

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C–P lyase incapable of degrading GP (C–P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C–P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C–P lyase II. O. anthropi GPK 3 also degraded MP via C–P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.

Ecologically safe destruction of the detoxification products of mustard–lewisite mixtures from the Russian chemical stockpile

An integrated chemical/biological method has been developed to create an environmentally safe technology for the destruction of the detoxification products of mustard–lewisite mixtures (MLM) from the Russian Stockpile of Chemical Warfare (CW) Agents. This method includes three sequential steps: (1) detoxification by alkaline hydrolysis, (2) electrochemical treatment of detoxification products including electrolysis and electrocoagulation for removing arsenic salts and for converting all organic components to bioutilized substances, and (3) bioutilization of the electrochemical products by a selected microbial association. This integrated system makes it possible to achieve an environmentally safe destruction of MLM. The approach provided for an effective engineering process that was able to biodegrade the organic materials resulting from the alkaline hydrolysis of sulfur mustard–lewisite mixtures once toxic arsenate was removed by electrochemical means. © 2000 Society of Chemical Industry

Biodegradation of glyphosate by soil bacteria: Optimization of cultivation and the method for active biomass storage

Conditions for obtaining the active biomass of Ochrobactrum anthropi GPK 3 and Achromobacter sp. Kg 16, bacteria which are able to degrade the herbicide glyphosate (N-phosphonomethylglycine), were investigated. In the batch culture, degradation was most effective in the medium with pH 6.0–7.0 and aeration at 10–60% of air saturation supplemented with glutamate and ammonium chloride as sources of carbon and nitrogen, respectively. Due to the adaptation of the cells and induction of the relevant enzymatic systems, the inoculum grown in the presence of glyphosate exhibited 1.5–2-fold higher efficiency of xenobiotic degradation than that grown with other sources of phosphorus (orthophosphate and methylphosphonic acid). The efficiency of the toxicant decomposition increased with an increase in a specific load of glyphosate, which the cells were subjected to during the initial stage of growth. The specific load was regulated both by the initial cell concentration and the concentration of the phosphorus source, and the effect was probably determined by its availability to microorganisms. Storage of the liquid biopreparation as a paste with stabilizers (ascorbate, thiourea, and glutamate) at room temperature for 50 days resulted in high level of bacteria viability and a degrading activity approximately equal to that obtained when the bacteria were maintained on the agar medium containing glyphosate at 4°C with monthly transfers to the fresh culture medium.

Sorption and microbial degradation of glyphosate in soil suspensions

Sorption and microbial destruction of glyphosate, the active agent of the herbicide Ground Bio, in suspensions of sod-podzol and gray forest soils has been studied. According to the adsorptive values (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 glyphosate is represented by extractable and bound (non-extractable) fractions. After long-term incubation of sterile suspensions, the ratio of these fractions reached 2: 1 for sod-podzol soil and 1: 1 for gray forest soil. Inoculation of a native suspension of sod-podzol soil with cells of a selected strain-degrader Ochrobactum anthropi GPK 3 resulted in a 25.4% decrease in the total glyphosate 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 in the glyphosate destruction processes in soil systems is demonstrated for the first time.

Organophosphonates utilization by soil strains of Ochrobactrum anthropi and Achromobacter sp.

Four bacterial strains from glyphosate- or alkylphosphonates-contaminated soils were tested for ability to utilize different organophosphonates. All studied strains readily utilized methylphosphonic acid and a number of other phosphonates, but differed in their ability to degrade glyphosate. Only strains Ochrobactrum anthropi GPK 3 and Achromobacter sp. Kg 16 utilized this compound after isolation from enrichment cultures with glyphosate. Achromobacter sp. MPK 7 from the same enrichment culture, similar to Achromobacter sp. MPS 12 from methylphosphonate-polluted source, required adaptation to growth on GP. Studied strains varied significantly in their growth parameters, efficiency of phosphonates degradation and characteristic products of this process, as well as in their energy metabolism. These differences give grounds to propose a possible model of interaction between these strains in microbial consortium in phosphonate-contaminated soils.

Strain Alcaligenes xylosoxydans subsp. denitrificans TD2 as the basis of a biosensor for determination of thiodiglycol

750 Thiodiglycol (β,β dioxydiethylsulfide, TDG) is the hydrolysis product of mustard gas (yperite), a chemical warfare agent with a blistering effect [1]. TDG biodegradation is carried out by the representa tives of several taxonomic groups of microorganisms, mainly representatives of the genera Alcaligenes and Pseudomonas [2–4]. Thiodiglycolic acid and bis(2 hydroxyethyl)sulfoxide were shown to be the products of TDG degradation by the strain Alcaligenes xylosox ydans subsp. denitrificans TD2 [5]. The latter com pound was formed when TDG was oxidized by molec ular oxygen [2]. Capacity of the microorganisms to oxidize low molecular organic compounds with the consumption of molecular oxygen can be used for analytical purposes. The aim of the present work was to develop a bio sensor model for determination of thiodiglycol using the immobilized cells of Alcaligenes xylosoxydans subsp. denitrificans TD2 as a receptor and the Clark type oxygen electrode as a converter. The strain A. xylosoxydans subsp. denitrificans TD2 was obtained by selection of the strain A. xylosoxydans subsp. denitrificans TD1 isolated from the soil con taminated with mustard gas reaction masses. Strain TD2 had better growth characteristics: a higher spe cific growth rate and a short lag phase. The bacteria were grown in flasks on a shaker in liquid mineral Strain Alcaligenes xylosoxydans subsp. denitrificans TD2 as the Basis of a Biosensor for Determination of Thiodiglycol T. N. Kuvichkina1, I. T. Ermakova, and A. N. Reshetilov1 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia Received April 2, 2012

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.

Chromatographic determination of morpholine and products of its microbiological degradation

A combination of thin-layer chromatography (TLC) with high-performance liquid chromatography (HPLC) was shown to be efficient in determining the intermediate products of the utilization of thiomorpholine with ligninolytic basidiomycete fungus Bjerkandera adusta VKM F-3477. The chromatographic mobility of the products of microbiological degradation of thiomorpholine was studied on Sorbfil PTSKh-P-V plates in the systems of chloroform-methanol-25% aqueous ammonia (80: 20: 2) for determining cyclic amines and isopropanol-25% aqueous ammonia (70: 30) for determining thio acids. The optimum conditions were selected for the separation of thiomorpholine and the formed metabolites by ion-exchange chromatography and reversed-phase chromatography.