Isolation and characterization of carbofuran and dicamba degrading bacteria

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Virginia Tech


This study was conducted to isolate and characterize bacteria that have the capacity to degrade both carbofuran and dicamba. The pathways of degradation for both pesticides were elucidated.

An aerobic, carbofuran-degrading bacterium was isolated from a high concentration carbofuran bioreactor. The isolate degrades carbofuran at the upper limit of carbofuran solubility (approximately 700 mg L⁻¹), to carbofuran phenol. In aqueous mineral salts medium with carbofuran as Furadan 4F (6 g L⁻¹ a.i.), degradation of carbofuran to undetectable levels required approximately 100 days. Although carbofuran phenol was not completely degraded, the cells remained viable in the presence of unusually high concentrations of both surfactant and carbofuran phenol. Additional nutrient sources had little effect upon the rate of degradation of carbofuran in pure culture.

A dicamba-degrading consortium enriched from wetland soil, using the batch culture method, was used to elucidate the pathway of dicamba degradation under anaerobic conditions. The consortium consisted of one sulfate reducing bacterium, one fermenter, and three methanogens. The sulfate reducing bacterium was isolated from the consortium using sulfate as a terminal electron acceptor and 2-bromoethanesulfonic acid was added to inhibit the growth of the methanogens. Since the fermenter is dependent upon the methanogens, elimination of these organisms caused the elimination of the fermenter. Three methanogens (Methanothrix, Methanosarcina and Methanospirillum sp) were isolated with acetate and headspace gas consisting of H₂-CO₂. Degradation of dicamba proceded through an initial demethylation reaction yielding 3,6-dichlorosalicylic acid, as determined by high performance liquid chromatography (HPLC) analysis of aqueous medium. This was followed by a reductive dehalogenation reaction at the meta position of 3,6-dichlorosalicylic acid forming 6-chlorosalicylic acid. The metabolites were isolated using thin layer chromatography. Confirmation of metabolite identity was achieved using HPLC, and mass spectrometry. It appears that the fermenter was responsible for mediating the demethylation reaction. The consortium was unable to mineralize the aromatic ring.

The substrate specificity of the dicamba-degrading consortium was investigated. The consortium was found to have the capacity to mediate the reductive dehalogenation of both 3-chlorosalicylic acid and 2,5-dichlorobenzoic acid at the meta position. The consortium was unable to dehalogenate either 3-chlorobenzoic acid, 4-chlorosalicylic acid, 5-chlorosalicylic acid, or 2,5-dichlorophenol. Addition of the reducing agent cysteine (0.025% and 0.050%) to a yeast extract amended (0.04%) mineral salts medium containing 3-chlorosalicylic acid reduced the rate of dehalogenation compared to medium containing sodium sulfide as the reducing agent. Only limited dehalogenation of 3- chlorosalicylic acid and 2,5-dichlorobenzoic acid was observed when the sulfate reducing bacterium was cultured alone in a yeast extract amended medium, suggesting that the mutualistic efforts of a mixed population of anaerobes were necessary to efficiently mediate reductive dehalogenation.