Isolation And Characterization Of Butachlor-Degrading Bacteria And Their Application In The Bioremidation Of Herbicide-Contaminated Soil

Chloroacetamide herbicides are among the most important class of pre-emergence herbicides used for the control of annual grass and broadleaf weeds. The most commonly used chloroacetamide herbicides in the world were acetochlor and butachlor. Chloroacetamide herbicides persist for a long time in soil, and the residues consistently injure subsequent rotation crops, especially in sandy soils with low organic matter. Several studies have demonstrated that these herbicides were highly toxic to some aquatic organisms and were carcinogenic in mammal:acetochlor and alachlor caused tumors in the nasal turbinates, butachlor caused stomach tumors, and metolachlor caused liver tumors. Thus, great concerns have been raised about the behavior and fate of chloroacetamide herbicides and their degradation metabolites in the environment.Studies have demonstrated that biodegradation was the most important factor in the dissipation of chloroacetamide herbicides in environment. Many microorganisms capable of degrading chloroacetamide herbicides have been isolated and the metabolic metabolites were also identified. However, up to now, most reported pure microbial strains co-metabolized chloroacetamide herbicides and only a partial biodegradation was achieved, resulting in the accumulation of their metabolites, which contaminated the soil, surface and ground water. Moreover, up to now, the influence of the molecular structure of chloroacetanilide herbicides on their biodegradability has not been studied extensively.In this study,14 butachlor-degrading strains were isolated form rice field soil and activated sludge. Among these strains,10 of the 14 strains could only grow and degrade butachlor in LB medium, indicating that these strains co-metabolized butachlor. Four strains were able to utilize butachlor as the sole carbon source for growth. All of these strains distributed in nine of bacteria genus, which imply the diversity of the butachlor-degradating bacteria. Two strains, designated DCA-1 and FLY-8, were selected for further study due to their high degradation efficiencies. Strain DCA-1 and FLY-8 were able to degrade about 80.3% and 68.5% of the initially added 100 mg L-1 butachlor in MSM medium within 5 d at 30℃.The taxonomic position of strain DCA-1 was determined using a polyphasic taxonomic approach. Cells of strain DCA-1 are non-sporulating, non-motile, strictly aerobic and Gram-negative. No diffusible pigments are produced. Vesicular internal membrane structures and photoheterotrophic growth were not observed. The major respiratory quinone was ubiquinone-10 and the major cellular fatty acids were C18:1ω7c and 11-methyl C18:1ω7c. The genomic DNA G + C content of strain DCA-1 was 62.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences comparison revealed that strain DCA-1 was a member of the family Rhodobacteraceae and was related most closely to the type strain of Catellibacterium aquatile (sequence similarity 96.5%). The combination of phylogenetic analysis, phenotypic characteristics and chemotaxonomic data supports the suggestion that strain DCA-1 represents a novel species of the genus Catellibacterium, for which the name Catellibacterium caeni sp. nov. is proposed.Strain FLY-8 is a non-spore-forming, gram-negative, nonmotile and rod-shaped bacterium. The DNA G+C content is 69.5 mol%. Phylogenetic analysis of the 16S rRNA gene sequences revealed that strain FLY-8 groupes among Paracoccus species and forms a subclade with Paracoccus kocurii JCM 7684T (similarity 99.4%) with a high bootstrap value of 100%. Thus, based on the results of phenotypic, genotypic and phylogenetic properties, strain FLY-8 was identified as Paracoccus sp.Strain DCA-1 was able to degrade alachlor, acetochlor, propisochlor and butachlor and utilized these herbicides as carbon source for growth. When the initial concentration of different chloroacetamide herbicides were 100 mg L-1,89.6% of alachlor,83.2% of acetochlor,36.4% of propisochlor and 75.9% of butachlor were degraded by strain FLY-8, respectively, after 5 d incubation at 30℃. Strain FLY-8 was able to degrade the six chloroacetamide herbicides used in study and utilized these herbicides as carbon source for growth; and the order of degradation rates was:alachlor> acetochlor> propisochlor> butachlor> pretilachlor> metolachlor. When the initial concentration of different chloroacetamide herbicides were 100 mg L-1,98.7% of alachlor,88.2% of acetochlor,78.3 % of propisochlor,65.2% of butachlor,35.9% of pretilachlor and 24.1% of metolachlor were degraded by strain FLY-8, respectively, after 5 d incubation at 30℃.The influence of the molecular structure of chloroacetanilide herbicides on their biodegradability was studied. The results indicated that the substitutions of alkoxymethyl side chains with alkoxyethyl side chain greatly reduced the degradation efficiencies; the length of amide nitrogen’s alkoxymethyl significantly affected the biodegradability of these herbicides, the longer the alkyl was, the slower the degradation efficiencies occurred; the phenyl alkyl substituents have no obviously influence on the degradation efficiency.The optimal pH and temperature for the butachlor degradation by the two strains were 6-9 and 20-35℃, respectively. The degradation efficiency was related positively to initial inoculum size and ventilation. Low concentrations of butachlor did not inhibit the butachlor degradation. However, high concentrations of butachlor (above 200 mg L-1) reduced the degradation rate.The pathway of butachlor degradation by strain DCA-1 and strain FLY-8 were studied by metabolite identification and enzymatic studies. In strain DCA-1, butachlor was degraded to N-hydroxymethyl-2-chloro-N(2,6-diethyl-phenyl)-acctamide, which then converted to 2-chloro-N-(2,6-diethyl-phenyl)-acetamide,2,6-diethyl-phenyl)-ethoxymethyl-carbamic acid or to N-(2,6-diethyl-pheny)-N-hydroxymethyl-acetamide. N-(2,6-diethyl-pheny)-N-hydroxymethyl-acetamide was transformed to N-(2,6-diethyl-pheny)-N-hydroxymethyl-formamide. In strain FLY-8, butachlor was degraded to alachlor by the partial C-dealkylation and then converted to 2-chloro-N-(2,6-dimethylphenyl) acetamide by N-dealkylation, which subsequently transformed to 2,6-diethylaniline,2,6-diethylaniline was further degraded via the metabolite aniline and catechol, and catechol was oxidized through an ortho-cleavage pathway.Inoculation of strain DCA-1 into soils was found to significantly promote the removal of butachlor residue in soil. The moderate pH, high concentration of organic matter and clay could promote the degradation efficiencies of DCA-1. The degradation of butachlor in the soils need an appropriate soil moisture, high content of soil water would inhibit the degradation efficiencies.