RESUMO
An Ochrobactrum anthropi bacterial strain named as NC-1, capable of utilizing phenmedipham (PMP) herbicide as the sole of carbon source and energy for growth was isolated from pesticide-contaminated soil sample by enrichment culture technique. The isolated bacterial strain was identified as Ochrobactrum anthropi NC-1 (MH 796134) based on its morphological, cultural, biochemical characteristics and analysis of 16S rRNA gene sequence. The strain NC-1 could degrade more than 98.5% of PMP (2 mM) within 168 h. The optimal degradation pH and temperature were 7.0 and 30-35 °C, respectively. The strain NC-1 degraded PMP by a pathway involving its initial hydrolysis of their central amide carbamate linkage to yield m-aminophenol via methyl-N-(3-hydroxyphenyl) carbamate and m-toluidine were the major intermediates. However, m-aminophenol was not further metabolized, because they neither supported the growth of organism nor stimulated oxygen uptake. But m-toluidine released by dealkylation was followed by hydrolysis. Further, results also revealed that degradation of 4-methyl catechol proceeded via 2-hydroxy-5-methyl-6-oxohexa-2, 4-dienoate through meta cleavage ring processes. The formation of these compounds was confirmed by UV, TLC, HPLC, IR, NMR, and GC-MS spectral analysis. The cell-free extracts of O. anthropi NC-1 grown on PMP contained the activities of PMP hydrolase, toluidine dioxygenase, and 4-methyl catechol 1, 2-dioxygenase. These results demonstrate the biodegradation of PMP and promote the potential use of strain NC-1 to bioremediate PMP-contaminated environment.
RESUMO
Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] (CIPC), an important phenyl carbamate herbicide, has been used as a plant growth regulator and potato sprout suppressant (Solanum tuberosum L) during long-term storage. A bacterium capable of utilizing the residual herbicide CIPC as a sole source of carbon and energy was isolated from herbicide-contaminated soil samples employing selective enrichment method. The isolated bacterial strain was identified as Bacillus licheniformis NKC-1 on the basis of its morphological, cultural, biochemical characteristics and also by phylogenetic analysis based on 16S rRNA gene sequences. The organism degraded CIPC through its initial hydrolysis by CIPC hydrolase enzyme to yield 3-chloroaniline (3-CA) as a major metabolic product. An inducible 3-CA dioxygenase not only catalyzes the incorporation of molecular oxygen but also removes the amino group by the deamination yielding a monochlorinated catechol. Further, degradation of 4-chlorocatechol proceeded via ortho- ring cleavage through the maleylacetate process. 3-Chloroaniline and 4-chlorocatechol are the intermediates in the CIPC degradation which suggested that dechlorination had occurred after the aromatic ring cleavage. The presence of these metabolites has been confirmed by using ultra-violet (UV), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Fourier transmission-infrared (FT-IR), proton nuclear magnetic resonance (1H NMR) and gas chromatography-mass (GC-MS) spectral analysis. Enzyme activities of CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase were detected in the cell-free-extract of the CIPC culture and are induced by cells of NKC-1 strain. These results demonstrate the biodegradation pathways of herbicide CIPC and promote the potential use of NKC-1 strain to bioremediate CIPC-contaminated environment with subsequent release of ammonia, chloride ions and carbon dioxide.
