Metabolism of 4-chloro-2-nitrophenol in a gram-positive bacterium, Exiguobacterium sp. PMA

Abstract

Background: Chloronitrophenols (CNPs) are widely used in the synthesis of dyes, drugs and pesticides, and constitute a major group of environmental pollutants. 4-Chloro-2-nitrophenol (4C2NP) is an isomer of CNPs that has been detected in various industrial effluents. A number of physicochemical methods have been used for treatment of wastewater containing 4C2NP. These methods are not as effective as microbial degradation, however.

Results: A 4C2NP-degrading bacterium, Exiguobacterium sp. PMA, which uses 4C2NP as the sole carbon and energy source was isolated from a chemically-contaminated site in India. Exiguobacterium sp. PMA degraded 4C2NP with the release of stoichiometeric amounts of chloride and ammonium ions. The effects of different substrate concentrations and various inoculum sizes on degradation of 4C2NP were investigated. Exiguobacterium sp. PMA degraded 4C2NP up to a concentration of 0.6 mM. High performance liquid chromatography and gas chromatography-mass spectrometry identified 4-chloro-2-aminophenol (4C2AP) and 2-aminophenol (2AP) as possible metabolites of the 4C2NP degradation pathway. The crude extract of 4C2NP-induced PMA cells contained enzymatic activity for 4C2NP reductase and 4C2AP dehalogenase, suggesting the involvement of these enzymes in the degradation of 4C2NP. Microcosm studies using sterile and non-sterile soils spiked with 4C2NP were carried out to monitor the bioremediation potential of Exiguobacterium sp. PMA. The bioremediation of 4C2NP by Exiguobacterium sp. PMA was faster in non-sterilized soil than sterilized soil.

Conclusions: Our studies indicate that Exiguobacterium sp. PMA may be useful for the bioremediation of 4C2NP-contaminated sites. This is the first report of (i) the formation of 2AP in the 4C2NP degradation pathway by any bacterium and (iii) the bioremediation of 4C2NP by any bacterium.

Figure 1

Growth and Degradation Studies. (a) Utilization of 4C2NP as a sole source of carbon and energy by Exiguobacterium sp. PMA. (b) Estimation of chloride and ammonia releases from 4C2NP by Exiguobacterium sp. PMA. (c) Effect of various substrate concentrations on degradation of 4C2NP by Exiguobacterium sp. PMA. (d) Effects on different inaculum sizes on degradation of 4C2NP.

Figure 2

HPLC elution profile of samples of degradation of 4C2NP by Exiguobacterium sp. PMA. HPLC confirmed complete depletion of 4C2NP by Exiguobacterium sp. PMA within 44 h. Metabolite I was detected in sample of 24 and 36 h whereas metabolite 11 was detected only in the sample of 36 h.

Figure 3

Mass spectra of metabolites and authenetic standards. (a) Metabolite I, (b) 4-Chloro-2-aminophenol (4C2AP), (c) Metabolite II, and (d) 2-Aminophenol (2AP).

Figure 4

Ring cleavage inhibition studies. Dipyridyl inhibits the ring cleavege to 2-aminophenol and as a result, 2-aminophenol and 4-chloro-2-aminophenol were accumulated in the media in significant amounts.

Figure 5

Microcosm studies. (a) Degradation of 4C2NP in sterile soil by Exiguobacterium sp. PMA. (b) Degradation of 4C2NP in non-sterile soil by Exiguobacterium sp. PMA, (c) Degradation of 4C2NP in control microcosm with sterile soil, (d) Degradation of 4C2NP in control microcosm with non-sterile soil.

Figure 6

Degradation of 4C2NP by bacteria. (a) Proposed pathway of degradation of 4C2NP for Exiguobacterium sp. PMA. (b) Degradation of 4C2NP by a genetically engineered bacterium, Pseudomonas sp. N31. (c) Degradation of 4C2NP by a co culture of two bacteria. (d) Biotransformation of 4C2NP by Bacillus sp. MW-1.