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. 2012;7(12):e52012.
doi: 10.1371/journal.pone.0052012. Epub 2012 Dec 12.

Decolourization of 4-chloro-2-nitrophenol by a soil bacterium, Bacillus subtilis RKJ 700

Pankaj Kumar Arora  1
Affiliations

Decolourization of 4-chloro-2-nitrophenol by a soil bacterium, Bacillus subtilis RKJ 700

Pankaj Kumar Arora. PLoS One. 2012.

Abstract

A 4-Chloro-2-nitrophenol (4C2NP) decolourizing strain RKJ 700 was isolated from soil collected from a pesticide contaminated site of India and identified as Bacillus subtilis on the basis of the 16S rRNA gene sequence analysis. Bacillus subtilis RKJ 700 decolourized 4C2NP up to concentration of 1.5 mM in the presence of additional carbon source. The degradation pathway of 4C2NP was studied and 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole (5C2MBZ) were identified as metabolites by high performance liquid chromatography and gas chromatography-mass spectrometry. Resting cell studies showed that Bacillus subtilis RKJ 700 depleted 4C2NP completely with stoichiometric formation of 5C2MBZ. This is the first report of (i) the degradation of 4C2NP at high concentration (1.5 mM) and, (ii) the formation of 5C2MBZ by a soil bacterium.

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Conflict of interest statement

Competing Interests: The author has declared that no competing interests exist.

Figures

Figure 1
Figure 1. Growth curves of Bacillus subtilis RKJ 700 in different conditions.
Growth of Bacillus subtilis RKJ 700 in (a) 10 mM glucose (as positive control), (b) 0.5 mM 4C2NP (negative control), (c) ) 10 mM glucose and 0.5 mM 4C2NP, (d) ) 10 mM glucose and 1 mM 4C2NP, (e) 10 mM glucose and 1.5 mM 4C2NP, and (f) 10 mM glucose and 2.0 mM 4C2NP.
Figure 2
Figure 2. Decolourization of 4C2NP by Bacillus subtilis RKJ 700 at various concentrations.
Decolourization of the minimal media containing 10 mM glucose and (a) 0.5 mM 4C2NP, (b) 1.0 mM 4C2NP, (c) 1.5 mM 4C2NP, and (d) 2.0 mM 4C2NP.
Figure 3
Figure 3. HPLC analysis of the samples collected from different intervals.
HPLC elution profiles of the sample collected from (a) 0 h, (b) 8 h, (c) 16 h and (d) 24 h.
Figure 4
Figure 4. Degradation curve of 4C2NP based on HPLC data.
Quantification of amounts of 4C2NP and its metabolites in sample collected from different intervals (0, 8, 16, 24 and 28 h).
Figure 5
Figure 5. Mass fragments of metabolites and authentic standards.
Mass fragments of the authentic 4C2AP (a), metabolite I (b), authentic 4C2AAP (c), metabolite II (d), authentic 5C2MBZ (e) and metabolite III (f).
Figure 6
Figure 6. Resting cell studies showing stoichiometric formation of 5C2MBZ from 4C2NP.
The resting cells of Bacillus subtilis RKJ 700 completely depleted 4C2NP with stoichiometric formation of 5C2MBZ within 40 minutes.
Figure 7
Figure 7. Proposed pathway of degradation 4C2NP for Bacillus subtilis RKJ 700.
4C2NP initially reduced to 4C2AP, which undergo acetylation to form 4C2AAP. After cyclization, 4C2AAP forms 5C2MBZ.
See this image and copyright information in PMC

References

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