Biodegradation of 4-chlorobiphenyl Mic~~c~cc~s species
B.G. Bevinakatti and H.Z. Ninnekar” A lbkrococcus sp., isolated by enrichment culture, grew on &chlorobiphenyl produced kzhlorobenzoic acid in the culture medium as a dead-end 4-chlorobipheny1 by 2,3-~~y~oxylation followed by meta-ring cleavage fragments for cell growth. Key words: Biodegradation,
Polychlorinated biphenyls (PCB) have been used in a variety of products and industrial applications. They are amongst the most recalcitrant of organic pollutants because of their high chemical inertness, low water solubility and toxicity. The degradation of PCB, recently reviewed by Commandeur & Parsons (1990; 1993), is mostly caused by Pseudomonas, Alcalige~es, ArfkrQb~cfer and Aci~ef~ba~~ spp. In this communication, the biodegradation of 4-chlorobiphenyl by a Micrococcw sp. isolated from soil is discussed. We believe this may be the first report of the involvement of a member of the genus Micrococcus in PCB degradation.
at 2 g/l as sole carbon source and metabolite. The organism degraded to yield kzhlorobenzoate and carbon
dependent 0, consumption in a Gilson oxygraph. 2,~ Dihydroxybiphenyi-~,2~ioxygenase was assayed by measuring the formation of ring-cleavage product at 432 nm.
The organism grew on 4-chlorobiphenyl, at I z g/l, as sole source of carbon and energy (Figure I). During this growth, the medium turned yellow and spectral analysis of the
~~crffcu~c~ sp., isolated from soil by biphenyl enrichment culture (Bevinakatti & Ninnekar 1992), was grown on mineral salts medium containing 4-chlorobiphenyl (O.Z%, w/v) as sole carbon source. Growth was determined turbidometrically at 660 nm. The degradation of 4-chlorobiphenyl was assayed by extraction of residual substrate from the media with diethyl ether, followed by ~ectrophotometr~c estimation at 252 nm. Isolation and identification of metabolites from the spent medium and oxygen uptake studies were performed as described previously (Bevinakatti & Ninnekar 1992). Cell-free extracts, prepared from the washed cells grown on 4-chlorobiphenyl by sonication, were used for the enzyme assays. 4-Cblorobiphenyl dioxygenase was assayed by substrateTime B.G. Bevinakatti and H.Z. Ninnekar are with the Biochemistry Department of Chemistry, Karnatak University, Dharwad-580 fax 0836 42464. “Corresponding author. @ 1993 Rapid
Division, 003, India;
during growth (0) 660 nm corresponded
of 4-chlorobiphenyl of Micrococcus to approx.
at 2 g/l.
sp. (An absorbance 35 mg dry cell wt/lOO
of 1.0 at ml.)
Ltd World ~ourml
.bficrobio/ogy and Biotechnology. Vol 9, 1993
B.G. Bevinakatti Table
Oxgen uptake by washed cell Micrococcus,sp. grown on various substrates.
CChlorobiphenyl Biphenyl 2,3-Dihydroxybiphenyl Benzoic acid CChlorobenzoic * Corrected
uptake* dry cells) 12 10 110 34 0
clarified supematants showed an absorption maximum at 430 nm. This indicated the accumulation of meta-cleavage products of 4-chlorobiphenyl. TLC analysis of an extract of a culture in which the yellow product had completely disappeared revealed a single spot with an Rf value 0.90, corresponding to that of authentic 4-chlorobenzoic acid. The HPLC retention time (2.18 min), A,,,, (237 nm) and NMR spectra of the isolated metabolite were also identical with those of authentic 4-chlorobenzoic acid. Mass spectral analysis of the compound showed the parent ion peak at m/z 156, which is in good agreement with the empirical formula C,H,O,Cl. The fragmentation pattern showed ion peaks at m/z 139 (M-OH) and 111 (M-COOH). These spectral data were again identical with those of 4-chlorobenzoic acid. Cells grown on 4-chlorobiphenyl readily oxidized 2,3-dihydroxybiphenyl and 4-chlorobiphenyl biphenyl, benzoic acid but not &chlorobenzoic acid (Table I). Glucose-grown cells failed to oxidize any of these compounds. The cell-free extracts of Micrococcus sp. grown on 4-chlorobiphenyl contained 4-chlorobiphenyl dioxygenase (0.01 units/mg) and 2,3-dihydroxybiphenyl-1,2dioxygenase (0.14 units/mg).
World Journal of Microbiolo~
and Biotechnology, Vol 9, 1993
The formation of 4-chlorobenzoic acid from 4chlorobiphenyl indicated that the bacterial dioxygenase exclusively attacked the unsubstituted aromatic ring to give carbon fragments, that were oxidized through the tricarboxylic acid cycle, and also 4-chlorobenzoic acid as a dead-end metabolite. The bacterium could neither grow on nor oxidize 4-chlorobenzoic acid. The formation of yellow product and the presence of 2,3-dihydroxybiphenyl-1,2dioxygenase clearly indicated the operation of a mefacleavage pathway for the degradation of 4-chlorobiphenyl by this Micrococcus sp., as found in other bacteria (Bedard et al. 1986; Commandeur & parsons 1990). Any 4-chlorobenzoic acid formed by this organism in the environment would be metabolized further by other soil bacteria.
Acknowledgement We thank assistance.
References Bedard, D.L., Unterman, R., Bopp, L.H., Brennan, MJ., Haberl, M.L. & Johnson, C. 1986 Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls. Applied and Environmenfal Microbiology 5 I, 761-768. Bevinakatti, B.G. & Ninnekar, H.Z. 1992 Degradation of biphenyl by a Micrococcw species. Applied Microbiology and Biotechnology 38, 273-275. Commandeur, L.C.M. & Parsons, J.R. 1990 Degradation of halogenated aromatic compounds. Biodegradation 1, 207-220. Commandeur, halogenated Degradation,
L.C.M. & Parsons, J.R. 1993 Biodegradation of aromatic compounds. In Biochemistry of Miuobial ed Ratledge, C. Dondrecht: Kluwer. In press.
(Received in revised form