Degradation of alpha, beta, gamma and
delta-hexachlorocyclohexanes by
Sphingomonas paucimobilis
Atul K. Johri*, Meenakshi Dua, Dipika Tuteja, Renu Saxena, D.M. Saxena
and Rup Lal
Department of Zoology, University of Delhi, Delhi-7, India
Fax : 91–11–7256541; Email : akjohri@himalaya.du.ac.in
Sphingomonas paucimobilis degrades aerobically α, β, γ and δ-hexachlorocyclohexane. With α-HCH, complete
degradation occurred after 3 days but with β and γ ,and with δ-HCH, 98 and 56 % degradation occurred after 12 and
8 days of incubation, respectively. Pentachlorocyclohexene was formed as the primary metabolite during the
degradation of all the HCH isomers.
Keywords : Hexachlorocyclohexane, Sphingomonas paucimobilis, pentachlorocyclohexene
Introduction
Hexachlorocyclohexane (HCH) is an organochlorine
insecticide extensively in use in the tropical countries and
in some advanced countries (Bachmann et al., 1988, Marks
et al., 1989, Johri et al., 1996, Mougin et al., 1996). In
addition, HCH isomers are highly persistent and the
residues have been reported from water, vegetables and
food commodities (Johri et al., 1996, Lal and Saxena 1982).
In fact the removal of -HCH, (formed as a byproduct
during the synthesis of -HCH) is a serious problem in the
production of -HCH.
Anaerobic degradation of HCH isomers and the formation
of metabolites have been reported by several workers and
recently reviewed (Johri et al., 1996) However, to our
knowleldge only four reports are available on the aerobic
degradation of -HCH by isolated microbes (Tu 1976,
Matsumura et al., 1976, Imai et al., 1989, Sahu et al.,
1990) two in a soil slurry (Bachmann et al., 1988, Bhuyan
et al., 1993), two on -HCH (Bhuyan et al., 1993,
Nagasawa et al., 1993) and one on -HCH (Bhuyan et al.,
1993). No report is as yet available on the aerobic degrada-
tion of -HCH. Rapid aerobic degradation of , , and
-HCH by the same bacterium Sphingomonas paucimobilis
as demonstrated in this study, has not been reported
previously.
Materials and methods
Microoganisms and culture conditions
Sphingomonas paucimobilis obtained from Prof. N.
Sethunathan Central Rice Research Institute (CRRI)
Cuttack, Orrisa, India, was grown in 20 ml LB medium of
pH 7.5, containing the following (in g l
-1
distilled water)
:tryptone (10), yeast extract (5), glucose (1), NaCl (5), to
an optical density of 0.5 (550 nm). Cells were harvested,
washed with 0.1 M phosphate buffer (pH 7.2), and resus-
pended in 50 ml of minimal medium containing following
(in g l
-1
distilled water), solution A: MgSO
4
.7H
2
O
(0.050), pH 7.2, solution B (NH
4
)
2
SO
4
(20), Na
2
HPO
4
(64.5), KH
2
PO
4
(18.75), solution C (filter sterilized)
Casamino acids (15), L-tryptophan (0.1), solution D trace
elements, ZnCl
2
(0.4), FeCl
3
.6H
2
O (2.0), CuCl
2
. 2H
2
O
(0.1), MnCl
2
4H
2
O (0.1), Na
2
B
4
O
7
(0.1), (NH
4
)
6
Mo
7
O
24
.
4H
2
O (0.1). The working solution contained 100 ml A +
5 ml B + 2 ml C + 0.2 ml D. The medium was
supplemented with HCH isomer at 5 mg l
-1
. Simultan-
eously cultures without HCH isomers were also kept as
control. Test flasks were incubated at 30°C with shaking.
One ml of the culture was withdrawn from the flasks at
intervals. The cell numbers were determined by plating
serial dilutions on agar plates containing minimal medium
and the respective HCH isomer. For degradation studies,
1 ml culture was extracted with 5 ml ethyl acetate and
subjected to GLC, equipped with an electron capture
detector (
63
Ni) and a glass column (2.6 mm inner diameter
2 m) packed with Silicon OV 17 (at 2% on 80–100
mesh Chromosorb W). The column, the injector and the
detector were maintained at 200, 220 and 210°C, respect-
ively and the N
2
carrier gas was at 1 ml/min.
Identification and characterization of metabolites
Metabolites formed during the degradation process were
identified from crude extract of cells. 50 ml culture, grown
overnight at 30°C was harvested, treated with ice-cold
Biotechnology Letters, Vol 20, No 9, September 1998, pp. 885–887
© 1998 Chapman & Hall Biotechnology Letters ⋅ Vol 20 ⋅ No 9 ⋅ 1998 885