World J Microbiol Biotechnol (2009) 25:1539–1546
DOI 10.1007/s11274-009-0039-x
ORIGINAL PAPER
Specific dechlorinase activity in lindane degradation
by Streptomyces sp. M7
Sergio A. Cuozzo Æ Graciela G. Rollán Æ
Carlos M. Abate Æ Marı́a J. Amoroso
Received: 23 December 2008 / Accepted: 31 March 2009 / Published online: 18 April 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Synthesis of dechlorinase in Streptomyces sp.
M7 was induced when the microorganism was grown in the
presence of lindane (c-hexachlorocyclohexane) as the only
carbon source. Activity of cells grown with lindane was
about four and half times higher compared to cells grown
with glucose. Maximum dechlorinase activity was
observed at 30°C in alkaline conditions pH (7.9) and the
enzyme did not show cation dependency. Sodium dodecyl
sulfate polyacrylamide gel electrophoresis revealed one
differential band with a molecular weight similar to serum
albumin (Mr 66,200), which corresponded to polynucleo-
tide phosphorylase, an enzyme that plays an important role
in the regulation system and could be involved in the
regulation of the dechlorinase gene. Detected in cell-free
extracts were c-pentachlorocyclohexene and 1,3,4,6-tetra-
chloro-1,4-cyclohexadiene, both being products of the de-
chlorinase activity. This is the first time that the presence of
an enzyme with dechlorinase activity has been demon-
strated in an actinomycete strain isolated in Tucumán,
Argentina. Characteristics of this enzyme revealed that
S. A. Cuozzo
C. M. Abate
M. J. Amoroso (&)
Planta Piloto de Procesos Industriales y Microbiológicos-
CONICET, Tucumán, Argentina
e-mail: amoroso@proimi.org.ar
S. A. Cuozzo
e-mail: sergio_cuozzo@yahoo.com.ar
G. G. Rollán
Centro de Referencia para Lactobacilos-CONICET, Tucumán,
Argentina
S. A. Cuozzo
C. M. Abate
M. J. Amoroso
Facultad de Bioquı́mica, Quı́mica y Farmacia, Universidad
Nacional de Tucumán, Avenida. Belgrano y Pje. Caseros T4001
MVB, San Miguel de Tucumán, Argentina
Streptomyces sp. M7 could be useful in the future in bio-
remediation of soil or as a biosensor.
Keywords Streptomyces
Dechlorinase
Lindane

Biodegradation
Introduction
Xenobiotic compounds such as organochlorine pesticides
constitute a major environmental problem due to their
extensive use in the past, pronounced resistance to chem-
ical and biological degradation (Miglioranza et al. 2002)
and their trend to bioaccumulate in the food chain (Mertens
et al. 2006). Contamination with halogenated aliphatic
compounds is often considered to be relatively recalcitrant
due to the gamma isomer of hexachlorocyclohexane
(c-HCH or lindane). There are many reports indicating that
this toxic compound is present in soil, water, air, plants,
agricultural products, animals, food, microbial environ-
ments and humans (Albanis et al. 1998; Hura et al. 1999;
Chaile et al. 1999; Waite et al. 2001; Botella et al. 2004).
c-HCH, commercially known as lindane, is highly chlori-
nated and has been extensively used as a broad-spectrum
insecticide against a wide range of soil-dwelling and her-
bivorous insects (Phillips et al. 2005). Lindane is consid-
ered a potential carcinogen and listed as a priority pollutant
by the US EPA (Walker et al. 1999; Tao et al. 2005).
c-HCH is a lipophilic compound and therefore tends to
accumulate and concentrate in the body fat of animals and
humans (Johri et al. 1996, 2000). It has been banned or
restricted in Argentina and in approximately other 50
countries. However, c-HCH continues to be a serious tox-
icological problem at industrial sites, where the past
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1540
production of lindane along with unsound disposal prac-
tices have led to serious contamination problems. In
addition, the presence of lindane residues is continually
reported in different environmental compartments (Rup
et al. 2006). Lindane contamination continues to be a
global issue, because its volatility is moderate and it can be
transported by air to remote locations (Galiulin et al. 2002;
Phillips et al. 2005). Due to its toxicity and persistence,
environments contaminated with this compound have been
targeted for bioremediation.
Since the toxicity of c-HCH is well established, it is
imperative to develop methods by which lindane can be
removed from the environment and to search for new
bioremediation agents (Singh and Kuhad 1999; Singh et al.
1999). There have been some reports regarding aerobic
degradation of c-HCH by Gram-negative bacteria like
Sphingomonas (Nagata et al. 2007) and by the white-rot
fungi Trametes hirsutus, Phanerochaete chrysosporium,
Cyathus bulleri and Phanerochaete sordida (Mougin et al.
1999; Singh and Kuhad 1999, 2000). However, little
information is available on the ability of biotransformation
of organochlorine pesticides by Gram-positive microor-
ganisms and particularly by actinomycete species, the main
group of bacteria present in soils and sediments (De
Schrijver and De Mot 1999).
Lindane (2.0 mg l-1), methoxychlor (1.3 mg 1-1),
aldrin and dieldrin (both 0.03 mg 1-1) were detected in
the main hydrographic system, the Salı́ River, in Tuc-
umán, Argentina (Amoroso et al. 1998; Chaile et al.
1999). In order to establish a strategy for bioremediation,
samples of river sediments and from other contaminated
local sources were collected to isolate and select wild-
type Streptomyces strains with the ability to tolerate and
remove organochlorine pesticides (Benimeli et al. 2003).
Nagata et al. (1999) demonstrated that Sphingobium
japonicum UT26 possesses a dechlorinase enzyme, LinA
(c-hexachlorocyclohexane dehydrochlorinase, EC 4.5.1),
encoded by the linA gene that catalyses two dehydro-
chlorination steps: c-HCH to 1,3,4,6-tetrachloro-1,4-
cyclohexadiene (1,4-TCDN) via c-pentachlorocyclohex-
ene (c-PCCH).
In addition to c-HCH and c-PCCH, a- and d-isomers of
HCH were also dehydrochlorinated by LinA, whereas
b-HCH was not (Nagata et al. 1993). Furthermore, it was
experimentally confirmed that dehydrochlorination of
c-HCH proceeds by a 1,2-ante dehydrochlorination reac-
tion (Nagata et al. 2007).
Regarding the environmental problems caused by lin-
dane and the current lack of information about the presence
of dechlorinase activity in Streptomyces, the aim of this
work was to demonstrate, for the first time, a specific
dechlorinase activity in Streptomyces using lindane as
substrate.
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World J Microbiol Biotechnol (2009) 25:1539–1546
Materials and methods
Microorganism and culture conditions
Streptomyces sp. M7 was previously isolated from waste-
water sediment samples from a copper filter plant (Beni-
meli et al. 2003, 2004).
Three different growth conditions of Streptomyces sp.
M7 were assayed in a liquid-defined medium (MM) con-
taining (in g per l): L-asparagine, 0.5; K2HPO4, 0.5;
MgSO4
7H2O, 0.20; FeSO4
7H2O, 0.01, at pH 7.0 (Kieser
et al. 2000). M1 condition: 0.1 g l-1 of glucose was added
to MM and used as control, M2 condition: MM supple-
mented with 100 lg ml-1 lindane as the only carbon
source and M3 condition: MM with 0.1 g l-1 of glucose
during 20 h of culture prior to addition of 100 lg ml-1
lindane.
Reagents and chemicals
Ethylenediaminetetraacetic acid (EDTA), N-[2-hydroxy-
ethyl]piperazine-N0 -[2-ethanesulphonic acid] (HEPES),
Coomassie brilliant blue reagent and bovine serum albumin
standard II (1.38 mg ml-1) were purchased from Bio-Rad.
Lindane (99.7%) was purchased from Sigma–Aldrich
Argentina. Solvents and other chemicals used were
reagent-grade and toluene and chloroform were purchased
from Sintorgan–Argentina.
Enzyme assays
Colorimetric assay for dechlorinase activity
Bacterial cells from 100 ml culture were aseptically har-
vested by centrifugation at 4,0009g for 5 min at 4°C. The
cell pellet was washed once with 1 ml sterile disruption
buffer: 50 mM Tris–sulphate (pH 7.5), 1 mM EDTA and
1 mM dithiothreitol (DTT). Washed cells were centrifuged
again as before and the pellet stored at -20°C. Prior to the
dechlorination assay, the pellet was thawed, resuspended in
twice its volume in sterile disruption buffer and lysed with
a French press. Cell debris were removed from the lysates
by centrifugation at 10,0009g for 10 min at 4°C and the
cell-free extract was used immediately for the dechlorina-
tion assay.
The following reaction mixture was used in the colori-
metric assay to determine haloalkane dechlorinase activity:
HEPES buffer 0.5 mM, sodium sulphate 10 mM and
EDTA 0.5 mM; the pH indicator, phenolsulphonaphtha-
lein, was dissolved in ethanol (Phillips et al. 2001) and
added to the buffer prior to pouring it into the wells of a 96-
well microtitre plate to give a final concentration of
20 lg ml-1. Each well was filled with 194 ll of buffer,
World J Microbiol Biotechnol (2009) 25:1539–1546
1 ll substrate stock solution and 1 ll of toluene, before
addition of 6 ll cell-free extract, in order to solubilize
lindane. The microtitre plate was covered to prevent vol-
atilization of the reaction mixture and incubated at 30°C
for 6 h.
A colour change from red to yellow in the presence of
cell-free extract was indicative of lindane dechlorination
and, therefore, a positive result.
A standard chloride curve was obtained by measuring
the absorbance at 540 nm in the wells containing known
concentrations of HCl in the reaction buffer, lindane and
toluene (in proportions equal to dechlorination assay wells)
using a Beckman Coulter AD200 microplate reader. The
specific activity was expressed as lmol Cl/h/mg protein.
The culture showing dehalogenase activity on the
microtitre plate was tested for c-HCH clearance on agar
plates to confirm degradation activity. Spray plates were
prepared with 1.5% agar in MM on Petri dishes and the
culture was streaked onto the agar. The agar surface of the
plate was sprayed with 0.5% c-HCH in diethyl ether
(Manickam et al. 2008).
Protein determination
The protein concentration was determined according to the
Bradford method using bovine serum albumin (Bio-Rad) as
standard.
Effect of pH and temperature on enzyme activity
The pH effect on the enzyme activity was determined at
30°C in a range between 6.8 and 8.2. The buffers used
were: HEPES buffer 0.5 mM, sodium sulphate 10 mM and
EDTA 0.5 mM. The composition of the buffers at different
pH was the same varying the quantity of each buffer
component in the adjustment of the pH value for each test.
The temperature effect was determined at pH 7.9 with a
temperature range of 25–35°C.
Effect of chemical reagents and cations
The effect of different cations (Ca2?, Mg2?, Na?, K?,
Zn2? and Hg2?) was determined adding the chloride salts
to the enzyme mixture at a final concentration of 0.5 and
50.0 mM, except for Zn2? and Hg2?, which were added at
a final concentration of 0.1 and 1 mM, respectively.
Protein profile detection by SDS–PAGE
The protein profile detection at M1, M2 and M3 condition
was determined using SDS–PAGE (Schägger and von
Jagow 1987), which roughly separates proteins according
to their molecular weight. The molecular weight of the
1541
differential band was determined by using the following
standards (BioRad): myosin (Mr, 200,000), b-galactosidase
(Mr, 116,250), posphorylase b (Mr, 97,400), serum albumin
(Mr, 66,200), ovalbumin (Mr, 45,000), carbonic anhydrase
(Mr, 31,000), trypsin inhibitor (Mr, 21,500), lysozyme (Mr,
14,400) and aprotinin (Mr, 6,500). Gels were stained with
0.1% (wt/v) Coomassie blue R250 in 50% (v/v) methanol-
7.5% (v/v) acetic acid and destained with 30% (v/v) eth-
anol-10% (v/v) acetic acid.
MALDI-TOF (matrix-assisted laser desorption/
ionization-time-of-flight)
Mass spectrometric data were obtained using an Ultraflex
II MALDI-TOF/TOF mass spectrometer (Bruker) at the
CEQUIBIEM mass spectrometry centre, Buenos Aires,
Argentina, according to the following protocol. The stained
protein spots were excised from the gel and destained with
10 mM ammonium bicarbonate-50% acetonitrile; gel pie-
ces were air-dried and further reduced with 10 mM DTT
and alkylated with 55 mM iodoacetamide, both in 50 mM
ammonium bicarbonate. Gel pieces were dehydrated with
acetonitrile and swollen in a minimum volume of digestion
buffer containing 10 ng ll-1 trypsin in 50 mM ammonium
bicarbonate. Digestion was performed by overnight incu-
bation at 37°C. The peptides were recovered from the
digestion mixture followed by a further extraction and
sonication with 30% acetonitrile-0.1% trifluoracetic acid
(TFA). The resulting peptide extracts were pooled and
concentrated in a Speed Vac, redisolved by sonication in a
matrix solution of a-cyano-4-hydroxycinnamic acid
(3 mg ml-1) and dissolved in 70% acetonitrile-0.1% TFA.
Sample peptides were analysed by TOF-MS and TOF-MS/
MS on an Ultraflex II MALDI-TOF/TOF with a 355 nm
neodymium-doped yttrium aluminium garnet (NdYAG)
laser with a 200 Hz laser repetition rate. MS (mass spec-
trometers) and MS/MS spectra were analysed using the
Mascot program and compared with the National Center
for Biotechnology Information (NCBI) database.
Lindane and metabolites characterization by GC–MS
Streptomyces sp. M7 was grown in flasks with 250 ml MM
containing 100 lg ml-1 c-HCH and incubated at 30°C at
100 rev min-1 for 48 and 96 h. At the beginning of the
experiment, the inoculum was started with 150 ll con-
centrated spore suspension (109 c.f.u. ml-1). Flasks with-
out inoculum served as control. After incubation pellets
were aseptically harvested by centrifugation at 4,0009g for
5 min at 4°C. The cell-free extract was used for extraction
of c-HCH, c-PCCH and 1,4-TCDN as follows. Cell-free
supernatants were extracted by solid phase extraction
(SPE) using C18 columns, evaporated until dry under
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reduced pressure and the residue was resuspended in

hexane. Routine quantitative determinations of c-HCH,
c-PCCH and 1,4-TCDN were carried out with gas chro-
matograph-electron capture detection-mass spectrometry
(GC–MS) analysis, using a HP 6890 gas chromatograph
equipped with a 63Ni electron capture detector and a HP5
capillary column (cross-linked 5% diphenyl-95% meth-
ylsiloxane; length, 30 m; internal diameter, 0.53 mm; film
thickness, 1.5 lm), a split/splitless HP 7694 injector and
Chem Station Vectra XM software. Nitrogen was used as
carrier gas at a flow rate of 25 cm s-1; initial oven tem-
perature was 90°C and increased to 180°C at 30°C min-1
and subsequently increased to 290°C at 20°C min-1.
Detector temperature was 320°C and the injection volume
1 ll. Under the given conditions the lindane retention time
was 8.01 min and the detection limit was 0.1 lg l-1. Fig. 2 Growth and lindane degradation in MM agar plates of
Streptomyces sp. M7 sprayed with 0.5% c-HCH in diethyl ether and
incubated for 4 days at 30°C

Results
Effect of temperature, pH and cations on dechlorinase
activity
Dechlorinase activity from Streptomyces sp. M7 displayed
thermostability up to 50°C for 1 h. Maximum enzyme
activity of this microorganism grown in lindane (M2 con-
dition) was at 30°C and at pH 7.9 (Fig. 1); in the other two
conditions (M1, M3) the dechlorinase activity was similar
to pH 6.8 for M1 and pH 7.1, 7.4 and 7.9 for M3. Fol-
lowing these results, pH 7.9 and 30°C were chosen to
determine enzyme activity in further experiments. The
inhibitory/stimulatory effect of several cations on the de-
chlorinase activity was examined, but had no effect on the
140
enzyme activity (data not show). Specific enzyme activities
obtained at pH 7.9 and at 30°C in the three different growth
conditions were
(U/mg): M1 = 31.14, M2 = 140, M3 = 70. These
results show that the enzyme activity at M2 condition was
four and a half times higher than M1 and double that of
M3, indicating that the enzyme synthesis was induced by
lindane. Also Streptomyces sp. M7 was inoculated into
MM medium (without glucose), sprayed with 0.5% c-HCH
in diethyl ether and incubated for 4 days at 30°C, the
results obtained are shown in Fig. 2, indicating the capacity
of this strain to degrade lindane because of the presence of
a clear halo around the colony.
Differential protein expression in SDS–PAGE
and MALDI-TOF

130
In order to identify lindane-induced proteins from Strep-
120 tomyces sp. M7 comparative analyses were performed
using glucose as the only carbon source.
Specific Activity

110
A major protein band could be detected in SDS–PAGE
100
when Streptomyces sp. M7 was grown in the presence of
90 with lindane as the only carbon source. This band was not
80
observed in the culture without lindane (Fig. 3). The band
intensity was stronger when the strain was cultured with
70
lindane that when it was preinduced with glucose prior to
60 addition of lindane. This result shows induction of
expression of dechlorinase due to the presence of lindane in
6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4
pH the culture medium (Fig. 3). The molecular weight of the
enzyme was estimated at 66 KDa.
Fig. 1 Specific dechlorinase activity of Streptomyces sp. M7 at This extra band was only detected on the 10% poly-
different pH values in three different growth conditions: j M1:
acrylamide SDS gel (pH 8.6). The amount of protein was
growth in MM supplemented with glucose 0.1 g l-1, d M2: MM with
lindane 10 mg l-1, m M3: MM with glucose 0.1 g l-1 and after 20 h enough to be analysed with MALDI-TOF mass spectrom-
addition of lindane 10 mg l-1 etry. The protein was selected and in-gel digested with

123
World J Microbiol Biotechnol (2009) 25:1539–1546
1 2 3 4
Bovine Serum Albumin 66,2 KD
Fig. 3 (SDS)–polyacrylamide gel electrophoresis. Streptomyces sp.
M7 grown in: 1 M1 condition. 2 M3 condition, 3 M2 condition, 4
broad range SDS–PAGE molecular weight standards (BioRad)
trypsin for MS/MS analysis. It was successfully analysed
and identified by sequence homology using the elucidated
amino acid sequence. Interestingly, the mass spectrum
revealed three intense peaks that were fragmented with
MS/MS. The Mascot search results confirmed that the
peptide viewed by MS/MS was a fragmentation of
DQLDFFPLTVDVEER corresponding to a Streptomyces
protein, a polynucleotide phosphorylase from Streptomyces
antibioticus with Carbamidomethyl (C) Ions Score: 98
Expect: 1.2e-06. Our results showed that only one differ-
ential band could be observed in SDS–PAGE when
Streptomyces sp. M7 was grown in the presence of lindane
as carbon source (Fig. 3).
Metabolites of lindane degradation
Gas chromatography results of the cell-free extracts
obtained at 48 and 96 h of growth of Streptomyces sp. M7
Table 1 Relative abundance analysis by GC-Mass of c-HCH and intermediates c-PCCH, and 1,4-TCDN during aerobic degradation of c-HCH
by Streptomyces sp. M7
Lindane and metabolites
MM with lindane at 0 h c-HCH
Cell-free extract at 48 h c-HCH
c-PCCH
1,4-TCDN
Cell-free extract at 96 h c-HCH
c-PCCH
1,4-TCDN
a
The relative abundance is referred to the ions of different atomic or molecular mass (mass-to-charge ratio) within a sample. It frequently refers
to the measured relative abundances of isotopes of a given element
1543
in M2 condition revealed the appearance of c-PCCH (Rt
6.26 min) and 1,4-TCDN, (Rt 5.29 min), the first and
second product of the lindane catabolism by the specific
dechlorinase in the catabolic way proposed by Nagata et al.
(2007). The relative abundance of c-PCCH and the 1,4-
TCDN increased one and half times, at 96 h compared to
48 h of growth (Table 1), these metabolites were not found
in MM with lindane (control) (Fig. 4).
Discussion
There are various reports on dehalogenases in different
microorganism species: such as: Pseudomonas sp., Xant-
hobacter sp. and Moraxella sp. (Nagata et al. 2007),
Sphingobium japonicum (Nagata et al. 1993, 1999, 2007),
Mycobacterium tuberculosis (Jesenska et al. 2000),
Bacillus and Micrococcus (Olaniran et al. 2004), Rhodo-
coccus sp. (Sallis et al. 1990) and Corynebacterium sp.
(Yokota et al. 1987). The specific substrate of these de-
halogenases was 1-chlorobutane and the enzymes were
able to utilize a range of halogenated aliphatic compounds
as sole carbon and energy sources. However, this it is the
first report on dehalogenase activity in an actinomycetes
with lindane as specific substrate. This has only been
reported in Sphingomonas (Nagata et al. 2007) and Nor-
mand et al. (2007) detected a putative 2,5-dichloro-
2,5cyclohexadiene-1,4-diol dehydrogenase (2,5-DDOL
dehydrogenase) in Frankia. Genetic studies of this strain
are necessary for a proper understanding of the principle
of its ability to degrade different chlorinated hydrocarbon
compounds.
Our results demonstrate that synthesis of the dechlo-
rinase enzyme was induced by the presence of lindane and
this activity was optimal in the culture medium at pH 7.9
(Fig. 1). Also, this specific dechlorinase activity was
increased in M2 condition compared with M1 or M3
conditions. These results make Streptomyces sp. M7 an
Retention time Relative abundancea
7.78 732750.96 ± 1041.37
7.78 674895.41 ± 1961.84
6.26 38270.56 ± 650.79
5.29 14795.47 ± 451.97
7.78 561952.29 ± 2310.10
6.26 70372.05 ± 3268.05
5.29 25988.30 ± 992.09
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123
Fig. 4 GC–MS total ion chromatogram (TIC) analysis of metabolites during c-HCH degradation at 48 and 96 h of growth of Streptomyces sp. M7 in MM 2 condition. a Relative abundance of
c-HCH, c-PCCH and 1,4-TCDN in: A1: MM ? 100 lg l-1 of c-HCH (control), A2: extract free cell of Streptomyces sp. M7 at 48 h of growth, A3: extract free cell of Streptomyces sp. M7 at
96 h of growth. b Electron-Impact fragmentation spectra (fingerprint pattern) of the compounds formed: B1: Rt 7.76 min (c-HCH), B2: Rt 6.26 min (c-PCCH), B3: Rt 5.29 min (1,4-TCDN)
World J Microbiol Biotechnol (2009) 25:1539–1546
World J Microbiol Biotechnol (2009) 25:1539–1546
ideal candidate for lindane bioremediation because the
maximum removal (70% approximately) was observed
when the microorganism was incubated in soil extract
liquid medium at 30°C (Benimeli et al. 2006) and, this
enzyme can function at alkaline pH, whereas dechlorinase
activity of Rhodanobacter lindaniclasticus and Sphingo-
monas paucimobilis UT26 were optimal in acidic pH
conditions (Phillips et al. 2001).
Furthermore a differential protein band corresponding to
polynucleotide phosphorylase appeared only when Strep-
tomyces sp. M7 was cultured in MM with lindane.
Bralley and Jones (2003) suggested a possible relation-
ship between the modification of the 30 -ends of RNA and
the regulation of antibiotic production in Streptomyces
antibioticus and S. coelicolor. Also, Andrade and Arraiano
(2008) found that the polynucleotide phosphorylase in
Escherichia coli plays a key role in the regulation of small
RNA molecules that control the expression of outer
membrane proteins. This enzyme would probably be
implied in the regulation of the dechlorinase synthesis in
Streptomyces sp. M7.
It is important to note that the two-first metabolites
(c-PCCH, 1,4-TCDN) produced by the action of dechlo-
rinase over lindane were detected in the cell free extract of
Streptomyces sp. M7 at 48 and 96 h at M2 growth condi-
tion (Fig. 4). Both metabolites increased after 96 h of
growth (Table 1). This result could prove the presence of a
specific lindane dechlorinase in this microorganism.
It is known that streptomycetes are metabolically
diverse and relatively resistant to adverse environmental
conditions in soil. The previously isolated and character-
ized Streptomyces sp. M7 (Benimeli et al. 2006, 2007)
could be considered an attractive solution for in situ lin-
dane degradation. Further studies are currently being car-
ried out for optimization of environmental conditions to
enhance bioremediation of contaminated soil containing
c-HCH by Streptomyces sp. M7. Meanwhile, the existence
of certain indigenous organisms able to degrade chlori-
nated compounds is a sign of a promising low cost biore-
mediation strategy for the clean-up of sites contaminated
with these pollutants.
Acknowledgments The authors gratefully acknowledge financial
support of CIUNT, ANPCyT and CONICET, Argentina. They are
also grateful to Dr. Claudia Benimeli and Dr. Jean-Guy Leblanc for
critically reviewing the manuscript and her invaluable advice as well
as Sr. Guillermo Borchia for technical assistance.
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