Isolation of pesticide-degrading actinomycetes from a contaminated site: Bacterial growth, removal and dechlorination of organochlorine pesticides

International Biodeterioration & Biodegradation 64 (2010) 434e441

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Isolation of pesticide-degrading actinomycetes from a contaminated site: Bacterial growth, removal and dechlorination of organochlorine pesticides M.S. Fuentes a, C.S. Benimeli a, b, *, S.A. Cuozzo a, M.J. Amoroso a, b, c a

Planta Piloto de Procesos Industriales y Microbiológicos (PROIMI-CONICET), Avenida Belgrano y Pasaje Caseros, 4000, Tucumán, Argentina Universidad del Norte Santo Tomás de Aquino (UNSTA), 9 de Julio 165, 4000, Tucumán, Argentina c Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán (UNT), Ayacucho 465, 4000, Tucumán, Argentina b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 March 2010 Received in revised form 30 April 2010 Accepted 2 May 2010 Available online 7 June 2010

Organochlorine pesticides are notorious, due to their high toxicity, persistence in the environment and their tendency to bioaccumulate. Their extensive use in the northwest of Argentina has left residues in the environment. Microbial degradation is an important process for pesticide bioremediation and actinomycetes have a great potential for that. The current study examined organochlorine pesticides in contaminated soil. Indigenous actinomycetes were isolated from contaminated samples to evaluate bacterial growth as well as pesticide removal and release of chloride ions as a result of degradation. Most of the isolated microorganisms belonged to the Streptomyces genus, except one, which belonged to Micromonospora. Bacterial growth depended on the microorganism and the pesticide present (chlordane, lindane or methoxychlor). Highest growth and pesticide removal were observed with chlordane. Twelve out of 18 studied strains released chloride into culture supernatants, and percentages were higher with chlordane as carbon source than with lindane or methoxychlor. These results are supported by principal component analysis. This is the first report about actinomycetes isolated from an illegal storage of organochlorine pesticide in Argentina with capacity to growth, remove and use different organochlorine pesticide. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Organochlorine pesticides Actinomycetes Dechlorination

1. Introduction Organochlorine pesticides (OPs) constitute a major environmental problem, because of their high toxicity, persistence in the environment and ability to bioaccumulate in the food chain (Ntow, 2005; Xue et al., 2006). Although most developed countries established bans and restrictions on the use of several OPs during the 1970s and 1980s, they are still being used in certain countries for agricultural and public health purposes because of their low cost and versatility as pest control (Itawa et al., 1993; Xue et al., 2006). These compounds reach aquatic environments through effluent release, atmospheric deposition and runoff among other ways (Itawa et al., 1993; Yang et al., 2005). Because of the low water solubility, OPs have a strong affinity for particulate matter, and consequently, sediments can serve as an ultimate sink (Strandberg et al., 1998; Fatoki and Awofolu, 2003; Pazou et al., 2006).

* Corresponding author. Tel.: þ54 381 4344888; fax: þ54 381 4344887. E-mail address: [email protected] (C.S. Benimeli). 0964-8305/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2010.05.001

The extensive use of OPs in the northwest of Argentina has left residues in the environment. Chaile et al. (1999) detected OPs such as chlordane, lindane and methoxychlor in the Salí River, the main hydrographical system of Tucumán, Argentina. Lindane (g-hexachlorocyclohexane) is a cyclic, saturated, chlorinated insecticide, which has been used worldwide for crop protection and the control of vector-borne diseases like malaria (Manickam et al., 2008). Because of its high toxicity and persistence, it has caused serious environmental problems since its production in the beginning of the 1940s (Turnbull, 1996). Chlordane (1,2,4,5,6,7,8,8-octachlor-2,3,3a,4,7,7a-hexahydro-4,7-methanoindane) is a toxic fumigation agent, which has been used for many years and is now ubiquitously found in air, soil and water resources (Moradas et al., 2008). Technical chlordane was used in USA (1948e1988) and worldwide as a pesticide for farmlands, home lawns and gardens, and as a termiticide for house foundations (Li et al., 2007). Many of the chlordane components and its metabolites are toxic (Hayes and Laws, 1990), suspected to be carcinogenic (WHO/IARC, 1991) and may have estrogenic activities (Colborn et al., 1993).

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Methoxychlor (2,2,2-trichloro-1,1-bis (4-methoxyphenyl) ethane) is a pesticide developed for the use as a replacement for DDT, which has been prohibited internationally since the 1970s due to its high toxicity (Lee et al., 2006). However, in the early 1990s about 300e500 thousand pounds of methoxychlor were still used per year only in the USA (EPA, 1999). Traditional technologies routinely used for remediation of contaminated environmental soil include excavation, transport to specialized landfills, incineration, stabilization and vitrification. However, bioremediation technologies, which use plants or microorganisms (including bacteria) to degrade toxic contaminants in soil into less-toxic and/or non-toxic substances, have become the focus of interest (McGuinness and Dowling, 2009). Microbial degradation is regarded as an important process of OPs removal from sediments, and to date many researchers have studied this issue (Chang et al., 2001; Yuan et al., 2001; Fava et al., 2003; Miyoshi et al., 2004). There is little information available about microbial chlordane and methoxychlor degradation (Lee et al., 2006). Actinomycetes have a great potential for bioremediation of toxic compounds (Ravel et al., 1998). Benimeli et al. (2003, 2006, 2007) and Benimeli (2004) isolated and selected wild type Streptomyces strains which were able to tolerate and remove lindane from river sediments and other local contaminated sources. Cuozzo et al. (2009) detected dechlorinase activity and lindane catabolism products as a result of microbial lindane degradation by Streptomyces sp. M7, isolated in Tucumán, Argentina. Halogenation of xenobiotics is often carried out to create persistence. The use of OPs as carbon and energy source by degrading bacteria is quite common. A number of soil microorganisms, which synthesize dehalogenase, have been found to utilize halogenated alkanoic acids and also biodegrade chlorinated polycyclic hydrocarbons (Olaniran et al., 2001). Dehalogenation is the first and most important step in OPs degradation and it plays a central role in the biodegradation of many chlorinated compounds (Cuozzo et al., 2009). The aim of this work was to isolate and characterise actinomycete strains from OP-contaminated soil, able to remove and dechlorinate lindane, chlordane and methoxychlor from the culture medium. 2. Materials and methods 2.1. Chemicals Lindane (99% pure), technical-grade chlordane and methoxychlor (99.8% pure) were purchased from SigmaeAldrich Chemical, MO, US. All other chemicals used throughout the study were of analytical grade and were purchased from standard manufacturers. 2.2. Microbial strains and media The actinomycete strains used in this study were isolated as described below (see 2.4.). Streptomyces coelicolor A3 (2) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and Streptomyces sp. M7, M15, M30, M50 and C39 were previously isolated by Benimeli (2004). Starch-casein medium (SC) used for isolation of actinomycetes consisted of (g l1): starch, 10.0 g; casein, 1.0 g; K2HPO4, 0.5 g; agar, 15.0 g. The pH was adjusted to 7.0 prior to sterilization. Actinomycetes were grown in liquid minimal medium (MM), containing (g l1): L-asparagine, 0.5; K2HPO4, 0.5; MgSO4.7H2O, 0.20; FeSO4.7H2O, 0.01 (Hopwood, 1967). All media were sterilized by autoclaving at 121  C for 20 min.

435

2.3. Soil samples Soil samples were collected from a village named Argentina in the province of Santiago del Estero, Argentina (Fig. 1), where more than 30 t of obsolete pesticides were found in 1994. Chlorinated pesticides such as DDT and g-HCH constituted a large part of the pesticides stockpiled at this hazardous site. Each soil sample was aseptically collected using sterile test tubes and kept at 5  C until drying at 30  C to constant weight. Samples were diluted with sterile 145 mM NaCl solution prior to inoculation onto isolation plates in duplicate. Soil samples were also analysed for the presence of OPs. The procedure for extraction of OP residues was performed according to Quintero et al. (2005) and the organic phase obtained was analysed by Gas Chromatography with Electron Capture Detector (GC/ECD). 2.4. Isolation of microorganisms Isolation of actinomycetes was carried out in SC, supplemented with 10.0 mg ml1 final concentration of nalidixic acid (NA) and cycloheximide to inhibit growth of Gram negative bacteria and fungi, as previously reported by Ravel et al. (1998). Plates were incubated at 30  C and colonies were purified without antibiotics by streaking them onto agar medium. Colonies on SC medium revealed a tough appearance with leathery characteristics typical of vegetative actinomycete mycelium (and in some cases with aerial mycelium and spore formation). Microbial selection was based on colony morphology and colour and presence of diffusible pigments of isolates according to Bergey’s Manual (Lechevalier, 1989). 2.5. Isolation of chromosomal DNA, PCR amplification of the 16S ribosomal DNA (rDNA) and sequencing Total DNA from the isolated actinomycete strains was prepared according to the method described by Hoffman and Winston (1987). Oligonucleotide primers with specificity for eubacterial 16S rDNA genes [forward primer 8e27: 50 -AGA GTT TGA TCC TGG CTC AG-30 (Weisburg et al., 1991) and reverse primer 1492: 50 -GGT TAC CTT GTT ACG ACT T-30 (Heuer et al., 1997)] were used to amplify 16S rDNA. Amplification reactions were carried out in an automated thermal cycler (PerkineElmer, model 9700, Applied Biosystems). PCR products were run on a 1.0% agarose gel, stained with ethidium bromide and then visualized using an Image Analyzer Gel Doc, BIORAD. The amplicons obtained were purified and sequenced by Macrogen (Korea). The 16S rDNA gene sequences of actinomycete strains have been deposited in GenBank. Sequence data were analysed by comparison with 16S rDNA genes in the GenBank databases using the MEGA4 programme package (Tamura et al., 2007). An evolutionary tree was constructed using the neighbourjoining algorithm (Saitou and Nei, 1987). Evolutionary distance matrices for the FitcheMargoliash method were generated as described by Jukes and Cantor (1969). 2.6. Screening of actinomycete strains able to grow in the presence of lindane, chlordane or methoxychlor Spore suspensions of the actinomycete strains were inoculated in MM. Chlordane, lindane and methoxychlor were dissolved in methanol (Pesticide grade, Merck, Argentina), filter-sterilized with Millipore (0.22 mm pore size) and then added aseptically to the autoclaved medium at a final concentration of 1.66 mg l1. All cultures were incubated on a rotatory shaker (100 rpm) at 30  C, for 7 days. Centrifuged culture supernatants (9000  g, 10 min, 4  C) were used to determine residual OPs using GC/ECD and release of chloride ions by a colorimetric assay (Phillips et al., 2001). Biomass

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Fig. 1. Sampling site of OP-contaminated soil in Santiago del Estero, Argentina.

was estimated by washing the pellets with 25 mM Tris-EDTA buffer (pH 8.0) and drying to constant weight at 105  C. 2.7. Gas chromatography Residual lindane, chlordane (cis-chlordane and trans-chlordane) and methoxychlor in cell-free supernatants were extracted by solid phase extraction (SPE) using a C18 column (Varian, Lake Forest, USA). Extracts were then quantified in a Gas Chromatograph (HewlettePackard 6890, Wilmington, DE) equipped with an HP 5

capillary column (30 m  0.53 mm  0.35 m) and 63Ni ECD detector, split/splitless injector HP 7694 and ChemStation Vectra XM software. Quantitative sample analysis was performed using appropriate calibration standards (ULTRA Scientific, North Kingstown, RI). 2.8. Colorimetric assay for dechlorination activity Cell-free supernatant samples were immediately used for indirect determination of the release of chloride ions using a modification of the procedure by Phillips et al. (2001), in which Phenol

M.S. Fuentes et al. / International Biodeterioration & Biodegradation 64 (2010) 434e441 Table 1 Organochlorine pesticides assayed in soil samples from Santiago del Estero, Argentina. Organochlorine pesticides

Concentration (mg g1)

Limit of quantification (LQ)

g-HCH HEPTACHLOR ALDRIN HEPTACLOR EPOXIDE A HEPTACHLOR EPOXIDE B DDE CHLORDANE DDD DDT METHOXYCHLOR

1.96 ND 0.06 ND 0.57 7.19 0.22 0.16 ND 0.46

0.007 0.007 0.001 0.003 0.003 0.007 0.003 0.007 0.007 0.007

437

Red Sodium Salt was added to 1 ml of supernatant at a ratio 1/10 as a pH indicator. A change in colour from red to orange to yellow in the presence of chloride in the supernatant was indicative of dechlorination of lindane, chlordane or methoxychlor and, therefore, a positive result. Culture medium with pH indicator was used as a blank. Chloride concentrations were determined colorimetrically at 540 nm using a Beckman spectrophotometer and compared with standard HCl solutions. 2.9. Statistical analysis A multivariate analysis of variance (MANOVA) was applied to the experimental data. Afterwards, the HotellingeBonferroni test was used to determine significant differences; data were A12 (GQ867056) A11 (GQ867055) A8 (GQ867053) S. odorifer DSM 40347 (NR_026535) S. xiamenensis MCCC 1A01550 (EF012099) A1 (GU085102)

Cluster I

A3 (GU085104) A2 (GU085103) A13 (GQ867057) A7 (GQ867052) A6 (GQ867051) M30 (GU085106) M15 (GQ867058) M50 (GQ867059) C39 (AY741282) A14 (GU085105) A5 (GQ867050) S. huangiae NRRL 8180 (EU170122)

Cluster II

S. aldersoniae NRRL 18513 (EU170123) S. zhihengliuii NRRL 11180 (EU170125) S. wellingtoniae DSM 40632 (EU170124) S. ascomycinicus DSM 40822 (EU170121) S. aureolacrimosus NRRL 5739 (EU170126) S. rimosus subsp. rimosus JCM 4667 (NR_024762) 75

S. hygroscopicus subsp. NRRL B-3822 (EU170118) S. platensis JCM 4662 (NR_024761)

71

S. venezuelae JCM 4526 (NR_024764) S. peucetius JCM 9920 (NR_024763) S. phaeochromogenes ATCC 3338 (EU594469)

79

S. hygroscopicus subsp. decoyicus NRRL 2666(EU170127)

100

A10 (GQ867054) M. saelicesensis type strain: Lupac 09T (AJ783993) N. farcinica DSM 43665 (AY756551)

99

Cluster III

N. asteroides DSM 43757 (AF430019)

87 89

M. aurum ATCC 23366 (FJ172298)

Fig. 2. Phylogenetic tree of the actinomycete strains isolated. Accession numbers of 16S rDNA sequences are given in parenthesis. Numbers at the nodes indicate the level of bootstrap support based on a neighbour-joining analysis of 40,000 resample datasets; only values more than 70% are given.

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considered different if P < 0.05. Principal components analysis (PCA) was used to simplify the interpretation of the results and was presented in biplot graphs. Each experiment was carried out in triplicate and the results were arithmetic means. Student’s t-test was used for statistical evaluation and one-way ANOVA for variance analysis. All statistical analyses were performed using a professional version of Infostat software.

0.30 0.25

Dry weight (g L- 1)

438

0.20 0.15 0.10 0.05 0.00

C3 9

S .co e lico lo r A 3

M5 0

M3 0

M1 5

S tre p to myce s M7

A13

A14

A11

3.4. Dechlorination activity To determine and compare the OP-degrading ability of the actinomycetes assayed, release of chloride ions into the culture medium was examined. The highest release determined for each pesticide was considered as 100%, the rest of the results were compared as relative percentages of chloride ion release for each strain (Fig. 5). Not all cell-free supernatants revealed chloride ions, although the microorganisms were able to remove OPs. Twelve of 1.4 1.2 1.0 0.8 0.6 0.4 0.2

S .co e lico lo r A 3

C3 9

M5 0

M3 0

M1 5

S tre p to myce s M7

A14

A13

A12

A11

A10

A8

A7

A6

0.0 -0.2

A5

Growth of the isolated actinomycetes with 1.66 mg l1 lindane, chlordane or methoxychlor was studied in liquid minimal medium (MM). Two criteria (dry weight and residual OP) were analysed according to Benimeli et al. (2006): the first was measured to establish the population behaviour of the microorganisms and the second for determination of pesticide removal. Dry weight in the presence of each OP varied between 0.02 and 0.09 g l1 with lindane, between 0.01 and 0.23 g l1 with chlordane and between 0.01 and 0.12 g l1 with methoxychlor (Fig. 3). Strains A2, A5, A6, A7 and A13 presented highest growth in the presence of chlordane. With lindane, the dry weight was relatively similar for all strains assayed, with a maximum value of 0.09 g l1 for A3, A5, A6, A8, A13

Methoxychlor

and Streptomyces M7. In the presence of methoxychlor strain A14 showed highest growth with a value of 0.12 g l1. Residual values of lindane and methoxychlor were 0.30e0.72 mg l1 and 0.01e1.20 mg l1, respectively. Cis and transchlordane, the two most abundant compounds in technical chlordane, were not detected with the method selected (Fig. 4); these compounds must have been removed from the culture medium or the residual concentration was below the detection limit of the method used (0.107 mg l1). All actinomycete strains assayed were able to remove the three OPs employed. In the presence of lindane, highest removal corresponded to M50 (82%). Most actinomycetes (A1, A2, A3, A5, A6, M15, M30, M50 and C39) could remove almost 100% of methoxychlor and use this pesticide as carbon and energy source.

A3

3.3. Screening of actinomycetes able to grow in the presence of lindane, chlordane or methoxychlor

Lindane

Fig. 3. Effect of lindane, chlordane and methoxychlor on the growth of the actinomycete strains assayed.

A2

Twelve indigenous colonies belonging to the actinomycetes group were isolated from the soil samples contaminated with OPs. The strains were identified as A1, A2, A3, A5, A6, A7, A8, A10, A11, A12, A13 and A14 and showed typical actinomycete characteristics such as vegetative mycelium and in some cases aerial mycelium and spore formation. Phylogenetic analysis was carried out to elucidate the taxonomic position of the 12 new isolates as well as four strains previously isolated in our laboratory (M15, M30, M50 and C39). 16S rDNA sequence was determined for all 16 strains and they were compared with the corresponding sequences of 19 culture collection strains (Fig. 2). All strains were classified into two clusters belonged to Streptomyces genus, except A10, which was classified into a separate cluster (III) and belonged to a different genus: Micromonospora. In cluster I, isolates A11, A12 and A8 were closely related to each other (99.1e99.5%) and to Streptomyces odorifer DSM 40347 (99.1e100%). Isolates A1, A2 and A3 were 100% related to Streptomyces xiamenesis MCCC 1A01550. Strains A6 and A13 were slightly less related 99.5% to S. xiamenesis MCCC 1A01550 and A7 only for 93.9%. In cluster II, isolates M30, M50 and C39 were 100% related to each other and 98.6% to Streptomyces rimosus subsp. rimosus JCM 4667. In cluster III, isolate A10 showed 100% similarity with Micromonospora saelicesensis Lupac 09T. The general G þ C content was 55.8e60.2 mol%, except strain A5 with 50.2 mol%.

Chlordane

A1

3.2. Isolation and molecular identification of actinomycetes

Strains

Residua l p estici de (mg L-1)

Chlorinated pesticides found in the soil samples are shown in Table 1. Lindane and DDE were the dominant contaminants. Although DDT was not detected, its metabolites (DDE and DDD) were present in significant quantities, indicating DDT degradation in these soils, which must have been contaminated a long time ago. Other OPs identified were chlordane, methoxychlor and aldrin, although at minor concentrations.

A12

A8

3.1. Analysis of OPs in soil samples from Santiago del Estero, Argentina

A10

A7

A6

A5

A2

-0.10

A3

3. Results

A1

-0.05

Strains Chlordane

Lindane

Methoxychlor

Fig. 4. Residual concentration of lindane, chlordane and methoxychlor in supernatants of culture medium.

M.S. Fuentes et al. / International Biodeterioration & Biodegradation 64 (2010) 434e441

A1 S.coelic. 100 M7 80 60 C39 40 20 M50 0 M30

M30, M50, C39 and S. coelicolor A3 (2) were related to chloride ion release. Most of the strains grown in the presence of lindane were positively related to residual pesticides and therefore lindane removal was low. Strains grown with methoxychlor principally released chloride ions.

A2 A3 A5 A6

Lindane Methoxychlor

A7

M15

4. Discussion

Chlordane

The soil samples analysed showed the presence of chlordane (0.22 mg g1), methoxychlor (0.46 mg g1) and lindane (1.96 mg g1). Zhang et al. (2009) studied two pesticide factories in Southeast China and measured chlordane and lindane concentrations of 8.43 mg Kg1 and 595 mg Kg1, respectively, which are about 26-fold (chlordane) and 3-fold (lindane) lower than our findings. The soil samples of the current study were obtained from an illegal OPs storage site, which could be the reason of the high pesticide concentrations. Soil pollution in the vicinity of pesticide deposits is typically dominated by mixtures of DDE and DDD, HCH isomers (especially g-HCH, or lindane) and methoxychlor. Naturally-occurring pesticide-degrading microorganisms may be relatively rare in pristine environments and non-exposed agricultural soils (Bartha, 1990). In the current study twelve indigenous actinomycetes were isolated from OP-contaminated soil, and the strains could have induced characteristics for growth in the presence of organochlorine pesticides. Therefore, the use of these wild type actinomycetes for bioremediation of soils is an attractive approach, since these microorganisms have already adapted to the habitat (Shelton et al., 1996). Pesticide-degrading actinomycetes belonging to Arthrobacter, Brevibacterium, Clavibacter, Corynebacterium, Micromonospora, Mycobacterium, Nocardia, Nocardioides, Rhodococcus and Streptomyces genera have been described previously (De Schrijver and De Mot, 1999). In Argentina, Santiago del Estero, actinomycetes isolated from soil belonged to Streptomyces and Micromonospora genera. 16S rDNA sequencing carried out in the current study confirmed that the actinomycetes isolated can be

A8

A14

A10 A13

439

A11 A12

Fig. 5. Percentage of chloride ions released into the culture medium by the actinomycete strains isolated in the presence of lindane, methoxychlor or chlordane.

the eighteen actinomycetes assayed for chlordane were able to release chloride ions, with A1 and A6 giving highest rates: 100% and 93.1%, respectively. Five isolates released chloride ions with methoxychlor: A3, A11, M15, M30 and M50. A3 and M30 showed the highest percentage: 100% and 21.96%, respectively. In the presence of lindane, chloride ions could only be detected with A14 (100%) and M50 (27.32%). 3.5. Principal component analysis (PCA) Fig. 6 explains 81.1% of the total variance of the data analysed. The first principal component (PC1) explains 48.5% of the total variance of data and groups the dry weight and chloride release on the right side of the chart and the residual pesticide on the left side of the chart. The second principal component (PC2) explains 32.6% of the total variance of data and is positively related to dry weight. Isolates A1, A2, A5, A6, A7, A12 and A13 were positively related to growth in the presence of chlordane. A3, A8, A10, A14, M7, M15,

3.5 Dry weight

3.0

C:A13

2.5 C:A2 C:A6

2.0

PC2 (32.6%)

1.5

C:A5

M:A14

Residual pesticide L:A6

1.0 M:A8

0.5 M:A11

0.0 M:A13

L:S. M7 L:A5 L:A13

L:A3 L:A2 L:A1 L:A12

M:A7

L:A8

M:A6

L:A14 L:A10 L:A7

M:S. coelicolor A3 M:A10

-0.5

L:C39

L:A11 L:S. coelicolor A3

C:A8

M:A12

M:A5

M:S. M7

C: S.M7

C:A3

M:M30 M:A2 M:M50

-1.5

M:M15

-2.0

-1.5

-1.0

-0.5

Chloride released

C:A14 M:A1

0.0

M:A3

C:C39

M:C39

C:A11

-2.5

C:A1

C:A12

L:M15

L:M30

L:M50

-1.0

-2.0 -3.0

C:A7

C:S. coelicolor A3 C:M30 C:M50 C:A10 C:M15

0.5

1.0

1.5

2.0

2.5

3.0

3.5

PC1 (48.5%) Fig. 6. Biplot obtained by principal component analysis of released chloride, residual pesticide and dry weight from pure cultures of actinomycete isolates grown in the presence of three organochlorine pesticides as sole carbon source. L: lindane, M: methoxychlor, C: chlordane.

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classified into several clusters. Three strains, A7, A14 and M15, are likely to be defined as new species based on their nucleotide similarity. Morphological, biochemical and physiological assaying is necessary to confirm this. Besides their potential metabolic diversity, Streptomyces strains may be well suited for inoculation in soil due to certain other characteristics: mycelial growth, their relatively rapid growth rate, colonization of semi-selective substrates and their ability to be genetically manipulated (Shelton et al., 1996). The Micromonospora genus is a prolific source of various bioactive metabolites such as antibiotics and enzyme inhibitors, second only to Streptomyces, belonging to the Actinomycetales order (Qiu et al., 2008). Members of Micromonospora are widely distributed in a variety of habitats, especially in soil rich in humus, and play an important role in the decomposition of organic matter. However, they do not usually constitute the majority of the actinomycete population (Qiu et al., 2008). The growth behaviour of the actinomycetes towards the pesticides assayed was strain-and OP-dependent. All current isolates were able to remove the three pesticides, but highest removal was obtained for chlordane, which was not detected in the culture supernatant. These results confirm previous findings by Benimeli et al. (2003), who found that 78 out of 93 wild type actinomycetes showed abundant growth in the presence of chlordane compared with other OPs such as lindane and methoxyclor. Elimination of halogens from halogenated xenobiotics is a key step in their degradation, because the carbon-halogen bond is relatively stable (Fetzner and Lingens, 1994). Nagata et al. (1999) determined that two different types of dehalogenases are involved in the early steps of g-HCH degradation by Sphingobium japonicum UT26: dehydrochlorinase and halidohydrolase. Because dehalogenation plays a central role in biodegradation of many chlorinated compounds, the current study assayed the release of chloride ions to assess microbial degradation of the three OPs. Manickam et al. (2008) used specific and rapid dechlorinase activity assays to screen bacteria from contaminated soils for HCH-degrading activity. They identified a bacterium able to grow on a-, b-, c- and d-HCH as sole carbon and energy source. Benimeli et al. (2006) reported on the release of chloride ions from lindane by a streptomycete strain. Cuozzo et al. (2009) demonstrated that synthesis of dechlorinase in Streptomyces sp. M7 was induced when the microorganism was grown in the presence of lindane as only carbon source. The current study showed the release of chloride ions in cell-free supernatants and highest dechlorination by the twelve actinomycete strains was obtained with chlordane as carbon source. The twelve strains showed a positive relationship with growth, chloride release and removal of chlordane in the presence of this pesticide and these findings were supported by PCA. 5. Conclusion Twelve actinomycetes, isolated from sites contaminated with organochlorine pesticides and belonging to the Streptomyces and Micromonospora genera, were able to grow in the presence of chlordane, lindane and methoxychlor. They were also able to remove these compounds from the culture medium or degrade them. These results favour application of actinomycetes as potential agents for bioremediation of polluted environments with different organochlorine pesticides. Acknowledgements The authors gratefully acknowledge financial support of CIUNT, ANPCYT and CONICET. They are also grateful for statistical assistance by Lic. Elena Bru de Lavanda.

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