Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 20058 16068Original ArticleHexachlorocyclohexane

degradersW. W. Mohn
et al.

Environmental Microbiology (2006) 8(1), 6068

doi:10.1111/j.1462-2920.2005.00865.x

Distribution and phylogeny of hexachlorocyclohexanedegrading bacteria in soils from Spain

William W. Mohn,1* Birgit Mertens,2 Josh D. Neufeld,1 Willy Verstraete2 and Victor de Lorenzo3 1 Department of Microbiology and Immunology, University of British Columbia, University Blvd., Vancouver, BC, V6T 1Z3, Canada. 2 Laboratory of Microbial Ecology and Technology, University of Ghent, 9000 Ghent, Belgium. 3 Centro Nacional de Biotecnologa, CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. Summary Hexachlorocyclohexane (HCH)-degrading bacteria are believed to mediate natural attenuation of HCH contamination and have potential for active bioremediation processes. This study addressed the very limited understanding of the distribution, diversity and substrate specicity of such bacteria from 13 soil samples, varying in levels of HCH contamination, from four sites in Spain. Hexachlorocyclohexane removal occurred in 16 of 36 enrichment cultures. Hexachlorocyclohexane-degrading populations were clearly associated with HCH-contaminated soils, and populations growing on the d-HCH isomer were only found in soil contaminated with d-HCH. bHexachlorocyclohexane was persistent in enrichment cultures, and there was no evidence for populations growing on b-HCH. From a- and g-HCH enrichment cultures, nine HCH-degrading isolates were obtained, which were all Sphingomonas spp. Attempts to isolate organisms from d-HCH enrichment cultures failed. None of the isolates grew on HCH as a sole organic substrate in pure culture. All isolates degraded a- and g-HCH, and most degraded b-HCH. dHexachlorocyclohexane inhibited growth of most isolates, but could be degraded by cell suspensions of at least four strains. Denaturing gradient gel electrophoresis indicated that the isolates represented predominant populations in the enrichment cultures, but additional predominant populations, including some Pseudomonas spp., could not be isolated. Introduction Hexachlorocyclohexane (HCH) remains a common pollutant despite the fact that its commercial use in most regions has been banned for two or more decades (Simonich and Hites, 1995). In particular, the g-isomer of HCH, lindane, was widely used as an insecticide and remains widely distributed in the biosphere. Other isomers, particularly a-, b- and d-HCH, were by-products of lindane production and occur at high concentrations in soil at specic locations where they were disposed. Despite its persistence, HCH can be biodegraded, and bioremediation is a potential solution to the challenge of cleaning up HCH-contaminated sites. There are numerous reports of HCH biodegradation in various environments, including aerobic and anaerobic soils (Doelman et al., 1985; Bachmann et al., 1988). Some studies suggest that HCH degraders were enriched in aerobic soils experiencing repeated application of lindane (Wada et al., 1989). However, the occurrence of HCH degraders in noncontaminated soils does not seem to have been systematically examined, and we lack a clear understanding of how HCH affects microbial communities in soil. Specically, it is unclear whether communities adapt to the contamination in any way that stimulates HCH biodegradation or otherwise reduces its harmful effects. Diverse HCH-degrading microorganisms have been reported, including fungi (Tu, 1976), cyanobacteria (Kuritz and Wolk, 1995) and anaerobic bacteria (MacCrae et al., 1969; Jagnow et al., 1977). However, only a few HCH degraders have been phylogenetically identied. These are aerobic Proteobacteria, including members of the genera Sphingomonas (formerly identied as Pseudomonas) (Senoo and Wada, 1989; Sahu et al., 1990), Rhodanobacter (Thomas et al., 1996; Nalin et al., 1999) and Pandoraea (Okeke et al., 2002). Additional aerobic HCH-degrading bacteria have been reported but not phylogenetically characterized (Tu, 1976; Gupta et al., 2000; 2001). Thus, very little is understood about the diversity of HCH degraders, and we do not know which organisms are responsible for any adaptation of soil communities to HCH contamination. Hexachlorocyclohexane degraders are typically grown in the laboratory on complex media, and it is questionable whether most HCH degraders use HCH as a sole organic substrate for heterotrophic growth. Alternatively, it is

Received 27 September, 2004; revised 25 March, 2005; accepted 28 March, 2005. *For correspondence. E-mail wmohn@interchange. ubc.ca; Tel. (+1) 604 822 4285; Fax (+1) 604 822 6041.

2005 Society for Applied Microbiology and Blackwell Publishing Ltd

Hexachlorocyclohexane degraders 61 possible that organisms use HCH simultaneously with other growth substrates or that HCH degradation is primarily a detoxication mechanism. Finally, it is unclear whether isolation procedures yield the various HCH degraders that can be enriched. It is possible that certain organisms have important roles in HCH degradation in soil and in mixed cultures but are incapable of growth under currently used conditions for axenic cultures. Thus, we do not know whether the organisms that have been studied to date are representative of HCH degraders in contaminated soils. The pathway for HCH degradation in Sphingomonas paucimobilis UT26 has been characterized in considerable detail (Nagata et al., 1999). Sphingomonas paucimobilis B90 has a number of lin genes encoding HCH degradation that are highly similar to those in UT26 (Kumari et al., 2002). Rhodanobacter lindaniclasticus was found to have a linA gene highly similar to those in the above Sphingomonas strains (Thomas et al., 1996), despite the fact that these genera are in different subdivisions of the Proteobacteria, a and g respectively. Although three Sphingomonas strains contain very similar lin genes, the genes were found to be associated with an insertion element and to vary substantially in gene arrangement and copy number (Dogra et al., 2004). Thus, the available evidence indicates horizontal transfer and rearrangement of the lin genes. This raises the question of how this genetic plasticity might contribute to the response of populations and communities to HCH contamination and to the environmental fate of HCH. A more complete understanding of the occurrence and characteristics of HCH degraders will help to better understand the environmental fate of HCH and may lead to practical technology for bioremediation of HCH contamination. The objectives of this study were: (i) to characterize the occurrence of HCH degraders in contaminated and uncontaminated soils, (ii) to evaluate whether enrichment of HCH degraders occurred in situ at contaminated sites, (iii) to determine which rRNA gene phylotypes are enriched in laboratory cultures and subsequently isolated and (iv) to compare isolates obtained with respect to phylogeny and the range of HCH isomers degraded. This is the rst report of a systematic examination of the occurrence of HCH degraders and of attempts to enrich and isolate microorganisms on b- and d-HCH.

Results and discussion Soil samples The soil samples varied greatly in levels of HCH contamination and presence of the various HCH isomers (Table 1). During sampling, HCH was apparent in all of the highly contaminated soils (>1 mg HCH per gram of soil). White HCH solids varying in size were visible and the odour of HCH was apparent. The three contaminated sites differed in composition of HCH isomers. Samples from Ansio had predominantly a-HCH. Those from Portugalete had relatively low total HCH concentrations, mainly a- and g-isomers. The contaminated samples from Artxanda mainly had high HCH concentrations, comprised of highly variable relative amounts of a-, g-, d- and e-isomers. All of the samples studied were from disturbed areas. In many cases, the soils contained rubble, such as broken tile and other foreign matter. The soil samples varied substantially in all of the characteristics measured (Table 1). Samples from Ansio (S1S3) all had visibly high clay content, while the remaining samples were silty. Samples from Artxanda (S10S16), unlike the other samples, were from an area with heavy plant cover and contained visibly high amounts of plant material. The Canto Blanco sample

Table 1. Characteristics of soil samples. Aerobic heterotrophs (cfu per gram dry soil) 1.44E+05 7.80E+06 3.08E+07 5.14E+05 9.20E+06 2.17E+06 1.07E+07 4.01E+06 1.82E+05 5.43E+05 6.61E+06 4.30E+06 Organic matter (% dry weight) 3.5 4.4 7.37 3.96 3.27 21.2 9.1 31.8 89.4 96.4 18.6 31.9

Sample S1, Ansio S2, Ansio S3, Ansio S4, Ansio S5, Portugalete S10, Artxanda S11, Artxanda S12, Artxanda S13, Artxanda S14, Artxanda S15, Artxanda S16, Artxanda

Sample depth (cm) 1020 3545 2535 90100 010 315 010 08 010 010 010 010

a-HCH (mg g-1) 15 500 158 5 920 3 580 4.36 ND ND 0.17 6 160 1 270 1 900 17.7

b-HCH (mg g-1) 140 108 131 144 4.19 0.01 ND 0.01 17.3 793 263 7.81

g-HCH (mg g-1) 447 0.87 43.9 15.3 0.08 ND ND 0.03 6110 298 98.6 1.01

d-HCH (mg g-1) 73.7 0.5 7.7 1.2 0.05 0.02 0.01 0.04 5 520 9 750 18 300 18.0

e-HCH (mg g-1) 77.2 8.72 21.9 14.2 0.27 ND ND 0.06 780 1438 1224 8.7

Total HCH (mg g-1) 16 200 276 6 130 3 750 8.95 0.03 0.01 0.40 18 600 13 500 21 800 53.3

Fungi (cfu per gram dry soil) 3.75E+03 1.94E+04 4.26E+03 3.51E+03 2.20E+04 7.43E+03 9.31E+03 6.41E+03 1.75E+05 4.18E+04 4.63E+04 1.05E+04

pH 7.8 7.9 7.8 5.9 8.4 5.4 8.1 7.6 2.9 3.6 4.4 7.2

cfu, colony-forming units; ND, not detected. 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 6068

62 W. W. Mohn et al. (S0) was not characterized like the other samples. This sample, serving as an uncontaminated control, consisted of dark, silty soil. Among the soils, there was a negative correlation (r = -0.62, P < 0.01) between pH and HCH concentration and a strong and signicant negative correlation (r = -0.81, P < 0.0001) between pH and organic matter content. Release of protons during HCH biodegradation may have contributed to low pH in contaminated soils, but high organic content seems a more important factor contributing to low pH. Particularly extreme samples were S13 and S14, which had very low pH values, very high organic content and among the highest HCH concentrations. As has been previously observed (Bth and Anderson, 2003), acid soil conditions appeared to select for fungal populations, as there was a very signicant negative correlation (r = -0.66, P < 0.005) between pH and fungal populations. There was a less signicant positive correlation (r = 0.52, P < 0.05) between pH and aerobic heterotrophs (which are presumably bacteria). Occurrence of HCH degraders Of 36 enrichment cultures varying in soil inocula and in HCH isomers used as sole organic substrates, HCH was removed in 16 (Table 2). All of these 16 cultures were transferred to secondary cultures in which HCH was also removed, indicating growth of organisms on the corresponding HCH isomers. Enrichment results suggest that HCH degraders are rare in soils not contaminated with HCH and that contamination selects for HCH-degrading populations in situ. None of the three soils without substantial HCH (S0, S10, S11) yielded an HCH-degrading enrichment culture. On the other hand, HCH-degrading enrichment cultures resulted from seven of nine HCHcontaminated soils (Table 2). Further, the only two contaminated soils that did not yield HCH degraders were S13 and S14, which had extremely low pH values, high HCH concentrations and low total heterotroph densities (Table 1). If acidication of these soils was a result of HCH degradation, the process may have become self-limiting. Buffering of soil pH might be an important means to promote in situ HCH biodegradation. Interestingly, the only two enrichment cultures in which d-HCH was removed were inoculated with Artxanda soil, which had high proportions of the d-isomer, relative to the other isomers. This suggests that the presence of d-HCH selected for population(s) that can degrade d-HCH. Enrichment success varied considerably with the different HCH isomers. The most persistent isomer was b-HCH, which was not degraded in any of six cultures (Table 2). This suggests that organisms capable of growth on bHCH are rare or require growth factors not present in the mineral medium used. The b-isomer of HCH is typically more persistent than other isomers in natural environments as well (Tatsukawa et al., 1972), which is generally attributed to the relative chemical stability of this isomer. There does not appear to be conclusive evidence for microbial growth on b-HCH, despite two reports making this claim. In one case the medium contained a high concentration of Casamino acids relative to the concentration of b-HCH (Johri et al., 1998). In the second case, the evidence for growth was optical density, but there were no data showing that optical density had increased and no control showing that the optical density did not result from the insoluble b-HCH in the medium (Siddique et al., 2002). Additionally, d-HCH was relatively persistent in our enrichment cultures (Table 2), being degraded in only two of seven cultures. Results with a- and g-HCH were very similar. A majority of soils yielded organisms that degraded both these isomers, and most of the soils that failed to yield a-HCH degraders also failed to yield g-HCH degraders. These observations suggest that there are frequently occurring populations capable of growth on both a- and g-HCH, but not on b- and d-HCH under conditions in the enrichment cultures. Isolates Secondary enrichment cultures that degraded HCH were streaked on solid medium containing the corresponding HCH isomer, and very small (<1 mm diameter) colonies

Table 2. Outcome of enrichment cultures. HCHs used for enrichment a Soil S0 S1 S2 S3 S4 Canto Blanco Ansio Ansio Ansio Ansio a ND a1-2 ND ND a4-2 a4-5 + + b ND ND ND ND ND g g1-7 + + + + g12-7 g12-8 g12-10 + g16-1 g16-7 g16-9 d ND ND ND ND ND ND -

S5 Portugalete S10 Artxanda S11 Artxanda S12 Artxanda

S13 S14 S15 S16

Artxanda Artxanda Artxanda Artxanda

+ a16-10 a16-12

ND ND -

+ +

a. ND indicates enrichment not performed with the specied soil and HCH isomer; - indicates enrichment not successful (no HCH removal from primary enrichment culture in excess of removal in uninoculated control after 3-month incubation); + indicates enrichment successful (HCH removal in primary and secondary cultures) but no isolate obtained; otherwise, strain designations are given for isolates obtained from the specied culture.

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Hexachlorocyclohexane degraders 63 formed very slowly (>2 weeks). Putative HCH-degrading isolates were indicated by clearing of precipitated HCH in zones around the colonies. Many of the isolates failed this test and were likely growing on the agar, organic contaminants in the agar or volatile organic matter in the air. Many colonies that cleared HCH failed to grow in subsequent transfers, suggesting that they had nutritional requirements provided by other organisms in the enrichment cultures. Multiple HCH-clearing isolates were obtained from enrichment cultures whenever possible, and when these isolates differed in cell or colony morphology, representatives of each discernible type were maintained. Additional efforts to isolate HCH degraders involved streaking enrichment cultures on solid medium with HCH plus 10% LB medium, but these strains never had HCH degradation activity. A total of nine HCHclearing isolates were obtained from six of the 16 enrichment cultures, and these were maintained and characterized. Notably, isolates were only obtained on aand g-HCH, and none were obtained from the two enrichment cultures that degraded d-HCH. Organisms appear to have grown on d-HCH in the enrichment cultures, but they did not grow as readily in pure culture as did organisms enriched on a- and g-HCH. All of the isolates obtained are members of the genus, Sphingomonas, on the basis of their 16S rRNA gene sequences (Fig. 1). The sequences of ve isolates from Artxanda soil all cluster together (>99% identical) and are closely related (>98% identical) to S. paucimobilis UT26. The sequences of three isolates obtained on a-HCH from Ansio soil form a second cluster (>99% identical), and the sequence of the remaining isolate obtained on g-HCH
Table 3. Per cent removal of HCH isomers by nine bacterial isolates during growth on 10% LB broth during 8-day incubations. Strain a1-2 a4-2 a4-5 g1-7 a16-10 a16-12 g12-7 g16-1 g16-9 a-HCH 86 96 36 82 95a 49 100 17 15 b-HCH 21 72 45 87 9 33 3 10 33 g-HCH 100 63 16 100 84a 37 69a 25 13 d-HCH 4a 99 1a 66 2a 2a 3a 0a 1a

a. Hexachlorocyclohexane apparently inhibitory cultures not visibly turbid by the end of the incubation period.

from Ansio soil was distinct from the other isolates. Thus, despite efforts to obtain isolates with different cell or colony morphologies and particularly to obtain relatively slow-growing strains, the isolates had very little phylogenetic diversity. While distinct populations were enriched and isolated from the different sites, all of the isolates were members of the genus Sphingomonas, like the majority of HCH degraders identied in other studies (Senoo and Wada, 1989; Sahu et al., 1990). All of the nine isolates removed HCH while growing on a complex, rich medium (10% LB broth). Therefore, HCH degradation by Sphingomonas spp. is unlikely to be repressed by sugars or other readily used substrates. Most of the isolates removed a-, b- and g-HCH (Table 3). All but two isolates failed to degrade d-HCH, but this resulted from inhibition of growth, rather than the inability to transform d-HCH. Individual suspensions of four iso-

Fig. 1. Neighbour-joining tree of the full-length 16S rDNA sequences of nine HCH-degrading isolates. In addition to Sphingomonas paucimobilis strain UT26 (Accession No. AF039168), sequences with the highest BLAST hit in GenBank for each of the isolates were included in the alignment. The tree was rooted with the 16S rDNA sequence of Rhodanobacter lindaniclasticus (L76222) strain 1021. Letter codes on the right indicate the clusters with identical 132 bp regions corresponding to the DGGE band sequences and match the band letter codes in Fig. 2. 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 6068

64 W. W. Mohn et al. lates (a1-2, a4-2, a4-5 and g1-7), previously grown on 10% LB plus a-HCH, were all able to completely remove 50 mg l-1 d-HCH in 24 h (not shown). None of the cell suspensions, isolate cultures or enrichment cultures yielded detectable HCH metabolites, although the analytical method used would not be expected to detect polar metabolites. The present study and other reports (Sahu et al., 1990; Johri et al., 1998; Siddique et al., 2002) suggest that it is typical for HCH-degrading Sphingomonas spp. to degrade multiple HCH isomers. It is important to note that the HCH removal observed here and in other studies does not equate to complete degradation. In one study, mineralization of a-, b- and gHCH by a Sphingomonas sp. ranged from only 5% to 12% (Sahu et al., 1995). The isolates in the present study differed substantially in their abilities to remove the four HCH isomers tested (Table 3). In almost all cases, the relative ability of each strain to remove a- and g-HCH was similar. Whereas, removal of b-HCH was more variable, and three strains failed to substantially remove that isomer (removed less than 10%). Within both the phylogenetic clusters (Fig. 1), individual strains differed substantially in HCH degradation ability, indicating important catabolic differences between closely related organisms. Differences in the arrangements and copy numbers of lin genes (Dogra et al., 2004) may account for the observed variability of HCH degradation capacity within closely related strains. None of the isolates from the present study grew in dened liquid medium with HCH as a sole organic substrate. Aerobic growth of a Sphingomonas sp. on g-HCH as a sole organic substrate has been demonstrated (Sahu et al., 1990). However, such use of HCH has not been rigorously demonstrated for most HCH degraders. The ability to enrich aerobic HCH degraders in the present and other studies strongly suggests that HCH is used as a source of reductant and carbon. The question remains as to whether most HCH degraders have additional nutritional requirements that are not provided by typical mineral media. Enrichment and isolation of denaturing gradient gel electrophoresis (DGGE) phylotypes Consistent trends were observed in the denaturing gradient gel electrophoresis (DGGE) ngerprints of the soils, enrichment cultures and isolates. In most cases, soil DNA ngerprints were faint smears (Fig. 2), which suggests very diverse bacterial communities lacking predominant phylotypes. Soil S1 was an exception, as its ngerprint contained a single, very dense band (band C). This band was excised and sequenced (132 bp), and its sequence was most similar to, but not identical to that of Sphingomonas sp. strains in cluster D (Fig. 1). This band appears to represent a predominant population, possibly selected by the HCH contamination in the soil. Interestingly, this population was not selected in the enrichment culture from that soil. The ngerprints reected selection for successively fewer populations in the sequential enrichment cultures. After incubation, each primary enrichment culture had a ngerprint with 512 discreet bands, and each secondary enrichment culture had fewer bands than the corresponding primary culture (Fig. 2). Consistent with enrichment, nearly all of the bands in the secondary cultures matched bands visible in the corresponding primary culture. Each isolate yielded a single DGGE band matching one of a few predominant bands in the corresponding secondary enrichment culture (bands B, D or E). Sequence analysis indicated that the bands from enrichment cultures were identical to the corresponding regions from the respective isolates. Thus, highly enriched populations were the ones ultimately isolated on solid medium. However, despite isolation of many strains from each secondary enrichment culture, isolates corresponding to only a single DGGE phylotype were obtained from each enrichment culture. Several of the enriched populations, evident as DGGE phylotypes, were not obtained in pure culture. The phylotypes not isolated may have been substantially less abundant (10-fold or more) than the ones isolated or may be incapable of growing on HCH (e.g. may grow on metabolites generated by another organism). Members of the genus Sphingomonas seem to be the HCH degraders most readily isolated from many soils, but our results suggest that there are additional populations with an important, unidentied role in HCH-degrading consortia. The DGGE ngerprints of the enrichment cultures and the phylogeny of the isolates obtained were clearly a function of both the sites where soil was sampled and the HCH isomers were used as substrates. The two soils from Ansio (S1, S4) enriched with a-HCH yielded highly similar ngerprints with many common bands (Fig. 2), and the three isolates from these enrichment cultures had tightly clustering rRNA gene sequences (Fig. 1). One of these soils (S1) enriched with g-HCH yielded an entirely different ngerprint and an isolate with an rRNA gene sequence distinct from all the other isolates. Similarly, the two soils from Artxanda (S12, S16) enriched with g-HCH yielded highly similar ngerprints with many common bands, which were distinct from the ngerprint of one of those soils (S16) enriched with a-HCH. All of the isolates from Artxanda soil had tightly clustering rRNA gene sequences, despite the differing ngerprints of the a- and g-HCH cultures from which they were obtained. Each of the two groups of multiple isolates tting into two rRNA gene sequence clusters yielded a single, common DGGE band (D and E). Interestingly, predominant upper bands of the same mobility (A) were common to the cultures from

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Hexachlorocyclohexane degraders 65
Fig. 2. Normalized (Bionumerics) DGGE gel for bacterial 16S rDNA gene fragments of original soil DNA, enrichment culture DNA and isolate DNA. Lanes are grouped according to the soil on which the enrichment was started. S indicates soil samples (e.g. S1). Enrichment cultures are identied by HCH isomer, soil inoculum number and primary or secondary cultures (e.g. a1-1). Isolates are identied by HCH isomer for enrichment, soil inoculum number and number of isolate (e.g. a1-2). Band letter codes on the left match the cluster letter codes in Fig. 1. Black triangles indicate bands that were excised and sequenced.

D E

Ansio and Artxanda soil. Sequence analysis indicated that these bands (A) in the a1, a4 and g16 enrichments were afliated with the genus Pseudomonas, and so, these bands may represent a population common to these soils that could be enriched but could not be isolated. The corresponding band (A) in the g12 enrichments was afliated with uncultured b-Proteobacteria. An additional lower band (F) from the g16 enrichment was afliated with the genus Methylobacterium. The above results indicate that geographically distant sites (>10 km) contain distinct bacterial populations capable of enrichment and isolation on HCH. Within these sites (within 10100 m) the soils share common bacterial populations capable of enrichment and isolation on HCH. Although all of the isolates can degrade both a- and gHCH, these two isomers select for different populations that likely differ in the relative efciency with which they can use the isomers and may differ in tolerance of inhibition by the two isomers. Important questions about HCH-degrading bacteria remain. In particular, it would be useful to determine the

relative importance of members of Sphingomonas and the possible role of uncultured microorganisms to in situ HCH degradation. Further, it would be useful to elucidate the nutritional requirements for members of Sphingomonas growing on HCH. Experimental procedures Soil samples
Soil samples were obtained from three HCH-contaminated sites near Bilbao in northern Spain and from one uncontaminated site in Canto Blanco in the region of Madrid. Samples were taken with an Endelman coring device or a stainless steel shovel, both surface-sterilized with alcohol, from specied depths (Table 1). The single sample from Canto Blanco was taken from a depth of 05 cm from a grass lawn at the Universidad Autonoma de Madrid. In the eld samples were divided for microbiological analyses and soil analyses. Samples for microbiological analyses were transported to the laboratory in sterile plastic bottles on ice and homogenized thoroughly, with subsamples for DNA extraction frozen at -80C and subsamples for inoculating enrichment cultures

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66 W. W. Mohn et al.
stored at 4C. Samples for soil analyses (analyses shown in Table 1) were transported to GAIKER, Centro Technologico, a certied commercial laboratory, and analysed using standard methods. Organic matter was determined by combustion. Aerobic heterotrophs were determined on Plate Count Agar (Oxoid) incubated at 30C for 48 h. Fungi were determined on Sabouraud agar with chloramphenicol (Oxoid) incubated at 25C for 96 h.

Denaturing gradient gel electrophoresis

DNA was extracted from soil samples and enrichment cultures using Bio 101 Soil DNA Extraction kits (Q-Biogene) according to the manufacturers instructions. Solids including cells were recovered from 10 ml samples of enrichment cultures by centrifugation at 3220 g for 20 min at 4C. Pellets were stored at -80C until DNA extraction. DNA was extracted from pure cultures by boiling (10 min). All DNA was stored at -20C. General polymerase chain reaction (PCR) primers targeting all bacteria (P388fP518r) were used (Ovreas et al., 1997). The nal concentrations of the different components in the mastermix were: 0.2 mM of each primer, 200 mM of each deoxynucleoside triphosphate, 1.5 mM MgCl2, 1 thermophillic DNA polymerase 10 reaction buffer (MgCl2 free), 1.25 U/50 ml of Taq DNA polymerase (Promega, Madison, WI, USA), 400 ng ml-1 bovine serum albumin (Hoffman-La Roche, Basel, Switzerland) and DNase and RNase free lter sterilized water (Sigma-Aldrich Chemie, Dteinheim, Germany). To 48 ml of PCR mastermix, 2 ml of DNA template was added. After each PCR, the size of the amplication product was veried on a 1.2% agarose gel. Denaturing gradient gel electrophoresis was performed using a DCode System (Bio-Rad, Hercules, CA, USA) as described previously (Muyzer et al., 1993; El-Fantroussi et al., 1999). Briey, the above amplicons were loaded (10 ml) onto 8% (w/v) polyacrylamide gels in 1 TAE [20 mM Tris, 10 mM acetate, 0.5 mM EDTA (pH 7.4)]. The gels had a denaturing gradient ranging from 45% to 60%. Electrophoresis was for 16 h at 60C at 38 V. After electrophoresis, the gels were soaked for 20 min in SYBR green I nucleic acid gel stain (1:10 000 dilution; FMC BioProducts, Rockland, ME, USA). The stained gel was immediately photographed on an UV transillumination table with a video camera module (Vilbert Lourmat, Marne-la Vall, France). Denaturing gradient gel electrophoresis bands selected for sequencing were excised from the gel and placed in 20 ml of sterile water. To verify their purity, these bands were reamplied (as above) and the amplicons analysed by DGGE. Amplicons were additionally puried with a Wizard Genomic DNA purication kit (Promega, Madison, WI, USA) and subsequently sequenced by ITT Biotech (Bielefeld, Germany). Partial sequences of approximately 132 bp (primer sequences removed) were aligned to 16S rRNA gene sequences obtained from the National Center for Biotechnology Information database by using BLAST (version 2.0).

Cultures
All cultures were grown on PAS mineral salts (Bedard et al., 1986) with an initial pH of 7.0 and incubated at 30C. Liquid cultures were incubated on a rotary shaker at 200 r.p.m. Solid medium contained 1.5 g of puried agar (USB) per litre and was in standard Petri dishes. Hexachlorocyclohexane was added to solid medium in 5 ml overlays containing 300 mg of HCH per litre. Hexachlorocyclohexane isomers were from Dr Ehrenstorfer, GmbH with the following purities: a, 99.5%; b, 98%; d, 99%; g, 99%. Hexachlorocyclohexane was added to solid or liquid media from stock solutions of 20 g of HCH per litre of lter-sterilized DMSO, with the exception of enrichment cultures, to which HCH was added directly as crystals (to avoid DMSO as a possible substrate). Uninoculated control enrichment cultures proved that the HCH reagents did not contain microorganisms capable of growth on HCH. Growth on HCH was evaluated by microscopy, based on the number of cells per eld of view. Enrichment cultures were in 250 ml asks containing 100 ml of PAS plus 100 mg of a specied HCH isomer per litre. Primary enrichment cultures were inoculated with 1.0 g of a specied soil, and secondary cultures, 100 ml of the corresponding primary culture. After isolation, strains were grown on solid or liquid media with 10% LB broth (Ultrapure, USB) in addition to HCH. To measure HCH removal by isolates, cultures were grown in glass tubes on 1.5 ml of PAS medium (15 ml of inoculum) with 10% LB plus 100 mg l-1 of the indicated HCH isomer. Percent removal of HCH was determined relative to the amount remaining in uninoculated control cultures. Each test was performed at least twice.

Hexachlorocyclohexane analyses
Soil HCH concentrations were measured by GAIKER, Centro Technologico, a certied commercial laboratory, using a standard method. Briey, that method involves soil extraction by sonication with hexane-acetone and analysis with a gas chromatograph-electron-capture detector. To measure HCH removal in cultures, the cultures were thoroughly mixed to suspend particles, and 0.50 ml samples were removed to microfuge tubes. Samples were extracted twice with 0.50 ml of ethyl acetate. Hexachlorobenzene (50 mg l-1) was added as an internal standard. Samples were analysed by gas chromatography-mass spectroscopy. Hexachlorocyclohexane removal was monitored weekly in enrichment cultures, which were considered positive for HCH removal when they had less than 10% of the HCH concentration measured in simultaneously incubated, uninoculated control cultures.

DNA sequence analysis

In order to obtain full length 16S rDNA sequences from HCHdegrading isolates, colony PCR was performed as described previously (Neufeld et al., 2004) but with primer set 27f-1492r (Lane, 1991). The PCR amplicons were cloned using the TOPO-TA cloning kit (Invitrogen). For each isolate, plasmids from three transformant colonies were extracted using a plasmid purication kit (Qiagen). Sequencing reactions and gels were prepared as previously described (Neufeld et al., 2004). All plasmids were sequenced once with primer 907r (5-CCG TCA ATT CMT TTG ACT TT) to verify that for each isolate, identical sequences were obtained from transformants. The

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complete 16S rRNA gene was sequenced from one plasmid for each isolate using plasmid specic primers, -21 M13f (5TGTAAAACGACGGCCAGT) and M13r (5-CAGGAAACAGC TATGACC), and the gap was lled between the sequences using the sequence data from 907r. For multiple fold coverage and additional condence, inserts were also sequenced with primers 27f and 1492r. Isolate full-length sequences were aligned in CLUSTALX (Thompson et al., 1997) with default settings. A neighbourjoining phylogenetic tree was generated with 100 bootstraps using TREECON for Windows 1.3b (Van de Peer and De Wachter, 1994). All alignment positions were used without taking insertions and deletions into account while calculating JukesCantor distance. The ~130 bp DGGE band sequences and correspondingly cropped rRNA sequences from isolates and reference strains were aligned in CLUSTALX before generating an unrooted phylogenetic tree as described above but without bootstrapping. Sequences were deposited in GenBank for all isolates (AY771794AY771802), other enrichment bands (AY771804AY771806), and for S1 soil band C (AY771803). Gupta, A., Kaushik, C.P., and Kasushik, A. (2000) Degradation of hexachlorocyclohexane (HCH; alpha, beta, gamma and delta) by Bacillus circulans and Bacillus brevis isolated from soil contaminated with HCH. Soil Biol Biochem 32: 18031805. Gupta, A., Kasushik, C.P., and Kasushik, A. (2001) Degradation of hexachlorocyclohexane isomers by two strains of Alcaligenes faecalis isolated from a contaminated site. Bull Environ Contam Toxicol 66: 794800. Jagnow, G., Haider, K., and Ellwardt, P.-C. (1977) Anaerobic dechlorination and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria. Arch Microbiol 115: 285292. Johri, A.K., Dua, M., Tuteja, D., Saxena, R., Saxena, D.M., and Lal, R. (1998) Degradation of alpha, beta, gamma and delta-hexachlorocycohexanes by Sphingomonas paucimobilis. Biotechnol Lett 20: 885887. Kumari, R., Subudhi, S., Suar, M., Dhingra, G., Raina, V., Dogra, C., et al. (2002) Cloning and characterization of lin genes responsible for the degradation of hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain B90. Appl Enivron Microbiol 68: 60216028. Kuritz, T., and Wolk, C.P. (1995) Use of lamentous cyanobacteria for biodegradation of organic pollutants. Appl Environ Microbiol 61: 234238. Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics. Stackebrandt, E., and Goodfellow, M. (eds). Chichester, UK: John Wiley & Sons, pp. 115175. MacCrae, I.C., Raghu, K., and Bautista, E.M. (1969) Anaerobic degradation of the insecticide lindane by Clostridium sp. Nature 221: 859860. Muyzer, G., de Waal, E.C., and Uitterlinden, A. (1993) Proling of complex microbial populations using denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplied genes coding for 16S rRNA. Appl Enivron Microbiol 59: 695700. Nagata, U., Miyauchi, K., and Takagi, M. (1999) Complete analysis of genes and enzymes for g-hexachlorocyclohexane degradation in Spingomonas paucimobilis UT26. J Indust Microbiol Biotech 23: 380390. Nalin, R., Simonet, P., Vogel, T.M., and Normand, P. (1999) Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium. Int J Syst Bacteriol 49: 19 23. Neufeld, J.D., Yu, Z., Lam, W., and Mohn, W.W. (2004) Serial analysis of ribosomal sequence tags (SARST): a highthroughput method for proling complex microbial communities. Environ Microbiol 6: 131144. Okeke, B.C., Siddique, T., Camps Arbestain, M., and Frankenberger, W.T. (2002) Biodegradation of g-hexachlorocyclohexane (Lindane) and a-hexachlorocyclohexane in water and a soil slurry by a Pandoraea species. J Agric Food Chem 50: 25482555. Ovreas, L., Forney, L., Daae, F.L., and Torsvik, V. (1997) Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplied gene fragments coding for 16S rRNA. Appl Enivron Microbiol 63: 33673373. Sahu, S.K., Patnaik, K.K., Sharmila, M., and Sethunathan, N. (1990) Degradation of alpha-, beta-, gamma-hexachloro-

Acknowledgements
We thank Iaki Gorostiza and Ana Isabel Diaz of GAIKER for managing the soil sampling and providing the soil analysis data. This research was partially funded by EU contract QLK3-CT-2002-01933 (LINDANE) of the 5th Framework Programme.

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