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Arch Microbiol. 1999 Feb;171(3):189-97

Diversity of chlorophenol-degrading  bacteria isolated from contaminated  boreal groundwater

Minna K. Männistö, Marja A. Tiirola, Mirja S. Salkinoja-Salonen, Markku S. Kulomaa, J. A. Puhakka

Abstract

Chlorophenol-degrading bacteria from a long­term polluted groundwater aquifer were characterized. All isolates degraded 2,4,6-trichlorophenol and 2,3,4,6-tetra­chlorophenol at concentrations detected in the contami­nated groundwater (< 10 mg l^–1). Pentachlorophenol was degraded by three isolates when present alone. In two gram-positive isolates, 2,3,4,6-tetrachlorophenol was re­quired as an inducer for the degradation of pentachlo­rophenol. The gram-positive isolates were sensitive to pentachlorophenol, with an IC₅₀ value of 5 mg/l. Isolates belonging to the Cytophaga/Flexibacter/Bacteroides phy­lum had IC₅₀ values of 25 and 63 mg/l. Isolates belonging to a-, (3- and y-Proteobacteria generally tolerated the high­est pentachlorophenol concentrations (> 100 mg/l). Poly­chlorophenol-degrading capacity was found in strains of Nocardioides, Pseudomonas, Ralstonia, Flavobacterium, and Caulobacter previously not known to degrade poly­chlorophenols. In addition, six polychlorophenol-degrad­ing sphingomonads were found.

Key words Bacterial diversity · Chlorophenol degradation · Groundwater · Toxicity

Introduction

A mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetra­chlorophenol (TeCP), and pentachlorophenol (PCP) was used for several decades for wood protection in Finland. This has led to severe groundwater contamination around sawmills (Kitunen et al. 1987). Chlorophenol degradation and bioremediation by mixed and pure bacterial cultures have been thoroughly investigated [for reviews see, e.g. Haggblom and Valo (1995), McAllister et al. (1996), and Puhakka and Melin (1996, 1998)]. Gram-negative and gram-positive polychlorophenol-degrading bacterial species of the genera Mycobacterium (Haggblom et al. 1994), Rhodococcus (Briglia et al. 1996), Streptomyces (Golovleva et al. 1992), and Sphingomonas (Nohynek et al. 1995) have been described.

Subsurface microbial ecology has systematically been studied only since the 1980s, after the development of sampling and laboratory techniques. These studies have accumulated evidence of vast microbial diversity in the subsurface environments [for a review, see Dobbins et al. (1992)]. Contamination may selectively enrich for specific degraders, or lead to genetic changes or induction of spe­cific enzymes in polluted sites (Van Der Meer et al. 1992). High contaminant concentrations may be toxic to the mi­croorganisms. These questions are of great importance when considering in situ biodegradation and bioremedia­tion of the contaminants, but they have been studied little so far.

In Karkola, in southern Finland, a large-scale chloro­phenol contamination was observed in 1987. Groundwa­ter adjacent to a sawmill was found to contain up to 190 mg of chlorophenols ^l–1 (Lampi et al. 1990). The stratifi­cation of chlorophenols in sediments of a nearby lake re­ceiving a flow of groundwater indicated that chlorophenol pollution had been occurring for at least 25 years (Lampi et al. 1992). This groundwater aquifer offered an opportu­nity for studying the responses of a groundwater micro­bial community to a severe, long-term exposure to chloro­phenols.

We analyzed bacteria in this groundwater and found that the ability to degrade chlorophenols was common in the aquifer. Chlorophenol-degrading bacterial isolates were characterized and identified by 16S ribosomal RNA gene restriction fragment length polymorphism (RFLP) pat­terns, partial 16S rRNA gene sequencing, whole-cell fatty acid patterns, G+C content of DNA, tolerance to PCP, and capacity to degrade 2,4,6-TCP, 2,3,4,6-TeCP, and PCP. Materials and methods

Chemicals and media

2,4,6-TCP (99% pure) was obtained from Fluka Chemie (Buchs, Switzerland), 2,3,4,6-TeCP (80% pure with 20% PCP) was a prod­uct of Tokyo Kasei Kogyo (Tokyo, Japan), and PCP (99% pure) was from Dow Chemical (USA).

The bacteria were isolated on peptone yeast extract glucose agar containing 0.025% w/v of each, 5 ml of vitamin solution ^l–1 and 1.5% agar (PYGV) medium (Staley 1968), maintained in PYGV or R2 A-agar (Reasoner and Geldreich 1985), and stored in liquid nitrogen. Whole-cell fatty acids were analyzed from cells grown on trypticase soy broth agar containing 3% w/v of trypticase soy broth and 1.5% agar (TSBA) (BBL, Becton Dickinson, Cockeys­ville, Md., USA) or double-strength PYGV medium. Chlorophenol degradation was tested in DSM 465 mineral salts medium contain­ing (g/l): Na2HPO4 · 2 H₂O (3.5), KH₂PO₄ (1.0), (NH4)2SO4 (0.5), MgCl2 · 6 H₂O (0.1), Ca(NO3)2 · 4 H₂O (0.05), EDTA (0.0005), and trace elements ( g/l): FeSO4 · 7 H₂O (200), ZnSO4 · 7 H₂O (10), MnCl2 · 4 H₂O (3), _H3BO3 (30), CoCl2 · 6 H₂O (1), NiCl2 · 6 H₂O (2), and Na₂MoO₄ · 2 H₂O (pH 7.2).

Isolation of bacteria

 Highly contaminated groundwater (10 mg of total chlorophenols l^–1; 75% 2,3,4,6-TeCP, 15% 2,4,6-TCP, and 10% PCP) was sam­pled from a contaminated aquifer (Kark6la, Finland). Samples were collected from a pumping well that pumps groundwater to an on-site biological treatment system (Puhakka and Melin 1998) via a sampling port inside the treatment plant into sterile bottles trans­ported on ice to the laboratory and processed within 24 h. Serial di­lutions of groundwater samples were spread-plated on PYGV agar. After 4 weeks at 20°C and at groundwater temperature (8°C), the colonies were counted; 50 colonies were randomly picked from the plates incubated at 8°C, and 52 colonies from those incubated at 20°C. The purity of the isolates was achieved by replating at least ten times on PYGV agar.

Phenotypic characterization

The strains grown on PYGV or R2A medium for 3–7 days were gram stained using the Hucker method (Gerhardt et al. 1994) and were examined under an Olympus BH-2 light microscope. Live cells were examined as wet mounts using phase-contrast microscopy.

Whole-cell fatty acid analysis

Cells were grown on PYGV agar at 20°C for 6 days. Fatty acid methyl esters were extracted and analyzed as described by Vaisa­nen et al. (1994). Microbial identification system (library version 3.9; MIDI, Newark, Del., USA) was used for identification of the isolates grown on TSBA.

DNA extraction

Isolates were grown in PYGV medium to the stationary phase; cells were collected by centrifugation, resuspended in TE buffer [0.1 M Tris-HCl (pH 8.0) and 1.0 mM EDTA], and homogenized with 0.1-mm-diameter glass beads [1 :1 (w/v); 1,600 rpm for 3 min; Braun Cell Homogenizer; B. Braun Instruments, Messlungen, Germany); the DNA was purified with phenol and chloroform­isoamyl alcohol extractions and by isopropanol precipitation (Wil­son 1990). The amount of DNA was measured by fluorospectrom­etry (Fluoroskan; Labsystems, Helsinki, Finland) using Hoechst Dye H33258 (Sigma) as recommended by the manufacturer of the fluorometer. For the measurement of DNA base composition, the RNA impurities in the samples were removed by treating the sam­ples with RNAse A (0.1 mg/l; Sigma) at 37°C for 3 h followed by ethanol precipitation.

RFLP patterns

The total 16S rRNA gene was amplified with universal primers corresponding to Escherichia coli positions 8–27 (5 -AGAGTTT­GATCCTGGCTCAG) (Brosius et al. 1978) and positions 1542– 1525 (5 -AAGGAGGTGATCCAGCC). The PCR amplification system consisted of 100–200 ng DNA, 10 pl 10 x reaction buffer (supplied by the manufacturer), 2 U DynaZyme polymerase F-501 (Finnzymes, Espoo, Finland), 200 pM each of the four deoxynu­cleotides, and 300 nM of each primer in a total volume of 100 µl. The cycling program was as follows: initial denaturation at 95°C for 5 min, 30 rounds of 94°C for 30 s, 55°C for 60 s, and 72°C for 120 s; the final extension step at 72°C for 20 min. The ethanol­precipitated PCR product was cut with restriction enzyme MspI and double-cut with HaeIII and HinfI (Promega). Restriction frag­ments were visualized on a 12% polyacrylamide gel followed by ethidium bromide staining, and were photographed with a Polaroid MP-4 LAND camera (Cambridge, Mass., USA) and Polaroid 667 Pack Film.

DNA base composition

The G+C content of the DNA was determined by hydrolyzing the purified DNA and by measuring individual nucleosides with re­versed-phase HPLC as described by Gerhardt et al. (1994). Hy­drolyzed A, DNA (Sigma) was included as a base composition stan­dard.

16S rRNA gene analysis

A region of the16S rRNA gene was sequenced in both directions using solid-phase sequencing with Pharmacia Alfexpress se­quencer (Pharmacia, Uppsala, Sweden) and primers corresponding to bases 8–27 and 536–518 (Escherichia coli numbering). The 16S rRNA gene sequences were compared with sequences available in the EMBL Nucleotide Sequence Database using the FastA pro­cedure in the GCG8.1 program package (Genetic Computer Group, Madison, Wis., USA). Sequences were aligned with the CLUSTAL X program, the revised version of CLUSTAL W (Thompson et al. 1994); gaps were edited with the SEAVIEW pro­gram (Galtier et al. 1996). The final data set comprised 410 nu­cleotide positions from the newly isolated strains and from strains of various phylogenetic groups, beginning with nucleotide 101 ac­cording to E. coli numbering. The sequence similarities were de­termined using the DNA Maximum Likelihood analysis in the PHYLO_WIN program (Galtier et al. 1996), and the final phylo­genetic tree was drawn with the TREETOOL program (Ribosomal Database Project, University of Illinois, Urbana, Ill., USA). Nu­cleotide sequences of the following reference strains were used for comparison in the phylogenetic tree: Arthrobacter ilicis x83407, Bacillus subtilis x60646, Caulobacter crescentus m83799, Clostri­dium butyricum m59085, E. coli j01695, Flavobacterium johnso­niae m59053, Nocardioides plantarum z78211, Pseudomonas amygdali z76654, Ralstonia eutropha m32021, Rhodopseudomo­nas palustris d25312, Sphingobacterium thalpophilum m58779, Sphingomonas subarctica x94102, and Zoogloea ramigera x74913. The strains were type strains with the exception of Clostridium bu­tyricum m59085, E. coli j01695, and F. johnsoniae m59053.

Table 1 Isolate characteristics and comparison of phylogenetic position of the groundwater isolates based on partial 16S rRNA se­quences

Nucleotide sequence accession numbers

The 16S rRNA gene nucleotide sequences obtained in this study were submitted to the EMBL database, and the accession numbers are listed in Table 1.

Chlorophenol degradation

Degradation of 2,4,6-TCP, 2,3,4,6-TeCP, and PCP was measured in 120-ml serum bottles capped with teflon-lined septa and alu­minum seals. Bacteria were grown on PYG broth containing (g/l) peptone (1.0), glucose (1.0), and yeast extract (1.0) and were sus­pended in DSM 465 medium with 0.01% yeast extract (pH 7.0). DSM 465 medium with chlorophenols (60 ml) was inoculated with 5 ml of bacterial suspension. Controls killed with formaldehyde (2%) were set up likewise. A known chlorophenol degrader, Sphingomonas chlorophenolica ATCC 39723 (Nohynek et al. 1995), was used as a positive control. The bottles were incubated on a gyratory shaker (200 rpm) at room temperature for 2 weeks. Chlorophenols were analyzed from samples (1–2 ml), derivatized with 40 pl acetic anhydride using 40 pl K2CO3 (5.2 M) as buffer and 2,3,6-TCP as an internal standard. The acetylated chlorophe­nols were vortexed (2 min) to 1 ml hexane and were determined using a Hewlett Packard 5890 Series II gas chromatograph (GC) equipped with an electron capture detector and an HP-5 column [25 m, 0.32 mm (i.d.)] with an oven temperature program ramped from 60°C (1 min) at 20°C/min to 200°C and held at 200°C for 6 min. The injection temperature was 225°C, and the detector tem­perature was 350°C.

Inorganic chloride was measured using a chloride-sensitive electrode (model 94–178; Orion Research) and an Orion reference electrode (model 90–02) or by titrating filtered samples by the ar­gentometric method (Greenberg et al. 1992).

Toxicity of PCP

Toxicity of PCP to the isolates was tested using an automatic Bio­screen (Labsystems, Finland) multiwell turbidity analyzer. Each isolate was grown on PYG broth. The cells were then centrifuged and diluted with mineral salts medium _(OD600 0.1). To each of the the 200 wells in the Bioscreen analyzer, 300 pl PYG broth con­taining 0, 1, 5, 10, 40, or 100 mg PCP ^l–1 (pH 7.0) was added. Trip­licate wells were inoculated with 50 pl of biomass suspension. Cultures were then incubated at 20°C for 7 days, and growth was measured as turbidity using a wide-band filter (450–580 nm). Bi­olink (Labsystems, Helsinki, Finland) software was used to record the turbidity measurements and draw the growth curves. PCP con­centration causing 50% inhibition of growth (IC₅0) was interpo­lated from the growth curves.

Results

Responses of the bacterial isolates from the contaminated aquifer towards chlorophenols

Bacterial isolates from highly contaminated Karkola groundwater were screened for their ability to degrade chlorophenols. A total of 102 isolates were screened, 59 of which degraded 1 mg of 2,3,4,6-TeCP l^–1. RFLP analy­sis was used to group the chlorophenol-degrading isolates and to select representative strains from different groups for further characterization. The 58 chlorophenol-degrad­ing strains were subjected to RFLP analysis, and 11 groups with different patterns were obtained using the re­striction endonuclease MspI. Of each group, 1–3 strains were selected for further studies, 17 in total. All 17 iso­lates degraded 2,3,4,6-TeCP at concentrations of 1 mg l^–1. Degradation of 2,4,6-TCP and PCP and release of inor­ganic chloride as an indication of chlorophenol mineral­ization was additionally studied with 11 isolates. All of these isolates completely degraded 7 mg of 2,4,6-TCP _l–1 and at least partially (> 50%) 5 mg of 2,3,4,6-TeCP ^l–1 in 2 weeks. PCP that was present in the 2,3,4,6-TeCP prepa­ration was completely degraded by four isolates (K44, K101, K103, and K112) and partially degraded (57–59%) by isolates K74 and K104. When PCP was present alone at 2 mg l^–1, only three of the isolates (K6, K101, and K112) degraded PCP in 4 weeks. The gram-positive iso­lates K44 and K103 did not degrade PCP (2 mg l^–1), al­though they degraded it completely when present in the 2,3,4,6-TeCP preparation that contained 80% 2,3,4,6-TeCP and approximately 20% of PCP. This indicates that degradation of PCP may have been induced by 2,3,4,6-TeCP. On the other hand, isolate K6 degraded PCP alone, but not in the mixture with 2,3,4,6-TeCP.

Table 2 Degradation of 2,4,6-TCP, 2,3,4,6-TeCP, and PCP and toxicity of PCP to the isolates. ++ >90%, 30–90%, and –30% of the chlorophenol degraded as indicated by removal of the parent compound (ND not done)

Release of inorganic chloride was measured after incu­bation to demonstrate mineralization of chlorophenols. Chloride release from 2,4,6-TCP and 2,3,4,6-TeCP was stoichiometric only for three of the isolates (K13, K44, and K112). No chloride release was detected from PCP degradation, suggesting that PCP was not mineralized.

The toxicity of PCP to groundwater isolates was deter­mined by measuring their growth in the presence of PCP. The PCP concentrations causing 50% inhibition on the growth (IC₅₀) are listed in Table 2. The gram-positive iso­lates K44 and K103 were most sensitive to PCP; 5 mg PCP ^l–1 resulted in a lag phase of 40 h, and 10 mg PCP _l–1 inhibited their growth completely. Strains K6, K16, and K31 were sensitive to PCP with IC₅₀ values of 4, 48, and 66 mg/l respectively. The growth of these isolates was in­hibited with even lower PCP concentrations, which was observed as 20- to 60-h lag phases. The growth of strains K8, K13, K27, K33, K39, K40, K74, and K101 was not affected by 100 mg PCP/l. Isolate K1 was inhibited by 100 mg PCP/l. The isolates K66 and K112 had IC₅₀ values of 25 and 63 mg/l, respectively.

Characterization of the chlorophenol-degrading isolates

Of the selected 17 isolates, 15 were gram-negative and 2 were gram-positive, both of which had characteristics of nocardioform Actinomycetes and a G + C content of 70%. The isolates that stained gram-negative varied in mor­phology from coccoid to long rods (Table 1). Isolates K66 and K112 had distinctly lower G+C contents (36 and 41%, respectively), while those of other gram-negative isolates varied between 57.8 and 68.5%.

The whole-cell fatty acid composition of all 17 strains was analyzed. Only four of the isolates could be cultured under the standard conditions described for the MIDI database. Therefore, we grew the strains on PYGV agar at 20°C. The main fatty acids of the isolates grown on PYGV medium are shown in Table 3. Isolates K6, K13, K16, K31, K39, K40, K74, and K101 contained high amounts (> 30%) of 18:1 fatty acids and considerable amounts of 16:1 and 16:0 fatty acids, which are typical for a-Pro­teobacteria (Busse et al. 1996). The main fatty acids of isolates K1, K8, and K33 were 16:0, 16:1, 18:1, and 17:0 cyclopropane fatty acids. Different a- and (3-hydroxy fatty acids were also found in these isolates. Isolate K1 con­tained both 10:0-3OH and 12:0-3OH fatty acids. Isolate K8 had 14:0-3OH fatty acid, which is diagnostic, for ex­ample, for the genera Alcaligenes, Burkholderia, and Ral­stonia (Urakami et al. 1995; Vancanneyt et al. 1996). In addition, isolate K8 contained a rare fatty acid, 14:0-2OH, which is found, for example, in lipids from Ralstonia eu­tropha (Busse and Auling 1992). Isolate K33 had 10:0-3OH as the sole (3-hydroxy fatty acid, which is typical among the members of the family Comamonadaceae con­taining such genera as Comamonas, Variovorax, Acidovo­rax, and Hydrogenophaga (Willems et al. 1989, 1990;

Suzuki et al. 1993; Busse et al. 1996; Vancanneyt et al. 1996). Fatty acid composition of isolate K104 supported its assignment to the genus Pseudomonas. It contained mainly 16:1, 18:1, 16:0, and 17:0 cyclopropane fatty acids and additionally hydroxy fatty acids (12:0-2OH, 12:0-3OH and 14:0-3OH) characteristic for pseudomonads (Busse et al. 1996; Vancanneyt et al. 1996).

Table 3 Whole-cell fatty acid composition of the chlorophenol-degrading isolates

The gram-positive isolates contained both straight- and branched-chain fatty acids. The predominant fatty acid was iso 16:0 fatty acid, although significant amounts (20%) of 10-methylated 16–18 carbon fatty acids were also present. The 10-methylated fatty acids are exclu­sively found in actinomycetes (Kroppenstedt 1985). Iso­lates K27, K66, and K112 contained mainly iso-branched and anteiso-branched fatty acids that are typical in the Cy­tophaga/Flexibacter group and in the genera Xantho­monas and Stenotrophomonas and their relatives (Kaneda 1991; Busse et al. 1996).

Four isolates (K1, K8, K101, and K104) were addi­tionally grown on the standard MIDI conditions and com­pared to the known bacteria in the MIDI database. Isolate K8 matched Ralstonia pickettii (formerly Burkholderia pickettii) with a similarity of 0.47. The matches for the other three isolates were poor and included Yersinia (0.13) for isolate K1, Flavobacterium resinovorum (0.21) for isolate K101, and Pseudomonas syringae (0.24) for iso­late K104.

Fig.1 Phylogenetic tree inferred by maximum-likelihood analysis based on alignment of partial 16S rRNA sequences, showing the phylogenetic affiliation of chlorophenol degrading isolates from Kark6la groundwater (bar 0.1 change per nucleotide position)

Phylogenetic analysis of the chlorophenol-degrading strains

The phylogenetic relationships of the isolates as inferred from comparison of partial sequences (404–486 bp) of the 16S rRNA genes were determined (Fig.1, Table 1). The isolates fell into five major lineages of the Bacteria do­main: the a-, (3- and y-Proteobacteria, gram-positive bac­teria with high G + C content, and the Cytophaga/Flex­ibacter/Bacteroides phylum (Fig.1). Eight isolates were placed in the a-subdivision of Proteobacteria, three in the (3-subdivision of Proteobacteria, and the remaining three phyla contained two isolates each.

Table 1 shows the nucleotide identity percentages of individual isolates to the closest validly identified phylo­genetic neighbor in the EMBL database as compared by partial 16S rRNA gene sequences. From the eight isolates belonging to the a-subdivision of Proteobacteria, six iso­lates (K6, K16, K39, K40, K74, and K101) matched se­quences of Sphingomonas or Rhizomonas species with a sequence identity of 94.5–99.3%, isolate K31 had a 98% identity to Caulobacter species, and isolate K13 a 93.6% identity to Rhodopseudomonas palustris. The six isolates whose sequences matched closely with sequences of Sphingomonas strains contained also 14:0-2OH, the sig­nature fatty acid for this genus (Yabuuchi et al. 1990).

The (3-Proteobacteria K1 and K8 had sequence identi­ties of 94.5% to Zoogloea sp. BAL15 and 94.5% to Ral­stonia sp. CT12, respectively. This confirms the identifi­cation of isolate K8 obtained with the whole-cell fatty acid analysis using the MIDI library. Isolate K33 did not have close relatives with identified strains of the EMBL database. The most similar (97–99.5%) sequences for this isolate were from genomic clones from activated sludge (Bond et al. 1995) and from unidentified denitrifying Fe(II)-oxidizing bacteria (sequences u51101, u51104, and u51105 in the EMBL database). Isolate K104 was 99.2% identical with the type strain of Pseudomonas amygdali, and in addition was at least 98% identical with five other Pseudomonas type strains.

The gram-positive isolates had a sequence identity of 99.6% to each other and 97% to Nocardioides sp. OS4 (accession no. u61298). They also had 96% identity with the cloned 16S rRNA gene sequence from a picric acid (2,4,6-nitrophenol)-degrading consortium (accession no. u27857). Isolates K66 and K112 fell into the Cytophaga/ Flexibacter phylum. No close relatives were found for iso­late K112, and isolate K66 matched best with Flavobac­terium sp. ATCC 51468 with a sequence identity of 94.6%.

These results show high diversity among the 17 chloro­phenol-degrading isolates. Seven of the isolates matched the closest identified strain with less than 95% identity, and two of them had even less than 90% identity to any sequences in the database. This suggests that they may represent novel or unknown bacterial strains.

Discussion

Our results show widespread chlorophenol degradation among bacteria from contaminated groundwater. A total of 102 isolates were isolated from contaminated ground­water, 59 of which degraded 2,3,4,6-TeCP and/or PCP.

Partial 16S rRNA gene sequences and whole-cell fatty acid composition of the isolates revealed that the poly­chlorophenol-degrading bacteria present in groundwater formed a diverse group. Phylogenetic analysis placed the isolates in five major lineages of the Bacteria domain, that is the a-, R-, and y-Proteobacteria, the Cytophaga/Flex­ibacter/Bacteroides phylum, and the gram-positive bacte­ria with high G+C content. Our results showed that the ability to degrade polychlorophenols in low concentra­tions was found in many phylogenetic branches.

The a-Proteobacteria consisted of eight isolates. Six of them were close phylogenetic relatives of the genera Sphingomonas/Rhizomonas. The fatty acid and DNA G+C content of these isolates supported their classifica­tion to the sphingomonads (Van Bruggen et al. 1990; Yabuuchi et al. 1990). Sphingomonads have frequently been detected in samples from subsurface environments (Balkwill et al. 1997) and are known to possess unique biodegrading potential for various environmental contam­inants (Schmidt et al. 1992; Wittich et al. 1992; Nohynek et al. 1995, 1996). Isolates K16 and K39 matched closely with Sphingomonas stygia and Sphingomonas aromati­civorans, respectively. These strains, which degrade a va­riety of aromatic compounds, were isolated from deep subsurface sediments (Balkwill et al. 1997).

Recent work by Nohynek et al. (1995, 1996), Karlson et al. (1995), and Ederer et al. (1997) has suggested that PCP degradation by gram-negative bacteria may occur only within the genus Sphingomonas. Our results indi­cated the presence of an additional chlorophenol-degrad­ing cluster within the a-Proteobacteria. Isolates K13 and K31 grouped most closely with Rhodopseudomonas and Caulobacter species, respectively (Table 1). Chlorophe­nol degradation by these genera has not been reported.

The isolates K1, K8, and K33 fell into the (3-subdivi­sion of Proteobacteria. This group includes several genera that are known to contain xenobiotic-degrading species (Chaudhry and Chapalamaduku 1991; Busse et al. 1992). The closest relative for isolate K1 was a Zoogloea species. The base composition of this isolate did not, however, match the G + C content described for this genus (Unz 1984). The best match for the 2,3,4,6-TeCP degrader iso­late K8 was observed with a Ralstonia species. FAME analysis and G+C content of this strain agreed with this match. The closest match, Variovorax paradoxus, for iso­late K33 had less than 90% identity. The fatty acid com­position and somewhat lower G+C content of isolate K33 suggested that it did not belong to the genus Variovorax. Ralstonia and related genera, Alcaligenes, and Burkholde­ria are well-known degraders of chlorinated aromatic sub­stances (Chaudhry and Chapalamaduku 1991; Valenzuela et al. 1997), but strains K1, K8, and K33 may represent the first TeCP and PCP degraders among (3-proteobacteria.

The y-subdivision of Proteobacteria contained isolates K104 and K27. The first one (K104) is a Pseudomonas, closest to Pseudomonas amygdali. Phylogenetic and chemotaxonomic data indicated that K27 belongs to the Xanthomonas/Stenotrophomonas/Microbulbifer group (Kaneda, 1991; Busse et al. 1996; Vandamme et al. 1996; Gonz·lez et al. 1997). This isolate is also likely to repre­sent a new chlorophenol-degrading taxon.

Isolates K66 and K112 were placed among the Cy­tophaga/Flexibacter/Bacteroides group based on 16S rRNA gene sequences and whole-cell fatty acid analyses. Both isolates contained high amounts of branched fatty acids that are typical for this group (Kaneda 1991; Busse et al. 1996). Moreover, isolate K66 was related most closely to a Flavobacterium species, whereas no close rel­atives were found for isolate K112. We are not aware of any chlorophenol-degrading strains from this phylum. Therefore, these isolates may represent new chlorophe­nol-degrading taxa.

The gram-positive isolates that degraded 2,4,6-TCP, 2,3,4,6-TeCP and PCP were nocardioform Actinomycetes. All the measured parameters showed them to be close to each other, possibly as members of the same species. The closest relative for both was Nocardioides sp. OS4, which has been described as a degrader of pyridine. Nocardioides simplex strain 3E has been reported to degrade 2,4,5-tri­chlorophenoxyacetic acid via 2,4,5-TCP (Golovleva et al. 1992). Our results indicate that also 2,3,4,6-TeCP and PCP belong to chemicals degraded by Nocardioides.

All of the 17 isolates degraded 2,4,6-TCP and 2,3,4,6-TeCP whereas the PCP degradation was not as efficient. PCP as the sole chlorophenol was degraded by only three isolates that represent members of the a-Proteobacteria and Cytophaga/Flexibacter/Bacteroides phylum. The gram­positive isolates degraded PCP only when it was present with 2,3,4,6-TeCP.

As an uncoupler of oxidative phosphorylation, PCP is toxic to microorganisms (Escher et al. 1996). In most of the isolates belonging to the Proteobacteria, 100 mg PCP l–1 had no effect on growth. In some a- and (3-Proteobac­teria, the toxicity of PCP was observed as lag phases, lower cell yields, or decreased growth rate. The gram-pos­itive bacteria K44 and K103 were most sensitive to PCP; 5 mg of PCP ^l–1 resulted in 40-h lag phases, and 10 mg _l–1 inhibited growth completely. The Cytophaga/Flexibac­ter/Bacteroides isolates were between the gram-positive bacteria and the Proteobacteria in their PCP tolerance. PCP-degrading bacteria have often been isolated after en­richment at high concentrations of PCP (e.g., Stanlake and Finn 1982). This may have led to a selection of PCP-tolerant organisms, for instance genera such as Sphin­gomonas, and to a limited view of the total diversity. In this study, bacteria were isolated on a general complex medium. In chlorophenol degradation tests, nontoxic con­centrations of PCP were used. This procedure led to isola­tion of the phylogenetically diverse group of chlorophenol degraders in the contaminated groundwater.

The chlorophenol degraders described above were iso­lated from a long-term chlorophenol-contaminated aquifer that was cold (7–8°C), oxygen-deficient, iron-rich, slightly acidic (pH 6–6.5), humic (4–23 mg DOC/l), and consisted of a nutrient-poor silt and clay environment (Nysten 1994). All strains were isolated from the aqueous phase and were capable of growth at 8°C. Of the recognized chlorophenol degraders, Caulobacter (Poindexter 1981) and Sphingomonas (Balkwill et al. 1997) are known for their capability of growing in oligotrophic environments. Bacteria of the Flavobacterium/Cytophaga group are known inhabitants of subsurface and natural water (Re­ichenbach 1992). The genera Burkholderia, Ralstonia, Nocardioides, Sphingomonas, and Pseudomonas are known degraders of aromatic substances. These properties may explain their presence in the contaminated groundwater.

In conclusion, our results show that the ability to de­grade chlorophenols is widely distributed among phyloge­netically very different bacteria in long-term contami­nated groundwater. The next step is to study whether the degradation is due to similar genes in these isolates, and how these genes have spread in the contaminated ground­water.

Acknowledgements This work was supported by grants from the Academy of Finland. We thank L. Nohynek and R. Boeck for help with the whole-cell fatty acid analyses.

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