Environ. Sci. Technol. 2000, 34, 1056-1061
Microbial Community Changes Associated with a Shift from Reductive Dechlorination of PCE to Reductive Dechlorination of cis-DCE and VC S H A N N O N J . F L Y N N , * ,‡ FRANK E. LO ¨ F F L E R , †,‡ A N D J A M E S M . T I E D J E ‡,§,# Center for Microbial Ecology, Department of Microbiology, and Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824
Subcultures that reductively dechlorinate cis-dichloroethene (cis-DCE) or vinyl chloride (VC) were derived from three independent enrichments that completely dechlorinated tetrachloroethene (PCE) to ethene in order to study the reductive dechlorination of the lesser chlorinated ethenes. These subcultures completely dechlorinated cis-DCE and VC and could be transferred indefinitely in basal salts minimal medium with H2 as the electron donor. After 10 transfers (1% V/V) the cis-DCE and VC-dechlorinating subcultures from two of the PCE enrichments failed to dechlorinate PCE, but the subcultures from the third PCE enrichment maintained the ability to dechlorinate PCE. Analysis of the 16S rRNA genes from these enrichments by terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) demonstrated shifts in the community composition of the subcultures that had lost the PCE-dechlorinating activity but not in the subcultures that maintained the PCE-dechlorinating activity. Analysis of the changes in community composition of the different enrichments suggested that at least two populations were responsible for the sequential dechlorination of PCE to ethene in these cultures and that consortia can cooperate in the complete dechlorination of PCE.
Introduction Tetrachloroethene (PCE) is among the most serious groundwater pollutants as a result of widespread use in dry cleaning and as a solvent. PCE has been shown to be reductively dechlorinated to ethene by sequential dechlorination through the intermediates trichloroethene (TCE), cis-dichloroethene (cis-DCE), and vinyl chloride (VC) (1-10). PCE appears to be resistant to aerobic metabolism (11) and although its dechlorination intermediates can be cometabolized aerobically (8, 9), they commonly accumulate under anaerobic * Corresponding author phone: (314)615-6915; fax: (314)615-6901; e-mail:
[email protected]. Present address: DNA Polymerase Technology, Inc., 4041 Forest Park Avenue, St. Louis, MO 63108. † Present address: Georgia Institute of Technology, School of Civil and Environmental Engineering, 200 Bobby Dodd Way, Atlanta, GA 30332-0512. ‡ Center for Microbial Ecology. § Department of Microbiology. # Department of Crop and Soil Sciences. 1056
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conditions (11, 12). Complete dechlorination of PCE has been observed in the field, and in laboratory experiments involving microcosms, reactors, and enrichment cultures, however, the site to site variability of dechlorination activities and rates is very high (see ref 13 for numerous examples). The dechlorination intermediates cis-DCE and VC commonly accumulate under anaerobic conditions, and their dechlorination appears to be rate limiting in the reductive dechlorination of PCE (2-4, 6, 7). VC is the most toxic and only proven carcinogen of the chlorinated ethenes, thus, accumulation of VC is of particular consequence for remediation of PCE contaminated sites. In contrast to the reports of complete dechlorination performed by microbial communities and mixed cultures, pure bacterial isolates from these sources commonly do not completely dechlorinate PCE (14-21). Many of these isolates appear to use chlorinated ethenes as terminal electron acceptors to derive energy from reductive dechlorination by coupling the reaction to the oxidation of organic compounds or molecular hydrogen (14-18). Although Gibbs free energy estimates show that energy is available from the reductive dechlorination of each of the chlorinated ethenes (22, 23), most isolates only dechlorinate PCE to TCE or cis-DCE. Only one known isolate, Dehalococcoides ethenogenes, completely dechlorinates PCE to ethene; however, the reduction of VC to ethene is rate limiting, and the reduction of VC does not appear to support the growth of this organism (24). Rosner et al. (25) obtained an enrichment culture on VC from a PCE enrichment that no longer dechlorinated PCE, suggesting that different populations do exist which dechlorinate specific chlorinated ethenes in the conversion of PCE to ethene. The compositions of microbial communities are not well described by classical microbiological techniques because of cultural biases (26). These cultural biases, such as the difficulty in isolating cis-DCE or VC dechlorinating bacteria, can be circumvented by the use of molecular approaches targeting the SSU rRNA gene to assess microbial community composition (27). Two such methods, terminal restriction fragment length polymorphism (T-RFLP) (28-31) and denaturing gradient gel electrophoresis (DGGE) (32), were used in this study to determine the response of PCE enriched communities to further enrichment on cis-DCE and VC. Subcultures were derived from enrichments that completely dechlorinated PCE to investigate the dechlorination of cis-DCE and VC intermediates. In two of the enrichments transfers in medium containing cis-DCE or VC enriched for populations that dechlorinate cis-DCE and/or VC and eliminated those that could only dechlorinate PCE. Here we report the effect of these different enrichment conditions on the bacterial composition of the dechlorinating communities and their ability to reduce chlorinated ethenes.
Experimental Section Chemicals. The chlorinated ethenes PCE and cis-DCE were obtained from Aldrich Chemical Co. (Milwaukee, WI). Gaseous VC was obtained from Fluka (Fluka Chemical Corp., Ronkonkoma, NY). Enrichment Cultures. The sediments were collected from three Michigan rivers, the Au Sable, the Pe`re Marquette, and the Red Cedar and used to construct microcosms which completely dechlorinated PCE. Sediment-free nonmethanogenic enrichment cultures capable of complete dechlorination of PCE were derived from these microcosms by successive transfers to fresh medium in the presence of 2-bromoethanesulfonate (33). Subcultures were then derived from the PCE enrichments by successive transfers of the 10.1021/es9908164 CCC: $19.00
2000 American Chemical Society Published on Web 02/15/2000
cultures (1% V/V) to fresh medium containing cis-DCE or VC. The parental enrichment was similarly maintained by transfer to medium containing PCE. The enrichment cultures were incubated at 25 °C in 100 mL of reduced anaerobic basal salt medium in sealed 160-mL serum bottles. The medium contained 5 mM acetate as the carbon source and electron donor; hydrogen was also supplied as an electron donor as previously described (34). The chlorinated ethenes PCE, cis-DCE, or VC were added as the only electron acceptors besides bicarbonate. The concentrations of PCE or cis-DCE were 0.2 or 0.24 mM, respectively. Gaseous VC (1 mL) was added to give an initial aqueous concentration of 0.185 mM. PCE dechlorination in the subcultures was measured to determine if enrichment on cis-DCE or VC had resulted in the loss of populations that could dechlorinate PCE. A 1% inoculum(vol/vol) from each of the subcultures was transferred to fresh medium amended with 0.2 mM PCE. These cultures were incubated for 2 months and monitored weekly for dechlorination of PCE by gas chromatography. After 2 months the cultures received another 1% inoculum, and chlorinated ethenes were monitored for an additional 2 months. For rate measurements, cultures that had completely dechlorinated the initial amount of chloroethene were further supplemented with either 0.2 mM PCE, 0.24 mM cis-DCE, or 0.185 mM VC. The formation of daughter products was monitored (see below), and the rates of removal of one chlorine were calculated and expressed as µmol/L/d. Hydrogen (82 µM) was added to these cultures and added again when depleted. Hydrogen consumption was monitored using a GC equipped with a reduction gas detector (34). Detection of Chlorinated Ethenes. Chlorinated ethenes in the enrichment cultures were detected by gas chromatography. Headspace samples of 200 µL at 25 °C were analyzed using a Varian GC (model 3700) equipped with a Megabore column model DB-624 (45 m × 0.543 mm) and a flame ionization detector as previously described (33). A temperature of 50 °C was held for 4 min followed by an increase of 50 °C/min to 200 °C. Extraction of DNA. Cells were harvested from 20 mL of medium by centrifugation at 12 000g for 30 min. The cells were lysed, and the DNA was extracted using the QIamp Tissue DNA kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. The resulting community DNA was quantified, and an equal amount of DNA was used for each subsequent molecular procedure. Terminal Restriction Fragment Length Polymorphism (T-RFLP). The T-RFLP analysis was performed as previously described (30) with the following modifications. PCR was performed with bacterial domain specific SSU rRNA primers 8-27 forward labeled with hexachlorofluorescene (Hex) at the 5′-end (synthesized by Operon Technologies, Inc. Alameda, CA) and 1510-1492 reverse each having the sequences AGAGTTTGATCMTGGCTCAG (35) and RGYTACCTTGTTACGACTT, respectively (36). In the reverse primer the degenerate positions R and Y employed the pyrimidine and purine derivatives dK and dP, respectively (Glen Research, Sterling, VA) (37). The SSU rRNA genes were amplified using approximately 40 ng of DNA and a Perkin-Elmer 9600 thermocycler (Perkin-Elmer, Norwalk, CT). The PCR reactions were performed under the following conditions: 95 °C 3 min initial denaturation followed by 30 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72° for 3 min followed by an extension at 72 °C for 7 min. The resulting PCR products were purified using the Wizard PCR purification kit (Promega, Madison, WI) and digested overnight at 37 °C with 20 units of HhaI, MspI or RsaI separately (Life Technologies, Gibco BRL, Gaithersburg, MD). The digest fragments were then resolved on an ABI 373A sequencer running in the gene scan mode with 6% urea-containing polyacrylamide gels (PEApplied
Biosystems, Foster City, CA). Size calibration was performed with the Tamara 2500 standard (PEApplied Biosystems). The resulting gel patterns were analyzed using the Genescan software version 2.1 (PEApplied Biosystems). Denaturing Gradient Gel Electrophoresis (DGGE). The PCR amplification for DGGE was performed using domain specific SSU rRNA primers for Bacteria; 968-983 forward and 1406-1392 reverse with the sequences AACGCGAAGAACCTTAC (38) and ACGGGCGGTGTGTACA (39), respectively. The forward primer included a GC clamp described by Muyzer et al. CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGG (32). The SSU rRNA genes were amplified from approximately 40 ng of DNA using a Perkin-Elmer 9600 thermocycler. The PCR reactions were performed using a hot start technique that employs AmpliTaq gold modified Taq polymerase (Perkin-Elmer, Norwalk, CT) under the following conditions: 95 °C, 8 min initial denaturation followed by 32 cycles at 95 °C for 30 s, 57 °C for 45 s, and 72 °C for 1 min followed by an extension at 72 °C for 7 min. Resolution of PCR amplified SSU rDNA fragments was performed with the Bio-Rad D-gene system (Bio-Rad, Hercules, CA) as previously described with the following modifications (32). The PCR amplified samples were loaded on a 35-60% denaturing gradient 6% acryamide gel and run at 200 V for 300 min at 60 °C with a 1x TAE buffer. The gels were then silver stained to visualize the DNA fragments (40). The gel image was digitized using a Biophotonics Gelprint 2000i system (Ann Arbor, MI), cropped, labeled, and the contrast was adjusted using Canvas 5 (Deneba Software, Miami, FL). Cloning of PCR Amplified SSU rRNA Genes. The SSU rRNA genes were amplified using PCR and cloned as previously described (41). PCR was performed using domain specific SSU rRNA primers for Bacteria 49-68 forward and 1510-1492 reverse each having the sequences TNANACATGCAAGTCGRRCG and RGYTACCTTGTTACGACTT, respectively, with modifications by Moyer et al. (42, 43) of primers by Weisburg et al. (35). The PCR reactions were performed under the following conditions: 95 °C 3 min initial denaturation followed by 30 cycles of 95 °C for 1 min, 57 °C for 1 min, and 72 °C for 3 min followed by an extension at 72 °C for 7 min. The resulting PCR products were purified using the Wizard PCR purification kit (Promega, Madison, WI), then ligated into the pCR II T vector (Invitrogen, Carlsbad, CA), and transformed into Escherichia coli. Sequence Analysis of SSU rRNA Clone Libraries. The cloned SSU rRNA genes were amplified from individual clones for restriction analysis using a primer set specific to the polylinker of the pCR II vector (44). The resulting amplicons were then amplified using the primers described above for T-RFLP. The resulting fluorescently labeled products were screened by T-RFLP to determine terminal fragment length. Partial DNA sequence of select clones was determined using J529R primer CGCGGCTGCTGGCAC (35) and automated fluorescent sequencing using the ABI catalyst 800 and ABI 373 (Applied Biosystems, Foster City, CA). Sequence similarity was determined using the Ribosomal Data Base Project II (45). The sequences of the SSU rRNA genes used to determine the similarity index have been deposited in GenBank under the accession numbers AF158712-AF158721.
Results Enrichment of PCE dechlorinating mixed cultures with cisDCE or VC. Sediment free nonmethanogenic enrichment cultures that completely dechlorinated PCE, cis-DCE, and VC were derived from three river sediments. Each of these enrichments displayed different rates of dechlorination and different responses to various electron donors. However, all of these enrichments produced the intermediates cis-DCE and VC prior to the formation of ethene. These original enrichments were further subcultured by more than 10 serial VOL. 34, NO. 6, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Dechlorination Rates and Products of Enrichments and Subcultures Amended with Various Chlorinated Ethenes enrichment/electron acceptor Au Sable PCE Au Sablecis-DCE Au Sable VC Pe` re Marquette PCE Pe` re Marquette cis-DCE Pe` re Marquette VC Red Cedar PCE Red Cedar cis-DCE Red Cedar VC
end product of dechlorination dechlorination rates µmol/L/da PCEd cis-DCEd VCd 13.9 24.2 43.6 288b 53.9 82.5 23.8 47.2 42.5
ETH PCE PCE ETH PCE PCE ETH ETH ETH
ETH ETH NDc ETH ETH NDc ETH ETH NDc
ETH ETH ETH ETH ETH ETH ETH ETH ETH
a The rates were normalized to the removal of one chlorine. Data were averaged from duplicate cultures from two independent experiments. Acetate and H2 were supplied as electron donors. All cultures had undergone at least 10 serial transfers on the indicated electron acceptor, which was also the substrate for the rate assays. b Small amounts of TCE accumulated transiently; the rate indicated is for PCE to cis-DCE dechlorination. c ND, not determined. d Test substrates.
transfers (1% V/V) to fresh medium in the presence of either cis-DCE or VC. Table 1 shows the activities of the parental PCE dechlorinating enrichments and the cis-DCE and VC dechlorinating subcultures and the rates of the dechlorination reactions. The Pe`re Marquette PCE enrichment had the highest overall rate of dechlorination from PCE to ethene. This was largely due to the rapid dechlorination rate to cisDCE in this enrichment of 288 µmol/L/d. The cis-DCE and VC intermediates were most rapidly dechlorinated by the Pe`re Marquette enrichments as well. The Au Sable PCE enrichment had the lowest rate of complete dechlorination of PCE to ethene due to the low rate of dechlorination of PCE to TCE (13.9 µmol/L/d). However the dechlorination of cisDCE and VC in the Au Sable enrichments was higher (24 and 43.6 µmol/L/d, respectively) than dechlorination of PCE to TCE (Table 1). Changes in Subculture Activities and Community Composition. The cis-DCE and VC subcultures derived from the Au Sable and Pe`re Marquette PCE enrichments lost the ability to dechlorinate PCE, but the Red Cedar subcultures retained PCE-dechlorinating activity (Table 1). T-RFLP and DGGE were used to determine if the community composition of the enrichments had changed. The fluorescently labeled terminal restriction fragments (TRF) from Msp I digests revealed changes in community composition associated with the shift from PCE to cis-DCE or VC in the Au Sable and Pe`re Marquette enrichments but not in the Red Cedar enrichments (Figure 1). T-RFLP also revealed several differences in the communities dependent on the sediment source. Each electropherogram shows the overlaid profiles of three independent PCR products digested with MspI. The shifts in the community composition in the Au Sable subcultures were characterized by the loss of several TRFs, and a corresponding increase in the area of TRFs at 438 and 490 nucleotides, and the appearance of a TRF at 83 nucleotides in the VC subculture. Community profiles of the Pe`re Marquette enrichments showed a decrease in the area of TRFs at 440, 490, and 525 nucleotides and the appearance of two ribotypes with TRFs at 83 and 435 nucleotides in both the cis-DCE and VC subcultures. The profiles of the Red Cedar enrichments are all very similar with the exception of two minor TRFs that appear in the VC subculture (Figure 1). Digests of the fluorescently labeled PCR products with HhaI and RsaI resolved fewer ribotypes than MspI most likely because different bacterial species in the enrichments displayed the same TRF length (data not shown). The data from all three restriction enzyme digests was summarized using a similarity index (Table 2). The Au Sable 1058
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FIGURE 1. Electropherograms showing the 5′ terminal MspI fragment lengths of the SSU rRNA PCR products from the PCE enrichments and the cis-DCE and VC subcultures. The sediment origin of the parental PCE enrichments are shown across the top, and the respective chlorinated ethene used in each enrichment is shown along the right. The horizontal axis shows the length of the terminal restriction fragments in nucleotide bases, and the vertical axis shows the relative level of fluorescence intensity. Each electropherograms shows the overlaid digest profiles of three independent PCR products from the same community. cis-DCE and VC subcultures had the two lowest similarity indices relative to the parental PCE enrichment with indices of 0.72 and 0.59, respectively. However, these subcultures
TABLE 2. Similarity Indices of the Enrichments Based on T-RFLP Community Profilesa enrichment comparison
Au Sable
Pere Marquette
Red Cedar
PCE vs cis-DCE PCE vs VC cis-DCE vs VC
0.72 0.69 0.59
0.73 0.75 0.97
1.00 0.92 0.92
a The similarity index was calculated as a ratio of the number of common terminal restriction fragment peaks divided by total number of terminal restriction fragment peaks between two enrichments for HhaI, MspI, and RsaI digests. Minor peaks were scored as half peaks.
were nearly as dissimilar to each other as each was to the parental PCE enrichment (similarity index ) 0.69). In contrast, the Pe`re Marquette cis-DCE and VC subcultures were very similar to each other with an index of 0.97 and moderately different from the parental PCE enrichment (0.73 and 0.75). The Red Cedar enrichments all displayed high similarity indices relative to each other, which drop only to 0.92 when compared to the VC subculture due to minor TRFs that consistently appeared in all three digests. Similarity indices determined across sites were low ranging from 0.25 to 0.59 (data not shown). The phylogenetic affiliation of different ribotypes was determined by sequence analysis of cloned SSU rRNA genes from the Au Sable VC subculture and the Pe`re Marquette cis-DCE and VC subcultures. Representative clones with the 83 nucleotide TRF ribotype were isolated from all three enrichments. The sequences of all of the 83 nucleotide TRF ribotype clones are closely related to Azoarcus sp. strain S5b2 a member of a nitrogen-fixing genus in the β Proteobacteria. Additionally, clones representing ribotypes common to all of the enrichments with 490 and 545 nucleotide TRF were isolated; they represent members of the Clostridiaceae (Table 3). DGGE was used as an independent method to determine whether there were changes in community composition. Changes in the communities were detected in the Au Sable and Pe`re Marquette cis-DCE or VC subcultures relative to the parental enrichments but not the Red Cedar enrichments (Figure 2). This observation was consistent with the T-RFLP results in that they demonstrated shifts in the community composition associated with a shift in electron acceptor from PCE to cis-DCE or VC as electron acceptors, although they were not identical. DGGE also demonstrated that the different sediment sources produced unique community profiles in the enrichments. The DGGE profile of the Au Sable PCE enrichment was the most complex of all the enrichments, and the subcultures derived from this enrichment were much less complex than their parental counterpart. The Pe`re Marquette subcultures were also distinct from the parental enrichments since one major band was lost and new bands appeared. In contrast, all three Red Cedar enrichments displayed very similar DGGE profiles. The same DGGE profiles were obtained from an additional independent amplification of DNA extracted from each of the enrichments (data not shown).
Discussion The T-RFLP and DGGE results suggest that different sites have different chloroethene dechlorinating communities and that communities specialized in cis-DCE and VC dechlorination can be different from those involved in PCE dechlorination. Furthermore, the rates of PCE-dechlorination relative to cis-DCE and VC-dechlorination varied in these different communities. Hence this study may provide an explanation for why different PCE dechlorination activities can be seen in the field (13), namely that different organisms at different sites may have different rates and activities.
Differences have begun to emerge in the biochemistry of halorespiration by different species. Rosner et al. observed VC and cis-DCE dechlorination that appeared to be independent of corrinoids (25) unlike the processes described for the dechlorination PCE and TCE which rely upon vitamin B12. Both the PCE- and TCE-reductive dehalogenases of D. ethenogenes are corrinoid dependent (46). Hence there appears to be a diversity of dechlorinating organisms and enzymes (47). The failure of the Au Sable and Pe`re Marquette subcultures to dechlorinate PCE provides physiological evidence suggesting that the changes in these communities, detected by both T-RFLP and DGGE, are functionally relevant. PCE dechlorination could not be recovered in the Au Sable and Pe`re Marquette subcultures even after a 4 month incubation with PCE. Thus the loss of the PCE-dechlorinating activity was likely due to a change in the community composition and not a lag in induction of the activity. In contrast, the Red Cedar cis-DCE and VC subcultures readily dechlorinated PCE. This type of response could be explained by the presence of an organism like D. ethenogenes (24), which completely dechlorinates PCE. Indeed, D. ethenogenes like sequences have been detected in the Red Cedar enrichments using PCR primers specific for D. ethenogenes (48). However, the predicted TRF from D. ethenogenes was not observed in these enrichments. It is possible that the domain specific primers used for T-RFLP did not compete well for this strain in the presence of a complex mixture of genomic DNAs. Additionally, this population may not have been numerically dominant within the enrichment community. Both of the molecular methods used to describe the microbial community composition of the enrichments demonstrated changes in community composition. However, there were some differences between the results obtained with the two techniques. The subcultures from the Au Sable enrichment appear similar by DGGE, but the T-RFLP profiles show a dominant ribotype (83 nucleotides by Msp I digest) in the VC subculture that is not present in the cis-DCE subculture. Interestingly, this ribotype also appears in the cis-DCE and VC subcultures from the Pe`re Marquette. In contrast the DGGE profiles of the Pe`re Marquette subcultures demonstrated differences between them, but the T-RFLP profiles were very similar (Figures 1 and 2). The Red Cedar enrichments demonstrated little if any change in the community composition. These two molecular techniques are complementary but not directly comparable since they rely upon analysis of separate regions of the SSU rRNA genes. Additionally, both techniques have limitations such as the formation of hetroduplexes for DGGE (49, 50) and common TRF lengths shared by many bacterial species and partial digest products for T-RFLP (30). Both techniques however share the bias of PCR amplification of genes from communities which excludes some members of microbial communities (49, 51, 52). The appearance of the 83 nucleotide TRF ribotype correlates with the dechlorination of VC in the Au Sable VC subculture and the Pe`re Marquette cis-DCE and VC subcultures. SSU rRNA genes representing this ribotype were cloned from these three enrichments, and the six clones sequenced were very similar to each other and to Azoarcus sp. S5b2, a strain cultured from Kallar grass roots (Table 3) (53). Azoarcus strains fix nitrogen, and many denitrify and degrade aromatic compounds both aerobically and anaerobically. Although phylogenetic affiliation is only suggestive of physiology, these results indicate that nitrogen-fixing bacteria are present in these enrichments in association with VC dechlorinating activity. Knowledge of consortia members, especially those whose presence correlates with activity, can potentially be valuable in aiding in the detection and cultivation of the relevant dechlorinating populations. VOL. 34, NO. 6, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Phylogenic Affiliation of Ribotypes from Dechlorinating Enrichments terminal fragment size
clone number
clone origin
phylogenic affiliationa
83 83 83 83 83 83 490 490 490 545
PM4-2 PM5 PM8-1 A36 A42 A49 PM3 A32 A33 A41
PM VC PM VC PM cis-DCE AuS VC AuS VC AuS VC PM VC AuS VC AuS VC AuS VC
β proteobacteria β proteobacteria β proteobacteria β proteobacteria β proteobacteria β proteobacteria clostridiaceae clostridiaceae clostridiaceae clostridiaceae
closest relativea
Azoarcus sp. S5b2 Azoarcus sp. S5b2 Azoarcus sp. S5b2 Azoarcus sp. S5b2 Azoarcus sp. S5b2 Azoarcus sp. S5b2 Acidaminobacter hydrogenoformans Acidaminobacter hydrogenoformans Acidaminobacter hydrogenoformans Clostridium limosum Clostridium butyricum
% SSU rRNA similarityb 97.8 99.8 99.6 99.6 99.3 99.8 96.8 94.1 95.6 87.8
a Phylogenic affiliation and closest relatives for partial sequences were determined from SSU rRNA sequences using the Ribosomal Database Program II and BLAST searches. b The percent similarities were determined using the similarity matrix program of the RDP II.
When we analyzed the community composition of these subcultures we observed unique communities from each location with different responses to enrichment on different chlorinated ethenes. Each location appears to have yielded unique populations of dechlorinating bacteria, some of which only dechlorinate the lesser chlorinated ethenes. Those subcultures that failed to dechlorinate PCE appear to contain specialist organisms that occupy a distinct niche within the sequential dechlorination pathway of PCE, cis-DCE and/or VC exclusively. Since cis-DCE and VC tend to accumulate in the field, more insight into these latter populations and the detection and isolation of the most efficient dechlorinators could improve our understanding of natural attenuation at chloroethene-contaminated sites.
Acknowledgments FIGURE 2. DGGE resolution of PCR amplified SSU rDNA fragments from PCE-dechlorinating enrichment cultures and cis-DCE or VCdechlorinating subcultures. The individual lanes are labeled with the respective chlorinated ethene used in the enrichment. The separately labeled sections of the gel show the sediment origin of the parental PCE enrichment. The complexity detected in these communities appears to account for more ribotypes than can be ascribed to the dechlorinators, especially those ribotypes common to all of the enrichments. Three common TRFs appear at 440, 490, and 545 nucleotides for all of the enrichments (Figure 1). In all of the DGGE profiles at least four bands (two major and two minor) appeared to be common (Figure 2). A stoichiometric accumulation of chlorinated ethenes and ethene from dechlorination was detected; bacteria in the enrichments did not appear to be degrading ethene. Two of the ribotypes common to all of the enrichments were identified through cloning and sequence analysis of SSU rRNA genes. Clones representing the 490 nucleotide TRF ribotype were obtained from two separate enrichments; their nearest relative was Acidaminobacter hydrogenoformans. The nearest relative of a clone representing the 545 nucleotide TRF ribotype from the Au Sable VC subculture was novel, having only 87.5% similarity to the corresponding region of the SSU rRNA gene of its closest matches, Clostridium limosum or C. butyricum. These organisms may not be associated with dechlorination but performing some other anaerobic process. The enrichment conditions using CO2 and H2 with 2-bromoethanesulfonate to inhibit methanogens were designed to be selective for hydrogenotrophic chloroethene-respiring bacteria. However, these conditions are also selective for acetogenic bacteria which includes many of the Clostridiaceae (54). Furthermore, acetate accumulation was observed in these enrichments (data not shown). 1060
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This work was supported by the SERDP Bioconsortium, NSF Grant DEB#9120006 to the Center for Microbial Ecology, and by a Feodor-Lynen-fellowship from the Alexander von Humbolt-Stiftung to F.E.L. We would like to thank James Cole, Robert Sanford, and Benjamin Griffin for helpful discussions and Stephen Nold for critically reading this manuscript.
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Received for review July 19, 1999. Revised manuscript received December 16, 1999. Accepted December 17, 1999. ES9908164
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