Bacterial Diversity of an Acidic Louisiana Groundwater

Bacterial diversity ofan acidic Louisiana groundwater contaminated by dense nonaqueous-phase liquid containing chloroethanes and other solvents Kimberly S. Bowman1, William M. Moe1, Brian A. Rash2, Hee-Sung Bae2 & Fred A. Rainey2

1Department of Civil and Environmental Engineering, Louisiana State University, Baton Rouge, LA, USA; and 2Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021

Correspondence: William M. Moe, 3418G Abstract CEBA Building, Department of Civil and Environmental Engineering, Louisiana State Bacterial concentration and diversity was assessed in a moderately acidic (pH 5.1) University, Baton Rouge, LA 70803, USA. anaerobic groundwater contaminated by chlorosolvent-containing DNAPL at a Tel.: 1225 578 9174; fax: 1225 578 8652; Superfund site located near Baton Rouge, Louisiana. Groundwater analysis e-mail: [email protected] revealed a total aqueous-phase chlorosolvent concentration exceeding 1000 mg L1, including chloroethanes, vinyl chloride, 1,2-dichloropropane, and Received 21 November 2005; revised 1 March hexachloro-1,3-butadiene as the primary contaminants. Direct counting of stained 2006; accepted 8 March 2006. cells revealed more than 3 107 cells mL1 in the groundwater, with 58% intact First published online 8 May 2006. and potentially viable. Universal and ‘Dehalococcoides’-speciﬁc 16S rRNA gene libraries were created and analyzed. Universal clones were grouped into 18 DOI:10.1111/j.1574-6941.2006.00146.x operational taxonomic units (OTUs), which were dominated by low-G1C Gram-

Editor: Max Haggblom¨ positive bacteria (62%) and included several as yet uncultured or undescribed organisms. Several unique 16S rRNA gene sequences closely related to Dehalococ- Keywords coides ethenogenes were detected. Anaerobically grown isolates (168 in total) were bioremediation; chlorosolvents; also sequenced. These were phylogenetically grouped into 18 OTUs, of which only ‘Dehalococcoides’; DNAPL; reductive three were represented in the clone library. Phylogenetic analysis of isolates and the dechlorination. clone sequences revealed close relationships with dechlorinators, fermenters, and hydrogen producers. Despite acidic conditions and saturation or near-saturation chlorosolvent concentrations, the data presented here demonstrate that large numbers of novel bacteria are present in groundwater within the DNAPL source zone, and the population appears to contain bacterial components necessary to carry out reductive dechlorination.

Biotransformation has been widely studied and applied Introduction for in situ remediation of chloroethanes and chloroethenes Chlorinated aliphatic ethanes and ethenes have been widely in cases where contaminants are present at relatively low used as industrial solvents and are produced on a large scale concentrations in groundwater plumes (Lorah & Olsen, as intermediates for the production of industrially impor- 1999; Hendrickson et al., 2002). Under anaerobic condi- tant chemicals (De Wildeman et al., 2003). Owing to spills tions, biotransformation of chloroethenes occurs through and inappropriate past disposal methods, these chlorinated dehalorespiration, whereby the chlorinated ethenes serve compounds are prevalent groundwater and soil contami- as electron acceptors, resulting in successive reductive de- nants throughout the world (Pankow & Cherry, 1996). chlorination from perchloroethene to trichloroethene, di- Because of their high speciﬁc gravity and relatively low water chloroethene, vinyl chloride, and ﬁnally the nontoxic solubility, many chlorinated solvents are present in the endproduct ethene. Chlorinated ethanes can also undergo environment as dense nonaqueous-phase liquids (DNAPLs) successive reductive dechlorination reactions, although with that serve as long-lasting and continuous sources of ground- more diverse pathways (Chen et al., 1996; Lorah & Olsen, water contamination (Pankow & Cherry, 1996; Carr et al., 1999). Because chloroethenes and chloroethanes serve as 2000; Cope & Hughes, 2001; Yang & McCarty, 2000, 2002; terminal electron acceptors, an electron donor such as

Adamson et al., 2003, 2004). molecular hydrogen (H2) is required in dehalogenating

c 2006 Federation of European Microbiological Societies FEMS Microbiol Ecol 58 (2006) 120–133 Published by Blackwell Publishing Ltd. All rights reserved Bacterial diversity in DNAPL-contaminated groundwater 121 microorganisms’ energy metabolism (Cupples et al., 2003). 2004). Because of these selective pressures, the microbial In recent years, several bacteria capable of reductive de- populations are not necessarily representative of in situ chlorination have been isolated in pure culture. These populations (Macbeth et al., 2004). Little research has been include Dehalobacter restrictus (Hollinger et al., 1998), published on the characterization of in situ microbial Sulfurospirillum multivorans (formerly Desulfuromonas mul- populations at chlorosolvent-contaminated sites in general, tivorans) and Sulfurospirillum halorespirans (Luijten et al., and even less information is available regarding microbial 2003), Desulfuromonas chloroethenica (Krumholz, 1997), populations from areas contaminated by chloroethanes and and ‘Dehalococcoides’ sp. (Maymo-Gatell´ et al., 1999; He solvent mixtures (as opposed to only chloroethenes). et al., 2003), the only group of microorganisms isolated in Because traditional methods (i.e. pump and treat) to pure culture that can reductively dechlorinate vinyl chloride remove DNAPL contamination in aquifers have historically

to ethene. been expensive, slow, and often ineffective, alternative ap- Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 Until recently, microbial degradation in zones where proaches, such as monitored natural attenuation (MNA), are chlorinated DNAPLs are present was generally assumed to increasingly being investigated as remediation technologies be negligible due to the toxicity of high concentrations of (Pankow & Cherry, 1996). Microcosm studies of sediment chlorinated compounds (Yang & McCarty, 2000, 2002). and groundwater at the Petro Processors of Louisiana, Inc. Recent laboratory-scale experiments have demonstrated that Superfund Site indicate that microbially mediated reductive some anaerobic perchloroethene-dechlorinating bacteria can dechlorination is occurring in areas outside of the DNAPL reductively dechlorinate chloroethenes in the presence of free- source zone (i.e. in the contaminant plume) (Truex et al., phase DNAPL (Nielsen & Keasling, 1999; Carr et al., 2000; 2001; Clement et al., 2002). The research described here was Yang & McCarty, 2000, 2002; Cope & Hughes, 2001; Dennis conducted using culture-dependent and culture-independent et al., 2003). Furthermore, recent research using perchlor- techniques to characterize the bacterial community within oethene as a model compound suggests that the rate of the DNAPL source zone in support of an effort to assess DNAPL dissolution may be biologically enhanced by reduc- whether MNA is also feasible for the DNAPL source zone. tively dechlorinating microorganisms (Yang & McCarty, 2000; Cope & Hughes, 2001; Adamson et al., 2004). This Materials and methods suggests that in situ bioremediation may be feasible for clean- up at some sites where DNAPL is present. This has important Sample collection ramifications for clean-up of contaminated sites, because Groundwater samples were collected from a waste recovery source zone removal is often one of the most expensive well (W-1024-1) located in the DNAPL source zone at the aspects of remediation (Pankow & Cherry, 1996). Brooklawn site, one of two areas collectively known as the During in situ anaerobic bioremediation of chlorinated Petro Processors of Louisiana, Inc. (PPI) Superfund Site, aliphatic compounds, it is hypothesized that syntrophic located approximately 10 miles north of Baton Rouge, LA. interactions such as interspecies hydrogen transfer among Operations at the Brooklawn site, opened in 1969 and microbial community members play a critical role in con- operated until 1980, involved disposal of petrochemical taminant biotransformation. For example, growth of ‘De- waste, including free-phase chlorinated organics, by direct halococcoides ethenogenes’ strain 195 apparently requires discharge to earthen ponds. Portions of the Brooklawn area unknown growth factors contained in anaerobic sludge were capped in the early 1990s, and starting in 1994, an array supernatant as well as molecular hydrogen (Maymo-Gatell´ of recovery wells was installed to recover free-phase organic et al., 1997). Although microbial community structure has contaminants. Recovery of DNAPL in the source zone area is been reported for several enrichment cultures capable of ongoing. Well W-1024-1 has a screened interval extending reductively dechlorinating chloroethenes (Duhamel et al., from 16.5 to 76.5 feet below ground surface in an area 2002; Richardson et al., 2002; Dennis et al., 2003; Rossetti containing alternating layers of clay, silt, and sand. Addi- et al., 2003; Gu et al., 2004), chloropropanes (Schlotelburg¨ tional details regarding contaminant hydrology for the site et al., 2002) or chlorobenzenes (von Wintzingerode et al., has been reported elsewhere (Clement et al., 2002). For 1999), structure–function relationships remain poorly un- microbial analyses, sterile 1.0 L glass bottles with Teflon- derstood. With a few exceptions (e.g. Macbeth et al., 2004), lined lids were filled with groundwater, leaving little or no microbial populations in reductively dechlorinating systems headspace, and then placed on ice during transport to the reported to date have been enriched over long time periods laboratory (approximately 1 h). in laboratory systems with chlorinated solvent concentrations far less than saturation levels (i.e. in the absence of Chemical analyses DNAPL) and in the presence of a readily available supply of electron donors (e.g. H2) (Duhamel et al., 2002; Richardson Concentrations of volatile organic compounds were mea- et al., 2002; Dennis et al., 2003; Rossetti et al., 2003; Gu et al., sured using US EPA method 624. Dissolved ethene, ethane

FEMS Microbiol Ecol 58 (2006) 120–133 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 122 K.S. Bowman et al. and methane were measured using method RSK 175. Nitrate buffered at pH 5.0 with 20 mM acetate buffer prepared in and nitrite were measured using US EPA method 353.2. the same manner but were also supplemented with Chloride was measured using US EPA method 325.2. Sulfate 1.0 mg L1 resazurin (as a redox indicator) and 0.25 g L1 was measured by ion chromatography using US EPA meth- cysteine hydrochloride (as a reducing agent) prior to od 300.0. Sulﬁde was measured using US EPA method 376.2. solidiﬁcation with agar. All plates were incubated at 30 1C. Ferrous iron was measured using US EPA method 3500-Fe Anaerobic plates were prepared and incubated in an anae- D. Total organic carbon and total inorganic carbon were robic chamber (Coy, Grass Lake, MI) containing approxi- measured using US EPA method 5310B. Detailed descrip- mately 2% H2, 10% CO2, balance N2. To estimate the tions of the US EPA analytical methods referenced above are number of organisms represented by spores in the original available elsewhere (National Environmental Index, http:// sample, a pasteurization technique was employed in the

www.nemi.gov/). The pH of groundwater samples was anaerobic plate counting procedure (Rosencrantz et al., Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 measured using an Orion model 290A pH meter. 1999). In this method, following serial dilution, groundwater samples were pasteurized by heating in a water bath at Microscopy 80 1C for 15 min prior to plating. The number of colonies on the plates was counted at 7, 14, 21 and 28 days. Total microbial numbers were determined by direct counting following staining with 40,6-diamidino-2-phenylindole (DAPI). In this procedure, groundwater samples were first DNA extraction preserved by addition of 4% glutaraldehyde in 0.1 M caco- For clone 16S rRNA gene library analysis, groundwater dylate buffer (pH 7) to an equal volume of groundwater. samples were transferred to sterile 15-mL polypropylene Samples were preserved within 2 h after sample collection tubes, which were centrifuged at 4000 g for 10 min. Super- and were subsequently stored at 20 1C until further natant was decanted, leaving a sediment pellet that was processing. Samples were sonicated for 45 s (Branson Ultra- resuspended in 0.5 mL of TE buffer (10 mM Tris, 1 mM sonic 170 V 50/60 Hz), stained for 1 h in the dark at a final EDTA, pH 8) and transferred to sterile 1.5-mL tubes, which DAPI concentration of 5 mgmL1, and then collected on were centrifuged at 16 000 g for 15 min. The supernatant was 0.20-mm-pore black Nuclepore polycarbonate filters. Filters decanted, and the pellet (approximately 0.3 mL) was resus- were mounted on microscope slides and imaged using a pended in fresh TE buffer (to a total volume of 2 mL) prior Nikon Microphot-FXA epifluorescent microscope at to freezing at 20 1C until DNA extraction. 80 magnification and a Nikon DM400 filter set (365 nm DNA was extracted using modifications of the method BP excitation, 400 nm LP dichroic, 400 nm LP barrier). Cells described by Herrick et al. (1993). In this process, 100 mg of were counted in two separate groundwater samples each polyvinylpolypyrilidone (Agros Organics, Geel, Belgium) with 20 fields of view. was added to 2 mL of resuspended groundwater sediment The fraction of live cells was estimated using a LIVE/ and 10 mL of TE buffer, vortexed, and centrifuged at DEAD BacLight Bacteria Viability Kit (Molecular Probes, 11 500 g for 10 min. Supernatant was decanted, the pellet Eugene, OR) according to the manufacturer’s recommended was resuspended in 5 mL of SET buffer (200 g L1 sucrose, protocol. In this protocol, a green-fluorescent nucleic acid 0.05 M EDTA, 0.05 M Tris-HCl, pH 7.6), and 500 mLof stain, SYTO 9, is added to label all bacterial cells, and red- lysozyme solution (c.15mgmL1 lysozyme in water) was fluorescent propidium iodide to stain only those cells with added. The sample was incubated at 37 1C for 2 h with compromised cell membranes. Viable cells fluoresce bright mixing by shaking at 5–15 min intervals. Then, 500 mLof green, while dead or compromised cells fluoresce red. 10% sodium dodecyl sulfate (filter sterilized with a 0.2-mm Samples were imaged using a Nikon Microphot-FXA epi- filter) was added, and three freeze–thaw cycles were per- fluorescent microscope at 80 magnification and a Nikon formed (5 min at 80 1C, then 5 min at 70 1C). The sample multiband DAPI-FITC-Rhodamine cube filter set (Boulos was centrifuged at 11 500 g for 10 min. The supernatant et al., 1999). Red and green cells were counted in 30 fields of was stored at 4 1C. The pellet was resuspended in 5 mL of view. The fraction of live cells was calculated as the average 0.12 M Na HPO and 50 mL of proteinase K. The resus- ratio of green cells to total (red1green) cells. 2 4 pended pellet was incubated in a 37 1C water bath for 30 min, and then at 65 1C for 1 h, with periodic mixing by Plate counts shaking (every 5–15 min). The sample was centrifuged at Groundwater was serially diluted, and 500 mL aliquots were 11 500 g for 30 min. The new supernatant was added to spread on R2A plates. Plates incubated under aerobic refrigerated supernatant from the previous step. The com- conditions were buffered at pH 5.0 with 20 mM acetate bined supernatant was centrifuged at 8000 g for 30 min. buffer prior to solidification with agar (EMD, Gibbstown, Polyethylene glycol 8000 (PEG) (10 mL; 30% w/v in NJ). Plates incubated under anaerobic conditions were deionized water) was added and mixed by shaking. NaCl

c 2006 Federation of European Microbiological Societies FEMS Microbiol Ecol 58 (2006) 120–133 Published by Blackwell Publishing Ltd. All rights reserved Bacterial diversity in DNAPL-contaminated groundwater 123 solution (1.5 mL, 5 M) was added and mixed well, and the and sequences were manually checked for chimeric struc- sample was refrigerated overnight at 4 1C. The sample was tures. Phylogenetic analyses were performed using ARB centrifuged at 22 500 g for 30 min. Supernatant was dis- (Strunk & Ludwig, 1995) (http://www.arb-home.de/). The carded and the pellet was resuspended in 4 mL of TE buffer neighbor-joining algorithm was used to build the phyloge- and vortexed. Four 1 mL aliquots were each extracted with netic tree, with Jukes–Cantor correction (Jukes & Cantor, 500 mL of phenol, and this was followed by centrifugation 1969) followed by bootstrap analysis with Phylip 3.62 for 10 min at 14 000 g and removal of the organic layer. An (Felsenstein, 2004). (http://evolution.genetics.washington. identical extraction procedure was carried out using chloro- edu/phylip.html). The nearest cultured relative to each 16S form in place of phenol. Clean-up was performed using a rRNA gene sequence was determined from the phylogenetic MoBio UltraClean PCR Clean-up Kit (Carlsbad, CA). position and from the similarity matrix generated using the

same algorithm used to make the tree. Clones having Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 16S rRNA gene PCR, cloning, and sequencing sequence similarity greater than or equal to 97% were defined as an operational taxonomic unit (OTU), considered Two sets of oligonucleotide primers, one set consisting of to represent a taxon at or below the genus level (i.e. species/ 27f (50-GAGTTTGATCCTGGCTCA-30) and 1525r (50- strain) (Stackebrandt & Goebel, 1994; Palys et al., 1997). AGAAAGGAGGTGATCCAGCC-30), universal to the 16S rRNA gene of all bacteria (Lane, 1991), and the other, Bacterial culture isolation DHC1f (50-GATGAACGCTAGCGGCG-30) and DHC 1377r (50-GGTTGGCACATCGACTTCAA-30), specific to variable Genomic DNA was extracted using a MoBio UltraClean regions of the 16S rRNA gene of ‘Dehalococcoides’ group Microbial DNA kit (Carlsbad, CA) from 168 colonies bacteria (Hendrickson et al., 2002), were used in separate isolated under anaerobic conditions on a variety of agar PCR reactions. For ‘Dehalococcoides’-specific primers, PCR media, including: R2A (pH 5), Columbia anaerobic sheep reactions were performed under the conditions reported by blood agar (BD), plate count agar (Difco), nutrient agar Hendrickson et al. (2002). For universal bacterial primers, a (Difco), peptone–yeast extract–fructose (PYF) medium hot start protocol was utilized, using minor modifications of (Engelmann and Weiss, 1985) solidified with 15 g L1 agar, the method described by Rainey et al. (1996). Each reaction and SRB medium (Widdel & Bak, 1992) supplemented with (100 mL) contained 1 Taq buffer with Mg21, 2.5 U of Taq fatty acids (lactate, acetate and pyruvate, 10 mM each) DNA Polymerase and 0.75 TaqMaster PCR Enhancer modified by replacing bicarbonate buffer with phosphate (Brinkmann, Westbury, NY), as well as 200 mM each dNTP buffer (30 mM) and sulfide with L-cysteine. PCR was (Applied Biosystems, Forster City, CA) and 1 mL of purified performed using universal bacterial primers, and amplified community DNA. For the universal bacterial primers, 0.5 mg 16S rRNA genes were then sequenced and analyzed as of each primer was added. PCR products were verified by gel described above. electrophoresis prior to cloning. For the ‘Dehalococcoides’- specific primers, 0.3 mg of each primer was added. Rarefaction curves, diversity indices, and 16S rRNA gene products were cloned using a TOPO TA LIBSHUFF analyses Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA). Estimates of microbial diversity within the clone library PCR-amplified inserts were sequenced using a BigDye were further investigated using rarefaction analysis con- Terminator Cycle Sequencing Ready Reaction Kit (Applied ducted using the analytical approximation algorithm aRare- Biosystems, Foster City, CA). Sequencing reactions asso- factWin (Analytic Rarefaction, version 1.3, S. Holland, ciated with inserts corresponding to the universal primers http://www.uga.edu/strata/software/) with 95% confi- were performed as described by Rainey et al. (1996). dence limits. Clone coverage and Shannon diversity index Sequencing reactions associated with inserts corresponding values were calculated as described by Good (1953) and to ‘Dehalococcoides’-specific primers employed the DHC1f Muller¨ et al. (2002), respectively. primer for one reaction and the DHC774f primer (50- Comparisons between a partial section of the bacterial GGGAGTATCGACCCTCTC-30) (Hendrickson et al., 2002) 16S rRNA gene corresponding to bases 134–596 in Escher- in a second reaction. The temperature program for both ichia coli numbering (c. 500 bp) were tested using the reactions was as described by Hendrickson et al. (2002). webLIBSHUFF version 0.96 program (Singleton et al., Sequencing was performed using an ABI 377 Automated 2001) (http://LIBSHUFF.mib.uga.edu), which incorporates DNA Sequencer. the coverage formula of Good (1953) to generate homologous and heterologous coverage curves. Sequence libraries Phylogenetic analysis between samples were shuffled 999 times. The measured DNA sequences were manually verified using BioEdit ver- distance between curves was calculated using the Cramer–´ sion 4.7.8 (http://www.mbio.ncsu.edu/BioEdit/page2.html), von Mises test statistic (Pettitt, 1982). Distance matrices

FEMS Microbiol Ecol 58 (2006) 120–133 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 124 K.S. Bowman et al. submitted to webLIBSHUFF were generated using the Table 1. Chlorosolvent concentrations measured in groundwater from DNADIST program of Phylip, utilizing the Jukes–Cantor well W-1024-1 at the PPI site model for establishing nucleotide substitution rates. Concentration (mg L1)

Analyte Range AverageÃ Nucleotide sequence accession numbers 1,1,2,2-Tetrachloroethane 30.1–89.2 57.3 (0.341 mM) Representative sequences from the clone library constructed 1,1,2-Trichloroethane 239–530 367 (2.75 mM) using universal bacterial primers and ‘Dehalococcoides’- 1,2-Dichloroethane 363–756 540 (5.46 mM) speciﬁc primers have been deposited in GenBank under 1,2-Dichloropropane 54.5–83.9 67.3 (0.596 mM) accession numbers DQ196601–DQ196618 and DQ196591– Vinyl chloride 33.1–75.2 52.2 (0.835 mM) Hexachlorobutadiene 5.26–7.89 6.58 (0.0252 mM) DQ196600, respectively. Representative sequences from cul- w

Total 767–1492 1090 Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 tured isolates have been deposited in Genbank under Ã accession numbers DQ176646 and DQ196619–DQ196635. n =3. wThe total is the summation of individual constituents listed, measured during each sampling event. Results Table 2. Geochemical parameters measured in groundwater from well Contaminant concentrations and geochemistry W-1024-1 at the PPI site 1 The results of analysis of aqueous-phase organic contami- Concentration (mg L ) nants in the groundwater are summarized in Table 1, and Analyte Range AverageÃ geochemical constituents are summarized in Table 2. As Ethene 7.15–12.4 9.57 (0.342 mM) w shown in the tables, groundwater from the DNAPL source Ethane o 0.6–0.305 0.302 (1.00 102 mM) zone contained high aqueous-phase concentrations of a Methane 1.53–1.78 1.66 (0.104 mM) variety of chlorinated compounds, including chloroethanes Total inorganic carbon 59.6–103 81.3 (6.78 mM) (average of 57.3 mg L1 1,1,2,2-tetrachloroethane, 367 mg L1 Total organic carbon 358–543 478 (39.8 mM) 1,1,2-trichloroethane, and 540 mg L1 1,2-dichloroethane), Chloride 3710–5010 4510 (127 mM) 4 1 Nitrate o 0.05 o 0.05 ( o 8.06 10 mM) chloroethenes (52.2 mg L vinyl chloride), chloropropanes 3 1 Nitrite 0.296–0.640 0.428 (8.91 10 mM) (67.3 mg L 1,2-dichloropropane) and hexachloro-1,3-bu- Sulfate 632–660 644 (6.71 mM) 1 w tadiene (6.58 mg L ). Other chlorinated compounds may Sulﬁde o 0.02–0.036 0.022 (6.86 104 mM) also have been been present; however, because of the high Ferrous iron 901–1110 990 (17.7 mM) concentrations, detection levels were high (10–25 mg L1 for pH 5.1 5.1 most chlorinated volatile organic compounds) due to the Ãn =3. large dilution factor necessary for analysis. Historical data wAverage concentration was calculated using one half of the method from production wells indicate that additional chloro- detection limit (MDL) for measurements below the MDL. ethenes, aromatics and polycyclic aromatic hydrocarbons are also constituents in the DNAPL at the site (Clement 7 1 7 1 et al., 2002). ( 0.4) 10 cells mL and 3.7 ( 0.7) 10 cells mL . The presence of vinyl chloride (average concentration LIVE/DEAD BacLight microscopy counts revealed that 52.2 mg L1), ethene (9.57 mg L1), ethane (0.302 mg L1), 58% of the total cells were intact and potentially viable. and chloride (4510 mg L1), all products of reductive dechlor- Plate count results are from 28 days of incubation, ination, suggests that dechlorination is occurring in the source corresponding to the highest counts observed. No growth zone. The fact that these concentrations are substantially was observed on pH 5 R2A plates incubated under aerobic conditions. On pH 5 R2A plates incubated under anaerobic higher than background levels previously reported for wells 4 1 hydraulically upgradient of the DNAPL source zone (Clement conditions, 1.3 ( 0.2) 10 CFU mL were observed. et al., 2002) further supports this notion. Microcosm studies Spores made up a minor fraction (2.5%) of the population conducted using groundwater from within the DNAPL source enumerated on plates incubated under anaerobic condi- zone indicate that the microbial population is able to reduc- tions. These results conﬁrm that viable bacteria were present tively dechlorinate 1,1,2-trichloroethane, 1,2-dichloroethane within the groundwater. and vinyl chloride to ethene (unpublished data). Phylogeny of clone libraries and cultured Microbial enumeration isolates Direct counting of DAPI-stained cells in two independent Nearly complete (c. 1200 bp) 16S rRNA gene sequences of groundwater samples collected 17 days apart revealed 3.2 223 clones created using PCR amplicons produced using

Table 3. Phylogenetic summary of bacterial community based on 16S rRNA gene sequences ampliﬁed using universal bacterial primers Clone OTU GenBank No. of Identity ID no. accession no. clones Closest cultured phylogenetic relativeÃ (accession no.) (%) Putative taxon BLUC-A DQ196617 47 Megasphaera micronuciformis strain CCUG 45952 (T) (AF473834) 94 Low-G1C Gram-positive bacteria BLUC-B DQ196616 33 Bacterium isolate ZF3 (AJ404681) 95 Low-G1C Gram-positive bacteria BLUC-C DQ196618 31 Actinomyces meyeri strain ATCC 35568 (T) (X82451) 94 Actinobacteria BLUC-D DQ196610 28 Olsenella profusa strain DSM 13989 (T) (AF292374) 95 Actinobacteria BLUC-E DQ196614 15 Dialister pneumosintes strain ATCC 33048 (T) (X82500) 91 Low-G1C Gram-positive bacteria BLUC-F DQ196606 12 Clostridium sporosphaeroides strain ATCC 25781(T) (M59116) 94 Low-G1C Gram-positive bacteria BLUC-G DQ196611 7 Slackia heliotrinreducens strain ATCC 29202 (T) (AF101241) 87 Actinobacteria BLUC-H DQ196604 5 Sanguibacter keddieii strain ATCC 51767 (T) (X79450) 92 Actinobacteria

BLUC-I DQ196612 4 Selenomonas sputigena strain DSM 20758 (T) (AF287793) 91 Low-G1C Gram-positive bacteria Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 BLUC-J DQ196613 3 Solobacterium moorei strain JCM 10646 (AB031057) 89 Low-G1C Gram-positive bacteria BLUC-K DQ196615 2 Trichococcus pallustris strain DSM 9172 (T) (AJ296179) 94 Low-G1C Gram-positive bacteria BLUC-L DQ196608 2 Oscillospira guillermondi (AB040497) 93 Low-G1C Gram-positive bacteria BLUC-M DQ196603 1 ‘Dehalococcoides’ sp. strain VS (AY323233) 99.8 ‘Dehalococcoidetes’w BLUC-N DQ196607 1 ‘Brachymonas petroleovorans’ strain CHX (AY275432) 99.6 Betaproteobacteria BLUC-O DQ196605 1 ‘Desulfuromonas michiganensis’ strain BB1 (AF357915) 97.2 Deltaproteobacteria BLUC-P DQ196602 1 Mogibacterium pumilum strain ATCC 700696 (T) (AB021701) 97 Low-G1C Gram-positive bacteria BLUC-Q DQ196601 1 ‘Desulfovibrio ferrireducens’ strain CY1 (AJ582755) 96 Deltaproteobacteria BLUC-R DQ196609 1 Thermoanaerobacterium aotearoense strain DSM 10170 (T) (X93359) 82 Low-G1C Gram-positive bacteria

Ã(T) denotes type strains. wInformally proposed class (Hugenholtz and Stackebrandt, 2004). universal bacterial primers were sequenced. Of these, 28 designated as BLUC-C (where the prefix ‘BLI’ (Brooklawn clones (12.6%) were identified as putative chimeric se- Isolate) denotes isolate sequences and the prefix ‘BLUC’ quences and excluded from further analysis. Based on (Brooklawn Universal Clone) denotes clone library se- phylogenetic analysis of 16S rRNA gene sequences from the quences). These are most closely related to Actinomyces remaining 195 clones, the environmental 16S rRNA genes georgiae (X80413) and Actinomyces meyeri (X82451), respec- were tentatively grouped into 18 OTUs (Table 3). The 18 tively. Isolate OTU BLI-C was 98.3% similar to clone OTU OTUs in the universal clone library spanned four phyla, with BLUC-Q, which were both most closely related to ‘Desulfovi- low-G1C Gram-positive bacteria (62%) and Actinobacteria brio ferrireducens’ strain CY1 (AJ582755). Additionally, iso- (36%) having the largest representation, distantly followed late OTU BLI-Q was 98.1% similar to clone OTU BLUC-P, by Proteobacteria (1.5%) and Chloroflexi (0.5%). both of which were most similar to Mogibacterium pumilum Eighty-five clones derived from PCR products produced (AB021701) in terms of previously cultured isolates. using primers specific to ‘Dehalococcoides’ 16S rRNA gene fragments were sequenced (c. 1300 bp). Of these, 23 clones Clone library comparisons had identical sequences, and 62 clone sequences were unique. Clones were closely related to ‘Dehalococcoides A comparison of phyla represented in the universal clone ethenogenes’ strain 195, with similarity ranging from 99.5% library from the PPI site to three previously published clone (7 bp difference) to 99.8% (3 bp difference). Thus, while libraries of reductively dechlorinating microbial consortia is ‘Dehalococcoides’ was represented by a single sequence in the shown in Fig. 2. The first comparison library (Fig. 2b) is universal clone library, there appears to be an appreciable from a trichloroethene DNAPL-contaminated aquifer at Test amount of minor variation within the ‘Dehalococcoides’ Area North (TAN) at the US Department of Energy’s Idaho population at the PPI site. National Engineering and Environmental Laboratory, where The 168 16S rRNA gene sequences determined for lactate addition has been used to stimulate in situ reductive cultured isolates grouped into 18 OTUs (Table 4). The dechlorination of trichloroethene to ethene (Macbeth et al., isolates were distributed among three phyla: Actinobacteria 2004). The second and third comparison libraries (Fig. 2c (73%), low-G1C Gram positive bacteria (20%), and Proteo- and d) are from functionally stable reductively dechlorinat- bacteria (7%). The phylogenetic positions of the universal ing enrichment cultures in which trichloroethene (Richard- clone OTUs and the isolate OTUs based on 16S rRNA gene son et al., 2002) and dichloroethene (Gu et al., 2004) were sequences are shown in Fig. 1. Only three of the 18 isolate biodegraded to ethene. At the PPI and TAN sites, both in situ OTUs were represented in the clone library. The isolate OTU environments, only a small percentage of the universal designated as BLI-A was 99.8% similar to the clone OTU bacterial 16S rRNA clone libraries (1% at the PPI site and

Table 4. Phylogenetic summary of bacterial community based on 16S rRNA gene sequences from anaerobic isolates Repre- Isolate sentative OTU strain GenBank No of Closest cultured phylogenetic relativeÃ Identity ID no. ID no. accession no. isolates (accession no.) (%) Putative taxon BLI-A BL-96 DQ196624 75 Actinomyces georgiae strain DSM 6843 (T) (X80413) 94 Actinobacteria BLI-B BL-34 DQ196625 45 Propionibacterium propionicum strain ATCC 14157 (T) 95 Actinobacteria (X53216) BLI-C BL-157 DQ196634 11 ‘Desulfovibrio ferrireducens’ strain CY1 (AJ582755) 96 DeltaProteobacteria BLI-D BL-3 DQ196627 7 Clostridium thiosulfatireducens strain DSM13105 (T) 99.6 Low-G1C Gram-positive bacteria (AF317650)

BLI-E BL-164 DQ196632 5 Baccilus cereus strain ATCC 14579 (T) (AF290547) 100 Low-G1C Gram-positive bacteria Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 BLI-F BL-17 DQ196620 3 Clostridium sporogenes strain ATCC 3584 (T) (M59115) 99.7 Low-G1C Gram-positive bacteria BLI-G BL-26 DQ196630 3 Clostridium puniceum strain DSM 2619 (T) (X73444) 99.5 Low-G1C Gram-positive bacteria BLI-H BL-30 DQ196622 3 Clostridium tetanomorphum, strain NCIMB 11547 (S46737) 97.7 Low-G1C Gram-positive bacteria BLI-I BL-20 DQ196623 3 Clostridium frigidicarnis strain DSM 12271 (T) (AF069742) 97 Low-G1C Gram-positive bacteria BLI-J BL-14 DQ196619 3 Clostridium histolyticum strain ATCC 19401 (T) (M59094) 96 Low-G1C Gram-positive bacteria BLI-K BL-8 DQ196629 2 Clostridium diolis strain SH1 (T) (AJ458418) 99.0 Low-G1C Gram-positive bacteria BLI-L BL-10 DQ176646 2 Propionicimonas paludicola strain Wd (T) (AB078858) 97 Actinobacteria BLI-M BL-21 DQ196628 1 Clostridium bifermentans strain ATCC 638 (T) (X75906) 99.6 Low-G1C Gram-positive bacteria BLI-N BL-24 DQ196631 1 Clostridium paraputriﬁcum strain M-21 (AB032556) 97.1 Low-G1C Gram-positive bacteria BLI-O BL-22 DQ196626 1 Clostridium acetylbutylicum strain ATCC 824 (T) (X78070) 97 Low-G1C Gram-positive bacteria BLI-P BL-28 DQ196621 1 Clostridium sartagoformum strain DSM 1292 (T) (Y18175) 95 Low-G1C Gram-positive bacteria BLI-Q BL-152 DQ196635 1 Mogibacterium pumilum strain ATCC 700696 (T) (AB021701) 94 Low-G1C Gram-positive bacteria BLI-R BL-169 DQ196633 1 Sutterella stercoricanis strain CCUG 47620 (T) (AJ566849) 92 Betaproteobacteria

Ã(T) denotes type strains.

3% at the TAN site) were closely related to known dechlor- bacterial clone libraries determined in both the PPI site and inators (‘Desulfuromonas michiganensis’ and ‘Dehalococ- the TAN site were subjected to rarefaction analysis. Rarefac- coides’ sp. strain VS at the PPI site and Sulfurospirillum tion curves for the libraries approached, but did not reach, a multivorans and Trichlorobacter thiogenes at the TAN site). clear saturation, suggesting that analysis of additional clones Additionally, the clone libraries at each site were dominated would probably reveal further diversity (Fig. 3). Compar- by low-G1C Gram-positive bacteria, with 62% and 65% ison of rarefaction curves suggests that the bacterial popula- representation at the PPI site and TAN site respectively. In tion at the PPI site is less diverse than that at the TAN site, contrast, clone libraries constructed from enrichment cul- even though there was a more extensive clone-sampling tures reported by Richardson et al. (2002) and Gu et al. effort for the PPI site (195 clones) than for the TAN site (93 (2004) both contained a high proportion of clones clones). The null hypothesis that there is no difference (34–37%) closely related to ‘Dehalococcoides ethenogenes’, a between the species richness at the two sites was rejected known dechlorinator phylogenetically affiliated with a por- because the 95% confidence interval for each site did not tion of the Chloroflexi phylum that has been informally overlap at high sample size. Clone coverage was calculated to proposed as class ‘Dehalococcoidetes’ (Hugenholtz & Stack- be 91% at the PPI site and 76% at the TAN site, indicating ebrandt, 2004). The enrichment culture clone libraries were that a larger portion of diversity was captured at the PPI site. composed of 33–36% low-G1C Gram-positive bacteria, a In addition, there was less diversity at the PPI site as lower proportion than in the PPI (62%) and TAN (65%) measured by the Shannon diversity index (3.59 with an sites. Another similarity between the bacterial community evenness of 0.80 reported by Macbeth et al. (2004) for the compositions at the PPI and the TAN sites was the low TAN site vs. 2.19 with an evenness of 0.76 for the PPI site) representation of Proteobacteria, present at levels of 2% and and fewer universal clone OTUs (22 OTUs for the TAN site 3%, respectively. Other than the previously described simi- vs. 18 OTUs for the PPI site reported here). larities between the PPI site and the TAN site bacterial phyla, LIBSHUFF comparisons of PPI and TAN data indicate the phyla at each site were different: the PPI site contained that the communities were derived from significantly differ- 36% Actinobacteria, and the TAN site contained 14% ent bacterial populations (Table 5). The DC test statistic, a Bacteriodetes, 13% OP.11, 4% Spirochaetes, and 1% OP.3. measure of the overall distance between the homologous (X) To further compare the PPI and TAN clone libraries, and heterologous (XY) curves, was highest when the PPI sequence data and OTU designations from the universal clone or isolate libraries were compared to the TAN site

c 2006 Federation of European Microbiological Societies FEMS Microbiol Ecol 58 (2006) 120–133 Published by Blackwell Publishing Ltd. All rights reserved Bacterial diversity in DNAPL-contaminated groundwater 127 Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021

Fig. 1. Neighbor-joining tree generated with Jukes–Cantor correction showing relationship between OTUs from this study and reference organisms. Labels corresponding to sequences from this study are shown in bold. Clone library sequences are denoted by the prefix ‘BLUC’ (Brooklawn Universal Clone) and isolates are denoted by the prefix ‘BLI’ (Brooklawn Isolate). Bootstrap values of 95% or greater are indi- cated by solid circles at branch points. T, type species. Bar represents 10 substitutions per 100 nucleotide positions. clone libraries (comparisons 1 and 2). A significant portion Discussion of this difference is due to low heterologous coverage estimates of D 0.20 (data not shown), indicating that the Collectively, the enumeration results indicate that a large clones generated from the PPI site are composed of many number of microorganisms (43 107 cells mL1) are pre- different higher-order taxonomic groups than the TAN sent in the groundwater, and 58% of the cells are apparently aquifer site. LIBSHUFF comparisons of the PPI clone and viable in spite of the high chlorosolvent concentrations and isolate libraries were significantly different, although DC acidic pH observed at the sampling location. For compar- values were lower (comparison 3). Furthermore, poor ison purposes, the total cell concentration observed in heterologous coverage, D 0.15, indicates that these li- groundwater from the PPI site DNAPL source zone is braries do share deep-branching taxa, but overall differences roughly one-third of that observed in the laboratory-grown are driven by the dominance of clones related to the enrichment culture employed in a recent bioaugmentation Acidaminococcaceae family (clones in OTUs BLUC-A, -E, effort (Lendvay et al., 2003). The fact that direct cell counts -I, and -J) and the Clostridium isolates. It is likely that poor via microscopy were four orders of magnitude higher than heterologous values in this comparison are the result of plate counts demonstrates that a relatively low percentage known biases in using culture-dependent and culture- (o0.1%) of the microbial community can be cultured using independent techniques (Amann et al., 1995). the techniques employed in this study. This is further

Table 5. LIBSHUFF comparisons of isolate and clone libraries A Homologous (X) Heterologous (Y) coverage data coverage data B Comparison no. Library n Library P DC 1 PPI clones 195 TAN clones 0.001 16.6 C TAN clones 93 PPI clones 0.001 18.2 2 PPI isolates 168 TAN clones 0.001 17.7 D TAN clones 93 PPI isolates 0.001 17.4 3 PPI clones 195 PPI isolates 0.001 11.7 PPI isolates 168 PPI clones 0.001 3.75 0% 20% 40% 60% 80% 100% Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 Low G+C Gram positives 'Dehalococcoidetes' Actinobacteria Proteobacteria OP 11 OP 3 to ethene as part of its energy metabolism (Cupples et al., Bacteroidetes Spirochaetes 2003). In a study of 24 chloroethene-dechlorinating sites Nitrospira Others/not sequenced throughout North America and Europe, at locations where ‘Dehalococcoides’ was detected, complete degradation of Fig. 2. Comparison of putative bacterial phylotype distribution for 16S chloroethenes was observed. At locations where ‘Dehalococ- rRNA gene libraries from reductive dechlorinating environments. (a) Environmental sample from aquifer at the PPI site (this study). (b) coides’ was not detected, incomplete dechlorination was Environmental sample from DNAPL-contaminated aquifer at the TAN observed (Hendrickson et al., 2002). This suggests that the site (Macbeth et al., 2004). (c) Trichloroethene-degrading enrichment presence of ‘Dehalococcoides’ is a necessary prerequisite for culture (Richardson et al., 2002). (d) Dichloroethene-degrading enrich- complete degradation of chloroethenes (i.e. formation of ment culture (Gu et al., 2004). ethene). The results described herein further expand the pH habitat and geographic range over which ‘Dehalococcoides’ sp. have been detected. 25 The second clone OTU most similar to a previously cultured dechlorinating microorganism, BLUC-O, was 20 97.2% similar to ‘Desulfuromonas michiganensis’, an aceto- 15 trophic anaerobe isolated from freshwater sediment that reductively dechlorinates 1,1,2,2-tetrachloroethane (Sung 10 et al., 2003), a contaminant detected at high concentrations PPI clone library in the PPI groundwater measured in this study, to an 5 TAN clone library endproduct of cis-dichloroethene. Laboratory studies have

Expected number of OTUs 0 also demonstrated that ‘Desulfuromonas michiganensis’ can 0 20 40 60 80 100 120 140 160 180 200 grow in the presence of free-phase perchloroethene (reduc- Number of universal clones analyzed tively dechlorinating perchloroethene to an endproduct of Fig. 3. Rarefaction curves of the OTU diversity in the PPI and TAN site cis-dichloroethene), demonstrating that it is tolerant of high 16S rRNA gene universal clone libraries. chlorosolvent concentrations (Sung et al., 2003). Detection of sequences closely related to ‘Dehalococcoides’ and ‘Desulfuromonas michiganensis’ at the PPI site where supported by the fact that of the 18 OTUs in the universal DNAPL is present supports the notion that contaminant clone library, only three were represented by cultured degradation is probably occurring in groundwater near the isolates, and it is consistent with previous reports that the DNAPL source, a phenomenon previously demonstrated in majority of bacteria are not enumerated using standard plate laboratory studies (Nielsen & Keasling, 1999; Carr et al., count techniques (Amann et al., 1995). 2000; Yang & McCarty, 2000, 2002; Cope & Hughes, 2001; Of the 18 OTUs represented in the universal clone library, Dennis et al., 2003). This is particularly true in the case of only three (BLUC-M, -N, and -O), each consisting of just ‘Dehalococcoides‘ sp., because chlorosolvents are the only one clone, were greater than 97% similar to previously class of compounds known to serve as their terminal cultured microorganisms. Two of these, BLUC-M and electron acceptors. Provided that this can be further sub- BLUC-O, had high similarity to previously isolated dechlor- stantiated, it may broaden the possibility for implementa- inating microorganisms. The ﬁrst, BLUC-M, was 99.8% tion of monitored natural attenuation as a scientiﬁcally similar to ‘Dehalococcoides’ sp. strain VS, which is able to defensible remediation strategy for the DNAPL source zone reductively dechlorinate dichloroethene and vinyl chloride areas at this and other sites.

The third OTU with greater than 97% similarity to a chloromethane, along with several chloroethenes, including previously cultured bacterium, BLUC-N, grouped within perchloroethene, at high concentrations (0.9 mM) (Chang the b-Proteobacteria subdivision with 99.6% similarity to et al., 2000). Like the OTUs from the clone library, most of ‘Brachymonas petroleovorans’ strain CHX, an aerobic degra- the isolate OTUs are most closely related to genera with der of light hydrocarbons, including cyclohexane and to- fermentative capabilities. All but two isolate OTUs (BLI-Q luene, which was isolated from oil reﬁnery wastewater and BLI-R), or 166 of the 168 isolates (99% of the total), sludge (Rouvie`re & Chen, 2003). All of the universal were most closely related to fermentative organisms, with bacterial clones except for this one were most closely related eight OTUs (BLI-C, -F, -H, -I, -K, -M, -N and -O), covering to facultative or obligate anaerobes. 25 of the 168 isolates (15% of the total), representing The remaining 15 OTUs, covering 192 of the 195 uni- organisms most similar to previously described bacteria that

versal bacterial clone library sequences (98% of the total), produce hydrogen during fermentative processes. Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 were distantly related to previously cultured bacteria (less Representatives from the isolate OTU designated as BLI-B than 97% similar). Thus, it appears that the composition of were recently characterized and described as Propionicicella the microbial population in the DNAPL source zone at the superfundia gen. nov., sp. nov. (Bae et al., 2006). This new PPI site is relatively novel and probably includes several new bacterial genus grows at pH levels as low as 4.5, tolerates species and even new genera. Consequently, it is impossible high chlorosolvent concentrations, and produces propio- to fully elucidate what function bacteria represented by these nate and acetate as fermentation products both in the OTUs may serve within the microbial population and what presence and absence of chlorosolvents. The closest cultured contribution, if any, they may play in biotransforming phylogenetic relatives of several additional OTUs in both contaminants found at the PPI site. It is interesting to note, isolate and universal clone libraries are also known to be however, that of these 15 OTUs less than 97% similar to acid tolerant. For example, members of the Megasphaera previously cultured organisms, four (BLUC-B, -K, -L, and (Haikara & Helander, 2002) and the genera Actinomyces -G), comprising a total of 44 clones (23% of the library), (Takahashi & Yamada, 1999) and Clostridium (Kuhner et al., phylogenetically grouped somewhat closely with uncultured 2000; Flythe & Russell, 2005) have been reported to grow at or uncharacterized bacteria from dechlorinating popula- the pH level observed in groundwater at the PPI site. tions (Fig. 1). The dominance of low-G1C Gram-positive bacteria Additionally, 10 of the 18 universal clone OTUs (BLUC- observed in the PPI clone library and cultured isolates can A, -B, -C, -D, -F, -I, -J, -K, -Q and -R), comprising 162 be found in other anoxic or anaerobic environments such as clones (83%), were most closely related to genera known to termite guts (Kudo et al., 1998, Schmitt-Wagner et al., exhibit fermentative and/or hydrogen-producing capabil- 2003), mammal intestinal microbial communities (i.e. Suau ities, namely Clostridium (Li et al., 2003; Wang et al., 2003), et al., 1999; Hold et al., 2002; Leser et al., 2002), and deep- Trichococcus (Liu et al, 2002), Desulfovibrio (Bryant et al., water sediments (Humayoun et al., 2003), where fermenta- 1977; Traore et al., 1981), Megasphaera (Doyle et al., 1995; tive metabolism is common. It is important to note, how- Miller & Wolin, 1979), Selenomonas, Olsenella (Dewhirst ever, that the high representation of low-G1C Gram- et al., 2001), Solobacterium (Kageyama et al., 2000), Actino- positive bacteria in the PPI clone library may be partially myces (Slack, 1974; Slack & Gerencser, 1975), and Thermo- due to PCR bias, because Bacillus and Clostridium species anaerobacterium (Liu et al., 1996). Fermentative bacteria are are known to have high rRNA gene copy numbers (Klap- generally thought to play an important syntrophic role in penbach et al., 2001). the biodegradation of chlorinated solvents by producing As statistically shown by LIBSHUFF comparisons, the PPI compounds used as electron donors by dechlorinating site clone and isolate libraries were signiﬁcantly different. bacteria. Speciﬁcally, some previously isolated reductively Owing to limitations imposed by culturing conditions dechlorinating bacteria, including ‘Desulfuromonas michiga- (media, pH, carbon source, etc.) that do not closely replicate nensis’, utilize fermentation products such as acetate, lactate in situ environments, isolates that grow on commonly used or pyruvate as electron donors (Gerritse et al., 1996; Sanford culture media such as those employed in this study are not et al., 2002; Sung et al., 2003, and Sun et al., 2000). Others likely to represent dominant species from in situ populations

(e.g. ‘Dehalococcoides’) apparently utilize only H2 (Maymo-´ (Amann et al., 1995). Gatell et al., 1997; Adrian et al., 2000; He et al., 2003). Drawing deﬁnitive conclusions regarding the novelty of Of the cultured isolates, one OTU (BLI-M) was 99.6% the microbial population at the PPI site in comparison to similar to Clostridium bifermentans strain ATCC 638 populations at other sites is difﬁcult because few clone (X75906), and 99.5% similar to Clostridium bifermentans libraries have been constructed for similar sites (i.e. those strain DPH-1 (Y18787), a known dechlorinator. Strain with chloroethanes as dominant contaminants, acidic pH, DPH-1 is able to dechlorinate a number of chloroethanes, or presence of DNAPL). However, current data suggest that including 1,1,2-trichloroethane, dichloropropane and di- the bacterial phylotypes collected at the PPI site contrast

FEMS Microbiol Ecol 58 (2006) 120–133 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 130 K.S. Bowman et al. with those from the few comparable environments that have ‘Dehalococcoides’ 16S rRNA gene sequences (AF388531, been studied. The PPI site and TAN site populations were AF388532, and AF388533). They also reported that ground- statistically different based on LIBSHUFF analysis, partly water from an industrial site in Niagara Falls, NY contained due to the abundance of Actinobacteria (36%) in the PPI two unique 16S rRNA gene sequences (AF388544 and clone library, compared to no Actinobacteria in the TAN AF388545). Duhamel et al. (2002) reported that an enrich- clone library. Based on the high representation of Actino- ment culture, referred to as KB-1, derived from soil and bacteria in both the PPI clone and isolate libraries, it is groundwater from a contaminated site in southern Ontario, assumed that they play a large role in the in situ bacterial Canada, contained ﬁve distinct sequences closely related to community function; however, few or no Actinobacteria ‘Dehalococcoides ethenogenes’. Richardson et al. (2002) re- representatives have been reported for clone libraries ported 10 unique 16S rRNA gene sequences closely related to

from perchloroethene-, trichloroethene-, vinyl chloride- ‘Dehalococcoides’ in an anaerobic enrichment culture derived Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 and dichloropropane-dechlorinating enrichment cultures from contaminated soil at the Alameda Naval Air Station, CA. (Richardson et al., 2002; Schlotelburg¨ et al., 2002; Dennis Members of the ‘Dehalococcoides’ group can have a high et al, 2003; Rossetti et al., 2003; Gu et al., 2004). degree of similarity in the 16S rRNA gene but use different Potential reasons why the PPI bacterial community may chlorinated compounds as terminal electron acceptors. For be novel and less diverse than the TAN bacterial community example, three isolated ‘Dehalococcoides’ strains, strain VS, include the unique mixture of chlorinated solvents, low-pH strain 195, and strain CBDB1, are phylogenetically similar conditions and high sulfate concentrations at the PPI site. In on the basis of 16S rRNA gene sequences (498.8% similar), a study of soil bacterial communities at numerous locations but each meets its energy needs using different chlorinated worldwide, Fierer & Jackson (2006) found that environ- compounds as terminal electron acceptors (Maymo-Gatell´ mental pH was the best predictor of bacterial diversity and et al., 1999; Adrian et al., 2000; Cupples et al., 2003; richness, with peak diversity and richness values at a pH of Holscher¨ et al., 2004). Because of the relatively small number approximately 7, and decreased values at higher and lower of ‘Dehalococcoides’ strains isolated and characterized to pH. Community differences, however, may also result from date, genetic variability within the ‘Dehalococcoides’ 16S differences in geographic location, differences in geochem- rRNA clone library from the PPI site cannot yet be corre- istry, or the fact that there was addition of an exogenous lated with specific functional roles in contaminant transfor- electron donor (lactate) at the TAN site. Addition of electron mation. Future study of specific functional genes may donors has previously been shown to affect microbial provide an indication of metabolic diversity. community structure (North et al., 2004). This latter effect Despite the acidity and the saturation or near-saturation may be the reason for the higher portion of dechlorinators chlorosolvent concentrations observed at the PPI sampling observed in some enrichment cultures (Richardson et al., location, the data presented here suggest that a number of 2002; Gu et al., 2004) compared to in situ systems (Macbeth bacterial types, many of them novel, can grow in this et al., 2004; this study). It should be noted, however, that not environment. Among these OTUs were microorganisms all enrichment cultures follow this trend, and many clone closely related to known dechlorinators, fermenters, and libraries contain sequences from as yet uncultured bacteria hydrogen producers. These field data, supported by micro- with unknown functions (Dennis et al., 2003; Rossetti et al., cosm data and chemical analysis that revealed degradation 2003). In the study described here, 98% of the sequences products, suggest that dechlorination is probably occurring belong to taxonomic groups that are distinct at the species in spatial locations in close proximity to DNAPL. level or higher (less than 97% similar) and have not been described previously. It is anticipated that as the body of knowledge increases, the data presented herein will contri- Acknowledgements bute to a better understanding of bacterial structure–func- The authors gratefully acknowledge the Hazardous Sub- tion relationships in situ at this and other sites. stance Research Center South and Southwest (through the The significance of the fact that the clone library con- HSRC Environmental Biotechnology Initiative) and NPC structed using ‘Dehalococcoides’-specific PCR primers revealed Services for financial support. The authors thank Ms Cindy many closely related but unique 16S rRNA gene sequences is Henk of the LSU Socolofsky Microscopy Center for assis- unclear at this point. There is some evidence that multiple tance with microscopy. strains closely related to ‘Dehalococcoides ethenogenes’maybe responsible for the degradation of chlorinated compounds in some locations. For example, Hendrickson et al.(2002) References reported that the population in a microcosm constructed Adamson DT, McDade JM & Hughes JB (2003) Inoculation of from sludge collected from the bottom of a pond at an DNAPL source zone to initiate reductive dechlorination of industrial site in Beaumont, TX contained three unique PCE. Environ Sci Technol 37: 2525–2533.

Adamson DT, Lyon DY & Hughes JB (2004) Flux and product reclassiﬁcation of Lactobacillus uli as Olsenella uli comb. nov. distribution during biological treatment of tetrachloroethene and description of Olsenella profusa sp. nov. Int J Syst Evol dense non-aqueous-phase liquid. Environ Sci Technol 38: Microbiol 51: 1797–1804. 2021–2028. De Wildeman S, Diekert G, van Langenhove H & Verstraete W Adrian L, Szewzyk U, Wecke J & Gorisch H (2000) Bacterial (2003) Stereoselective microbial dehalorespiration with dehalorespiration with chlorinated benzenes. Nature 408: vicinal dichlorinated alkanes. Appl Environ Microbiol 69: 580–583. 5643–5647. Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic Doyle L, McInerney J, Mooney J, Powell R, Haikara A & Moran A identiﬁcation and in situ detection of individual microbial (1995) Sequence of the gene encoding the 16S rRNA of the cells without cultivation. Microbial Rev 59: 143–169. beer spoilage organism Megasphaera cerevisiae. J Ind Microbiol Bae HS, Moe WM, Yan J, Tiago I, da Costa MS & Rainey FA 15: 67–70. (2006) Propionicicella superfundia gen. nov., sp. nov., a Duhamel M, Wehr SD, Yu L, Rizvi H, Seepersad D, Dworatzek S, Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 chlorosolvent-tolerant propionate-forming, facultative Cox EE & Edwards EA (2002) Comparison of anaerobic anaerobic bacterium isolated from contaminated dechlorinating enrichment cultures maintained on groundwater. Systematic and Appl Microbiol, in press tetrachloroethene, trichloroethene, cis-dichloroethene and (doi:10.1016/j.syapm.2005.11.004). vinyl chloride. Water Res 36: 4193–4202. Boulos L, Prevost´ M, Barbeau B, Coallier J & Desjardins R (1999) Engelmann U & Weiss N (1985) Megasphaera cerevisiae sp. nov.: a LIVE/DEADs BacLightTM: application of a new rapid staining new Gram-negative obligately anaerobic coccus isolated from method for direct enumeration of viable and total bacteria in spoiled beer. System Appl Microbiol 6: 287–290. drinking water. J Microbiol Methods 37: 77–86. Felsenstein J (2004) PHYLIP (Phylogeny Inference Package) Bryant MP, Campbell LL, Reddy CA & Crabill MR (1977) Growth version 3.6. Distributed by the author. Department of Genome of Desulfovibrio in lactate or ethanol media low in sulfate in Sciences, University of Washington, Seattle.

association with H2-utilizing methanogenic bacteria. Appl Fierer N & Jackson RB (2006) The diversity and biogeography of Environ Microbiol 33: 1162–1169. soil bacterial communities. Proc Nat Academy Sciences, USA Carr CS, Garg S & Hughes JB (2000) Effect of dechlorinating 103: 626–631. bacteria on the longevity and composition of PCE-containing Flythe MD & Russell JB (2005) The ability of acidic pH, growth nonaqueous phase liquids under equilibrium dissolution inhibitors and glucose to increase the proton motive force and conditions. Environ Sci Technol 34: 1088–1094. energy spilling of amino acid-fermenting Clostridium Chang YC, Hatsu M, Jung K, Yoo YS & Takamizawa K (2000) sporogenes md1 cultures. Archives of Microbiol 183: 236–242. Isolation and characterization of a tetrachloroethylene Gerritse J, Renard V, Gomes TMP, Lawson PA, Collins MD & dechlorinating bacterium, Clostridium bifermentans DPH-1. Gottschal JC (1996) Desulfitobacterium sp. strain PCE1, an J Biosci Bioeng 89: 489–491. anaerobic bacterium that can grow by reductive Chen C, Puhakka JA & Ferguson JF (1996) Transformations of dechlorination of tetrachloroethene or ortho-chlorinated 1,1,2,2-tetrachloroethane under methanogenic conditions. phenols. Arch Microbiol 165: 132–140. Environ Sci Technol 30: 542–547. Good IJ (1953) The population frequencies of species and the Clement TP, Truex MJ & Lee P (2002) A case study for estimation to the population parameters. Biometrika 40: demonstrating the application of US EPA’s monitored natural 237–264. attenuation screening protocol at a hazardous waste site. Gu AZ, Hedlund BP, Staley JT, Strand SE & Stensel HD (2004) J Contam Hydrol 59: 133–162. Analysis and comparison of the microbial community Cope N & Hughes JB (2001) Biologically-enhanced removal of structures of two enrichment cultures capable of reductively PCE from NAPL source zones. Environ Sci Technol 35: dechlorinating TCE and cis-DCE. Environ Microbiol 6: 45–54. 2014–2021. Holscher¨ T, Krajmalnik-Brown R, Ritalahti KM, von Cupples AM, Spormann AM & McCarty PL (2003) Growth of a Wintzingerode F, Gorisch¨ H, Loffler¨ FE & Adrian L (2004) Dehalococcoides-like microorganism on vinyl chloride and cis- Multiple nonidentical reductive-dehalogenase-homologous dichloroethene as electron acceptors as determined by genes are common in Dehalococcoides. Appl Environ Microbiol competitive PCR. Appl Environ Microbiol 69: 953–959. 70: 5290–5297. Dennis PC, Sleep BE, Fulthorpe RR & Liss SN (2003) Haikara A & Helander I (2002) Pectinatus, Megasphaera and Phylogenetic analysis of bacterial populations in an anaerobic Zymophilus. The Prokaryotes: An Evolving Electronic Database microbial consortium capable of degrading saturation for the Microbiological Community. 3rd edn, release 3.5 concentrations of tetrachloroethylene. Can J Microbiol 49: (Dworkin M, ed), pp. 1–24. Springer Verlag, New York. 15–27. He J, Ritalahti KM, Yang K, Koenigsberg S & Loffler¨ FE (2003) Dewhirst F, Paster B, Tzellas N, Coleman B, Downes J, Spratt D & Detoxification of vinyl chloride to ethane coupled to growth of Wade W (2001) Characterization of novel human oral isolates an anaerobic bacterium. Nature 424: 62–65. and cloned 16S rDNA sequences that fall in the family Hendrickson ER, Payne JA, Young RM, Starr MG, Perry MP, Coriobacteriaceae: description of Olsenella gen. nov., Fahnestock S, Ellis DE & Ebersole RC (2002) Molecular

analysis of Dehalococcoides 16S ribosomal DNA from for chlorinated solvent remediation. Environ Sci Technol 37: chloroethene-contaminated sites throughout North America 1422–1431. and Europe. Appl Environ Microbiol 68: 485–495. Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M & Herrick JB, Madsen EL, Batt CA & Ghiorse WC (1993) Mller K (2002) Culture-independent analysis of gut bacteria: Polymerase chain reaction amplification of naphthalene- the pig gastrointestinal tract microbiota revisited. Appl Environ catabolic and 16S rRNA gene sequences from indigenous Microbiol 68: 673–690. sediment bacteria. Appl Environ Microbiol 59: 687–694. Liu SY, Rainey FA, Morgan HW, Mayer F & Wiegel J (1996) Hold GL, Pryde SE, Russell VJ, Furrie E & Flint HJ (2002) Thermoanaerobacterium aotearoense sp. nov., a slightly Assessment of microbial diversity in human clonic samples by acidophilic, anaerobic thermophile isolated from various hot 16S rDNA sequence analysis. FEMS Microbiol Ecol 39: 33–39. springs in New Zealand, and emendation of the genus Hollinger C, Hahn D, Harmsen H, Ludwig W, Schumacher W, Thermoanaerobacterium. Int J Syst Bacteriol 46: 388–395. Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 Tindall B, Vazquez F, Weiss N & Zehnder AJB (1998) Liu J, Tanner RS, Schumann P, Weiss N, McKenzie CA, Janssen Dehalobacter restrictus gen. nov. and sp. nov., a strictly PH, Seviour EM, Lawson PA, Allen TD & Seviour RJ (2002) anaerobic bacterium that reductively dechlorinates tetra- and Emended description of the genus Trichococcus, description of trichloroethene in an anaerobic respiration. Arch Microbiol Trichococcus collinsii sp. nov., and reclassification of 169: 313–321. Lactosphaera pasteurii as Trichococcus pasteurii comb. nov. and Hugenholtz P & Stackebrandt E (2004) Reclassification of of Ruminococcus palustris as Trichococcus palustris comb. nov. Sphaerobacter thermophilus from the subclass in the low-G1C gram-positive bacteria. Int J Syst Bacteriol 52: Sphaerobacteridae in the phylum Actinobacteria to the class 1113–1126. Li H, Morimoto K, Kimura T, Sakka K & Ohmiya K (2003) A new Thermomicrobia (emended description) in the phylum type of beta-N-acetylglucosaminidase from hydrogen- Chloroflexi (emended description). Int J Syst Evol Microbiol 54: producing Clostridium paraputrificum M-21. J Bioscience 2049–2051. Bioeng 96: 268–278. Humayoun SB, Bano N & Hollibaugh JT (2003) Depth Lorah M & Olsen L (1999) Natural attenuation of chlorinated distribution of microbial diversity in Mono Lake, a volatile organic compounds in a freshwater tidal wetland: field meromictic soda lake in California. Appl Environ Microbiol 69: evidence of anaerobic biodegradation. Water Resources Res 35: 1030–1042. 3811–3827. Jukes TH & Cantor CR (1969) Evolution of protein molecules. Luijten MLGC, de Weert J, Smidt H, Boschker HTS, de Vos WM, Mammalian Protein Metabolism (Munro NH, ed), pp. Schraa G & Stams AJM (2003) Description of Sulfurospirillum 121–132. Academic Press, New York, NY. halorespirans sp. nov., an anaerobic, tetrachloroethene- Kageyama A & Benno Y (2000) Phylogenic and phenotypic respiring bacterium, and transfer of Dehalospirillum characterization of some eubacterium-like isolates from multivorans to the genus Sulfurospirillum as Sulfurospirillum human feces: description of Solobacterium moorei gen. nov., sp. multivorans comb. Int J Syst Evol Microbiol 53: 787–793. nov. Microbiol Immunol 4: 223–227. Macbeth TW, Cummings DE, Spring S, Petzke LM & Sorenson Klappenbach JA, Saxman PR, Cole JR & Schmidt TM (2001) KS (2004) Molecular characterization of a dechlorinating rrndb: the ribosomal RNA operon copy number database. community resulting from in situ biostimulation in a Nucleic Acids Res 29: 181–184. trichloroethene-contaminated deep, fractured basalt aquifer Krumholz L (1997) Desulfuromonas chloroethenica sp. nov. uses and comparison to a derivative laboratory culture. Appl tetrachloroethylene and trichloroethylene as electron Environ Microbiol 70: 7329–7341. acceptors. Int J Syst Bacteriol 47: 1262–1263. Maymo-Gatell´ X, Chien Y, Gossett J & Zinder S (1997) Isolation Kudo T, Ohkuma M, Moriya S, Noda S & Ohtoko K (1998) of a bacterium that reductively dechlorinates Molecular phylogenetic indentification of the intestinal tetrachloroethene to ethene. Science 276: 1568–1571. anaerobic microbial community in the hindgut of termite, Maymo-Gatell´ X, Anguish T & Zinder SH (1999) Reductive Reticulitermes speratus, without cultivation. Extremophiles dechlorination of chlorinated ethenes and 1,2-dichloroethane 2: 151–161. by ‘Dehalococcoides ethenogenes ’ 195. Appl Environ Microbiol Kuhner CH, Matthies C, Acker G, Schmittroth M, Gossner AS & 65: 3108–3113. Drake HL (2000) Clostridium akagii sp. nov. and Clostridium Miller TL & Wolin MJ (1985) Methanosphaera stadtmaniae gen. acidisoli sp. nov.: acid-tolerant, N(2)-fixing clostridia isolated nov., sp. nov.: a species that forms methane by reducing from acidic forest soil and litter. Int J Syst Evol Microbiol 50: methanol with hydrogen. Arch Microbiol 141: 116–122. 873–881. Muller¨ AK, Westergaard K, Christensen S & Srensen SJ (2002) Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic Acid The diversity and function of soil microbial communities Techniques in Bacterial Systematics (Stackebrandt E & exposed to different disturbances. Microbial Ecology 44: 49–58. Goodfellow M, eds), pp. 115–175. Wiley, Chichester. Nielsen RB & Keasling JD (1999) Reductive dechlorination of Lendvay JM, Loffler¨ FE, Dollhopf M, et al. (2003) Bioreactive chlorinated ethene DNAPLs by a culture enriched from barriers: a comparison of bioaugmentation and biostimulation contaminated groundwater. Biotechnol Bioeng 62: 160–165.

North NN, Dollhopf SL, Petrie L, Istok JD, Balkwill DL & Kostka Slack JM & Gerencser MA (1975) Actinomyces, Filamentous JE (2004) Change in bacterial community structure during in Bacteria: Biology and Pathogenicity. Burgess, Minneapolis, MN. situ biostimulation of subsurface sediment cocontaminated Stackebrandt E & Goebel BM (1994) Taxonomic note: a place for with uranium and nitrate. Appl Environ Microbiol 70: DNA–DNA reassociation and 16S rRNA sequence analysis in 4911–4920. the present species definition in bacteriology. Int J Syst Palys T, Nakamura LK & Cohan FM (1997) Discovery and Bacteriol 44: 846–849. classification of ecological diversity in the bacterial world: the Strunk O & Ludwig W (1995) ARB—a software environment for role of DNA sequence data. Int J Syst Bacteriol 47: 1145–1156. sequence data. [Online] http://[email protected] Pankow JF & Cherry JA (1996) Dense Chlorinated Solvents and muechen.de. Department of Microbiology, Technical Other DNAPLs in Groundwater: History, Behavior, and University of Munich, Germany. Remediation. Waterloo Press. Suau A, Bonnet R, Sutren M, Godon JJ, Gibson GR, Collins MD Pettitt AN (1982) Cramer-von Mises statistic. Encyclopedia of & Dore´ J (1999) Direct analysis of genes encoding 16S rRNA Downloaded from https://academic.oup.com/femsec/article/58/1/120/468855 by guest on 29 September 2021 Statistical Sciences (Kotz S & Johnson NL, eds), pp. 220–221. from complex communities reveals many novel molecular Wiley-Interscience, New York, N.Y. species within the human gut. Appl Environ Microbiol 65: Rainey FA, Ward-Rainey N, Kroppenstedt RM & Stackebrandt E 4799–4807. (1996) The genus Nocardiopsis represents a phylogenetically Sun B, Cole J, Sanford R & Tiedje J (2000) Isolation and coherent taxon and a distinct actinomycete lineage: proposal characterization of Desulfovibrio dechloracetivorans sp. Nov., of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46: 1088–1092. a marine dechlorinating bacterium growing by coupling Richardson RE, Bhupathiraju VK, Song DL, Goulet TA & the oxidation of acetate to the reductive dechlorination Alvarez-Cohen L (2002) Phylogenetic characterization of of 2-chlorophenol. Appl Environ Microbiol 66: microbial communities that reductively dechlorinate TCE 2408–2413. based upon a combination of molecular techniques. Environ Sung Y, Ritalahti K, Sanford R, Urbance J, Flynn S, Tiedje J & Sci Technol 36: 2652–2662. Loffler¨ F (2003) Characterization of two tetrachloroethene- Rosencrantz D, Rainey FA & Janssen PH (1999) Culturable reducing, acetate-oxidizing anaerobic bacteria and their populations of Sporomusa spp. and Desulfovibrio spp. in the description as Desulfumonas michiganensis sp. nov. Appl anoxic bulk soil of flooded rice microcosms. Appl Environ Environ Microbiol 69: 2964–2973. Microbiol 65: 3526–3533. Takahashi N & Yamada T (1999) Effects of pH on the glucose and Rossetti S, Blackall LL, Majone M, Hugenholtz P, Plumb JJ & lactate metabolisms by the washed cells of Actinomyces Tandoi V (2003) Kinetic and phylogenetic characterization of naeslundii under anaerobic and aerobic conditions. Oral an anaerobic dechlorinating microbial community. Microbiol and Immunology 14: 60–65. Microbiology-SGM 149: 459–469. Traore AS, Hatchikian CE, Belaich JP & Le Gall J (1981) Rouvie`re PE & Chen MW (2003) Isolation of Brachymonas Microcalorimetric studies of the growth of sulfate-reducing petroleovorans CHX, a novel cyclohexane-degrading beta- bacteria: energetics of Desulfovibrio vulgaris growth. J Bacteriol proteobacterium. FEMS Microbiol Lett 227: 101–106. 145: 191–199. Sanford R, Cole J & Tiedje J (2002) Characterization and Truex MJ, Skeen RS, Butcher MG & Clement TP (2001) description of Anaeromyxobacter dehalogenans gen. nov., sp. Microcosm Studies for Evaluating Chlorinated Ethene and nov., an aryl-halorespiring facultative anaerobic Ethane Degradation Kinetics at the Brooklawn Site. Batelle, myxobacterium. Appl Environ Microbiol 68: 893–900. Pacific Northwest Division, Richland, WA. Schlotelburg¨ C, von Wintzingerode C, Hauck R, van von Wintzingerode F, Selent B, Hegemann W & Gobel¨ UB (1999) Wintzingerode F, Hegemann W & Gobel¨ UB (2002) Microbial Phylogenetic analysis of an anaerobic, trichlorobenzene structure of an anaerobic bioreactor population that transforming microbial consortium. Appl Environ Microbiol continuously dechlorinates 1,2-dichloropropane. FEMS 65: 283–286. Microbiol Ecol 39: 229–237. Wang CC, Chang CW, Chu CP, Lee DJ, Chang BV & Liao CS Schmitt-Wagner D, Friedrich M, Wagner B & Brune A (2003) (2003) Producing hydrogen from wastewater sludge by Phylogenetic diversity, abundance and axial distribution of Clostridium bifermentans. J Biotechnol 102: 83–92. bacteria in the intestinal tract of two soil-feeding termites Widdel F & Bak F (1992) Gram-negative mesophilic sulfate- (Cubitermes spp.). Appl Environ Microbiol 69: 6007–6017. reducing bacteria. The Prokaryotes. 2nd edn (Balows A, Truper¨ Singleton DR, Furlong MA, Rathbun SL & Whitman WB (2001) HG, Dworkin W, Harder W & Schleifer K-H, eds), pp. Quantitative comparisons of 16S rRNA gene sequence libraries 3352–3378. Springer-Verlag, Berlin, Germany. from environmental samples. Appl Environ Microbiol 67: Yang YR & McCarty PL (2000) Biologically enhanced dissolution 4374–4376. of tetrachloroethene DNAPL. Environ Sci Technol 34: Slack JM (1974) Family Actinomycetaceae Buchanan 1918 and 2979–2984. Genus Actinomyces Harz 1877. Bergey’s Manual of Yang YR & McCarty PL (2002) Comparison between donor Determinative Bacteriology. 8th edn (Buchanan RE & Gibbons substrates for biologically enhanced tetrachloroethene DNAPL NE, eds), pp. 659–667. Williams & Wilkins, Baltimore, MD. dissolution. Environ Sci Technol 36: 3400–3404.