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 LÀ1, 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 Â 107 cells mLÀ1 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.