Environ Geochem Health (2013) 35:535–549 DOI 10.1007/s10653-013-9513-3 ORIGINAL PAPER Microbiota associated with the migration and transformation of chlorinated aliphatic hydrocarbons in groundwater Xiangyu Guan • Fei Liu • Yuxuan Xie • Lingling Zhu • Bin Han Received: 7 November 2012 / Accepted: 10 February 2013 / Published online: 19 February 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Pollution of groundwater with chlorinated monooxygenases and methane monooxygenases capa- aliphatic hydrocarbons (CAHs) is a serious environ- ble of degradation of PCE and TCE were detected, mental problem which is threatening human health. demonstrating the major mechanism for PCE and TCE Microorganisms are the major participants in degrad- degradation and possibility for in situ remediation by ing these contaminants. Here, groundwater contami- addition of oxygen in this study. nated for a decade with CAHs was investigated. Numerical simulation and field measurements were Keywords Chlorinated aliphatic hydrocarbons Á used to track and forecast the migration and transfor- Degradation Á Microbiota Á 16S rRNA Á Remediation mation of the pollutants. The diversity, abundance, and possible activity of groundwater microbial com- munities at CAH-polluted sites were characterized by molecular approaches. The number of microorgan- Introduction isms was between 5.65E?05 and 1.49E?08 16S rRNA gene clone numbers per liter according to Chlorinated aliphatic hydrocarbons (CAHs) are ideal quantitative real-time PCR analysis. In 16S rRNA chemical agents for use as industrial solvents, dry gene clone libraries constructed from samples along cleaning agents, and degreasers (Ellis et al. 2000; the groundwater flow, eight phyla were detected, and Barnes et al. 2010). However, due to irresponsible Proteobacteria were dominant (72.8 %). The micro- storage, spills, and disposal practices, CAHs have bial communities varied with the composition and permeated soil and groundwater systems throughout concentration of pollutants. Meanwhile, toluene the world over the past decades (Middeldorp et al. 1998; Aulenta et al. 2010; Hendrickson et al. 2002). As the most prevalent CAHs, tetrachloroethene (PCE), X. Guan Á F. Liu (&) Á Y. Xie Á L. Zhu Á B. Han trichloroethene (TCE), dichloroethene (DCE), and Beijing Key Laboratory of Water Resources and vinyl chloride (VC) are the chief contaminants Environmental Engineering, School of Water Resources affecting water quality (Moran et al. 2007). They and Environment, China University of Geosciences, No.29 Xueyuan Road, Haidian District, Beijing 100083, threaten human health because of their toxicity and People’s Republic of China carcinogenicity (McCarty 1997; Yeh and Kastenberg e-mail: [email protected] 1991; Toraason et al. 1999). Biodegradation contributed to the migration and X. Guan School of Ocean Sciences, China University of transformation of CAHs in groundwater (Zhang and Geosciences, Beijing 100083, People’s Republic of China Bennett 2005; Griffin et al. 2004; Loffler and Edwards 123 536 Environ Geochem Health (2013) 35:535–549 2006). In general, biological degradation of PCE or study bacterial communities and distribution along the TCE occurs by sequential dechlorination from PCE to path of CAH-polluted groundwater. Combined with TCE to cis-DCE, trans-DCE, 1,1-DCE, and then to the environmental parameters and microbiological VC and finally to non-toxic ethene (Ritalahti et al. analysis, it will allow us to better understand mech- 2006b; Mattes et al. 2010). However, the reductive anisms that may contribute to the migration and dechlorination of parent contaminants (PCE and TCE) transformation of CAHs. is not complete, yielding the more toxic intermediates DCE (trans-orcis-DCE) and VC in anaerobic contaminated groundwater (Cheng et al. 2010; Smidt and de Vos 2004; Maymo-Gatell et al. 2001; Mattes Materials and methods et al. 2010). In addition, degradation products and their proportions are closely related to microbial Study area and sampling composition and environmental factors (Bhatt et al. 2007; Mattes et al. 2010). The study area is located in the southwest of North To date, many microbes capable of dechlorination China Plain. The region annual average temperature have been isolated, such as Sulfurospirillum multivo- and rainfall are 11.6 °C and 627.2 mm, respectively. rans, Pseudomonas, Methylomonas methanica, Rainfall between June and September accounts for Dehalococcoides ethenogenes (Dhc), Nitrosomonas 80 % of the annual precipitation. Average annual europaea, and Burkholderia kururiensis (Luijten et al. water surface evaporation is 1,711.8–1,807.1 mm. 2003; Utkin et al. 1994; MaymoGatell et al. 1997; The sampling sites are located in the second terrace of Arciero et al. 1989; Zhang et al. 2000a). Under alluvial plain, which are wide and flat on terrace anaerobic conditions, Dhc 195 was the first strain surface. The study area belongs to the upstream of found capable of complete reductive dechlorination of alluvial fan. Quaternary strata are mainly clayey sand PCE/TCE and conversion beyond DCEs to VC and and gravel, whose lithology is single. The lithology is ethene, and Dhc GT and Dhc VS were capable of mainly clayey sand on surface. The aquifer in this area dechlorinating TCE to ethene as the major end product is a single gravel layer with high permeability. The (Johnson et al. 2009; Nijenhuis and Zinder 2005; thickness is generally 35–45 m with flow from west to Krajmalnik-Brown et al. 2004; He et al. 2003). The east. The overlying soil is permeable clayey sand, with functional reductive dehalogenase (RDase) genes in low thickness; thus, it is an advantage to groundwater Dhc code for enzymes that catalyze special dechlori- supply. Dispersed exploitation of groundwater nation steps directly. The genes include pceA (for PCE through domestic wells is the main means of con- to TCE), tceA (for TCE to VC), and bvcA and vcrA (for sumption, followed by evaporation and groundwater DCEs to ethene) (Magnuson et al. 2000; Krajmalnik- outflow to the downstream side. The removal rate is Brown et al. 2004; Sung et al. 2006). Under aerobic 34.3 9 104 m3/a/km2. Study area groundwater cone and hypoxic conditions, there are also some other of depression was not apparent. enzymes such as biphenyl dioxygenase, toluene Groundwater samples (25 L) were individually dioxygenase, toluene monooxygenases (tMMO), and obtained from four sampling sites, Shuangfeng methane monooxygenases (sMMO) that are involved (SF), Shuizhan (SZ), Fajicun (FJC), and Humuxi in biodegrading CAHs and related contaminants (HMX), along groundwater flow in winter of 2010. co-metabolized with CAHs (Kikuchi et al. 2002; Five-liter water samples were kept in darkness in Ryoo et al. 2001; Leahy et al. 1996). glass bottles at 4 °C prior to being used for Recent studies focused on the characteristics of measuring water quality. Bacteria from 20 L of CAH degradation in certain bacterial species or genus, water from each sample were harvested by mem- but few bacterial communities and degradation mech- brane filtration with 0.22 lm-pore-size Millipore anism in natural CAH-polluted groundwater were GSWP filters and then suspended in 10 mL phys- revealed. In the southwest of North China Plain, an iological saline within 6 h. After centrifugation of area contaminated with CAHs for over a decade was the above mentioned suspension, the concentrated detected based on monitoring data collected by our microbial samples were retained for further use. All laboratory (Li et al. 2005). It supplied a good area to tools were autoclaved. 123 Environ Geochem Health (2013) 35:535–549 537 Environmental parameter analysis strength is 2 9 105m3/a/km2. Precipitation recharge was only 0.002 m/day and ignored due to hardened Analysis on volatile organic compounds (VOCs) of surface. In the flow model, the chemical concentrations the groundwater samples was performed with an found in the investigation sites in 2002 were used as the automatic static headspace sampler. Purge & Trap- initial conditions and as analogs to simulate the Gas Chromatography–Mass Spectrometry (P&T-GC– movement of PCE and TCE. Parameter calibration MS) with internal standards was used to quantify was carried out based on the data from 2005 (Table 1). VOCs (HP Tekmar 3100, Hewlett Packard), 6890 GC Degradation of PCE occurred by sequential dechlori- (Agilent, Santa Clara, CA, USA), DB-VRX MS nation from PCE to TCE to cis-DCE, trans-DCE, and Capillary Column (60 m 9 0.25 mm 9 1.4 lm, then to VC in this study. The partition ratios of PCE and Hewlett Packard), and 5973 MS (Agilent). Purge & TCE were from equilibrium experiment using vadose Trap conditions: injection sample volume: 10 mL. soil medium of this area. PCE attenuation rate fits first- Transfer line and valve temperatures are 150 °C. order kinetic model (Ferrey et al. 2004;Luetal.2006); Purge temperature: 40 °C, purge time: 11 min, and a PCE degradation rate (*10-4) was gained in this area purge flow: 40 mL/min. Desorb temperature: 180 °C, through the concentration of same site in 2002 and and desorb time: 2 min. Bake temperature: 225 °C, 2005. and bake time: 12 min. GC oven temperature: initially 40 °C held for 5 min, gradually increased to 140 °Cat Total DNA extraction and PCR amplification the rate of 6 °C/min, and then gradually increased to 210 °C at the rate of 5 °C/min. Injection temperature: DNA extractions were performed using the FastDNA 150 °C. Column flow rate: 1.0 mL/min. Split ratio: Kit for Soil (MP Biomedicals, Solon, OH, USA). DNA 10:1. MS conditions: ion source: EI 70 eV. Acquisi- concentration and quality was measured using a tion mode: SIM. The reporting limits were 1.0 mg/L NanoDrop ND-1000 spectrophotometer (Thermo Sci- for all the analyses. Total organic carbon (TOC) entific, Wilmington, DE, USA) and electrophoresis. was analyzed using a TOC-VCPN Carbon Analyzer 16S rRNA gene was amplified by the universal primers (Shimadzu, Kyoto, Japan). The reporting limit was 16S-27F (50-AGAGTTTGATCATGGC-30) and 16S- 0.5 mg/L.