Microbiota Associated with the Migration and Transformation of Chlorinated Aliphatic Hydrocarbons in Groundwater

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 used to track and forecast the migration and transfor- Degradation Microbiota 16S rRNA Remediation mation of the pollutants. The diversity, abundance, and possible activity of groundwater microbial communities 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 F. Liu (&) Y. Xie L. Zhu 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. Fe2? and total Fe concentrations were 1492R (50-TACCTTGTTACGACTT-30) (Sangon, determined by o-phenanthroline spectrophotometric Shanghai, China) to measure the diversity of microbes. method using a HP 8453 UV–Visible Spectrophotom- Twenty-five microliters of reaction contained 1 ng eter (Hewlett Packard, Palo Alto, CA, USA) (APHA purified DNA template, 5 lL 1X GoTaq Flexi buffer,

1998). The reporting limit was 0.1 mg/L. pH value and 2.5 lL MgCl2 solution, 0.5 U Taq DNA polymerase, temperature were measured by a Quanta water quality monitoring system (Hydrolab, Austin, TX, USA). Table 1 Solute transport model parameter calibration

Numerical simulation Parameter Values Hydraulic conductivity (m/day) 100 RT3D in Visual MODFLOW and WinPEST (Doherty Specific yield 0.250 2005) was used to establish the migration and Soil density (g/cm3) 1.524 transformation model of PCE and TCE, from which Extent of porosity (n) 0.400 the concentrations of PCE and TCE in the ground- 3 Partition ratio Kd (cm /g) water of the study area were predicted. Simulation area TCE 0.086 comprised a single-layer aquifer of 60 m in vertical PCE 0.211 thickness and divided to grids of 100 9 100 m size. 2 Dispersion coefficient (m /day) Western boundary of area was simulated as a row of Longitudinal (Dx) 10.000 injection boundary, and eastern boundary was treated as Horizontal (Dy) 1.000 a row of pumping boundary. A total water injection rate Vertical (Dz) 0.100 was determined by the hydraulic gradient of the study Advection velocity (m/day) 0.903 area, the hydraulic conductivity, and boundary length. PCE attenuation rate (1/day) 0.0001 The region was set as planar mining mode, and mining 123 538 Environ Geochem Health (2013) 35:535–549 and 1 lM each primer (Promega, Madison, WI, USA). rRNA sequences and possible chimeras were identified PCR was performed in a Veriti PCR apparatus via the RDP (http://rdp.cme.msu.edu/) (Altschul et al. (Applied Biosystem, Carlsbad, CA, USA) under the 1997; Cole et al. 2009). Sequences identified as chi- following conditions: 95 °C for 5 min, followed by meras were excluded from further analyses (Cole et al. 95 °C for 30 s, 54 °C for 30 s, 72 °C for 60 s, and a 2009). The operational taxonomic units (OTUs) num- final extension step at 72 °C for 10 min (Braker et al. ber was determined using DOTUR by defining the 2001). Primers tMMO-F (50-TGGGTAACYGTAN sequences sharing 98 % or greater similarity as one YGCCAATCG-30) and tMMO-R (50-GGATACTGG OTU (Schloss and Handelsman 2005). Taxonomic AGCAACGTCCTTA-30), sMMO-F (50-ATGATTG results were shown with MEGAN (Huson et al. 2007). 0 0 ATTCAG CCAACAGAG-3 ) and sMMO-R (5 -T OTU richness values SChao1 and SACE, as well as the GAGGTTATTGGCCAGGATG-30) (Sangon, Shang- Shannon diversity index (H), were calculated using hai, China) were designed to amplify the genes coding Estimate S version 7.5 (Colwell 2005). Evenness tMMO and sMMO, respectively. PCR of both enzymes (E) indices were calculated as follows: E = H/ln(n), genes was performed as same as 16S rRNA gene where n is the number of OTUs. Coverage (C) was except annealing step of 55 °C. Successful PCR calculated as follows: C = 1 - (n1/N), where n1 is the amplicons were purified by using a PCR Purification number of OTUs that occurred once and N is the total Mini Kit (Bio Basic Inc., Markham, Canada) and number of clones (Singleton et al. 2001; Li et al. 2008). cloned using the pEASY-T1 cloning kit (TransGen The most similar reference sequences to sequences in Biotech, Beijing, China). Verified transformants with our clone libraries were retrieved from the GenBank 16S rRNA, tMMO, and sMMO coding genes frag- database, and phylogenetic trees were constructed ments were sequenced, assembled, and did BLASTx using MEGA4 with neighbor-joining (NJ) and maxi- on NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi)(BGI, mum parsimony (MP) methods (1,000 replications) Beijing, China) using primers M13F (50-GTAAAA (Tamura et al. 2007). Canonical correlation analysis CGACGGCCAG-30) and M13R (50-CAGGAAACA (CCA), which is an approach to measure the relation- GCTAT GAC-30). ship between environmental parameters and bacterial community structure, was performed using Canoco for Bacterial quantification Windows 4.5 (Li et al. 2008). Relationships were examined among the main environmental parameters Quantitative PCR was performed on an ABI Prism 7300 (Eh, temperature, concentrations of chlorinated eth- (Applied Biosystems, Foster City, CA, USA) with a enes, TOC, and so on) and the bacteria including seven reaction mixture containing 10 lL SYBR Green PCR dominant phyla in this research and orders such as Master Mix (Applied Biosystems, Foster City, CA, Gallionellales and Rhodocyclale in reported polluted USA), 1 lM each primer, 2.0 lL DNA diluted template water. corresponding to 10.0 ng of total DNA, and Rnase-free water to complement a 20 lL volume. Thermal cycling Nucleotide sequence accession numbers conditions used for each reaction were as follows: 95 °C for 15 min, 95 °C for 15 s, 40 cycles of 60 °C for 30 s, The 16S rRNA nucleotide sequence data collected from 72 °C for 30 s, 80 °C for 15 s, and 1 cycle of 95 °Cfor this study were deposited in the GenBank database 15 s, 60–95 °C (Ritalahti et al. 2006a; Hallin et al. under the accession numbers JQ278751 to JQ279056. 2009). Primers were 341F (50-CCTACGGGAGGCAGC AG-30) and 534R (50-ATTACCGCGGCTGCTGGCA-30). Standard curves were obtained with serial dilutions of a Results known amount of plasmid DNA containing a fragment of the 16S rRNA (Ritalahti et al. 2006a; Hallin et al. 2009). Current water quality

Phylogenetic analysis and statistical analysis The tests of chemical properties showed that there were CAH pollutants in these groundwater sampling The assembled sequences were checked by BLAST in sites along groundwater ﬂow (Fig. 1). The contami- NCBI (http://www.ncbi.nlm.nih.gov/), and the 16S nants at site HMX were more complex than at the other 123 Environ Geochem Health (2013) 35:535–549 539

Fig. 1 Map of groundwater levels and ﬁeld measured contaminant concentration

sites, and CCl4 was only detected in site HMX. PCE Numerical simulation of the migration concentration was highest at site SF. TCE concentra- and transformation of CAHs tion was highest at site HMX, and following were sites SF, SZ, and FJC. However, VC and DCE were not Numerical simulation showed that the center of the detected except at site HMX, and the concentration of PCE and TCE plume would migrate eastward (Fig. 2). VC and DCE as degradation products of PCE/TCE All sampling points for PCE and TCE will be was both lower than the sites mentioned above eliminated within 9,000 days later from year 2002 if (Table 2) (Lowe et al. 2002; Carreo´n-Diazconti et al. there is no new input of pollutants (Fig. 3). In 2009; van der Zaan et al. 2010). simulated results of four sampling points of this study, Compared to the data from other sites in the same PCE concentrations increased and then decreased after city (TOC B 10 mg/L, Cl- between 17 and 20 mg/L, 4,500 days. TCE concentration changes along time 2- - SO4 between 32 and 61 mg/L, and NO3 between 5 scale can be divided into two patterns: (1) continued and 9 mg/L) (Wang et al. 2009; Aji et al. 2008). Not decline in concentration at sites SF and HMX from 2- only the concentration of TOC and SO4 was high, year 2002, and (2) an initial increase in concentration - - but also Cl and NO3 was higher than general data in followed by a decrease at sites SZ and FJC (Fig. 3). this study area, mainly due to garbage dumps around it The concentrations of PCE and TCE predicted at each in previous years. TOC content and the numbers of site by numerical simulation were lower than mea- microbial colonies were highest in groundwater from sured values from groundwater samples except for site site FJC compared to the other sites. Nitrate and HMX (Table 2). sulfate ions, which are the major electron acceptors in groundwater with organic environmental pollution, Bacterial community composition and degradation - - were ubiquitous at the sampling sites. NO3 /Cl ratio of enzyme was 1.22 at site SZ and 0.8–0.9 at the other study sites. The percentage of cis-DCE/total DCE was 37.2 % at In total, 328 sequences of 16S rRNA genes were site HMX, but more than 98 % at the while that of the obtained from four groundwater samples (SF, SZ, FJC, remaining sites (Table 2). and HMX) and grouped into 137 OTUs. The majority 123 540 Environ Geochem Health (2013) 35:535–549 /

- of sequences were identiﬁed as Proteobacteria 3 - (72.8 %), with representatives of the a-proteobacte- Cl NO ria, b-proteobacteria, and c-proteobacteria classes. The remaining sequences were classiﬁed as members

-DCE/ of the phyla Nitrospirae (7.5 %), Acidobacteria cis Total DCE (5.5 %), Actinobacteria (3.1 %), Bacteroidetes (3.1 %), 05 0.37 0.82 07 0.98 0.89 06 0.99 0.88 08 0.99 1.22 Planctomycetes (2.8 %), Firmicutes (2.4 %), and Chla- ? ? ? ? mydiae (\ 1%).InFig.4, bacteria from the families

Clones (No./L) Nitrospiraceae, Gallionellaceae,andRhodocyclaceae were common in each of the groundwater samples.

C) Nonetheless, abundance and composition among bacte- o Temp ( rial communities of the four sites were obvious, such as Azospirillum, Brevundimonas, Desulfuromonas,

(mV) Gemella, Nitrospiraceae, Rhodocyclaceae,andSulfu- ricurvum (Bae et al. 2007; Carvalho et al. 2006;Lofﬂer pH Eh et al. 2000).

- As shown in Table 3, the Shannon diversity indices 2 4 for all groundwater samples ranged from 4.54 to 6.03. (mg/L) SO The values were higher than values for other CAH- -

3 polluted groundwater sites (Lowe et al. 2002; Macbeth (mg/L) NO et al. 2004). Among these four sites, site FJC had the highest Shannon, Simpson diversity, and Evenness

- indices, and the lowest concentration of pollutants. (mg/L) Cl Although concentrations of PCE and cis-DCE at site SF were higher than concentrations at other three sites, the Shannon, Simpson diversity, and Evenness values TOC (mg/L) were not the lowest.

4 The genes coding tMMO were detected in sites SZ g/L) l ( CCl and HMX, and sMMO were detected in sites FJC and

3 HMX. Sequences of gene coding tMMO and sMMO

g/L) were *550 and 455 bp and have 98 and 97 % identity l ( CHCl with sequences of accession numbers FJ713039 and J04996.1, respectively. g/L) l PCE ( Phylogenetic analysis g/L) l TCE ( g/L) A phylogenetic tree was constructed with the 51 l unassigned sequences from the four sites and with 0.5 g/L) B l

1,1- DCE ( closely related 16S rRNA sequences from GenBank representing known species from more than 10 different - g/L) environments (Fig. 5). The tree comprised eight clus- l cis DCE ( ters. Most of the unassigned sequences clustered with

- reference sequences representing bacteria from envi- g/L) l

trans DCE ( ronments such as soil and water polluted with oil or TCE and uranium, a saturated zone, an iron-oxidation

g/L) bioﬁlm, marine sediment, and karst groundwater. In Summary of water quality parameter values for four groundwater samples l ( cluster I, sequences from the four samples were

not detected (i.e., concentration associated with EU638362 (Symbiobacterium), Table 2 Sites VC HMX 5.85 4.55 5.50 4.75 16.08 11.83 8.84 7.62 88.45 215.01 176.17 185.53 7.40 39.6 12.50 5.65E FJC ND ND 0.27 ND 0.77 3.28 0.93 ND 95.13 206.15 183.98 193.60 7.40 67.7 11.20 3.96E SF ND ND 13.26 ND 5.88 81.69 2.00 ND 90.62 180.21 159.39 175.71 7.20 92.8 6.90 2.01E SZ ND ND 2.18 ND 4.37 12.46 3.53 ND 81.62 120.31 147.14 149.43 7.31 116.3 14.13 1.49E ND HM448253 (candidate division OP1 bacterium), and 123 Environ Geochem Health (2013) 35:535–549 541

(m) A

SF FJC

HMX 0 1100 2200 3300 4400 5500 6600 7700 (m)

(m) B

60 SF 40 FJC 20 10 HMX 5 1 800 1600 2400 3200 4000 4800 5700 800 1600 2400 3200 4000 4800 5700 0 1100 2200 3300 4400 5500 6600 7700 (m)

Fig. 2 Simulation of the migration and transformation of the PCE (a) and TCE (b) plumes in year 2011

FJ205349 (Verrucomicrobia), which are thermophilic KP23, Methylosinus trichosporium OB3b, Methylococ- bacteria. In cluster IV, JQ278983 from site SF and cus capsulatus, Pseudomonas putida F1, and Pseudo- JQ278842 from site HMX were assembled with Dhc monas sp. CF600. These bacteria are capable of strains, which are the most effective TCE-degrading degrading PCE and TCE through co-metabolism with bacteria known. In cluster V, sequences from the site SF hydrocarbons, cyclohexanol, and other organic pollu- groundwater sample had a close relationship with tants. In cluster VI, JQ278950 had a close relationship another group of PCE/TCE-degrading bacteria, namely with a strictly anaerobic PCE-respiring bacterium, Dehalobacter restrictus, Burkholderia kururiensis Sulfurospirillum halorespirans. 123 542 Environ Geochem Health (2013) 35:535–549

Discussion 250 A Sampling date The pollution of groundwater by chlorinated aliphatic compounds, such as PCE and TCE, has become a serious environmental problem that is threatening

150 200 human health (Ohlen et al. 2005). In this study area, the pollution sources were complex, and the source of pollution is still not very clear after more than 10 years 100 of investigation. Fortunately, nearly all sources of the

PCE Concentration (µg/L) pollution have been eliminated since 2005 thanks to 50 strict emission control and environmental monitoring.

0 However, the current situation of our study area is as 0 1500 3000 4500 6000 7500 9000 (day) follows: The region is a residential area now, buildings on the ground ﬂoor are intensive, monitoring data is 70 B only from domestic wells, and it is impossible to

60 establish a formal system of monitoring wells, so the acquisition of monitoring data and the analysis of groundwater flow are difficult. To track and forecast the migration of the previous pollution, a strategy combining numerical simulation with field measurements was employed in this study. SZ Computer models of the migration and transformation SF of CAHs in groundwater flow showed that the peak TCE Concentration (µg/L) FJC concentration of the pollutant plume decreased as the 10 HMX area of the plume gradually expanded. Over time, the 020304050 0 1500 3000 4500 6000 7500 9000 (day) plumes of both PCE and TCE showed regular face-like Year Year distributions, with their center eastward. Firstly, at 2002 2011 sites SZ, SF, and FJC, the measured concentrations Fig. 3 Simulation of the PCE (a) and TCE (b) concentration were all lower than the predicted values. Microorgan- curve from year 2002. Zero means the first field sampling day in isms living in the soil and groundwater may have 2002. Circles the concentrations of PCE and TCE field sampling accelerated pollutant degradation around these sites days in 2011 (He et al. 2005; Liu et al. 2006). Secondly, the Canonical correspondence analysis (CCA) exploitation level of the different wells will also affect the small-scale groundwater flow in study area. From Canonical correlation analysis with water quality the spatial analysis, site SZ and site HMX certainly parameters and some bacterial taxa in samples from had an obvious relationship of upstream and down- sites SF, SZ, FJC, and HMX is shown in Fig. 6. The stream. Only site HMX had degradation products VC, a- and b-proteobacteria were positively related with and PCE was much higher in the SZ site than the trans-DCE, TCE, CCl4, and CHCl3 and negatively downstream site HMX. The PCE concentration associated with Eh. Actinobacteria, Nitrospira, and declined from site SZ to site HMX. Although the Bacteroidetes were closely associated with TOC, concentrations of PCE and TCE were higher than - 2- - NO3 ,SO4 , and Cl and negatively relative with numerical simulation in HMX, degradation of PCE PCE and cis-DCE. Rhodocyclales and Gallionell- and TCE was also happened. We speculated that new ales (both orders within the b-proteobacteria) and inputs of pollutants permeated into the groundwater c-proteobacteria were correlated with cis-DCE/total near this site, creating the discrepancy between DCE ratios and Eh. d-proteobacteria and Acidobac- predicted and observed values. - - teria were positively associated with NO3 /Cl ratios, Chlorinated ethene biodegradation can be classified - PCE, and cis-DCE and negatively related with NO3 , into four types of metabolism: anaerobic reductive 2- - SO4 ,Cl , and pH values. dechlorination, anaerobic oxidation, aerobic co- 123 Environ Geochem Health (2013) 35:535–549 543

Fig. 4 Bacteria at the sites SF, SZ, FJC, and HMX based on 16S rRNA genes metabolism, and aerobic assimilation (Mattes et al. the microbial diversity and community variation along 2010). To describe the microbes associated with this the large-scale groundwater ﬂow. Concentrations and CAH-contaminated environment as well as to explore complexity of pollutants have inﬂuences on diversity the potential CAH-degrading bacteria, we analyzed level of bacterial communities. Site FJC had the 123 544 Environ Geochem Health (2013) 35:535–549

Table 3 Coverage and diversity indices of bacterial 16S rRNA gene libraries for four groundwater samples

Sites No. of No. of Schao1 SACE Shannon Simpson Evenness Coverage clones OTUs value value (2, Shannon) index (1/D) index index (%)

FJC 110 58 243.1 271.9 6.03 146.25 0.98 47.3 HMX 92 37 134.5 113.1 4.76 24.94 0.89 59.8 SF 61 43 82.6 90.1 5.22 67.78 0.97 29.5 SZ 65 35 59.1 83.9 4.54 22.86 0.91 46.2 lowest concentrations of pollutants and the highest speculated that the microbial degradation was active in diversity and abundance of bacteria. In contrast, the early stage of pollution, but slowed down when bacterial diversity was lowest at site HMX, which cis-DCE accumulated to a certain level. had mixture of pollutants, and at site SF, which had the On the other hand, CAH degradation in the study highest concentration of PCE. Proteobacteria was the area may occur through co-metabolism. A number of dominant phylum across the study area, followed by bacteria with relatives known to co-metabolize Nitrospirae (7.5 %), Acidobacteria, Actinobacteria, organic compounds were found. For example, Flavo- Bacteroidetes, Planctomycetes, Firmicutes, and bacterium strains may degrade pentachlorophenol, Chlamydiae. Dhc strains were not detected, and the chlorinated compounds, and herbicides (Saber and genes pecA, tecA, vcrA, and bvcA were not success- Crawford 1985; Soares et al. 2003); Rhodocyclaceae fully amplified from our sampling sites (data not strains have been shown to degrade halogenated shown). This was in agreement with findings from compounds, denitrify, and reduce (per)chlorates (Song TCE-polluted groundwater in remote Altamont Hills, et al. 2000; Bae et al. 2007). Sulfate-reducing Desulf- USA. Proteobacteria was the abundant phylum, and uromonas strains can oxidize acetate, while using PCE Dhc was not detectable. Dhc was not the reason of and TCE as respiratory electron acceptors for growth CAH degradation in study area (Lowe et al. 2002). (Sung et al. 2003). Meantime, Desulfuromonas is One major mechanism for CAH degradation in the capable of dechlorinating occurred at 10 °C close to study area was through special enzymes tMMO and temperature of our study area groundwater; it is sMMO in the site. The following species were found in indicated that Desulfuromonas may play an important our sampling sites, such as Pseudomonas strains, role in the degradation process. Burkholderia strains, Methylococcus capsulatus, and Among environmental parameters, CAH pollutants so on. Pseudomonas strains capable of using a wide were the dominating factor influencing bacterial range of aromatic hydrocarbons and degrading chlo- biodiversity and species, and there were obviously roethenes through biphenyl dioxygenase and biphenyl different degradation levels in our sampling sites. Site dioxygenase (Suyama et al. 1996); Burkholderia and FJC had the lowest concentrations of pollutants and Pseudomonas strains producing tMMO that may the highest diversity and abundance of bacteria. In degrade 36–67 % of the TCE (Ryoo et al. 2001; contrast, bacterial diversity was lowest at site HMX, Zhang et al. 2000b); Methylococcus strains containing which had mixture of pollutants, and at site SF, which sMMO were found in both HMX and FJC (Zahn and had the highest concentration of PCE. Concentrations DiSpirito 1996). Both tMMO and sMMO were and complexity of pollutants have a strong influence responsible for PCE and TCE degradation in this on diversity level of bacterial communities. At site SZ, study under hypoxic conditions. In addition, the the cis-DCE/total DCE ratio was the highest among cis-DCE to total DCE ratio at site SZ was the highest the sites, and the concentration of sulfate (an electron among the sites, and the concentration of sulfate was acceptor) was the lowest. This implied that microbial the lowest. This implied that microbial degradation degradation may have been highest at site SZ may have been highest at site SZ compared to the other compared to the other sites, and thus, PCE and TCE sites, and thus, PCE and TCE degradation products degradation products accumulated at this site. At the accumulated at this site. At the same time, NO3- at same time, NO3- at site SF was lower than HMX site SF was lower than HMX and FJC, which would and FJC, which would result in the shortage of this result in the shortage of this electron acceptor. We electron acceptor. We speculated that the microbial 123 Environ Geochem Health (2013) 35:535–549 545

Fig. 5 Phylogenic trees of unassigned 16S rRNA sequences

123 546 Environ Geochem Health (2013) 35:535–549

In conclusion, our characterization of the microbial communities and diversity across groundwater habits polluted for a decade provides insights into complexity of this ecosystem under CAH stress. Enzymes tMMO and sMMO were detected and were the main reason for PCE and TCE degradation in this study. The identiﬁcation of CAH-associated bacteria in this area also indicates a possibility for in situ microbiological remediation through increase of oxygen (Kao et al. 2003; Semprini 1995; Kao et al. 2001). Given current environmental protection trends around the globe, it is important to document microbiota and adopt environment-friendly approaches for in situ pollution treatment.

Fig. 6 CCA analysis of water quality parameters and bacterial Acknowledgments This research was supported by The taxon abundance in four groundwater samples (SF, SZ, FJC, Fundamental Research Funds for the Central Universities and HMX). The abundance of bacterial taxa was based on (2010ZY05), National Program of Control and Treatment of the number of 16S rRNA gene sequences in each clone Water Pollution (2009ZX07424-002), and National Natural library. (Abbreviations were followed as Acidobac: Acidobac- Science Foundation (40972162). The authors would like to teria, Actinoba: Actinobacteria, Alphapro: Alpha-proteobacteria, thank the contributions of Mingzhu Liu, Yonggen Zhang, Bacteroi: Bacteroidetes, Betaprot: Beta-proteobacteria, Ni Yan, and Minghan Wang for their assistance in this paper. Deltasub: Delta-proteobacteria, Gallione: Gallionellales, Gammapro: Gammaproteobacteria, Nitrospi: Nitrospira, Rhodocyc: Rhodocyclale) References degradation was active in the early stage of pollution, Aji, K., Tang, C., Song, X., Kondoh, A., Sakura, Y., Yu, J., et al. but slowed down when cis-DCE accumulated to a (2008). Characteristics of chemistry and stable isotopes in groundwater of Chaobai and Yongding River basin, North certain level. VC was only detected at site HMX, and China Plain. Hydrological Processes, 22, 63–73. ethene and ethane were not detected in any of the study Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, sites. The dechlorination of DCE to VC may only Z., Miller, W., et al. (1997). Gapped BLAST and happen at site HMX, as indicated by numerical PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389–3402. simulation results. The results were shown that the APHA. (1998). Standard methods for the examination of water a-, b-, and d-proteobacteria were positively related and wastewater (20th ed.). Washington, DC: American with CAH pollutants. It is speculated the species Public Health Association. capable of dechlorination mostly belong to a-, b-, and Arciero, D., Vannelli, T., Logan, M., & Hooper, A. B. (1989). Degradation of trichloroethylene by the ammonia-oxidiz- d-proteobacteria, and certain dechlorinating species ing bacterium Nitrosomonas europaea. Biochemical and survived in polluted environments selectively. Biophysical Research Communications, 159(2), 640–643. Rhodocyclales and Gallionellales were correlated Aulenta, F., Maio, V. D., Ferri, T., & Majone, M. (2010). The with cis-DCE/total DCE ratios (as an indicator of humic acid analogue antraquinone-2,6-disulfonate (AQDS) serves as an electron shuttle in the electricity- microbial degradation) and Eh, suggesting that Rho- driven microbial dechlorination of trichloroethene to docyclales and Gallionellales play an important role in cis-dichloroethene. Bioresource Technology, 101(24), degradation of CAHs and suitable to anaerobic 9728–9733. environment. Both Rhodocyclales and Gallionellales Bae, H. S., Rash, B. A., Rainey, F. A., Nobre, M. F., Tiago, I., da Costa, M. S., et al. (2007). Description of Azospira restricta may be indicators of pollutants biological degradation sp. nov., a nitrogen-ﬁxing bacterium isolated from ground- in groundwater. Nitrospira as nitrite-oxidizing bacte- water. International Journal of Systematic and Evolutionary - ria were closely associated with NO3 . As the Microbiology, 57, 1521–1526. products of Nitrospira biological activity, NO - was Barnes, R. J., Riba, O., Gardner, M. N., Singer, A. C., Jackman, 3 S. A., & Thompson, I. P. (2010). Inhibition of biological the electron acceptors proﬁtably for degradation of TCE and sulphate reduction in the presence of iron nano- CAHs. particles. Chemosphere, 80(5), 554–562. 123 Environ Geochem Health (2013) 35:535–549 547

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