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Microbial Diversity and Changes in the Distribution of Dehalogenase Genes during Dechlorination with Different Concentrations of cis-DCE Kotaro Ise,* Koichi Suto, and Chihiro Inoue Department of Environmental Studies, Graduate School of Environmental Studies, Tohoku University, 6-6-20 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan
bS Supporting Information ABSTRACT: A dechlorinating consortium (designated as TES-1 culture) able to convert trichloroethene (TCE) to ethene was established from TCE-contaminated groundwater. This culture had the ability of complete dechlorination of TCE within about one month. From the clone library analysis of 16S rRNA gene, this culture was mainly composed of fermentation bacteria, such as Clostridium spp., and Desulfitobacterium spp. known as facultative dechlorinator. PCR using specific primers for Dehalococcoides spp. and the dehalogenase genes confirmed that the culture contained the Dehalococcoides spp. 16S rRNA gene and three dehalogenase genes, tceA, vcrA and bvcA. Dechlorination experiments using cis-dichloroethene (cis-DCE) at concentrations of 37146 μM, revealed that the gene copy numbers of tceA, vcrA, and bvcA increased up to 107 copy/mL, indicating that Dehalococcoides spp. containing these three dehalogenase genes were involved in cis-DCE dechlorination. However, in the culture to which 292 μM of cis-DCE was added, only the tceA gene and the Dehalococcoides spp. 16S rRNA gene increased up to 107 copy/mL. The culture containing 292 μM of cis-DCE also exhibited about one tenth slower ethene production rate compared to the other cultures.
’ INTRODUCTION In recent years, the contamination of soil and groundwater has been reported as a widespread environmental problem.1 This type of contamination causes economic standstill, like brownfield issues, that needs to be resolved rapidly and cost-effectively. Bioremediation for the contamination of chlorinated solvents has already been applied at many sites. However, a major problem is the accumulation of toxic intermediate products, such as cis-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), trans-dichloroethene (trans-DCE) and vinyl chloride (VC).2,3 Because Dehalococcoides spp. have highly similar 16S rRNA gene sequences, the dehalogenase gene sequence could be used to estimate the dechlorination capability of a microbial consortium.4 To date, tceA, vcrA, and bvcA have been detected as chloroethene dehalogenase genes in Dehalococcoides sp.4 The tceA gene was the first dehalogenase gene identified in Dehalococcoides strains 195 and FL2 that can dechlorinate TCE to ethene, but the final step of conversion of VC to ethene is slow due to a cometabolic reaction.57 The vcrA gene found in strains VS and GT can dechlorinate all DCE isomers and VC metabolically,8,9 and the bvcA gene is a putative dehalogenase gene of VC found in strain BAV1.10,11 These dehalogenase genes are present in a single copy, therefore it is thought that quantifying these genes may represent the relative abundance of Dehalococcoides spp. harboring these genes. Because these dehalogenases have different substrate specificities, these distributions may strongly affect the dechlorination rate. r 2011 American Chemical Society
To date, many dechlorination experiments have been conducted using dechlorinating consortium or isolated Dehalococcoides sp.;12,13 however, in many of these cases the dehalogenase genes were not analyzed or the species involved only contained one or two of the three dehalogenase genes. For example, KB-1 culture,14 a well-known TCE-dechlorinating consortium, harbors only the vcrA and bvcA genes, and ANAS culture,15 another well-known TCE-dechlorinating consortium, contains only the tceA and vcrA genes. There are some reports of quantification of these three dehalogenase genes in TCE-contaminated sites,4,1618 but because of certain variables it is difficult to analyze and determine which factors critically affect the dynamics of the Dehalococcoides spp. population in a contaminated site. In addition, in many cases, even if all three types of dehalogenase genes are present, complete dechlorination may rarely occur and cis-DCE and VC may accumulate especially in highly contaminated sites, such as those near DANPL source zones.19 Therefore, the reason why these dechlorinaters do not function effectively remains unclear. In this study, we constructed a TCE dechlorinating consortium that contains three types of dehalogenase genes, tceA, vcrA and bvcA, by repetitive subculturing using TCE as the electron acceptor. To analyze the effect of the cis-DCE concentration on the diversity of Dehalococcoides spp., we examined the dynamics Received: December 16, 2010 Accepted: May 2, 2011 Revised: April 29, 2011 Published: May 24, 2011 5339
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Environmental Science & Technology of Dehalococcoides spp. bearing the tceA, vcrA, and bvcA genes, in this TCE-dechlorinating culture at different initial cis-DCE concentrations.
’ MATERIALS AND METHODS Chemicals. The chemicals TCE (98%), cis-DCE (97%), transDCE (99%), 1,1-DCE (99%), ethene (99.5%), and methane (99.9%), were purchased from GL Sciences Ltd., Japan. Gaseous VC was obtained from DOWA Eco-System Co., Ltd., Japan. Other chemicals for cultivation, such as methanol, ethanol, and sodium acetate, were purchased from Wako Pure Chemical Industries, Ltd., Japan. All chemicals used in this study were of analytical grade. Microorganisms, Media, and Cultivation Conditions. A source of anaerobic enrichment culture was obtained from the groundwater of a TCE-contaminated site in Japan in October 2004. The groundwater contained TCE, cis-DCE and VC at concentrations of 0, 220, and 12.5 mg/L, respectively. The groundwater sample was kept in a low temperature room (NK system Ltd., CMC-1S, Japan) at 4 °C before use. The groundwater sample was incubated in media containing inorganic salt solution, trace elements, an electron donor and TCE as an electron acceptor. The inorganic salt medium component was comprised of KH2PO4, 0.27 g; K2HPO4, 0.35 g; NH4Cl, 0.53 g; CaCl2 3 2H2O, 0.075 g; MgCl2 3 6H2O, 0.1 g; FeCl2 3 4H2O, 0.02 g and yeast extract, 0.2 g and 1 mL of trace element solution20 per liter. The concentration of TCE was 69 μM. In this cultivation, 100 mL of medium was added into a 125 mL glass vial bottle. The inorganic salt solution was sterilized by autoclaving at 121 °C for 15 min, and then cooled to room temperature. Yeast extract and sodium acetate were sterilized through a syringe filter (0.2 μm pore size) and added to the medium at a final concentration of 0.2 g/L and 7.4 mmol/L, respectively. As reducing agents, Na2S and L-cysteine were added at a final concentration of 0.2 mM. The final pH value was about 7.0. Deoxygenated nitrogen gas was introduced into the medium for 30 min to remove dissolved oxygen. The vials were then sealed with Teflon-lined butyl rubber septums and aluminum seals. One milliliter of the groundwater sample was inoculated through the septum with a syringe. Then, each vial was incubated upside down in the dark at 30 °C on a bioshaker (Taitec Co., Ltd., Japan) at 130 rpm. The subculture and dechlorination experiments were carried out as described above for the first cultivation. Analysis of the Gas Components during the Dechlorination Experiments. Headspace gas in experimental vials was collected using a micro syringe through the septum and the concentrations of chloroethenes, ethene, methane and hydrogen were analyzed periodically using two GC-FID systems (GC-390, GC-353; GL Science Co., Ltd., Tokyo, Japan) and the GC-TCD system (GC-323; GL Science Co., Ltd.). The measured concentrations of gases were converted into aqueous concentrations and the amount of substances per bottle were based on Henry’s law constants (mol/kg 3 Pa), and were 3.9 107, 4.8 108, 1.4 108 and 7.8 109 for VC, ethene, methane, and hydrogen, respectively.2123 The GC-390 system is equipped with a TC-5 capillary column (GL Science Co., Ltd.; 30 m in length, 0.32 mm inside diameter and 25 μm film thickness), which was able to quickly analyze TCE and DCEs. The GC-353 system has a SilicaPLOT
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(CHROMPAC Co.; 30 m in length, 0.53 mm inside diameter and 4 μm film thickness) for measuring VC, ethene and methane. The GC-323 system has a Prapak Q50/80 packed column (GL Science Co., Ltd.) for measuring hydrogen. The analytical conditions of the GC390 system were an injection port temperature of 200 °C; oven temperature, 50 °C; detector temperature, 200 °C; carrier gas, He; gas flow rate, 42.5 cm3/min. The analytical conditions of the GC353 system were an injection port temperature of 200 °C; oven temperature, 40 °C; detector temperature, 200 °C; carrier gas, He; gas flow rate, 60.9 cm3/min. Finally, the analytical conditions of the GC323 system were an injection port temperature of 120 °C; oven temperature, 60 °C; carrier gas, N2. DNA Extraction. Total DNA from the microbial consortium in the dechlorination experiments were extracted from the subcultures.24 Following extraction, the DNA samples were treatment with phenol/chloroform/isoamyl alcohol extraction, and 2-propanol precipitation, and ethanol precipitation. The concentration of DNA was quantified on spectrophotometer at a wavelength of 260 nm. PCR Amplification of the Eubacterial and Archeal 16S rRNA Gene and the Reductive Dehalogenase Genes. EX Taq DNA polymerase (TAKARA Bio. Inc., Japan) was used for the amplification of DNA fragments. Amplification was carried out in a Gene Amp PCR System 9700 (Applied Biosystems Ltd., U.S.) using several primer sets, as shown in Supporting Information Table S1. The eubactarial 16S rRNA genes were amplified with primers Eu10F and Eu1492R. The 16S rRNA gene of Dehalococcoides sp. was amplified using the primer set Fp DHC 1 and Rp DHC 1377. In addition, reductive dehalogenase genes, such as tceA, bvcA, and vcrA, were also amplified using specific primer sets, as shown in Supporting Information Table S1. Cloning and Sequencing Procedure. PCR products were purified using GenEluteTM Agarose Spin Columns (Sigma Aldrich Co., MO). A slice of 2% agarose gel containing the DNA was filtered through the spin columns to remove the agarose gel. Collected DNA was purified by the ethanol precipitation method. The purified DNA was ligated into the TA cloning vector pCR2.1 and transformed into E. coli top10 cells according to the manufacturer’s protocol (Invitrogen, U.S.). The sequences of each of the DNA fragments were analyzed on an ABI PRISM 3013 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s protocol. Clones were sequenced using the M13 Forward and M13 Reverse primers, which bind to sites flanking the insertion site on the plasmid. The obtained gene sequences have been deposited in the GenBank. The obtained sequences were analyzed with the DNA alignment software MEGA4,25 and phylogenetic trees were constructed based on the 16S rRNA gene (Supporting Information Figure S1). Classifications of Clostridium sp. were conducted by dendrogram analysis with 435 sequences of Clostridiales-type strains. qPCR. The copy numbers of the chloroethenes dehalogenase genes and the Dehalococcoides sp. 16S rRNA gene were estimated using the 7500 Real-Time PCR System (Applied Biosystems) with the primers TceA1270F and TceA1336R for tceA; VcrA1022F and VcrA1093R for vcrA; BvcA925F and BvcA1017R for bvcA; and Dhc1200F and Dhc1271R for the Dehalococcoides sp. 16S rRNA gene.4 qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems). The reactions were performed using the following thermal program: 95 °C for 5 min, 5340
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Table 1. Phylogenetic Summary of the TCE Dechlorination Consortium Clone Library Analysis Using Bacterial Primers Eu10F and Eu1492R order Clostridiales
Bacteroidales
family and genus
followed by 40 cycles of denaturing (10 s at 95 °C) and annealing (60 s at 58 °C). Total bacterial 16S rRNA gene copy numbers in the extracted DNA were estimated using 0.5 μM of the primers 341f and 518r, with the following thermal program: 95 °C for 5 min, followed by 40 cycles of denaturing (10 s at 95 °C) and annealing (90 s at 58 °C). Melting curve analysis from 6595 °C was performed with a plate read every 5 °C, and all samples were run in triplicate. Calibration curves of qPCR were obtained using serial dilutions of plasmids carrying a single, cloned target gene, that is, the bacterial and Dehalococcoides 16S rRNA gene for plasmids carrying the Dehalococcoides sp. 16S rRNA gene PCR product amplified with Fp DHC 1 and Rp DHC 1377. The tceA, vcrA, and bvcA gene-carrying plasmid were constructed using the PCR product amplified by the same primer pairs using real-time PCR from the TES-1 culture. A quantification equation was used to calculate the number of gene copies in a known amount of DNA using the Avogadro number.4
’ RESULTS Dechlorination of TCE to Ethene by the Microbial Consortium. Figure 1 shows the time course of TCE dechlorination
using the microbial consortium with sodium acetate as the electron donor. TCE was dechlorinated to ethene through cisDCE and VC. Figure 1(B) shows the amount of hydrogen and methane in the bottle. Hydrogen was generated and consumed rapidly in the early part of the experiment. Methane was
accession no.
Clostridium Cluster XIa
46% (36/79) AB596881
Clostridium Cluster I
41% (32/79) AB596882
unclassified Clostridiales
1% (1/79)
AB596888
Desulfitobacterium sp.
4% (3/79)
AB596883
unclassified Ruminococcaceae
1% (1/79)
AB596889
unclassified Veillonellaceae 1% (1/79)
AB603498
Parabacteroides sp.
3% (2/79)
AB596884
unclassified Rikenellaceae
3% (2/79)
AB596885
1% (1/79)
AB596883
Desulfovibrionales Desulfovibrio sp.
Figure 1. (A) Dechlorination pattern of 0.076 mM TCE to ethene in this dechlorinating culture. (B) Amount of hydrogen and methane in the bottle. Symbols: TCE (0), cis-DCE (O), VC (4), ethene (b), hydrogen (9), methane (2).
frequency
produced mainly after hydrogen consumption after about 100 h of cultivation. At this time, the culture showed 350-fold increase in Dehalococcoides spp. 16S rRNA gene numbers, from 7.3 104 copy/mL (initial condition) to 2.6 107 copy/mL (after 727 h incubation). In this study, subcultivation of the culture was carried out using sodium acetate as an electron donor and carbon source by routinely transferring 1 mL of the culture solution to new medium (99 mL) containing TCE. This microbial consortium (designated the TES-1 culture) has been maintained since August 2004. Microbial Community in the TES-1 Culture. To investigate the microbial community in the TES-1 culture, clone library analysis of the eubacterial 16S rRNA gene was carried out using culture solutions at 473 h of incubation. At that time, the dechlorinating culture transformed almost all of the cis-DCE and started to produce ethene. A total of 79 clones carrying 16S rRNA gene were sequenced. Table 1 shows the summary of the TES-1 culture clone library analysis. From the phylogenetic tree, the TES-1 culture was found to be composed mainly of two types of Clostridium spp., belonging to Clostridium Cluster I and XIa, according to the phylogeny of the genus Clostridium proposed by Collins et al.;26 and clones 32 and 36 were detected. One unclassified Clostridiales was also detected. These Clostridiumrelated species represented 87% of the consortium. Clostridium spp. are well-known as a high hydrogen-yielding bacteria when they ferments organic compounds like amino acids, so may play an important role in the production of hydrogen from the fermentation of yeast extracts. Three clones of Desulfitobacterium spp. known as PCE- and TCE-dechlorinators27 were detected. The other detected clones were unclassified Ruminococcaceae, unclassified Veillonellaceae, Parabacteroides spp., unclassified Rikenellaceae and Desulfovibrio spp. Other sequence classifications were conducted using the classifier program supplied by the Rebosormal database project.28 Detection of Dehalococcoides spp. and Reductive Dehalogenase Genes. Although the TES-1 culture showed dechlorination activity from TCE to ethene, Dehalococcoides spp. were not detected in the analysis of the clone library. To confirm the existence of Dehalococcoides spp., target PCR using specific primer sets, shown in Supporting Information Table S1, for the 16S rRNA gene of Dehalococcoides spp. and the reductive dehalogenase genes was carried out. 5341
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Figure 2. Dechlorination profile of different cis-DCE concentrations: (A) 37 μM (0.0038 mmol/bottle), (B) 73 μM (0.0076 mmol/bottle), (C) 146 μM (0.0152 mmol/bottle), and (D) 292 μM (0.0304 mmol/bottle). Symbols: cis-DCE (O), VC (4), ethene (closed circleb). The symbols indicate averages based on triplicate determinations. The error bars indicate standard deviations; error bars are not shown if they are smaller than the symbols.
A single band PCR amplicon of the 16S rRNA gene was detected and its sequence (AB495355) showed 100% similarity (1377/1377) to the corresponding sequences of strains FL2 and CBDB1, and 99.9% similarity (1376/1377) to the corresponding sequence of strain BAV1, known as the Pinellas Sequence Subgroup29 of Dehalococcoides. The reductive dehalogenase genes, tceA and vcrA 7,8 and the putative dehalogenase gene, bvcA,11 were also detected as single bands by the target PCR. These sequences showed high similarity with each gene (vcrA (442/442), bvcA (830/841), tceA (1710/1724) (99%). The results showed that each amplicom was the same as the reported dehalogenase gene and that this culture contained all three of these dehalogenases. We then conducted clone library analysis on Dehalococcoides spp. 16S rRNA gene with specific primers Fp DHC 1 and Rp DHC 1377, shown in Supporting Information Table S1. The 17 clones showed over 99% (1364/1377) similality with each other and the constructed phylogenetic tree showed that they belonged to the Pinellas subgroup Dehalococcoides spp. (data not shown). These findings confirmed that this culture contained three types of chloroethenes dehalogenases, tceA, vcrA, and bvcA. However, it cannot be confirmed whether these dehalogenases were distributed individually or together. Effect of Different cis-DCE Concentrations on the Dechlorination Rate and Distribution of Dehalogese Genes. After incubation with 69 μM TCE, the culture was transferred to fresh medium containing different concentrations of cis-DCE. Figure 2 shows the dechlorination time course of the culture with different
amounts of cis-DCE. The cultures containing 37 μM (0.0038 mmol/bottle), 73 μM (0.0076 mmol/bottle) and 146 μM (0.0152 mmol/bottle) of cis-DCE showed almost the same dechlorination rate and about 800, 1600, and 3000 h were required for complete dechlorination, respectively. The culture containing 292 μM (0.0304 mmol/bottle) of cis-DCE exhibited a slow dechlorination rate, and almost all of the cis-DCE transformed and remained as VC at 6000 h. The dechlorination rate of this culture dropped obviously, especially during VC dechlorination. From Figure 2, there were no obvious differences in the rates among these cultures in the case of VC production. However, in the case of ethene production, the culture containing 292 μM of cis-DCE showed about a one tenth production rate compared to the other cultures. Figure 3 shows the gene copy numbers of the tceA, vcrA, bvcA, and 16S rRNA gene of Dehalococcoides spp. and eubacteria after cultivation with different cis-DCE concentrations. The initial set of columns represents the source of the microbial consortium that was obtained from cultivation with 69 μM TCE. Regarding the source of the consortium, the copy number of the 16S rRNA gene of Dehalococcoides spp. was 4.8 105 copies/mL, which was one tenth of that of eubacteria. The copy numbers of each the dehalogenase genes, tceA, vcrA, and bvcA, were 3.1 105, 2.1 105 and 9.9 103 copy/mL, respectively. After cultivation with different cis-DCE concentrations, 16S rRNA gene copy number of total eubacteria increased by 1 or 2 orders of magnitude. The copy numbers of each dehalogenase genes, tceA, vcrA and bvcA, increased by 2 orders of magnitude during the cultivations with 5342
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Figure 3. Chloroethene dehalogenase gene, Dehalococcoides sp. 16S rRNA gene and the total eubacterium 16S rRNA gene abundance in each culture after cultivation with different cis-DCE concentrations. Gene copy numbers were determined by real-time PCR using the absolute calibration curve method. Each bar represents the average of triplicate real-time PCRs performed on triplicate cultures. The error bars indicate standard deviations.
73146 μM of cis-DCE. In these cases, the copy number of each dehalogenase gene was almost equal, and the total copy number of all dehalogenase genes was about three times larger than that of the 16S rRNA gene of Dehalococcoides spp. This may be due to the plasmids used as calibration standards. We used four individual plasmid standards, so these might have caused variability.15 By contrast, in the cultivation with 292 μM of cis-DCE, the copy numbers for the 16S rRNA gene of Dehalococcoides spp. and the tceA gene were increased about 20 and 50 times during the cultivation, respectively, whereas the copy numbers of the vcrA and bvcA genes decreased to 1/10 compared to the initial condition.
’ DISCUSSION A TCE dechlorinating consortium was established using TCE-contaminated groundwater. This consortium designated as TES-1, showed dechlorination ability converting TCE to ethene. The TES-1 culture has been maintained for over five years by repeated subculturing using acetate as a carbon source. Dehalococcoides spp. were detected from the dechlorination culture by target PCR using a specific primer set to detect the 16S rRNA gene of Dehalococcoides spp. Clone library analysis on 16S rRNA genes showed that the TES-1 culture had narrow eubacterial diversity, which was dominated by fermentation bacteria such as Clostridium Cluster I and XIa. Clostridium Cluster I constitutes the core Clostridium genus, the characteristic features of which are endospore-forming, obligate anaerobic energy metabolism, nonsulfate reducing and a Gram-positive-type cell wall. Cluster XIa is classified within the proposed “Peptostreptococcaceae”. It is known that these Clostridium species do not metabolize acetate as an energy source. However, these bacteria might have fermented the yeast extract in the culture media. This suggests that at the beginning of the cultivation, hydrogen was produced rapidly from the fermentation of the yeast extract as shown in Figure 1(B). Then hydrogen consumption exceeded hydrogen production because of the depletion of yeast extract. Clostridium spp. are sporeforming and able to efficiently produce hydrogen from the fermentation of organic compounds.
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The well-known dechlorination culture ANAS consisted of mostly fermentation bacteria such as Clostridium spp. and Bacteroides spp., as shown in this study and a previous study,30 on the other hand, the KB-1 culture, another well-known dechlorination culture, mainly comprised of dehalogenators, such as those from Dehalococcoides spp. and Geobacter spp.31 Desulfitobacterium spp. were detected as a dechlorinator in the clone library analysis in this study. Desulfitobacterium spp. are a known facultative dechlorinator of PCE and TCE and dechloritates TCE to cis-DCE. These bacteria may therefore play an important role in TCE dechlorination. However, Desulfitobacterium spp. comprised only 4% of the total clones. Dehalococcoides spp. have only one copy of the16S rRNA gene per cell, whereas Clostridium spp. have 214 copies of the 16S rRNA gene.32 These different copy numbers may affect the results of the clone library. When different concentrations of cis-DCE were added to the culture as an electron acceptor, the time period until completion of dechlorination varied depending on the initial concentration of cis-DCE. The time period became longer with increased initial concentrations of cis-DCE. This was especially clear in the case of 292 μM of cis-DCE, when the dechlorination reaction was unfinished even after 6000 h of cultivation and almost all of the chlorinated organics remained as VC. The production rates of VC remained almost the same and did not vary with the concentration of cis-DCE, but those of ethene differed in the case of 292 μM of cis-DCE. The copy numbers of the Dehalococcoides spp. 16S rRNA gene increased with the concentration of cis-DCE from 37 to 146 μM, but decreased at 292 μM of cis-DCE. In addition, the gene copy numbers of tceA, vcrA, and bvcA increased up to 107 (copy/mL) in the cultures containing 37, 73, and 146 μM of cis-DCE. But in 292 μM condition, only tceA increased. Magnuson et al. reported that purified TceA dechlorinated TCE, all DCE isomers and VC, but the VC dechlorination rate was very low.6 In another report, VcrA was shown to dechlorinate TCE, all DCE isomers and VC, but TCE dechlorination was very slow.8 Muller et al. reported that Dehalococcoides sp. strain BAV1 which contains bvcA gene was estimated to be mainly related to dechlorination of all DCE isomers and VC.10 These data support the finding that the copy numbers of all three dehalogenase genes increased when cis-DCE was used as an electron acceptor. Figure 3 shows the distribution of tceA, vcrA, and bvcA gene copy numbers in each culture. These three genes ratios changed dramatically after cis-DCE dechlorination. Under the initial culture conditions, tceA, vcrA, and bvcA accounted for 59%, 39%, and 2% of the gene ratios, respectively, but after cis-DCE (0.0038 mmol/bottle) dechlorination, tceA, vcrA, and bvcA accounted for 27%, 38%, and 35% of the gene ratios, respectively, with the ratio of bvcA clearly increasing. This same trend was seen in the cultures with 73 and 146 μM of cis-DCE, but the ratio of tceA increased slightly with increased cis-DCE. In the 292 μM culture, this trend changed significantly, and the tceA distribution ratio was 99.9%. The reason for this increase in the tceA gene was not clear, but there may be a threshold concentration between 146 μM and 292 μM of cis-DCE that selectively inhibited the growth of Dehalococcoides spp. bearing vcrA and bvcA. Similar results were obtained with the BDI culture when this culture was tested for its resistance to oxygen exposure.33 Oxygen-exposed BDI lost VC dechlorination activity compared with the nonexposed culture and accumulated VC. A study into the geochemical effects on dehalogenase abundance, reported that the tceA gene 5343
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Environmental Science & Technology preferred more oxidized conditions compared with the vcrA and bvcA genes.18 From these reports, the stress tolerance of Dehalococcoides spp. bearing tceA may be stronger to that of vcrA and bvcA. And according to the report of Maymo-Gattell et al., commercial cis-DCE frequently contaminated with chloroform, inhibited activity of Dehalococcoides spp.34 From this, Dehalococcoides sp. bearing vcrA and bvcA might be inhibited by elevated concentration of cis-DCE. In view of these data, either toxicity of the high concentration of cis-DCE or the toxicity of the contaminants in commercial cis-DCE may inhibit vcrA- and bvcAbearing Dehalococcoides spp. These results imply that when we apply bioremediation to sites contaminated with high concentration of cis-DCE, we should consider pretreatment such as physicochemical way to reduce cis-DCE concentration.
’ ASSOCIATED CONTENT
bS
Supporting Information. Figure S1 and Table S1. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: þ81-(0)22-795-7404; fax: þ81-(0)22-795-7404; e-mail:
[email protected].
’ ACKNOWLEDGMENT This reseach was partially supported by a Grant-in-Aid for Scientific Research (17206089) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by EcoCycle Corporation. ’ REFERENCES (1) Abelson, P. H. Inefficient remediation of groundwater pollution. Science 1990, 250 (4982), 733–733. (2) Lendvay, J. M.; Loffler, F. E.; Dollhopf, M.; Aiello, M. R.; Daniels, G.; Fathepure, B. Z.; Gebhard, M.; Heine, R.; Helton, R.; Shi, J.; Krajmalnik-Brown, R.; Major, C. L.; Barcelona, M. J.; Petrovskis, E.; Hickey, R.; Tiedje, J. M.; Adriaens, P. Bioreactive barriers: A comparison of bioaugmentation and biostimulation for chlorinated solvent remediation. Environ. Sci. Technol. 2003, 37 (7), 1422–1431. (3) Sharma, P. K.; McCarty, P. L. Isolation and characterization of a facultatively aerobic bacterium that reductively dehalogenates tetrachloroethene to cis-1,2-dichloroethene. Appl. Environ. Microbiol. 1996, 62 (3), 761–765. (4) Ritalahti, K. M.; Amos, B. K.; Sung, Y.; Wu, Q. Z.; Koenigsberg, S. S.; Loffler, F. E. Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Appl. Environ. Microbiol. 2006, 72 (4), 2765–2774. (5) He, J.; Sung, Y.; Krajmalnik-Brown, R.; Ritalahti, K. M.; Loffler, F. E. Isolation and characterization of Dehalococcoides sp strain FL2, a trichloroethene (TCE)- and 1,2-dichloroethene-respiring anaerobe. Environ. Microbiol. 2005, 7 (9), 1442–1450. (6) Magnuson, J. K.; Romine, M. F.; Burris, D. R.; Kingsley, M. T. Trichloroethene reductive dehalogenase from Dehalococcoides ethenogenes: Sequence of tceA and substrate range characterization. Appl. Environ. Microbiol. 2000, 66 (12), 5141–5147. (7) Magnuson, J. K.; Stern, R. V.; Gossett, J. M.; Zinder, S. H.; Burris, D. R. Reductive dechlorination of tetrachloroethene to ethene by twocomponent enzyme pathway. Appl. Environ. Microbiol. 1998, 64 (4), 1270–1275.
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