Chlorinated electron acceptor availability selects for specific Dehalococcoides populations in dechlorinating enrichment cultures and in groundwater

Individual Dehalococcoides mccartyi (Dhc) strains differ primarily from one another by the number and identity of the reductive dehalogenase homologous catalytic subunit A (rdhA) genes contained within their respective genomes. While thousands of rdhA genes have been sequenced, the activity of the corresponding proteins have been identified in only a handful of cases. Most effort has focused on identifying the enzymes that dechlorinate substrates including trichloroethene (TCE), cis-dichloroethene (cDCE) and vinyl chloride (VC) relevant to groundwater remediation. The associated rdhA genes, namely tceA, bvcA, and vcrA, along with the D. mccartyi 16S rRNA gene are often used to track growth and dechlorinating activity in DNA extracted from field samples. In this study, we augmented the typical suite of three characterized rdhA genes to include an additional 12 uncharacterized rdhA sequences identified in the metagenome in the mixed Dhc-containing culture KB-1 to track population shifts within the culture and at two bioaugmented field sites. Quantitative PCR assays were developed for the 15 selected D. mccartyi rdhA genes and evaluated using 11 different sub-cultures of KB-1, each enriched on different chlorinated ethenes and ethanes. The proportion of rdhA gene copies relative to Dhc 16S gene copies indicated the presence of multiple distinct Dhc populations in each culture. The specific electron acceptor amended to each culture had a major influence on the distribution of D. mccartyi populations and their associated rdhA genes. We also surveyed the abundance of rdhA genes in samples obtained from two bioaugmented field sites. Growth of the dominant D. mccartyi population in the KB-1 inoculum was detected in the UK site samples. At both field sites, the measurement of relative rdhA abundances revaled significant D. mccartyi population shifts over time as dechlorination progressed from TCE through cDCE to VC and ethene, indicating that the selective pressure of the most abundant chlorinated electron acceptor that was observed in lab cultures was also occurring in the populations in the field. Understanding driving forces behind D. mccartyi population selection and activity is improving predictability of remediation performance at chlorinated solvent contaminated sites.


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There are thousands of public and private sites with chlorinated solvent related groundwater 59 contamination problems (1). Chlorinated volatile organic compounds (cVOCs) such as 9 Files containing all amino acid and nucleotide sequences as well as matching of tree 182 nomenclature with protein names can be found in a folder labelled rdhA_Dhc_seq150420 at the 183 following address: 184 https://docs.google.com/folder/d/0BwCzK8wzlz8ON1o2Z3FTbHFPYXc/edit.

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Groundwater sampling and site description. Groundwater was collected from two TCE-187 contaminated sites prior to and after bioaugmentation with KB-1 ® . The first site was located in 188 Southern Ontario (ISSO site) and was characterized by contaminants in fractured bedrock. where groundwater from the three extraction wells was combined ( Figure S1b). Additional 202 information on this site can be found in a previous publication that surveyed community 203 dynamics at the site (22). 10 The second site is located in the U.K. and consisted of a pilot test cell (30x7x4 m) for treatment 206 of a DNAPL source area (~ 1000 kg of DNAPL within the cell). The cell was conceived as an 207 "in situ laboratory" for investigating source area bioremediation (SaBRE project - ii) within the source zone (SW70); iii) within the plume (SW75); and iv) at the effluent (EFF), 216 also within the plume. The test cell was operated initially for a 90-day baseline period to 217 establish steady-state pre-treatment conditions. Groundwater was extracted at an average of 1.4 218 liters per minute, corresponding to an average residence time within the cell of 45 days. A total 219 of 2,400 Kg of SRS™ at a 5% concentration was used as the electron donor and injected along 220 the test cell. Two weeks after donor injection approximately 65 L of KB-1 ® was added using the 221 same injection ports that were used to add electron donor. Within the test cell, both TCE and 222 cDCE were the main cVOCs. A dissolved phase plume emanating from the source and extending 223 more than 400 m away was further characterized by the presence of VC and ethene.

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The starting point (Day 0) was defined as the day following the end of the emulsified oil 225 injection, which took approximately one week to complete. Further details on the SaBRE site can 226 be found in technical bulletins freely available on the SaBRE-CL:Aire website (see above).

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Nucleic Acid Extraction. Samples were collected from eleven different enrichment cultures at 229 one or two times each during 2011 (Table S1). Archived samples from the 230 TCE/ME_2001_SiREM culture spanning 5 years were obtained from SiREM. DNA was also 231 extracted from groundwater samples at from the two field sites described above. For extraction   With the exception of five genes (KB1-6/bvcA, KB1-14/vcrA, KB1-27/tceA, KB1-1 and KB1-5), 249 new primers were designed for all other rdhA genes in this study. The specificity of the probes 12 was tested in silico against the public NCBI NR nucleotide database and tested in vitro against 251 DNA extracted from mixed dechlorinating and non-dechlorinating cultures. Table S4 252 (Supporting Information) shows the number of mismatches for each primer pair with sequences 253 belonging to the ortholog group of the targeted rdhA gene. The PCR reaction (20 µL) consisted 254 of: 10 µL of SsoFast™ EvaGreen® Supermix (Biorad, CA), 0.5 µL of each primer (10 mM), 7 255 µL of UltraPure™ DNase/RNase-Free distilled water (Invitrogen, CA) and 2 µL of template.

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The amplification program included an initial denaturation step at 98 °C for 2 min followed by 257 40 cycles of 5 s at 98 °C and 10 s at the corresponding annealing temperature. A final melting 258 curve from 70 to 95 °C degrees at increments of 0.5 °C per second was performed. Since two 259 concentrations were tested per template (undiluted and 10-fold diluted) for assessment of 260 potential matrix-associated inhibitory effects, reactions for each dilution were performed in 261 duplicate. The undiluted sample generally contained between 10-20 ng of DNA per µL as 262 measured using a Nanodrop spectrophotometer (Thermo Scientific, DE). Generally, there was 263 good agreement between the two measurements. Ten-fold serial dilutions of plasmid DNA 264 containing one copy of the 16S rRNA gene or the rdhA gene (~1000-1500 bases) were used as 265 calibrators. Plasmids for calibration were prepared by PCR amplifying the desired rdhA from   identity within an ortholog group can be substantially lower than 90%. The terminology 310 reductive dehalogenase homologous genes (rdhA) was adopted years ago, and perhaps implies 311 more knowledge of common ancestry than is in fact known, but it is not unreasonable to suspect 312 that these genes arose from either speciation (orthologous) or duplication (paralogous) events.

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Herein we use the term "ortholog" to refer to rdhA genes that group together according to the  Furthermore, KB1-5 (OG 15) is orthologous to DET1545 from Strain 195, which is expressed 342 upon starvation (55, 56). KB1_14, KB1_6 and KB_27 correspond to the functionally-343 characterized vinyl chloride and trichloroethene reductases VcrA, BvcA and TceA. We designed 344 qPCR primers to these 10 uncharacterized genes, and used previously designed primers for 345 remaining genes (Table S4). Next, we monitored the abundance of this suite of rdhA genes in 346 DNA samples from 11 different KB-1 enrichment sub-cultures maintained over years on 347 different chlorinated electron acceptors (Table S1). contains that gene) to less than 0.001 (where the gene is present in fewer than 0.1% of the D. 356 mccartyi populations) ( Figure 2). We color-coded these ratios (x) into one of five abundance 357 categories (x≥0.6 (dark red); 0.6 >x≥0.1 (red); 0.1>x≥0.01 (orange); 0.01>x≥0.001 (yellow) and 358 x<0.001 (pale yellow)). Although the primers were designed to target specific KB-1 rdhA 359 sequences, most sequences within the same ortholog group would likely also be amplified. Table   360 S4 (Supporting Information) compiles all mismatches found between primers used and other 361 sequences in the corresponding OG.  (Tables S6 and S7), as expected since Geobacter only dechlorinates PCE or TCE 388 as far as cDCE. In control DNA samples from cultures without D. mccartyi, copies of rdhA 389 genes were all below the MDL (data not shown).  The results from the Canadian ISSO site revealed that the two most abundant rdhA genes in 469 native populations (monitored prior to any treatment) were orthologs to KB1-4 and KB1-6/bvcA, 470 but that their concentrations were barely above detection limits, at approximately 3x10 5 copies 471 per L (Table S8; Supporting Information). Although the bvcA-encoded dehalogenase is known to  other rdhA genes increased to above 10%, including genes similar to 501 and especially vcrA (Figure 4a). This parttern of rdhA genes was also seen in the KB-1 VC 502 enrichments. Three months after bioaugmentation (Month 22), the proportion of ethene relative 503 to VC and cDCE further increased. Here, the bvcA ratio decreased to less than 10% while the 504 vcrA ratio remained at approximately 1. At Month 30, when there was almost no VC or cDCE 505 left, the vcrA ratio returned to below 0.6. At this time, relative abundance of KB1-12 decreased 506 substantially; these rdhA was also found to vary between enrichment cultures. starting from below detection limit (< ~10 5 copies L -1 ) up to 0.9-3x10 6 copies L -1 (Table S8). The The rdhA gene suite also was used to investigate groundwater from the SaBRE bioaugmentation 519 trial within a test cell at a TCE-contaminated site in the U.K. Prior to any treatment, D. mccartyi 520 gene copy numbers were below the detection limit (<6x10 4 gene copies L -1 ) in all wells studied 521 except the effluent (EFF) location where D. mccartyi titers of about 10 5 gene copies L -1 were 522 detected (data not shown). No significant VC and ethene concentrations were measured in these wells prior to treatment. Unfortunately, no samples were collected during biostimulation prior to 524 inoculation of KB-1 (all data provided in Table S8). Two weeks after bioaugmentation with 525 KB-1 and about a month after a single donor addition event, D. mccartyi titers increased by three 526 orders of magnitude in SW70, reaching 10 8 gene copies L -1 (Table S8). In a sample from this 527 well, all KB-1 biomarkers characteristic of the original inoculum, that is KB1-4, KB1-11, KB1-528 12, KB1-14/vcrA, and KB-23 were found to have rdhA ratios greater than 0.6 ( Figure 4b). The  (Table S1); Summary of rdhA genes in KB-1 (Table S2); Timeline and 593 sketch of the ISSO site in Canada ( Figure S1); Timeline and sketch of the SaBRE site in the UK 594 ( Figure S2); Phylogenetic analysis of cultures (Text S1); Clustering analysis of rdhA genes (Text 595 S2); Phylogenetic tree of rdhA genes based on nucleotide sequences ( Figure S3); Primer pairs for 596 qPCR quantification (Table S3); Mismatches between primers and sequences from OG groups 597 (Table S4); qPCR standard curves (Table S5); qPCR data for Figures 2 and 3 (Tables S6-S8).

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Detection limits for qPCR (Table S9).    Table   645 S3 or Figure 1 for correspondence between OG and KB-1 rdhA numbers.        c) through f) most abundant rdhA genes detected in the respective enrichment culture samples, with evidence for considerable strain variation within and between enrichment cultures. OG numbers that are grey correspond to genes that are abundant in only some of the samples from the respective enrichments. See Table  S3 or Figure 1 for correspondence between OG and KB-1 rdhA numbers.