A Dehalogenimonas Population Respires 1,2,4-Trichlorobenzene and

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A Dehalogenimonas Population Respires 1,2,4Trichlorobenzene and Dichlorobenzenes Wenjing Qiao, Fei Luo, Line Lomheim, Elizabeth Erin Mack, Shujun Ye, Jichun Wu, and Elizabeth A. Edwards Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04239 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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Environmental Science & Technology

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A Dehalogenimonas Population Respires 1,2,4-

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Trichlorobenzene and Dichlorobenzenes

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Wenjing Qiao1,2, Fei Luo2, a, Line Lomheim2, E.Erin Mack3, Shujun Ye1*, Jichun Wu1, Elizabeth

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A. Edwards2*

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1Key

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Engineering, Nanjing University, Nanjing 210023, China;

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2Department

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M5S 3E5, Canada

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3DuPont

Laboratory of Surficial Geochemistry, Ministry of Education; School of Earth Sciences and

of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto

Corporate Remediation Group, Newark, Delaware 19711, United States

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*Corresponding Authors:

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Shujun Ye: Tel. (+86) 2589684150; Fax (+86) 2583686016; email: [email protected];

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Elizabeth A. Edwards: Tel. (+1) 4169463506; Fax (+1) 4169788605; email:

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[email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Chlorobenzenes are ubiquitous contaminants in groundwater and soil at many

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industrial sites. Previously, we demonstrated the natural attenuation of chlorobenzenes and

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benzene at a contaminated site inferred from a five-year site investigation and parallel laboratory

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microcosm studies. To identify the microbes responsible for the observed dechlorination of

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chlorobenzenes, the microbial community was surveyed using 16S rRNA gene amplicon

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sequencing. Members of the Dehalobacter and Dehalococcoides are reported to respire

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chlorobenzenes, however neither were abundant in our sediment microcosms. Instead, we

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observed a significant increase in the relative abundance of Dehalogenimonas from 50 bp overlap were joined together using the default mismatch (8%). The joined reads with

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quality score ≥19 were merged together to be screened to remove chimeras using Usearch61

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method22 and Ribosomal Database Project (v11).23 The filtered sequences were then clustered into

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Operational Taxonomic Units (OTUs) against the Greengene Database (v13.8)24 using Usearch

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61. The most abundant sequence in each cluster was chosen as the representative sequence for

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each OTU. The α diversity was calculated for each sample using both richness and Shannon

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metrics. The β diversity was also calculated using the weighted UniFrac method which measures

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phylogenetic distances between samples based on the lineages that they contain.25 An Unweighted

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Pair Group Method with Arithmetic (UPGMA) clustering26 was implemented subsequently to

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cluster the samples using the calculated phylogenetic distance matrix.

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Quantitative PCR (qPCR) Analysis and Yield Calculation. Given that the Illumina

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sequencing results only provide information of relative abundance, we next used qPCR to

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enumerate the absolute numbers of 16S rRNA gene copies of Dehalococcoides, Dehalobacter,

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Dehalogenimonas, and total Bacteria in the DNA samples shown Table S2 using previously

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published primers listed in Table S3A. The primer sets were checked with the

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Ribosomal Database Project (RDP) platform27 to confirm that the

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primer sets are specific to Dehalococcoides, Dehalobacter or Dehalogenimonas.

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Standard curves were generated using serial dilutions of plasmids containing corresponding 16S

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rRNA gene fragments. Each sample was run in duplicate. All the manipulations were conducted

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in a PCR cabinet (ESCO Technologies, Hatboro, PA). The reactions were completed with a Bio-

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Rad CF96 Touch real-time modular thermal cycler platform and CFX Manager software. Each


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qPCR mixture contained 10 μL of SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules,

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CA), 1 μL of each 10 μM forward and reverse primers, 2 μL of DNA template (blank or plasmid

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dilutions) and 6 μL of sterile UltraPure distilled water. The thermocycling program was as follows:

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initial denaturation at 98°C for 2min, followed by 40 cycles of denaturation at 98°C for 5s,

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annealing at Tm (see Table S3A in SI) for 10s and chain extension at 65°C for 10s.

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In order to further demonstrate growth, yields in gram dry mass of cells per moles chloride

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released were calculated from qPCR and concentration data. Cell volume was estimated from

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known cell dimensions and converted to wet mass using an estimated cell density of 1.03 g/mL,

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and then to dry mass assuming 70% water.28 Measured copies of 16S gene copies were converted

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to cell numbers assuming three 16S copies per genome for Dehalobacter and one for

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Dehalococcoides and Dehalogenimonas (Table S4).29

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Sanger Sequencing to Obtain Complete 16S rRNA Genes. Two primer sets (A and B)

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with overlapping sequence regions were used to obtain the full-length Dehalogenimonas 16S

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rRNA gene sequence in our samples via Sanger sequencing. The primer sequences were as

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follows: for primer pair A, 8f (5’- AGA GTT TGA TCM TGG CTC AG-3’) and 1111r (5’-ATC

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GCC AGA GAA TAT AAC TGGC-3’); for primer pair B, 944f (5’-CCT CAC CAG GGY TTG

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ACA TGT TAG AAG-3’) and 1409r (5’-CTT GCG GGT TAG CCY ATC GAC TTC AG-3’).

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The primer 8f was designed and used in previous study.30 The remaining primers were designed

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based on an alignment of 16S rRNA gene sequences from Dehalobium DF-1, Dehalococcoides,

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Dehalogenimonas and other relevant sequences from clones listed in Table S5. The representative

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sequences of Dehalogenimonas OTUs obtained from 16S rRNA gene amplicon sequencing in this

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study were also aligned using MUSCLE version 3.631 in Geneious (v8.1.9) to assist in selecting

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the best primers manually. The forward and reverse primer sets (A, B) were designed to anneal at


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the same temperature and to avoid forming hairpin loops, self-dimers or heterodimers. The primer

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sets were checked with the Ribosomal Database Project (RDP) platform27 to confirm that the

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primer sets were specific to Dehalogenimonas. The designed primers were synthesized by Sigma

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(Toronto, ON, Canada). PCRs were conducted using a PTC-200 Peltier Thermal Cycler (MJ

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Research Inc., Waltham, MA). Each PCR mixture contains 12.5 µL of Taq DNA polymerase in

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PCR Master Mix (2×) (Thermo Scientific, MA), 1.25 µL of 10 µM forward and reverse primers,

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8 µL of sterile UltraPure distilled water, and 2 µL of template. No template controls (NTC) were

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also prepared for each reaction. The PCR thermal cycling protocols were as follows: initial

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denaturation at 95°C for 2 min, and then 35 cycles at 95°C for 30s, the desired annealing

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temperature (47°C, 54.5°C were tested) for 30s, and extension at 72°C for 60s, followed by a final

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extension at 72°C for 7 min.

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PCR products (5 µL) were separated on 1% agarose gels containing Tris/Acetate/EDTA

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(TAE) buffer, run for 30 min at 100V. The remaining volume of PCR products were purified using

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a GeneJET PCR purification kit (ThermoScientific, MA) according to the manufacturer’s protocol.

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The purified DNA samples were then submitted to the Center for Applied Genomics (Toronto,

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Canada) for Sanger sequencing using an ABL 3730 xl DNA analyzer with the same primers used

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for PCR.

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Phylogenetic Tree Building. Selected 16S rRNA gene sequences for known chlorobenzenes

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dechlorinators: Dehalococcoides and Dehalobacter, as well as published Dehalogenimonas

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sequences were aligned and trimmed to the same sequence length using MUSCLE. Subsequently,

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a maximum likelihood phylogenetic tree was built with PHYML32, 33 using the Jukes-Cantor (JC-

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69) genetic distance model with 1000 bootstrap in Geneious.


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Accession Numbers. The 16S rRNA gene amplicon sequence data for this study have been

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deposited at the NCBI under the short-read archive (SRA) project accession no. SRR5753358 to

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SRR5753368 as part of bioproject no. PRJNA390777. The 16S rRNA gene sequence of the

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identified Dehalogenimonas population (Dhb_CB) has been deposited to NCBI under the

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accession number MF356602.

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3. Results

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The laboratory microcosm study demonstrated that the 1,2,4-TCB was dechlorinated

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primarily to 1,4-DCB (88%) with lesser amount of 1,2- and 1,3-DCBs after a lag of approximately

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60 days, and that 1,2- and 1,3-DCBs were dechlorinated to MCB after a lag of approximately 200

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days. The more recalcitrant 1,4-DCB and MCB were dechlorinated much later in the study, only

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after supplementation with or transfer into defined vitamin-containing mineral medium. The

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additions of electron donor did not alter dechlorination patterns, but only resulted in higher

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methane production.

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provided in Table 1. Dechlorination profiles were presented in our previous paper;4 this

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paper mainly focuses on the microorganisms responsible for the previously reported dechlorination

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processes.

4

The overall mole changes by compound and per bottle are

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Microbial Community Composition. Illumina 16S rRNA gene amplicon sequencing was

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performed on samples collected from the original site soil and from active chlorobenzenes-

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dechlorinating microcosms twice over the 19 months of incubation. After processing, the final

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number of sequences per sample ranged from 18,575 to 44,090 (Table S6); the corresponding

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phylogenetic assignments of OTUs, as well as representative sequences are provided in Table S7.

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The 926f/1392r primers used for amplicon sequencing do not match well to all archaeal


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sequences34, possibly resulting in lower numbers of Archaeal OTUs. Nevertheless, the cumulative

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methane produced, even in the electron donor amended microcosms, was less than 200 μmol per

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bottles after 19 months of incubation, suggesting that methanogenic Archaea were not a large

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fraction of the community anyway. The OTUs with relative abundances greater than 1% were all

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affiliated with bacteria and are represented in Figure 1. A distance tree (UPGMA using

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weighted Unifrac) is shown above the heat map in Figure 1. The samples clustered based on

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incubation time, but not based on electron donor addition, consistent with the finding that there

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was abundant electron donor already present in the groundwater at the site, as previously

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concluded.4 Unexpectedly, the known chlorobenzenes dechlorinators Dehalobacter and

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Dehalococcoides were not as abundant as Dehalogenimonas. The latter became enriched over time

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increasing from less than 1% measured in site soil to 6~9% after 12 months and to 16~30% after

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19 months of incubation. These data strongly suggested that Dehalogenimonas was responsible

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for the dechlorination of 1,2,4-TCB, 1,2-DCB, and 1,3-DCB. The amplicon sequencing data also

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revealed that the microbial diversity and species richness decreased with time (Figure S2).

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Quantitative PCR and Yield Calculations. All growth yields determined in this study are

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summarized in Table 2, based on data calculations provided in Table S9. Over the first 19 months

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of incubation, Dehalobacter never represented more than 0.3% of the

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total bacteria (Table S8, Figure S3) and corresponding yields (as

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g cells/mol Cl- released) were 100 times lower (Table S9) than

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reported

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Dehalobacter was not responsible for the observed dechlorination

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in this time period. Considering Dehalococcoides, its relative

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abundance increased from 0.2% (12 months) to approximate 1.5% (19

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months),

growth

but

yields

growth

of

yields

Dehalobacter,

were

also

much

suggesting

lower

than

that

those


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reported

typically

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Dehalogenimonas gene copies increased by 2 orders of magnitude

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(Table S8 and Figure S3) to become the most abundant microbe in

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the

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bacteria by 19 months. An average yield of 3.9 g Dehalogenimonas cells per mol Cl-

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released was obtained, ranging from 1.5 to 8.1 g/mol (N=12) (Table 2). No other microbe had such

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high yields, and this yield is comparable to typical yields reported for organohalide respiring

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bacteria. These results demonstrate that Dehalogenimonas growth was coupled to the

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dechlorination of 1,2,4-TCB to the three DCB isomers, and to the dechlorination of 1,2-DCB, and

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1,3-DCB to MCB as these processes were significant in this time period (Figure 2A). We noted

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that Dehalobacter gene copies, although not significant compared to Dehalogenimonas, were a

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little higher (4.5 times) in the electron donor amended treatments compared to the unamended

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bottles after 19 months of incubation, suggesting that amending donor stimulated the growth of

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Dehalobacter. In the poisoned control bottles that contained sodium azide and mercuric chloride

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we observed nonspecific amplification using the Dehalogenimonas primers. In these samples, we

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observed an unusual melt-curve (Figure S4) that likely resulted in overestimation of the signal in

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these bottles. This non-specific amplification fortunately did not affect conclusions, because the

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gene copies measured in samples from these poisoned control bottles were still over an order of

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magnitude lower than in samples from active microcosms.

microbial

for

Dehalococcoides.11

217

community,

accounting

for

11-26%

In

of

contrast,

the

total

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As reported previously, dechlorination of recalcitrant 1,4-DCB was speculated to be nutrient-

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or vitamin-limited and began in our microcosms only after supplementation with vitamin-amended

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mineral medium to Bottle 11.4 We used qPCR to track the growth of Dehalobacter (Table S8).

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The 10% transfer cultures from Bottle 11 prepared on Day 817 in triplicate (Bottles 11-T1, reps

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1/2/3) also confirmed the role of Dehalobacter in dechlorinating 1,4-DCB (Figure 2B and S3) with


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a growth yield of 4.5 ± 0.9 g Dehalobacter cells per mol Cl- released (N=3), particularly evident

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in the three transfer cultures (Table 2 and S9).

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The dechlorination of MCB was observed in a single transfer culture (Bottle 10-T3) supplied

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with mineral medium, nevertheless dechlorination was observed following 2 feedings of MCB.

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After 155 days of incubation, the gene copies of Dehalobacter, Dehalococcoides, and

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Dehalogenimonas all increased (Figure 2C and S3), but Dehalococcoides and Dehalogenimonas

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accounted for less than 2% of the total bacteria, and their corresponding yields were very low

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(96% sequence identities (Table S10). A 16S rRNA gene phylogenetic tree constructed

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with the known chlorobenzenes-dechlorinators Dehalobacter, Dehalococcoides, as well as the


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other known Dehalogenimonas species (Figure 3) supported Dhg_CB’s affiliation with the

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Dehalogenimonas genus.

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4. Discussion

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Prior to this work, known chlorobenzene-dechlorinating bacteria were restricted

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Dehalococcoides, Dehalobium, and Dehalobacter (Table S1) and were reported to transform

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1,2,4-TCB primarily to 1,4-DCB and/or 1,3-DCB. In our microcosm study, we found that 1,2-

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DCB, 1,3-DCB and 1,4-DCB were generated in approximately 2%/10%/88% molar proportions,4

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hinting at the possibility of novel microbe(s). We have now shown that indeed, a Dehalogenimonas

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population was responsible for the observed activity. This research broadens the diversity of

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microorganisms that can respire chlorinated benzenes.

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Dehalogenimonas lykanthroporepellens, D. alkenigignens, D. formicexedens have been

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shown to dechlorinate polychlorinated aliphatic alkanes.35-38 Dehalogenimonas sp. strain WBC-2

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was shown to dechlorinate trans-dichloroethene.39 Further, a recent study demonstrated that

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Dehalogenimonas etheniformans strain GP could respire vinyl chloride (VC).40 This was

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particularly notable because it was previously believed that only certain strains of

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Dehalococcoides could respire VC. Dehalogenimonas strain CG3 was able to metabolize a

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mixture of polychlorinated biphenyl (Arochlor 1260).41 Now we demonstrated that some

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Dehalogenimonas can also dechlorinate chlorinated benzenes. A summary of known halogenated

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substrates for Dehalogenomonas is provided in Table S11. The research expanded the metabolic

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diversity of Dehalogenomonas.

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The importance of Dehalogenimonas was revealed in a survey of 1173 groundwater wells

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from chlorinated solvent-impacted sites.40 The authors found that the distribution of

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Dehalogenimonas was similar to that of Dehalococcoides, and that Dehalogenimonas even


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outnumbered the Dehalococcoides in the majority of wells where both were detected.40 At present,

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Dehalococcoides is the most commonly tracked dechlorinator at field sites, and is abundant in

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commercial consortia used for bioaugmentation to clean up chlorinated solvent contaminated sites.

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However, Dehalogenimonas are closely related to Dehalococcoides and are also obligate

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organohalide respiration bacteria (OHRB) with a diverse substrate range. Thus, the contributions

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of Dehalogenimonas to dehalogenation at contaminated sites is probably underappreciated. More

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research is needed to discover the breadth of reductive dehalogenases from Dehalogenimonas to

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augment the suite of biomarkers for site characterization and risk abatement.

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ASSOCIATED CONTENT

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Supporting Information. Figures S1-S5 and Tables S1-S11, including information on microcosm

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and transfer culture history, community diversity, amplification of Dehalogenimonas by qPCR in

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DNA samples from poisoned controls, Dehalogenimonas 16S rRNA gene bands, primers used in

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this study, Illumina sequencing data, qPCR raw data and calculation, yield calculations, summary

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of known chlorobenzenes dechlorinators and the dechlorinating substrates of Dehalogenimonas.

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Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT

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We gratefully acknowledge funding from E.I. du Pont de Nemours and Company, the National

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Natural Science Foundation of China (NSFC) grant 41472212, Key Program for International

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S&T Cooperation Projects of China (Ontario-China Research and Innovation Fund,

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2016YFE0101900), and the Government of Canada through Genome Canada and the Ontario

309

Genomics Institute (OGI-102). W.Q. was supported by the China Scholarship Council.


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of high-throughput community sequencing data. Nat. Methods 2010, 7, (5), 335-336. 22. Edgar, R. C., Search and clustering orders of magnitude faster than BLAST. Bioinformatics 2010, 26, (19), 2460-2461. 23. Cole, J. R.; Wang, Q.; Fish, J. A.; Chai, B.; McGarrell, D. M.; Sun, Y.; Brown, C. T.; Porras-Alfaro, A.; Kuske, C. R.; Tiedje, J. M., Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucl. Acids Res. 2014, 42, (D1), D633-D642. 24. McDonald, D.; Price, M. N.; Goodrich, J.; Nawrocki, E. P.; DeSantis, T. Z.; Probst, A.; Andersen, G. L.; Knight, R.; Hugenholtz, P., An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 2012, 6, (3), 610-618. 25. Lozupone, C.; Knight, R., UniFrac: a new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 2005, 71, (12), 8228-8235. 26. Felsenstein, J., Inferring phylogenies. Sinauer associates Sunderland, MA: 2004; Vol. 2. 27. http://rdp.cme.msu.edu/probematch/search.jsp. 28. Rittmann, B. E.; McCarty, P. L., Environmental biotechnology: principles and applications. Tata McGraw-Hill Education: 2012. 29. Adrian, L.; Löffler, F., Organohalide Respiring Bacteria. In Springer: 2016. 30. Zhou, J. Z.; Fries, M. R.; Cheesanford, J. C.; Tiedje, J. M., Phylogenetic analyses of a new group of denitrifiers capable of anaerobic growth on toluene and description of azoarcus tolulyticus sp. nov. Int. J. Syst. Bacteriolo. 1995, 45, (3), 500-506. 31. Edgar, R. C., MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 17921797. 32. Kasai, Y.; Takahata, Y.; Manefield, M.; Watanabe, K., RNABased Stable Isotope Probing and Isolation of Anaerobic BenzeneDegrading Bacteria from Gasoline-Contaminated Groundwater. Appl. Environ. Microbiol. 2006, 72, (5), 3586-3592. 33. Ramospadrón, E.; Bordenave, S.; Lin, S.; Bhaskar, I. M.; Dong, X.; Sensen, C. W.; Fournier, J.; Voordouw, G.; Gieg, L. M., Carbon and Sulfur Cycling by Microbial Communities in a Gypsum-Treated Oil Sands Tailings Pond. Environ. Sci. Technol. 2011, 45, (2), 439. 34. Luo, F.; Devine, C. E.; Edwards, E. A., Cultivating microbial dark matter in benzene‐degrading methanogenic consortia. Environ. Microbiol. 2016, 18, (9), 2923-2936. 35. Bowman, K. S.; Nobre, M. F.; da Costa, M. S.; Rainey, F. A.; Moe, W. M., Dehalogenimonas alkenigignens sp. nov., a


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chlorinated-alkane-dehalogenating bacterium isolated from groundwater. Int. J. Syst. Evol. Microbiol. 2013, 63, (4), 14921498. 36. Moe, W. M.; Yan, J.; Nobre, M. F.; da Costa, M. S.; Rainey, F. A., Dehalogenimonas lykanthroporepellens gen. nov., sp. nov., a reductively dehalogenating bacterium isolated from chlorinated solvent-contaminated groundwater. Int. J. Syst. Evol. Microbiol. 2009, 59, (11), 2692-2697. 37. Yan, J.; Rash, B.; Rainey, F.; Moe, W., Isolation of novel bacteria within the Chloroflexi capable of reductive dechlorination of 1, 2, 3‐trichloropropane. Environ. Microbiol. 2009, 11, (4), 833-843. 38. Key, T. A.; Bowman, K. S.; Lee, I.; Chun, J.; da Costa, M.; Albuquerque, L.; Rainey, F. A.; Moe, W. M., Dehalogenimonas formicexedens sp. nov., a chlorinated alkane respiring bacterium isolated from contaminated groundwater. Int. J.of Syst. Evol. Microbiol. 2017, 67, 1366-1372. 39. Manchester, M. J.; Hug, L. A.; Zarek, M.; Zila, A.; Edwards, E. A., Discovery of a trans-dichloroethene-respiring Dehalogenimonas species in the 1,1,2,2-tetrachloroethanedechlorinating WBC-2 consortium. Appl. Environ. Microbiol. 2012, 78, (15), 5280-7. 40. Yang, Y.; Higgins, S. A.; Yan, J.; Simsir, B.; Chourey, K.; Iyer, R.; Hettich, R. L.; Baldwin, B.; Ogles, D. M.; Loffler, F. E., Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes. ISME J. 2017, 11, (12), 2767-2780. 41. Wang, S.; He, J., Phylogenetically distinct bacteria involve extensive dechlorination of Aroclor 1260 in sedimentfree cultures. PloS one 2013, 8, (3), e59178.

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480

Figure 1. Relative abundance of major OTUs (>1%) from 16S rRNA gene amplicon Illumina

481

sequencing analysis. The taxonomical assignments and OTU numbers in the brackets are on the

482

right; and the culture names and incubation time (372, 379 and 568 days) of each DNA sample are

483

shown on the top of each column. The samples from Bottles 5 and 7 were incubated under

484

simulated in situ conditions, while the other two (Bottles 9 and 10) were grown with electron donor

485

(e-) amended. The UPGMA tree on top of the heat map represents the β diversity among different

486

microbial communities, and the distance between samples was computed using the weighted

487

Unifrac method. The scale bar represents the branch distances of microbial communities.

488


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489 490

Figure 2. Increase of Dehalogenimonas (Dhg - blue bars) and Dehalobacter (Dhb - green bars)

491

copies per mL during dechlorination of 1,2,4-TCB, 1,2-/1,3-DCB (panel A), 1,4-DCB (panel B)

492

and MCB (panel C), respectively. In Panel A, data shown are cumulative 1,2,4-TCB, 1,2-DCB and

493

1,3-DCB dechlorinated in representative Bottles 5, 7, 9 and 10. Dechlorination profiles for all the

494

replicate sediment microcosms were available in our previous paper4, and all the qPCR data were

495

in Table S8 and Figure S3. In Panel B and C, data are measured concentrations in transfer cultures

496

and arrows indicate corresponding feeding events; the numbers (817 and 804) next to zero on the

497

X-axis represent the day when the transfer cultures were set up from parent microcosms. Data in

498

panel B are averages of duplicates (Bottles 11-T1-Rep2 and 3) because the other replicate was

499

amended with a higher concentration of 1,4-DCB. Data in panel C were from Bottle 10-T3.

500


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501 502

Figure 3. Maximum likelihood tree showing the phylogenetic relationship of Dehalogenimonas

503

spp. and the other related chlorobenzene-respiring bacteria based on nearly complete 16S rRNA

504

genes. Tree was created using the PHYML plugin in Geneious under the JC69 mode of evolution.

505

Bootstrap support values (out of 1000 bootstraps) are indicated. The scale bar represents mean

506

substitutions per site. The numbers in parenthesis are the GenBank accession numbers. Different

507

symbols next to the strain names represent different dechlorination substrate groups. See Table S1

508

and S11 for detailed dechlorination substrates.

509


21


510

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TOC Cl Cl

HCl 2H

Cl

Cl

Cl

Cl Cl Cl

Cl

Cl

511

Dehalogenimonas Dehalobacter


22

Page 23 of 24

512


Table 1. Overall changes by compound in sediment microcosms and transfer cultures Microcosms description In situ conditions

Donor Amended In situ conditions Donor Amended

Bottle No. 5 6 7 8 9 10 11 12 5 7 9 10

10-T3 (amended with MCB) 11-T1 (amended with 1,4-DCB)

513 514

Rep1 Rep2 Rep3

Overall changes by compound (µmol/bottle) 1,2,4-TCB 1,4-DCB 1,2-DCB 1,3-DCB MCB Benzene -5.21 5.04 -0.99 -0.97 2.74 -0.17 -6.16 6.32 -0.47 -0.47 0.89 -3.36 -6.14 6.26 -0.46 -0.47 1.19 -3.78 -6.1 5.27 -0.43 -0.45 0.2 -3.08 -6.39 6.82 -0.48 -0.5 1.17 -1.06 -4.4 3.22 -0.97 -0.99 2.36 -0.19 -6.03 4.63 -0.4 -0.41 1.75 -3.31 -6.55 5.81 -0.5 -0.54 1.38 -3.87 -9.8 7.9 -0.62 1.1 1.1 -0.3 -22.9 20.27 0.15 2.2 0.77 0.01 -22.5 18.5 0.4 1.9 1.9 0.02 -12.1 10.7 -0.2 0.77 1.5 -0.5

Δ∑ 0.61 0.11 0.38 -1.51 0.62 -0.78 -0.46 -0.40 -0.62 0.50 0.22 0.17

Recovery (%) 103% 101% 103% 87% 105% 95% 96% 97% 117% 113% 106% 114%

Time Period (days)

355 (Day 0355) (to 12 months)

231 (Day 355586) (to 19 months)

n.a.

n.a.

n.a.

n.a.

-19

17

-1.1

94%

153 (Day 804958)

n.a. n.a. n.a.

-90.6 -71.8 -71.9

n.a. n.a. n.a.

n.a. n.a. n.a.

82 68 70.8

0.13 0.3 0.2

-8.2 -3.3 -0.9

91% 95% 99%

31 (Day 817848)

Note: Negative values represent compounds that were consumed; positive values represent compounds that were produced. n.a.: not applicable.

515 516 517



Page 24 of 24

518

Table 2. Yield calculations of Dehalococcoides (Dhc), Dehalobacter (Dhb) and Dehalogenimonas

519

(Dhg) determined by qPCR and the corresponding halogenated substrates (n.d.: negative change

520

in copy number over interval; see Table S9 for yield calculations) Microcosm description In situ conditions (no amendments other than site soil and groundwater and chlorobenzenes or benzene)

Bottle No. Bottle 5 Bottle 6 Bottle 7 Bottle 8 Bottle 9

Electron donor amended microcosm bottles

Bottle 10 Bottle 12 Bottle 11

11-T1: Transfers Rep.1 from Bottle 11 Rep.2 (amended with 1,4Rep.3 DCB) 10-T3: Transfer from Bottle 10 (amended 2X with MCB)

Day 372 568 372 372 568 372 379 568 379 568 379 379 941 836(19) 836(19)

Yield (g cell/mol of Cl- released) Dhb Dhc Dhg n.d. 0.14 3.59 0.06 1.57 3.90 0.01 0.1 7.75 n.d. 0.07 4.09 0.02 0.07 3.38 0.00 0.02 2.09 0.15 0.19 8.06 0.06 0.04 1.52 0.01 0.10 2.99 0.18 0.49 1.99 0.01 0.03 3.14 0.02 0.05 4.07 0.63 0.00 n.d. 5.09 0.00 0.01 3.52 0.00 0.00

Substrate and Average Yield

1,2,4-TCB, 1,2-/1,3-DCB Yield (Dhg): 3.9  2.1 g/mol (N=12) (Period up to 19 months)

836(19)

4.86

n.d.

0.01

1,4-DCB Yield (Dhb): 3.5  2.1 g/mol (N=4)

959(155)

0.83

0.01

0.01

MCB (N=1)

521


A Dehalogenimonas Population Respires 1,2,4-Trichlorobenzene and

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