Functional Genes and Bacterial Communities During Organohalide Respiration of Chloroethenes in Microcosms of Multi-Contaminated Groundwater

Abstract

Microcosm experiments with CE-contaminated groundwater from a former industrial site were set-up to evaluate the relationships between biological CE dissipation, dehalogenase genes abundance and bacterial genera diversity. Impact of high concentrations of PCE on organohalide respiration was also evaluated. Complete or partial dechlorination of PCE, TCE, cis-DCE and VC was observed independently of the addition of a reducing agent (Na₂S) or an electron donor (acetate). The addition of either 10 or 100 μM PCE had no effect on organohalide respiration. qPCR analysis of reductive dehalogenases genes (pceA, tceA, vcrA, and bvcA) indicated that the version of pceA gene found in the genus Dehalococcoides [hereafter named pceA(Dhc)] and vcrA gene increased in abundance by one order of magnitude during the first 10 days of incubation. The version of the pceA gene found, among others, in the genus Dehalobacter, Sulfurospirillum, Desulfuromonas, and Geobacter [hereafter named pceA(Dhb)] and bvcA gene showed very low abundance. The tceA gene was not detected throughout the experiment. The proportion of pceA(Dhc) or vcrA genes relative to the universal 16S ribosomal RNA (16S rRNA) gene increased by up to 6-fold upon completion of cis-DCE dissipation. Sequencing of 16S rRNA amplicons indicated that the abundance of Operational Taxonomic Units (OTUs) affiliated to dehalogenating genera Dehalococcoides, Sulfurospirillum, and Geobacter represented more than 20% sequence abundance in the microcosms. Among organohalide respiration associated genera, only abundance of Dehalococcoides spp. increased up to fourfold upon complete dissipation of PCE and cis-DCE, suggesting a major implication of Dehalococcoides in CEs organohalide respiration. The relative abundance of pceA and vcrA genes correlated with the occurrence of Dehalococcoides and with dissipation extent of PCE, cis-DCE and CV. A new type of dehalogenating Dehalococcoides sp. phylotype affiliated to the Pinellas group, and suggested to contain both pceA(Dhc) and vcrA genes, may be involved in organohalide respiration of CEs in groundwater of the study site. Overall, the results demonstrate in situ dechlorination potential of CE in the plume, and suggest that taxonomic and functional biomarkers in laboratory microcosms of contaminated groundwater following pollutant exposure can help predict bioremediation potential at contaminated industrial sites.

Keywords: chloroethenes (CEs); contaminated groundwater; dehalogenase genes; organohalide respiration; perchloroethylene (PCE).

FIGURE 1

Organohalide respiration pathways of chloroethenes, associated bacteria and reductive dehalogenase genes (based on Futagami et al., 2008; Hug, 2016; Zhao et al., 2017; Saiyari et al., 2018).

FIGURE 2

Dissipation of CEs in microcosms spiked with PCE and acetate. Concentrations of PCE (triangles), TCE (squares), cis-DCE (circles), VC (diamonds), and total chlorinated ethenes (white circles), in microcosms spiked with 10 μM PCE (A) or 100 μM PCE (B). Represented values are the average of three replicates. Error bars are not shown for clarity and were below 100% (see Supplementary Figure S1). Data from microcosms that received acetate but no Na₂S are shown as a representative example. Results were very similar for other experimental conditions tested.

FIGURE 3

Chlorinated ethene dissipation in microcosms spiked with PCE and acetate. Dissipation rates estimated for PCE (triangles), cis-DCE (circles), VC (diamonds), and total chlorinated ethenes (white circles) are given for microcosms spiked with 10 μM PCE (A) or 100 μM PCE (B).

FIGURE 4

Biomarker genes in microcosms. Quantification of 16S rRNA gene (white circles), pceA(Dhc) (diamonds), pceA(Dhb) (squares), vcrA (black circles) and bvcA (triangles) genes in microcosms amended with acetate and spiked with 10 μM (A) or 100 μM (B) PCE. The tceA gene was not detected. Values represent the mean (±standard deviation) of three replicates. Quantification limits of qPCR analysis for functional genes are indicated by red lines. nd, not detected.

FIGURE 5

Relative abundance of the 17 most abundant genus-level OTUs during CEs dissipation. Only data obtained for microcosms with acetate amendment were analyzed in detail. A total of 412,231 sequences of the V4–V5 region of the 16S rRNA gene were obtained by Illumina MiSeq and analyzed with Qiime software. 10 μM, 100 μM: concentrations of PCE added. OTUs with <1% abundance were clustered together.

FIGURE 6

Principal component analysis (PCA) ordination plot of bacterial communities during CEs dissipation. Largest contributions of bacterial taxa are represented by red arrows. Significant physico-chemical and biomolecular explanatory variables were fitted a posteriori on the PCA and are represented by gray arrows. Values on the axes indicate the percentage of the total variation explained by the corresponding axis (PC1, principal component axis 1; PC2, principal component axis 2). δ-prot., Deltaproteobacteria; Arc, Arcobacter; Dem, Dechloromonas; Dhc, Dehalococcoides; Dsv, Desulfovibrio; Gb, Geobacter; Hgp, Hydrogenophaga; Sul, Sulfurospirillum.

FIGURE 7

Phylogenetic affiliation of OTU935 within genus Dehalococcoides. The tree was obtained by the Neighbor-Joining and Jukes and Cantor methods from a 923 nucleotides alignment of 16S rRNA gene sequences. OTU935 of the microcosm is displayed in bold. Dehalococcoides subgroup affiliation is also shown. Bootstrap values are expressed as the percentage of 1000 replications.