Coupling Bioflocculation of <i>Dehalococcoides mccartyi</i> to High-Rate Reductive Dehalogenation of Chlorinated Ethenes

Continuous bioreactors operated at low hydraulic retention times have rarely been explored for reductive dehalogenation of chlorinated ethenes. The inability to consistently develop such bioreactors affects the way growth approaches for <i>Dehalococcoides mccartyi</i> bioaugmentation cultures are envisioned. It also affects interpretation of results from in situ continuous treatment processes. We report bioreactor performance and dehalogenation kinetics of a <i>D. mccartyi</i>-containing consortium in an upflow bioreactor. When fed synthetic groundwater at 11–3.6 h HRT, the upflow bioreactor removed >99.7% of the influent trichloroethene (1.5–2.8 mM) and produced ethene as the main product. A trichloroethene removal rate of 98.51 ± 0.05 me^– equiv L^–1 d^–1 was achieved at 3.6 h HRT. <i>D. mccartyi</i> cell densities were 10¹³ and 10¹² 16S rRNA gene copies L^–1 in the bioflocs and planktonic culture, respectively. When challenged with a feed of natural groundwater containing various competing electron acceptors and 0.3–0.4 mM trichloroethene, trichloroethene removal was sustained at >99.6%. Electron micrographs revealed that <i>D. mccartyi</i> were abundant within the bioflocs, not only in multispecies structures, but also as self-aggregated microcolonies. This study provides fundamental evidence toward the feasibility of upflow bioreactors containing <i>D. mccartyi</i> as high-density culture production tools or as a high-rate, real-time remediation biotechnology

Bacterial composition at the class level as determined by 454 pyrosequencing of the V2-V3 region of the 16S rRNA gene.

<p>The outer pie charts (A–C) represent the relative abundance of select classes in the Cuzdrioara uncontaminated soil, (B) Carolina uncontaminated sediment, and (C) Parris Island contaminated sediment. The inner pie charts (A'–C') show the five most abundant classes in the respective soil/sediment-free enrichment cultures, ZARA-10, LINA-09, and ISLA-08. The classified taxa presented contributed to at least 1% of the total relative abundance and are organized in alphabetical order.</p

Enumeration of <i>Dehalococcoides mccartyi</i> in enrichment cultures.

<p>qPCR tracking <i>Dehalococcoides</i> 16S rRNA genes and their reductive dehalogenase genes, <i>tceA</i>, <i>vcrA</i>, and <i>bvcA</i> in the enrichment cultures after three consecutive additions of 0.5 mmol L⁻¹ TCE. The plot is representative of triplicate cultures and the error bars show standard deviations of triplicate qPCR reactions.</p

Environmental inocula and enrichment conditions of chlorinated ethene-dechlorinating cultures.

<p>Enrichment originating <i>Dehalococcoides mccartyi</i> strain ^a195, ^bBAV1, ^cVS, and ^dFL2.</p

Alpha and beta microbial diversity analyses.

<p>(A)–(C) Rarefaction plots for PD Whole Tree measurements from the 454 analysis using trimmed, equal sequencing depth OTUs (1,486) per sample. (D) Weighted UNIFRAC distance calculated after trimming the samples to equal sequence depth in QIIME. The PCoA plot was generated by grouping the samples into two categories (soils/sediments vs. enrichment cultures). The color blue corresponds to the soil/sediment samples, while green corresponds to the soil/sediment-free enrichment cultures.</p

Interpreting Interactions between Ozone and Residual Petroleum Hydrocarbons in Soil

We evaluated how gas-phase O₃ interacts with residual petroleum hydrocarbons in soil. Total petroleum hydrocarbons (TPH) were 18 ± 0.6 g/kg soil, and TPH carbon constituted ∼40% of the dichloromethane-extractable carbon (DeOC) in the soil. At the benchmark dose of 3.4 kg O₃/kg initial TPH, TPH carbon was reduced by nearly 6 gC/kg soil (40%), which was accompanied by an increase of about 4 gC/kg soil in dissolved organic carbon (DOC) and a 4-fold increase in 5-day biochemical oxygen demand (BOD₅). Disrupting gas channeling in the soil improved mass transport of O₃ to TPH bound to soil and increased TPH removal. Ozonation resulted in two measurable alterations of the composition of the organic carbon. First, part of DeOC was converted to DOC (∼4.1 gC/kg soil), 75% of which was not extractable by dichloromethane. Second, the DeOC containing saturates, aromatics, resins, and asphaltenes (SARA), was partially oxidized, resulting in a decline in saturates and aromatics, but increases in resins and asphaltenes. Ozone attack on resins, asphaltenes, and soil organic matter led to the production of NO₃^–, SO₄^2–, and PO₄^3–. The results illuminate the mechanisms by which ozone gas interacted with the weathered petroleum residuals in soil to generate soluble and biodegradable products

Biostimulation of chlorinated ethene-respiring communities containing <i>Dehalococcoides</i>.

<p>Dechlorination of TCE in microcosms (left panels), first transfers from microcosms (middle panels), and enriched soil/sediment-free cultures (right panels). The microcosms (left panels) were setup with (A) uncontaminated garden soil, (B) uncontaminated mangrove sediment, and (C) PCE-contaminated groundwater sediment. A total of 26 microcosms were established. (A)–(B) (left panels) Cuzdrioara and Carolina microcosm replicates exhibited the same pattern for reductive dechlorination product formation and one replicate is shown. Eight Parris Island replicate microcosms from different core depths formed VC and ethene within 30 days after microcosms were established. (C) (left panel) One representative VC and ethene-producing microcosm is presented. The dashed arrows represent an additional transfer not shown. The time-course experiments from the right panels (A–C) are from the third consecutive addition of 0.5 mmol L⁻¹ TCE. The error bars in the right panels show standard deviation of triplicate cultures. Note the time scale differences between left, middle, and right panels.</p

Characterization of <i>Dehalococcoides mccartyi</i>-containing cultures enriched in this study.

a<p>Rates calculated between two consecutive sampling points. The transient rates were highest for all cultures on the third addition of 1 mmol L⁻¹ TCE.</p>b<p>Conversion times reported from independent experiments for the third consecutive addition of TCE.</p>c<p>Final densities after three consecutive additions of TCE.</p>d<p>Yields were calculated from the change in the 16S rRNA gene copies measured by qPCR divided by the change in concentration of TCE reduced to ethene.</p

Bioaugmentation of microcosms with their respective enrichment cultures.

<p>Dechlorination of TCE in (A) in Cuzdrioara soil microcosms bioaugmentated with ZARA-10 enrichment culture and in (B) Carolina sediment microcosms bioaugmented with LINA-09 culture. The inoculum used for these experiments was 1% vol/vol.</p

Selective Enrichment Yields Robust Ethene-Producing Dechlorinating Cultures from Microcosms Stalled at <i>cis</i>-Dichloroethene

<div><p><i>Dehalococcoides mccartyi</i> strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates <i>cis</i>-dichloroethene (<i>cis</i>-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of <i>Dehalococcoides</i>, biostimulation sometimes fails to promote dechlorination beyond <i>cis</i>-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at <i>cis</i>-DCE, albeit <i>Dehalococcoides mccartyi</i> containing the reductive dehalogenase genes <i>tceA, vcrA</i> and <i>bvcA</i> were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond <i>cis</i>-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation. However, transfers from microcosms stalled at <i>cis</i>-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 10⁹<i>Dehalococcoides mccartyi</i> cells mL⁻¹. <i>Proteobacterial</i> classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of <i>Dehalococcoides</i> in the <i>cis</i>-DCE-stalled microcosms was impeded by other microbes present at higher abundances than <i>Dehalococcoides</i> and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting <i>Dehalococcoides</i> for H₂. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing <i>Dehalococcoides</i> in high abundance were able to compete for H₂ for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of <i>Dehalococcoides mccartyi</i> and occasional stalling of dechlorination at <i>cis</i>-DCE; thus, bringing a new perspective to better assess biological potential of different environments and to understand microbial interactions governing bioremediation.</p></div