2005 Progress Report: Continuous-Flow Column Studies of Reductive Dehalogenation with Two Different Enriched Cultures: Kinetics, Inhibition, and Monitoring of Microbial Activity

EPA Grant Number: R828772C012
Subproject: this is subproject number 012 , established and managed by the Center Director under grant R828772
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: HSRC (2001) - Western Region Hazardous Substance Research Center for Developing In-Situ Processes for VOC Remediation in Groundwater and Soils
Center Director: Semprini, Lewis
Title: Continuous-Flow Column Studies of Reductive Dehalogenation with Two Different Enriched Cultures: Kinetics, Inhibition, and Monitoring of Microbial Activity
Investigators: Semprini, Lewis , Dolan, Mark E. , Spormann, Alfred M.
Institution: Oregon State University , Stanford University
EPA Project Officer: Lasat, Mitch
Project Period: September 1, 2001 through August 31, 2006
Project Period Covered by this Report: September 1, 2004 through August 31, 2005
RFA: Hazardous Substance Research Centers - HSRC (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management

Objective:

This project is evaluating the transformation of chlorinated ethenes in continuous-flow column studies with the Evanite (EV) culture and the Victoria Strain (VS) culture that have been developed and kinetically characterized by Yu and Semprini, 2004; Yu, et al., 2005; Cupples, et al., 2004a; and Cupples, et al., 2004b. The overall goals of the project are to: (1) determine if kinetic parameters that were derived under batch conditions can be used to model the sequential transformation of chlorinated ethenes spatially in the columns; (2) evaluate if the predicted performance of the two enrichment cultures is achieved and to test methods that may distinguish the VS from the EV culture; (3) apply molecular methods such as FISH and Real-Time PCR to determine the spatial distribution of the cultures and quantify the dehalogenating biomass within the column; (4) apply RNA-based methods to determine energetically based TCE and VC-dehalogenating activity temporally and spatially within the column; (5) apply molecular based activity tests, such as transformation of fluorinated analogs, to determine dehalogenating activity that develops within the column; (6) study toxicity and inhibition that may result from the presence of co-contaminants, such as chloroform or acetylene; and (7) compare the results from modeling, molecular, and activity based results.

Rationale

Biologically driven reductive dehalogenation is becoming a commonly used process for remediating groundwater contaminated with chlorinated ethenes and mixtures of other chlorinated aliphatic hydrocarbons. Several studies have now demonstrated that engineered systems of enhanced reductive dehalogenation can result in complete dehalogenation of PCE and TCE to ethene. Bioaugmentation of microbial consortium that contain phylogenetic relatives of Dehalococcoides ethenogenes has promoted the complete dehalogenation of PCE or TCE to ethene. Remediation of source zones containing high concentrations of PCE and TCE via reductive halogenation is also being considered. Few studies have been performed that have evaluated changes in community structure and function under flow conditions where spatial and temporal changes in transformation and community structure can result. Column studies to date have not been performed with cultures with well defined kinetic parameters or have employed RNA-based methods to characterize the microbial activity. This study will therefore compare results of modeling, molecular, and activity based measurements in a series of continuous flow column studies.

Progress Summary:

Part I: Continuous Flow Column Studies (Lewis Semprini, PI, and Mark Dolan, Co-PI, Oregon State University)

Experimental Approach. Studies are being conducted in continuous flow columns that are packed with aquifer solids from Hanford, Washington. The size of the columns allow packing and unpacking of the columns within an anaerobic glove box. Initial studies were conducted with glass columns connected in series. These initial tests helped determine the column size needed to observe all the steps of the transformation within one column. We have now fabricated three columns from stainless steel, with sampling ports along the columns to permit spatial sampling. Three continuous flow column experiments can now be performed simultaneously. The experimental approach for these column studies was to study the transport of the CAHs prior to biostimulation; add the cultures and biostimulate through electron donor addition; and continue electron donor and CAH addition until desired spatial transformations were observed. During the course of the experiments the aqueous concentrations of the CAHs, the electron donor, fermentation products, sulfate, iron, methane, and hydrogen are being monitored. In addition, the redox status of the columns is being monitored through Dr. Ingle’s Center Project. After the desired spatial distribution of CAH transformation is achieved, the column’s aquifer material will then be sampled in an anaerobic glove box and molecular analyses will be performed at Stanford University, under the direction of Dr. Spormann, using FISH, Real-Time PCR, and RNA-based methods.

Status. Anaerobic continuous-flow column experiments initiated last year with the Evanite (EV) enrichment culture in the presence of Hanford aquifer solids were completed. Three columns were connected in series for these tests. At the highest injected lactate concentration of 1.34 mM, PCE was completely transformed to ethene with a hydraulic residence time of 4.6 days. Within a hydraulic residence time of 1.5 days, PCE was transformed to VC and ethene. Electron mass balances showed that about 70 percent of the lactate added could be accounted for, with about 2 percent going to CAH reduction, 5 percent iron reduction, 16 percent sulfate reduction, and 38 percent to the production of acetate and propionate. The 2 percent of the electron donor associated with dehalogenation reactions is consistent with microbial enumerations described below that show Dehalococcoides sp. gene copy numbers represented 0.5 to 4.0 percent of the Eubacterial 16S rRNA genes present.

Redox capacity measurements (see report for Grant No. R828772C011), showed more reducing conditions were associated with the stage of the experiment when VC was being reduced to ethene. During the stage of the test when PCE transformation was stalled at cis-DCE, reducing conditions in the column were associated with iron reduction.

At the end of the tests the columns were destructively sampled, and solid samples were obtained spatially from columns. The solids were used in microcosm construction to evaluate for rate of PCE and VC transformation, as well as rates of lactate fermentation. Spatial samples for molecular analysis were sent to Stanford for molecular methods analysis described below. Microcosm tests showed that PCE was most rapid transformed by microbes attached to aquifer solids near the entrance to the column 1, and rates rapidly decreased with distance from the column entrance. The results are consistent concentration observations that showed PCE was rapidly transformed to VC and ethene in this column. Rates of VC transformation in the microcosms were similar spatially in column 1, with slightly higher rates near the column entrance. The results were consistent with the column concentration measurements that indicated VC was being transformed to ethene in all three columns.

A second series of column tests were initiated this year in columns fabricated with stainless steel. The columns were sized so that samples could be obtained for concentration measurements on three spatial locations along the column as well as the column exit. The columns were also fabricated so that solid coupons could be collected during the course of the study at three different spatial locations to enumerate microbial activity. The columns were again packed with Hanford aquifer solids and bioaugmented with the Evanite culture.

Prior to bioaugmenting the culture, the aquifer solids were reduced by injecting groundwater that contained sulfide. Upon bioaugmentation of the Evanite culture, the transformation of PCE to VC and ethene proceeded rapidly, and no stall with cis-DCE formation was observed. Injected lactate concentration was also greatly reduced compared to the amount required in the previous tests to achieve VC transformation to ethene. A series of transient tests have been performed with gradual increases in PCE concentration from 10 mg/L to 50 mg/L. Complete transformation of VC to ethene is achieved with a hydraulic residence time of 4.5 days, and PCE is transformed to VC and ethene within a hydraulic residence of 1.5 days. In order to effectively transform PCE to ethene, increased lactate addition was needed when PCE concentrations were raised.

Part II. Real-Time (RT-)PCR for Monitoring of Reductive Dehalogenation of Chloroethenes (Alfred Spormann, PI, and Sebastian Behrens, postdoctoral researcher, (Stanford University)

Spatial Distribution of the Genus Dehalococcoides and Species Subpopulations in a Continuous Flow Column Bioreactor. We analyzed the microbial community composition of the first column study with special focus on the genus Dehalococcoides. Aquifer solids from the column were sampled for molecular analysis after 170 days of column operation. The column was split in six 5 cm sections and solids of each section were used for DNA extraction. DNA of each section served as template in real-time PCR assays to quantify Dehalococcoides organisms along the column profile. Figure 1 shows the abundance of Dehalococcoides species as percentage of total Eubacteria indicating an increase in relative species abundance from 0.5 percent in the first 5 cm to about 4 percent towards the column outflow.

We further analyzed the population composition of the genus Dehalococcoides with the help of functional gene primer specific to certain Dehalococcoides strains, e.g., strain VS, and strain BVA-1. This is possible because advanced sequencing efforts revealed that reductive dehalogenases despite their functional homogeneity contain diverse nucleotide sequences regions that allow for highly specific gene probing. We chose to target key genes of reductive dehalogenation that have been genetically and biochemically characterized to catalyze the complete dechlorination of trichloroethene (TCE) and/or vinyl chloride (VC) to ethene. Primer sets were designed to target the vinyl chloride reductase of Dehalococcoides sp. strain VS (vcrA_VS) (4), the vinyl chloride reductase of Dehalococcoides sp. strain BVA-1 (vcrA_BVA-1) (1), and the trichloroethene reductase of Dehalococcoides ethenogenes strain 195, Dehalococcoides sp. strain FL2, and Bacterium PM-VC1, RC-VC2, and YK-TCE1 (tceA_195+) (2). The distribution of the selected functional genes over the vertical profile of column 1 is shown in Figure 2.

Figure 1. Relative Abundance and Spatial Distribution of Dehalococcoides sp. Over the Vertical Profile of Column 1.

Figure 1. Relative Abundance and Spatial Distribution of Dehalococcoides sp. Over the Vertical Profile of Column 1.

The reductive dehalogenase gene abundances are displayed as percentage of the 16S rRNA gene pool of all Dehalococcoides species present in the respective section. These gene comparisons are possible because Dehalococcoides cells have only one ribosomal RNA operon per genome. The three Dehalococcoides subpopulations differ dramatically in abundance along the column profile. Whereas strain BVA-1 makes up to two-thirds of all Dehalococcoides cells in the last 10 cm closest to the column outflow, tceA containing relatives of Dehalococcoides ethenogenes strain 195 decrease in abundance from 6 percent to 0.2 percent and 1 percent towards the column end. The vcrA gene of Dehalococcoides strain VS showed a more equal distribution over the column profile decreasing from 20 percent to about 10 percent with an numerical low of only 4 percent around 25 cm from the column inflow (Figure 2).

Figure 2. Relative Abundance of Selected Genes Catalyzing the Stepwise Dechlorination of Trichloroethene to Ethene on Bioreactor 1.

Figure 2. Relative Abundance of Selected Genes Catalyzing the Stepwise Dechlorination of Trichloroethene to Ethene on Bioreactor 1. The tracked genes are specific to subpopulations of the genus Dehalococcoides indicating functional and spatial differences in the localization of different Dehalococcoides strains along the columns vertical profile.

Monitoring of Gene Expression Associated With Reductive Dehalogenation Under Continuous Flow Conditions. The primer for the TCE and VC reductive dehalogenases was further used in real-time reverse transcription (RT-) PCR experiments to study gene expression along the vertical horizon of the first reactor. Therefore, RNA was extracted from each 5 cm section. The RNA was reverse transcribed into cDNA, which served as a template in real-time PCR assays to estimate the relative expression of the described reductive dehalogenases. Figure 3 shows the expression profile of the three reductive dehalogenases followed in this study. The expression data for each gene were normalized to the gene abundance determined in the DNA quantification experiments as described in the previous paragraph. Despite their low gene abundance, the trichloroethene reductase (tceA) showed the highest expression of all three dehalogenases under investigation. The relative expression of the tceA from Dehalococcoides ethenogenes strain 195 and others peaked 10 to 15 cm from the column inflow. With the highest PCE reduction rates measured within the 5 cm from the column inflow, TCE reduction starts further up the column, where most of the PCE has already been reduced. Vinyl chloride reduction, as measured by the vcrA expression of strain VS and BVA-1, showed a more uniform activity pattern over the column profile with slightly elevated expression of the vcrA of strain BVA-1 towards the reactor outflow (Figure 3).

Figure 3. Relative Expression of Reductive Dehalogenases on Column 1. The expression data was normalized to the gene abundance based on genomic DNA extractions from each section.

Figure 3. Relative Expression of Reductive Dehalogenases on Column 1. The expression data was normalized to the gene abundance based on genomic DNA extractions from each section.

Our results show that real-time (RT-)PCR can be used to quantify abundance and activity of genes involved in reductive dehalogenation of tetrachloroethene under bioremediation conditions. The protocol and oligonucleotide primer developed in this study assemble a powerful tool for the in-situ monitoring and evaluation of laboratory scale bioreactors and field sites undergoing bioremediation.

Students Working on the Project

Sebastian Behrens, post-doctorial student, Stanford University.

Andrew Sabolowsky, Ph.D. student, Department of Civil, Construction, and Environmental Engineering, Oregon State University.

References:

1. Krajmalnik-Brown R, Hölscher T, Thomson IN, Saunders FM, Ritalahti KM, Löffler FE. Genetic identification of a putative vinyl chloride reductase in dehalococcoides sp. strain BAV1. Applied and Environmental Microbiology 2004;70:6347-6351.

2. Magnuson JK, Romine MF, Burris DR, Kingsley MT. Trichloroethene reductive dehalogenase from dehalococcoides ethenogenes: sequence of tceA and substrate range characterization. Applied and Environmental Microbiology 2000;66:5141-5147.

3. Major DW, McMaster ML, Cox EE, Edwards EA, Dworatzek SM, Hendrickson ER, Starr MG, Payne JA, Buonamici LW. Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene. Environmental Science & Technology 2002;36:5106-5116.

4. Mueller JA, Rosner BM, von Abendroth G, Meshulam-Simon G, McCarty PL, Spormann AM. Molecular identification of the catabolic vinyl chloride reductase from dehalococcoides sp. strain VS and its environmental distribution. Applied and Environmental Microbiology 2004;70:4880-4888.

5. Yu S, Dolan ME, Semprini L. Kinetics and inhibition of reductive dechlorination of chlorinated ethylenes by two different mixed cultures. Environmental Science & Technology 2005;39:195-205.

6. Amann RI. In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes. In: Akkerman ADL, van Elsas DJ, de Bruijn FJ, eds. Molecular Microbial Ecology Manual. Dordrecht, Netherlands: Kluwer Academic Publishers, 1995, pp. 1-15.

7. Cupples AM, Spormann AM, McCarty PL. Comparative evaluation of chloroethene dechlorination to ethene by dehalococcoides-like microorganisms. Environmental Science & Technology 2004A;38:4768-4774

8. Cupples AM, Spormann AM, McCarty PL. Vinyl chloride and cis- dichloroethene dechlorination kinetics and microorganism growth under substrate limiting conditions. Environmental Science & Technology 2004b;38:1102-1107.

9. Yu S, Semprini L. Kinetics and modeling of reductive dechlorination at high PCE and TCE concentrations. Biotechnology Bioengineering 2004;88(4):451-464.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 5 publications 2 publications in selected types All 2 journal articles
Other center views: All 158 publications 63 publications in selected types All 60 journal articles
Type Citation Sub Project Document Sources
Journal Article Pon G, Semprini L. Anaerobic reductive dechlorination of 1-chloro-1-fluoroethene to track the transformation of vinyl chloride. Environmental Science & Technology 2004;38(24):6803-6808. R828772 (2003)
R828772 (2004)
R828772 (Final)
R828772C012 (2005)
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  • Journal Article Yu S, Dolan ME, Semprini L. Kinetics and inhibition of reductive dechlorination of chlorinated ethylenes by two different mixed cultures. Environmental Science & Technology 2005;39(1):195-205. R828772 (2003)
    R828772 (Final)
    R828772C012 (2005)
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    Progress and Final Reports:

    Original Abstract
  • 2002
  • 2003
  • 2004 Progress Report
  • Final

  • Main Center Abstract and Reports:

    R828772    HSRC (2001) - Western Region Hazardous Substance Research Center for Developing In-Situ Processes for VOC Remediation in Groundwater and Soils

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R828772C001 Developing and Optimizing Biotransformation Kinetics for the Bio- remediation of Trichloroethylene at NAPL Source Zone Concentrations
    R828772C002 Strategies for Cost-Effective In-situ Mixing of Contaminants and Additives in Bioremediation
    R828772C003 Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbon Compounds with Butane-Grown Microorganisms
    R828772C004 Chemical, Physical, and Biological Processes at the Surface of Palladium Catalysts Under Groundwater Treatment Conditions
    R828772C005 Effects of Sorbent Microporosity on Multicomponent Fate and Transport in Contaminated Groundwater Aquifers
    R828772C006 Development of the Push-Pull Test to Monitor Bioaugmentation with Dehalogenating Cultures
    R828772C007 Development and Evaluation of Field Sensors for Monitoring Bioaugmentation with Anaerobic Dehalogenating Cultures for In-Situ Treatment of TCE
    R828772C008 Training and Technology Transfer
    R828772C009 Technical Outreach Services for Communities (TOSC) and Technical Assistance to Brownfields Communities (TAB) Programs
    R828772C010 Aerobic Cometabolism of Chlorinated Ethenes by Microorganisms that Grow on Organic Acids and Alcohols
    R828772C011 Development and Evaluation of Field Sensors for Monitoring Anaerobic Dehalogenation after Bioaugmentation for In Situ Treatment of PCE and TCE
    R828772C012 Continuous-Flow Column Studies of Reductive Dehalogenation with Two Different Enriched Cultures: Kinetics, Inhibition, and Monitoring of Microbial Activity
    R828772C013 Novel Methods for Laboratory Measurement of Transverse Dispersion in Porous Media
    R828772C014 The Role of Micropore Structure in Contaminant Sorption and Desorption