Grantee Research Project Results

2004 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: Solutions for Energy, AiR, Climate and Health Center (SEARCH)
Center Director: Bell, Michelle L.
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: Aja, Hayley
Project Period: September 1, 2001 through August 31, 2006
Project Period Covered by this Report: September 1, 2003 through August 31, 2004
RFA: Hazardous Substance Research Centers - HSRC (2001) Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management

Objective:

This project is evaluating the transformation of chlorinated ethenes in sequencing batch reactors and continuous-flow column studies with the Point Mugu (MU) and the Evanite (EV) cultures that have been developed and kinetically characterized in our earlier WRHSRC project (Yu and Semprini, 2004; Yu, et al., in press). 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 MU culture 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 dehaogenation of PCE and TCE to ethene. Bioaugmentation of microbial consortium that contain phylogenetic relatives of Dehaloccoides etheneogenes 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 anerobic 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 were conducted with the Evanite (EV) enrichment culture in the presence of Hanford aquifer solids to evaluate the sequential reductive dechlorination of chlorinated ethenes. Three glass columns (30 cm length x 4.5 cm i.d.) with Teflon endcaps were connected in series. Each column was packed with about 1 kg Hanford soil, resulting in a porosity of 0.35 with 190 m L pore volume, and sequentially connected under anaerobic conditions. After packing the columns, synthetic Hanford groundwater was pumped into the bottom of the first column using a gradient HPLC pump. Tracer tests were performed by pumping 0.6 mM Br and 0.07 mM PCE through the column at flow rate of 0.1 mL/min and measuring breakthrough of bromide and PCE. PCE retardation factors of 6.8 to 7.4 were calculated from bromide and PCE tracer breakthrough curves by using CXTFIT two-site-non-equilibrium model. After four weeks of PCE and bromide addition, the columns were amended with nutrients, PCE 0.07 mM, lactate 0.34 mM, and 0.2 mM sulfate in synthetic Hanford groundwater. Little microbial activity was observed during this period with indigenous microbes from the Hanford soil, based on the absence of sulfate reduction and lactate removal. After six weeks, the columns were inoculated with the EV enrichment culture by direct injection of the culture into the bottom, inlet end, of each column. The injected lactate concentrations were increased from 0.34 mM to 0.67 mM, and to 1.34 mM, after six, eight, and sixteen weeks, respectively.

PCE dechlorination to TCE, and cis-DCE were observed when lactate concentrations increased from 0.35 mM to 0.67 mM on week eight. Rapid increases in cis-DCE concentration in the effluent compared to influent PCE concentration suggested enhanced PCE desorption and consequent reduction to cis-DCE. All the PCE was transformed to cis-DCE in the first column. When the lactate concentration was increased to 1.34 mM, propionate production was observed from lactate fermentation, and cis-DCE reduction to vinyl chloride (VC) occurred. VC transformation to ethene was observed in column three once the cis-DCE concentrations were reduced to low levels, and enhanced cis-DCE desorption was also indicated. The results are consistent with previous laboratory and modeling studies with the EV culture (Yu, et al., in press) that showed cis-DCE strongly inhibits VC transformation. The reductive capacity increased over time as the columns became more biologically active and production of VC and ethene was observed. These results suggest that the EV culture is able to transform PCE to ethene under controlled reducing conditions in a continuous-flow system. Methane production has not been detected in the columns.

CAH recoveries were 98.8%, 90.4% , and 113.5% of the PCE injected in columns #1, #2 and #3, respectively. Lactate mass recoveries were 43%, 59%, and 45.7%, and sulfate recoveries were 36.1%, 31.1%, and 26.1%. Mass balances on lactate reduction as an electron donor were calculated using chemical reactions for CAH and sulfate reduction, and the production of propionate. Results show that only 31.1% of reduced lactate could be accounted for by the reduction of CAHs, sulfate, and production of propionate. We have not observed acetate production, and are currently determining if we have had an analytical problem with our acetate measurement.

Part II: Development and application of molecular methods for the detection and quantification of microbial populations capable of reductive dehalogenation (Alfred M. Sporman, PI, and Sebastian Behrens, postdoctoral researcher and Co-PI, Stanford University)

A. Monitoring and Quantifying Dehalogenating Microorganisms. We developed and evaluated the specificity of a new primer set targeting the 16S rRNA gene of Dehalococcoides sp. These primers will be used in real-time quantitative PCR studies to quantify 16S rRNA gene copy numbers in total DNA from different sections of the column and in the aqueous phase.

B. Determining the Spatial Distribution of Dehalococcoides sp. on the Bioreactor Columns. Fluorescence in situ hybridization (FISH) with rRNA-targeted probes is, among other things, a staining technique that allows phylogenetic identification of bacteria in mixed assemblages without prior cultivation by means of epifluorescence and confocal laser scanning microscopy (Armann, 1995). In theory, each ribosome within a bacterial cell, containing one copy each of 5S, 16S and 23S rRNA, is stained by one probe molecule during the hybridization procedure. For FISH with mono-labeled fluorescent oligonucleotide probes, this is often the critical point because the majority of environmental bacteria are small, slowly growing or starving, and contain only low amounts of ribosomes. Therefore, signal intensities are frequently below detection limits or lost in high levels of background fluorescence.

These limitations can be overcome by the use of horseradish peroxidase (HRP) labeled oligonucleotide probes and tyramide signal amplification (TSA), also known as catalyzed reporter deposition (CARD) (Pernthaler, et al., 2002). CARD is based on the deposition of a large number of fluorochrome-labeled tyramine molecules by peroxidase activity. In this way, numerous fluorescent molecules can be introduced at the hybridization site in situ. This results in greatly enhanced FISH sensitivity compared to probes with a single fluorochrome.

We developed a CARD-FISH protocol for the identification of Dehalococcoides sp. in environmental samples. The protocol uses HRP-labeled 16S rRNA targeted oligonucleotide probes specific for the genus Dehalococcoides sp. (Figure 1). The probe targets all 16S rRNA gene sequences of Dehalococcoides species currently in public databases, comprising isolated strains and environmental clones. All 16S rRNA gene sequences are closely related and form a unique cluster of Dehalococcoides species.

Figure 1. Photomicrograph of Dehalococcoides sp. Strain VS Stained with DAPI and Hybridized with HRP-Labeled Oligonucleotide Probes. A. DAPI image. B. phase contrast image. C. Dehalococcoides sp. genus-specific probe image. Scale bar 10 μm.

We could show that our HRP-labeled probes can be used to detect the Dehaloccoccoides sp. subpopulation within the Evanite enrichment culture (Figure 2). By using the CARD-FISH protocol and the evaluated Dehalococcoides-genus specific probes, we will map the spatial distribution of these organisms throughout the length of the column.

Figure 2. Photomicrograph of the Evanite Enrichment Culture Hybridized with HRP-Labeled Oligonucleotide Probes. A. Dehalococcoides sp. genus-specific probe image. B. general Eubacteria probe image. Scale bar 10 μm.

C. Determining the Spatial Distribution of vcrA. Müller, et al. (2004) showed a strong correlation of the presence of a vcrA homolog with reductive VC dehalogenation. They used the vcrAB sequence as molecular probe to test for in situ VC reduction potential in environmental samples. We were able to demonstrate the presence of the vcrAB gene in total DNA extractions of the Evanite enrichment culture using the primer published by Müller, et al. (2004)

We developed two new primer sets that target the vcrA gene of Dehalococcoides sp. strain VS for application in real-time quantitative PCR. The specificity of the primers was tested by standard PCR. The primer will be used in real-time quantitative RT-PCR assays to quantify vcrA copy numbers in total RNA from column matrix and pore water samples. This is a “straightforward ” approach since we showed the presence of vcrA in the Evanite enrichment culture that has been used to inoculate the flow columns.

In parallel we are working on developing a protocol for the detection of vcrA mRNA gene transcripts by fluorescence in situ hybridisation (Pernthaler and Amann, 2002). By combining the sensitivity of the CARD-FISH approach with the use of polyribonucleotide transcript probes of vcrA, our aim is to visualize microorganisms that are actively transcribing vcrA in the column material.

Students Working on the Project

Sebastian Behrens, post-doctoral student, Stanford University.

SeunghoYu, post-doctoral student, Department of Civil, Construction, and Environmental Engineering, Oregon State University.

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

References:

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.

Müller JA, Rosner BM, von Abendroth G, Simon-Meshulam 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(8):4880-4888.

Pernthaler A, Amann R. Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Applied and Environmental Microbiology 2004;70(9):5426-5433.

Pernthaler A, Pernthaler J, Amann R. Fluorescence in situ hybridisation and catalyzed reporter deposition for the identification of marine bacteria. Applied and Environmental Microbiology. 2002;68(6):3094-3101.

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

Yu S, Dolan ME, Semprini L. Kinetics and inhibition of reductive dechlorination of chlorinated ethylenes by two different mixed cultures. Environmental Science & Technology (in press, 2004).

Future Activities:

Future Plans for Part I—Aquifer solids from the three columns will be sampled for molecular analysis as soon a VC is completely transformed to ethene in the third column. Of particular interest will be the first column where PCE is added and is currently being transformed to TCE, cis-DCE and VC. We have also fabricated three stainless steel columns that will permit simultaneous experiments with the EV and MU cultures. In the third columns, we may study a mixture of the EV and MU cultures, or the Dehalococcoides sp. strain VS that has been studied in detail at Stanford University in Dr. Spormann’s laboratory.

Journal Articles:

No journal articles submitted with this report: View all 5 publications for this subproject

Relevant Websites:

http://wrhsrc.oregonstate.edu/ Exit

Progress and Final Reports:

Original Abstract

2002

2003

2005 Progress Report

Final

Main Center Abstract and Reports:

R828772    Solutions for Energy, AiR, Climate and Health Center (SEARCH)

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
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