The Influence of In-Situ Activated Carbon on Biodegradation of Chlorinated Solvents

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8-2018 The nflueI nce of In-Situ Activated Carbon on Biodegradation of Chlorinated Solvents Kameryn McGee Clemson University, [email protected]

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Recommended Citation McGee, Kameryn, "The nflueI nce of In-Situ Activated Carbon on Biodegradation of Chlorinated Solvents" (2018). All Theses. 2925. https://tigerprints.clemson.edu/all_theses/2925

This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact [email protected]. THE INFLUENCE OF IN-SITU ACTIVATED CARBON ON BIODEGRADATION OF CHLORINATED SOLVENTS

A Thesis Presented to the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree Master of Science Environmental Engineering and Earth Sciences

by Kameryn McGee August 2018

Accepted by: Dr. Kevin Finneran, Committee Chair Dr. David Ladner Dr. Sudeep Popat ABSTRACT

Chlorinated solvents have been a contaminant of interest in the remediation field for many years because they have been manufactured in large amounts and released into the environment due to improper storage and disposal. Since they are produced in such large quantities and there has been so many cases of uncontrolled releases, corrective actions are necessary and many remediation strategies have been explored. In- situ bioremediation of chlorinated solvents (TCE) is an intricate respiratory process which combines the addition of electron donors, chemical reactions, and the presence of microorganisms which directly access contaminants. Microbes metabolize the electron donor and utilize the energy for complete dechlorination of TCE to ethene. Vendors suggest the addition of activated carbon to the system should be used to provide an absorptive surface for both contaminants and microbes and is thought to act as an electron shuttle to increase the rate of reduction.

Each microcosm was set up in triplicate with TCE contaminated sediment and surface water from the same site, an electron donor amendment (lactate, acetate + hydrogen, or emulsified oil substrate (EOS)), 20umol of neat TCE, and activated carbon.

For both granular activated carbon (GAC) and powdered activated carbon (PAC) the high mass loading rate, suggested by the vendor, was added at 78 mg/mL of water and the low mass loading rate was added at 26 mg/mL of water, one-third the suggested amount. Each electron donor amended series was evaluated at high and low loading rates of GAC and

PAC as well as a no activated carbon control. There were also 2 additional controls, a no amendment series control and sterile series control. A gas chromatograph (GC) was used

ii to determine the mass of TCE, its dechlorination products, and methane present in each microcosm. Data suggest that the series with low PAC and no activated carbon have the most dechlorinating activity regardless of the electron donor amendment and the series with high mass loading of GAC and PAC are showing no dechlorination. However, amongst the amendments, EOS is showing the highest activity but it is also producing more methane than any other amendment.

iii TABLE OF CONTENTS

Page

Abstract ...... ii

Table of Contents ...... iv

List of Figures ...... vi

Abbreviations and Acronyms ...... xi

1.0 Introduction ...... 1

2.0 Materials and Methods ...... 8

2.1 Site location and sample collection ...... 8 2.2 Time zero preparations and measurements ...... 8 2.3 Monitoring of TCE and degradation products ...... 9 2.4 Additional amendments ...... 10 2.5 Analysis of TCE and degradation products ...... 11

3.0 Results and Discussion ...... 11

3.1 Influence of GAC on TCE removal ...... 11 3.2 Influence of PAC on TCE removal ...... 13 3.3 Methane increases in the presence of activated carbon ...... 15 3.4 Does electron donor make a difference in TCE reduction ...... 16 3.5 Partitioning of dechlorination products and how it can impact analyses ...... 18

4.0 Conclusions ...... 19

4.1 TCE reduction ...... 19 4.2 Methane production ...... 20 4.3 Future research ...... 20

Appendices.…...... 24

Appendix A: Data Plots for Initial Series of Bath Incubations ...... 22 Appendix B: Data Plots for Second Series of Bath Incubations ...... 48

iv Table of Contents (Continued) Page

References…… ...... 64

v LIST OF FIGURES

Figure Page

A-1 Initial series batch incubations, no electron donor amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 23

A-2 Initial series batch incubations, no electron donor amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 24

A-3 Initial series batch incubations, no electron donor amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 25

A-4 Initial series batch incubations, no electron donor amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 26

A-5 Initial series batch incubations, no electron donor amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 27

A-6 Initial series batch incubations, lipid amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 28

A-7 Initial series batch incubations, lipid amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 29

A-8 Initial series batch incubations, lipid amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 30

A-9 Initial series batch incubations, lipid amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 31

vi List of Figures (Continued)

Figure Page

A-10 Initial series batch incubations, lipid amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 32

A-11 Initial series batch incubations, lactate amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 33

A-12 Initial series batch incubations, lactate amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 34

A-13 Initial series batch incubations, lactate amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 35

A-14 Initial series batch incubations, lactate amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 36

A-15 Initial series batch incubations, lactate amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 37

A-16 Initial series batch incubations, hydrogen gas plus acetate amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 38

A-17 Initial series batch incubations, hydrogen gas plus acetate amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 39

A-18 Initial series batch incubations, hydrogen gas plus acetate amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 40

vii List of Figures (Continued)

Figure Page

A-19 Initial series batch incubations, hydrogen gas plus acetate amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 41

A-20 Initial series batch incubations, hydrogen gas plus acetate amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 42

A-21 Initial series batch incubations, sterile series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 43

A-22 Initial series batch incubations, sterile series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 44

A-23 Initial series batch incubations, sterile series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 45

A-24 Initial series batch incubations, sterile series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 46

A-25 Initial series batch incubations, sterile series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 47

B-1 Second series batch incubations, no electron donor amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 49

B-2 Second series batch incubations, no electron donor amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 50

viii List of Figures (Continued)

Figure Page

B-3 Second series batch incubations, no electron donor amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 51

B-4 Second series batch incubations, no electron donor amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 52

B-5 Second series batch incubations, no electron donor amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 53

B-6 Second series batch incubations, lipid amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 54

B-7 Second series batch incubations, lipid amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 55

B-8 Second series batch incubations, lipid amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 56

B-9 Second series batch incubations, lipid amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 57

B-10 Second series batch incubations, lipid amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 58

B-11 Second series batch incubations, lactate amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 59

B-12 Second series batch incubations, lactate amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 60

ix List of Figures (Continued)

Figure Page

B-13 Second series batch incubations, lactate amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 61

B-14 Second series batch incubations, lactate amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20µmol of TCE...... 62

B-15 Second series batch incubations, lactate amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20µmol of TCE...... 63

x ABBREVIATIONS AND ACRONYMS

Cis-DCE cis-dichloroethene Dhc Dehalococcoides DIET direct interspecies electron transfer DOM dissolved organic matter EOS emulsified oil substrate GAC granular activated carbon GC gas chromatograph MNA monitored natural attenuation PAC powdered activated carbon RPI Remediation Products Inc. TCE trichloroethylene VC vinyl chloride VOCs volatile organic compounds

xi 1.0 Introduction

Environmental chemicals such as chlorinated solvents are volatile organic compounds (VOCs) and have been contaminants of interest in the remediation field for many years because they are the third most prevalent groundwater contaminant in the

United States [1, 2]. These chlorinated organic compounds, including chloroethylenes, chloromethanes, and chloroethanes, are colorless, highly volatile liquids at room temperature and have a low aqueous solubility [3]. They are manufactured in large amounts to be used for industrial, military, agricultural, and household applications and can be found in many environmental mediums including soil, air, groundwater and surface waters [4]. Chlorinated organics are commonly associated with dry cleaning operations, used as a solvent to for degreasing metals, used in the production of some refrigerants and paint removers [4, 5].

Trichloroethylene (TCE) a member of chloroethylenes is widely used as a solvent for waxes, fats, and oils and has been used extensively for dry cleaning, equipment maintenance and metal degreasing since the 1920s. It is not consumed through its various uses and as a result of limited regulations during its heavy use in the mid to late 20th century it was released to the environment through improper handling and the disposal of waste. As a result of environmental release, TCE and its transformation products, cis- dichloroethene (cis-DCE), vinyl chloride (VC), and ethene, are commonly found in the at industrial, commercial, and military sites where they persist in the groundwater and can be transported long distances [1, 4-7]. Accumulation of these compounds is troubling because these subsurface contaminants are a threat to our drinking water source and

1 unless TCE is completely dechlorinated the degradation products are still toxic [6, 8-10].

Due to the presence in many environmental mediums, there are many routes of exposure to humans such as inhalation, ingestion, and dermal absorption. Exposure to chlorinated solvents is linked to adverse reproductive outcomes and many types of cancers. TCE is linked to liver cancer, biliary cancer, and non-Hodgkin’s lymphoma [4, 7].

Due to the potential adverse and carcinogenic health effects it is important to find effective removal technologies for volatile chlorinated solvents including TCE [2]. Some of the bioremediation approaches include biological and chemical processes[7].

Bioremediation is a process which relies on biological mechanisms to degrade, detoxify, or transform pollutants to an innocuous state and can be carried out either ex-situ or in- situ. However, some strategies are inefficient to be carried out as a large-scale solution

[3, 11]. Ex-situ strategies can be costly because it is difficult to access these contaminants in the subsurface and remove them [4]. In-situ strategies can also be expensive because of the equipment that needs to be installed onsite and another concern is the inability to visualize and control the subsurface [11, 12].

Ex-situ remediation approaches include pump and treat, excavation followed by incineration, and ozonation. The pump and treat method can be effective in hydraulic containment but rarely lead to concentrations required for site closure [1]. Excavation and incineration can destroy TCE in a short period of time. There is a high cost associated with this strategy because TCE has low flammability and elevated temperatures, up to

1000 oC, are needed to destroy it. Ozonation is an oxidation process in which TCE is oxidized through direct contact with ozone molecules. This strategy is not practical

2 because ozone is not very soluble in water; there is also humic substances and carbon dioxide present which can restrict the ability of ozone to target TCE [4].

In-situ remediation approaches include zero valent metal reduction and chemical and biological degradation [4, 12]. Zero valent metal reduction utilizes iron as an electron donor to reduce TCE to chlorine ions and non-chlorinated products; however, this process has a low reaction rate [4]. One of the most cost-effective approaches is monitored natural attenuation which relies on both natural biotic and abiotic processes; these processes include sorption, dilution, volatilization and bioremediation, the natural microbial degradation processes [13, 14]. This approach requires documentation of efficacy of microbial degradation to gain regulatory and public approval. Bioremediation occurs through anaerobic reductive dechlorination or aerobic co-oxidation pathways and is a key component in MNA [13, 15].

The use of in situ bioremediation as a remediation strategy allows microbes to directly access the contaminants in the subsurface and convert them to the innocuous byproducts [1, 16, 17]. Most anaerobic dechlorinating bacteria, Desulfuromonas,

Sulfurospirillum multivorans, and Dehalobacter, are able to reduce TCE to the toxic intermediate cis-DCE. However, complete reductive dechlorination, the replacement of the chlorine substituents with a hydrogen atom, to non-toxic ethene can be achieved by using a microbe of the genus Dehalococcoides (Dhc) [1, 3, 9, 15, 18]. Each dechlorination step consumes two electrons and two protons and released one proton and one chlorine ion. Dehalococcoides are naturally occurring, slow growing, strict anaerobes that require certain halogenated organic compounds as electron acceptors. Some strains

3 of Dhc are known to reductively dechlorinate cis-DCE to ethene; these strains contain the bvcA and vcrA genes [3, 19].

In-situ bioremediation of TCE requires an intricate respiratory process which combines the addition of electron donors, chemical reactions and microbial respiration with TCE as the electron acceptor [10, 15, 16]. Common electron donors are acetate, molecular hydrogen, lipids, and lactate [10, 20]. Molecular hydrogen as a single electron donor is a limitation because methanogens and acetogenic bacteria complete for it. If the concentration of hydrogen is ideal, Dhc will carry out the more thermodynamically favorable reductive dechlorination reactions and can outcompete the methanogens for the electron donor that is present [3]. The microbes are able to utilize the electron donor to produce a carbon source, usually acetate, therefore the addition on acetate is not needed unless it is being used as the donor. TCE is the electron acceptor and through this metabolism process the microbes can gain the energy required for complete reductive dechlorination from trichloroethene (TCE) to cis-dichloroethene (cis-DCE) to vinyl chloride (VC) to ethene [5, 21].

In the case where endogenous microbes are not present, it is possible to introduce an inoculation of exogenous bacterial degraders to the contaminated soil [7, 22]. Dhc cultures are grown in the lab using strictly anoxic techniques. Mixed cultures generally do better in reducing TCE than pure cultures because other members of the microbial community can protect the Dhc from toxic oxygen or they are able to provide the required growth factor vitamin B12. This cobalt-containing corrinoid cofactor is essential in reductive dechlorination and can be supplied by natural occurring or bioaugmented

4 iron reducers. They are also able to maintain the pH around 6.8-7.2 which is the level that is optimal for Dhc organisms to thrive [1, 3].

Activated carbon is an adsorption technology that is well known and has been used for decades [23]. This technology is used for industrial applications, agriculture, medical purposes, and environmental applications [24-27]. It is also one of the best available techniques for removal of small molecular organic contaminants from drinking water [28]. Activated carbon is a product of combustion or incomplete combustion of hydrocarbons resulting in a saturation of hydrogens at the edges of the layers. Elements other than hydrogen are also found in the surface including oxygen which is taken up during formation or storage. Oxygen is important because it can have a large impact on the surface properties and behavior in practical applications of the activated carbon [29].

Activated carbon is porous and provides a large surface area and therefore has a high affinity for many organic pollutants [30]. Activated carbon can be found in either powder or granular forms. The granular form has a large internal surface area and small pores and the powder form with particle sizes ranging from 10 to 50µm, has larger pore diameters and a smaller internal surface area [28]. Adsorption of contaminants to activated carbon can be characterized as either chemical or physical. The physical adsorption is mainly attractive forces known as van der Walls forces and they are reversible [18].

Since TCE is produced in large quantities, which results in many cases of uncontrolled releases corrective actions are necessary and many remediation strategies have been explored. A technology has emerged in the last two years for dissolved plume control at chlorinated solvent contaminated sites; it is referred to as in situ activated

5 carbon amendment. The use of in situ activated carbon amendment is marketed by two major vendors, Remediation Products Inc. (RPI) and Regenesis, although there are several smaller market vendors that also provide the material.

RPI’s BOS200, a modified PAC Trap & Treat Bacteria Concentrate, primarily used for the treatment of hydrocarbons, contains a blend of naturally occurring bacteria, fungi, selected nutrients and a variety of electron acceptors that can be utilized under aerobic and anaerobic conditions [31]. The product is distributed when injected at high pressures into identified contaminated zones creating an intertwined network of fractures within the clay or silt [32]. The hydrocarbons absorb to the carbon which triggers the activated carbon to come in contact with the contaminant. The microbes then digest the hydrocarbons which reactivates the spent carbon and pulls in more contaminant to be digested. This cyclic action regenerates the carbon for continued use rather than it being depleted after the initial injection. RPI states that treatment is accomplished when microorganisms, electron donors, electron acceptors, and nutrients are present and that treatment results in metabolic by-products, energy, and new microorganisms. The driving force for the biodegradation is the transfer of electrons from the hydrocarbon donor to the electron acceptor [32-34].

RPI’s BOS100, a modified GAC Trap & Treat Bacteria Concentrate, contains a blend of naturally occurring bacteria and fungi and is used primarily for the treatment of chlorinated contaminants. It is impregnated with elemental iron and heated to high temperatures which creates a highly active metallic iron surface area [35]. The delivery methods include direct push injection, soil mixing techniques and trenching and is

6 applied at a number of points using a triangular grid pattern. The TCE adsorbs to the

GAC particles and is reduced to ethene and methane as it effectively controls any toxic daughter products through the reduction process [32].

Regenesis’ PlumeStop liquid activated carbon is composed of 1-2 µm particles and suspended in water using organic polymer dispersion chemistry. PlumeStop is injected into the subsurface through low pressure injections without fracturing the formation[32, 36]. The dispersive flow provides a thin layer that coats the aquifer matrix and acts as a colloidal biomatrix to remove the contaminants from the groundwater. Once they sorb to the surface and naturally present or an inoculum of contaminant degrading bacteria is added, permanent biodegradation of the contaminant takes place [36, 37].

The vendor’s literature claims activated carbon readily adsorbs chlorinated solvents contaminants, PCE, TCE, and the degradation products, removing them from the aqueous medium and then facilitates long-term biological and chemical degradation processes that transform the contaminants to innocuous end products. Activated carbon uses a combination of chemical and biological processes to adsorb contaminants from the groundwater and promote bioremediation with pre-saturated microbes and nutrients [32].

TCE does sorb to activated carbon; however, despite vendor marketing claims, data suggests VC and ethene do not sorb and cis-DCE has a low affinity for activated carbon so mass transfer would be to the aqueous solution not to the activated carbon. If there were active complete dechlorination of TCE we would expect to see VC and ethene in solution as well as some cis-DCE.

7 There are two critical reasons for investigating this remediation technology. First, it has been readily adopted by regulators so it is incumbent upon the research community to investigate this technology so it is applied in the most productive manner. Second, and more importantly, there are no peer reviewed data indicating that the strategy works as marketed – that microbial activity will degrade contaminants that sorb to activated carbon in situ.

2.0 Materials and Methods

2.1 Site locations and sample collection

Surface water and sediment samples were collected from various locations along a small stream at a known TCE contaminated site in South Carolina. The surface water and the sediment were both kept in an incubator set at 30oC until they were ready to be used.

2.2 Time zero preparations and measurements

Three electron donors were tested in triplicate using batch incubations: acetate plus hydrogen, lactate, and lipid. A no electron donor control was also tested in triplicate.

Each incubation was set up in glass serum bottles and filled with 10 g of sediment, with an assumed porosity of 0.3, and 10 mL of water from the site. High and low loading rates of GAC and PAC were added to each electron donor amended series as well as including a no activated carbon control. The PAC used was from Fisher Scientific, DARCO G-60 and the GAC used was from US Filter Westates, AC1250C. For both GAC and PAC the high mass loading rate, suggested by the vendor, was added at 78 mg/mL of water and

8 the low mass loading rate was added at 26 mg/mL of water, one-third the suggested amount. Then each bottle will be sealed with a thick rubber butyl stopper and crimped.

The headspace was flushed with high purity nitrogen gas, which was passed through a heated reduced copper column to remove any oxygen; this created an anoxic environment. Once sealed, each electron donor amendment was added at 1x stoichiometry and 20 µmol of neat TCE was added to each bottle. An additional sterile series control was also prepared. The sterile series bottles were autoclaved for 1 hour, for

3 days in a row to ensure that there will not be any spore forming organisms that survive the first sterilization. This control group was used to observe any reduction that may have resulted from non-biological processes.

A second series of batch incubations were prepared in triplicate using only lactate,

EOS, and no electron donor amendment. Each incubation was set up with 10 g of sediment, with an assumed porosity of 0.3, and 20 mL of water from the site. High and low loading rates of GAC and PAC was added to each electron donor amended series as well as including a no activated carbon control. Then each bottle was sealed with a blue rubber butyl stopper and the headspace was flushed with high purity nitrogen gas to create an anoxic environment. Once sealed, each electron donor amendment was added at

1x stoichiometry and 20 µmol of neat TCE was added to each bottle.

2.3 Monitoring of TCE and degradation products

After letting the experimental bottles equilibrate for 24-48 hours at room temperature, time 0 data were collected. A Shimadzu 2014 gas chromatograph (GC) with

9 a flame ionization detector and a 30m GS-Q column was used to quantify the mass, recorded as µmol per bottle, of TCE and the dechlorination daughter products present in each microcosm. The carrier gas was ultra-high purity helium at 128.5 cm/second and the initial oven temperature was held at 40 oC for 1.5 minutes before ramping up to 200 oC at a rate of 40 oC/minute. The injection port temperature was 200 oC and the detector temperature was set to 260 oC. A pressure-lok VICI precision sampling glass syringe was flushed with high purity nitrogen gas and 0.2 mL of nitrogen was added to the headspace of the experimental bottle. Then a sample of 0.2 mL of headspace was removed with the glass syringe and injected, using an injection needle, into the GC. In between samples, the syringe and the injection needle were placed on a vacuum.

2.4 Additional amendments

After 57 days the second series of batch incubations was amended to have a total of 20 mM of lactate and 1 g/L of EOS in each bottle in their respective series. The bottles were kept sealed and the amendment was added directly to the surface water through the rubber butyl stopper using a 24-gauge needle and syringe. Data continued to be collected using the same method.

After 113 days, all of the second series batch incubations were amended with a crossover culture from another set of experiments that used lactate as an electron donor.

The setup for these experiments used the same contaminated sediment and surface water from the Easley site. This culture contained DHC organisms that were completely reducing TCE to ethene. Freshwater media was added to completely liquefy the contents

10 of the bottle as well as add necessary nutrients for the microbes. Using an 18-gauge needle, 1 mL of liquid was added to each of the experimental bottles except the sterile control. Data continued to be collected using the same method.

2.5 Analysis of TCE and degradation products

The peak area was used to calculate the mass present in the sample at time zero using the slope from standard curves that were created using known concentrations of

TCE, cis-DCE, VC, and ethene. The same process was used to collect subsequent data points.

3.0 Results and Discussion

3.1 Influence of GAC on TCE removal

A GC was used to determine the concentrations of TCE, cis-DCE, VC, ethene, and methane present in both high mass loading and low mass loading of GAC. The high mass loading of GAC incubations contain 78 mg of GAC per 1 mL of water and the low mass loading of GAC incubations contain one-third of the high mass loading, 26 mg of

GAC per 1 mL of water. In all incubations with both high and low mass loading of GAC,

TCE concentrations reach 0 µmol/bottle within days to weeks of its introduction into the system. In all incubations with high mass loading of GAC there is almost no evidence of dechlorination of TCE. In the low mass loading of GAC, the data indicate there was some dechlorination of TCE to cis-DCE, VC, and ethene. There is about 5 µmol/bottle of cis-

DCE detected in the lipid and the hydrogen gas + acetate electron donor amended series,

11 seen in Figures A-7, B-7 and A-17. VC and ethene is detected at minimal concentrations, below 1µmol/bottle, in Bottle 3 of the hydrogen gas + acetate electron donor amended series, seen in Figure A-17. Data for Bottle 1 in Figure B-7 is missing because it broke 4 weeks into data collection and no significant data had been recorded. All other incubations with low mass loading of GAC have either little or no evidence of dechlorination.

GAC particles contain a variety of pore sizes, ranging from the largest to the smallest, macropores, mesopores, and micropores. The smallest and the deepest pore spaces are called micropores. There is minimal evidence of TCE reduction in the presence of GAC which indicates the TCE sorbs into these pore spaces which are too small to allow direct contact between the TCE and the Dhc. However, in some cases there is evidence of dechlorination from TCE to cis-DCE which suggests that the Dhc are either able to access the TCE before it reaches the micropore sorption sites or access the

TCE after it desorbs and diffuses out of the micropores [38].

Environmental factors also play a major role in the adsorption capacity of contaminants to the activated carbon because they can change the physiochemical properties of the activated carbon. Other compounds in the system, such as dissolved organic matter (DOM), can compete for adsorption sites on the activated carbon leaving a limited number of sites available for the TCE and its degradation products to sorb [38].

Not only is DOM a competitor for sorption sites on the activated carbon but it is also an electron donor as well as a sorbate for TCE [39, 40]. There are a few instances, Figures

A-1, A-7, and A-17, where the data indicate a decrease in TCE concentration followed by

12 a brief increase in TCE concentration. This could be a result of the TCE sorbing to the

DOM and then being released back into the aqueous phase as the DOM is utilized by the microbes present as an electron donor.

TCE is not completely reduced to ethene at stoichiometric levels in any incubations which suggests that the sorbed TCE may not be fully available for microbial respiration, or it is possible that the presence of oxygen on the surface of the activated carbon provides an unsuitable environment for Dhc to thrive. These microbes are strict anaerobes and oxygen is present on the surface of activated carbon [29]. This could lead to their inability to utilize the activated carbon and access the sorbed TCE. There are many known genera that can reduce TCE to cis-DCE, if these organisms are present this could be why we are seeing partial dechlorination, from TCE to cis-DCE, but no complete dechlorination [9].

3.2 Influence of PAC on TCE removal

A GC was used to determine the concentrations of TCE, cis-DCE, VC, ethene, and methane present in both high mass loading and low mass loading of PAC. The high mass loading of PAC incubations contain the same loading rates as the GAC incubations,

78 mg of PAC per 1 mL of water and the low mass loading of PAC incubations contain one-third of the high mass loading, 26 mg of PAC per 1 mL of water. The trends seen in the PAC amended incubations are similar to those discussed in the GAC amended incubations. In all incubations with both high and low mass loading of PAC, TCE concentrations reach 0 µmol/bottle within days to weeks of its introduction into the system. In all incubations with high mass loading of PAC there is almost no evidence of

13 dechlorination of TCE. In the low mass loading of PAC there is dechlorination of TCE to cis-DCE and in some cases complete dechlorination to ethene however, only low concentrations of less than 1 µmol/bottle were detected. In the no electron donor amended series, Figure A-4, and hydrogen gas + acetate electron donor amended series,

Figure A-19, concentrations of cis-DCE were detected between 10 and 20 µmol/bottle. In the lipid electron donor amended series, Figure A-9 and B-9, and the lactate electron donor amended series, Figure A-14, data indicate stoichiometric reduction of TCE to cis-

DCE, concentrations of about 20 µmol/bottle and some complete dechlorination to ethene. In the sterile control low mass loading of PAC, Bottle 1, Figure A-24, has a concentration of cis-DCE of about 20 µmol/bottle. This suggests that there is some microbial activity despite the 3-day heat sterilization technique of the sediment and surface water, it is possible that some microbes survived and became active over time

[41].

The trends are similar between the influence that both GAC and PAC have on

TCE removal. They both remove TCE within a few days to weeks, the high mass loading show little to no evidence of dechlorination of TCE while the low mass loading shows dechlorination to cis-DCE and in some cases complete dechlorination to ethene. The data indicate that incubations with low mass loading of GAC show dechlorination of TCE to cis-DCE but the incubations with low mass loading of PAC show stoichiometric dechlorination of TCE to cis-DCE. These findings are not surprising because PAC particles are a result of grinding up GAC particles into smaller particles. Therefore, the chemistry of the particles is similar but the physical properties are different. GAC has a

14 lot of its surface area contained in internal pore spaces and PAC has a larger external surface area giving it more external sorption sites [29]. The presence of more external sorption sites allows for greater availability of TCE to be accessed by either anaerobic dechlorinators or Dhc present.

3.3 Methane increases in the presence of activated carbon

Throughout all activated carbon incubations regardless of electron donor amendment, methane values were high relative to the no activated carbon amended incubations. The highest methane concentrations were detected in the lipid amended incubations and were found to be between 1500 and 2000 µmol/bottle, seen in Figures A-

6 through A-9, compared to 200 µmol/bottle in the no carbon control, Figure A-10. The other electron donor amended incubations show the same trend of increased methane production in activated carbon amended bottles compared to the no activated carbon controls, even in the no electron donor control amended series seen in Figures A-1 through A-5.

High production of methane can be a potential problem in terms of remediation because not only is it a known greenhouse gas but it is also flammable. Methane is colorless and odorless allowing it to go unnoticed. Full scale application of in-situ activated carbon in the field could potentially lead to vapor intrusion problems. In residential areas with underground structures such as basements a buildup of methane can be an explosion hazard. Activated carbon has been shown to facilitate interspecies electron transfer; the activated carbon assists in the movement of electrons from non-

15 methanogens, either the Dhc or other non-methanogen organisms present in the environment, to methanogens and the electron donor become less relevant [42]. The non- methanogen would oxidize the electron donor present and the electrons would be transferred through the carbon to the methanogens who then produce methane through a process called direct interspecies electron transfer (DIET). It is suggested that DIET is a more effective mechanism for interspecies electron exchange under anaerobic conditions than the electrical connections generated by microbes [43, 44]. GAC has a high electrical conductivity and is possibility an electron acceptor for anaerobic respiration and because of these properties it has been added to methanogenic digesters to accelerate the production of methane in digesters [45]. Methanogens are able to receive electrons indirectly through conductive materials such as GAC and studies show that DIET is promoted. Liu et al. shows that in the presence of GAC and when Methanosaeta are the dominant methanogen, methane is produced 2.5 times faster than in the GAC-free controls [43, 46].

3.4 Does electron donor make a difference in TCE reduction

Although hydrogen gas is the sole electron donor for Dhc, other organic donors are generally used in-situ where they can be fermented to suplly the hydrogen for Dhc.

Once the hydrogen is produced the Dhc must compete against other microbes that can also utilize the hydrogen. Various electron donors that are commonly used for in-situ bioremediation of TCE were used in the batch experiments; lipid in the form of EOS,

Lactate, and hydrogen gas + acetate. There was also a no electron donor control series

16 which is referred to as the “unamended series.” In the no activated carbon control incubations all electron donor amended series performed the same each reaching stoichiometric concentrations of cis-DCE, close to 50 µmol/bottle. The unamended series data also indicated TCE reduction to cis-DCE but at lower concentrations of about 30

µmol/bottle seen in Figure A-5. Lipid amended incubations outperformed the other electron donor amended incubations in both the initial series batch incubations and the second series batch incubations. The initial series the lipid amended incubations produced the most methane as already discussed but they also had the highest detected concentrations of cis-DCE of about 5 µmol/bottle in the low GAC bottles and 20

µmol/bottle in the low PAC bottles. The other two electron donor amended series performed similarly to the lipid amended series but overall, not as well. The hydrogen gas

+ acetate electron donor data indicate TCE dechlorination to cis-DCE in the low GAC bottles at slightly greater than 5 µmol/bottle but in the low PAC bottles between 10 and

20 µmol/bottle. The lactate electron donor data indicate TCE dechlorination to cis-DCE in the low GAC bottles at less than 2 µmol/bottle but in the low PAC bottles greater than

20 µmol/bottle. In the second series incubations the difference between the lipid amended series and the lactate amended series is more notable and can be seen in Figures B-7, B-9,

B-12, and B-14. The cis-DCE concentrations in the lactate amended series are less than 5

µmol/bottle compared to between 5 and 20 µmol/bottle in the lipid amended series.

17 3.5 Partitioning of dechlorination products and how it can impact analyses

The data suggest there is no evidence of dechlorination in incubations with high mass loading of both PAC and GAC. The presence of these particles at such high concentrations suggests the possibility of more sorption sites for TCE and the dechlorination products to sorb to. Log Kow is a partition coefficient used to predict the distribution of a compound between the octanol phase and the aqueous phase. The octanol phase, in this case is used as a proxy for activated carbon. The larger the Kow value, the more likely a compound is to sorb to activated carbon. In this case, as dechlorination occurs, the affinity of the dechlorination products to stay sorbed to the activated carbon decreases. TCE has the highest value at 2.53 and is more likely to sorb and VC has the lowest value at 1.38 and is the least likely to sorb. These values indicate that TCE will sorb to the activated carbon and stay sorbed unless there is a change in the system. However, these values also indicate that cis-DCE and VC are not as likely to sorb. If there was dechlorination of TCE we should be able to measure cis-DCE and VC in the aqueous phase.

Chlorinated ethene Log Kow

TCE 2.53

Cis-DCE 1.86

VC 1.38

Figure 1. Log Kow values, partitioning coefficients between the octanol phase and the aqueous phase, for TCE, Cis-DCE, and VC [47].

18 4.0 Conclusions

4.1 TCE reduction

In the presence of high mass loading of both GAC and PAC amended bottles, there is no evidence of dechlorination, or very minimal as seen with the lipid amended initial series batch incubations. In the cases where minimal dechlorination is seen, there is no production of VC or ethene. The production of some cis-DCE but no further reduction could be a result of sorption phenomenon, the dechlorination products also being sorbed to the activated carbon especially in the incubations with high mass loading of carbon, or a limitation of TCE availability to the Dhc. In the case of high mass loading of PAC and

GAC, there is little or no evidence of cis-DCE production. This could be a result of having so much activated carbon in the system that any cis-DCE being produced is getting sorbed to the carbon. Cis-DCE is present in both low mass loading rates of PAC and GAC in the electron donor series but is present in higher concentrations in the no carbon amendments. VC and ethene are present in small concentrations in the low mass loading of PAC and in higher concentration in the no carbon amended incubations for all electron donor incubations, however there is no complete dechlorination in any bottles.

The presence of cis-DCE but no other or very little other dechlorination products in the activated carbon amended bottles could be a result of electron transfer between the active carbon and the chlorinated solvents which would only facilitate the reduction from TCE to cis-DCE. The data also indicate that there is more microbial activity in the incubations with low mass loading of PAC than there is in the incubations with low mass loading of

GAC. GAC has a larger surface area and more pore space and therefore provides more

19 sorption sites for TCE to bind to. This results in less microbial access to the TCE for metabolism.

4.2 Methane production

The data indicate methane concentrations are highest in the batches with high mass loading rates of both PAC and GAC amended incubations. Methane production is highest in EOS electron donor amended incubations compared to all other electron donor amended bottles at over one thousand micromoles per bottle in the initial series of batch experiments. The same trend can also be seen in the second series of batch incubations however; the concentrations are not as high. The trends are the same even though the concentrations are different magnitudes because there are probably differences in initial microbial community due to sediment collected during different collection dates and seasons. There is very little methane production in the no activated carbon amended bottles independent of the electron donor amendment.

4.3 Future research

Current data suggests that TCE has a high affinity to sorb to activated carbon.

However, it also suggests that there is a low affinity for cis-DCE sorption and that VC does not sorb to activated carbon. It would be beneficial to find the sorption isotherms for

TCE, cis-DCE, and VC with activated carbon. This will give a better explanation as to why there are no dechlorination products seen in the presence of high mass loading of

GAC and PAC.

Despite any ongoing microbial activity, all residual TCE should be extracted from the activated carbon to determine extent of contaminant still sorbed. This will allow for

20 stoichiometric calculations of the TCE present on the activated carbon. It will also give information on the mass of TCE that is sorbed to the natural organic matter in the sediment. This will give an insight into the availability of the TCE and the ability of Dhc to reduce it.

The phylogenetic and functional genes, bvcA, vcrA, tceA, and Dhc specific 16s rRNA genes, relevant to complete dechlorination should be quantified to determine if which Dhc communities are present. It is known that specific Dhc organisms need to be present for complete reductive dechlorination from TCE to ethene. There are many microbes that are able to reduce TCE to cis-DCE but they do not completely reduce it to ethene. If Dhc are present in the no activated carbon amended bottles but not in the carbon amended bottles, this could suggest that the presence of activated carbon provides a toxic environment for the essential Dhc organisms. If this is the case further testing can be done to reduce the activated carbon to get rid of any oxygen present on the surface and see if this provides a suitable environment for Dhc to completely dechlorinate TCE to ethene.

21 Appendix A

Data Plots for Initial Series of Batch Incubations

22 Figure A-1: Initial series batch incubations, no electron donor amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

23 Figure A-2: Initial series batch incubations, no electron donor amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

24 Figure A-3: Initial series batch incubations, no electron donor amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

25 Figure A-4: Initial series batch incubations, no electron donor amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

26 Figure A-5: Initial series batch incubations, no electron donor amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

27 Figure A-6: Initial series batch incubations, lipid amended series with high mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

28 Figure A-7: Initial series batch incubations, lipid amended series with low mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

29 Figure A-8: Initial series batch incubations, lipid amended series with high mass loading of PAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

Figure A-9: Initial series batch incubations, lipid amended series with low mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

31 Figure A-10: Initial series batch incubations, lipid amended series with no carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

32 Figure A-11: Initial series batch incubations, lactate amended series with high mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

33 Figure A-12: Initial series batch incubations, lactate amended series with low mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

Figure A-13: Initial series batch incubations, lactate amended series with high mass loading of PAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

35 Figure A-14: Initial series batch incubations, lactate amended series with low mass loading of PAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

36 Figure A-15: Initial series batch incubations, lactate amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

37 Figure A-16: Initial series batch incubations, hydrogen gas plus acetate amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

Figure A-17: Initial series batch incubations, hydrogen gas plus acetate amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

39 Figure A-18: Initial series batch incubations, hydrogen gas plus acetate amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

40 Figure A-19: Initial series batch incubations, hydrogen gas plus acetate amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

41 Figure A-20: Initial series batch incubations, hydrogen gas plus acetate amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

42 Figure A-21: Initial series batch incubations, sterile series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

43 Figure A-22: Initial series batch incubations, sterile series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

Figure A-23: Initial series batch incubations, sterile series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

45 Figure A-24: Initial series batch incubations, sterile series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE.

46 Figure A-25: Initial series batch incubations, sterile series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE.

47 Appendix B Data Plots for Second Series of Batch Incubations

48 Figure B-1: Second series batch incubations, no electron donor amended series with high mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

49 Figure B-2: Second series batch incubations, no electron donor amended series with low mass loading of GAC. Concentration of degradation products from initial concentration of 20 µmol of TCE. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

50 Figure B-3: Second series batch incubations, no electron donor amended series with high mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

51 Figure B-4: Second series batch incubations, no electron donor amended series with low mass loading of PAC. Concentration of degradation products from initial concentration of 20 µmol of TCE. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

Figure B-5: Second series batch incubations, no electron donor amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

53 Figure B-6: Second series batch incubations, lipid amended series with high mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

54 Figure B-7: Second series batch incubations, lipid amended series with low mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

Figure B-8: Second series batch incubations, lipid amended series with high mass loading of PAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first sample 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

56 Figure B-9: Second series batch incubations, lipid amended series with low mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

57 Figure B-10: Second series batch incubations, lipid amended series with no carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first sample 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

Figure B-11: Second series batch incubations, lactate amended series with high mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

59 Figure B-12: Second series batch incubations, lactate amended series with low mass loading of GAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

60 Figure B-13: Second series batch incubations, lactate amended series with high mass loading of PAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

61 Figure B-14: Second series batch incubations, lactate amended series with low mass loading of PAC amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

62 Figure B-15: Second series batch incubations, lactate amended series with no activated carbon amendment. Concentration of degradation products from initial concentration of 20 µmol of TCE. The blue arrow indicates day 68, the first data point taken 1 week after the bottles were reamended with electron donor. The red arrow indicates day 113, the first data point taken 1 week after the bottles were amended with the crossover culture.

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