THE HEALTH EFFECTS AND A BIOREMEDIATION PROCESS FOR VINYL CHLORIDE

by

Meng-Chen Lee

B.S. M.S., University of Iowa, 2010, 2012

Submitted to the Graduate Faculty of

Department of Environmental and Occupational Health

Graduate School of Public Health in partial fulfillment

of the requirements for the degree of

Master of Public Health

University of Pittsburgh

2014

ii

UNIVERSITY OF PITTSBURGH

GRADUATE SCHOOL OF PUBLIC HEALTH

This essay is submitted

by

Meng-Chen Lee

on

April 28 , 2014

and approved by

Essay Advisor:James Peterson, PhD ______________________________________Associate ProfessorDepartment of Environmental and Occupational HealthGraduate School of Public HealthUniversity of Pittsburgh

Essay Reader:Na Wei, PhD ______________________________________Assistant ProfessorDepartment of Civil and Environmental EngineeringSwanson School of EngineeringUniversity of Pittsburgh

iii

Copyright © by Meng-Chen Lee

2014

James Peterson, PhD

THE HEALTH EFFECTS AND A BIOREMEDIATION PROCESS FOR VINYL CHLORIDE

Meng-Chen Lee, MPH

University of Pittsburgh, 2014

ABSTRACT

Vinyl chloride is a colorless organic gas with high volatility. The carcinogen is the secondary

pollutant from chlorinated ethenes that have been extensively used in plastic industries and

facilities during manufacturing. Since 1970s numerous reports have appeared regarding a distinct

relationship between high level of exposure to vinyl chloride and angiosarcomas of the liver

(ASL), in which occupational exposure is of high concern for potential risk of public health.

Improved bioremediation for dense non-aqueous phase liquid (DNAPL) contamination has been

utilized to reduce vinyl chloride concentration in groundwater. To understand the factors

controlling VC degradation in the presence of both substrates (methane and ethene) and both

microbial groups (methanotrophs and etheneotrophs), cometabolic activities of methanotrophs,

etheneotrophs, and both microbial groups are discussed respectively in this essay.

iv

TABLE OF CONTENTS

1.1 DNAPL AND VINYL CHLORIDE APPLICATIONS.....................................1

1.2 VINYL CHLORIDE ENVIRONMENTAL CONTAMINATION AND

REMEDIATION...................................................................................................................2

1.2.1 Physical characteristics of vinyl chloride.......................................................2

1.2.2 Regulation of vinyl chloride.............................................................................4

2.1 VINYL CHLORIDE EXPOSURES...................................................................6

2.1.1 General population exposure..........................................................................6

2.1.2 Occupational exposure.....................................................................................8

(Source: Kielhorn, Melber et al. 2000)......................................................................10

2.2 ADVERSE HEALTH EFFECTS OF VINYL CHLORIDE...........................11

2.2.1 Effects of short-term exposure......................................................................11

2.2.2 Liver cancer observed from long-term exposure to vinyl chloride............12

2.2.3 Angiosarcomas of the liver observed from long-term exposure to vinyl

chloride.........................................................................................................................14

2.2.4 Hepatocellular carcinoma observed from long-term exposure to vinyl

chloride.........................................................................................................................16

2.2.5 Other malignant neoplasms observed from long-term exposure to vinyl

chloride.........................................................................................................................16

v

3.1 VC COMETABOLISM BY METHANOTROPHS........................................18

3.2 COMPETITIVE INHIBITION AND PRODUCT TOXICITY OF CAHS TO

METHANOTROPHS.........................................................................................................20

3.3 VC COMETABOLISM BY ETHENEOTROPHS..........................................21

3.4 VC COMETABOLISM IN THE PRESENCE OF BOTH MICROBIAL

GROUPS..............................................................................................................................22

3.4.1 Microbial behavior with VC and methane mixtures..................................25

3.4.2 Microbial behavior with VC and ethene mixtures......................................25

3.4.3 Microbial behavior with methane and ethene mixtures without VC........26

3.4.4 Microbial behavior with mixtures of methane, ethene, and VC................26

BIBLIOGRAPHY........................................................................................................................31

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LIST OF TABLES

Table 1. Occupational exposure: reported levels of vinyl chloride in workplace air samples in

VC/PVC production plants............................................................................................................10

Table 2. Results of Poisson Regression Analysis of Liver Cancer, Angiosarcoma, and

Hepatocellular Carcinoma by Time Since First Employment, Duration of Employment, and

Cumulative Exposure.....................................................................................................................13

vii

LIST OF FIGURES

Figure 1. Formation of vinyl chloride from higher chlorinated ethenes by microorganisms..........4

Figure 2. Log relative risk and 95% confidence interval of all liver cancer (N=71), by cumulative

exposure to vinyl chloride (ppm-years).........................................................................................14

Figure 3. Log relative risk and 95% confidence interval of liver angiosarcoma (N=37), by

cumulative exposure to vinyl chloride (ppm-years)......................................................................15

viii

PREFACE

I would like to thank my family for their selfless support of my education in the past nine years.

The path from studying Microbiology to Environmental Science, and to Environmental &

Occupational Health has been the journey to discover what my heart truly desires. It is their

love that has motivated me to keep on going when times were rough, it is the faith they have in

me so that I always know that I can do it.

I would like to thank the professors who have given me opportunities to learn. Without the

training you have given me, I would never know what I am capable of, and what I am lack of.

I am also grateful that I was never alone on this journey. I have always had great friends

surrounded me to keep me warmed and accompanied. I shall always cherish the time we spent

together and the laughter we shared with each other. I would like to thank each one of you who

helped me when I was down, encouraged me when I was low, and gave me a push when I was

weak. I know I could not make it to this far without all of my dear friends.

Finally, I thank god. Thank you for giving me your blessing so that I was never left alone. Thank

you for leading me to the path that enriches me. Thank you for always giving me what is the best

for me.

I know I should do a lot better and I promise you I will. I know I still have much to learn on this

path to pursue my goal, and with your love and faith I shall keep on going and never stop.

ix

1.0 INTRODUCTION

1.1 DNAPL AND VINYL CHLORIDE APPLICATIONS

Chlorinated ethenes are the most common source of dense non-aqueous phase liquid (DNAPL)

contamination in groundwater, including tetrachloroethene (perchloroethene or PCE),

trichloroethene (TCE), cis-1,2-dichloroethene (cis-DCE), trans-1,2-dichloroethene (trans-DCE),

1,1-dichloroethene (DCE), and vinyl chloride (VC). All of these compounds, with the exception

of VC, have densities greater than water and therefore can create a distinct sinking layer of the

compound.

Large quantities of chlorinated ethenes are still used in numerous industries and facilities

during manufacturing. The advantage of being non-flammable and highly evaporable had made

tetrachloroethene and trichloroethene popular dry cleaning solvents after the petroleum shortages

caused by World War II in the 1940s . Polymerization of VC to produce polyvinyl chloride

(PVC) homopolymer/copolymer resins was also developed early in the 20 th century, and its

production capacity has almost doubled since the 1980s for a wide variety of applications. At a

global level, demand for PVC exceeds 35 million tonnes per year. PVC manufacture represents

99% of the total vinyl chloride applications. The production of Western Europe is estimated for

1996 to be 5,209 thousand tonnes with an annual growth of 2% compared to 4% in North

America and 9.5% in Asia (WHO, 1999).

1

PVC is one of the most commonly used thermoplastic materials in daily short-life

products, such as PVC packaging materials used in food, cleaning materials, textiles, beverage

packaging bottles, and medical devices. It is also extensively utilized in long-life products such

as pipes, window frames, cable insulation, floor coverings, roofing sheet, etc .

1.2 VINYL CHLORIDE ENVIRONMENTAL CONTAMINATION AND

REMEDIATION

1.2.1 Physical characteristics of vinyl chloride

Vinyl chloride is a colorless organic gas with high volatility, this characteristic acts as a

significant transport mechanism in the environment. The high volatility and low solubility (1.1

g/litre) at 25 °C in water of VC has made the removal of VC from surface water to be considered

the most rapid route. Therefore, the concentrations measured in water and sediment generally do

not exceed 10g/L. However, volatilization of VC is unlikely to occur in groundwater, where

there’s no direct exposure to air. As a result, much higher levels of up to 56000g/L have been

found in groundwater samples from contaminated sites. The half-lives of VC measured in

models for disturbed or quiescent water range from <1h to 5h and from 2.5 to 43h for natural

water bodies. Conversely, VC may remain for months or years in groundwater, which poses a

threat to the health of animals.

When DNAPLs other than vinyl chloride are released into the subsurface, they migrate

downwards below the level of the groundwater table. Due to the slow dissolution rate of

2

DNAPLs, areas in the subsurface containing DNAPL serve as lasting sources of groundwater

contamination .

Under anaerobic conditions the reductive dechlorination biodegradation process carried

out by bacteria such as Dehalococcoides can converts PCE (perchloroethene ) to TCE

(trichloroethene) ,TCE to c-1,2-DCE (dichloroethene), c-1,2-DCE to VC, and VC to ethene

(Figure. 1). However, when these dominant anaerobic processes fail to go to completion, the

more toxic secondary pollutant VC will accumulate in groundwater zones, where significant

amount of methane and ethene may also be generated from methanogenesis and reductive

dechlorination of chlorinated ethenes. In some situations, the groundwater returns to aerobic

conditions down gradient of the source with methane, ethene, VC, and possibly cis-DCE still

present. Natural attenuation of VC could occur via oxidation by aerobic microorganisms as a VC

plume intersects with groundwater containing molecular oxygen .

3

Figure 1. Formation of vinyl chloride from higher chlorinated ethenes by microorganisms (Source:

1.2.2 Regulation of vinyl chloride

The evidence showing vinyl chloride to be carcinogenic in both animal studies and in human

case reports first came out in the early 1970s. In 1974, Congress passed the Safe Drinking Water

Act and vinyl chloride is one of the Hazardous Air Pollutant (HAP) that is regulated by the

Environmental Protection Agency (EPA) in order to ensure protection of public health and the

environment. The environmental statutes that are associated with VC include the Clean Air Act

(CAA), the Safe Drinking Water Act, the Resource Conservation and Recovery Act. The

4

regulation of vinyl chloride became effective in 1989. Under Section 112 of the Clean Air Act,

The EPA requires that VC emissions from VC/PVC production facilities not exceed an average

of 10 ppm over a 3-hour period. Under the Safe Drinking Water Act, the EPA has set an

enforceable regulation for VC, called a maximum contaminant level (MCL), at 0.002 mg/L or 2

ppb. Furthermore, EPA determined that the zero maximum contaminant level goals (MCLG) and

0.002 mg/L or 2 ppb MCL for vinyl chloride are still considered adequately protective of human

health in the review of VC as part of the Six Year Review (OECD SIDS 2001).

Overall, vinyl chloride contamination of groundwater, on which 50% of Americans

developed for their potable supply, may cause prolonged human and environmental health

problems since it is very difficult to locate and treat DNAPL source areas . Chlorinated solvent

remediating technologies have shifted over the last 30 years. Currently, aggressive source zone

in situ bioremediation (ISB) of chlorinated ethenes has proven to be successful for degrading

chlorinated ethenes to the levels that are ideal for natural attenuation to occur. This paper

summarizes the epidemiologic studies on adverse health effects associated with vinyl chloride

exposure, as well as the limitation and progress of technologies in DNALP source zone

remediation .

5

2.0 REVIEW ARTICLES

In 1999, Toxic Release Inventory (TRI) data reported 848,576 lb of vinyl chloride was emitted to

the air, 106 lb to water and 405 lb to land or underground injection in the US. The partitioning

tendency of VC in different environmental compartments based upon the Mackay Level 1 model

is as followed: 99.99% air, 0.01% water, <0.01% soil and ,<0.01% sediment. However, high

levels of vinyl chloride contamination was reported to present in soil, groundwater, aquifers, and

wells near landfill and industrial waste disposal sites from several countries (Euro Chlor 1999). It

is crucial to understand the potential exposure routes to vinyl chloride and the adverse health

effects results from contacting the carcinogen.

2.1 VINYL CHLORIDE EXPOSURES

2.1.1 General population exposure

Inhalation of air polluted with VC, mainly in the vicinity of VC/PVC plants or waste disposal

sites, is of greater concern than atmospheric concentration of vinyl chloride in ambient air. As

the ambient concentration of vinyl chloride is usually less than 3 µg/m3, much higher

concentration- up to 8000 µg/m3 and 100 µg/m3 of vinyl chloride had been observed near

6

industrial plants and waste disposal sites, respectively in the past. Indoor air concentration in the

residential house near to landfills could reach a maximal concentration of 1000 µg/m3. However,

such exposure is likely to be transient, so there is very little exposure of the general population to

vinyl chloride in the air (IPCS, 1999).

The contaminating concentrations of vinyl chloride in surface waters vary from 10 µg/L

to 200,000 µg/L due to its high volatility. One study reported high concentrations, up to 200 000

µg/litre of vinyl chloride were detected in well water in the vicinity of a PVC plant in Finland 10

years after leakage . Areas contaminated with chlorinated hydrocarbons contained approximately

60 000 µg/litre of vinyl chloride in the leachate and 56 000 µg/litre in a contaminated sand

aquifer . In 2000, a vinyl chloride concentration of 27 000 µg/litre was detected in site

groundwater above a drinking-water aquifer (Peterson et al., 2000).

Unplasticized polyvinyl chloride (uPVC) tubing and bottles are possible sources of vinyl

chloride in drinking-water. Malck (1999) studied the migration of vinyl chloride from uPVC

pipies into water under different parameters. The study showed that after 30 days of exposure

exposure of double distilled water at 45 oC, the uPVC pipes released more than 2.5 ppb of VC

into the contact water, which is just above the permissible level in drinking water set by the U.S.

Environmental Protection Agency, 2.0 ppb . Other studies into PVC-bottled drinking-water have

identified vinyl chloride at levels of 0.06–0.18 µg/litre and <0.6 µg/litre . In 1975, vinyl chloride

has been identified in peanut oil samples packaged in PVC bottles at levels ranging from 0.3 to

3.3 ppm . Overall, the frequency of occurrence of

vinyl chloride is expected to be higher in such water than in tap water since bottled-water is

always in contact with new PVC packaging material.

7

2.1.2 Occupational exposure

Before vinyl chloride was recognized as a human carcinogen, no regular workplace monitoring

and precautions were taken against contact with the toxic gas while the autoclaves or reactor

vessel cleaners emptied the PVC reactors manually with spatulas and chisels. In the 1950s and

earlier, the VC workers were estimated to be exposed to as much as 1000 ppm (2600 mg/m3). By

the mid-1970s, the exposure level was reduced to one-tenth of this, the levels were generally

lower than 1-5 ppm (2.6-13 mg/m3) after 1975 in many countries .

In the United States, the number of workers exposed to vinyl chloride has been estimated

to be approximately 80,000 during 1981-1983 and more than 5000 in Sweden during 1975-1980.

Factories in many countries that failed to reduce the emission of vinyl chloride below the

regulated level in the early 1970s were forced to close. However, in other countries where the

VC polymerization plants were not forced to close for socioeconomic reasons, the workers

continued to be exposed to high levels of vinyl chloride emitted from the old-fashioned

technologies .

Table 1 summarizes the occupational exposure of vinyl chloride in VC/PVC production

plants in the following countries: China, Croatia, Egypt, Finland, Italy, Poland, Russia, and

Taiwan. Thirty years (1949-1987) of the reported levels of vinyl chloride in VC/PVC plants

from Croatia revealed that employees were exposed to an average vinyl chloride concentration

of 543 mg/m3 with an occasional peak concentration of 1300 mg/m3. Vinyl chloride exposure

levels reported from Italy from 1950 to 1985 showed a wide range of concentrations from 13-

1300 mg/m3. The data collected after 1980 from countries like Finland, Poland, and Russia

showed significant reduced vinyl chloride exposure levels, where the mean concentrations

ranged from 0.2 to 400 mg/m3. The VC/PVC production plants from Asian countries such as

8

China and Taiwan also showed high levels of vinyl chloride exposure levels ranging from 0.13 to

1009 mg/m3 within the specified investigation years .

Table 1. Occupational exposure: reported levels of vinyl chloride in workplace air samples in VC/PVC production plants

(Source: Kielhorn, Melber et al. 2000)

9

2.2 ADVERSE HEALTH EFFECTS OF VINYL CHLORIDE

After vinyl chloride was extensively produced in the late 1930, various effects caused by

exposure to vinyl chloride were noticed. The reported symptoms include hepatic abnormalities in

vinyl chloride workers, acroosteolysis, Raynaud-type-phenomenon and sclerodermoid skin

lesions (Lelbach & Marsteller, 1981). Starting from 1974, numerous reports have appeared

regarding a distinct relationship between high level of exposure to vinyl chloride (as much as

2600 mg/m3, 1000 ppm) and angiosarcomas of the liver (ASL), a type of rare tumor that

constitutes only 2% of all primary tumors of the liver in the general population. . Furthermore,

studies have shown that the average latent period between starting work in an occupation

involving VC exposure and ASL diagnosis/death for 99 cases was 22 years .

Several further cases series and small epidemiologic studies also indicated that vinyl

chloride may also be connected with other (non-ASL) cancer sites and types, including

hepatocellular carcinoma, respiratory system, digestive system other than the liver,

lymphopoietic and hematopoietic tissue, brain and other central nervous system and malignant

melanoma .

2.2.1 Effects of short-term exposure

For the workers who exposed to high level concentration of vinyl chloride (about 1000 pm) for

more than 1 month to several years prior to 1974, several specific pathological syndromes were

reported and defined as “vinyl chloride illness”. Symptoms described were earache and

headache, dizziness, unclear vision, fatigue and lack of appetite, nausea, sleeplessness,

breathlessness, stomachache, pain in the liver/spleen area, pain and tingling sensation in the

10

arms/legs, cold sensation at the extremities, loss of libido and weight loss (Thiess & Versn,

1974). Clinical findings includes scleroderma-like changes in the fingers with subsequent bony

changes in the tips of the fingers described as acroosteolysis, peripheral circulatory changes

similar to Raynaud’s diseases, and enlargement of the liver and spleen with a specific

histological appearance, and repiratory manifestations (Lange et al., 1974, Veltmann et al.,

1975).

2.2.2 Liver cancer observed from long-term exposure to vinyl chloride

Two large cohort studies of male factory workers who had been exposed to vinyl chloride for

more than a year from the United States and Europe were conducted to determine the adverse

health effects associated with long-term vinyl chloride exposure. Wong et al. conducted a cohort

study (covered 1942-1982) included 10,173 male who had been exposed with vinyl chlorides in

37 plants for at least 1 year in United Sates before January 1973. On the other hand, the update

study (Ward et al. 2001) on a major European cohort study of mortality and cancer incidence

among VC/PVC workers (Simonato et al. 1991) was published in 2001. The observation period

was different for different factories, starting for most in1955, extended until 1986, then updated

from 1993 through 1997. The multicentric study comprised a total of 12,700 male workers with

at least 1 year of employment in 13 factories in three countries (Norway, Sweden, and the United

Kingdom). These two studies provide the most informative data on the health effects associated

with exposure to VC.

The increase in standardized morality ratio (SMR) for liver cancer reported from the two

European cohort studies showed similar results: 2.86 (95% CI= 1.86-4.25) in the original

analysis by Simonato (1991) and 2.40 (95% CI= 1.80-3.14) in the updated investigation by Ward

11

in 2001. The data from the international programme on chemical safety (IPCS) task group

indicated a 5-fold excess risk for liver cancer observed among workers exposed to vinyl chloride

(WHO, 1999).

A strong association between liver cancer and time since first exposure to vinyl chloride,

duration of employment, and estimated ranked and quantitative exposures was confirmed in both

original and follow-up investigation . Poisson regression analysis of liver cancer performed by

Ward et al. showed strong positive trends for “time since first employment”, duration of

employment, and cumulative exposure (Table2).

Table 2. Results of Poisson Regression Analysis of Liver Cancer, Angiosarcoma, and

Hepatocellular Carcinoma by Time Since First Employment, Duration of Employment, and

Cumulative Exposure .

Table 2. Results of Poisson Regression Analysis of Liver Cancer, Angiosarcoma, and Hepatocellular Carcinoma by Time Since First Employment, Duration of Employment, and Cumulative Exposure

(Source: Ward, Beffetta et al. 2001)

12

In this study, the cumulative dose was subdivided into 13 categories, and found a fairly

monotonic increase in rate ratios, with a rate ratio (RR) of 82 (95% CI= 21-323) in the highest-

dose category of > 18,300 ppm-years. The (natural) logarithmic transformation of cumulative

exposure provided the best fit when the midpoints of the dose categories was used to model dose

as a continuous variable. The RR for one logarithmic unit of cumulative exposure was 2.0 (95%

CI= 1.7-2.4). The estimated categorical rate ratios for cumulative exposure and liver cancer

along with the continuous curve and CIs were shown in Figure 2.

Figure 2. Log relative risk and 95% confidence interval of all liver cancer (N=71), by cumulative

exposure to vinyl chloride (ppm-years) (Source: Ward, Boffetta et al. 2001)

2.2.3 Angiosarcomas of the liver observed from long-term exposure to vinyl chloride

In the USA study, a total of 15 angiosarcomas were recorded on the death certificate. Although

no expected numbers were calculated, this must be a very marked excess, since the annual

incidence of liver angiosarcoma is approximately 2 per 10 million, which would in this cohort

lead to an expected number of approximately 0.05. The authors noted that when these 15

angiosarcomas were excluded, there remained 22 other liver and biliary tract tumours; this

13

represents an excess (5.7 expected, SMR 3.86, p<0.02). In addition, this result is likely to be an

overestimate, since some of these other liver cancers were likely to be angiosarcomas.

Simonato et al. reported a 45-fold excess risk for ASL in workers exposed to VC >

10,000 ppm-years, and the updated study (Ward et al., 2001) estimated an approximately 200

times greater risk of ASL than the general population (Table 2).

Exposure response was estimated for 37 angiosarcomas in a model including 12 dose

categories in the updated European study. Compared to the exposure-response relation for liver

cancer, a sharper relation is observed for ASL, where the RR for a logarithmic unit of cumulative

exposure was 2.9 (95% CI= 2.2-3.9). Figure 3 shows the estimated categorical rate ratios for

cumulative exposure and liver angiosarcomas along with the continuous curve and CIs.

Figure 3. Log relative risk and 95% confidence interval of liver angiosarcoma (N=37), by cumulative

exposure to vinyl chloride (ppm-years) (Source: Ward, Beoffetta et al. 2001)

14

2.2.4 Hepatocellular carcinoma observed from long-term exposure to vinyl chloride

A noticeable exposure-response trend with both duration of employment and cumulative vinyl

chloride exposure was present for the ten known cases of hepatocellular carcinoma, suggesting

an association between vinyl chloride and this tumor. Two updates of parts of the Italian cohort

have shown that an elevated mortality of liver cancer (11 observed, 5.7 expected cases, SMR

1.93) was observed in Marghera. In an extended survey for hepatic cancers, four further cases

were uncovered. There were 5 angiosarcomas, 5 hepatocellular carcinomas, 3 cirrhotic

carcinomas, and in two cases the histology was not known . The studies have shown that the risk

for hepatocellular carcinoma is not as great as the risk for ASL.

2.2.5 Other malignant neoplasms observed from long-term exposure to vinyl chloride

Statistically significant increase of brain and CMS tumours after occupational exposure to vinyl

chloride were reported in USA surveys by Waxwiler et al. (1997), as well as in Sweden (Byron

et al., 1976) and Germany (Greiser et al., 1982).

In the cohort study conducted by Wong et al. (1991) in USA showed that the relative risk

was elevated for cancers of the brain and other CNS cancers (SMR 1.80, CI 1.14–2.70).

Mortality was highest in the group with the longest duration of exposure (SMR 1.64, 1.20 and

3.85, for workers with the duration of exposure < 10 years, 10–20 years and > 20 years,

respectively), while no trend with latency was observed (SMR 1.83, 1.58 and 2.10, for latencies

of < 20, 20–30 and > 30 years).

An update of the study by Wong et al. was completed in 1999, in which specific cancers

reported to be associated with vinyl chloride exposure both through 1982 and through 1985 had

15

shown no excess values, including lung cancer, cancers of lymphatic and hematopoieic tissue,

emphysema and pneumoconiosis. . The same conclusion was confirmed by the European

study. The statistical analysis suggested no clear pattern with duration of employment, ranked

level of exposure, or cumulative exposure to vinyl chloride; also, vinyl chloride exposure was

not strongly related to other cause of death, or incident cancer, including brain cancer, lung

cancer, soft-tissue sarcoma, non-Hodgkin’s lymphoma and malignant melanoma .

16

3.0 COMETABOLIC BIODEGRADATION OF VINYL CHLORIDE IN THE

PRESENCE OF METHANE AND ETHENE

Cometabolic biodegradation processes have been extensively studied in the past thirty years

because of their potential usefulness in biotransforming many of the chlorinated aliphatic

hydrocarbons (CAHs) found in naturalsystems. However, cometabolic activity is also an

unavoidable side reaction that may be harmful to the microorganisms. To understand the factors

controlling VC degradation in the presence of both substrates (methane and ethene) and both

microbial groups (methanotrophs and etheneotrophs), cometabolic activities of methanotrophs,

etheneotrophs, and both microbial groups are discussed respectively. In addition, competitive

inhibition between the primary (carbon and energy source) and secondary substrates (pollutants),

and the toxic effects from the intermediate compounds on methanotophs and etheneotophs are

reviewed in this chapter.

3.1 VC COMETABOLISM BY METHANOTROPHS

Several reports showed that chlorinated ethenes are not degraded in the absence of methane in

groundwater (Wilson et al., 1983). In the presence of oxygen and methane, methanotrophs can

cometabolize chlorinated aliphatic compounds very rapidly, on the order of days.

17

In small-inoculum experiments, a particulate methane monooxygenase (pMMO)

expressing methanotrophic mixed culture was added to the sample bottles, in which 34% of

methane and 94 to 99% of the chlorinated aliphatic hydrocarbons (CAHs) (t-DCE, c-DCE, and

VC) were in solution. The transformation yield ((Ty), defined as the mass of CAH degraded per

unit mass of growth substrate consumed, is useful in characterizing the finite value of CAH

cometabolism that occurs in a culture system). The highest observed Ty expressed as moles of

CAH per mole of methane for VC was 0.20 mol of VC/ mol of methane when the equilibrium

aqueous concentration of VC and methane were 17 µM and 30 µM respectively. However,

MMO failed to oxidize both VC and methane when the VC concentrations were high at 86 µM

and 340 µM. This can be due to competitive inhibition, the availability of reducing power and/or

the toxic effect of the transformation product (Anderson et al., 1997).

Large-inoculum experiments were done under different growth condition in order to

confirm the high yields for VC found in the small-inoculation experiments. The maximum Ty

values for VC was 0.25 mol of VC/mol of methane, which is very similar to the result obtained

from small-inoculum experiments. However, when comparing the two experiments, maximum

Ty value of small-inoculums was found in the bottle containing the lowest methane

concentrations (30 µM) and the lowest VC concentration (17 µM), while with the large

inoculums, much higher methane concentration (180 µM) together with highest VC

concentration (260 µM) gave the maximum transformation yield. The results suggested that

factors such as reducing-energy availability, competitive inhibition, and transformation toxicity

need to be corrected to reach its balance, so that the maximum values of Ty could be obtained

(Anderson et al., 1997).

18

Fogel et al. utilized a mixed culture containing methane-oxidizing bacteria (CL-M)

isolated from lake sediment to demonstrate its ability to degrade TCE and other chlorinated

ethenes to CO2. It is observed that acetylene inhibits 100 ng/ml of TCE degradation completely,

suggesting that it is methanotrophs that are responsible for TCE oxidation among the methane-

utilizing culture since acetylene is a specific inhibitor of methane monooxygenase. The fact that

12mM methanol can cause 50% inhibition, indicating that methanotrophs are very sensitive to

inhibition by methanol. This mixed culture of methanotrophs is observed to degrade VC from

600 ng/ml to less than 0.2 ng/ml in 1 day. Obligate methanotrophs are typically the major

component in the mixed culture, although one or more heterotrophic organisms could be present

as well. Therefore, methane-oxidizing bacteria are benefited by the presence of these

heterotrophic organisms since they can consume the potential inhibiting organic compounds

produced by the methanotrophs (Fogel et al., 1986). The authors conclude that mixed cultures

are more stable than pure cultures in terms of environmental remediation efforts.

3.2 COMPETITIVE INHIBITION AND PRODUCT TOXICITY OF CAHS TO

METHANOTROPHS

Transformation capacity (Tc) is a defined as the amount of cometabolic compound degraded

(dSc,µmol/L) divided by the amount of cells that are inactivated during this process(dx, mg/L).

Chang et al. (1996) conducted a study in which four methane-oxidizing cultures (a mixed

methanotrophs culture grown under both chemostat and batch conditions, a pure culture of

CAC1, and a pure culture of M. trichosporium OB3B grown under batch conditions) and 18

individual chlorinated and nonchlorinated aliphatics compounds were utilized to test the

19

hypothesis that “the product toxicity of chlorinated aliphatic hydrocarbons to methane-oxidizing

cells decreases in proportion to their chlorine content” (Chang et al. 1996).

In an effort to accurately reflect the product toxicity associated with cometabolic

degradation reactions, formate (20 mM) was amended into the cultures to secure the availability

of reducing energy without exerting competitive inhibition effects that would occur if methane is

added during the reaction. Similar results observed with the four different cultures supported the

hypothesis, indicating that within similar carbon structure groups (methane, ethanes, and

ethenes), the highest Tc values were found in nonchlorinated compounds. However, 1,1-DCE

appeared to have lower Tc values /higher toxicity than TCE, cDCE and tDCE. In addition, the

results of degradation kinetics for TCE, cDCE, and VC showed no direct relationship between

the degradation rate and chlorine content (Chang et al. 1996).

3.3 VC COMETABOLISM BY ETHENEOTROPHS

Numerous organisms are known to grow on ethene according to previous research, including

several strains of Mycobacterium (Hartmans et al, 1985, Hartmans et al. 1992). To date,

Mycobacterium, Nocardioides, and Pseudomonas strains are reported to grow on VC and

assimilate ethene with the same enzymes (Jin and Mattes, et al. 2008). In one study, an ethene

enrichment culture was used to evaluate aerobic biodegradation of VC in the presence of ethene,

ethane and VC. VC removal was most extensive in the bottles containing both ethene +VC,

followed by ethane+ethene+VC, and only VC. Ethene biodegradation was fastest when it was

the sole substrate, and the highest rate of ethane oxidation occurred in bottles that contained both

ethene and ethane. The results indicated that ethene is essential for ethene-utilizing organisms to

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induce the alkene monooxygenase in order to cometabolize VC and catabolize ethane (Jin and

Mattes, et al. 2008). This work also confirmed that ethane culture was able to degrade both

ethene and VC since alkane monooxygenase has a broader substrate range than AkMO. VC

biodegradation by the ethene and ethane grown cultures were reported to be roughly equivalent.

Interestingly, ethene was still consumed first by ethane-grown culture fed with ethane, ethene,

and VC. This result is consistent with the fact that alkane monooxygenases have broader

substrate range than AkMO reported in the literature (Jin and Mattes, et al. 2008).

The effect of varying ratios of VC to ethene on the rate of removal of each compound

was investigation as follows: (1) VC only; (2) ethene only; (3) VC: ethene = 1:3; (4) VC: ethene

= 1:1; (5) VC : ethene = 3:1. Initially, VC removal rates were uniformly high, and started to

diverge as the experiment progressed. By day 64, the bottles that received equimolar amounts of

VC and ethene displayed the highest VC removal rate. In contrast,the rate of VC degradation in

the bottles that contained unequal amount of VC and ethene are significantly reduced;

approximately 90% reduction occurred in the bottle that contained only VC. The results

suggested that at ethene to VC levels greater than a 1:1 molar ratio, VC degradation was

competitively inhibited by primary substrate that have higher affinity to AkMO (Jin and Mattes,

et al. 2008).

3.4 VC COMETABOLISM IN THE PRESENCE OF BOTH MICROBIAL GROUPS

The first microcosm studies comparing methane and ethene as primary substrates for

cometabolism of VC by native methanotrophs and etheneotrophs were done by Freedman et al in

2001 (Freedman et al., 2001). The sediment and groundwater were taken from monitoring well

21

#3037 at a Superfund site in California. The initial concentration of methane, ethene, and VC

were equivalent to what was measured in the anaerobic portion of the groundwater that was

closest to the interface with the aerobic zone, where ethane and methane were an order of

magnitude lower than that of the VC: 200µg/l methane, 112µg/l ethene, and 2600 µg/l VC.

The whole period of microcosm operation lasted 150 days, during the first 76 days of

microcosm operation a noticeably slower rate of VC consumption occurred when just CH4 was

present. Beyond day 75, VC biodegradation was fastest with just ethene present in the bottle. The

results indicated that ethene may be a more effective primary substrate for alkene

monooxygenase(s). The results of cumulative amounts of VC, ethene and methane consumed in

the microcosm bottles indicated the presence of methane alone with ethene inhibited ethene

biodegradation, thereby inhibiting VC consumption. The fact that VC alone did not inhibit

ethene consumption, but did inhibit methane uptake suggests that methanotrophs are more

susceptible to the presence of VC than are etheneotrophs. The results from these microcosms

confirm that ethene and methane promoted cometabolism of VC even when VC aqueous phase

concentration was an order of magnitude higher than that of the ethene and methane (Freedman

et al., 2001).

A series of microcosm studies evaluating ethene’s role in VC cometabolism were done

by Begley in 2011. The purpose of this study was to establish the feasibility of a full-scale

aerobic biostimulation treatment system at a demolition debris landfill in Carver, Massachusetts.

By 2002, the site was assessed to have a detached VC plume 3000’ long, 40’ wide and 30’ thick,

located 50’ below the water table with persistent low levels of VC (2 to 27 μg/L) extending

4600’ downgradient from the landfill.

22

A mixed site groundwater sample was used to generate a series of microcosms to test for

aerobic VC degrading activity. The groundwater contained 50 µM of methane and was amended

with 1 ml of 100% O2, MSM, 13µM ethene and 0.5 µM VC. VC degradation was complete in 21

days when groundwater was amended with minerals and 13µM ethene, compared to 9% VC

degradation from unamended groundwater, and 25% VC degradation from the groundwater that

was amended with mineral nitrogen, phosphorus, and trace elements after 21 days. In this

ethene-supplemented microcosm study, VC degradation lagged in the first 12 days, then the

concentration of VC started to decrease to nondetectable levels by day 21 after ethene

consumption. The result indicated that VC- degrading activity could be simulated by the addition

of ethene, and VC is consumed slower than ethene in this groundwater sample. However, rapid

degradation of methane was observed in both unamended and amended groundwater samples,

but the cometabolism of VC was limited. The results suggested that the addition of oxygen did

not stimulate the native methanotrophic bacteria to degrade VC in the site groundwater (Begley

et al., 2012).

Another microcosm experiment was done with the groundwater from well 63-I at the

Carver site in MA. The concentrations of methane, ethene and VC contained in the groundwater

are 195, 0.35 and <2 µg/L respectively. The relative abundance of etheneotroph and

methanotroph were estimated by qPCR analysis of functional genes, showing that methanotophs

and etheneotrophs are both present in the samples, and the methanotrophs are more than

etheneotrophs by 2-3 orders of magnitude. The screening also showed that methanotrophs,

etheneotrophs and VC-assimilators were all present in the groundwater sample (Findlay et al.)

Relevant observations from this study are described below.

23

3.4.1 Microbial behavior with VC and methane mixtures

The lag time for VC utilization decreased from 14 days to 10 days, shorter than that of VC as

sole substrate (13days). This suggested that cometabolic oxidation of VC (0.47 µM) was carried

out by methanotrophs rather than by VC-assimilators in the presence of 1µM methane (Findlay

et al.).

The lag time for initial methane utilization and the time for 50% methane degradation

both increased when 0.47µM VC and 0.45µM of methane were in the aqueous phase of the

sample, suggesting that methane utilization by methanotrophs was inhibited by similar aqueous

concentration of VC. This phenomenon was overcome if increasing amount of methane (0.45

µM, 1.28 µM, 3.6 µM) was fed to the microcosms. However, VC degradation is enhanced in the

presence of higher methane concentration, which supports the idea that VC is primarily oxidized

cometabolically (Findlay et al.).

3.4.2 Microbial behavior with VC and ethene mixtures

The initiation of VC consumption was about 16 days for microcosm experiments with VC (0.47

µM) alone and with both VC and ethene (3.0 µM). However, etheneotrophs started to oxidize

intensively after day 32, resulting in 77% of VC degradation by day 43, which was the time

required for VC-assimilator to oxidize just 50% of VC. Increasing concentration of ethene (1.3

µM, 3.8 µM,10.7 µM) did not affect the lag time for VC use significantly, but did increase the

time for 50% VC degradation. These results suggest that there is competition between VC and

ethene for the alkene monooxygenase binding site. However, the presence of ethene enhanced

24

VC cometabolic oxidation because in each test VC was degraded faster in the presence of ethene

than when alone (Findlay et al.).

3.4.3 Microbial behavior with methane and ethene mixtures without VC

The time required for methanotophs in the groundwater sample to degrade 50% of 1 µM

methane was 20 days, after lagging for about 9 days. When 3 µM of ethene was fed into the

bottle contained 1 µM of methane, both lagging period and times for 50% degradation increased

to 12 days and 26 days respectively. On the contrary, the times for 50% of 3 µM of ethene

decreased significantly from 39 days to 20 days when 1 µM of methane was fed into the

groundwater samples. The results are expected because methanotrophs can cometabloize ethene

using MMO, which impacts its ability to oxidize methane since methane and ethene are

competing for the active site. Another reason for faster ethene loss could be due to the inducing

of AkMO gene expression by epoxyethane generated by methanotrophs (Findlay et al.).

3.4.4 Microbial behavior with mixtures of methane, ethene, and VC

In 2010 experiments, VC degradation took only 19 days to reach 50% degradation when tested

with 1.3 µM methane and 3.8 µM ethene, it is faster than with only 1.3 µM methane (32 days) or

with only 3.8 µM ethene (23 days). It took 44 days to obtain 50% VC degradation with VC

alone. VC and ethene oxidation are shown to be a synchronized event, whilst great variability

were shown in terms of times to reach 50% methane degradation. For example, it took more than

70 days for methnotrophs to oxidize 50% of methane in the microcosm containing only 0.5 µM

25

methane and 3.8 µM ethene, which indicated that low concentration made methantophs

susceptible to VC inhibition (Findlay et al.).

Similar time frames for VC degradation were shown in 2011 experiments, where VC

degradation took only 20 days to reach 50% degradation when tested with 1 µM methane and 3

µM ethene, it is faster than with only 1.3 µM methane (31 days) or with only 3.8 µM ethene (38

days). It also confirmed that VC degradation did occur simultaneously with ethene use (Findlay

et al.).

From these microcosm results, groundwater scenarios were created with relatively low

concentrations of methane, ethene and VC. It is shown that methane utilization by

methanotrophs was inhibited when similar or low concentrations of VC and methane were

contained in the groundwater samples. In addition, the presence of ethene also impacted the

methane utilization by MMO. Nevertheless, ethene degradation is enhanced by the presence of

methane. VC degradation is observed to be proceeded via cometabolism by methanotrophs and

ethenenotrophs, and it has the highest potential to be fully degraded when methane and ethene

are both present (Findlay et al.). This work concluded that VC cometabolic activity in

groundwater system is shaped by the relative abundance of native methanotrophs and

etheneotrophs as well as the aqueous concentrations of methane, ethene, and VC at the specific

site.

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4.0 CONCLUSION

U.S. EPA classified vinyl chloride as a known human carcinogen by inhalation exposure (EPA

2000). The carcinogen is one of the 188 hazardous air pollutants (HAPs) listed under Section 112(b)

of the 1990 Clean Air Act Amendments, and its emissions are regulated from more than 170

industrial air pollutant source categories. The primary target organ for vinyl chloride exposure is

the liver. The association between angiosarcoma of the liver and vinyl chloride exposure is well

documented for occupational exposures. Noncancer liver pathologies have also been associated

with vinyl chloride exposure, including liver necrosis and cysts.

General population are at risk for exposure to vinyl chloride from ambient air contaminated

with vinyl chloride by emissions released from polyvinyl chloride plastics production and

manufacturing facilities, as well as some incinerators. Contamination of groundwater and drinking

water with vinyl chloride-contaminated run-off from such manufacturing facilities is also a concern

for public health. Reports of effects of chronic exposure to vinyl chloride focus primarily on

occupational adult exposures and experimental animal studies. Evidences of chronic vinyl chloride

exposure causing angiosarcoma of the liver in occupationally-exposed adults have been shown from

cohort studies worldwide. In addition, other cancers of the liver, as well as lung and brain cancer,

have been reported in occupational exposure studies and experimental animal studies .

In Situ bioremediation is a destruction technology that takes advantage of

microorganisms natural metabolism to break down contaminants to less toxic end products.

27

Under anaerobic condition higher chlorinated ethenes like PCE and TCE will be converted to

less chlorinated ethenes by microorganisms through reductive dechlorination process. When the

groundwater returned to aerobic condition downgradient of the source with methane, ethene, VC,

cis-DCE and several other compounds still present, natural attenuation of VC could occur more

readily via oxidation by aerobic microorganisms as a VC plume intersects with groundwater

containing molecular oxygen . As the results, a complete degradation of chlorinated ethenes shall

be achieved if a two-stage treatment system is proposed, which consisting anaerobic as first

stage, and aerobic as the second.

Biostimulation is the process of stimulating active bioremediation through the addition of

substrates that encourage microbial growth. Aerobic bioremediation of chlorinated ethenes can

be enhanced by injecting methane or ethene and oxygen into a dilute VC plume to stimulate

growth of the ethenotrophs/methanotrophs and subsequent cometabolic oxidation of VC .

However, one of the disadvantage of growth-linked bioremediation process is that the target

bacteria may not be populous enough for biodegradation to proceed at an acceptable rate, and

thus bioaugmentation would be needed at most sites. In addition to suitable condition s for

bacteria growth (i.e., redox conditions, pH range, supply of electron donors, and presence of

nutrients), it is equally critical to measure the presence or absence of methanotrophs and

etheneotrophs.

Even more promising than cometabolism, future research can be focusing on

investigating the role of VC-assimilators, the bacteria are widespread and readily isolated from

contaminated and uncontaminated environments, for example Nocardioides sp. strain JS614

(ATCC BAA-499) that can directly degrade VC and ethene as a growth substrate .

28

29

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