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°“√„™â‡∑§π‘§‰Õ‚´‚∑ª‡æ◊ËÕµ‘¥µ“¡°“√‡ª≈’Ë¬π·ª≈ß¢Õß “√Õ‘π∑√’¬å√–‡À¬„ππÈ”„µâ¥‘π„π∫√‘‡«≥π‘§¡Õÿµ “À°√√¡¿“§‡Àπ◊Õ ®—ßÀ«—¥≈”æŸπUse of Isotope Techniques to Trace the Transformation of VolatileOrganic Compounds in Polluted Ground Water in the Area ofNorthern Region Industrial Estate, Lampoon Province

*»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ àß‡ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ ‡∑§‚π∏“π’ µ.§≈ÕßÀâ“ Õ.§≈ÕßÀ≈«ß ®.ª∑ÿ¡∏“π’ 12120 ‚∑√. 0-2577-1136 ‚∑√ “√. 0-2577-1138Environmental Research and Training Center, Department of Environmental Quality Promotion. Technopolis. Klong 5 Klong Luang, Pathumthani 12120 e-mail: [email protected]

Mr. Panja Yaithavorn*,Mr.Srihanart Charnnarong*

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“√‰µ√§≈Õ‚√‡Õ∏‘≈’π ‰¡à·µ°µà“ß°—π

ABSTRACTTwo factories were investigated to

be polluters of toluene and TCE in ground

water and 7 wells were installed inside and

outside TCE contaminated area. Two water

samples from TCE contaminated wells and

one from wastewater were measured bacterial

population. The result shows that samples

having higher TCE contamination have less

bacterial population. Bacteria in water samples

from 7 wells were tested for toluene and TCE

degradability. After 11 days of inoculation

samples from 6 wells shows ability in degrading

toluene but are not able in degrading TCE.

Measuring isotopic ratio of 13C and 18O of

the dissolved carbon dioxide indicated that

bacteria in waste water and ground water uses

dif ferent carbon sources and generates

isotopically different carbon dioxide. However

isotope ratio of dissolved carbon dioxide in

wells contaminated with and without TCE can

not be differentiated.

1. IntroductionBy the survey of Thailand Environ-

ment Inst i tute in 1998 on hazardous

chemicals used by industry, it was found

that volatile organic solvents has been

increasingly imported and intensively used by

industry in Thailand. The solvents, consisting

primarily of chlorinated aliphatic hydrocarbons

have been widely used as degreasing agents

of aircraft engines, automobile parts, electronic

components, and metal products. In many

cases the solvents are also used as raw

materials for producing electronic components.

After uses, the solvents are dirty and have

often been disposed into refuse sites, waste

pits and storage tanks and sometimes directly

on soil. Because of their relative low solubility

in water and their poor sorption to soil, they

tend to migrate downward through soil,

contaminating groundwater with which they

come into contact. By improper handling,

storage, disposal and lack of legislation

enforcement, it was estimated that the

chemicals have extensively contaminated soil

and groundwater of the country.

In 1998 Environmental Research

and Training Centre (ERTC) conducted a

survey to search in the area of Northern

Industrial Estate of Thailand for potential

contamination of the solvents in soil and

groundwater and found that organic solvents

used in the industrial complex such as

trichloroethylene (TCE), toluene, ethyl methyl

ketone etc. have already contaminated the

soil and shallow groundwater of the estate

at many locations. As shown by the cases of

developed countries, groundwater once

contaminated is not easy to clean up and

need long time and high cost for remediation.

Thailand like many developing countries

does not have technology to cope with the

problem. The research to search for appropriate

technology to remediate soil and ground water

has therefore been initiated.

In treating contaminated soil and

groundwater, many remediation techniques

have been employed. Among these are pump

and treat, soil venting, photooxidation etc.

These techniques need sophisticated and

expensive technology. However bioremediation

or using microorganisms to degrade or to

convert harmful contaminants to less harmful

species is one of attractive technology

because it is less expensive. In order for

these biodegradable processes to occur,

microorganisms require the presence of

¢-18 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ àß‡ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

certain minerals, referred to as nutrients and

several other conditions i.e. oxidized-reduced

condit ion, temperature, pH etc. These

parameters can impact the effectiveness of

these processes.

This project is initiated to explore

microorganisms having capability in converting

toxic contaminants in the group of both

hydrocarbon and chlorinated hydrocarbon to

less toxic species and carbon dioxide which

is a final and nonharmful products in many

degradation pathways. Finally, the project is

intended to use isotope technique such as

measuring isotope ratio of 13C and 18O of

oxygen used and carbon dioxide generated

by bacteria to differentiate biodegradation

pathways and to measure degradation rate

of toluene (representative of aromatic hydro-

carbon) and TCE (representat ive of

chlorinated aliphatic hydrocarbon) at the

contaminated sites (Aggarwal et al, 1997).

In 1998 ERTC has conducted an

invest igat ion to search for potent ia l

contaminated sites by volat i le organic

compounds by selecting Northern Industrial

Estate in Lumphun province as a target. The

estate has been operated for 20 years to

run about 60 factories, most of them are

electronic appliance type production. The

survey was conducted by applying soil gas

method to find the possibility of contami-

nation of VOCs in soil in the estate area.

The method was done by drilling small holes

1 m. depth, 20x20 m grid spacing and

inserting absorbent tubes or detector tubes

into the holes, then soil vapor was withdrawn

through the tubes. By this method trace of

the organic compounds such as TCE, toluene

etc. contaminated in soil or groundwater was

trapped by the tubes. In case of detector

tube, the type of organic compounds and

content of soil vapor can be detected by

observing color development in the tubes.

In the other hand adsorbent tubes can be

read by a GC equipped with a thermal

desorption unit. By this method 2 factories

could be identified as TCE soil polluter while

one is a polluter for TCE and toluene. The

factory polluting both TCE and toluene was

selected as a auger drill site to evaluate

further subsurface contamination and auger

wells were installed to collect contaminated

groundwater samples. The plume of TCE

soil gas and locations of auger wells in the

selected factory can be shown in Figure 1

and 2.

Figure 1. Map of ground water sampling wells at site B showing plume of TEC soil vapor

»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ àß‡ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ ¢-19

Figure 2. Map of Ground water sampling wells at site C showing plume of TCE soil vopour.

2. Methods and MaterialsDetermining bacterial population

density in CFU form

Water collected from 2 contaminated

wells and wastewater of the estate were

used to measure bacterial population density

by viable count method which detects living

microorganisms by their ability to form colonies

on agar plates. One millilitre of each sample

was transferred onto surface of a sterile

petri dish agar plate by using a sterile bent

glass rod. Inoculum was distributed over

surface of the media by rotating the dish by

hand to completely adsorb on the medium

before incubating at 28 ÌC for 7 days. The

suitable dilution was selected so that the

total number of colonies on plate will be

between 30-300. The colonies were counted

by a colony counter. The heterotrophic plate

count was computed in unit CFU/ml and

multiplies average total number of colonies

per plate by the reciprocal of the dilution

used.

Screening bacteria having capability

in degrading toluene and TCE

The isolates from water samples

were obtained by filtering through a coarse

filter (No.5 What man) and fine filter (pore size

of 0.22 µm) cellulose acetate (Millipore). The

recovered cell were cultured in erlenmayer

flasks containing M-R2A medium (Fries et al,

1994) at 30 ÌC for 7 days. To evaluate toluene

and TCE transformation rate, selected

samples were transferred into three 20 ml

sterile vials per sample. The vials were

incubated without shaking after addition of

9 ml of basal salts (BS) medium (Owens et al,

1969) amended with 5 mM KNO3 and sealed

with teflon-lined stoppers. Stock of toluene

and TCE were added to BS medium to make

concentrat ion of toluene and TCE in

the vials to be 50 and 10 mg/l respectively.

Control from the same batch of medium but

without the inoculum were incubated at the

same time to determine any nonbiological

loss of toluene and TCE. Three vials were


sacrificed at each sampling time of 0, 1, 2, 3,

6, 11, 17 and 20 days by adding 10 M HCl.

The vials were frozen at 4 ÌC for analysis of

toluene and TCE concentrations. Toluene

and TCE were equilibrated at 30 ÌC and

measured by headspace analysis with a gas

chromatograph equipped with photoionization

detector and an ultra alloy stainless steel

capillary column (UAC-504-30V-3.0F). The

GC column was set at 80 ÌC and injector

and detector at 110 ÌC. Nitrogen was used as

the carrier gas. All reported data on toluene

and TCE degradation rate were averaged by

three replications.

Study toluene and TCE degradation

pathways of bacteria by applying isotope

technique

Eight samples were collected from

wells, 4 from contaminated and other 4

from uncontaminated wells. They were fixed

and sent to the laboratory of International

Atomic Energy Agency for performing 13C and18O analysis in carbon dioxide gas dissolved

in the water sample. The ratio of these 2 stable

isotopes will be used to differentiate the

pathways of organic compound transfor-

mation.

3. Results and DiscussionsCultivation of microbes and counting

population density in CFU form

The population of bacteria from B-1,

B-5 and waste water were counted on plate

is show in table 1. From the table population

density of bacteria in wastewater is higher

than B-1 and B-5 because wastewater

contains many types of organic compounds

which can be used by many types of bacteria

as carbon sources while samples from B-1 and

B-5 wells might have less amount of carbon

sources. Comparing between B-1 and B-5

samples, B-1 has less abundance of bacteria

because B-1 well is located closer to the hot

spot of toluene and TCE contamination.

The higher concentration of the compounds

will get rid of general bacteria that cannot

use the compounds as carbon source or both

compounds might be toxic to them. Therefore

indigenous bacteria that will be able to

transformed both toluene and TCE is limited.

The tolerated species will be used in the

toluene and TCE degradation study.

Table 1. Population density of bacteria by

heterotrophic plate count

Sample CFU/ml

B-1 29 X 104

B-5 139 X 105

WA 151 X 107

Testing degrading capability of

microbes on toluene and TCE

Degradation of toluene and TCE

by bacteria collected from well B-1, well

BH 1, well BH 2, well BH 3, well AIWA, and well

LP 3 are shown in Fig 3, 4, 5 and 6

respectively. With 50 and 10 mg/l of toluene

and TCE at starting concentration, degradation

rate of the 2 compounds by bacteria seems

to be not to much different from control.

However the degradation of toluene by

bacteria from well B-1, well LP 3, well AIWA,

well BH 1, well BH 2, well BH 3 and the

waste water are significantly different from

control after 11 days from inoculation. This

might be because of in the first period,

bacterial population are not enough to

degrade toluene. After 11 days bacteria from

isolates well LP3, well Bl, well BHl, well BH2

can decrease toluene 23-29% comparing to

control, while bacteria from well BH3, well

AIWA and the waste water can decrease

TCE 12-14% and bacteria from well LP3,

well Bl, BHl and BH2 can decrease TCE 20-

35% if these bacteria are cultured in media

containing toluene.

The transformation of toluene and

TCE is supposed to be either anaerobic or


aerobic pathways. The anaerobic pathways

of transformation cannot be tested in

this experiment because of the lack of

anaerobic culturing apparatus. However both

Figure 3. Degradation of 50 mg/l toluene by bacteria filtered from water sample AIWA,LP3 and B1 collected in Northern Industrial Estate.

transformation pathways will be measured

by measuring isotopic ratio of carbon

dioxide generated by bacteria.

Figure 4. Degradation of 50 mg/l toluene by bacteria filtered from water sample BH1, BH2,BH3 and waste water (WA) collected in Northern Industrial Estate.


Stable isotope of dissolved carbon

dioxide in contaminated and uncontaminated

water samples

The stable isotopes ratios of dissolved

carbon dioxide in 7 water samples, 6 ground

waters and 1 waste water, were analyzed at

the laboratory of International Atomic Energy

Agency. Both carbon and oxygen isotope

ratios are reported relative to Pee Dee

Belemite (PDB). The ratios are given using

the per mil notation where

Figure 5. Degradation of 10 mg/l TCE in solution of 50 mg/l toluene by bacteria filtered fromwater sample AIWA, LP3 and B1 collected in Northern Industrial Estate.

Figure 6. Degradation of 10 mg/l TCE in solution of 50 mg/l toluene bybacteria filteredfrom water sample BH1, BH2, BH3 and waste water (WA) collected in Northern Industrial Estate.


δ13C = [(13C/12C) sample

- (13C/12C)PDB

] X 1000

(13C/12C)PDB

δ18O = [(18O/16O) sample

- (18O/16O)PDB

] X 1000

(18O/16O)PDB

The carbon isotope ratio of δ13C of

3 uncontaminated and 4 contaminated

ground waters is in the range of -13.31

to -16.24 and -12.76 to -20.32 .

respectively. It looks like contaminated

ground water are slightly more depleted

in δ13C than the uncontaminated ones.

However the dif ferences are not clear.

If the sample from well AIWA is not included

in the comparison, the range of δ13C ratio

of the two groups are same. Landmeyer

et.al. (1996) found that the carbon isotope

signature of uncontaminated aquifer is

-24.6 . This indicates active respiration of

bacteria, fungi and plants in temperate

climates. And the atmospheric carbon dioxide

is - 8.5 . Our data of isotope ratio of δ13C

in groundwater are in the range which

indicated that dissolved carbon dioxide

in ground water collected from our wells is

from the mixing of carbon dioxide generated

from activity of bacteria decomposing plant

material and carbon dioxide from atmosphere.

Because of no difference between isotopic

signature of dissolved carbon dioxide in

contaminated and uncontaminated wells,

the special signature of carbon dioxide from

TCE degradation is not significantly different

from other sources of bacterial respiration.

The intrinsic degradation of TCE in the

sites occurs by anaerobic pathway which

is confirmed by anaerobic degraded products

such as 1,2-cis-dichloroethylene and vinyl

chloride found in well AIWA, Bl (a), Bl (b), and

LP3.

The plot between δ13C and δ18O of 7

groundwater samples and one wastewater

sample is shown in Figure 7. By this figure

groundwater has different isotopic signature

of δ13C and δ18O in dissolved carbon dioxide.

This might be attributable to different carbon

sources for bacterial respiration. While carbon

sources in waste water comes from variety

of mixture such as carbohydrate, fat, protein

and other organic compounds from industry,

the carbon sources in groundwater composes

mainly of humic acid, the final products of

degraded plants. Unfortunately we do not

have any data on the isotopic signature in

carbon dioxide from these carbon sources.

Comparing between waste water and ground

water, it is shown that carbon dioxide derived

from waste water has more depleted δ13C

and δ18O than of dissolved carbon dioxide

in ground water. This may indicate the

different carbon sources used by bacteria.

4. ConclusionsThe bacteria from 7 ground water

wells were tested for capability in degrading

toluene and TCE by 2 toluene concentration

level 50 and 10 mg/l, it was found that a

sample tended to be able to decrease

toluene and TCE. These samples will be used

to isolate the pure cultures and the cultures

will be characterized and tested to be used

for treating ground water contaminated with

toluene and TCE.

By the results of the study on isotopic

signatures of carbon dioxide, it is clearly


shown that dissolved carbon dioxide in

ground water in the area of Nor thern

Industrial Estate and waste water have

different δ13C and δ18O which indicates

that bacteria in waste water and ground

water use different carbon sources for their

respiration. However the different of δ13C

and δ18O in dissolved carbon dioxide in ground

water contaminated and uncontaminated

with TCE can not be seen.

5. References1. Aggarwal, P.K., M.E. Fuller, M.M. Gurgas, J.F.

Manning and M.A. Dillon. 1997. Use of stable

oxygen and carbon isotope analyses for

monitoring the pathways and rates of

intrinsic and enhanced in situ biodegrada-

tion. Env. Sci. and Technol. 31:590-596.

2. Fries, M.R., J. Zhou. Chee-Sanford, and J.M.

Tiejde.1994. Isolation, characterization

and distribution of denitrifying toluene

degraders from a variety of habitats.

Appl.Environ.Microbiol.60:2802-2810.

3. Owens, J.D.., and R.K. Keddies. 1969. The

nitrogen nutrition of soil and herbage

coryneform bacteria. J. Appl. Bacteriol.

32:338-347.

Figure 7. Isotopic composition of 13C and 18O in dissolved carbon dioxide from TCE contaminatedand uncontaminated ground water wells and waste water of Northern Industrial Estate


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