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*»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ ‡∑§‚π∏“π’ µ.§≈ÕßÀâ“ Õ.§≈ÕßÀ≈«ß ®.ª∑ÿ¡∏“π’ 12120 ‚∑√. 0-2577-1136 ‚∑√ “√. 0-2577-1138 Environmental Research and Training Center, Department of Environmental Quality Promotion. Technopolis. Klong 5 Klong Luang, Pathumthani 12120 e-mail: Panja@deqp.go.th
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ABSTRACT Two 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 different 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. Introduction By the survey of Thailand Environment Institute 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 ¢-18
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 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
certain minerals, referred to as nutrients and several other conditions i.e. oxidized-reduced condition, 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 hydrocarbon) and TCE (representative of chlorinated aliphatic hydrocarbon) at the contaminated sites (Aggarwal et al, 1997). In 1998 ERTC has conducted an investigation to search for potential contaminated sites by volatile 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 contamination 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¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
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Figure 2. Map of Ground water sampling wells at site C showing plume of TCE soil vopour.
2. Methods and Materials Determining 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. ¢-20
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 concentration 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 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
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 Ì C 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 and 18 O 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 transformation.
3. Results and Discussions Cultivation 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. »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
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 B-1 B-5 WA
CFU/ml 29 X 104 139 X 105 151 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 2035% if these bacteria are cultured in media containing toluene. The transformation of toluene and TCE is supposed to be either anaerobic or ¢-21
aerobic pathways. The anaerobic pathways of transformation cannot be tested in this experiment because of the lack of anaerobic culturing apparatus. However both
transformation pathways will be measured by measuring isotopic ratio of carbon dioxide generated by bacteria.
Figure 3. Degradation of 50 mg/l toluene by bacteria filtered from water sample AIWA, LP3 and B1 collected in Northern Industrial Estate.
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.
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Figure 5. Degradation of 10 mg/l TCE in solution of 50 mg/l toluene by bacteria filtered from water 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 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 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
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
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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 δ 13 C than the uncontaminated ones. However the differences 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. ¢-24
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. Conclusions The 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 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
Figure 7. Isotopic composition of 13C and 18O in dissolved carbon dioxide from TCE contaminated and uncontaminated ground water wells and waste water of Northern Industrial Estate shown that dissolved carbon dioxide in ground water in the area of Northern Industrial Estate and waste water have different δ 13C and δ 18O 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. References 1. 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 biodegradation. 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.
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