Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.

Remediation of TCE-contaminated groundwater using integrated biosparging and enhanced bioremediation system
Kuo Y.C., 1 Liu J.K., 2 Chien H.Y.,1 Chen C.C.3 and Kao C.M. 1*
1. Institute of Environmental Engineering, National Sun Yat-Sen University, Kaohsiung City, TAIWAN 2. Department of Biological Science, National Sun Yat-Sen University, Kaohsiung City, TAIWAN 3. Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung City, TAIWAN *jkao@mail.nsysu.edu.tw

Abstract A biosparging well (BSW) was installed at the upgradient area of the test site for air injection. Brown sugar, which was used as the primary substrate, was pressure-injected into the TCE plume through the BSW to enhance the rate of TCE co-metabolism. To evaluate the effectiveness of TCE bioremediation, monitor wells were installed in series down gradient of the BSW along the groundwater flow path. Polymerase chain reaction (PCR) and nucleotide sequence analyses indicate that TCE-degrading enzymes (e.g. toluene monooxygenase, toluene dioxygenase, phenol monooxygenase and particulate methane monooxygenase) were identified in field groundwater samples after the air and substrate supplement.
Results from the pilot-scale study indicate that the aerobic co-metabolism was the major cause of the decrease in TCE concentrations. Without primary substrate supplement, intrinsic bioremediation and injection of air and nutrients alone could not enhance the aerobic co-metabolic mechanism and cause the decrease in TCE concentrations within the plume. The occurrence of aerobic TCE co-metabolism could be confirmed by the following investigations within the plume: (1) significant decrease in TCE concentrations; (2) increase in chemical oxygen demand (COD) concentrations and microbial populations; (3) increase in dissolved oxygen (DO) and oxidation-reduction potential (ORP) in the BSW; (4) significant depletion of DO and decrease in ORP after the supplement of substrate; (5) production of CO2 and decrease in pH value and (6) detected specific TCE-degrading genes. Approximately 97 and 92% of TCE was removed in BSW and monitor well located 5 m down gradient of the BSW. The operation of biosparging caused the shifting of low oxygen conditions inside the plume to aerobic conditions. Results indicate that the integrated in situ biosparging and enhanced aerobic bioremediation is a feasible and promising technology to remediate TCE-contaminated groundwater.
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Keywords: Bioremediation, biosparging, co-metabolism, gene analysis, groundwater contamination, TCE.

Introduction
Groundwater at many existing and former industrial sites and disposal areas is contaminated by halogenated organic compounds that were released into the environment. The chlorinated solvent trichloroethylene (TCE) is one of the most ubiquitous compounds. One cost-effective approach for remediation of contaminated aquifers that is attracting increased attention is the application of enhanced bioremediation for contaminant biodegradation. Current evidence suggests that TCE can be degraded under aerobic co-metabolic conditions or under anaerobic reductive dechlorinating conditions by supplying an alternate primary substrate3,7,21. Because the biodegradation of TCE is generally faster under aerobic conditions, introduction of DO into the plume will increase the TCE biodegradation (co-metabolism) rate and will significantly reduce the TCE mass flux if bioavailable primary substrates are sufficient. Moreover, due to the recalcitrant characteristics of the chlorinated solvents, unlike the petroleum-hydrocarbon contaminated sites, most of the TCE-contaminated site groundwater is under oxidized conditions. Therefore, in situ aerobic bioremediation is a feasible technology to clean up TCE contaminated aquifers if oxygen can be provided to the subsurface economically. Several aerobic microorganisms or microbial communities have the ability to synthesize oxygenase enzyme systems that catalyze the initial step in oxidation of their respective primary or growth substrates and have the potential for initiating the oxidation of TCE and other chlorinated aliphatic hydrocarbons4,13,14. The groups of aerobic bacteria include oxidizers of the following compounds: methane, propane, ethylene, toluene, phenol, acetic acid, propionic acid, cresol, ammonia and isoprene7. Methane oxidizer, phenol degrader and toluene degrader are the main bacteria, which are able to perform the aerobic co-metabolic process of TCE6,15,20. In situ bioremediation is a feasible technology to clean up TCE contaminated sites if oxygen and biodegradable primary substrates can be provided to the subsurface efficiently. Brown sugars are products of sugar industry. They

Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
have the following characteristics that make them good candidates for using as the primary substrates: (1) they are rich in carbon, an essential energy source for biodegradation; (2) they have the potential to exhibit sufficient carbon bioavailability for aerobic co-metabolism to occur; and (3) they are relatively inexpensive. The above discussion suggests that the brown sugar injection system installed in the upgradient area of the plume is a practical method to enhance TCE biodegradation. Biosparging is an effective mechanism for removal of volatile organic compounds (VOCs) including TCE under aerobic biodegradation mechanism1,5,10,22. Biosparging functions by injecting air at a low rate into the aquifer below the zone of contamination. The injected air promotes oxygenation of the aquifer as necessary to promote aerobic biodegradation. In this study, the purpose of the biosparging system is to stimulate aerobic co-metabolism of TCE. Thus, development of an aerobic co-metabolic reactive zone containing both sufficient oxygen and primary substrates would be a feasible alternative to enhance in situ bioremediation of TCE cost-effectively. Recently, molecular biology technologies have been applied in site remediation studies to confirm the effectiveness of the bioremediation11,12,15. Results from other studies reveal that polymerase chain reaction (PCR) and nucleotide sequence analysis techniques provide a guide for microbial ecology, which can be used as an indication of the trend of biodegradation process9,10,15. Thus, total bacterial DNAs of representative groundwater samples were extracted in this study for detecting the community dynamics in the process of TCE degradation8,11. In this study, the dominant microorganisms and existence of bacteria responsible for aerobic co-metabolism of TCE were verified using a series of molecular biology techniques including DNA extraction, PCR amplification and DNA analysis. The objectives of this pilot-scale study were to (1) evaluate the effectiveness of the integrated biosparging and enhanced in situ aerobic co-metabolism technology on the remediation of TCE-contaminated groundwater and (2) determine the dominant native microorganisms at different locations of the contaminated aquifer through microbial identification during the remediation process. average groundwater elevation within the shallow aquifer is approximately 6 to 7 m below land surface. Groundwater in the unconfined aquifer, according to the groundwater elevation in site monitor wells, flows to northwest. The measured effective porosity is 0.31 and the average hydraulic conductivity for the surficial, unconfined aquifer is 0.006 cm/s. The calculated site groundwater flow velocity is 9.1 cm/d. The measured groundwater temperature in the surficial aquifer varies from 19 to 27C. The preliminary site investigation results indicate that the TCE contamination has become a diffuse pollution which has resulted in a very dispersed TCE plume in the site. Since the year of 2007, a pilot-scale study has been applied within the TCE plume to evaluate the effectiveness of applying in situ bioremediation technology for plume control and TCE removal. System design: In this study, a biosparging well (labelled as BSW) was installed in the upgradient area for air, primary substrate (brown sugar) and nutrients injection and groundwater monitoring. Three monitor wells (labelled as W1, W2 and W3) were installed in series downgradient of the BSW along the groundwater flow path for groundwater sampling and analyses. A background monitor well (labelled as BW) located in the uncontaminated area was used for the background sample collection. Three monitor wells W1, W2 and W3 were located 5, 10 and 15 m downgradient of the BSW respectively. All wells were screened from 7 to 17 m below land surface (bls). Figure 1 presents the site map showing the locations of representative monitor wells and groundwater flow direction. The biosparging system consisted of a BSW (injection points) (well screen at 8 to 10 m bls), air compressor, flow indicator, inline regulator and pressure gauge. For the BSW, the air flow was approximately 0.06 to 0.17 m/min so the TCE volatilization can be minimized. In this study, different substrates were injected during the 250-operational period. Three operational stages were performed during the pilot-scale study. From day 0 to day 79 (Stage 1), no substrates were supplied. This stage was to evaluate the effectiveness of intrinsic bioremediation on TCE plume control. From day 80 to day 139, biosparging system was operated for air supplement in BSW to increase the DO concentrations in groundwater and nutrient solution [300 g (NH4)2SO4 and 30 g Na2HPO4 in 1,000 L of groundwater] was injected into the BSW to evaluate the effectiveness of nutrient addition on TCE removal (Stage 2). From day 140 to day 250 (Stage 3), biosparging system remained active and brown sugar (3 kg for each injection) was also injected in BSW for primary substrate supplement. Biosparging system was operated daily and nutrient injection was performed monthly during the stages 2 and 3 operations. Brown sugar injection was performed monthly during the stage 3 operation. Table 1 shows the three-stage operation during the operational period.
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Material and Methods

Site description: A government owned industrial park site located in southern Taiwan was selected for this in situ bioremediation study. In early 2000, leakage from a TCE storage tank resulted in the groundwater contamination by TCE. During the following four-year investigation period, more than 230 soil samples were collected and 25 monitor wells were installed for site characterization and TCE-plume delineation. On-site borings encountered up to 22 m of mostly brownish and fine to medium sandy loam. The

Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
PCR analysis and microbial identification: In the field study, groundwater samples collected from GW, BSW and monitor wells were used for the PCR analysis to evaluate the occurrence of genes responsible for the aerobic co-metabolism of TCE before and after bioremediation process. Total bacterial DNAs from 4 L of each well were extracted with DNA Purification kits (GeneMark Co., Taiwan) for detecting the community dynamics. Bacterial 200-bp fragments of 16S rDNA V3 region were amplified with the primer sets (341f, forward: 5-CCTACGGGAGGCA GCAG-3 containing a GC clamp of 40-nucleotide GC-rich sequence; 534r, reversed: 5-ATTACCGCGGCTGCTGG-3)12. The individual primer sets were allowed to amplify of fragments of toluene dioxygenase, toluene monooxygenase, particulate methane monooxygenase (pMMO) and phenol hydroxylase9,15,19. These primer sets were listed in table 2. The mixtures of PCR contained 10 ng of DNA extract, 4 pmol of each primer and 5 U of Taq polymerase (Takara, Shiga, Japan) in final concentrations of 2.5 mM of MgCl2 and 0.12 mM of deoxyribonucleoside triphosphates in PCR buffer. The PCR amplification was conducted for 35 cycles: denaturation at 94oC for 1 min, annealing temperature was initially 65.8C and it was decreased by 1C per cycle until it was 55.8C, after which 25 additional cycles were carried out at 55.8 oC; and extension at 72 oC for 2 min. The 10% polyacrylamide gel with a 30-60% denaturant gradient was used and electrophoresis was performed at 60 oC and 70 V for 14 h. The gels were then stained with SybrGreenand photographed. The PCR-amplified products were electro-eluted from gel and then sequenced by MdBio Inc. in TaBSWan. Those sequences were evaluated by using the basic local alignment search tool (BLAST) to determine the closest relatives in the GenBank databases (http://www.ncbi.nlm.nih.gov). Groundwater samples were collected on specific days during the three stages of treatment period. Groundwater sample analyses: Groundwater samples were collected bimonthly and analyzed for organic compounds and geochemical indicators including TCE, CH4, CO2, inorganic nutrients (ammonia, nitrate, phosphate), Fe(II), pH, Eh, DO and chemical oxygen demand (COD). Organic compound analyses were performed in accordance with U.S. EPA Method 602, using a Tekmer Purge-and-Trap Model LSC 2000 with a Perkin-Elmer Model 9000 Auto System Gas Chromatograph (GC). Methane was analyzed on a Shimadzu GC-9A GC using headspace techniques. Ion chromatography (Dionex) was used for inorganic nutrients and anions analyses. COD measurements were conducted in accordance with the dichromate reflux method described in Standard Methods2. DO, Eh, pH, CO2 and temperature were measured in the field. An Accumet 1003 pH/Eh meter (Fisher Scientific) was used for pH and
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Eh measurements, an Orion DO meter (Model 840) was used for DO and temperature measurements and a Hach digital titrator cartridge was used for CO2 measurements. The analytical procedures for groundwater analyses are described in Standard Methods2.

Results and Discussion

Stage 1 operation: Groundwater samples were collected and analyzed from BW, BSW and three monitor wells (W1 to W3) before the start of the air and substrate injection. Table 3 presents the averages of groundwater analytical results during the stage 1 operational period to evaluate the intrinsic bioremediation mechanisms on TCE plume control. Results show that the pH, DO and ORP varied from 6.9 to 7.2, 0.7 to 1.0 mg/L and 71 to 127 mv respectively, within the TCE plume. The averaged pH, DO and ORP in BW during the stage 1 operation were approximately 7.2, 1.1 mg/L and 132 mv respectively. Results show that DO and ORP values in BW were only slightly higher compared to the DO and ORP values inside the plume area. This indicates that the intrinsic groundwater was in oxidative conditions in both contaminated and uncontaminated areas and intrinsic bioremediation was not significant within the plume area. Low nutrients (e.g. ammonia, phosphate) concentrations and microbial populations reveal that nutrient and substrate additions might be required to proliferate the indigenous microbial populations and activate the microbial activity (Table 3). No methane was detected and ferrous iron concentration was less than 0.2 mg/L at this site (Table 3). Significant amounts of sulfate (varied from 29 to 35 mg/L) and nitrate (varied from 1.5 to 2.3 mg/L) were observed in both background and contaminated areas. Results from nitrate, sulfate, methane and ferrous iron analyses reveal that DO was used as the electron acceptor and aerobic oxidation was the main degradation trend. No significant variations in CO2 production between the background and plume areas were observed. This indicates that the microbial activity within the contaminated area was low. Figures 2 to 4 present the variations in TCE concentrations, COD concentrations and total viable bacterial counts in BSW and three monitor wells (W1 to W3) during the three-stage operational period respectively. Results from fig. 2 and table 3 show that the averaged TCE concentrations in BSW, W1, W2 and W3 were 207, 195, 178 and 166 g/L respectively during the stage 1 operational period. Results indicate that approximately 7, 6, 1 and 3% of TCE were removed from stage 1 operation. Thus, no significant TCE removal was observed in stage 1 during the 79-day operational period in BSW and three monitor wells. This reveals that intrinsic bioremediation of TCE was not a possible remedial option at this site. Results from table 3 and fig. 3 indicate that averaged COD concentrations were below 8 mg/L in stage 1 in both background and contaminated areas. This implies that the existence of natural carbon-contained substrate was insignificant.

Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
Thus, the low COD concentration would become the limiting factor for the occurrence of aerobic TCE co-metabolism. The averaged total viable bacterial counts in stage 1 varied from 9.6 102 CFU/mL in W3 to 5.1 103 CFU/mL in BSW (Fig. 5). The low total bacterial counts and low CO2 production indicate that the intrinsic microbial activity was low and this could be due to the low biodegradable substrate in the subsurface. Results indicate that the occurrence of intrinsic bioremediation of TCE was not significant based on the following observations: (1) insignificant decrease in TCE concentrations along the transport path from BSW to W2; (2) insignificant depletion of DO, nitrate and sulfate within the plume; and (3) insignificant production of dissolved ferrous iron, CO2 and methane. Stage 2 operation: On day 80, nutrients [300 g (NH4)2SO4 and 30 g Na2HPO4 in 1,000 L of groundwater] were injected into BSW for nutrient supplement. Biosparging system was operated in stage 2 to enhance the enhanced aerobic bioremediation process. Table 4 shows the averages of groundwater analytical results during the stage 2 operation to evaluate the intrinsic bioremediation mechanisms on TCE plume control. Results show that the averaged pH, DO and ORP varied from 6.7 to 7.2, 1.2 to 6.5 mg/L and 127 to 305 mv respectively, within the TCE plume during the operational period. Results show that pH, DO and ORP values in BW had no significant changes compared to the results measured in stage 1. This indicates that the groundwater was in oxidative conditions in both contaminated and uncontaminated areas. Results show that nutrient and air injection caused the significant increase in nutrient (e.g. ammonia, phosphate) concentrations and proliferation of microbial populations (Table 4). No methane and ferrous iron were detected and significant amounts of sulfate (varied from 27 to 49 mg/L) and nitrate (varied from 1.1 to 1.8 mg/L) were observed in both background and contaminated areas. Results from nitrate, sulfate, methane and ferrous iron analyses reveal that aerobic oxidation was the main degradation trend. Slightly higher CO2 production was observed inside the plume area compared to the CO2 measurements in stage 1 period. This indicates that the nutrient and air injection slightly activate the microbial activity within the contaminated area. The variations in TCE concentrations, COD concentrations and total viable bacterial counts in BSW and three monitor wells (W1 to W3) during the stage 2 operational period are presented in Fig. 2 to 4, respectively. Results from fig. 2 and table 4 show that the averaged TCE concentrations in BSW, W1, W2 and W3 were 179, 180, 168 and 157 g/L, respectively during the stage 2 operational period. Results indicate that approximately 13, 12, 11 and 9% of TCE were removed through the stage 2 operation. Results show that only slight TCE removal was observed in BSW in stage 2. This reveals that nutrient and air injection could not effectively enhance the TCE bioremediation.
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Results from table 4 and fig. 3 indicate that averaged COD concentrations were below 7.5 mg/L in the contaminated area. The low COD concentration would become the limiting factor for the occurrence of aerobic TCE co-metabolism. The averaged total viable bacterial counts in stage 2 varied from 1.8 103 CFU/mL in W3 to 9.1 104 CFU/mL in BSW (Fig. 5). The low total bacterial counts and low CO2 production indicate that the enhanced microbial activity was low and this could be due to the low biodegradable substrate in the subsurface. In BSW, the averaged TCE concentrations dropped from 207 g/L in stage 1 to 179 g/L in stage 2 after the operation of biosparging system (Tables 3 and 4). However, no significant variations in TCE concentrations were observed in the downgradient monitor wells. In the biosparging process, air was injected at a relatively low rate compared to the traditional air sparging process which applied a higher air injection rate. Thus, only 14% of TCE was removed in BSW from stage 1 to stage 2. TCE removal trend from stage 1 indicates that intrinsic TCE co-metablism was insignificant due to the limited primary substrate in the subsurface. Thus, TCE removal in BSW was mainly due to the vaporization process. Natural organic contents and inorganic compounds (e.g. ammonia) might be consumed by native bacteria for carbon and energy sources respectively which caused the increase in total bacterial counts. Results from the stage 2 operation reveal that the natural occurring organic carbon was not significant enough to enhance the in situ co-metabolic mechanism with the air supplement process (biosparging). Thus, primary substrate injection was a necessity to enhance the aerobic co-metabolism for in situ TCE bioremediation. Stage 3 operation: The stage 3 operation was applied to enhance the aerobic co-metabolism for TCE biodegradation. In stage 3, brown sugar (3 kg for each injection) was injected in BSW for primary substrate supplement and air and nutrients (same as stage 2) were supplied through the biosparging system which was operated since the stage 2 operation. Table 5 shows the averages of groundwater analytical results during the stage 3 operation. Results show that the pH, DO and ORP varied from 6.5 to 7.0, 1.3 to 2.9 mg/L and 155 to 245 mv respectively, within the TCE plume. The pH, DO and ORP values in BW had no significant changes compared to the results measured in stages 1 and 2. Results reveal that the groundwater was in oxidative conditions in both contaminated and uncontaminated areas. Compared to the pH, DO and ORP values in stage 2, slightly decrease in pH, DO and ORP were observed in BSW in stage 3. This reveals that the occurrence of aerobic biodegradation caused the drops of the three indicating parameters. Similar to the stage 2, because nutrients (ammonia and phosphate) addition was performed monthly, nutrient concentrations remained high compared to

Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
the concentrations measured in stage 1. No significant variations in nitrate, sulfate, methane and ferrous iron concentrations were observed compared to the measurements in stage 2. This might be due to the fact that aerobic biodegradation process was the dominant degradation process. The variations in TCE concentrations, COD concentrations and total viable bacterial counts in BSW and three monitor wells (W1 to W3) during the stage 3 operational period are presented in fig. 2 to 4 respectively. Results from fig. 2 and table 5 show that the averaged TCE concentrations in BSW, W1, W2 and W3 were 15, 34, 89 and 116 g/L respectively during the stage 3 operational period. Results indicate that approximately 96, 90, 73 and 38% of TCE were removed from stage 3 operation only. However, approximately 97, 92, 75 and 45% of TCE removal was observed for the three-stage of operation from Day 0 to Day 250. No significant difference of the TCE removal was observed between the stage 3 and three-stage operation. This indicates that the major TCE removal was due to the carbon injection process in stage 3. The averaged COD concentrations in BSW, W1, W2 and W3 were 167, 73, 16 and 9 mg/L respectively during the stage 3 operational period. In BSW, the TCE concentrations dropped to 38 g/L after 10 days of operation. The COD concentrations in BSW samples jumped up to 258 mg/L after 19 days of operation. This indicates that the injection of brown sugar caused the increase in COD concentrations and the added brown sugar was used as the primary substrate and energy source for the enhancement of TCE co-metabolism which resulted in TCE biodegradation. In BW, no significant variations in COD concentrations were observed. Compared to the DO concentrations observed in BSW during the stage 2 operation, drops of DO and ORP were detected which were due to the aerobic biodegradation of released carbon substrate from brown sugar. The averaged total viable bacterial counts in BSW, W1, W2 and W3 were approximately 6.7 108, 1.1 107, 4.5 105 and 4.3 104 CFU/mL respectively (Fig. 5). The produced CO2 in BSW and W1 during the stage 3 period were approximately 181 and 167 mg/L respectively. The high total bacterial counts and CO2 production in BSW and W1 indicate that the injected carbon and air sources activate the microbial activity and increase the microbial population which enhanced the aerobic co-metabolism of TCE in around the injection area. Compared to BW, only slight increase in COD concentration was observed in W2 and W3. This indicates that the injection of brown sugar caused the increase in COD concentrations in BSW and also transported to the downgradient wells (W2 and W3). However, due to the slow groundwater flow velocity (approximately 9.1 cm/d) and longer distance from BSW to W3 (15 m), lower COD increase in W3 was observed which would result in lower TCE co-metabolic rate. This caused the slightly drop of TCE
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concentrations in W3 compared to the TCE results in stage 2. In BW, no TCE was detected and no significant variations in COD and microbial population were observed during the treatment period from stage 1 to stage 3. Results show that significant TCE removal was observed in BSW indicating that high concentrations of COD and DO played important roles in TCE co-metabolism. However, less TCE removal efficiency was observed in W3. This could be due to the lower COD concentrations which could not cause significant proliferation of microbial populations for subsequent TCE-degrading enzymes. The aerobic TCE co-metabolism could be confirmed by the following investigations: (1) decrease in TCE concentrations along the transport path from BSW to W3; (2) increase in COD concentrations and microbial populations along the transport path from BSW to W3; (3) depletion of DO and decrease in BSW and W1 compared to the results in stage 2; and (4) production of CO2 and decrease in pH value in BSW and W1. Gene analysis for the screening of TCE-degrading enzymes: Figure 5 presents the gel showing the PCR-amplified fragments (206 bp) produced using the PHE primer on DNA extracted from the groundwater samples of the TCE-spill site. Results indicate that except for W3, all five groundwater samples (BW, BSW, W1 and W2) contained phenol monooxygenase in stage 1 (day 50) process before the start of nutrient or air/substrate injection. The observed phenol monooxygenase reveals that the enhanced in situ aerobic co-metabolism of TCE is feasible at this site. Results indicate that the phenol monooxygenase was not evenly distributed in the entire site. Variations in environmental conditions (e.g. natural organic substrate concentration, contaminant concentration, oxidation-reduction trend) would cause the change in the appearance of specific TCE-degrading enzymes. Results from fig. 5 also show that phenol monooxygenase was detected in all four groundwater samples (BSW, W1, W2 and W3) inside the plume after the injection of nutrients in stage 2 process on day 130. This indicates that the nutrients and air injection caused the increase in microbial population (Fig. 4) including the bacteria, which could produce phenol monooxygenase15. Figure 5 also shows that phenol monooxygenase was observed in all four groundwater samples (BSW, W1, W2 and W3) during the stage 3 treatment on days 200 and 250. This indicates that phenol monooxygenase remained inside the plume area during the aerobic co-metabolism treatment process and phenol monooxygenase might play a role in the TCE biodegradation during the stage 3 process. Figure 6 presents the gel showing the PCR -amplified fragments (466 bp) produced using the RMO primer on DNA extracted from the groundwater samples of the TCE-spill site. Results indicate that among the five groundwater samples, only W1 and W2 samples contained

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toluene monooxygenase during the stage 1 intrinsic bioremediation process before the start of nutrient or air/substrate. This indicates that toluene monooxygenase was not evenly distributed in the subsurface. The observed toluene monooxygenase also reveals that the enhanced in situ aerobic co-metabolism of TCE is feasible at this site. Toluene monooxygenase was detected in all four groundwater samples (BSW, W1, W2 and W3) inside the plume after the injection of nutrients and air in stage 2 process on day 130. This indicates that the nutrients injection caused the increase in microbial population (Fig. 4) including the specific bacteria e.g. Pseudomonas mendocina KR1, Ralstonia pickettii PKO118, which could produce toluene monooxygenase. During the stage 3 treatment, toluene monooxygenase was also observed in all four groundwater samples (BSW, W1, W2 and W3) on days 200 and 250 after air and substrate injection. This indicates that toluene monooxygenase, which was distributed inside the plume might activate the aerobic co-metabolic process and cause the TCE biodegradation.

Table 1 The three-stage operation during the operational period Stage 1 2 3 Operational period Day 0 to day 79 Day 80 to day 139 Day 140 to day 250 Injection/Objective No injection/intrinsic bioremediation Air (BSW), nutrients (BSW)/aerobic bioremediation Air (BSW), brown sugar (BSW), nutrients (BSW)/aerobic cometabolism

Table 2 Primer sets of four TCE-degrading enzymes Sequence F:5-GTGCTGAC(C/G)AA(C/T)CTG(C/T)TGTTC R:5-CGCCAGAACCA(C/T)TT(A/G)TC F:5-TCTC(A/C/G)AGCAT(C/T)CAGAC(A/C/G)GA CG R:5-TT(G/T)TCGATGAT(C/G/T)AC(A/G)TCCCA A-189 Particulate methane F: 5-GGNGACTGGGACTTCTGG A-682 monooxygenase19 R:5-GAASGCNGAGAAGAASGC TOD-F Toluene dioxygenase15 F:5-TGAGGCTGAAACTTTACGTAGA TOD-R R:5-CTCACCTGGAGTTGCGTAC a Forward (-F) and reverse (-R) primers are indicated. Table 3 Averages of analytical results for BW, BSW and monitor wells during the stage 1 operational period
Parameters BW BSW W1 W2 W3 pH 7.2 6.9 7.1 6.9 7.2 DO mg/L 1.1 0.7 0.8 1.0 0.9 ORP mv 132 71 98 127 118 sulfate mg/L 35 29 36 31 34 CO2 mg/L 138 142 159 143 133 Fe2+ mg/L < 0.1 0.2 < 0.1 < 0.1 < 0.1 nitrate mg/L 2.1 1.5 2.3 1.8 2.0 ammonia mg/L 1.7 1.2 1.1 1.3 0.9 phosphate mg/L 0.15 0.08 0.17 0.25 0.29 COD mg/L 7 5 5 6 7 bacterial count CFU/mL 7.2 103 5.1 103 1.9 103 3.4 103 9.1 102 TCE g/L BDL 207 195 178 166

Primera PHE-F PHE-R RMO-F RMO-R

Target Phenol monooxygenase15 Toluene monooxygenase9

Methane < 0.01 mg/L in all five wells; BDL: below detection limit. Table 4 Averages of analytical results for BW, BSW and monitor wells during the stage 2 operational period
Parameters BW BSW W1 W2 W3 pH 7.1 6.7 7.0 7.1 7.2 DO mg/L 1.9 6.5 3.4 1.9 1.2 ORP mv 121 305 211 165 127 sulfate mg/L 28 49 41 30 27 CO2 mg/L 128 181 167 159 145 Fe2+ mg/L < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 nitrate mg/L 1.8 1.2 1.1 1.6 1.3 ammonia mg/L 2.3 13.5 6.2 3.8 2.6 phosphate mg/L 0.08 1.8 1.07 0.55 0.33 COD mg/L 6 8 6 5 4 bacterial count CFU/mL 7.2 103 9.1 104 1.6 104 8.2 103 1.9 103 TCE g/L BDL 179 180 168 158

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Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
Methane < 0.01 mg/L in all five wells; BDL: Below detection limit. Table 5 Averages of analytical results for BW, BSW and monitor wells during the stage 3 operational period
Parameters BW BSW W1 W2 W3 pH 7.1 6.5 6.7 6.7 7.0 DO mg/L 1.5 2.9 2.1 1.7 1.3 ORP mv 142 245 199 167 155 sulfate mg/L 21 39 32 28 24 CO2 mg/L 121 269 221 189 177 Fe2+ mg/L < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 nitrate mg/L 1.2 1.0 1.1 1.2 1.2 ammonia mg/L 2.4 9.2 4.9 3.5 2.6 phosphate mg/L 0.06 1.2 1.0 0.5 0.3 COD mg/L 5 167 73 16 9 bacterial count CFU/mL 6.2 103 6.7 108 1.1 107 4.5 105 4.3 104 TCE g/L BDL 15 34 89 116

Methane < 0.01 mg/L in all five wells; BDL: below detection limit.

X BW

Upgradient Area Of the plume + BSW W1 W2 W3

Ground Water Flow

Estimated Plume Boundary W 1 : monitor well X BW: background well + BSW: biosparging and substrate injection well

Scale

Fig. 1: Site map showing the locations of representative monitor wells, air and substrate injection well and groundwater flow direction Figure 7 presents the gel showing the PCRamplified fragments (757 bp) produced using the TOD primer on DNA extracted from the groundwater samples of the TCE-spill site. Results indicate that none of the five samples (BW, BSW, W1, W2 and W3) contained significant amounts of toluene dioxygenase. This indicates that toluene dioxygenase was not significant at this TCE-spill site. During the stage 2 treatment process, toluene dioxygenase was not observed on day 130 although the microbial population were increased after air and nutrient injection. This might be due to the fact that site groundwater contained fewer microbial populations that could produce toluene dioxygenase. During the stage 3 treatment process, toluene dioxygenase was detected in all four samples (BSW, W1, W2 and W3) on days 200 and 250. This reveals that with the
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supplement of primary substrates, the production of specific microbial species and their associated TCE-degrading enzymes might occur, which caused the enhanced TCE co-metabolism. Results from fig. 7 also indicate that the increase in total microbial population does not necessarily correspond with the increase in specific bacteria e.g. Pseudomonas putida F116 for TCE-degrading enzyme production. Thus, microbial population could not be used as an indicator for the occurrence of TCE aerobic co-metabolism. Figure 8 presents the gel showing the PCR-amplified fragments (525 bp) produced using the A primer on DNA extracted from the groundwater samples of the TCE-spill site. Results indicate that none of the five samples (BW, BSW, W1, W2 and W3) contained significant amounts of pMMO on day 50 during the stage 1 operation.

Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
The production of pMMO was positively related to methane concentrations. Because the site groundwater was not under strictly anaerobic conditions, no methane was detected in groundwater samples (data not shown)17,19 and this caused insignificant pMMO production at this site. During the later part of stage 1, samples were collected on day 75 before the start of stage 2 treatment process. Results show that pMMO was observed on day 75 in BSW and W1. This might be due to the fact that pMMO was produced by some specific bacteria around the injection well due to the decrease in DO in the upgradient area of the plume. During the stage 2 treatment process, pMMO was slightly detected on day 130 in BSW and W1. The observed pMMO indicates that this TCE-degrading enzyme was decaying and only slight residual pMMO remained in the plume area after the start of stage 2 process. During the stage 3 treatment process, pMMO was not detected on days 200 and 250 from all groundwater samples. This reveals that with the injection of air into the subsurface, the production of specific microbial species and their associated pMMO might be inhibited due to the increase in DO in the plume. Results from fig. 8 indicate that the detection of pMMO can be used as an indicator of status of oxidation-reduction state in the subsurface. Under aerobic co-metabolic process, pMMO might not be detected within the aerobic treatment zone.
225 200 TCE concentration (ug/L) TCE concentration (ug/L) 175 150 125 100 75 50 25 0 0 25 50 75 100 125 150 175 200 225 250
IW MW-2 MW-1 MW-3

In this study, the effectiveness of aerobic TCE co-metabolism on TCE concentration reduction with the injection of nutrients, air and brown sugar was evaluated within the test area of a TCE-spill site. Conclusions of this pilot-scale study include the following: (1) Aerobic co-metabolism was the major cause of the decrease in TCE concentrations in groundwater in this study. Without primary substrate supplement, intrinsic bioremediation and injection of air and nutrients alone could not enhance the aerobic co-metabolic mechanism and cause the decrease in TCE concentrations within the plume. (2) The effectiveness of air sparging and substrate injection and occurrence of aerobic TCE co-metabolism could be confirmed by the following investigations within the plume: (a) significant decrease in TCE concentrations; (b) increase in COD concentrations and microbial populations; (c) increase in DO and ORP in the air sparging well; (d) significant depletion of DO and decrease in ORP within the TCE plume after the supplement of brown sugar; (e) production of CO2 and decrease in pH value and (f) detected specific TCE-degrading enzymes. Field results reveal that the operation of biosparging caused the shifting of low oxygen conditions inside the plume to aerobic conditions. (3) TCE-degrading enzymes, including toluene monooxygenase, toluene dioxygenase, phenol monooxygenase and pMMO were identified in field groundwater samples. This indicates that in situ enhanced bioremediation is a feasible technology for site groundwater remediation. (4) Identification of TCE-degrading enzymes using gene analysis is a useful tool to evaluate the feasibility of applying enhanced in situ bioremediation for TCE removal.
400 CODconcentration concentration (mg/L) COD (mg/L) 350 300 250 200 150 100 50 0 0 25 50 75 100 125
Day Day IW MW-2 MW-1 MW-3

Day Day

Fig. 2: Variations in TCE concentrations in BSW and three monitor wells (W1 to W3) during the three-stage operational period Because the detected phenol monooxygenase, toluene monooxygenase and toluene dioxygenase are able to activate the aerobic cometabolism of TCE, the observed specific enzymes at this site imply that in situ enhanced bioremediation is a feasible technology for site groundwater remediation. The results also indicate that a thorough gene analysis is necessary before in situ bioremediation is applied to site remediation. Gene analysis would be helpful in determining if the TCE-degrading enzymes exist at the site and if in situ bioremediation is a potential remedial option.

150

175

200

225

250

Fig. 3: Variations in COD concentrations in BSW and three monitor wells (W1 to W3) during the three-stage operational period

Acknowledgement
This project was funded in part by National Science Council in Taiwan. The authors would like to thank the personnel at Department of Biological Sciences of National
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Conclusion

Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
Sun Yat-Sen University and Guan Cheng Environment Technology Protection Co. Ltd. for their assistance and support throughout this project.
Total Total viable bacteria (CFU/mL) viable bacteria (CFU/mL)
1.E+10 1.E+09 1.E+08 1.E+07 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 0 25 50 75 100 125
IW MW-2 MW-1 MW-3

150

175

200

225

250

Day Day

Fig. 4: Variations in total viable bacterial counts in BSW and three monitor wells (W1 to W3) during the three-stage operational period

TCE-spill site Fig. 6: Gel showing the PCR-amplified fragments (466 bp) produced using the RMO primer on DNA extracted from the groundwater samples of the TCE-spill site

References
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Fig. 5: Gel showing the PCR-amplified fragments (206 bp) produced using the PHE primer on DNA extracted from the groundwater samples of the
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4. Han Y.L., Kuo M.C., Tseng I.C. and Lu C.J., Semicontinuous microcosm study of aerobic co-metabolism of trichloroethylene using toluene, J. Hazard. Mater, 148, 583-591 (2009)

Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.

200 day

50 day

130 day

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75 day

130 day

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250 day

Fig. 7: Gel showing the PCR-amplified fragments (757 bp) produced using the TOD primer on DNA extracted from the groundwater samples of the TCE-spill site
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Fig. 8: Gel showing the PCR-amplified fragments (525 bp) produced using the A primer on DNA extracted from the groundwater samples of the TCE-spill site
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Research Journal of Chemistry and Environment____________________________________Vol.16 (2) June (2012) Res. J. Chem. Environ.
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