Abstract: Performance of oxygen-carbon-inducer releasing materials (OCIRM) on co-metabolize TCE and degradation of its metabolites were studied. Potential of the orange peel as the inducer was evaluated as compared to limonene solution. The experiment was conducted in batch mode to determine the optimum size of OCIRM and suitable carbon source. Results showed a 1.0 cm3 cube was the optimal size and sugarcane bagasse was a suitable C source in which the maximum organic releasing rate of 0.30 mg COD day-1 g-1 cube and dissolved oxygen concentration of 11.95 mg O2 L-1 were generated. Limonene in orange peel and in solution successfully induced monooxygenase enzyme to co-metabolize TCE. Optimal concentration ratio of contaminant to limonene was 1:2.5. OCIRM containing sugarcane bagasse and limonene in orange peel using a biofilm of aerobic sludge as the inoculum effectively co-metabolize TCE, cis-DCE and VC with maximum percentage removal of 56, 61 and 72%, respectively.
INTRODUCTION
Chlorinated aliphatic hydrocarbons, especially trichloroethylene (TCE), are the most widespread contaminants in groundwater. TCE has been a concern due to its toxicity, especially carcinogenic effect and high persistence in the environment. Under anaerobic condition, such as in groundwater, TCE could be degraded via reductive dechlorination process resulting in the metabolites named cis-1,2-dichloroethylene (cis-DCE) and Vinyl Chloride (VC) which have the higher toxicity than TCE. Due to the slow degradation rate under anaerobic condition, cis-DCE and VC have been accumulated and detected in many TCE contaminated sites (Broholm et al., 2005; Shen and Sewell, 2005). Therefore, the interests have been paid to the aerobic bioremediation which is effective in degradation of TCE and its metabolites especially cis-DCE (Olaniran et al., 2008).
Under aerobic condition, the biodegradation of TCE and cis-DCE could occur via co-metabolism process in which the primary substrate mainly toluene and phenol are used as their growth substrates and act as the inducer of monooxygenase enzyme capable of co-metabolic oxidation of TCE and its metabolites (Ferhan, 2003; Kim et al., 2008). However, toluene and phenol are toxic to the environment. Therefore, there are the attempts to search for the plant terpenes such as cumene, limonene, carvone and pinene which have analogous structures to toluene and phenol for inducing microbial degradation of TCE (Singer et al., 2003; Suttinun et al., 2004, 2009). In this study, orange peel was used to induce the production of monooxygenase enzyme in microorganisms since it composes of limonene (>90%) that might effectively induce the cometabolism degradation of TCE (Raeissi and Peters, 2004).
In order to sufficiently supply the primary substrate and oxygen throughout the in situ remediation process of TCE in groundwater, researchers have developed the organic-oxygen- releasing materials containing different sources of oxygen and/or primary substrates to be used as the passive oxygen and primary substrate supplier during TCE remediation in groundwater (Borden et al., 1997). The results clearly demonstrated that with the presence of these slow release materials, the aerobic co-metabolism of TCE and its metabolites could be efficient for long term of remediation process and the contaminant plume in groundwater could be effectively confined (Borden et al., 1997; Kao et al., 2001).
The objective of this study is to construct the oxygen-carbon-inducer-releasing material (OCIRM) and evaluated its performance for co-metabolism of TCE and degradation of its metabolites. The OCIRM contained CaO2, sugarcane bagasse or compost, limonene as the oxygen, carbon and inducer sources, respectively. It is hoping that the OCIRM might be able to continuously release oxygen, carbon and limonene when contact with groundwater to stimulate and induce the microorganisms to completely degrade TCE contamination in groundwater.
MATERIALS AND METHODS
Research location: Construction of the OCIRM and evaluation of its performance for co-metabolism of TCE were carried out at Department of Biotechnology, Faculty of Technology, Khon Kaen University, Thailand. Study on the performance of OCIRM on degradation of cis-DCE and VC was carried out at Department of Environmental Engineering and Science, Chia Nan University of Pharmacy and Science, Tainan, 71710, Taiwan, Republic of China. This study was conducted between June, 2008-April, 2009.
Chemicals: TCE, 98%, was purchased from Riedel-deHa¸n, Germany. cis-DCE, 97%, was purchased from Sigma Aldrich Chemical, USA VC, 2000 mg L-1 in methanol, was purchased from Chem Service, USA R-(+)-limonene, 98%, was purchased from Fluka, Switzerland. All other chemicals and reagents are analytical grade and purchased from BDH, England.
Synthetic groundwater: Synthetic groundwater consisted of (mg L-1 ): KH2PO4, 326.4; Na2HPO4, 1263.8; Mg2SO4.7H2O, 98.6; CaCl2.2H2O, 44.1; NH4Cl, 10.7 and trace elements including FeSO4.7H2O, 1; MnSO4.4H2O, 1; (NH4)6Mo7O24.4H2O, 0.25; Na2B4O7.10H2O, 0.25; CoCl2.6H2O, 0.25; CuCl2.2H2O, 0.25; ZnCl2, 0.25; NH4VO3, 0.1. The pH of the synthetic groundwater was 7.5 (Kao et al., 2001).
Potential inducer: Orange peel, the waste from orange juice drink shop, was used as potential inducer for co-metabolism of TCE and cis-DCE by Rhodococcus gordoniae P3 and aerobic sludge in comparison to limonene solution. It was dried at room temperature, chopped into small pieces, passed through 2 mm sieve and kept at -20°C prior the usage. One gram dry weight of orange peel contains approximately 13 mg of limonene determined by the method followed by Chotratanadilok et al. (2002).
Organic amendment: Compost and sugarcane bagasse were used as carbon source in this experiment. Compost was obtained from Faculty of Agriculture, Khon Kaen University. It was composted from yard waste. The compost sample was pulverized and passed through a 2 mm sieve. It consists of 0.76% total N and 7.44% organic carbon.
Sugarcane bagasse, the fibrous residue of sugarcane stalk which remain after the juice was extracted, was collected from sugar factory, Mitara Phoo Weang Co Ltd., Khon Kaen Province. Before the usage, sugarcane bagasse was dried at 60°C, milled and sieved through a 2 mm sieve. The dry milled sugarcane bagasse consists of 0.19% total N and 49.93% organic carbon.
Support material: Corncob was used as the support material to immobilize the microorganisms. It was obtained from Faculty of Agriculture, Khon Kaen University, Thailand. Organic carbon and total nitrogen of corncob were 45.84 and 0.23%, respectively. It was shredded by knife into small pieces (approximately 0.5x0.5x0.5 cm). After that, it was delignified by boiling in 1% NaOH for 3 h to remove lignin which might be toxic to microorganisms and then thoroughly washed under tap water and soaked in distilled water overnight (Iconomou et al., 1995). This process was done 2 times and kept at -20°C prior the usage.
Inoculum
Pure culture: TCE degrader, R. gordoniae P3 (GenBank accession
number EF450777), a gram positive aerobic bacteria, was isolated from petroleum-contaminated
soil collected from Bangkok area. It was kindly provided by Dr. Ekawan Luepromchai,
Department of Microbiology, Chulalongkorn University, Thailand.
Aerobic sludge: Aerobic sludge was collected from wastewater treatment plant of Lardkabang Industrial Sector, Bangkok, Thailand. Wastewater treated at this wastewater treatment plant is from electronic part industries where TCE and toluene are used as solvent. Aerobic sludge was acclimatized with 10 mg L-1 TCE and 10 mg L-1 limonene solution, shaken at 150 rpm and incubated at the room temperature for 21 days (Ozbelge et al., 2007) to enhance TCE degradation ability of the microorganisms in sludge. After acclimatization, the aerobic sludge was harvested by centrifugation at 6,000 rpm for 10 min and the pellets were re-suspended in synthetic groundwater before using as the seed inoculum.
Immobilization of R. gordoniae P3 on corncob: Cell immobilization by adsorption mechanism was conducted by adding 75 g of delignified corncob into 300 mL Minimal Salt Medium (MSM) (Focht, 1994) and autoclaved at 121°C for 15 min twice before inoculating with 10% (V/V) of R. gordoniae P3 (106 cfu mL-1 ) or aerobic sludge (20,600 mg VSS L-1 ). Then, 172 mg L-1 of toluene, a primary substrate, was added into the bottle before incubating at room temperature, shaken at 200 rpm on orbital shaker for 24 h. After that, the immobilized cells were transferred to a fresh MSM containing 172 mg L-1 toluene and incubated as described previously for 2 times before harvesting by filtration through Buchner filter funnel. The immobilized cells on corncob were washed twice with 0.85% NaCl using aseptic technique. The immobilized cells were furthered cultivated in MSM containing 10 mg L-1 limonene for 7 days, shaken at 200 rpm on orbital shaker, at room temperature. Immobilized cells were harvested and washed as previously described then kept at 4°C until the usage. The internal cell density on the corncob was approximately 106 cells g-1 dry wt of support materials determined by viable plate count technique.
Biofilm: Biofilm of R. gordoniae P3 and aerobic sludge was developed by coating the inoculum on a 1.0 cm3 cube oxygen-carbon-inducer-releasing material (OCIRM) using the method adapted from the protocol of Tremoulet et al. (2002). Briefly, five gram of 1.0 cm3 cube OCRM was placed in a 50 mL serum bottle containing 20 mL of synthetic groundwater. The bottles were inoculated with 10% inoculum (R. gordoniae P3 (106 cfu mL-1) or aerobic sludge (20,600 mg VSS L-1) and further incubated for 3 weeks at room temperature. After 3 weeks, the synthetic groundwater was removed and biofilm was used in the experiment. Extraction and quantification of bacteria in biofilm was conducted following the method of Kadurugamuwa et al. (2003).
Oxygen-carbon-releasing material (OCRM): The purpose of OCRM was to continuously supply the dissolved oxygen (DO) and carbon for microorganisms to use in co-metabolism of the contaminants. It was comprised of binding cement, calcium peroxide (CaO2), sand, carbon source (compost or sugarcane bagasse), fly ash and water at the ratio of 1.5:1.5:0.2:0.2:1.3:2 by weight (modified from Kao et al., 2001). When the inducer (limonene in orange peel or limonene solution) was added into the OCRM, it was called the Oxygen-Carbon-Inducer-Releasing Material (OCIRM).
Effect of OCRM size on supplying dissolved oxygen and carbon: Various sizes of OCRM of 1x1x1, 2x2x2 and 3x3x3 cm3 cube were evaluated in order to test the effect of OCRM size on supplying dissolved oxygen and carbon over time. Sugarcane bagasse and compost were assessed its effectiveness as carbon source. Inducer was not added. The experiment was conducted in a 1 L enclosed glass bottle containing 125 g of cube and 250 mL of DI water. Cube without CaO2 and carbon source was used as a control. Water samples were taken every day for 100 days and analyzed for the Dissolved Oxygen (DO) and COD. DO was measured by DO meter (DO 551, Digital, Japan). COD analysis was conducted by dichromate reflux method (APHA, 1992; Nkegbe et al., 2005).
Effect of concentration ratio of TCE to limonene on co-metabolism efficiency of TCE by R. gordoniae P3 and aerobic sludge: The effect of concentration ratio of TCE to limonene on the co-metabolism efficiency of TCE by R. gordoniae P3 and aerobic sludge was investigated. In this experiment, limonene in orange peel and limonene solution were added into OCIRM as the inducers at various concentrations ratio of TCE to limonene of 1:0.5, 1:1, 1:2.5 and 1:5. A suitable carbon source was added into the OCIRM. Experiment was conducted in a 50 mL serum bottle containing 20 mL synthetic groundwater, 10 mg L-1 TCE, 5 g OCIRM and 106 cfu mL-1 of inoculum (R. gordoniae P3 or aerobic sludge). The bottle was tightly sealed with Teflon-lined rubber septa and aluminum cap to prevent the leakage of TCE into the air. Controls were non-inoculated and inoculated microcosms without OCIRM. Water samples were taken at day 0, 5, 7, 14, 21 and 35 to analyze for the TCE residues by GC-FID.
Effect of inoculum form on co-metabolism of TCE: Efficiency of various forms of aerobic sludge and R. gordoniae P3 i.e., free cell, immobilized cell on corn cob and biofilm coated on OCIRM to co-metabolism TCE were conducted. Suitable concentration ratio of TCE to limonene and suitable carbon source were added into the OCIRM. Microcosms were constructed in a similar manner as previous experiment except the inoculum was added in various forms.
Biodegradation of cis-DCE, VC and a combination of cis-DCE and VC: Co-metabolism of cis-DCE, VC and a combination of cis-DCE and VC by the most effective type and form of inoculum were conducted. Suitable concentration ratio of TCE to limonene and suitable carbon source were added into the OCIRM. Microcosms were constructed in a similar manner as previous experiment except the synthetic groundwater contained 10 mg L-1 cis-DCE or 10 mg L-1 VC or a combination of 10 mg L-1 cis-DCE and 10 mg L-1 VC. The samples were sacrificed at day 0, 5, 7, 14 and 21 and analyzed for cis-DCE and VC by GC-FID and chloride ion by IC.
Enumeration of toluene degrading bacteria: One gram wet weight of immobilized support material was blended by blender into small particles under aseptic condition. Then, serial 10-fold dilutions of each suspension were plated on Minimal Salt Agar (MSA) that coated with 10 mg L-1 of the contaminants and incubated at room temperature in the box fumigated with toluene as a primary substrate for one week. The number of colony forming unit (cfu) between 30-300 colonies in each plate were counted.
Analytical method: TCE, cis-DCE and VC concentrations in the synthetic groundwater were measured by analyzing 50 μL headspace sample on Agilent 4820D gas chromatography equipped with flame ionization detector (GC-FID) and Rtx-624 capillary column (30x0.53 mm ID; 3 μm). The GC oven temperature of 60°C was held for 6 min and then ramped at 8°C min-1 to 200°C where the oven was held for 1 min. Percentage of recovery for this analyzing method is 87%. The detection limit of GC-FID is 0.1 mg L-1 for TCE, cis-DCE and VC.
Anions i.e., bromide and chloride were determined by Ion Chromatograph (Dionex DX-120) equipped with RFC-30 EGCII (KOH), Autosampler Thermo Finnigan Spectra SYSTEM model AS 1000 with 20 μL injection volume, IonPac®AG11 guard column (4x50 mm), IonPac®AS11 analytical column (4x250 mm), ASRS®-ULTRA II (4 mm) suppressor and conductivity detector. The column temperature was controlled at 30°C. IC was operated at the flow rate of 1.0 mL min-1 with gradient of 0.5 mM KOH 0-4 min, 0.5-35 mM KOH 4-22 min, 18 mM KOH 22-26 min and 0.5 mM KOH 26-30 min.
RESULTS AND DISCUSSIONS
Performance of OCRM: The carbon and oxygen released from the OCRM into the DI water were determined as COD and DO, respectively. The results showed that the overall COD releasing rate of each carbon source at all sizes dropped rapidly within 12 days and then stable after day 15 (Fig. 1). Every size of the OCRM containing sugarcane bagasse gave COD releasing rates higher than that of compost (Fig. 1a).
| Fig. 1: | (a) COD releasing rate from oxygen releasing material and (b) DO concentration from oxygen releasing material |
However, there was no significantly different on COD releasing rates obtained among the size of the OCRM containing sugarcane bagasse or compost. The maximum COD releasing rates of 0.30 and 0.12 mg COD day-1 g-1 cube were obtained from a 1.0 cm3 of OCRM containing sugarcane bagasse and compost, respectively.
Size and carbon source in the OCRM did not affect DO concentration (Fig. 1b). The average maximum oxygen concentrations over 100 days were 13.55 mg O2 L-1 and 11.95 mg O2 L-1 obtained from a 1.0 cm3 cube OCRM containing compost and sugarcane bagasse as C source, respectively.
Since the results indicated that a 1.0 cm3 cube with sugarcane bagasse as a C source showed a maximum COD releasing rates and DO concentration, therefore a 1.0 cm3 was used in a further experiment to supply oxygen and carbon.
Effect of concentration ratio of TCE to limonene on degradation efficiency of TCE: In abiotic control (Table 1) (treatment with no inoculum and inducer), 15% of TCE was dissipated which might be the result from volatilization of TCE or sorption of TCE on OCIRM.
| Table 1: | Percentage of TCE removal using different concentration ratios of limonene and concentration of TCE at day 35 |
| *Comparisons between treatments within each column are significantly different (LSD, p<0.05) if marked with different small letters. Comparisons between treatments within each row are significantly different (LSD, p<0.05) if marked with different capital letters | |
Without the presence of OCIRM, the addition of R. gordoniae P3 or aerobic sludge did not improve the efficiency of TCE removal which indicated that microorganisms needed oxygen, carbon and especially inducer to degrade TCE under aerobic condition. In contrast, it was obviously seen that limonene in orange peel and limonene solution had significantly improved the degradation of TCE by R. gordoniae P3 and aerobic sludge with the removal efficiency in the range of 18.03 to 42.31% (Table 1). The results clearly demonstrated that limonene in orange peel and limonene solution has a potential to be used as the inducer for aerobic co-metabolism of TCE.
The effect of concentration ratio of TCE to limonene in orange peel on TCE removal by R. gordoniae P3 was not significant while the highest percentage of TCE removal (31.03%) was obtained when limonene solution was used at the ratio of TCE to limonene solution of 1:5 (Table 1).
Aerobic sludge could remove TCE more efficient than R. gordoniae P3. When the orange peel was used as the inducer, the percentages of TCE removal (23.21-23.25%) (Table 1) at the concentration ratio of TCE to limonene in orange peel of 1:0.5 and 1:1 were not significantly different. The increase in limonene concentration in orange peel to the ratio of 1:2.5 significantly improved the TCE degradation by aerobic sludge resulting in the increase in percentage of TCE removal to 33.58%. Further increase in the concentration ratio of TCE to limonene in orange peel to 1:5 did not affect the TCE degradation ability of aerobic sludge suggesting that the concentration ratio of TCE to limonene in orange peel of 1:2.5 was optimal for TCE degradation by aerobic sludge.
When limonene solution was used as the inducer, the optimum concentration ratio of TCE to limonene solution was 1:1 indicating by the percentage of TCE removal of 40.61 (Table 1). Increase in concentration of limonene solution to the ratio of 1:2.5 and 1:5 did not increase or worsen the efficiency of TCE removal by aerobic sludge.
Limonene solution gave a higher percentage of TCE removal compared to orange peel. These might be due to the fact that limonene in orange peel was inside the orange peel thus it was difficult for microorganisms to access in comparison to limonene solution. When considered that the orange peel is the waste and has no value, it can be a good inducer for TCE remediation process.
The induction of co-metabolic degradation of TCE by various microorganisms using plant limonene and limonene solution had been studied. Suttinun et al. (2004) reported that limonene solution was able to induce TCE co-metabolic pathway of R. gordoniae P3 resulting in the 10% higher percentage of TCE removal as compared to the treatment without inducer. In addition, lemon oil or lemon grass oil containing approximately 32% of limonene was able to increase TCE removal capability of Rhodococcus sp. L4 by 23% in comparison to the treatment without inducer (Suttinun et al., 2009).
Since the concentration ratio of TCE to limonene solution at 1:1 was not significantly different than other ratios and the optimum ratio of TCE to limonene in orange peel was 1:2.5, thus in order to be able to make a comparison between the efficiency of limonene source, the ratio of 1:2.5 was used in the further experiment.
Co-metabolism of TCE: This experiment examined the co-metabolism of TCE by R. gordoniae P3 and aerobic sludge in free cells, immobilized cells and biofilm forms using the optimum concentration ratio of TCE to limonene in orange peel and limonene solution of 1:2.5. Percentages of TCE removal after 35 days of incubation were shown in Table 2. When limonene in orange peel was used as the inducer, there was no significantly different among the TCE degradation efficiency of free cell, immobilized cell on corncob and biofilm of inocula (Table 2). The percentage of TCE removal by every forms of R. gordoniae P3 were in the range of 31.22-36.99, while the percentage of TCE removal by every forms of aerobic sludge were in the range of 38.93-40.41. Result implied that the efficiency of aerobic sludge to co-metabolite TCE is better than pure culture. The findings were similar to the study of Coyle et al. (1993) whom reported that 80-85% of TCE removal could be accomplished by mixed cultures which was greater than that of pure culture, Psuedomonas putida (55%) under aerobic condition using phenol as the primary substrate.
| Table 2: | Percentage of TCE removal at day 35 of incubation |
| *Comparisons between treatments within each column are significantly different (LSD, p<0.05) if marked with different letters | |
When limonene solution was used as the inducer, the TCE removal efficiency by free and immobilized cell forms of the inoculum was not significantly different. Seed inocula in biofilm form showed a better performance of TCE degradation as compared to free and immobilized cell forms with the highest percentage of TCE removal of 45.80 for R. gordoniae P3 and 55.89 for aerobic sludge (Table 2). The improvement of TCE degradation by biofilm might be because during the formation of biofilm, some of organic compound such as surfactants have also been produced, which could lead to more bioavailability and better biodegradation of TCE (Singh et al., 2006).
Number of TCE degraders i.e., R. gordoniae P3 and TCE degrader in aerobic sludge increased from approximately 105 cfu mL-1 to approximately 107 cfu mL-1 at 3 days of incubation (Fig. 2). After day 5, number of TCE degraders decreased continuously until the end of incubation. The decrease in number of TCE degraders might result in a decrease in TCE degradation efficiency.
pH of synthetic groundwater increased from 7.0 to 11.0 after 35 days of incubation (Fig. 3) which might inhibit the growth and cause the adverse effect to the TCE degrader. The increase in pH of synthetic groundwater during incubation was due to the release of OH- from binding cement. The use of the other binding compound which had no effect on pH or the addition of pH maintaining substances may aid the TCE removal ability of microorganisms.
As the result indicated that biofilm form of aerobic sludge is the most efficient inoculum for TCE removal from synthetic groundwater, it was further used to remediate cis-DCE, VC and a combination of cis-DCE and VC in the next experiment.
| Fig. 2: | Cell number of TCE degrader (a) R. gordoniae P3 and (b) aerobic sludge |
Biodegradation of cis-DCE, VC and a combination of cis-DCE and VC under aerobic condition: Biodegradation process of cis-DCE, VC and a combination of cis-DCE and VC by biofilm of aerobic sludge on OCIRM in comparison to free cell was studied. In the abiotic control, 28-34% of cis-DCE and VC were degraded but no chloride ions were generated which indicated that some amount of cis-DCE and VC were dissipated directly from synthetic groundwater via volatilization process.
The percentage of contaminants removal in the synthetic groundwater was shown in Table 3. The maximum percentage of cis-DCE removal of 61.36 was achieved when the biofilm was used as inoculum while the maximum percentage of cis-DCE removal was 50.65 when free cell was used, which confirmed that biofilm technique could improve the co-metabolism of TCE and cis-DCE.
The maximum percentage of VC removal by the free cell of aerobic sludge and biofilm were 60.69 and 71.89, respectively. Results indicated that the biofilm could degrade VC more efficient than the free cell. It was found that, there was no significantly different among the treatments that had the inducers (limonene from orange peel and limonene solution) and the treatments without inducer which implied that the degradation of VC is not from co-metabolism process which was in correlation to the results in the previous published reports (Freedman et al., 2001; Coleman et al., 2002; Elango et al., 2006).
| Fig. 3: | pH value in microcosm study versus time (a) R. gordoniae P3 and (b) aerobic sludge |
| Table 3: | Percentage of contaminants removal in synthetic groundwater containing acclimatized aerobic sludge with different types of organic releasing material after 21 day of incubation |
| Comparisons between treatments within each column are significantly different (LSD, p<0.05) if marked with different letters | |
Biofilm could degrade a combination of cis-DCE and VC solution in the synthetic groundwater better than free cell. The maximum percentage of cis-DCE and VC removal by biofilm in a combination of cis-DCE and VC solution were 66.14 and 75.67, respectively, which were higher than cis-DCE and VC removal by free cell (56.50 and 65.12%) (Table 3). Percentage of cis-DCE removal in a combination of cis-DCE and VC solution was higher than the percentage of cis-DCE in treatment of cis-DCE alone. Broholm et al. (2005) reported that the removal of cis-DCE occured concurrently with VC, suggesting that the biodegradation of cis-DCE depended on the biodegradation of VC.
cis-DCE and VC were dechlorinated by the inocula resulting in the increase of chloride ion during incubation (Fig. 4a). Chloride ion was found to increase rapidly from day 0 to 7, then gradually decreased. The same phenomena were also observed in a combination of cis-DCE and VC solution. The maximum percentage of chloride generated of 32.54, 39.22 and 34.75 were achieved when cis-DCE, VC and a combination of cis-DCE and VC solution were treated by biofilm when limonene solution was used as inducer.
The variation of pH and cell number of contaminant degraders in the microcosm were monitored as the important factors affecting the efficiency of cis-DCE and VC removal in the microcosm. Initially (day 0-7), pH in microcosm was in the range of 7.5 to 8.5 (Fig. 4b), after that pH increased to approximately 11.
| Fig. 4: | (a) Percentage of chloride generated and (b) pH in microcosm study |
| Fig. 5: | Cell number of contaminants degrader (a) cis-DCE (b) VC and (c) combination of cis-DCE and VC solution |
These results coincided with the cell number of contaminant degraders in microcosm which initially increased, during day 0-7, from 105 to 108 cfu mL-1 and then decreased over time (Fig. 5) which suggested that an increase in the pH in microcosms caused the adverse effect on the degraders.
CONCLUSIONS
This study successfully constructed the OCIRM to supply oxygen, carbon and inducer for the co-metabolism of TCE and degradation of its metabolites. The optimal size of the OCIRM was 1 cm3 cube. OCIRM containing sugarcane bagasse as a C source gave a maximum COD releasing rates and DO concentration of 0.30 mg COD day-1 g-1 cube and 11.95 mg O2 L-1 over 100 days. Limonene in orange peel and limonene solution successfully induce a co-metabolism of TCE and the optimum concentration ratio of TCE to limonene in orange peel and limonene solution was 1:2.5 and 1:1, respectively. Biofilm of R. gordoniae P3 and aerobic sludge coated on OCIRM could co-metabolize TCE better than free and immobilized cell forms. cis-DCE was effectively degraded by biofilm of aerobic sludge with the presence of limonene as the inducer while VC could be metabolized directly without the addition of inducer. The presence of VC in the system could enhance the degradation of cis-DCE by aerobic degrader via co-metabolism process.
ACKNOWLEDGMENTS
The authors would like to express our sincere gratitude to the Research Centre for Environmental and Hazardous Substance Management, Khon Kaen University, Thailand, Chia Nan University of Pharmacy and Science, Taiwan and Groundwater Research Center, Khon Kaen University, Thailand for funding and facilitating the research. We appreciated the travel funds from Rajamangala University and Technology-Isan -Kalasin Campus and the Graduate College, Khon Kaen University, for MT.