Chemosphere 75 (2009) 837842

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Chemosphere
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Technical Note

Removal of spilled petroleum in industrial soils by spent compost of mushroom Pleurotus pulmonarius
Siu-Wai Chiu *, Ting Gao, Cissy Sze-Sze Chan, Carmen Kar-Man Ho
Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China

a r t i c l e

i n f o

a b s t r a c t
Two batches of oil-contaminated soil collected from an industrial area and one pile of oil-contaminated soil in a power plant were treated by the spent compost of mushroom Pleurotus pulmonarius (SMC). SMC contained macronutrients for biostimulation, possessed 1.01.5 U mg1 laccase and 0.80.9 U mg1 manganese peroxidase for biodegradation and harboured (11 3) 107 cfu g1 bacteria and (56 9) 104 cfu g1 fungi for bioaugmentation. In off-site ex situ bioremediation, the industrial area soil was contaminated with organic 5.46.9 g kg1 total petroleum hydrocarbons (TPH), 14.519.0 g kg1 oil and grease and 9599 mg kg1 di(2-ethylhexyl) phthalate (DEHP) and inorganic 104136 mg kg1 Cu, 430691 mg kg1 Pb and 477578 mg kg1 Zn. The removal by 3% SMC amendment applied twice accounted for 5664%, 3133% and 5154% disappearance of the TPH, oil and grease and DEHP contaminants, respectively. For the latter soil, one 0.3% SMC application removed 4045% of the initial 1.2 0.2 g kg1 TPH and 4.0 0.6 g kg1 oil and grease in 22 d. Further using four bacteria and four fungi inoculated onto the sterilized soil samples, samples with greater removal of the pollutants bore larger microbial populations. Thus SMC simultaneously degrades petroleum residues and reduces toxicity in less than a month. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 18 August 2008 Received in revised form 17 December 2008 Accepted 17 December 2008 Available online 21 January 2009 Keywords: Spent mushroom compost (SMC) Total petroleum hydrocarbons (TPH) Di(2-ethylhexyl) phthalate (DEHP) Biodegradation Bioremediation Lignolytic enzymes

1. Introduction Oil and plasticizers are two major contaminants in contemporary world. Di(2-ethylhexyl) phthalate (di-2-ethylhexyl phthalate, bis(2)-ethylhexyl phthalate, DEHP), being the most commonly used plasticizer and in the priority list for treatment, is a suspected human carcinogen, a hepatic, reproductive and developmental toxicant and an endocrine disruptor (Moore et al., 2001; LovekampSwan and Davis, 2003; Hokanson et al., 2006). The widespread use of plastics and the persistent nature of plasticizers made domestic sewage sludge contaminated with 15346 mg kg1 DEHP residues (Alatriste-Mondragon et al., 2003; Beauchesne et al., 2008). Bioremediation, in terms of landfarming, biopiling, biostimulation by adding fertilizer or other amendments to boost up the microbial growth and metabolism, and/or bioaugmentation by enriching the biota, has successfully been applied (Mishra et al., 2001; Juteau et al., 2003; Rivera-Espinoza and Dendooven, 2004; Bento et al., 2005; Coulon et al., 2005; Di Gennaro et al., 2005; Baek et al., 2007; Fallgren and Jin, 2008; Machn-Ramrez et al., 2008). Yet improvement is needed for shortening the time and reducing the cost. The spent composts (SMCs) of mushrooms Agaricus and Pleurotus as wastes of mushroom industry, still con-

tain many residual enzymes, e.g. proteases, cellulases, hemicellulases, lignin peroxidase, manganese peroxidase and laccase (Lau et al., 2003). The last three enzymes belonging to lignolytic enzymes act as Fenton reagents to produce reactive radicals for non-specic cleavage of a wide variety of highly recalcitrant organopollutants (Chiu et al., 1998; Hestbjerg et al., 2003; Gong et al., 2006). Thus SMCs have been tested or applied to treat atrazine, creosote, pentachlorophenol and polycyclic aromatic hydrocarbons (PAHs) (Masaphy et al., 1996; Eggen, 1999; Lau et al., 2003; Law et al., 2003; Kadian et al., 2008). The present study examines the bioremediation of oil-contaminated industrial soils, one of which was also mixed polluted with DEHP and heavy metals, by SMC of Pleurotus pulmonarius. In addition to monitoring pollutant removal and toxicity reduction, the impact of this bioremediation on the indigenous soil bacterial and fungal populations was assessed.

2. Materials and methods SMC of mushroom P. pulmonarius strain Pl-27 was produced in the Department of Biology at The Chinese University of Hong Kong after the harvest of edible crops using sawdust based fermented compost (Chiu et al., 1998; Lau et al., 2003; Law et al., 2003). The physico-chemical properties of SMC are listed in Table 1.

* Corresponding author. Tel.: +86 852 2609 6101; fax: +86 852 2603 5646. E-mail address: SWChiu@cuhk.edu.hk (S.-W. Chiu). 0045-6535/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.12.044

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Table 1 The physico-chemical properties (mean s) of the spent compost of mushroom Pleurotus pulmonarius (SMC) and the SMC-treated soils collected from a recycling factory. Pile SMC Batch 1 Control Incubation period (days) TOC (%) NKjeldahl (mg kg1) NO3N (mg kg1) NH3N (mg kg1) PTotal (mg kg1) PO3P (mg kg1) Electrical conductivity (lS cm1) K+ (mg kg1) pH Soil moisture (%) TPH (g kg1) Oil and grease (g kg1) DEHP (mg kg1) 22 2 2227 381 231 21 276 51 769 162 205 26 2397 321 2826 396 7.07.1 ND ND ND ND 98 40 655 186 14 0 50 228 27 93 8 206 17 652 27 7.88.0 12 2 5 1* 14 2* 87 5* Treatment 98 5 0* 1002 99* 14 0 7 0* 341 49* 117 4 293 59* 742 60* 7.77.8 23 1* 10 10 1 27 6 Batch 2 Control 28 40 651 231 14 0 40 186 58 65 1 187 4 609 19 8.78.8 13 0 5 1* 15 4* 93 7* Treatment 28 51 1062 243* 14 0 7 1* 324 63* 63 2 249 302 698 49* 7.47.6 25 4* 10 81 44 1

ND, not detected. Signicant difference between control and treatment groups at p < 0.05 by student t-test.

2.1. Off-site ex situ bioremediation of mixed contaminated soil 21 m3 batches of mixed contaminated soils were collected from an industrial area with mixed factories practising waste recycling, manufacturing of portable toilets and producing heavy instruments in Lau Fau Shan and then treated in The Chinese University of Hong Kong. The rst batch was contaminated with total petroleum hydrocarbons (TPH), 6.9 0.3 g kg1 19.0 0.7 g kg1 oil and grease and 99 17 mg kg1 DEHP. The second batch was contaminated with 5.4 0.1 g kg1 TPH, 14.5 1.5 g kg1 oil and grease and 95 6 mg kg1 DEHP. The contamination levels were above the Dutch intervention values (5000 mg kg1 TPH and 60 mg kg1 total phthalates). Besides, their heavy metal contents also exceeded the China EPA standard for industrial soil (GB 15618-1995; 100 mg kg1 Cu, 350 mg kg1 Pb and 300 mg kg1 Zn) having (mg kg1): Cu, 136 38 and 104 13; Pb, 430 46 and 691 251; and Zn, 477 24 and 578 96 for batch 1 and 2 soils, respectively. A soil batch was divided into two groups of three piles for control or treatment, respectively. In parallel to 3% SMC application manually to the treatment group, tilling was done to the control group for comparison. For the rst batch, tilling was also practised at days 35 and 77 to both groups to increase aeration, and its effect on pollutant removal and soil microbial community was monitored. The piles of soil were watered with 15 L water weekly during the incubation period. 610 g samples were collected randomly from a pile and mixed together to form a composite sample for chemical and microbial soil analyses. SMC was added twice to the treatment pile for speeding up biodegradation; For the rst batch, SMC was added at days 0 and 56 while for the second batch, SMC was added at days 0 and 15. 2.2. On-site ex situ bioremediation of oil-contaminated soil 80 m3 contaminated soils were excavated by a grabber in a power plant in Tuen Mun. The soil was contaminated with 1.2 0.2 g kg1 TPH, exceeding the Dutch target level (1000 mg kg1) and 4.0 0.6 g kg1 oil and grease. 0.3% SMC was applied once on-site as an ex situ treatment by mechanical mixing with a grabber. The pile of soil was wetted to 30% initial soil moisture by spraying with water for 1 h. 5100 g samples were collected randomly from the pile at 37 d interval and then transported in an ice box to the laboratory for chemical and microbial analyses.

2.3. Soil analyses Fresh samples were used for determination of soil moisture and microbial populations. Total cultivable bacteria and total cultivable fungi counts were measured on Luria broth (LB) agar plates for 3 d at 37 C and potato dextrose agar plates for 5 d at 28 C, respectively, and their populations were presented as colony forming unit (cfu) g1. Air-dried soil samples were sieved through 2 mm screens for other soil analyses. Soil texture was analyzed using the Bouyoucos hydrometer method (Chiu et al., 2006). TOC content was analyzed with a Shimadzu analyzer (model TOC-5000A). Soil pH and conductivity were measured in an aqueous solution (soil:ultrapure water, 1:2.5; w/v) with a pH and conductivity meter (Jenway 4330 pH) (Chiu et al., 2006). A soil sample was digested with nitric and sulfuric acid for measuring Kjeldahl N and total P, respectively; Available N was extracted with 1 M KCl and determined spectrophotometrically in ltered extracts. Available P was extracted with Trougs reagent (H2SO4-(NH4)2S2O8 buffer, pH 5.0). All the diluents were determined by an automated ion analyzer (Skalar AIA 4000, Holland) (Chiu et al., 2006). Mineral K was analyzed by AAS (Hitachi Polarized Zeeman Z-8100 model) after nitric acid digestion (Chiu et al., 2006). The oil and grease content was determined by a modied gravimetric method 5520E whereas TPH was determined by USEPA Method 1664A (modied from EPA 821/B-94-004b) (Juteau et al., 2003). The extraction solvent used in the former was ethyl acetate (ve volumes) while three volumes of n-hexane were used in the latter. Then the extracts were rotary dried and dehydrated with anhydrous sodium sulfate. To measure DEHP, 5 g freeze-dried soil samples were extracted twice with 10 mL dichloromethane (Analytical grade, BDH) shaking at 200 rpm for 2 h (Gong et al., 2006). The two extracts were pooled, rotary dried and redissolved in 1 mL acetone (HPLC grade). The sample was then ltered by a 0.45 lm lter (Acrodisc syringe lters 4CR PTFE) before GC-MS (Shimadzu GCMS-QP5050A) analysis using a 0.25 mm (i.d.) 30 m HP-1 methyl siloxane capillary column coated with 0.25 lm lm (Hewlett Packard HP19091Z413). The temperature proles started at 60 C, held for 2 min, then increased to 300 C at 8 C min1 and held for 12.5 min (Lau et al., 2003; Law et al., 2003). An authentic DEHP standard (Cat no.: 36735, Riedel-de HaF n, Seelze, Germany) was resolved at 27.1 min in the GC chromatogram. In this study, the extraction efciency of DEHP in samples by articial spiking and followed by

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extraction was 94 3%. Degradative removal of the organopollutant is represented by removal efciency which is the amount of pollutant removed over the initial pollutant amount and expressed in percentage. 2.4. Soil toxicity Three bacteria (namely: Bacillus cereus, Methylobacterium sp. and Pseudomonas aeruginosa) and three fungi (namely: Fusarium solani, Trichoderma asperellum and Trichoderma harzianum) isolated from a mixed and heavily contaminated site in an abandoned shipyard area in Hong Kong, and one bacterium (Flavobacterium sp.) and 1 fungus (Penicillium glabrum) isolated from the industrial area were used to assess the soil toxicity (Chiu et al., 2006). A bacterium was inoculated into the autoclaved soil sample and incubated at 30 C for 3 d. Their initial and nal bacterial population sizes were determined on LB agar plate by spread plate method. Similarly, the initial and nal fungal population sizes in soil samples were determined by measuring their ergosterol contents (Reeslev et al., 2003; Chiu et al., 2006). Soil samples were extracted with acetone/hexane mixture (1:3; v/v), and the extracts were concentrated by rotary evaporation. The residues were dissolved in 1 mL hexane (HPLC grade) for GCMS. The GC operating conditions and temperature proles were as follows: temperature was raised to 80 C for 1.5 min and heated to 290 C at 10 C min1 and then maintained at 290 C for 5 min. A relative scale (fold change in population growth) was adopted to compare the microbial population sizes in the soil type (control or treatment) before and after the incubation period. Three replicates were examined for one soil type.

Fig. 1. The changes of TPH, oil and grease and DEHP contents (mean s) of the rst batch soil collected from an industrial area under off-site ex situ bioremediation by spent compost of mushroom Pleurotus pulmonarius. *, SMC application in treatment or tilling in control; @, tilling.

3. Results and discussion SMC showed high nutrient contents in terms of TOC, N, P and K contents (Table 1). Besides, SMC harboured (11 3) 107 cfu g1 bacteria and (56 9) 104 cfu g1 fungi some of which are organopollutant degraders (Law et al., 2003). Further, SMC contained 1.7 2.0 U mg1 laccase and 1.81.9 U mg1 manganese peroxidase. These enzymes could act directly and immediately to degrade persistent organopollutants (Chiu et al., 1998; Hestbjerg et al., 2003; Lau et al., 2003; Law et al., 2003; Gong et al., 2006). 3.1. Off-site ex situ bioremediation of mixed contaminated soil Bacteria B. cereus and Flavobacterium sp. and fungi P. glabrum and F. solani were isolated from these industrial soils (unpublished results). Some of them could degrade DEPH, hydrocarbons and/or PAHs (Wunder et al., 1997; Kim et al., 2002, 2007; Rahman et al., 2002; Van Hamme et al., 2003; Abu and Dike, 2008). These sandy loam soils contained low-moderate amounts of TOC, N and P supporting microbial degradation if favourable environmental conditions were provided. For the rst batch soil, tilling was done at days 0, 35, 56 and 77 in the control piles which were weekly watered. These led to spontaneous degradation of 17% DEHP, 30% TPH and 24% oil and grease in 98 d (Fig. 1; Table 1). The total cultivable bacteria showed gentle decreasing population size. In contrast, the soil fungi showed a transient small rise in population 7 d after the rst tilling (Fig. 2). As with soil bacteria, the total cultivable fungi decreased at the end of the incubation period. This microbial die-down in the control piles may be a result of the nutrient depletion. Three percent SMC was applied at days 0 and 56 while tilling was applied at days 35 and 77 to the treatment piles. SMC rapidly removed the pollutants for 14 d and then slowed down (Fig. 1). Simultaneously, the rst SMC application raised total cultivable bacteria and total cultivable fungi by 3.3 folds and 1.6 folds, respecFig. 2. The changes in population sizes (means) of total cultivable bacteria and total cultivable fungi of the rst batch soil collected from an industrial area under off-site ex situ bioremediation by spent compost of mushroom Pleurotus pulmonarius. *, SMC application in treatment or tilling in control; @, tilling.

tively (Fig. 2). The subsequent tilling at days 35 and 77 did not enhance the removal except day 77 tilling for TPH (Fig. 1). Yet tilling raised transiently the soil communities to different extents (Fig. 2). Similarly, the second SMC application greatly increased the pollutant removal and increased transiently the soil fungal population and the soil bacterial population which later fell (Figs. 1 and 2). When the initial and nal population sizes in the treatment piles were compared, soil bacteria showed 2.7-fold increases while soil fungi were increased by 2.6 folds (Fig. 2). The introduced nutrients and micro-organisms by SMC amendment contribute to the soil community growth (Fig. 2; Table 1). SMC degraded 73% DEHP, 84% TPH and 57% oil and grease (Fig. 1). The net removal by two SMC applications deducting from that of spontaneous degradation was 56% DEHP, 54% TPH and 33% oil and grease. From the above results, tilling is found not a steady practice to increase the soil micro-organisms for speeding up biodegradation. Rather, SMC application possessed the lignolytic enzymes to cause the immediate degradation of the pollutants while the nutrients and micro-organisms introduced also contributed to the rapid

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S.-W. Chiu et al. / Chemosphere 75 (2009) 837842 Table 2 The physico-chemical properties (mean s) of the contaminated soil in a power plant before and after treatment by the spent compost of mushroom Pleurotus pulmonarius. Parameters TOC (%) NKjeldahl (mg kg1) NO3N (mg kg1) NH3N (mg kg1) PTotal (mg kg1) PO3P (mg kg1) Electrical conductivity (lS cm1) K+ (mg kg1) pH Soil moisture (%) Bacteria (105 cfu g1) Fungi (103 cfu g1) TPH (g kg1) Oil and grease (g kg1) Before treatment 31 25 6 14 0 51 281 85 13 3 503 418 767 180 7.98.6 63 17 5 61 1.2 0.2 4.0 0.6 After treatment 41 43 11 14 0 51 317 57 23 7 1051 283 866 87 7.78.1 91 24 12 10 7 0.7 0.1 2.3 0.3

decontamination seen in days 014 and 5670 periods (Figs. 1 and 2). For the second batch, SMC was applied twice at 14-d interval according to the experience gained with the rst batch and in previous studies (Lau et al., 2003; Law et al., 2003; Gong et al., 2006). With tilling done at days 0 and 15 to increase aeration and weekly watering to create favourable environment for spontaneous degradation, the entire 28-d incubation removed 12% TPH, 2% oil and grease and 2% DEHP but did not raise the population sizes of the soil micro-organisms in the control piles (Fig. 3; Table 1). When the soil samples taken at 1 h before and 1 h after SMC application at day 0 were compared, SMC raised the total cultivable bacterial population size by 11.3 folds and total cultivable fungal population by 1.7 folds. Meanwhile SMC showed immediate, rapid and large removal of organopollutants for 48 d, and then the removal slowed down (Fig. 3). This rst application of SMC removed 39% TPH, 15% oil and grease and 28% DEHP. The second SMC application at day 15 also raised the total cultivable bacterial population by 2.1 folds. At day 28, the treatment piles showed 6.7-fold and 2.0-fold increases for total cultivable bacteria and total cultivable fungi over the control piles, respectively. This SMC amendment further removed the pollutants and lowered them below the intervention values with the nal degradative removal efciencies of 76% TPH, 33% oil and grease and 53% DEHP. The net removal by SMC was 64% TPH, 31% oil and grease and 51% DEHP. Among the control piles, longer incubation time in the rst batch (98 d) than the second batch (28 d) led to higher spontaneous degradation because the soil-borne pollutant-degrading micro-organisms were present, and there were low-moderate nutrients available (Table 1). Low efciency of spontaneous degradation has also been reported in other studies (Baheri and Meysami, 2002; Coulon et al., 2005). In parallel, the two SMC applications in treatment piles performed similarly to rapidly degrade the organopollutants (Figs. 1 and 3; Table 1). 3.2. On-site ex situ bioremediation of oil-contaminated soil The power plant soil was contaminated by 1.2 0.2 g kg1 TPH and 4.0 0.6 g kg1 oil and grease. Its heavy metal contents (6 2 mg kg1 Cu and 76 28 mg kg1 Pb) did not exceed the China EPA standard for industrial soil (GB 15618-1995). It contained fewer nutrients (TOC, NKjeldahl, PO3P) and thus supported less soil micro-organisms (Tables 1 and 2). This sandy loam type soil was

decient in N and C unfavourable for biodegradation. Amending SMC at 0.3% did not affect the soil physico-chemical properties (Table 2). SMC raised total cultivable bacterial population by 5.1 folds transiently (Table 2). Such smaller increase in soil bacterial population might be owing to the smaller dose of SMC amended leading to little introduction of micro-organisms and less nutrients (Tables 1 and 2). In contrast, the total cultivable fungi upon SMC application dropped by 57% and then returned to the initial level gradually. Beside competition among soil micro-organisms, the introduced saprotrophic SMC fungi faced the infertile soil which might reduce the population size. In 22 d SMC amendment lowered the TPH and oil and grease contents by 40% and 45%, respectively; TPH dropped to 694 107 mg kg1 while the oil and grease content decreased to 2332 257 mg kg1 (Table 2). The treatment efciency is explained partially by the immobilized lignolytic enzymes in, and nutrients and micro-organisms introduced by SMC amendment (Table 1; Fig. 2). Bioaugmentation introduces competition and may affect the native microbial biota during bioremediation, and biostimulation also modulates the soil community (DAnnibale et al., 2005; Kirk et al., 2005; Federici et al., 2007). SMC amendments changed without a consistent pattern on the microbial communities of these industrial soils. Besides, SMC provided a favourable environment for bioremediation by retaining higher soil moisture and lowering pH to neutral (Tables 1 and 2). Further, SMC is known to have a relatively low bulk density (0.26 g cm3) and high porosity (86%) serving as a bulking agent (Law et al., 2003; Gong et al., 2006). Its application may increase oxygen diffusion favouring biodegradation as other amendments (Rivera-Espinoza and Dendooven, 2004; Molina-Barahona et al., 2005). 3.3. Soil toxicity The relationship between biodegradation and reduction in toxicity is controversial (Phillips et al., 2000; Brohon et al., 2001; Pelletier et al., 2003; Coulon et al., 2005). For instance, the bacterial degradation of DEHP generates more toxic metabolites, e.g. adipic acid and 2-ethylhexanol (Chang et al., 2004; Nalli et al, 2006; Beauchesne et al., 2008). In contrast, Molina-Barahona et al. (2004) reported that biostimulation caused diesel removal by 50.6% and signicantly reduced phytotoxicity by 915% and toxicity towards aquatic invertebrate Daphnia by 67%. Similarly, the treatment of contaminated soils by mushroom P. ostreatus or P. pulmonarius led to a reduction in toxicity (Baud-Grasset et al., 1993; Hestbjerg et al., 2003; Gong et al., 2006). Some fungal degradation pathways of DEHP were known different from the bacterial one (Kim et al., 2002, 2007).

Fig. 3. The changes of TPH, oil and grease and DEHP contents (mean s) of the second batch soil collected from an industrial area under off-site ex situ bioremediation by spent compost of mushroom Pleurotus pulmonarius. *, SMC application in treatment or tilling in control.

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In this study, a panel of six exogenous micro-organisms previously isolated from heavily and mixed contaminated soils and two indigenous micro-organisms isolated from the industrial area soil showed two patterns in the relative population growth test. In the control samples of the industrial area soil, the relative population growth had values around 1 (0.91.2 folds); the microbial growth rates were similar for the soil samples before and after the incubation period with spontaneous degradation. In contrast, the microbial population growth was mostly increased (range: 1.415.2 folds; mean: 7.2 folds) for the soil samples after SMC treatment. This is partially owing to the reduced toxicity of the soil samples after remediation having lower pollutant levels (Table 1). Also, the high dose of SMC used showed a residual nutrient effect for this industrial soil (Table 1). For the power plant soil, the population growth of the four bacteria was enhanced by SMC treatment (1.21.6 folds) while the four fungi did not change population growth rates. This may be because nil nutrient effect was observed with the low dose (0.3%) of SMC applied (Table 2). When the type of micro-organisms is compared, the bacteria showed higher sensitivities with greater fold changes in population growth (mean: 5.8 folds) than the fungi (mean: 1.5 folds). This difference may be because many fungi could consume the organopollutants as additional C-sources (Chiu et al., 2006; Gong et al., 2006; Kim et al., 2007). When organopollutants are removed, presumably less C source is available for fungal growth. Also, a bacterium requires less resource than a fungus for population growth. In brief, using microbial response as a biomarker, when greater removal of the organopollutants was observed in the SMC treated soil than the control soil with spontaneous degradation, the greater fold change in the population growth was. In brief, the present result conrms reduction of soil toxicity by SMC treatment as reported in previous studies using Microtox bioassay or seed germination test (Lau et al., 2003; Law et al., 2003; Gong et al., 2006). 4. Conclusions In addition to the multiple and integrated action mechanisms in removal of organopollutants, the merit of using SMC for bioremediation is the recycle of waste alleviating the disposal problem and lowering the cost in waste treatment. Further, its application does not introduce additional expense in disposal as the materials used are biodegradable. The eld application indicates that SMC treatment did not cause any nuisance to the environment. This SMC treatment not only degrades the persistent organopollutants but also reduces toxicity of the treated soil. Acknowledgements The authors thank Gammon Construction Limited and Wai Lung Recycling factory for providing the power plant site and contaminated soil for study, respectively. S.W.C. thanks the direct grants from The Chinese University of Hong Kong for partially sponsoring the present study. References
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