1,1,1-Trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) was a widely used organochlorine pesticide with high toxicity and residual levels. In 2001 the Governing Council of the United Nations Environment Programme issued a treaty to eliminate or restrict the production and use of persistent organic pollutants (POPs). Twelve offenders were listed and DDT is among them. In U.S. and some other countries a ban on DDT took effect earlier [1
]. Although the use of DDT for wide agricultural use has been banned in China since 1983, a large amount of DDT still remains in soils. In some areas the DDT concentration found in soil markedly exceeds the level set by the national soil quality standards (GB/T 18407-2001). For example, the concentration of DDT exceeded the national soil quality standards in the agricultural soils of North of Zhejiang Province [2
]. In Tianjin region, the concentration of p,p′-DDT and p,p′-DDE in the soils was 27.5 and 18.8 ng/g, respectively [3
]. Soil pollutants may adversely affect agricultural products. The rate of detection DDT in some vegetables produced in a Nanjing suburb was up to 100% [4
]. Therefore, remediation of the DDT-contaminated soils must be conducted to ensure the agricultural products safety and human health.
Recently, some physical, chemical and biological approaches have been researched to clean up DDT in the soils. For example, the physical approaches of bioventing and thermal treatment [5
] and chemical remediation of leaching with surfactants [6
], oxidation [7
], dechlorination by metallic reduction [8
] and light degradation [10
]. However, remediation using physical approaches is expensive, and chemical approaches adversely affect the soils physical-chemical properties, causing secondary pollution. Bioremediation methods, mainly phytoremediation [11
] and microbial bioremediation [15
], have been utilized to remove DDT from contaminated soils. Enzymatic remediation is a rapid and efficient method of removing pesticide residue from the environment [18
]. Laccase (EC 18.104.22.168) can catalyze the oxidation of organic compounds [19
], and has been widely used to degrade pollutants from environment [20
]. However, the environmental conditions of soil are complex. Enzymatic activity may be reduced or eliminated by degradation of proteases, adsorption on soil colloids and extreme acidity or alkalinity etc.
]. In this study, the effects of different soil oxygen conditions and different soil pH on remediation of DDT-contaminated soil by laccase were investigated in laboratory batch experiments.
2. Materials and Methods
White rot fungi (Panus conchatus) used for the production of laccase were purchased from Guangzhou Chemical Company Ltd of the Chinese Academy of Sciences (Guangzhou, China). Standards of the four components of DDT (p,p′-DDE, p,p′-DDT, p,p′-DDD and p,p′-DDT) were purchased from Supelco (Park Bellefonte, PA, USA). The experimental soil was woodland soil that was obtained from the arboretum of South China Agricultural University. The basic physical and chemical properties of the soil after air drying were measured. The soil texture was silty soil, the soil pH was 5.87, organic matter was 9.6 g/ kg, and total iron was 39 mg/kg. DDTs in this study stands for the total sum of p,p′-DDE, o,p′-DDT, p,p′-DDD and p,p′-DDT in each sample. DDT was not detected in these soils (the detection limit will be described later).
2.2. Preparation of Laccase and Enzymatic Activity Measurement
White rot fungi was cultivated for nine days on solid agar medium plates. The solid agar medium employed in the experiment contained (g/L): KCl, 0.1; MgSO4·7H2O, 0.3; KH2PO4, 1; NaNO3, 2.5; glucose, 20; agar, 20; potato flour, 20 and the following micronutrients (mg/L): FeCl3·6H2O, 10; CaCl2·2H2O, 10; VB1, 10. Final pH was 6.0. A mycelial mat of appropriate growth phase was cut from the solid agar medium plates by a 10 mm hole diameter puncher and blended with 50 mL potato liquid medium for precultures. The liquid medium in the experiment was made with 1,000 mL of potato extract and the following components (g/L): MgSO4·7H2O, 1.5; KH2PO4, 3; yeast extract, 5; glucose, 20; VB1, 0.01. Precultures were prepared for four days at 130 rpm and 28 °C. Crude laccase preparation was carried out in 1 L Erlenmeyer flasks with the same potato liquid medium for nine days at 130 rpm and 28 °C. The crude laccase was filtered and centrifuged. The supernatant was brought to 20% saturation ((NH4) 2SO4, overnight at 4 °C) and then centrifuged. The resulting supernatant was brought to 80% saturation ((NH4) 2SO4, overnight at 4 °C) and then centrifuged. The supernatant was loaded onto a Sephadex G-75 column equilibrated with 0.02 mol/L sodium acetate buffer (pH 4.6). Proteins were eluted with 0–0.5 mol/L NaCl at a flow rate of 0.5 mL/min. Eluted fractions containing laccase activity were pooled and kept (4 °C). Laccase activity was determined by monitoring the oxidation of ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] at 420 nm. 1 U of activity was defined as the amount of enzyme able to oxidize 1 μmol ABTS per min. Regardless of the initial amount of laccase added, the activity of laccase reduced gradually during a 20-d incubation under different conditions. Laccase lost 100% of the initial activity in any of the soil microcosms after a 20-d exposure to soil.
2.3. Preparation of DDT-contaminated Soils
Commercial DDT (0.0547 g) was dissolved in 100 mL acetone in a volumetric flask. The DDT solution was kept with a volumetric flask and stored at 4 °C for use; 5 mL of 0.547 mg/mL DDT solution was added into 50 g soil passed through a 2-mm sieve, stirred well and air-dried. Then the dried sample was put into 450 g soil, sieved at 2 mm, and mixed well. This was the experimental DDT-contaminated soil sample. The soil sample was kept at room temperature for four weeks, after that remediation experiments were carried out to assess the remedial potential of laccase. Throughout the experiment, the water content of all soil samples was kept at approximately 15%. The initial contents of DDT components and DDTs in the soil samples were 0.351 mg/kg (p,p′- DDE), 0.775 mg/kg (o,p′-DDT), 1.403 mg/kg (p,p′-DDD), 2.334 mg/kg (p,p′-DDT) and 4.863 mg/kg (DDTs), respectively.
2.5. Sample Pre-Treatment
The preparation of sample and the measurement of DDT concentration were conducted according to the standards described in national standards of P. R. China (GB/T14550-93) set for Soil Quality-Determination of BHC and DDT-Gas chromatography. Briefly, 10 g of soils were placed in a Soxhlet extraction apparatus and immersed with 100 mL petroleum ether-methanol (1:1) solution for 10 h, then extracted for 6 h. The extract liquid was transferred to a separating funnel and 10% aqueous sodium sulfate was added. After 1 min of shaking, the mixture stratified into two liquid phases on standing. The upper layer petroleum ether was separated and treated with concentrated sulfuric acid with a volume equivalent to 10% of the petroleum ether solution. The mixture of petroleum ether and sulfuric acid was shaken 3–4 times until both layers became colorless. 10% anhydrous sodium sulfate solution with a volume equivalent to half of the petroleum ether layer was added to the remaining petroleum ether solution for washing up until the petroleum ether solution became neutral. At the end, the petroleum ether solution was dehydrated through anhydrous sodium sulfate, concentrated and suspended in 10 mL volume for DDT measurement.
2.6. Gas Chromatography Analysis of DDT
The DDT standard solution contained components of p,p′-DDE, o,p′-DDT, p,p′-DDD and p,p′-DDT, in concentrations of 20, 20, 60.08, 60 mg/L, respectively. Chromatographic grade acetone was used to dilute the DDT solution to make four concentration levels for each component. The gas chromatographic instrument used was a HP5890. Column HP-5, 30 m × 0.320 mm (id) × 0.25 μm. High purity nitrogen (99.999%) was used as the carrier gas. Gasification temperature was 220 °C, column temperature was 195°C, and detector temperature (ECD) was 245 °C. The speed of gas flow was 2 mL/min. Splitless injections (2 μL) were made. 2,4,5,6-Tetrachloro-m-xylene was used as the recovery indicator to control the recovery rate during the whole operation procedure. The recovery standard was used to control the sample recovery rate. The recovery rate of the indicator ranged between 78% and 88.6%. The recovery rate of DDT ranged between 88.4% and 98.7%. The detection limit for p,p′-DDE, o,p′-DDT, p,p′-DDD and p,p′-DDT was 0.902, 3.869, 2.847l and 0.756 μg/L, respectively.
2.7. Statistical Analysis
One-way ANOVA analysis was performed to compare the residues of four components DDT and DDTs in soils between different treatments by using SPSS11.5 software.