Tourmaline combined with Phanerochaete chrysosporium to remediate agricultural soil contaminated with PAHs and OCPs

Journal of Hazardous Materials 264 (2014) 439–448

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Tourmaline combined with Phanerochaete chrysosporium to remediate agricultural soil contaminated with PAHs and OCPs Cuiping Wang ∗ , Li Yu, Zhiyuan Zhang, Baolin Wang, Hongwen Sun MOE Key Laboratory of Pollution Processes and Environmental Criteria, Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China

h i g h l i g h t s • • • • •

Tourmaline combined with microorganism could be applied to remove PAHs and OCPs. Tourmaline enhanced the degradation rates of PAHs and OCPs. Tourmaline increased soil invertase and hydrogen peroxidase activities. Tourmaline increased PAH and OCP-degraders and soil microorganism biodiversity. Tourmaline make soil humic acid decreased.

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Article history: Received 19 June 2013 Received in revised form 27 October 2013 Accepted 29 October 2013 Available online 7 November 2013 Keywords: Tourmaline Remediation PAHs and OCPs Soil humic acid Soil enzyme activities PCR–DGGE bacterial diversity

a b s t r a c t The potential application on tourmaline was explored. The combination of tourmaline and Phanerochaete chrysosporium was conducted to remediate the field soil from the Dagu Drainage River bank of Tianjin in China. The total PAH and OCP concentrations in the soil were 6.4 ± 0.05 and 145.9 ± 1.9 mg/kg, respectively. During the 60 day remediation program, the remediation degradation rates of all the 16 U.S. EPA priority PAHs and OCPs were 53.2 ± 4.7% and 43.5 ± 3.1%, respectively. The PAH and OCP removal rates were 31.9 ± 2.9% and 26.4 ± 1.8%, respectively, in soil with the addition of tourmaline, and the removal rates were 40.5 ± 2.3% and 34.2 ± 3.9%, respectively, in soil with the addition of P. chrysosporium. Thus, the combination of tourmaline and P. chrysosporium promoted the bioremediation rate of PAHs and OCPs in the soil, compared with the rates obtained using tourmaline or P. chrysosporium individually for the remediation of PAH and OCP degradation. In addition, tourmaline can promote the generation of soil hydrogen peroxidase and invertase enzyme, significantly increase the indigenous bacterial community and the number of PAH and OCP-degraders compared to those in the control, and reduce the soil humic acid content. Hence, the present study provides a potential alternative for the remediation of soils contaminated by PAHs and OCPs. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) are typical persistent organic pollutants (POPs). There is worldwide concern regarding the long-term effects of these contaminants due to their persistence, bioaccumulation, and potential negative impacts on biota and food chemistry. Because of their lipophilic nature, the PAHs and OCPs are easily adsorbed onto suspended particles, making soil a predominant repository for environmental POPs [1]. Bioremediation of soil contaminated by organic pollutants based on microbial degradation has been a preferred method

∗ Corresponding author. Tel.: +86 22 23504362; fax: +86 22 23509241. E-mail address: [email protected] (C. Wang). 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.10.073

because it is cost-effective, generates no secondary pollution and is friendly to ecosystems [2]. However, due to the high hydrophobicity of PAHs and OCPs, once they enter the soil, they may relocate into the micropores of the soil matrix or become irreversibly bound to the soil, where they are subject to reduced degradation and experience reduced or uptake by organisms. Furthermore, nutrients and electron acceptors are typically lacking in the field soil, which prohibits the application of bioremediation technology in this soil. Therefore, a major initiative of environmental science is to develop an effective technology that overcomes the challenges faced by current soil bioremediation techniques. Tourmaline is a complex borosilicate mineral with an intricate chemical composition. The general chemical formula of tourmaline is written as XY3 Z6 (T6 O18 )(BO3 )3 V3 W. In this formula, the following definitions apply: X = Na+ , K+ , Ca2+ or a vacancy;

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Y = Li+ , Fe2+ , Fe3+ , Al3+ , Mg2+ , Cr3+ , V3+ , (Ti4+ ), Z = V3+ , Fe3+ , Cr3+ , Al3+ , Mg2+ , Fe3+ ; T = Si4+ , Al3+ , (B3+ ), B = B3+ ; V = [O(3)] = OH− , O2− ; and W = [O(1)] = OH− , O2− , F− , with the metal ions in parentheses indicating minor or possible substitutions [3]. Therefore, the unique properties of tourmaline, such as its electrical, magnetic and calorific properties, are determined by its structure. In addition, tourmaline is capable of radiating far infrared energy, producing an electrostatic field, releasing rare microelements [4] and stimulating the growth and metabolism of microorganisms [5,6]. However, no data have been reported with regard to using tourmaline combined with microorganisms to remove POPs from contaminated field sites. In addition, the impact of tourmaline on soil enzyme activities and biodiversity of the active microorganisms has not been thoroughly evaluated. The activity of microorganisms or a specific active microorganism species is an important parameter in biodegradation, which can provide information to elucidate the degradation mechanisms of PAHs [7] and OCPs. In addition, the information on biodiversity can reveal the change in the microorganism population of the soil. In addition to the biodegradability of POPs, the combination state of the sorbed contaminants in soils is an important factor that influences their degradation [8]. The soil organic matter (SOM) content is a primary soil property that affects the combination state and bioavailability of organic contaminants [9]. A high content of SOM in soil can negatively influence the decontamination rate of organic contaminants by bacteria [9,10]. Additionally, humic acid (HA) is a principal component of humic substances, which are the major organic constituents of SOM. Therefore, changes in the level of HA of the soil during PAH and OCP degradation using tourmaline were evaluated, which might provide a good understanding of the remediation mechanisms of tourmaline. The purpose of the present study was to explore tourmaline potential application. A combination of white rot fungus (P. chrysosporium) and tourmaline was applied to remediate soil contaminated with PAHs and OCPs. We investigated the distribution of PAHs and OCPs in agricultural soil contained in the Dagu Drainage River (DDR) bank. Then, we remediated the PAH- and OCP- contaminated soil using the new technology described herein. The role played by tourmaline in the remediation process was elucidated via studies involving soil enzyme activities, soil bacterial diversity evaluation by polymerase chain reaction (PCR) and -denaturing gradient gel electrophoresis (DGGE), the activity of PAH and OCP degraders selectively enumerated in the soil, and soil humic acid analysis before and after soil remediation. This study provides a new method for the remediation of soils contaminated with POPs and reports useful information regarding the applicability of tourmaline in removing pollutants from the environment.

2. Materials and methods 2.1. Tested soil and materials The DDR is a primary drainage river in Tianjin, receiving effluents from domestic, agricultural and industrial wastewaters. The agricultural soil tested in this study was obtained from the top layer (0–10 cm) of the river bank at the sewage outfall of the DDR (117◦ 12 11.49 W, 38◦ 57 36.20 N). The soil was taken from depths ranging from 0 to 20 cm. All of the soil was air-dried, thoroughly mixed and passed through a 1-mm mesh sieve to remove gravel. Several physico-chemical properties of the soil were determined using Chinese standard methods [11]. The pH of the soil was measured with a pH meter (310P-02, Thermo-Orion, USA) in a 1:2.5 (w/w) soil–CaCl2 water suspension. The organic matter content was determined by the potassium dichromate-outside heating method.

Particle size distributions were determined by a particle size analyzer (BT-90, Dandong Bettersize Instrument Ltd, China). The soil texture was determined to be silt clay with the following composition: 33.8% clay, 46.4% silt, and 19.8% sand. The soil pH (1:2 soil/water by wet weight) was 7.45, and the organic carbon content was 2.23%. The cation exchange capacity (CEC), measured by the barium chloride method, was 8.28 C mol/kg. A mixture of 16 U.S. Environmental Protection Agency (EPA) priority PAH stock standards [naphthalene (Nap), acenaphthene (Ace), acenaphthylene (Acy), fluorine (Fl), phenanthrene (Phe), anthracene (Ant), fluoranthene (Flu), pyrene (Pyr), benz[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), dibenzo[a,h]anthracene (DBA), indeno[1,2,3-cd]pyrene (INP), and benzo[ghi]perylene (BgP)] was purchased from J&K Chemical (Pforzheim, Germany). A mixture of internal standards (naphthalene-d8, acenaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12) was obtained from J&K Chemical (USA). A mixed standard solution of 16 OCPs (including ␣-HCH, ␤-HCH, ␥-HCH, ␦-HCH, heptachlor, Heptachlor epoxideII, o,p DDT, p,p -DDD, p,p -DDE, aldrin, dieldrin, endrin, ␣-endosulfan, ␤-endosulfan, endrin aldehyde, and endosulfan sulfate) was purchased from Accustandard, Inc., USA. Other surrogates or internal standards were purchased from J&K Acros, USA. All of the solvents were of analytical grade and were redistilled to remove impurities prior to use. Analytical grade n-hexane and dichloromethane were obtained from Tianjin Reagent, China, and were purified by distillation. Granular anhydrous sodium sulfate, silica gel (100–200 mesh) and neutral alumina were purchased from Tianjing Reagent, China.

2.2. Tourmaline Iron-rich black tourmaline was produced in the Xinjiang Province, China. The tourmaline was processed to a particle size of 800 nm by Hongyan Mineral Products Co., (10.18%) Ltd., Tianjin City, China. The compositional analysis was accomplished via an electron microprobe analyzer (EMPA) using a Shimadzu 1600 electron microprobe equipped with four-channel wavelength dispersive spectrometers (WDS). The chemical composition of the tourmaline was as follows: SiO2 , 36.75%, Al2 O3 , 33.62%, Fe2 O3 , 10.18%, TiO2 , 0.57%, B2 O3 , 9.78%, FeO, 1.7%, CaO, 0.4%, MgO, 4.76%, K2 O, 0.14%, Na2 O, 0.74%, P2 O5 , 0.19%, H2 O+ , 1.0%, and MnO, 0.21%.

2.3. Microorganism and preparation of fungal suspension P. chrysosporium (collection number: 5.776) was obtained from the Institute of Microbiology, Chinese Academy of Science and was maintained on potato dextrose agar (PDA) slants at 4 ◦ C. P. chrysosporium was inoculated from the PDA slants into sterilized Petri dishes, then transferred by an inoculation loop to test tubes filled with 10 mL fresh liquid potato dextrose medium with rubber Cyp-riots caps. The tubes were shaken for 24 h on a reciprocating shaker at 150 rpm and 28 ± 1 ◦ C to fully activate P. chrysosporium. Then, 5 mL of the activated mycelial suspension was transferred into 250 mL conical flasks containing 45 mL of liquid potato dextrose medium for 24 h incubation under the same conditions as those for proliferation. Cultures were centrifuged at 3000 rpm, and the supernatant was removed. The flocculent mycelia were collected by centrifugation, weighed, washed twice with sterile mineral salt medium (MSM), and suspended in an appropriate volume of MSM. This suspension was used as the fungi inoculum in subsequent experiments.

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2.4. Remediation experiments The soil was remediated in the laboratory using tourmaline (T) alone, P. chrysosporium (P) alone, and P combined with T. A control treatment (C) with no T or P added was also prepared. Each treatment was carried out in triplicate. The mixtures were carefully homogenized. Each treatment included 12 aliquots of soil samples (500 g). Among them, every three aliquots were used as a group to measure the PAH and OCP residuals in the soil samples, enzyme activities including hydrogen peroxidase enzyme and invertase enzyme activities, and soil humic acid levels during soil remediation. For soil remediation using P combined with T, three aliquots of soil samples (500 g) were placed into 1.5-L brown wide-mouth bottles, and 25 mg of tourmaline and a 240 mL fungal suspension (0.016 g/mL) were added. At this point, all of the soil samples were supplemented with sterile deionized water to approximately 65% of the soil’s water-holding capacity. The bottles were sealed with gauzes and placed in an incubator at 35 ◦ C with periodic watering as necessary. The collection of 3-g soil samples from each of the three jars were collected at 0, 10, 20, 30, 40, 50 and 60 d for determination of their PAH and OCP concentrations, and a collection of 0.25 g at 60 d was obtained for measurement of their microbial community (see Section 2.6). Of the remaining rest of nine jars, soil samples in six jars were used for enzyme activity analysis, and the other three jars were used for soil humic acid analysis. For soil remediation using P or T, the remediation method and dose of P or T followed the same remediation methods used for P combined with T.

2.5. Enzyme extraction and analysis The hydrogen peroxidase activity was measured as previously described in the literature [12]. In brief, for soil remediation using P combined with T, a 3-g soil sample from each of the three jars among the six jars of the same treatment was mixed with 40 mL of distilled water and 5 mL of substrate (1% H2 O2 ). The reaction mixture was incubated at 25 ◦ C for 20 min. To stabilize the undecomposed H2 O2 in this mixture, 5 mL of H2 SO4 (3 mol/L) was added. The solid phase was separated by centrifugation, and the supernatant was centrifuged. The remaining H2 O2 in the supernatant was determined using a 0.02 mol/L KMnO4 solution. The oxidized H2 O2 content was used to calculate catalase activity. The activity of hydrogen peroxidase in the soil was expressed as IU/g dry soil/24 h. The values are the means obtained from triplicate samples. To determine the invertase activity, 3 g of soil from each of the other three jars among the six jars of the same treatment and 0.2 mL of methylbenzene were incubated at 25 ◦ C for 15 min. Then, 15 mL of an 18% sugar solution and 5 mL of phosphate–citrate buffer solution (pH = 5.5) were added successively, and the resulting solution was incubated at 30 ◦ C for 24 h. Five milliliters of the supernatant was collected in a 50 mL test tube and then added to 3 mL of 3,5-dinitrosalicylic acid solution. Finally, the tube was heated for 5 min in a boiling water bath. After centrifugation, the absorbance at 540 nm was determined using a spectrophotometer (T6, Purkinje General Instrument Co. Ltd., China). The invertase activity (IU) was defined as the amount of enzyme that liberates 1 mg of glucose/g dry soil/24 h under the assay conditions. For soil remediation using P or T, measurement methods on the hydrogen peroxidase and invertase activities followed the same the above enzyme activity methods used for P combined with T. Experiments were conducted in the absence of soil and substrate to provide a reference for enzyme activity assays. All of the

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enzyme activities were measured in triplicate, and the results were expressed as IU/g dry soil. 2.6. Bacterial community analysis 2.6.1. DNA extraction The total bacterial community DNA was extracted from 250 mg of soil aliquots from each soil microcosm after 60 days of treatment (including the control soil, tourmaline-added soil, and P. chrysosporium/tourmaline-added soil) using the Omega DNA Extraction Kit (Omega Biotek, Doraville, GA, USA) according to the manufacturer’s instruction. 2.6.2. PCR amplification Partial 16S rRNA sequences were amplified from the extracted genomic DNA by PCR using a PTC thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). The variable V3 region of the domain bacteria was amplified using the PRBA338F (5 -ACT CCT ACG GGA GGC AGC AG-3 ) and PRUN518R (5 -ATT ACC GCG GCT GCT GG-3 ) primers [13] with a GC clamp (5 -CGC CCG CCG CGC CCC GCG CCG GTC CCG CCG CCC CCG CCC-3 ) attached to the forward primer. Amplification by PCR was performed as follows: initial denaturation at 94 ◦ C for 5 min, then, 94 ◦ C for 1 min, 65 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by 10 repetitions of this cycle, each time lowering the annealing temperature by 1 ◦ C; then, 25 cycles at 94 ◦ C for 1 min, 55 ◦ C for 1 min, and 72 ◦ C for 1 min; and, finally, 72 ◦ C for 7 min. For DGGE analysis, the first PCR product was diluted 1:100 and used as a template for a second cycle PCR based on the bacterial primers P63F (CAG GCC TAA CAC ATG CAA GTC-3 ) and R1378r (5 CGG TGT GTA CAA GGC CCG GGA ACG-3 ) [14]. The program used for the second PCR cycle was the same as that used for the first PCR cycle. The resulting PCR samples were then stored at 4 ◦ C until further processing. 2.6.3. DGGE analysis DGGE was performed using 8% polyacrylamide gels (acrylamide: bisacrylamide, 37.5:1) with a gradient of 30% to 60% denaturants (100% denaturant was defined as 7 mol urea and 40% formamide). The gels were run at 60 ◦ C (140 V) for 4 h in a Dcode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA). After staining with ethidium bromide in 1 × TAE for 45 min, the gels were visualized under UV illumination and photographed. The DGGE banding patterns were digitized and subsequently processed using Quantity One image analysis software (version 4.3.1, Bio-Rad Laboratories, Hercules, CA, USA). 2.7. Joint effect experiments For degradation of a mixture of 16 PAHs using P combined with T, specific volumes of a mixture of 16 PAH stocks and 3% methanol were added to 20 mL of mineral salts media (MSM) in the 50 mL reaction vials to obtain an initial concentration of 20 mg/L PAHs. 0.02 g of tourmaline was added into reaction vials, then appropriate volumes of P. chrysosporium inocula were added to the MSM-PAHs culture to reach an initial population of 0.01 g (wet weight)/mL. For degradation of a mixture of 16 PAHs using P or T, the degradation method and dose of P or T followed the same degradation methods used for P combined with T. Control groups contained the same constitutes, except that the cells had been killed by pasteurization. All of the cultures were incubated at 150 rpm at 30 ◦ C on a rotary shaker. Replicate samples were periodically sacrificed and the entire samples were used to measure the PAH concentrations. MSM was prepared and used by following the methods of Tao et al. [15]. All chemicals that were used in preparing the media were

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of analytical grade. The liquid media was autoclaved at 121 ◦ C for 30 min. For degradation of a mixture of 16 OCPs, the degradation methods followed the above PAH degradation methods. 2.8. Most-probable-number (MPN) procedure A modified most-probable-number (MPN) procedure was used to selectively enumerate PAH and OCP degraders. The MPN method was carried out according to the study by Wrenn and Venosa [16] with minor modifications. PAH degraders were enumerated in separate 96-well microtiter plates. A mixture of PAHs was used as the selective growth substrate. The PAH substrate mixture was added to the microtiter plates as a solution in pentane (10 ␮L/well). PAHs were deposited onto the surfaces inside of each well after the pentane evaporated rapidly. Then, the plates were filled with Bushnell–Haas (B–H) medium supplemented with 2% NaCl as the growth medium (180 ␮L/well). One gram soil was resuspended in a saline buffer solution that contained 0.1% sodium pyrophosphate (pH 7.5) and 2% NaCl. A 5-tube MPN dilution series with nine dilutions (10−1 to 10−9 ) were used for each tube. We inoculated 96-well plates by adding 20 ␮L of each dilution to 1 of the 12 rows of the five wells. The first row of each plate was inoculated with 20 ␮L of undiluted sample, and Row 12 remained uninoculated to serve as a sterile control. PAH-degrader plates were incubated for 3 weeks before 50 ␮L of iodonitrotetrazolium violet (INT, 3 g/L) was used to identify the positive wells. In positive wells, INT was reduced to an insoluble formazan that deposited as a red precipitate. Positive wells were scored after an overnight, room-temperature incubation with INT. The MPN value was determined with an MPN table [17]. OCP degraders were enumerated according to the above PAH degraders methods. 2.9. Humic acid extraction Humic acid (HA) was fractionated and extracted from the control soil and the tourmaline -added soil following the methods of Kohl and Rice [18] and Kelsey et al. [19]. Briefly, 10 g of air-dried and sieved (2 mm) soil was mixed with 200 mL of 0.5 M NaOH and shaken under N2 for 24 h at 22 ◦ C and 200 rpm. Samples were centrifuged (5000g) for 15 min under cryogenic conditions, and the supernatants were isolated from the residual soil. Soil residues were extracted seven additional times by sonication in 50 mL of 0.5 mol/L NaOH. Alkaline extracts were combined and acidified to pH < 2 and centrifuged to isolate the precipitated HA. Demineralization of the humic samples was performed by repetitive washing with HCl and HF. Samples were washed with distilled water to achieve a neutral pH and were then stored in desiccators until further analysis. HA was weighed using the German BT124S Sartorius electronic analytical balance. 2.10. Chemical extraction Extraction, fractionation, and instrumental analyses of the PAHs and OCPs were performed as described in detail below. A 2.75-g homogenized and freeze-dried sample was spiked with surrogate standards of five deuterated PAHs with 0.5 mg/L and 0.25 mg/L DCB/TCMX, which were added as internal standards. Subsequently, the soil was extracted with dichloromethane in a Soxhlet extractor for 48 h. Activated copper was added for desulfurization. The extract was concentrated and solvent-exchanged to hexane and then injected on a 1:2 alumina/silica gel glass column for cleanup and fractionation. The glass column employed for clean-up consisted of a 10-mm i.d. modified column packed with, from top to

bottom, 1 cm of anhydrous sodium sulfate, 6 cm of 30% deactivated silica, and 12 cm of deactivated neutral alumina. The anhydrous sodium sulfate was heated to 420 ◦ C in a furnace for 4 h and stored in a sealed desiccator prior to use. The silica gel and neutral alumina were pre-cleaned for three times in an ultrasonic cleaner for 0.5 h each time and were then placed in a ventilation cabinet until no organic solvent volatility was evident. Afterwards, the silica gel was heated to 160 ◦ C for 16 h, deactivated using deionized water to obtain a 3% deactivated silica gel, and then stored in a sealed desiccator until use. The 3% deactivated neutral alumina was treated similarly. The column was eluted with hexane to obtain the aliphatic fraction (which was discarded); subsequently, the column was eluted with hexane/dichloro-methane (7:3) to yield the second fraction. The PAHs and OCPs were contained in the second fraction, which was divided into two equal parts. These partitioned fractions were concentrated to 0.5 mL under a gentle nitrogen stream. 2.11. Analyses of PAHs and OCPs The fractions were analyzed by GC–MS on an Agilent Packard HP 6890 series II gas chromatography system fitted with an HP 5975, which was used in the selective ion monitoring (SIM) mode or in the scanning mode. A J&W Scientific DB-5MS fused-silica capillary column (30 m × 0.25 mm i.d. × 0.25 ␮m film thickness) was used. The column flow was 1 mL/min (helium). The samples were injected in the splitless mode. Splitless injection of a 1 ␮L sample was performed using an automatic HP 7683b Series injector (Palo Alto). For analysis of the PAHs, the injector and detector temperatures were 280 and 300 ◦ C, respectively. The oven temperature was maintained at 70 ◦ C for 1 min, increased to 260 ◦ C at 10 ◦ C/min and maintained for 4 min, and then increased to 300 ◦ C at 5 ◦ C/min and maintained for 4 min. For OCP analysis, the injector and detector temperatures were 230 and 250 ◦ C, respectively. The oven temperature was maintained at 80 ◦ C for 2 min, increased to 190 ◦ C at 15 ◦ C/min and maintained for 4 min, then increased to 230 ◦ C at 10 ◦ C/min and maintained for 5 min and, finally, increased to 290 ◦ C at 10 ◦ C/min for 6 min. 2.12. Quality control Surrogate standards were added to all of the samples prior to extraction to quantify the procedural recoveries. The recoveries of the surrogate standards were as follows: 80.1 ± 1.3% naphthalened8 , 85.2 ± 2.1% acenaphthene-d10 , 92.1 ± 4.3% phenanthrene-d10 , 93.4 ± 1.6% chrysene-d12 , 90.8 ± 8.1% perylene-d12 , 79.8 ± 11.3% DCB, and 87.6 ± 8.9% TCMX. Quantitation was achieved using the internal calibration method (five-point calibration). Final PAH and OCP concentrations were corrected using the recoveries of the surrogate standards. For each batch of 24 samples, a procedural blank (solvent with a clean GF/F filter), a spiked blank (16 PAH and 16 OCP standards spiked into solvent with a clean GF/F filter), a matrix spiked sample (16 PAH and 16 OCP standards spiked into pre-extracted soil), a matrix spiked duplicate, and a sample duplicate (n = 3) were processed. Procedural blanks (n = 3) contained traces of target chemicals, but the levels were less than 1.5% of the sample mass; therefore, they were not subtracted from the sample extracts. The recoveries of the spiking blanks and matrix spikes ranged from 80.0% to 95.2% for the PAHs and from 75.3% to 100.5% for the OCPs. Student’s t test was used to determine significant differences between different remediation modes. The significance level was set at p = 0.05.

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3. Results and discussion 3.1. PAH and OCP contents Fig. 1 shows the PAH and OCP concentrations in the soil sampled from the DDR bank. The concentration of PAHs (n = 16) was 6.35 ± 0.045 mg/kg. The concentration of PAHs containing 4–6 rings was higher (0.27–0.88 mg/kg) than the concentration of PAHs with 2–3 rings (0.011–0.031 mg/kg, except for Phe, with a value of 0.92 mg/kg). These results may be because low molecular weight PAHs are able to degrade and easily evaporate into the air or leak into deep soil, whereas higher molecular weight PAHs are less easily lost from the soil. This effect led to the shift of PAH homologue distribution to high molecular weight PAHs [20]. The 4–6 ring PAHs are carcinogenic, which implies that the soil from the DDR of Tianjin in northern China exhibits a high health risk. In addition, the total PAH concentrations in the soil were above the effects range low of 4022 ng/g [21], suggesting that the PAH concentration levels in soil from the DDR have a negative effect on organisms. Therefore, remediation for this contaminated soil is extremely necessary. Several OCPs that were targeted were not detected in the soil. Only heptachlor, aldrin, endrin, p,p -DDE p,p -DDD, and o,p -DDT were detected (Fig. 1). The total concentration of these OCPs was 145.92 ± 1.92 mg/kg. The concentration of endrin was higher than that of all the other OCPs, reaching 77.11 ± 5.12 mg/kg, whereas the other OCPs, such as heptachlor, aldrin, p,p -DDE, p,p -DDD,

2-3 rings

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Different rings PAHs Fig. 2. Remediation percentage of the total PAHs (a) and different rings PAHs (b) in the soils by tourmaline (T), P. chrysosporium (P) and tourmaline combined with P. chrysosporium (T + P). The error bars indicate standard deviations.

and o,p -DDT, had concentrations lower than 28.05 mg/kg. In this study, DDTs were detected in the soil samples, whereas the HCHs (HCHs) were not detected. This result is consistent with previous observations regarding OCP soil contamination in China [22]. The low soil concentrations of HCHs most likely result from the fact that HCHs have higher water solubilities, vapor pressures and biodegradabilities, whereas they display lower lipophicilities and particle affinities compared with DDTs [23]. DDTs tend to remain in the particulate phase longer than HCHs [24]. Although the agricultural application of OCPs was terminated in 1983, the OCP level detected indicated that OCP residues remain in the soil of the DDR of Tianjin in northern China. This result suggested that this residue is a typical characteristic of a historically contaminated soil. Therefore, the agricultural soil obtained from the DDR bank exhibits high risk, and remediation of the contaminated soil is urgent. 3.2. Remediation of PAHs and OCPs by tourmaline Figs. 2 and 3 illustrate the enhancement of the degrees of removal of the PAHs and OCPs by tourmaline and P. chrysosporium. For example, after 60 days, approximately 18.0 ± 1.9% of the initial amount of PAHs was lost in the control soil, whereas 31.9 ± 2.9% was removed in the tourmaline-added soil (Fig. 2a). Fig. 2b shows the removal percentage of the PAHs with differing numbers of rings in the soil samples after 60 days of treatment. The removal percentages of low-ring PAHs were significantly higher

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with only 16.8 ± 2.2% removal in control soil samples on the 60th day (Fig. 3a). Mineralization of PAHs in non-sterile soil inoculated with P. chrysosporium is significantly higher than that observed in either the P. chrysosporium-added soil or the soil alone because P. chrysosporium acted synergistically with the indigenous microorganisms in soil [25]. With regard to the effect of aromaticity on PAH degradation, two- and three-ring PAHs were unstable compared with PAHs having more than four rings (Fig. 2b). The removal percentage of low-ring PAHs was significantly higher that of high-ring PAHs in both the P. chrysosporium and control soils. However, the removal of high molecular weight PAHs was enhanced to a greater extent than the removal of low molecular weight PAHs by the combined technique, with a degradation rate of 3 times for 6-ring PAHs and a rate of 2 times for low molecular weight (2–3 rings) PAHs compared with the control soil. For every OCP, 29.1 ± 2.7% to 44.4 ± 3.4% of the initial concentration was removed from the P. chrysosporium-added soil, and the removal rate of p,p -DDE was the highest, obtaining a significantly higher value than that in the control soils (p < 0.05) (Fig. 3b).

p,p'-DDE p,p'-DDD o,p'-DDT

Individual OCPs Fig. 3. Remediation percentage of the total OCPs (a) and individual OCPs (b) in the soils by tourmaline (T), P. chrysosporium (P) and tourmaline combined with P. chrysosporium (T + P). The error bars indicate standard deviations.

than those of high rings PAHs in both the tourmaline and control soil samples. However, the removal percentages of high molecular weight PAHs were enhanced by tourmaline to a greater extent than that of low molecular weight PAHs by tourmaline, with double the a removal rates for 6-ring PAHs and 1.6 times the removal rate for low molecular weight (2–3 rings) PAHs increased 1.6 times compared with the control soil. Fig. 3a shows that approximately 16.8 ± 2.2% of the initial amount of OCPs was lost in the control soil samples, whereas the removal was approximately 26.4 ± 1.8% in the tourmaline-added soil. Decreases in heptachlor, aldrin, endrin, p,p -DDE p,p -DDD, and o,p -DDT in tourmaline-added soil samples after 60 days of treatment ranged from 24.1 ± 2.8% to 38.0 ± 2.6%, with p,p -DDE having the highest removal rate, whereas the removal rates of while that 13.3 ± 5.1 to 21.5 ± 1.3% of the initial amount of other OCPs, except for aldrin, were achieved in the control soil samples (Fig. 3b). In addition, the removal rates of the individual OCPs in the tourmaline-added soil were significantly higher than those of the corresponding OCP compounds in the control soil (p < 0.05). Therefore, tourmaline enhanced the removal of PAHs and OCPs.

Figs. 2 and 3 show the degradation of PAHs and OCPs observed when soils containing P. chrysosporium and tourmaline. Higher PAH degradation rates were observed when the tourmaline-added soils were incubated with P. chrysosporium (53.2 ± 4.7%) than when only tourmaline was added to the soil (31.9 ± 2.9% removed) or when only P. chrysosporium was added (40.5 ± 2.3% removed) (Fig. 3). Similar differences were observed for the degradation of OCPs with both tourmaline- and P. chrysosporium- added soil, exhibiting a 43.5 ± 3.1% removal percentage compared with 26.4 ± 1.8% for tourmaline-only soil and 34.2 ± 3.9% for P. chrysosporium–only soil on the 60th day (Fig. 3). The removal percentage of low-rings PAHs was significantly higher than that of high-ring PAHs in the simultaneous presence of tourmaline and P. chrysosporium and in the control soils. However, the removal of high molecular weight PAHs was enhanced to a greater extent than that of low molecular weight PAHs by the combined technique, with removal rates of 4 times for 6-ring PAHs and of 2.7 times for low molecular weight (2–3 rings) PAHs compared with the control soil (Fig. 2b). In addition, the removal of OCPs with different structures significantly increased in the simultaneous presence of tourmaline and P. chrysosporium (Fig. 3b) compared with other treatments such as tourmaline-only soil and P. chrysosporium-only soil (p < 0.05). Therefore, we concluded that the combined effects of tourmaline and microorganisms increase the degradation of PAHs and OCPs. Most of previously published studies have focused on the removal of PAH and OCP contaminants in spiked soil. The present study is the first in which real concentrations of PAHs and OCPs in field agricultural soil have been remediated using this new technology that combines tourmaline with P. chrysosporium, and the results show that tourmaline can enhance both PAH and OCP remediation efficiencies. Although tourmaline is used widely in health care, behavior in soil is not well known. Therefore, we investigated the role of tourmaline during soil remediation. 3.5. Role of tourmaline during soil remediation

3.3. Bioremediation of PAHs and OCPs The total remediation rate of PAHs in the soil to which P. chrysosporium was added reached 40.5 ± 2.3%, which was higher than that obtained in the control soil on the 60th day (18.0%) (Fig. 2a). Approximately 34.2 ± 3.9% of the total OCP content was removed in soil samples to which fungus was added, compared

3.5.1. Effect of tourmaline on soil enzyme activities Certain studies have reported positive correlations between most soil enzyme activities and microbial biomass [26,27]. In the present study, soil enzyme activities were tested during the degradation of PAHs and OCPs in the soil to provide a better understanding of the link between tourmaline and soil microbial

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biomass. Fig. 4 depicts the changes in the activities of invertase and hydrogen peroxidase during the remediation of PAHs and OCPs. In general, the soil enzyme activities increased rapidly during the initial stage of remediation, then slowly decreased after the 20th day. The maximum invertase activity values obtained on the 20th day were 140.2 ± 6.2, 189.9 ± 12.2, 196.9 ± 4.2, and 246.9 ± 13.0 IU/g in the control soil, tourmaline-added soil, P. chrysosporium-added soil and P. chrysosporium and tourmalineadded soil, respectively (Fig. 4a). Similarly, the maximum hydrogen peroxidase activity values on the 20th day were 12.5 ± 1.1, 19.2 ± 0.4, 19.8 ± 0.8 and 25.9 ± 0.7 IU/g, respectively (Fig. 4b). The values in tourmaline-added soil contained tourmaline were significantly higher than those obtained in the control soil (p < 0.05). Thus, tourmaline can stimulate the generation of invertase and hydrogen peroxidase, which indicated that the number or growth of microorganisms caused an increase in the level of invertase and hydrogen peroxidase. Furthermore, the microorganisms directly involve or act synergistically with P. chrysosporium to enhance the degradation of PAHs and OCPs. 3.5.2. Effect of tourmaline on the composition of the soil bacterial community To further investigate the PAH and OCP removal mechanisms and evaluate the effect of tourmaline on the variation in the soil micro-ecosystem at the end of the experiment, the genetic structures of indigenous soil microorganism communities were evaluated by molecular methods based on DGGE fingerprinting of PCR-amplified 16S rDNA sequences from soil-extracted DNA. The

a

Fig. 5. DGGE fingerprint file (a) and lane comparison (b) of bacterial populations in soil samples of microcosms after 60 days of treatment: (1) control soil (C-s), (2) tourmaline-added soil (T-s).

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Invertase (IU/g)

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b Hydrogen peroxidase (IU/g)

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24 20 16 12 10

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Time (d) Fig. 4. Activity change time courses for invertase (a) and hydrogen peroxidase (b) enzymes during the PAH and OCP degradation processes.

PCR–DGGE technique is widely used to monitor changes in soil microflora due to pollution and changes in agricultural practices [28,29]. In the present study, DGGE profiles of the control soil (Cs) and tourmaline-added soil (T-s) after a 60-day remediation are shown in Fig. 5. The control soil showed six bands consisting of the main bacterial population (as seen in Fig. 5b), therefore, revealed a less diverse community. Significant increases in population structures were indicated for T-s microcosms, as demonstrated by the appearance of new bands (Fig. 5b). Upon analysis of the C-s and T-s microcosms, clearly differentiated clusters were observed. The results showed that the similarity value of the microorganism communities from the T-s and C-s samples was 69.5%. These results indicated a significant increase in the numbers of bands in the T-s microcosms compared with those present in the control soil, suggesting that tourmaline can greatly increase the indigenous bacterial community. The degradation data of PAHs and OCPs reveal that their degradation percentage in the P + T remediation group was higher than those tourmaline-only and P. chrysosporium-only groups, and the tourmaline-only group exhibited a higher degradation percentage than the soil control group. Therefore, we further concluded that the large population increase in soil bacterial communities after the addition of tourmaline may result in increases in PAH and OCP degradation in T-s remediation groups compared with control soil. These results were sufficient proof that tourmaline addition activated native microorganisms in the soil during remediation. These activated microorganisms were likely to act as direct strains or to act synergistically with P. chrysosporium to degrade PAHs and OCPs. However, specific organisms in the clusters of the PCR–DGGE profile responsible for PAH and OCP degradation or joint effects among activated native microorganisms, tourmaline and P. chrysosporium require further discussion. Zhang et al. [30] have reported that tourmaline could

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Fig. 6. Kinetics of PAH (a) and OCP (b) degradation in aqueous solutions by tourmaline (T), P. chrysosporium (P) and tourmaline combined with P. chrysosporium (T + P). The error bars indicate standard deviations.

30

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a

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Time (d) Fig. 7. Effect of the addition of tourmaline on PAH (a) and OCP (b) -degraders in the soil at different remediation days. The error bars indicate standard deviations.

enhance the number and activity of the aniline-degrading bacteria immobilized on the mycelial pellet. In the present study, specific organisms in the clusters of the PCR–DGGE profile responsible for PAH and OCP degradation were not discussed. However, these results indicate that tourmaline is effective for the remediation of PAHs and OCPs-contaminated soil and provides valuable information about the behavior of the native microorganism population during bioremediation, which can be used in the risk assessment of soil microorganism population and new technology of bioremediation. 3.5.3. Joint effects of tourmaline and P. chrysosporium The importance of the mutual relationships in the tourmaline and P. chrysosporium in the PAH and OCP degradation was demonstrated. Indeed, 66.2 ± 1.9% of the initial concentration was removed from the tourmaline and P. chrysosporium-added aqueous solutions after 192 h of incubation, only 38.3 ± 2.1% of the total OCP content was removed to which P. chrysosporium was added, compared with only 18.5 ± 1.3% removal in the tourmaline-added aqueous solution (Fig. 6a). Therefore, the sum of PAH removal percentage in the tourmaline-only and P. chrysosporium-only systems is lesser than that of tourmaline combined with P. chrysosporium, which proves that synergistic effect was present in the combined process. For every OCP, the total OCP removal was 45.2 ± 3.2% in the system added tourmaline and P. chrysosporium after 192 h of incubation (Fig. 6b). By comparison, the OCP removal of 19.5 ± 2.5% was

recorded in the tourmaline-only system, whereas the removal of 33.2 ± 2.1% took place in the P. chrysosporium-only system. Therefore, the tourmaline combined with P. chrysosporium treatment was more efficient in PAH elimination than either one alone. The disappearance of an amount of PAHs and OCPs in the system added tourmaline and P. chrysosporium could be explained by the addition of tourmaline enhancing the P. chrysosporium growth. 3.5.4. PAH and OCP degrader MPN The impact of the addition of tourmaline on the number of PAH and OCP organisms present at different days was investigated using the modified MPN technique in this study (Fig. 7). Fig. 7a shows the PAH-degraders in the tourmaline-added soil at different days ranged between 6.6 and 7.2 log 10 CFUs/g dry soil, while the control soil ranged between 6.0 and 6.3 log 10 CFUs/g dry soil. Thus, the population of PAH-utilising microorganisms also increased significantly in soil added tourmaline compared with the control soil (p < 0.05). Fig. 7b shows the OCP-degraders at different days ranged from 5.2 to 5.6 log 10 CFUs/g dry soil added tourmaline, while the control soil ranged between 4.8 and 5.1 log 10 CFUs/g dry soil. Therefore, the addition of tourmaline can effectively enhance the number of PAH and OCP- degrading microorganisms. 3.5.5. Effect of tourmaline on soil humic acid To gain further evidence that tourmaline plays a key role in this new remediation technique, we analyzed changes in soil HA levels due to tourmaline addition. Before and after remediation, soil

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demonstrate the great potential of tourmaline use in soil remediation.

1.2 control Tourmaline

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Acknowledgments

0.6

This work was supported by the Ministry of Science and Technology of China (2014CB441104) and Tianjin Key Programme of Basic Research (13JCYBJC20200 and 10JCZDJC24200), and National Natural Science Foundation of China (2090702 and 441225014).

m t /m

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Time (d) Fig. 8. Time courses of relative mass of humic acids in tourmaline-added and control soil samples during remediation. (Note: to evaluate the effect(s) of tourmaline on soil properties, all mt /mo values were obtained by calculating the ratios of extractable HA mass values at remediation time t to the HA mass of the initial soil sample(s).

HAs were extracted, and their relative contents (the ratio of the HA mass from the tourmaline-added soil to the HA mass in the initial soil sample) were determined (see Fig. 8). When the remediation time was 0, 20, 40 and 60 days, the HA relative contents in the tourmaline-added soil were 0.98, 0.84, 0.73 and 0.50, respectively, whereas these values ranged from 0.99 to 0.86 in the control soil. Thus, the soil HA level was decreased as the remediation time increased. These results suggested that tourmaline addition reduced the soil HA content. A decrease in the HA level might result from the microbial degradation of HA by the native microorganisms that were increased by tourmaline. Mishra and Srivastava [31] noted that the humic acid in a forest soil sample could be degraded by nine fungi isolated from a forest soil sample from Palamau (Bihar). Soils with higher SOM content typically contain more active sorption sites and combine more strongly with hydrophobic organic contaminants (HOCs), thereby limiting their mobility and bioavailability [9,31]. HAs are the most abundant and most chemically and biochemically active fractions of the SOM. Adsorption/desorption of POPs onto soil HAs is generally considered one of the most important processes that controls the behavior of POPs and their fate in the soil, including their mobility, transport, accumulation, and bioavailability [32]. Thus, the decrease in the HA level after tourmaline was added to the soil further facilitated the release of HOCs from the HAs in the soil. As a result, soil samples contaminated with PAHs and OCPs were easily remediated using tourmaline, as demonstrated by remediation rates that were higher than those obtained for control soil samples. Given that tourmaline is a novel material, further study is required to understand the mechanisms by which it improves the remediation efficiency of PAHs and OCPs.

4. Conclusions To our knowledge, this study is the first report demonstrating that the combination of tourmaline and a microorganism could be applied to remove PAHs and OCPs from field agricultural soil. The results showed that tourmaline may act synergistically with microorganisms to enhance the biodegradation of PAHs and OCPs at sites contaminated with these chemicals. Further studies have shown that tourmaline can improve the soil micro-ecosystem, enhance soil enzyme activities and PAH and OCP-degraders, and decrease the soil HA content. Thus, the results contained herein

[1] A. Zhang, L. Fang, J. Wang, W. Liu, H. Yuan, L. Jantunen, Y. Li, Residues of currently and never used organochlorine pesticides in agricultural soils from Zhejiang Province, Chin. J. Agric. Food Chem. 60 (2012) 2982–2988. [2] A. D Annibale, M. Ricci, V. Leonardi, D. Quarantino, E. Mincione, M. Petruccioli, Degradation of aromatic hydrocarbons by white-rot fungi in a historically contaminated soil, Biotechnol. Bioeng. 90 (2005) 723–731. [3] P.S.R. Prasad, Study of structural disorder in natural tourmalines by infrared spectroscopy, Gondwana Res. 8 (2005) 265–270. [4] C. Wang, J. Liu, Z. Zhang, B. Wang, H. Sun, Adsorption of Cd(II), Ni(II), and Zn(II) by tourmaline at acidic conditions: Kinetics, thermodynamics, and mechanisms, Ind. Eng. Chem. Res. 51 (2012) 4397–4406. [5] M. Xia, C. Hu, M. Zhang, Effects of tourmaline addition on the dehydrogenase activity of Rhodopseudomonas palustris, Process. Biochem. 41 (2006) 221–225. [6] H. Ni, L. Li, H. Li, Tourmaline ceramic balls stimulate growth and metabolism of three fermentation microorganisms, World J. Microb. Biot. 24 (2008) 725–731. [7] T. Iwabuchi, S. Harayama, Biochemical and molecular characterization of 1hydroxy-2-naphthoate dioxygenase from Nocardioides sp KP7, J. Biol. Chem. 273 (1998) 8332–8336. [8] Y. Yang, W. Hunter, S. Tao, J. Gan, Microbial availability of different forms of phenanthrene in soils, Environ. Sci. Technol. 43 (2009) 1852–1857. [9] Y. Yang, N. Zhang, M. Xue, S. Lu, S. Tao, Effects of soil organic matter on the development of the microbial polycyclic aromatic hydrocarbons (PAHs) degradation potentials, Environ. Pollut. 159 (2011) 591–595. [10] K. Nam, N. Chung, M. Alexander, Relationship between organic matter content of soil and the sequestration of phenanthrene, Environ. Sci. Technol. 32 (1998) 3785–3788. [11] G. Liu, N. Jiang, L. Zhang, Z. Liu, Soil Physical and Chemical Analysis and Description of Soil Profiles, Standards Press of China, Beijing, 1996, In Chinese. [12] K.L. Zhou, The Science of Soil Enzymes, The Science Press, Beijing, 1987, In Chinese. [13] T.M. LaParaa, C.H. Nakatsu, L.M. Pantea, J.E. Alleman, Stability of the bacterial communities supported by a seven-stage biological process treating pharmaceutical wastewater as revealed by PCR–DGGE, Water Res. 36 (2002) 638–646. [14] P. Garbeva, J. Van Veen, J. Van Elsas, Predominant Bacillus spp. in agricultural soil under different management regimes detected via PCR–DGGE, Microbiol. Ecol. 45 (2003) 302–316. [15] X. Tao, G. Lu, Z. Dang, C. Yang, X. Yi, A phenanthrene-degrading strain Sphingomonas sp. GY2B isolated from contaminated soils, Process Biochem. 42 (42) (2007) 401–408. [16] B.A. Wrenn, A.D. Venosa, Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure, Can. J. of microbial. 42 (1996) 252–258. [17] ISO 4831, Microbiology-General Guidance for the Enumeration of ColiformsMost Probable Number Technique, International Organization for Standardization, Geneva, 1991. [18] S.D. Kohl, J.A. Rice, The binding of contaminants to humin: a mass balance, Chemosphere 36 (1998) 251–261. [19] J.W. Kelsey, I.B. Slizovskiy, R.D. Peters, A.M. Melnick, Sterilization affects soil organic matter chemistry and bioaccumulation of spiked p,p -DDE and anthracene by earthworms, Environ. Pollut. 158 (2010) 2251–2257. [20] L. Ma, S. hu, X. Wang, H. Cheng, X. Liu, X. Xu, Polycyclic aromatic hydrocarbons in the surface soils from outskirts of Beijing, China, Chemosphere 58 (2005) 1355–1363. [21] E.R. Long, D.D. Macdonald, S.L. Smith, F.D. Calder, Incidence of adverse biological effects with in ranges of chemical concentrations in marine and estuary sediments, J. Environ. Manage. 19 (1995) 81–97. [22] A.S. Muhayimana, S. Qi, Y. Wang, X. Kong, O. Owago, J. Zhang, Distribution and sources of organochlorine pesticides (OCPs) in Karst Cave, Guilin, China, Acad. Arena 1 (2009) 47–56. [23] R. Yang, G. Jiang, Q. Zhou, C. Yuan, J. Shi, Occurrence and distribution of organochlorine pesticides in sediments collected from East China Sea, Environ. Int. 31 (2005) 799–804. [24] D.D. Nhan, F.P. Carvalho, N.M. Am, N. Tuan, N. Yen, J. Villeneuve, Chlorinated pesticides and PCBs in sediments and molluscs from fresh water canals in the Hanoi region, Environ. Pollut. 112 (2001) 311–320. [25] Z. Zheng, J.P. Obbard, Oxidation of polycyclic aromatic hydrocarbons (PAH) by the white rot fungus, Phanerochaete chrysosporium, Enzyme Microb. Technol. 31 (2002) 3–9.

448

C. Wang et al. / Journal of Hazardous Materials 264 (2014) 439–448

[26] J.E. Hassett, D.R. Zak, Aspen harvest intensity decreased microbial biomass and extracellular enzyme activity, and soil nitrogen cycling, Soil Sci. Soc. Am. J. 69 (2005) 227–235. [27] X. Tan, S. Chang, R. Kabzems, Soil compaction and forest floor removal reduced microbial biomass and enzyme activities in a boreal aspen forest soil, Biol. Fertil. Soils 44 (2008) 471–479. [28] G. Muyzer, E.C. de Waal, A.G. Uitterlinden, Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction genes coding for 16S rRNA, Appl. Environ. Microbiol. 59 (1983) 695–700. [29] H. Heuer, M. Krsek, P. Baker, K.W. Smalla, M.H. Elizabeth, Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and

gel-electrophoretic separation in denaturing gradients, Appl. Environ. Microbiol. 63 (1997) 3233–3241. [30] S. Zhang, A. Li, D. Cui, J. Yang, F. Ma, Performance of enhanced biological SBR process for aniline treatment by mycelial pellet as biomass carrier, Bioresour. Technol. 102 (2011) 4360–4365. [31] B. Mishra, L.L. Srivastava, Degradation of humic acid of a forest soil by some fungal isolates, Plant Soil 6 (1986) 413–416. [32] C. Wang, H. Sun, J. Li, Y. Li, Q. Zhang, Enzyme activities during degradation of polycyclic aromatic hydrocarbons by white rot fungus Phanerochaete chrysosporium in soil, Chemosphere 77 (2009) 733–738.