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Effects of electrokinetic operation mode on removalof polycyclic aromatic hydrocarbons (PAHs), and theindigenous fungal community in PAH-contaminated soilJian Wang a , Fengmei Li b , Xu Li a , Xiujuan Wang a , Xinyu Li a , Zhencheng Su a , HuiwenZhang a & Shuhai Guo ba State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, ChineseAcademy of Sciences , Shenyang , P. R. Chinab Institute of Applied Ecology, Chinese Academy of Sciences , Shenyang , P. R. ChinaPublished online: 15 Aug 2013.

To cite this article: Jian Wang , Fengmei Li , Xu Li , Xiujuan Wang , Xinyu Li , Zhencheng Su , Huiwen Zhang & ShuhaiGuo (2013) Effects of electrokinetic operation mode on removal of polycyclic aromatic hydrocarbons (PAHs), and theindigenous fungal community in PAH-contaminated soil, Journal of Environmental Science and Health, Part A: Toxic/HazardousSubstances and Environmental Engineering, 48:13, 1677-1684, DOI: 10.1080/10934529.2013.815500

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Journal of Environmental Science and Health, Part A (2013) 48, 1677–1684Copyright C© Taylor & Francis Group, LLCISSN: 1093-4529 (Print); 1532-4117 (Online)DOI: 10.1080/10934529.2013.815500

Effects of electrokinetic operation mode on removalof polycyclic aromatic hydrocarbons (PAHs), and theindigenous fungal community in PAH-contaminated soil

JIAN WANG1, FENGMEI LI2, XU LI1, XIUJUAN WANG1, XINYU LI1, ZHENCHENG SU1,HUIWEN ZHANG1 and SHUHAI GUO2

1State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang,P. R. China2Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, P. R. China

Electrokinetic remediation is an emerging physical remediation technology for the removal of heavy metals and organic chemicalsfrom contaminated soil. We set up a soil chamber (24 × 12 × 8 cm) with two stainless steel electrodes (12 × 0.5 cm), and a constantvoltage gradient of 1.0 v cm−1 or 2.0 v cm−1 was applied to study the effects of unidirectional and altered directional electric fieldoperation modes on the moisture content and pH, the removal rate of PAHs, and the abundance and diversity of indigenous fungiin a PAH-contaminated soil at the Benxi Iron and Steel Group Corporation (N41◦17′24.4′′, E123◦43′05.8′′), Liaoning Province,Northeast China. Electrokinetic remediation increased the PAH removal rate, but had less effect on soil moisture content and pH,in comparison with the control. In the 1 v cm−1 altered directional operation, in particular, the PAH removal rate by the end of theexperiment (on day 23) had increased from 5.2% of the control to 13.84% and 13.69% at distances of 4 and 20 cm from the anode,respectively, and to 18.97% in the middle region of the soil chamber. On day 23, the indigenous fungal 18S rRNA gene copy numbersand community diversity were significantly higher in a voltage gradient of 1 v cm−1 than in a voltage gradient 2 v cm−1. An altereddirectional operation was more conducive to the fungal community’s uniform distribution than was a unidirectional operation ofthe electric field. We found the major PAH-degrading fungi Fusarium oxysporum and Rhizophlyctis rosea to be present under EKremediation. We suggest that a 1 v cm−1 altered directional operation could be an appropriate electrokinetic operation mode for PAHremoval, and the maintenance of abundance and diversity of the indigenous fungal community.

Keywords: electrokinetic remediation, electric field, fungi, electric intensity, PAHs.

Introduction

Soil pollution is an increasing environmental problem.Polycyclic aromatic hydrocarbons (PAHs) are the com-mon environmental pollutants produced by incompletecombustion and pyrolysis of organic matter, and they havegained widespread attention because of their carcinogenic,teratogenic, and mutagenic effects,[1,2] and their highhydrophobicity and strong sorption to soil particles.[3]

Bioremediation, because of its cost-effectiveness andenvironmental acceptability, has been shown to be apractical method of remedying PAH-contaminated soil.[4,5]

However, this approach has many limiting factors, suchas the insufficient bioavailability of the contaminants.[6]

Address correspondence to Zhencheng Su, State Key Labora-tory of Forest and Soil Ecology, Institute of Applied Ecology,Chinese Academy of Sciences, Shenyang, P. R. China; E-mail:zhenchengsu@yahoo.com.cnReceived December 7, 2012.

Electrokinetic (EK) remediation can overcome some of thelimitations associated with bioremediation, and is usefulin treating organic pollutant-contaminated soils.

EK remediation is an emerging physical remediationtechnology for the removal of heavy metals and organicchemicals from contaminated sites.[7,8] Its process consistsof electroosmosis, electromigration, and electrophoresis.Electroosmosis can lead to the migration of soil moisture, [9]

whereas electromigration and electrophoresis together canresult in soil ions, ion complexes, and charged particles in-cluding microbes moving toward the field region oppositein polarity.[10–14] Under different EK operation modes, soilproperties such as pH, temperature, and moisture contentcan be changed, [15] and the direction of movement of soilmatters and microbes can be altered; encouraging the mi-crobes and contaminants in soils to contact and interactwith each other. [11–14]

EK remediation has been combined with other ap-proaches to enhance the removal efficiencies of both or-ganic contaminants and heavy metals,[16–18] and successful

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attempts have been made on clay soil, red soil, and kaolinsoil.[19–21]

Some studies have examined the effects of EK remedi-ation on soil bacterial communities,[22] cell number, anddiversity in contaminated soils.[23] However, the effects ofEK remediation on soil fungal activity have not been ascer-tained, and there has been little analysis of the changes inthe fungal community during EK remediation under dif-ferent electric field operation modes and electric intensitiesin PAH-contaminated soils. Many soil fungi can diffusethrough the soil and release extracellular ligninolytic en-zymes,[24] and may metabolize PAHs pollutants.

Our objective was to explore the effects of both uni-directional and altered directional electric field operationmodes in different voltage gradients, on the remediation ofPAH-contaminated soil and the soil fungal community ata laboratory scale.

Materials and methods

Soil

The test soil (clay loam) (0–10 cm in depth) was collectedfrom a woodland at Benxi Iron and Steel Group Corpo-ration (N41◦17′24.4′′, E123◦43′05.8′′), Liaoning Province,Northeast China. The soil had 20% moisture content, pH8.36, 1.77% organic matter, 0.09% total nitrogen, 0.04% to-tal phosphorus, 64.85% sand, 21.86% silt, 13.29% clay, anda total of 220.01 mg/Kg of PAHs (among which, four- tosix-ring PAHs and light PAH phenanthrene accounted for92.19% and 7.81% of total PAHs, respectively). The con-centration and composition of individual PAHs are listedin Table 1. The soil was air dried and sieved through a 2-mmmesh.

Table 1. Concentrations and composition of PAHs.

Polycyclic aromatic Content Percentagehydrocarbons Rings (mg kg−1) (%)

Phenanthrene (Phe) 3 17.19 ± 0.034 7.81Fluoranthene (Flu) 4 36.49 ± 0.225 16.59Pyrene (Pyr) 4 29.50 ± 0.541 13.41Benzo(a)anthracene (BaA) 4 9.41 ± 0.336 4.28Chrysene (Chr) 4 18.93 ± 0.185 8.61Benzo(b)fluoranthene (BbF) 5 29.52 ± 0.346 13.42Benzo(k)fluoranthene (BkF) 5 11.91 ± 0.432 5.41Benzo(a)pyrene (BaP) 5 21.92 ± 0.234 9.96Indeno(1,2,3-cd)pyrene

(IcdP)5 3.00 ± 0.196 1.36

Dibenzo(a,h)anthracene(DahA)

6 21.66 ± 0.098 9.85

Benzo(g,h,i)perylene (BghiP) 6 20.47 ± 0.076 9.3Total PAHs 220.01 mg/Kg

Fig. 1. Experimental setup and sampling positions.

Electrokinetic equipment setup

A soil chamber (24 × 12 × 8 cm) was set up as shownin Figure 1. The soil was packed into the chamber, with0.1 kg/cm2 compaction pressure for 12 h. Two stainlesssteel electrodes (12 × 0.5 cm) were installed, and constantvoltage gradients of 1.0 v cm−1 and 2.0 v cm−1 were ap-plied. The electric field modes consisted of uni- and altereddirectional operations, and the EK equipment was capableof polarity reversal intervals in the electric field for 12 h.The in situ soil was in a soil reactor 4, 8, 12, 16, 20 cm fromanode, for a total of five sampling sites. Fifteen soil sampleswere taken for analyses along the length of three parallelsampling lines on days 0 and 23. Samples at the same siteson the three sampling lines were mixed together to formone composite sample before analysis. The control soil inanother soil reactor was similarly set up, without applica-tion of electrical current. The temperature was controlledat 25 ± 1◦C by air conditioning.

Soil moisture content, pH, and PAH assays

Soil pH, moisture content, and PAH concentrations wereanalyzed in triplicate. Soil pH was measured using a pHprobe, and soil moisture content was determined by leav-ing the soil in an oven at 110◦C for 24 h. The extraction ofPAHs was performed following the EPA Standard Method3550C.[25,26] The concentrations of 16 PAHs (US EPA pri-ority PAHs) were determined using a high performance liq-uid chromatograph (HPLC, Waters, Milford, MA, USA)equipped with a variable wavelength fluorescence detector(FLD, Waters 2475) and a Waters PAH Column (250 ×4.6 mm internal diameter, 5-µm particle size). Prior to in-jection, the extracted PAHs were filtered through a 0.22-µmTeflon filter (Sigma, Munich, Germany). The injection vol-ume was set at 10.0 µL, and the column temperature was25.0◦C. The gradient elution program consisted of 60% wa-ter and 40% acetonitrile for 2 min, and then the programwas changed to 100% acetonitrile in 12 min at a flow rate of1.0 mL min−1. All data in the Figures 2, 3 and 4 are means± standard deviations, calculated using SPSS for Windowsv. 13.0 (IBM, Rosemead, CA, USA).

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Fig. 2. Soil moisture content on day 23 of the experiment.

Identification and quantification of PAH compoundswere based on matching their retention times with a mixtureof soil PAHs and individual PAH standards. The procedu-ral blank (individual PAH standards) was determined bygoing through the same extraction. The mean recovery ofPAHs in the mixture was from 78% (BbF) to 98% (BaP),and the standard curves were linear in the concentrationrange of 0.01–0.49 ng kg−1.

The percentage PAH removal rate (R%) was calculatedusing the formula: R% = 100% (MI − MF)MI−1, whereMI is the initial concentration of total PAHs, and MF is thefinal concentration of total PAHs in each treatment after23 days’ incubation.

Fig. 3. PAH removal rate on day 23 of the experiment.

Fig. 4. 18S rRNA gene copy number on day 23 of the experiment.

PCR and DGGE assays

The fungal community structure was examined using de-naturing gradient gel electrophoresis (DGGE) analysis ofPCR-amplified 18S rRNA gene fragments. The 18S rRNAgene fragments were PCR-amplified using the universalfungal primers FF390 (5′-CGA TAA CGA ACG AGACCT-3′) and FR1 (5′-AIC CAT TCA ATC GGT AIT-3′),containing a GC clamp.[27] For each sample being ampli-fied, the following were required: 5 µL 10 × PCR buffer,200 µM dNTP mixture, 10 µM of each primer, 1 µL tem-plate DNA, and 2.5 U Taq polymerase enzymes. The PCRcycle conditions were 94◦C for 8 min, then 30 cycles at95◦C for 30 s, 50◦C for 45 s, and 72◦C for 2 min, and afinal extension at 72◦C for 10 min. The PCR products wereanalyzed by DGGE, using a Biorad system. The sampleswere run on 8% polyacrylamide gels with a denaturing gra-dient from 40% to 60% (100% denaturant gels containing7 M urea and 40% formamide). Electrophoresis was runin a 1×TAE buffer (40 mM Tris–HCl, 40 mM acetic acid,1 mM EDTA, pH 8.0) at a voltage of 75 V for 16 h at aconstant temperature of 60◦C.

The PCR-DGGE banding patterns were analyzedusing Quantity-One image analysis software (Bio-rad,Hercules, CA, USA). The cluster analysis of DGGE pat-terns was completed using UPGMA between different sam-ples. The fungal 18S rRNA sequences of excised DGGEbands were searched for in the GenBank database us-ing the BLAST program (NCBI, Betnesda, MD, USA).On the basis of the BLAST results, highly similar Gen-Bank sequences and the sequences of excised DGGE bandswere added to the data set for multiple sequence align-ment, and a phylogenetic distance tree was constructedusing MEGA 4.0 (Arizona State University, Tempe, AZ,USA).

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Quantitative PCR assays

The total DNA was extracted from 0.5 g soil samples (wetweight) using the Fast DNA R© spin kit for soil (Bio 101,Santa Ana, CA, USA). The copy number of 18S rRNAgenes in soil DNA samples was estimated by quantitativePCR (qPCR), using the universal fungal primers FF390(5′-CGA TAA CGA ACG AGA CCT-3′) and FR1 (5′-AIC CAT TCA ATC GGT AIT-3′). [26] The qPCR wasperformed in a 25 µl volume using the SYBR R©PremixEx TaqTM II kit with SYBR green I (Takara, Japan) onan ABI 7000 Real-Time PCR detection system (AppliedBiosystems, Foster City, CA, USA). The PCR amplifica-tion procedure was 95◦C for 30 s, then 40 cycles at 95◦C for10 s, 50◦C for 30 s, and 72◦C for 45 s, with a final extensionat 72◦C for 5 min.

Results and discussion

Soil moisture content and pH

The soil moisture content in the control on days 1 and23 was 20% (w/w) and 19.3%, respectively. Different EKoperation modes induced changes in soil moisture content(Fig. 2). Under unidirectional operation, the soil moisturecontent increased with increasing distance from the anode,while under altered directional operation, the moisturecontent showed a uniform distribution. In a differentvoltage gradient, the soil moisture content near the cathodewas less changed by the same electric field modes. Under 2v cm−1 unidirectional operation, the soil moisture contenton day 23 changed no more than 5%, except in the region ofthe anode. No statistically significant difference (P = 0.068)was observed in the soil moisture content under differentEK operation modes, in comparison with the control.As a result of the strong buffering capacity, the soil pHunder different electric field operation modes and electricintensities showed less change by day 23 (data not shown).Our results suggest that different EK operation modes hadlesser effects on the test soil moisture content and pH.

PAH removal rate

Figure 3 shows that the removal rate in the electric fieldsof uni- and altered directional operations significantly in-creased, compared with the control (value is 5.2%). Duringincubation and electrokinetic process, high-ring PAHs weredegraded into low-ring PAHs, resulting in an increase inlow-ring PAHs. In addition to the complicated processes ofPAHs degradation under natural conditions, it is difficultto differentiate the effects of electrokinetic operation andmicrobial degradation. Therefore, the total removal rateof PAHs was determined; total PAH concentration after23 days’ incubation is shown in Table 2. Under altered di-rectional operation, the removal rate of PAHs was basicallythe same in the bilateral region, and peaked in the middleregion (near anode 12 cm).

Table 2. Total PAH concentrations after 23 days of incubation.

Electrokinetic Sample Total PAHsoperation site (mg kg−1)

1 v cm−1 4 cm 214.89 ± 0.314Unidirection operation 8 cm 197.65 ± 0.204

12 cm 187.82 ± 0.42216 cm 189.82 ± 0.37220 cm 200.02 ± 0.225

1 v cm−1 4 cm 189.56 ± 0.273Altered direction operation 8 cm 186.31 ± 0.125

12 cm 178.28 ± 0.30516 cm 181.73 ± 0.24920 cm 189.88 ± 0.358

2 v cm−1 4 cm 205.84 ± 0.266Unidirection operation 8 cm 196.57 ± 0.319

12 cm 193.36 ± 0.31916 cm 194.50 ± 0.34220 cm 191.63 ± 0.134

2 v cm−1 4 cm 196.66 ± 0.246Altered direction operation 8 cm 189.36 ± 0.304

12 cm 182.23 ± 0.11616 cm 191.51 ± 0.26720 cm 194.13 ± 0.448CK23 208.57 ± 0.037

Under unidirectional operation, the removal of PAHswas relatively small in the region near the anode, but grad-ually increased and peaked in the middle region. Under 1 vcm−1 altered directional operation, the PAH removal ratewas higher than in the other treatments, showing a highdegradation rate (18.97%) in the middle region, and about13.84% and 13.69% at 4 and 20 cm from the anode, re-spectively. This indicates that a 1 v cm−1 altered directionaloperation can effectively remove PAHs from the soil. Pre-vious studies have also revealed that using alternating po-larity electrokinetics can enhance the removal of phenolsin unsaturated soil. [14]

Soil fungal number

In the control, the log copy number of fungal 18S rRNAsequences per gram of soil on day 23 of the experiment was8.61. Figure 4 shows that in a voltage gradient of 1 v cm−1,the fungal 18S rRNA copy number was significantly higherthan that in a voltage gradient of 2 v cm−1, and increasedmarkedly with increasing distance from the anode under al-tered directional operation, but showed the opposite trendunder unidirectional operation. In a voltage gradient of 2v cm−1, the fungal 18S rRNA copy number showed lesserdifferences between uni- and altered directional operations.These findings suggest that higher electric intensity couldhave lethal effects on soil microbes, and that compared withunidirectional operation, an altered directional operationcould encourage fungi into a more uniform distribution.

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Fig. 5. A: ISU1 and ISR1 refer to the initial soil sample under 1 v cm−1 uni- and altered directional operations, ISU2 and ISR2 referto the initial soil sample under 2 v cm−1 uni- and altered directional operations, and CK0 and CK23 refer to the initial soil sample ondays 0 and 23, respectively, without application of electrical current; B: in a voltage gradient of 1 v cm−1; and C: in a voltage gradientof 2 v cm−1. B and C refer to the DGGE profiles of fungal 18S rRNA gene under uni- and altered directional operations on days0 and 23, respectively. The numbered DGGE bands were excised and sequenced. The distance from the anode is shown above eachlane. M refers to the marker. H′ refers to the Shannon–Weaver index of fungal diversity.

Soil fungal community

Figure 5 shows that the initial soil samples under 1 v cm−1-and 2 v cm−1 uni- and altered directional operations hadfewer DGGE bands and that the band intensity was weak.The CK0 and CK23 had some highly similar DGGE bands,whereas the soil samples under different EK operationmodes had higher fungal community diversity.

The DGGE bands consisted of the dominant fungalpopulations, showing an abundant and diverse community

in the electric field on day 23. An obvious shift in theDGGE banding pattern was detected in the unidirectionaloperation electric field. In the voltage gradient of 1 v cm−1,the band intensity gradually weakened from 4 to 20 cmof the anode. The DGGE profile at a distance of 20 cmfrom the anode contained fewer bands; some dominantbands disappeared, while the main dominant fungalpopulations increased rapidly. In the voltage gradientof 2 v cm−1, the band intensity quickly weakened at 16

Fig. 6. Cluster analysis of fungal community profiles: (a) unidirectional operation of electric field soil samples, UB refers to 1 v cm−1

unidirectional operation, and UC refers to 2 v cm−1 unidirectional operation; (b) altered directional operation of electric field soilsamples, RB refers to 1 v cm−1 unidirectional operation, and RC refers to 2 v cm−1 altered directional operation. 4, 8, 12, 16, 20 werethe distances from the anode (color figure available online).

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Fig. 7. Phylogenetic tree of the fungal 18S rRNA gene sequences. DGGE bands are distinguished by �, while the • signifies the speciesor strain names followed by GenBank accession numbers. The well-known PAH-degrading fungi are indicated with an asterisk (∗).

and 20 cm from the anode. In contrast, in the altereddirectional operation electric field, the bands were highlysimilar at a distance of 4 to 20 cm from the anode, andthe DGGE profiles showed very similar patterns in voltagegradients of 1 and 2 v cm−1. These results indicate that the

fungal community structure and diversity were changed inthe unidirectional operation electric field, but less changedin the altered directional operation electric field.

Figure 6 shows that the fungal community under unidi-rectional operation of the electric field could be clustered

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into two main groups, i.e., at 16 and 20 cm from the anodein voltage gradients of 1 and 2 v cm−1, and at 4, 8, and 12 cmfrom the anode in a voltage gradient of 1 v cm−1 (Fig. 6(a)).The fungal community under altered directional operationcould be clustered into three groups, i.e., at 20 cm from theanode in the voltage gradient of 2 v cm−1, all the samplesin the voltage gradient of 1 v cm−1, and all the samples inthe voltage gradient of 2 v cm−1 (Fig. 6(b)).

Forty-six bands in the DGGE profile were excised andsequenced, of which 3 were successful. A dendrogram il-lustrating the phylogenetic relationships of the bands ex-cised from the DGGE gel is shown in Figure 7. The 18SrRNA gene sequences from the DGGE bands and the rep-resentatives of different fungal communities formed threegroups (A, B, and C). Groups A and B comprised knownPAH-degrading fungi. Group A included the known PAH-degrading Fusarium oxysporum isolates K5 (JF807399),K9 (JF807401), and K4 (JF807398) and uncultured fungi.Group B included the PAH-degrading Rhizophlyctis roseastrains BK57-5 (AF164250) and EL318 (NG 017175) anduncultured fungi. Group C included non PAH-degradingfungi and uncultured fungus.

The phylogenetic tree shows the strains of unculturedfungi, and F. oxysporum and R. rosea, the major fungiable to degrade PAHs.[28] This suggests that some soil fungicould play roles in degrading soil PAHs, during an EKremediation process.

Conclusion

In comparison with the control, EK remediation had lesseffect on soil moisture content and pH, but increased PAHremoval rate. The 1 v cm−1 altered directional operation,in particular, could effectively remove soil PAHs. A volt-age gradient of 1 v cm−1 had little negative effect on thegrowth of soil fungi, and was able to increase the fungalcommunity diversity. The fungal 18S rRNA copy numbersand community diversity was higher than in a voltage gra-dient of 2 v cm−1. Altered directional operation was moreconducive to a higher indigenous fungal 18S rRNA genecopy number and the community’s uniform distribution,than was unidirectional operation. A voltage gradient of 2v cm−1 could have lethal effects on soil microbes, and com-pared with unidirectional operation, could encourage thefungal copy number to present a uniform distribution. Un-der EK remediation, we found F. oxysporum and R. rosea,the major fungi that are able to degrade PAHs.

Overall, we felt that a 1 v cm−1 altered directional oper-ation could be a promising EK remediation mode for theremoval of PAHs, and support the abundance and diversityof the indigenous fungal community.

Acknowledgments

This work was supported in part by the Knowledge In-novation Project Key Direction Project Sub-project of the

Chinese Academy of Sciences (No. KZCX2-EW-407), theNational High Technology Research and DevelopmentProgram (863) of China (No. 2012AA100603), and the Na-tional Science Foundation of China (Nos. 41271336 and41271336).

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