remediation of polychlorinated biphenyl-contaminated soil by using a combination of ryegrass,...

Chemosphere xxx (2014) xxx–xxx

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Chemosphere

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Remediation of polychlorinated biphenyl-contaminated soil by usinga combination of ryegrass, arbuscular mycorrhizal fungi and earthworms

0045-6535/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.12.089

⇑ Corresponding author at: College of Landscape and Art, Jiangxi AgriculturalUniversity, Nanchang 330045, China. Tel.: +86 791 83813461.

E-mail address: [email protected] (Y.-F. Lu).

Please cite this article in press as: Lu, Y.-F., et al. Remediation of polychlorinated biphenyl-contaminated soil by using a combination of ryegrass,cular mycorrhizal fungi and earthworms. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2013.12.089

Yan-Fei Lu a,b,⇑, Mang Lu c, Fang Peng b, Yun Wan b, Min-Hong Liao d

a College of Landscape and Art, Jiangxi Agricultural University, Nanchang 330045, Chinab College of Information and Engineering, Jiangxi Agricultural University, Nanchang 330045, Chinac School of Materials Science and Engineering, Jingdezhen Ceramic Institute, Jingdezhen 333403, Chinad Jiangxi Chenmin Paper Co., Ltd., Nanchang 330013, China

h i g h l i g h t s

� Ryegrass, AMF and earthworms were combined to remediate PCB-contaminated soil.� Planting ryegrass can significantly increase soil PCB removal.� The inoculation of AMF can significantly enhance PCB removal.� PCB degrading efficiency is correlated with the corresponding degrading community.

a r t i c l e i n f o

Article history:Received 28 September 2013Received in revised form 5 December 2013Accepted 30 December 2013Available online xxxx

Keywords:PCBsAMFPlant uptakeqPCR

a b s t r a c t

In this work, a laboratory experiment was performed to investigate the influences of inoculation with thearbuscular mycorrhizal fungus (AMF) Glomus caledoniun L. and/or epigeic earthworms (Eisenia foetida) onphytoremediation of a PCB-contaminated soil by ryegrass grown for 180 d. Planting ryegrass, ryegrassinoculated with earthworms, ryegrass inoculated with AMF, and ryegrass co-inoculated with AMF andearthworms decreased significantly initial soil PCB contents by 58.4%, 62.6%, 74.3%, and 79.5%, respec-tively. Inoculation with AMF and/or earthworms increased the yield of plants, and the accumulation ofPCBs in ryegrass. However, PCB uptake by ryegrass accounted for a negligible portion of soil PCB removal.The number of soil PCB-degrading populations increased when ryegrass was inoculated with AMF and/orearthworms. The data show that fungal inoculation may significantly increase the remedial potential ofryegrass for soil contaminated with PCBs.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction landfill and soil washing (Semple et al., 2001). However, these ap-

Polychlorinated biphenyls (PCBs) are among the neutral organiccompounds of the greatest concern as environmental contami-nants (Secher et al., 2013). PCBs are characterized by high lipophil-icity, low water solubility, and recalcitrance to oxidation, whichmake them highly resistant to biodegradation (Xu et al., 2010).Although the production and usage of PCBs has been banned sincethe 1970s, their persistence in soil/sediment continues to presentconsiderable environmental and human health risks because ofthe bioaccumulation and bioamplification of PCBs (Petrik et al.,2006; Xu et al., 2010). In this respect, there is an urgent need toremediate and restore soil ecosystem in PCB-contaminated soils.

Over the past decades, much effort has been spent onremediating PCB-contaminated soils. Traditionally, remediation ofPCB-contaminated soils has been performed by incineration,

proaches have high costs and/or involve destruction of the soil ma-trix, which renders them suitable unsuitable for the remediation ofagricultural soils.

A variety of PCB-degrading species have been found in contam-inated soils (Abraham et al., 2002). However, it is difficult to gener-ate sufficient numbers of microbial populations with considerablePCB-degrading activity in soil to obtain an acceptable removal rateof PCBs (Vasilyeva et al., 2010).

Microbe-assisted phytoremediation, including rhizoremedia-tion, may have potential as an effective and inexpensive meansto remove and/or degrade organic pollutants from impacted soils,and has been investigated under laboratory, greenhouse and fieldconditions (Gerhardt et al., 2009). Some studies have reported thatspecific plants are able to improve PCB biodegradation and en-hance the dissipation of PCBs in soil (Xu et al., 2010; Li et al.,2013; Secher et al., 2013).

Arbuscular mycorrhizal fungi (AMF), a group of ubiquitous soil-borne fungi, have drawn considerable attention in the context of

arbus-

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2 Y.-F. Lu et al. / Chemosphere xxx (2014) xxx–xxx

phytoremediation of organic compounds. AMF are indirectly in-volved in bioremediation processes through the so-called mycor-rhizosphere effect, which stimulate soil microbial activity,improve soil structure and contribute to the overall degradationof pollutants (Joner and Leyval, 2003). Many studies have thereforebeen performed on the phytoremediation of soil contaminatedwith polycyclic aromatic hydrocarbons (PAHs) with the beneficialparticipation of AMF (Wu et al., 2009; Yu et al., 2011; Arandaet al., 2013). Recently, Teng et al. (2010) reported that planting al-falfa inoculated with AMF Glomus caledonium and Rhizobium melil-oti produced 43.5% of PCBs removal relative to 8.1% in the case ofalfalfa alone.

Earthworms improve soil structure and contribute to organicmatter decomposition and nutrient cycling (Ortiz-Ceballos et al.,2007). Earthworms facilitate and increase the contact betweencontaminants and soil microorganisms, and thus can enhance bio-degradation of pollutants (Hickman and Reid, 2008). The impactsof earthworms on the removal of PAHs have been reported by sev-eral authors (Contreras-Ramos et al., 2008; Hernández-Castellanoset al., 2013). To the authors’ knowledge, however, no studies havebeen performed with earthworms to remediate PCB-contaminatedsoil.

The aims of this study were to investigate the effects of ryegrass(Lolium perenne L.) inoculated with AMF and/or epigeic earth-worms (Eisenia foetida) on the remediation of an agricultural soilcontaminated with PCBs in the laboratory and also to monitorthe shifts in soil microbial populations in the soil.

2. Materials and methods

2.1. Chemicals

The PCB standards were of the highest purity available (99%)and were purchased from Promochem (Wesel, Germany). All sol-vents used were of HPLC grade. Deionized water (>18.0 MX) wasused for all experiments. All other chemicals and reagents usedwere of analytical-reagent grade or higher purity.

2.2. Soil samples

Soil samples were collected from a former transformer andcapacitor storage site in Nanchang, Jiangxi Province, China. Previ-ously, the site was contaminated with PCBs due to some arbitrarydisposal of used electric appliances. Soil samples were excavatedfrom the top 15 cm of the soil profile, air-dried, sieved through2-mm sieve, and thoroughly mixed. Initial total concentration of21 PCB congeners in the soil was 475 lg kg�1. The soil had the fol-lowing basic properties: pH (1:2.5 water) 5.11, total organic carbon33.6 g kg�1, total N 1.45 g kg�1, total P 0.53 g kg�1, total K452 mg kg�1, exchangeable P 56 mg kg�1, and exchangeable K115 mg kg�1 (dry weight basis). The soil acidity was neutralizedthrough the lime amendment. The soil type was classified as horticanthrosols.

2.3. Host plants, earthworms and microbial inocula

Ryegrass seeds were surface-disinfected by soaking in 3% (v/v)solution of hydrogen peroxide for 15 min and rinsed with steriledistilled water.

The earthworms (E. foetida) were collected manually from thetop 20 cm soil layer at a farmer. The earthworms were cultivatedin laboratory conditions in an anthrosol soil with approximately40% of water content at room temperature for six months. Juvenileearthworms were used in this study. The earthworms were placed

Please cite this article in press as: Lu, Y.-F., et al. Remediation of polychlorinatcular mycorrhizal fungi and earthworms. Chemosphere (2014), http://dx.doi.o

on a moist filter paper for 24 h to allow them purge their guts, andthen rinsed with sterile water before use.

Original inoculum of the AMF Glomus caledoniun L. was propa-gated in pot culture on sorghum for 60 d in a zeolite–sand mixturein a greenhouse. Then the inoculum, consisting of spores, myce-lium, sand and root fragments, was air-dried and sieved (<2 mm).

2.4. Experimental design

The following six treatments were formed in porcelain pots,each containing 1.5 kg air-dried soil: (1) soil without any additionof plants, earthworms and AMF as control (CK); (2) soil withoutplanting of ryegrass but with addition of earthworms (E); (3) soilwith planting of ryegrass but without AMF inoculation or earth-worm addition (R); (4) soil with planting of ryegrass and withaddition of earthworms (R + E); (5) soil with planting of ryegrassand with AMF inoculation (R + A); and (6) soil with planting of rye-grass and with addition of both earthworms and AMF inoculum(R + E + A), resulting in 18 treatments with three replicates each.To account for abiotic loss, one pot of soil was mixed with 2 wt.%HgCl2 to serve as abiotic control.

The inoculum of AMF was thoroughly mixed into the soil(50 g kg�1 soil). Surface sterilized seeds of ryegrass were germi-nated on moist filter paper placed in Petri dishes at 25 ± 2 �C. Oneweek after germination, seedlings of similar size were transplantedto pots, each containing 12 seedlings. Three cleaned juvenile earth-worms were then added to the corresponding pots. In treatmentswith earthworms, the pots were covered with 1.5 mm mesh screento prevent the earthworms from escaping from the vessels. In thiscase, the seedlings were allowed to pass through the screen in ad-vance. After AMF inoculation, earthworm inoculation and trans-plant of ryegrass, all the pots were arranged randomly in agreenhouse with natural light and day/night temperature of 28/21 �C and humidity of 73/86%. The soils were fertilized with NPKfertilizer mixture (1 g kg�1 of soil) containing N:P2O5:K2O =1:0.35:0.8 ratio. The moisture of all pots was adjusted regularly toapproximately 60% of field water holding capacity with deionizedwater by weighing the pots.

2.5. Sampling, extraction and clean up

After 180 d of transplanting, the soils and plants were sampled.The shoots and roots of ryegrass were harvested separately, andwashed with deionized water to remove remaining soil and dustparticles. Portions of fresh root samples were randomly collectedfrom each pot to determine the AMF infection rate of roots. Otherroot and shoot sub-samples were then freeze-dried (for 72 h undervacuum with a collector temperature of �50 ± 2 �C) and ground, inpreparation for PCB analysis. The entire soil in each pot was thor-oughly homogenized, ground sufficiently to pass through a100-mesh sieve, and divided into two sets. One was stored at�20 �C for DNA extraction, and the other was stored at 4 �C forPCB and other biogeochemical analysis.

Approximately 10 g of soil sample was spiked with 10 lL of a400 lg L�1 solution of PCB 166 in hexane (as surrogate standard),and then mixed with 0.5 g copper powder and 10 g anhydrous Na2-

SO4. The mixture was then Soxhlet-extracted with hexane:acetone(1:1, v/v) for 16 h. The extract was condensed to approximately2 mL by rotary evaporation, loaded onto a silica-gel column(10 cm � 6 mm ID) and then eluted with hexane. The filtrate wasconcentrated by evaporating the solvent under N2 and the residuewas dissolved in hexane with a final volume of 1.0 mL. Then 10 lLof a solution containing 400 lg L�1 of PCB 30 and 400 lg L�1 PCB204 (as internal standards) was added to the condensed filtratesample before PCB analysis.

ed biphenyl-contaminated soil by using a combination of ryegrass, arbus-rg/10.1016/j.chemosphere.2013.12.089


Y.-F. Lu et al. / Chemosphere xxx (2014) xxx–xxx 3

Plant samples were carefully cut into shoots and roots, and theirfresh weights were recorded. Approximately 2 g of plant samplewas then ground with anhydrous Na2SO4 using a ceramic mortarand pestle, and then extracted and cleaned as described above.

All the earthworms in each pot were taken out and placed onfilter paper for 24 h to allow their guts to empty. Earthworms werethen washed with distilled water, freeze-dried, crushed andground using a mortar and pestle, mixed with 1.5 times theirweight of anhydrous Na2SO4 (Wagman et al., 2001). Afterwards,the earthworm samples were Soxhlet-extracted and cleaned as de-scribed above.

2.6. PCB analysis

PCB congeners were analyzed usingan Agilent 7890–5975cgas chro-matography–mass spectrometer (GC–MS) equipped with an AgilentHP-5MS fused silica capillary column (60 m� 0.25 mm� 0.25 lm).The injection volume was 1 lL. Helium was used as the carrier gas ata flow rate of 1 mL min�1. The column temperature was set to 50 �Cfor the first 1 min, increased 20 �C min�1 to a temperature of 120 �C,then increased 4 �C min�1 to 310 �C, and maintained at 310 �C for30 min. Mass spectrometer conditions were: electron impact, electronenergy 70 eV; filament current 100 lA; multiplier voltage, 1200 V; fullscan.

PCB congeners were identified by retention time matching tothe standard mixture and quantified by using peak area integra-tion. All the samples were analyzed in triplicate, and the relativestandard deviations (RSD) were all below 10%. Field and laboratoryblanks covering the entire analytical procedure (from the extrac-tion to GC analysis) were routinely monitored and no PCB contam-ination was detected.

2.7. Quantification of mycorrhizal root colonization

Mycorrhizal root colonization was quantified based on the gridline intersect method according to White et al. (2006). In brief, a 2-g (fresh weight) sub-sample of the fresh roots was cut into shortpieces (2–4 mm) and the root segments were soaked in 10% KOHfor 10 min at 90 �C. The roots were removed and sequentially im-mersed in tap water, 2.3% HCl, and 3% hydrogen peroxide. Theroots were then stained in 0.05% aniline blue for 8 min. The per-centage of root length colonized by mycorrhiza was assessed by vi-sual observation of fungal colonization and calculated byobservation at 200 �magnification.

2.8. DNA extraction and qPCR assay

Genomic DNA was extracted from frozen soil using a commer-cially available kit (Beyotime Biotechnology, China) according tomanufacturers’ protocols. The bphA and bphC genes encode dioxy-genases BphA and BphC respectively, which are considered as keyenzymes catalyzing critical reactions in PCB-transformation(Pieper, 2005). Thus, in this study, the bphA and bphC genes werequantified by quantitative polymerase chain reaction (qPCR) in aRotor-Gene RG-6000 real-time PCR machine (Corbett Research,Sydney, Australia), using primer sets 463f/674r and bphC(Rh)f/bphC(Rh)r + bphC(Ps)f/bphC(Ps)r, respectively (Petric et al., 2011).Calibration of qPCR was carried out with a serial dilution preparedwith the appropriate cloned target sequence. Data analysis wascarried out with the iQ5 Optical System Software (version1.0.1384.0 CR) (Bio-Rad).

2.9. Data analysis

In this study, all experiments were performed in triplicate to getreliable data, and the data were calculated and reported on


dry-weight basis. Statistical significance was evaluated using SPSSpackage (version 11.0) with two-way ANOVA and least significantdifference (LSD) was applied to test for significance at p < 0.05 be-tween the means.

3. Results and discussion

3.1. Mycorrhizal root colonization and plant biomass

Mycorrhizal root colonization rates after growth for 180 d arepresented in Table 1. No colonization of roots was found in theroots of uninoculated plants. The inoculation of AMF significantlyenhanced colonization (p < 0.05). Adding earthworms to AMFtreatment significantly (p < 0.05) increased the AMF colonizationrate of ryegrass roots. These results suggested that E. foetida raisedcolonization rate of ryegrass roots. This is consistent with the re-sults of Li et al. (2012) who reported positive effect of epigeicearthworms on the AMF colonization rate of maize roots. Positive,negative, and neutral impacts of earthworms on AMF root coloni-zation have been reported (Li et al., 2012).

As shown in Table 1, compared to the R treatment, the introduc-tion of AMF alone (R + A) or with earthworms (R + E + A) increasedryegrass yield. Ryegrass yield was significantly greater in R + E + Athan in the other treatments (p < 0.05). The R + E + A treatment sig-nificantly increased ryegrass shoot and root biomass compared tothe R treatment. However, R + A only significantly increased rye-grass root biomass (p < 0.05). Li et al. (2012) reported that earth-worms and AMF could enhance the availability of N, P, and Kcontents in the soil and their uptake by the plant, resulting in animproved plant growth.

3.2. Soil PCB concentrations and congener profiles

Fig. 1A shows total PCB concentrations and percent removalafter 180 d of experiment in each treatment. After 180 d of plantgrowth, the initial concentrations of PCBs across all treatments de-clined greatly, which ranged from 85.7 to 342.5 lg kg�1 dry soil.The removal of PCBs was the greatest in the dual inoculation plant-ing treatment (R + E + A) (79.5%), followed by planted soils withAMF inoculation (R + A) (74.3%) and earthworm addition (R + E)(62.6%), and planted alone soil (R) (58.4%). Contrastly, the totalconcentrations of PCBs decreased by 38.4% and 40.1% in non-planted control (CK), and earthworm added non-planted soil (E),respectively (Fig. 1A). The ryegrass planting has significant stimu-latory effects on PCB removal (p < 0.05), resulting in an increase of20% points in degradation efficiency relative to the non-plantedcontrol. The AMF inoculation enhanced the stimulatory effects ofplanting by 15.9% points, but the earthworms only raised PCB re-moval by 1.7–5.2% points.

The mean percentage composition of PCB homologues in thesoil under different treatments is depicted in Fig. 1B. In general,the depletion of lower chlorinated PCBs was greater than thoseof higher chlorinated PCB congeners. Planting with ryegrass andinoculation with AMF enhanced dissipation of the lower chlori-nated di-, tri- and tetra-chlorinated biphenyls and increased theproportion of more highly chlorinated penta- and hexa-chlorinatedbiphenyls relative to the average congener profile of the unplantedsoils (CK and E). Moreover, the concentration of tetra- and penta-CB in the soil was significantly lower in R + E + A or R + A than inother treatments (p < 0.05). Biodegradation resulted in the relativeenrichment of the higher-chlorinated congeners.

The present experiment suggested that ryegrass plantingplayed an important role in the remediation of PCB-contaminatedsoil. This result further confirmed the findings of Chekol et al.(2004) and Tu et al. (2011) in potted and field experiments



Table 1Mycorrhizal colonization rates of ryegrass roots, and shoot and root dry matter yield (freeze-dried basis) after 180 d (mean ± SD, n = 3).

Treatment

R R + E R + A R + E + A

Mycorrhizal colonization (%) – – 43.6 ± 3.4a 55.2 ± 4.2bShoot weight (g per pot) 6.33 ± 0.22a 6.55 ± 0.27a 6.42 ± 0.31a 6.85 ± 0.34bRoot weight (g per pot) 2.26 ± 0.15a 2.41 ± 0.18a 2.59 ± 0.13b 2.68 ± 0.20bShoot + root weight (g per pot) 8.59 ± 0.30a 8.96 ± 0.38a 9.01 ± 0.37a 9.53 ± 0.41b

Note: values within a row followed by different lowercase letters are significantly different.

0

100

200

300

400

500

cdc

bb

a

R+E+AR+AR+ERECK

Soil

PCB

conc

entra

tion

(µg

kg-1

)

(A)The 180th day

Initial

a

Treatment

0

20

40

60

80

100

PCB rem

oval (%)

0

20

40

60

80

100

Treatment

(B)

Rel

ativ

e ab

unda

nce

of P

CB

Hexa to Deca-CB Penta-CB Tetra-CB Tri-CB Di-CB

R+E+AR+AR+ERECKInitial

hom

olog

ues

(%)

Fig. 1. PCB concentration changes in the soil under different treatments over 180 d.(A) Concentration of total PCB in soil and percent removal. (B) The relativeabundance of PCB congeners before and after treatment. Values in (A) followed by adifferent lowercase letter between treatments are significantly different (p < 0.05).

0

20

40

60

80

100

bc

b

R+E+AR+EE

PCB

conc

entra

tion

(mg

kg-1)

(A)a

Treatment

20

40

60

80

100

e ab

unda

nce

of P

CB

cong

ener

s (%

)(B) Hexa to Deca-CB Penta-CB Tetra-CB Tri-CB Di-CB


respectively, that planting could promote soil PCB dissipation. Inthis study, planting with ryegrass also enhanced the removal ofcertain lower chlorinated biphenyls from the soils (Fig. 1B). Theinteractions of plants with bacteria or AMF may further improvethe dissipation of PCBs from soil. The secretion of root exudatesmay accelerate the growth of the degrading microorganisms andhelp to improved PCB availability for biodegradation (Xu et al.,2010).

0

Treatment

Rel

ativ

R+E+AR+EE

Fig. 2. PCB concentrations (A) and relative abundance of PCB congeners (B) in theearthworm biomass after 180 d of incubation. Values in (A) followed by a differentlowercase letter between treatments are significantly different (p < 0.05).

3.3. Bioaccumulation of PCBs by earthworms

In the worm–amended treatment, no mortality of the wormswas observed during the entire incubation period. In the presenceof the worms, the depletion of PCBs from the soils was slightly en-hanced compared to those without the worms (Fig. 1). Fig. 2A


shows the bioaccumulation of PCBs in the earthworm biomass.The concentrations of PCBs in the earthworms were 78.4, 56.3and 42.6 mg kg�1 dry weight for E, R + E and R + E + A, respectively.This demonstrated that the earthworms could accumulate a highlevel of PCBs while remaining viable. The result also demonstratedthat the accumulation level of PCBs in the earthworms was posi-tively correlated with the residual concentration of PCBs in the soil.

Fig. 2B shows the congener profiles of PCBs in earthworms after180 d of incubation. As shown, the percentage of PCB homologuestended to decrease with increasing the chlorination extent, and nosignificant differences were observed among treatments. This wasopposite to the congener profiles of soil PCBs after treatment asshown in Fig. 1B.

These results reflected the complex impacts of various factors inthe soil environment. The uptake and bioaccumulation of organic




pollutants by earthworms is an interactive process between soil,interstitial water and the worms themselves. Tharakan et al.(2006) found that the level of PCB accumulation by earthwormswas proportional to the PCB concentration in the surroundingsoil–sludge matrix. Additionally, soil characteristics such as TOCcontent, particle size, clay content, moisture content, and eventhe density of the worms in soil can affect the bioavailability ofcontaminants in the soil (Belfroid et al., 1996).

3.4. PCB accumulation in plants

PCB concentrations in plant shoots and roots are shown inFig. 3A and B, respectively. As shown, in both shoots and roots,the PCB concentrations decreased in the orderR + E + A > R + A > R + E > R, which was consistent with results ofsoil PCB dissipation demonstrated in Fig. 1A. The average PCB con-centrations in shoots of the R, R + E, R + A, and R + E + A treatmentwere 33.5, 34.2, 48.3 and 51.6 lg kg�1 dry biomass, respectively.The mean PCB concentrations were 148, 165, 273 and 306 lg kg�1

in roots of the R, R + E, R + A, and R + E + A treatment, respectively.The average PCB concentration in both shoots and roots of theR + E + A treatment was significantly higher than those of boththe R + E and R treatment (p < 0.05), but no significant differenceswere observed in PCB concentration between the R + E + A andR + A treatment (p > 0.05). These results indicated that AMF

0

10

20

30

40

50

60b

b

a

R+E+AR+AR+ETreatment

(A)

R

Shoots

a

0

100

200

300

400

b

b

aa

R+E+AR+AR+ETreatment

(B)

R

Roots

PCB

conc

entra

tions

(µg

kg-1

)PC

B co

ncen

tratio

ns (µ

g kg

-1)

Fig. 3. Concentrations of PCBs in shoots (A) and roots (B) of ryegrass in the differentvegetated trials. Values followed by a different lowercase letter between treatmentsare significantly different (p < 0.05).


and/or earthworm inoculation had a positive impact on ryegrassPCB acquisition.

Based on the data of Table 1 and Fig. 3, the percent removal ofPCBs by plants could be obtained as follows: 0.077, 0.087, 0.143and 0.165% for the R, R + E, R + A, and R + E + A treatments, respec-tively (data not shown). This demonstrated that AMF and/or earth-worm inoculation raised PCB uptake and phytoextraction. Forexample, the percentage of PCBs phytoextracted by ryegrass inoc-ulated with both AMF and earthworms was 2.14 times the uninoc-ulated ryegrass. Thereupon, plant uptake was a negligible removalpathway for PCB depletion in contrast to biodegradation in thepresent study. These findings were consistent with Zeeb et al.(2006) that plants accumulated 0.2–1.3% of total soil PCBs.

3.5. Mass balance of PCBs

Table 2 lists the mass balance and distributions of PCBs in dif-ferent compartments after 180 d. As shown, 6.3% of initial PCBswere lost due to abiotic factors such as evaporation. Additionally,the losses of PCBs in soils mainly resulted from biodegradationby soil microorganisms, and plant uptake or earthworm accumula-tion was a negligible removal pathway for PCBs in contrast tobiodegradation.

3.6. Abundance of the targeted gene sequences

The abundance of the PCB-degrading functional communitywas estimated by amplifying the bphA and bphC gene sequencesby qPCR. qPCR analyses of the targeted genes indicated that thecopy number of these genes was high (107–109 per g soil) in allof the soils and remained steady or increased in all treatments(Fig. 4). This result also suggested the presence of a native and ro-bust microbial community responsible for oxidation-dominatedbiodegradation of PCBs. It can be observed that the number of bphAgene copies was lower than that of bphC (Fig. 4A and B). Bioreme-diation treatment with the addition of inorganic nutrients resultedin increments in the number of both bphA and bphC genes. Rye-grass planting significantly increased the number of the two genes(p < 0.05), but AMF or earthworm inoculation did not. Additionally,the number size of targeted genes was in accordance with that ofPCB removal.

Dioxygenases BphA (encoded by the bphA gene) and BphC(encoded by the bphC gene) are considered as key enzymes for cat-alyzing critical reactions in PCB-transformation (Pieper, 2005). Bythe incorporation of two hydroxyl groups into the aromatic ring,BphA raises the reactivity of PCBs and renders them more suscep-tible to enzymatic ring fission reactions catalyzed by BphC(Bruhlmann and Chen, 1999). Several authors have assessed thegenetic potential of the PCB-degrading bacterial population incontaminated soil or sediment by developing qPCR assays to targetthe bphA or bphC genes (Singer et al., 2003; Kurzawova et al., 2012;Dudášová et al., 2013). These studies suggested a positive impact ofthe addition of different active PCB degraders, earthworms andplants on the abundance of biphenyl and/or monochlorobenzoate-degrading bacterial populations and the levels of the bphA and bphCgenes. These two genes generally belong to some well-known PCBdegraders such as Rhodococcus, Arthrobacter, Corynebacterium,Sphingomonas, Pseudomonas, and Acinetobacter. The results of thepresent study suggested the positive effects of the plants, AMF andearthworms on bioremediation of PCB contamination throughstimulating the growth of specific PCB degraders.



Table 2Mass balances and PCB distribution (% of initial mass applied) in the phytoremediation system after 180 d.

Treatment

CK E R R + E R + A R + E + A

Soil 61.6 ± 4.2 59.9 ± 4.5 41.6 ± 4.8 37.4 ± 3.6 25.7 ± 3.2 20.5 ± 2.7Earthworm – 0.125 ± 0.033 – 0.138 ± 0.029 – 0.121 ± 0.016Root – – 0.047 ± 0.006 0.056 ± 0.005 0.099 ± 0.010 0.115 ± 0.012Shoot – – 0.030 ± 0.008 0.031 ± 0.009 0.044 ± 0.006 0.050 ± 0.004Biodegradation loss 32.1 ± 2.9 33.7 ± 2.6 52.0 ± 4.7 56.1 ± 5.1 67.9 ± 5.4 72.9 ± 5.8Abiotic lossa 6.3 ± 0.5

a Calculated from the abiotic control (soil mixed with 2 wt.% HgCl2).

0

2

4

6

8

10

R+E+AR+AR+ERECK

(A) The 180th day

Initial

bbbba

Treatment

aa

0

2

4

6

8

10

R+E+AR+AR+ERECK

log

gene

cop

ies

g-1 s

oil

(B) The 180th day

Initial

ccccb

Treatment

ab

log

gene

cop

ies

g-1 s

oil

Fig. 4. Total putative bphA (A) and bphC gene copies (B) in soils before and after180-d incubation. Values followed by a different lowercase letter betweentreatments are significantly different (p < 0.05).


4. Conclusions

The present study demonstrated that ryegrass can play animportant role in the enhancement of PCB removal in soil andthe inoculation with AMF and/or earthworms may have a posi-tively synergistic effect on phytoremediation of PCBs by ryegrass.Mycorrhizal colonization caused an increase in PCB accumulationin plants. However, PCB uptake contributed negligibly to PCB dissi-pation in the soil. Contrastly, the effective microbiota in the mycor-rhizal association was deemed to be responsible for the reductionsin PCB concentrations in the soil. Further studies are needed toinvestigate the PCB metabolic pathway in plant–microbialassociations.


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