granulometry and surfactants, key factors in desorption ... · granulometry and surfactants, key...

Granulometry and Surfactants, Key Factors in Desorptionand Biodegradation (T. versicolor) of PAHsin Soil and Groundwater

P. Rodríguez-Escales & E. Borràs & M. Sarra &

A. Folch

Received: 23 July 2012 /Accepted: 18 December 2012 /Published online: 10 January 2013# Springer Science+Business Media Dordrecht 2013

Abstract High hydrophobicity of polycyclic aromatichydrocarbons (PAHs) is the most limiting factor forthe remediation of polluted soils and aquifers. Thepresent study analyzes the effect of three nonionicsurfactants (Tween 80, BS-400, and Gold Crew) andthe granulometry of soil (1 %, 5 %, 10 %, and 20 % ofclay and silt) on desorption of a PAH mixture (fluo-rene, phenanthrene, anthracene, and pyrene). As ageneral trend, decrease of fine material content andincrease of surfactant concentration raises desorption.However, some particularities have to be considereddepending on granulometry together with the surfac-tant applied. Furthermore, increase of fine materialcontent tends to reduce the importance of the PAHproperties, e.g., Kow and solubility, in desorption. To

complete the remediation process, biodegradation byTrametes versicolor was tested with the surfactantTween 80. Results indicate that a high concentrationof surfactant does not affect the efficiency of fungusbioremediation. Nevertheless, high fine material con-tent in soil/aquifer can reduce the degradation rate.Moreover, desorption and biodegradation used syner-gically guarantee better overall results in the remedia-tion of soils polluted by PAH mixtures than othermethods that separate desorption and remediation.

Keywords Polycyclic aromatic hydrocarbons (PAHs) .

Finematerial . Remediation . Ligninolytic fungus

1 Introduction

Pollution by Polycyclic Aromatic Hydrocarbons(PAHs) is a major environmental concern. PAHs accu-mulate in the environment primarily as a result ofanthropoghenic activities, mainly fuel combustion.Their broad distribution and their high carcinogeniccapacity (Bamforth and Singleton, 2005) make themone of the target pollutants in many environmentalpolicies (World Health Organization 1998; EuropeanEnvironment Agency 2000).

Due to their molecular characteristics, PAHs are hy-drophobic compounds with lowwater solubility (Lippoldet al., 2008). In soil and aquifer systems, PAHs are sorbedinto organic materials and clay fractions (Tersahima et al.2003) restricting their bioavailability. Although this be-havior makes them relatively harmless compounds,

Water Air Soil Pollut (2013) 224:1422DOI 10.1007/s11270-012-1422-z

P. Rodríguez-EscalesD’Enginy Biorem, Carrer Madrazo 68,08006 Barcelona, Spain

P. Rodríguez-Escales :A. Folch (*)Unitat de Geodinàmica Externa i Hidrogeologia,Departament de Geologia, Universitat Autònoma deBarcelona, 08193 Bellaterra, Spaine-mail: [email protected]

P. Rodríguez-Escales : E. Borràs :M. SarraDepartament d’Enginyeria Química, Escola d’Enginyeria,Universitat Autònoma de Barcelona,08193 Bellaterra, Spain

P. Rodríguez-Escales :A. FolchGHS, Departament d’Enginyeria del Terreny, Cartogràsficai Geofísica, Universitat Politècnica de Catalunya-BarcelonaTech, Jordi Girona 1-3, Modul D-2,08034 Barcelona, Spain

retention in the solid matrix limits the extraction andtreatment of PAHs by pump and treat (Sánchez-Martinet al. 2008) or other techniques as bioremediation.

Surfactant-enhanced remediation is an economicallyand technically feasible approach for the remediation ofPAH-contaminated sites (Garon et al. 2002; Zhao et al.2005; Kim et al. 2008, Zhou and Zhu 2008; Clark andKeller 2012). Surfactants are amphiphilic compoundspromoting desorption and the transfer of the pollutantfrom a solid to liquid phase, thus facilitating their treat-ment (Edwards et al. 1991; Jafvert et al. 1994). However,only when surfactant content achieves a concentrationthat promotes micelle formation, known as effectivecritical micelle concentration (CMCeff), can PAHs betransferred to the liquid phase (Holmberg et al. 2002;Zheng and Obbard 2002; Laha et al. 2009).

Achievement of CMCeff is conditioned by mediumproperties such as the presence of organic matter and/or cation exchange capacity (CEC). Interaction be-tween surfactants and organic matter has been widelystudied (Chu and Chan 2003; Cheng and Wong 2006;Lippold et al. 2008; Zhou and Zhu 2008). In contrast,CEC, which is closely linked with soil/aquifer gran-ulomtery, has been poorly studied (Mulligan et al.2001; Rodríguez-Escales et al. 2012). Nevertheless,in many soils and aquifer systems, organic mattercontent is very low (Schwarzenbach and Gier 1985)and sorption processes are limited by porosity. Hence,despite this relative lack of knowledge, in those envi-ronments with organic matter below 0.1 %, as is thecase with most aquifer systems, sorption and desorp-tion processes are controlled by fine soil materials,mainly clays (Schwarzenbach and Westall 1981;Karickhoff 1984; Teixeira et al. 2011).

In most real pollution episodes, several PAHs arepresent as a mixture, yet most published articles thatstudy desorption processes focus on only one hydro-carbon (Kim and Park 2001; Sartoros et al. 2005;Lippold et al. 2008; Sánchez-Martín et al. 2008; Zhouand Zhu 2008, Rodríguez-Escales et al. 2012). Studiesof the surfactant effect on a PAH mixture show that theresults can vary significantly depending on the numberand type of hydrocarbons (Guha et al. 1998; Alcántaraet al. 2009). The present study corroborates theseresults. A selective desorption of some of the com-pounds in the PAH mixture was observed in the pres-ence of both anionic and ionic surfactants, clearlyshowing the existence of the synergic effect of themixture.

Once PAHs are available in the liquid phase, aremediation strategy must be applied. For this, theefficiency of Trametes versicolor, a wood-rotting pol-ypore that grows on dead or dying trees, against sev-eral PAHs has been amply demonstrated (Majcherczyket al. 1998; Collins and Dobson 1998; Mougin 2002;Tekere et al. 2005; Borràs et al. 2010). Due to thenonspecificity of the fungal enzymes for the substrate,these enzymes are capable of degrading a wide varietyof highly recalcitrant and xenobiotic organic com-pounds (Cerniglia 1997). Borràs et al. (2010) reportedthe capacity of this fungal strain to degrade PAHs insoil systems. However, their degradation capacity isoften limited by the availability of the pollutant, show-ing much better degradation in a liquid media than insoils (Boyle et al. 1998). Therefore, the success ofbioremediation is often associated with the effect ofsurfactants on pollutant desorption.

Given the above considerations, our study proposedtwo main objectives. Firstly, to study how desorptionof a PAH mixture (fluorene, phenanthrene, anthracene,and pyrene) can be modified by different soil granul-ometry with nonorganic matter, surfactants (Tween 80,BS-400, and Gold Crew), and surfactant concentra-tions. The effect of the PAH mixture by comparing theobtained results with experiments realized with thesame conditions and the same soils but for a singlePAH such as pyrene (Rodríguez-Escales et al. 2012)also is checked. Second, the study also aims to studythe biodegradation capacity of this PAH mixture by T.versicolor after the desorption process with Tween 80.Several soil granulometry were chosen as representativeof most aquifer media in sedimentary environments and/or soils with a sufficient range of permeability to allowsurfactant application. The first surfactant (Tween 80) iswidely cited in scientific literature, while the other two(BS-400 and Gold Crew) are commercial surfactants.

2 Materials and Methods

2.1 Materials

2.1.1 Soil

The artificial soils were a mixture of inert sand andfine materials (silt and clay). Fine materials were col-lected from a silt and clay layer of the Neogene sedi-ments of the Vallès-Penedès graben (Catalonia, NE

1422, of 12 Water Air Soil Pollut (2013) 224:1422

Spain) with the following composition: quartz (10 %),microcline plus illite (62 %), chlorite (16 %), andmontmorillonite (1 %). This mineralogy correspondswith the classical mineralogy found in the tertiarysediments of the Ebro Basin (Martinez-Bofill et al.2008). Then, soil samples were washed with distilledwater, kept at 105 °C for 24 h, and then sieved. Finesand (0.25–0.50 mm) and fine materials, silt and clay(<63 μm), were separated. Four artificial soils of dif-ferent granulometry were prepared, covering a widerange of fine material content found in soils and inaquifer formations (Table 1).

2.1.2 PAH Mixture

Fluorene, phenanthrene, anthracene, and pyrene wereobtained from the Sigma-Aldrich Co. Their puritywere 95 %, 98 %, 99 %, and 95 %, respectively.Pollutants were mixed in the same proportions in astock solution of 2,500 mgL−1 in dichloromethane(DCM).

2.1.3 Surfactants

The surfactants were of the nonionic sort. Gold Crewwas obtained from Biotecnologías DescontaminantesS.L. (Spain), BS-400 from IEP Europe S.L. (Spain),and Tween 80 from Sigma-Aldrich Co. (Spain). Thesewere added, separately, at different concentrations inthe media.

2.1.4 Fungus

T. versicolor (ATCC# 42530) was maintained by sub-culturing on 2 % malt extract agar slants (pH 4.5) atroom temperature. Subcultures were routinely madeevery 30 days. Pellets were obtained as described inBlánquez et al. (2004). Medium was composed asdescribed in Borràs et al. (2008): 10 gL−1 glucose,2.1 gL−1 NH4Cl, 100 mLL−1 macronutrient solution

(Kirk et al. 1978), 10 mLL−1 micronutrient (Kirk et al.1978), 1,168 gL−1 2,2-dimethylsuccinate buffer, and10 mgL−1 thiamine.

2.2 Preparation of PAH-Spiked Soil

PAH-spiked soil was prepared in 1-L bottles adaptedfrom the methodology used by Zhou and Zhu (2008).Ten grams of each soil was poured into 400 mL ofdistilled water in a glass bottle. NaCl (0.01 M) andNa3N (1 gL−1) were added and the bottles were left ina shaker (130 rpm, 25 °C) for 24 h. NaCl (0.01 M) wasadded to balance the ionic strength. Bottles were ster-ilized for 30 min, 121 °C. After 24 h, PAHs werespiked into the soil preparation to make a final totalPAH concentration of 2,500 mgkg−1. PAH solutionwas prepared with DCM. The spiked soil was incu-bated for 48 h at room temperature and shaken at130 rpm to allow DCM mixtures to evaporate andmake sure that PAHs were completely sorbed to soil.Then, an initial aliquot was sampled.

2.3 Desorption of PAHs in Soil–Water System

The amount of surfactant applied was based on experi-ments carried out by Rodríguez-Escales et al. (2012),following previous studies and concentrations recom-mended by the manufacturer. After 48 h, surfactantswere added in 100 mL sterilized water solutions tobottles with spiked soil, reaching a final volume of500 mL. After 1 week of contact with surfactant,samples (2 mL) were pipetted into an Eppendorf de-vice and were centrifuged for 10 min at 15,000 rpm.The PAHs in supernatant were then analyzed as de-scribed in “Section 2.5.1”. Desorption of PAH mixtureis calculated as the sum of the proportions of eachpollutant in liquid phase.

2.4 Biodegradation of the PAH Mixturewith T. versicolor

2.4.1 Evaluation of the Effect of the Surfactanton T. versicolor

T. versicolor was added as pellets (0.25 g dry weight)in a 500-mL bottle. Then, 50 mL of the mediumdescribed in “Section 2.1.4” containing the surfactantwas added to a 500-mL bottle. Experiments werecarried out in triplicate and abiotic controls were

Table 1 Soil properties

Fine materials (%) Sand (%) foc

Soil A 1.00 99.00 2.6×10−4

Soil B 5.00 95.00 4.0×10−4

Soil C 10.00 90.00 5.8×10−4

Soil D 20.00 80.00 9.2×10−4

Water Air Soil Pollut (2013) 224:1422 of 12, 1422

prepared containing the same medium with the addi-tion of Na3N.

2.4.2 Biodegradation of the PAH Mixturewith T. versicolor

A 50-mL aliquot was taken from the bottles containingthe PAH mixture, soils, and surfactant in equilibriumpreviously sterilized, which was achieved 7 days afterthe surfactant addition. After that, the biodegradationexperiment was performed in 500-mL bottles for15 days, under sterile conditions.

Next, 10 mL of the medium was added as describedin “Section 2.4.1” and 0.25 g of T. versicolor pellets.The bottles were left in the orbital shaker at 130 rpmand at 25 °C. Every 4 days, a sample was taken tomonitor PAH concentration, glucose consumption,and laccase enzymatic activity. An analysis was madeof the supernatant of the previously centrifuged sam-ples at 15,000 rpm for 10 min. Finally, after 15 days, afinal extraction was made of the adsorbed pollutant inthe soil with DCM.

2.5 Analytical Methods

2.5.1 Analysis of PAHs and BiodegradationMetabolites

PAHs and biodegradation products were analyzedin a gas chromatograph (GC8690N, Agilent,Spain) equipped with a flame ionization detector(FID). The column used was the Zebron ZB-5HTInferno (Phenomenex, USA). The initial tempera-ture was maintained at 50 °C for 1 min; it wasincreased by 7 °C/min until it reached 320 °C andthen by 20 °C/min up to 400 °C. The detectionlimit was 0.010 mgL−1 for each PAH. The con-centrations of fluorene, phenanthrene, anthracene,and pyrene were determined after calibrating themethod using respective standards. Biodegradationproduct identification was carried out by matchingthe retention time of well-known PAH metabolitesusing standards.

2.5.2 Analysis of Glucose

Glucose was analyzed using the enzymatic glucoseand lactate analyzer YSI 2700 (Yellow Springs Instru-ments Co.).

2.5.3 Analysis of Laccase Activity

Laccase activity was measured using a modified ver-sion of the method by Wariishi et al. (1992) for thedetermination of manganese peroxidase. It was ana-lyzed in a Varian Cary 3 UV/Vis spectrophotometerequipped with a thermostat. The reaction mediumcontained 200 μL of sodium malonate 250 mM atpH 4.5, 50 μL of 2,6-dimethoxyphenol (DMP)20 mM, and 600 μL of sample.

3 Results and Discussion

3.1 Desorption of the PAH Mixture by Surfactants

As a general trend, the maximum desorption wasachieved when the concentrations of surfactant werehighest and fine material content of the soil was lowest(Fig. 1). This is due to clay minerals present in the finematerials, which have a high sorption force/capacity.Total mixture desorption of PAHs for the three surfac-tants tested was in the same range, with overall de-sorption ranging between 20 % and 50 %, except for“Soil A+Gold Crew” (totally desorbed) and “Soil D+Tween 80” (no desorption). As expected, an increaseof surfactant raised the amount of total PAHs des-orbed, but an increase of fine material content de-creased the efficiency of surfactants. However, thereis no specific trend or relation between surfactantconcentration applied/PAHs desorbed and/or PAHsdesorbed/proportion of fine material. Furthermore,for a given soil, an increase of Tween 80 concentrationimplies an increase in desorption. However, withBS400 and Gold Crew, once a significant percentageof desorption has been accomplished (20–50 %depending on the soil), an increase of surfactant con-centration does not imply a significant increase ofdesorption (Fig. 1).

Individual PAHs were not desorbed equallydepending on the surfactant employed (Online Re-source 1). In the case of Tween 80, all PAHs weredesorbed, with anthracene exhibiting the lowest de-sorption. As a general trend, anthracene and fluoreneare less desorbed than pyrene and fluorene. On theother hand, BS-400 and Gold Crew did not permitanthracene or phenanthrene desorption in most soilsand surfactant concentrations, while desorption of pyr-ene and fluorene was similar to or higher than BS-400


and Gold Crew than with Tween 80. With the maxi-mum concentration of Gold Crew, more than 90 %desorption of all PAHs was accomplished in soil A.These results indicate that the concentration of surfac-tant employed can modify not only the total PAHsdesorbed but also which PAHs of the mixture are

desorbed. Differently from Tween 80, the two highestconcentrations of BS400 and Gold Crew allowed de-sorption of more than 25 % of flourene and pyrene inall tested soils.

Several authors have studied other PAHmixtures withdifferent results. Bernal-Martínez et al. (2005) studiedthe desorption of a PAH mixture including fluorene,phenanthrene, anthracene, fluoranthene, pyrene, ben-zo[a]anthracene, chrysene, benzo[b]fluoranthene, ben-zo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene, and indeno[1,2,3,cd]pyrene using Tyloxapol, Tergitol NP10, and Brij 35 assurfactants. They found that the removal was similar forall surfactants, reaching up to 60 % for Brij 35, 60 % forTergitol NP10, and 65 % for Tyloxapol. In contrast,Fabbri et al. (2008), using Brij 35 as surfactant, treateda PAH mixture (2-naphtol, 2,4-dichloroaniline, 3,4-dichloroaniline, chlorobenzene, 1,4-dichlorobenzene,tetrachlorobenzenes, 9,10-anthraquinone, benzanthrone)with recovery ranging from 14 % to 69 % depending onthe PAH analyzed. Dhenain et al. (2006) showed thatTween 80 is the best surfactant for the treatment of aPAH mixture of fluoranthene, benzo[a]anthracene, ben-zo[b]fluoranthene, benzo[j]fluoranthene benzo[k]fluo-ranthene, and benzo[a]pyrene, with PAH removalranging from 35% to 50% for different PAHs. Alcántaraet al. (2009) studied desorption in a kaolin soil contain-ing different PAH mixtures of benzo(a)pyrene, fluoreneand pyrene using Tween 80 as a surfactant. The percent-age of removal for all these combinations was higherthan 80 %. In each PAH, an increase of desorptionfollows benzo(a)pyrene>fluorene>pyrene. Despitethese studies having different experimental conditions,different results obtained in all of them and in the presentwork seems to indicate that total desorption is alsoconditioned by the characteristic of the PAH mixture(hydrophobicity of the mixture), the characteristic ofeach PAH in the mixture, and the surfactant employed.

Hydrophobicity, and therefore desorption, dependson the octanol/water constant distribution and the sol-ubility of each PAH in the different types of soil andsurfactant (Fig. 2). Nevertheless, relation of desorptionbetween solubility and Kow seemed to trend quantita-tively different in each soil. Different proportions offine materials might displace the response but did notproduce any qualitative changes (except for GoldCrew which, for soil A, practically achieved 100 %desorption). Moreover, the slope between Kow andsolubility decreased with the increase of fine material

Fig. 1 Desorption (%) of the PAH mixture depending on theconcentrations of a Tween 80, b BS-400, and c Gold Crew fordifferent types of soil, where fine m. are fine materials


(Fig. 2). Hence, in soils with high fine material con-tent, molecular properties might become less impor-tant than soil properties, indicating that in soils withhigh fine material content, the desorption of PAHcould be more conditioned by the CEC of the soil thanby the characteristics of the PAHs.

Comparing the experimental data with previous stud-ies by Rodríguez-Escales et al. (2012) with the samesoils and surfactants but applying only to pyrene, theresults are very different despite maintaining the sameratio of surfactant concentration/total PAHs concentra-tion. When using only pyrene, maximum desorptionachieved was around 80 % for Tween 80, 40 % forBS-400, and 35% for Gold Crew. Therefore, our experi-ments indicate that surfactant efficiency is affected by

the presence of several PAHs. The reason for thesedifferences may be that, in spite of preserving the samerelation between pollutant and surfactant, there was notthe same relation between the PAH mixture hydropho-bicity and the surfactant, a factor conditioning the de-sorption process as pointed out by Alcántara et al.(2009). Furthermore, for a specific surfactant concentra-tion, it is possible to see that desorption efficiency forone PAH, over against the PAH mixture, changes foreach type of soil. For instance, 1,150 mgL−1 of Tween80, close to the CMCeff proposed by Peng et al. (2011),desorbs around 80 % of pyrene in all soils. Oppositely,although Tween 80 desorbs all PAHs, even with higherconcentration of the same surfactant (5,000 mg/L), totalPAH desorption ranges from around 1 % to 45 %,

Fig. 2 Desorption depending on solubility and Kow


depending on the soil employed. Likewise, when using65 mgL−1 of Gold Crew with pyrene, desorption isaround 1 % for soils B, C, and D and around 31 % forsoil A. The same concentration with the PAH mixturereaches an efficiency of 20 % for soils C and D andaround 45 % for soils A and B.

However, desorption process is also affected by thetype of surfactant employed. Considering Tween 80with the 150, 1,000, and 1,150 mgL−1 concentrations,pyrene desorption was generally lower in the mixturethan in the pure solution. However, for BS-400, theobserved behavior was the opposite. In the case ofGold Crew, for 16.25 and 32.65 mgL−1 of surfactantconcentration, desorption was a little bit higher that forthe pure pyrene system. However, for the 65 and150 mgL−1, it was the opposite.

Hence, according to our and previous results, acomplex desorption mechanism is shown. The desorp-tion capacity of a PAH mixture not only depends onthe compounds of the mixture and their interaction butalso on the type of surfactants and fine material pro-portion. As our results show, different desorption isobserved depending of the surfactant employed foreach soil granulometry.

3.2 Biodegradation of the PAH Mixtureby T. versicolor

The biodegradation of the PAH mixture previouslydesorbed with the most efficient concentration of sur-factant achieved with 5,000 mgL−1 of Tween 80 wasstudied.

3.2.1 Influence of Surfactant on T. versicolor

Although surfactants increase the bioavailability ofhydrophobic pollutants, some authors claim that theycan inhibit biodegradation (Zhou et al. 2007; Zhou andZhu 2008). On this assumption, a comparison wasmade between the influence of 5,000 mgL−1 of Tween80 on the glucose consumption by the fungus and thecontrol, without surfactant (Fig. 3). The resultsshowed that T. versicolor consumed the same glucosein both cases indicating no negative effect towards thefungus.

These results contrast with the study by Providentiet al. (1993), which reported that concentrations over700 mgL−1 of Tween 80 caused inhibition on degra-dation by some white-rot fungi. On the other hand,

Kotterman et al. (1998) found no negative effect onfungus growth in the presence of 1,000 mgL−1 ofTween 80.

3.2.2 Biodegradation by T. versicolor of the DesorbedPAH Mixture

Once the feasibility of the study had been determinedfor 5,000 mgL−1 with T. versicolor, a biodegradationexperiment of the desorbed PAH mixture was carriedout. In soils A, B, and C, both the concentration ofpollutants and of glucose in the liquid phase decreasedover time, indicating a possible degradation (OnlineResource 2). In all three soils and for each pollutant,after 1 week, a reduction in the concentration higherthan 90 % was achieved and this was not affected bythe different initial PAH concentrations. Consequently,the residual concentration in the liquid phase of allPAHs also decreased over time for all three soils(Fig. 4).

In soil D, which had the highest clay and siltcontent (20 %), no reduction of PAH concentrationwas observed. The high fine material content could beassociated to high content of a trace element that couldhave inhibited or limited the fungus’ degradative ac-tivity. Baldrian et al. (2003) reported that some heavymetals can interfere negatively, even at low concen-trations, with the white-rot fungi extracellularenzymes involved in degradation processes. For pyr-ene, in soil D, there was a marked increase in pyreneconcentration in the liquid phase over time, indicatingthe existence of desorption. The degradation of theother PAHs from the mixture seems to indicate adecrease in the concentration that promoted the dis-placement of the equilibrium and, therefore, pyrenedesorption, increasing its concentration in the liquidphase. Therefore, reduction or increase in the con-centration is not always synonymous of degrada-tion. To assess whether PAHs were degraded andto be able to make a balance of the pollutant matterthroughout the experiment, a final extraction usingDCM was carried out.

In the present study, the compound biodegraded themost was anthracene, followed by fluorene, pyrene,and phenanthrene, respectively. Biodegradation wasrelated to initial mass in experiments (Table 2). Over-all biodegradation was quite similar in soils A, B, andC but was lower in soil D (Table 2). Higher desorp-tion, on the other hand, does not guarantee greater


biodegradation. For instance, anthracene desorbed theleast yet it is the compound that biodegraded the most.A feasible explanation is that the low concentration ofanthracene in the liquid phase was rapidly degradedcausing continuous displacement of the solid-phaseequilibrium towards the liquid phase. In fact, a similarphenomenon can be observed in soil D. As fluorene,phenanthrene, and anthracene biodegraded, the con-centration of surfactant available to the pollutant in-creased and pyrene was desorbed and transferred tothe liquid phase, but it was not biodegraded. Theglucose concentrations and laccase activity in soil Dindicates very low metabolic activity, which agrees

with the minimum degradation of pyrene and the lowdegradation of the remaining PAHs.

Finally, the low laccase activity could indicate thatit is not the main enzymatic system responsible for theoxidation of PAHs and that another enzymatic system,such as CYT-P450, could be involved in the degrada-tion mechanism. Majcherczyk et al. (1998) state thatlaccase of T. versicolor is capable of degrading severalof the PAHs employed in our study, leading to theformation of the corresponding quinones. However,only for fluorene was a peak detected, which corre-sponded to 9-fluorenone. This was corroborated bymatching the retention time with a standard, which in

Fig. 3 Glucose consump-tion by Trametes versicolordepending on time

Fig. 4 Evolution over timeof the PAH mixture concen-tration in the liquid phase indifferent soils, where fine m.are fine materials


Tab

le2

Balance

ofPA

Hs(m

g)in

thesolid

phaseandliq

uidph

asethroug

hout

thedesorptio

nandbiod

egradatio

nexperiment;theinitial

massin

soils

forfluo

rene,ph

enanthrene,

anthracene,andpy

rene

are0.66

8±0.00

1mg,

0.66

±0.01

mg,

0.77

4±0.00

1mg,

and0.63

0±0.00

1mg,

respectiv

ely,and0mgforliq

uidph

ase

Fluorene

Phenanthrene

Anthracene

Pyrene

Solid

(mg)

Liquid(m

g)Solid

(mg)

Liquid(m

g)Solid

(mg)

Liquid(m

g)Solid

(mg)

Liquid(m

g)

SoilA

(1%

fine

materials,99

%sand

)

Soil–PA

H–surfactantsystem

0.44

3±0.04

30.22

5±0.02

20.36

8±0.03

20.29

2±0.02

60.73

7±0.14

60.03

7±0.00

90.22

2±0.03

70.40

8±0.06

0

After

15days

inpresence

ofT.

versicolor

0.110±0.01

60.01

0±0.00

60.27

3±0.01

50.01

0±0.00

90.011±0.00

10.00

1±0.00

10.24

1±0.01

60.00

8±0.07

Degradatio

n(%

)82.04±8.72

57.12±7.93

98.44±0.01

60.48±4.01

SoilB(5

%fine

materials,95

%sand

)

Soil–PA


0.52

1±0.08

10.14

7±0.02

30.44

2±0.06

50.21

8±0.03

20.74

1±0.07

30.03

3±0.00

40.36

6±0.08

50.26

4±0.00

7

After

15days

inpresence

ofT.

versicolor

0.09

2±0.00

70.01

2±0.00

40.23

6±0.00

20.011±0.00

10.00

2±0.00

10.00

1±0.00

10.16

6±0.00

40.011±0.02

Degradatio

n(%

)84.43±6.42

62.57±5.30

99.61±0.01

71.90±2.73

SoilC(10%

fine

materials,90

%sand

)

Soil–PA


0.51

6±0.113

0.15

2±0.03

30.52

6±0.31

40.13

4±0.08

00.74

9±0.00

00.02

5±0.00

10.47

6±0.29

60.15

4±0.09

0

After

15days

inpresence

ofT.

versicolor

0.04

4±0.00

60.01

8±0.00

60.25

1±0.01

30.011±0.00

10.00

2±0.00

10.00

1±0.00

10.16

1±0.00

30.00

5±0.01

Degradatio

n(%

)90.71±2.57

60.45±4.56

99.61±0.01

73.65±3.21

SoilD

(20%

fine

materials,80

%sand

)

Soil–PA


0.57

1±0.03

00.09

7±0.00

50.53

3±0.03

70.12

7±0.09

0.73

4±0.08

60.04

0±0.00

50.45

8±0.08

40.17

2±0.03

0

After

15days

inpresence

ofT.

versicolor

0.13

5±0.10

10.111±0.00

60.17

6±0.00

80.14

5±0.01

70.00

9±0.00

50.00

3±0.00

10.10

1±0.04

40.52

0±0.03

1

Degradatio

n(%

)63.17±4.22

51.37±6.87

98.44±0.01

1.43

±0.76

Total

degradationisin

bold


turn, coincided with the metabolite described by Collinset al. (1998). Although several authors (Field et al. 1992;Sack and Fritsche 1997; Tekere et al. 2005) havereported that laccase can degrade anthracene and pyr-ene, no other metabolites were detected in the liquidphase. It seems that the other intermediary productswere rapidly metabolized by the fungus.

4 Conclusions

As expected, the increase of fine material contentreduce PAH desorption when surfactants are applied.Likewise, the increase of surfactant concentration rai-ses the total PAH desorption. However, some particu-larities have to be considered depending on thegranulometry together with the surfactant applied.

Depending on which surfactant is applied, it caninteract only with certain PAHs of the mixture, thuslimiting the total amount of PAH desorbed. This be-havior will persist even with the increase of surfactantconcentration. However, in some cases, such as GoldCrew in soil A, the amount of surfactant employed canmodify not only the total amount of PAHs desorbedbut also which ones are desorbed. Hence, an increaseof the surfactant applied can modify substantially thetotal PAH desorbed.

The fine material content of the soil/aquifer (i.e.,the CEC) modifies the behavior of desorption for aspecific PAH mixture when surfactants are applied.According to our results, this behavior is due to thefact that with the increase of fine material content,molecular properties become less importance than soilproperties. With further research, we hope to showhow a mix of surfactants employed and/or the miner-alogy of the fine materials can modify total PAHdesorption for a given granulometry.

Considering the factors affecting desorption bymeans of surfactants, in real pollution episodes insoils and/or groundwater, specific laboratory testsare necessary in each case before applying surfac-tant at a field scale. Data from previous studies,even considering similar PAH mixtures, same sur-factants, and/or similar soils, can only be the start-ing point to design the surfactant application atfield scale. With this procedure, it is possible toimprove the efficiency of remediation and minimizethe financial and environmental impacts when sur-factants are employed.

Our experiments showed that bioremediation of aPAH mixture in different soil granulometry using T.versicolor is possible despite high concentrations ofTween 80 (5,000 mgL−1). In soils with less than 15 %of fine materials, degradation ranged between 57 and99 % depending of the PAH considered. However, insoil with 20 % of fine materials, a decrease in degra-dation was observed. Further research is also neededto learn how high fine material content in the sedimentreduces the effects of biodegradation by T. versicolor.

The component with the lowest desorption(antracene) is the most biodegraded PAH, indicatingthat greater desorption did not guarantee better bio-degradation. Nevertheless, desorption and biodegrada-tion used synergically guarantee better overall resultsin the remediation of soils polluted by PAHs thanother methods that separate desorption and remedia-tion such as pump and treat.

Acknowledgments This work was financed by the CICYTprojects CGL2008-06373-C03-01 and CGL2011-29975-C04-01from the Spanish Government, and projects 2009SGR00103 and2009SGR1199 from the Catalan Government.

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