Effects of Autoclaving and Mercuric Chloride Sterilization on PAHs Dissipation in a Two-Liquid-Phase

Pedophere 21(1): 56 64, 2011ISSN 1002-0160/CN 32-1315/PC 2011 Soil Science Society of ChinaPEDOSPHEREPublished by Elsevier B.V. and Science Presswww. elsevier. com/locate/pedosphereEffects of Autoclaving and Mercuric Chloride Sterilization onPAHs Dissipation in a Two Liquid-Phase Soil Slurry*1WANG CongYingl-2, WANG Fang' , WANG Tao', YANG Xing-Lun', BIAN Yong-Rong', F. 0. KENGARAS,LI Zeng Bo4 and JIANG Xin),*21 State Key Labonatory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008(China)2School of Envirnmental Science and Resources, Shanzi University, Taiyruan 030006 (China)3 Department of Chemistry, Maseno University, Private Bag Maseno (Kenya)4 Institute of Chemical and Biological Technology, Taiyuan University of Science and Technology, Taiyuan 030021 (China)(Received May 27, 2010; revised October 6, 2010)ABSTRACTA two liquid-phase (TLP) soil slurry system was employed to quantify the efficiencies of autoclaving and mercuric chloride 8teri-lization in the dissipation of polyeyclic aromatic bhydrocarbons (PAHs). The fates of 11 PAHs (naphthalene, fuorene, phenanthrene, an-thracene, fuoranthene, pyrene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fuoranthene, benzo(k)fuoranthene, dibenzo(a, h)anth-racene) were recorded over 113 days of incubation. No microorganisms were detected in the HgCl-sterilized soil slurries during thewhole incubation period, indicating very effective steriization. However, about 2%- 36% losses of PAHs were observed in the HgCl2-sterilized slurry. In contrast to the HgCla-sterilized soil slurry, some microorganisms survived in the autoclaved soil slurries. Moreover,significant biodegradation of 6 PAHs (aphthalene, fuorene, phenanthrene, anthracene, fuoranthene and pyrene) was observed in theautoclaved soil slurries. This indicated that biodegradation results of PAHs in the soil slurris, calculated on basis of the autoclavedcontrol, would be underestimated. It could be concluded that the sterilization fficiency and efctiveness of HgCl2 on soil slurry wasmuch higher than those of autoclaving at 121。C for 45 min.Key Words: adsorption, aqueous solubility, biodegradation, microorganisms, octanol-water partition coficientCitation: Wang, C. Y, Wang, F, Wang, T, Yang, X. L, Bian, Y. R, Kengara, F.0., Li, z. B. and Jiang, X. 2011. Efects ofautoclaving and mercuric chloride sterilization on PAHs dissipation in a two-liquid- phase soil slurry. Pedosphere. 21(1): 56- 64.INTRODUCTIONsamples, due to its low cost and ease of use. However,the sterilization effectiveness of autoclaving dependsIn laboratory experiments, sterilized soil is fre-on time. Trevors (1996) suggested that the minimumquently used as a control to investigate the biologicalautoclaving time for soil sterilization was 20 to 30 minand abiological processes in soil, including degradationat 121 °C and 0.1 MPa. Furthermore, the larger theof pesticides and other xenobiotics, sorption and mo-amount of soil, the longer the time that should bebility of contarninants in soils, plant biological test sys-used. However, the longer the time, the more the dam-tems, and microbiological tests such as enzyme assayage that the soil would be subjected to. Ideally, ster-(Kruger et al, 1997). Various sterilization procedures,ilization should cause no measurable changes in thesuch as autoclaving (Berns et al, 2008), ry-irradiation,physical and chemical properties of the soil while com-microwaves, amendment with poisons (e.g., HgC12,pletely eliminating all viable organisms (Bank et al,NaN3), and gaseous chemical (including ethylene ox-2008). Therefore, a short and efective time is required.ide, propylene oxide, chloroform and methyl bromide)It has been assumed that 20 to 30 min is enough forhave been employed in previous studies (Sheremata etsterilizing soil/sediment slurries in the study of pollu-al, 1997; Paguirigan and Beebe, 2007; Yamamoto ettant degradation (Siddique et al, 2002; Yu et al, 2005;al, 2008). Among them, autoclaving (steam steriliza-Wang et al, 2008). Sediment slurries and soil- watertion) is the most widely used method for sterilizing soil?claving for 20 min中国煤化工*1Supported by the National High Technology Research and Development FHCN M H Go.20010)0 andthe National Natural Science Foundation of China (Noe. 20707028, 4092106*2Corresponding author. E mail: jiangxin@issas.ac.cn.STERILIZATION EFECT ON PAHS DISSIPATION57(Melkonian, 2009). Nonetheless, Trevors (1996) recom-cocktail of PAHs was motivated by the fact that pol-mended that soil should not be saturated with wa-luted sites are usually contarinated with a mixture ofter during autoclaving because air-dried soil or soilcompounds (Thiele Bruhn and Brimmer, 2005; Yin etwith low water content (less than about 60% of wa-al, 2008). The objective of the study was to quantifyter holding capacity) would allow for better steriliza-the eficiencies of the two commonly used sterilizationtion. However, losses of the tested compounds wereprotocols by comparing the effects of the two steriliza-observed in the sterile treatment, although microor-tion methods on dissipation of PAHs in the TLP soilganism activity was not measured to determine theslurry system.effectiveness of the sterilization (Siddique et al, 2002;MATERIALS AND METHODSYu et al, 2005; Wang et al, 2008). Therefore, it isnecessary to find out whether or not the losses wereSoil materialssolely due to abiotic degradation in the autoclaved soilslurry.Two diferent soil samples were used; an agricul-HgCl2 sterilization, another commonly used sterili-tural soil without prior PAHs contamination (AS)zation method, generally results in minimal changes incollected from an agricultural field in the suburb ofsoil properties compared with other techniques (WolfBaguazhou in Nanjing of China, and a contaminatedet al, 1989; Trevors, 1996) and may obtain a sterilesoil (CS) ftom a long-term PAH contaminated site nearenvironment for a long time (Tuominen et al, 1994;a steel plant in Nanjing suburb. They were silt loams,Stephens et al, 2002). However, as a chemical treat-classified as Luvisol according to WRB (World Refer-ment, it targets specific biological processes and there-ence Base for Soil Resources). Soils were sampled fromfore may result in incomplete sterilization (Radloff etthe upper layer (0 20 cm), sieved ( < 2 mm) and storedah, 2008). Thus it is necessary to think through theat 4 °C. Prior to the start of laboratory experiments,fate pathways of target compounds in a biodegrada-the soil samples were equilibrated for one week at 28 °Ction study, so as to properly quantify the degradationwith 60% of the water holding capacity. Selected soilrates.properties are listed in Table I, and the concentrationsMany reports on the biodegradations of polycyclicof PAHs in soils are given in Table II.aromatic hydrocarbons (PAHs) in soils indicate thatChemicals and culture mediumsterilized soil is often used as a control (Yu et al, 2005;Wang et al, 2008). However, limited bioavailability ofNaphthalene (NAP), fuorene (FLR), phenanthre-PAHs in soil is an obstacle to understanding and ob-ne (PHE)， anthracene (ANT)， fuoranthene (FLN),jectively assessing the fficiencies of sterilization proto-pyrene (PYR), benzo(a)anthracene (BaA), benzo(a)cols. Two-liquid-phase (TLP) systems are thus widelypyrene(BaP), benzo(b)fuoranthene (BbF), benzo(k)used in PAHs degradation studies due to the meritsfuoranthene (BkF), dibenzo(a, h)anthracene (DBahA)of ignoring the inAuence of pollutants' aging and masswith 99.0% to 99.9% purity were obtained from Su-transfer on PAHs degradation, optimizing the microen-pelco Corporation, USA. Silicone oil (polydimethyl-vironment for the organisms, and facilitating the desiloxane, fiuid type; molecular weight, 2000; viscosity,termination of PAHs concentrations (Ascon-Cabrera et50 centistokes; density, 0.96 g cm- 3) was purchasedal, 1993). In the present study, a TLP soil slurry sys-from Sinopharm Chemical Reagent Company, Ltd.,tem was employed to investigate the efficiency of theShanghai, China. AIll the other chemicals and reagentstwo commonly used sterilization methods: autoclav-were of analytical grade and were purchased from Nan-ing and HgCl2 sterilization. PAHs were used a8 modeling Reagent Company, China.compounds for hydrophobic organic compounds. TheMineral medium (MM, per liter) consisted of 8.8behavior of a PAHs mixture in soil slurry, sterilized byg Na2HPO42H2O, 3.0 g KH2PO4, 1.0 g NH4Cl, 0.5gautoclaving and HgCl2, was monitored. The use of aNaCl, 1.0 mL 1 mol L-1 MgSO4, and 2.5 mL slightlyTABLE ISelected physico chemical properties of the soils studiedSoil)TOCb)Total NP2OsTotal KSitSandmg kggkg-1中国煤化工-%_AS23.051.3215.94YHCNMHG52.2331.30CS27.521.2017.6814.2214.8023.8257.0519.13)AS = soil from an agricultural site, CS = soil from a contaminated site; b)Total organie carbon; c)Cation exchange capacity.5C. Y. WANG et al.TABLE IISelected physico-chemical properties and initial concentrations of polycyclic aromatic hydrocarbons (PAHs) used in the studyPAHlogKowo)AqueousVaporInitial concentra-Initialsolubilityb)preseurec)tion in soild)concentration incontcontaminateA:Ssilicone oilmgL-1二mgkg-1，-Naphthalene (NAP)3.3732.010.40.0290.1217.92Fluorine (FLR)4.182.204.27 x 10-20.0270.03911.61Phenanthrene (PHE)4.501.209.07 x 10-2 .0.0070.41910.82Anthracene (ANT)2.27 x 10-3.0.042 .0.10110.55Fluoranthene (FLN)5.000.213.33x 10-40.0061.21411.36Pyrene (PYR)0.156.67 x 10-40.0051.1151.05Benzo(a)anthracene (BaA)5.800.012.93x 10-60.015 0.66211.9Benzo(b)fuoranthene (BbF)5.781.00x 10-36.67 x 10-50.005 0.9080.02Benzo(k)fuoranthene (BkF)6.801.28x 10-80.0040.3978.91Benzo(a)pyrene (BaP)6.006.00 x 10- 37.47 x 10-70.0080.8121.37han th6.00x 10-3Dibenzo(a,h)anthracene (DBahA)1.33 x 10-80.0090.12912.94”) Kow = octanol water partitin cofficient; b)Mackay et al, 2006;。) ATSDR, 1995; d) AS = soil from an agricultural site, CS = soilfrom a contaminated site.modified trace element solution (23 mg L-1CK-Hg contained 100 mL sterilized MM, 40 g con-MnCl2:2H2O, 31 mg L-1 H3BO3, 36 mg L-1 CoCl2. .taminated silicone oil and HgCl2 (1000 mg L- 1). The6H2O, 10 mg L-1 CuCl2.2H2O, 20 mg L-1 NiCl2.un-sterilized control, contained 5 g soil, 95 mL sterili-6H2O, 30 mg L-1 Na2MoO42H2O and 50 mg L-zed MM and 40 g contaminated silicone oil, but with-ZnCl2) at pH 7 (Bastiaens et al, 2000).out autoclaving or HgCl2. All the treatments wereperformed in triplicates and were incubated at 28。CContaminated silicone oil preparationon a rotary shaker at 160 r min-1 in dark. SiliconeThe purchased silicone oil was treated with acti-oil and soil suspension were sampled periodically forvated carbon to get rid of unpleasant smell. The con-PAH determination and cell counting. The PAHs con-taminated silicone oil was prepared as described bycentrations in water phase were not determined oW-Wang et al. (2010): certain amounts of each PAHing to low water solubility of most PAHs (Table II),were mixed homogenously with the treated silicone oilwhich could also be neglected compared to those in oilby stirring with Teflon-coated magnetic stirring barsphase (Table II) (Villemur et al, 2000). Furthermore,in a beaker. After that, the contaminated silicone oilto precisely measure the PAHs concentrations in water,was fltered through a 0.22 um polytetrafluoroethy-large amount of water had to be sampled, which wouldlene membrane to eliminate impurities and microor-change the ratio of water to oil phase and thereafterinfuence the dissipation behavior of PAHs in the TLPganisms.system. Therefore the concentrations of PAHs in theDissipation of PAHs in two-liquid-phase (TLP) systemoil phase, instead of the PAHs amount in the TLP sys-with different sterilization methodstems, were used to describe their dissipation behavior.Autoclaving of soil was performed as follows: 5 gCell countingequilibrated soil (AS or CS, dry weight equivalent) wastransferred into a 250 mL fAask containing 95 mL MM,The detection of microorganisms in the TLP slurryfollowed by autoclaving at 121 °C and 0.1 MPa for 45was performed using dilution plate counting. One mLmin. After cooling to room temperature, 40 g con-of soil suspension without silicone oil was sampled totaminated silicone oil was added to the fask. Thedilute with sterilized MM and then spread on the nu-treatments were labeled as AS au and CS au, respec-trient broth agar plates. Colony-forming units (cfu)tively. HgCl-sterilization was conducted as follows:were counted after incubating at 28 °C for 4 d.5 g soil was added to the 250 mL flask containing 95Dete中国煤化工lions in slicone oilmL MM, and then solid HgCl2 was introduced to ob-tain a final concentration of 1000 mg L-1. The sameYHC N M H G determined accor-amount of silicone oil was added as mentioned above.ding' to the procedure reported by Villemur et al.They were numbered as AS Hg and CS Hg. The blank(2000): 0.5 mL silicone oil sample was vortexed withSTERLIZATION EFFECT ON PAHS DISSIPATION591 mL of N,N dimethylformamide (DMF) for 2 minthat HgCl2 sterilization was much more eficient thanand centrifuged at 1150 x g for 1 min to separate thetwo phases. A volume of 0.5 mL of the upper phaseresults also contributed to the growing body of ev-was then mixed with 0.5 mL of acetonitrile contain-idence that autoclaving of soil slurry at 121 。C foring 0.1% acetic acid. The mixture was finally analyzed45 min is insuficient for complete sterilization (Sukulby a Shimadzu HPLC (LC-20A, Japan) with a fuo-and Spiteller, 2001; Chen et al, 2008). As reportedrescence detector and a Supelco (USA) PAHs specialby Razavi Darbar and Lakzian (2007), the bacterialchromatogram column (25 cm x 4.6 mm, 5 pum particlepopulations in the soil were only decreased from 207diameter) according to the program described by Yinx 108to1 x 108 g-1 soil with autoclaving at 121。Cet al. (2008).for 15 min, although autoclaving is much more eficientin sterilizing soil than chloroform, ultraviolet irradia-Quality control and data analysistion and microwave irradiation. Villemur et al. (2000)Experiments on recovery were carried out by spik-sterilized soil three times for 60 min at 1-day intervalsing clean silicone oil with known concentration PAHs.for their abiotic TLP slurry systems. In a recent reportThe extraction and purification of the sample were per-(Manning et al, 2006), the mixture of 3 g sediment andformed in the same procedure as above and average6 mL of artificial groundwater was autoclaved for 1 honce or on three consecutive days. Autoclaving in therecoveries for 3 replicates ranged from 88% to 105%.SigmaPlot (Version 10.0) was used for simple dataproposed way was successful in keeping the sedimentmanipulations and plotting graphs. All the statisticalsterile for 30 d, although it altered the geochemistry ofdata analysis was done by a software package, SPSSthe sediment. Accordingly, the autoclaving time shouldbe prolonged or the autoclaving frequency should be13.0, and the significance level was P < 0.05.increased to ensure a complete elimination of microor-RESULTS AND DISCUSSIONganisms from the soil slurry.Bacteria growth in the sterilized TLP soil slurryDissipation of PAHs in the TLP slurry with HgC-To test the effect of autoclaving and HgCl2-sterilizationsterilization on microbial growth in the biodegrada-tion experiment, aliquots of soil suspension withoutTo investigate the effects of HgCl-sterilization onsilicone oil were sampled for cell counting. No colonythe dissipation of PAHs in the TLP soil slurry, thwas detected during the 113 days' incubation in theconcentrations of 11 PAHs in silicone oil were followedHgCl2-sterilized TLP slurry both with and withoutfor 113d (Figs.1 and 2). The HgCl2-sterilized treat-soil, showing that the microorganisms in both soilsments with and without soils were compared. No sig-were killed by the HgCl2. The results indicated thatHgCl2 had a high and long eficiency of sterilization,was observed between the soil slurry (AS-Hg, and CS-which agreed with reports in literature (Tuominen etHg) and the control without soil (CK Hg) during theal, 1994; Radloff et al, 2008). In contrast to thewhole incubation period (Figs. 1 and 2), showing thatHgCl-sterilized TLP slurry, the number of microor-the presence of both sterile contarminated and uncon-ganisms in the AS-au and CS- au increased from 0 totaminated soils did not affect the PAHs dissipation in0.98 x 106 and to 6.33 x 106 cfu mL-', respectively,the TLP soil slurry. This indicated that PAHs degra-after 4 d incubation. The microorganisms reached 2.52dation did not occur during the 113 d incubation pex 107 and 2.79x 107 cfu mL-1 in the AS-au and CS_ .riod and this was confirmed by the fact that no mi-au, respectively, at the end of 113 d incubation. Thiscroorganisms were detected during the incubation asshowed that some of the microorganisms were killedmentioned above. However, for all the HgCl2 sterilizedduring the process of autoclaving, and that some oftreatments (AS-Hg, CS-Hg and CK-Hg), PAHs con-the surviving microorganisms revived during the incu-centrations did decrease during the incubation periodbation. To confrm the survival of the bacteria, theand only about 64.3% to 97.7% of the initial concentra-autoclaved soil slurries, without introduction of PAHs-tion of the PAHs were detected after 113 d incubation.polluted silicone oil, were also sampled for cell count-中国煤化工-urred in the HgCl2-ing. A few colonies were observed on the agar plates af-steril: loss was not causedter 3 days' incubation, showing that surviving microor-by biYHC N M H ispation.oganisms did exist in the autoclaved soil slurries andThe abiotic losses of PAHs in the HgClz-sterilizedthat they could survive without PAHs. This showedTLP soil slurries were determined by the difference be-60C. Y. WANG et al.1412 [ NAP。。FLFANT口AS10上=气020406080100120020 40608010012002040608010012014 [PHEPYR至10+++020406080100120020406080100120020406080100120Incubation period(d)Fig. 1 Dissipation of polycyclic aromatic hydrocarbons (PAHs), including naphthalene (NAP), fuorene (FLR), anthracene (ANT),phenanthrene (PHE), pyrene (PYR) and fuoranthene (FLN), in two-liquid phase (TLP) slurry. AS-Hg = HgCla-sterilized slurry withagricultural soil; CS-Hg = HgCl2 sterilized slurry with PAHg-contaminated soil; CK-Hg = soil free HgCla-sterilized slurry; AS-au= autoclaved slurry with agricultural soil; CS-au = autoclaved slurry with PAHs-contaminated soil; AS = un- sterilized slurry withagricultural soil; CS = un- sterilized slurry with PAHs contaminated soil. Vertical bars represent standard deviations of the means(n=3).1200 AS-Hg 0 CS-Hg 8 CK-Hg 0 AS-au 0 CS-au日AS四Cs86(4(2BaABaPBbF3KFDBahAPAHFig. 2 Reeidual amounts of polycyelic aromatic hydrocarbon (PAH8), including benzo(a)anthracene (BaA), benzo(a)pyrene (BaP),benzo(b)fuoranthene (BbF), benzo(k)Auoranthene (BkF) and dibenzo(a,h)anthracene (DBahA), in two-liquid-phase (TLP) slurryafter 113 days of incubation. AS-Hg = HgCl2-sterilized slurry with agricultural soil; CS Hg = HgCl2-sterilized slurry with PAH8- contarminated soil; CK-Hg = soil free HgCla-sterilized slurry; AS-au = autoclaved slurry with agricultural soil; CS-au = autoclaved slurrywith PAHs contaminated soil; AS = un-sterilized slurry with agricultural soil; Cs = un- sterilized slurry with PAHs contaminated soil.Vertical bars represent standard deviations of the means (n= 3).tween the initial concentrations and the mean valu-studies also showed similar results (Gauthier et al,es of the three HgCl-sterilized treatments (AS-Hg,2003). However, the possible reason for the abioticCS-Hg and CK-Hg). Most of the PAHs showed aboutlosses was not explained in the previous studies. It10%- 30% abiotic losses of the initial concentrationsis therefore necessary to elucidate the possible path-in the earlier period of 7-20 d incubation, and thenways of PAHs losses in the sterilized TLP slurry, asstabilized at this level. Benzo( k)fuoranthene and di-this would enable the comprehensive understanding ofbenzo(a, h)anthracene had the lowest losses of 2.3%the dissipation behavior of PAHs in the sterilized TLPand 3.1% respectively. The highest loss was 35.7%slufor NAP. Villemur et al. (2000) and Marcoux et中国煤化工hways for the abi-al. (2000) observed nearly 10%- -30% abiotic lossesotic[YHC N M H Grilized TLP slurryof high-molecular weight PAHs during the incubationsuch as sorption, voltluzaton and photolysis. Inof TLP slurry with sodium azide sterilization. Otherthe present study, the losses might not have resultedSTERILIZATION EFFECT ON PAHS DISSIPATION61from biosorption or photolysis. First of all, the adsorp-with higher vapour pressure, higher octanol water par-tion of the PAHs on the sterile soil was negligible duetition coefficient (logKow) and higher water solubilityto the fact that there were no significant diferences(Table I). This showed that volatilization of PAHs and(P > 0.05) in concentrations of the PAHs between thethe distribution of PAHs between silicone oil and wa-two TLP soil slurries (AS-Hg and CS-Hg) and the con-ter might be the possible pathways for the losses introl without soil (CK-Hg) (Figs.1 and 2). Secondly,the sterilized TLP slurries. Although some studies resince no microorganisms capable of degrading PAHsported that biphasic reactors reduce the volatilizationwere introduced into treatment CK-Hg, and no mi-of compounds (Ascon-Cabrera et al, 1993; Guieyssecroorganisms were found during the incubation in allet al, 2001), volatilization of PAHs could still occur.the HgCl2-sterilized TLP slurries, biosorption as theThis is because the silicone oil and water in the faskspossible cause of abiotic loss of PAHs was ruled out.were mixed continuously and therefore some readilyFnally, photolysis of PAHs should be excluded as wellvolatile PAHs, especially NAP, could escape from thebecause the fasks containing the TLP slurry were keptliquid phase. Moreover, the temperature employed inin the dark during the entire experimental period. Ad-the present study was 28 °C, which might facilitate theditionally, it has been reported that photodegradationvolatilization. Additionally, the loss could also be con-of PAHs in solution is an oxidative process and thattributed to the distribution of PAHs from silicone oilthe more polar the solvent, the faster the degradationto water phase. This was indicated by the fact thatprocess of PAHs (Lehto et al, 2000). Given that the sil-the loss rates of PAHs in the HgCl2-sterilized TLPicone oil used in the present study is a non-polar com-slurries negatively correlated with their logKow andpound, it might not have facilitated the photodegra-positively correlated with logarithm of water solubilitydation of PAHs. The adsorption of PAHs to the gla8s8(Fig. 3). Considering the relative poor water solubilitieisfAask's wall might also lead to the losses. However,of most of the compounds, especially the high molec-recent studies on PAHs sorption, conducted in glassular weight PAHs (Table II)， the amounts of PAHsflasks, showed that PAHs losses were less than 4%dissolved in the water phase cannot account for the re-of the initial concentrations (Lemic et al, 2007). Theduced PAHs concentrations in the silicone oil. PAHslimited amount of PAHs adsorbed to the glass wall ofmight be“adsorbed" at the interfaces between the wa-the fAask could thus not explain the large amount ofter and the silicone oil, since the concentration of PAHsPAHs losses, especially the low molecular weight PAHscould be accumulated on the interface due to the in-(Fig. 1 and Table II). Besides, bydrophobicity is theterfacial energy distribution heterogeneity (Benjamin,main factor controlling the sorption of PAHs to glass1997; Wachter, 2001; Benjamin, 2009). The solutionwalls (Lemic et al, 2007), meaning tbat the sorptioncan decrease its Gibbs free energy to the lowest to aC-capacity would increase with hydrophobicity. However,quire stability, and this would induce positive or neg-in this study, a completely opposite sequence was ob-ative adsorption of the solute in the interfaces (Ben-tained (Figs. 1 and 2 and Table I),，confirming the con-jamin, 1997; Wachter, 2001). A positive adsorption-clusion that sorption of PAHs on the glass-wall did notsolute is accumulated in the interface- -is characterizedplay a key role in the abiotic loss.by an excess of positive surface coverage and vice versaWhat then was the key factor contributing to the(Wachter, 2001; Benjamin, 2009). Yoon et al. (2009)loss of PAHs in the HgCl2- sterilized TLP slurry? Asfound positive values of surface coverage for hydropho-can be seen from Figs. 1 and 2, more losses of PAHsbic compounds such as benzene in the oil-water system,in the TLP slurries were detected for the compoundswhich is in agreement with our hypothesis.40「a40「35y= 5.2884x+ 22.2513030 tR* = 0.715225520?0 t10 y=00*+5.*42◆0tR'= 0.8017中国煤化工2logK.THCNMHGFig. 3 Relationships between the losse of 11 polycyclic aromatic hydrocarbons (PAHE) in HgCl2-sterilized slurriee and their (@)octanol- water partition coefficient (Kow) and (b) aqueous olubility (S). .C. Y. WANG et al62Dissipation of PAHs in the autoclaved TLP soil slurrytinguish between biological and abiological processes,it is necessary to reassess the biodegradation rates ofThe changes in concentrations of NAP, FLR, ANT,PAHs in the TLP soil slurry. Dissipation of PAHs in thePHE, PYR and FLN in the autoclaved TLP slurryHgCl-sterilized TLP slurry was caused, actually, by aare presented in Fig. 1. Compared with the HgCl-technical drawback. It is impossible to eliminate thesterilized TLP surries (AS-Hg, CS-Hg, and CK-Hg)interfacial efects between the two liquid phases. How-where there was no detectable biodegradation of allever, the substrates enriched in the interfaces would bethe PAHs, significant biodegradation of the 6 PAHsutilied by microorganisms when PAHs are fnally useddid occur in the AS-au and CS au. The results agreedup (Deziel et al, 1999). Therefore the biodegradationwith the facts that no bacteria were detected in theof PAHs in the TLP slurry would be underestimatedTLP slurry with Hgsterilization, whereas a lot of bac-when the calculation is based on the HgCl-sterilizedteria were observed in the autoclaved TLP slurry. Thecontrol.results also showed that autoclaving for 45 min did notCompared with HgCla-sterilization, which is akill all the bacteria in the soil, and that microorgani-toxic seriling method, autoclaving is more often 1usedsms able to degrade NAP, FLR, ANT, PHE, PYR andto investigate biodegradation behavior of compoundsFLN did exist in the autoclaved soil slurry.in the soil/sediment. However, our results showedHowever, the degradation of all the 6 PAHs in thethat high degradation of PAHs occurred in the 8l-autoclaved TLP slurries was much slower than that intoclaved TLP slurry because the surviving microor-both control treatments of AS and CS without steri-ganisms maintained their degradation abilities afterlization (Fig. 1). For example, complete disappearancethe autoclaving. Dissipation of chemical compoundsof NAP occurred in 7 d in both the autoclaved treat-has been observed in autoclaved soil/8ediment slurryments of AS-au and CS au, whereas the concentrations(Sukul and Sieller, 2001). Chen et al. (2008) auto-of NAP decreased to nearly detection limits in onlyclaved 100 g fresh sediment with 100 mL water for4 d in the un-sterilized TLP slurries. A similar trend20 min and found that the loss of phenanthrene waswas observed with the other PAHs (FLR, ANT, PHE,about 1% after 6 d incubation. Yu et al (2005) rePYR and FLN). The results showed that the microbialported around 30% lo8s of fluorine in sterile sedimentpopulation capable of degrading PAHs in the surrisslurry autoclaved for 30 min. Nevertheless, the descrip-(AS-au and CS-au) was decreased by autoclaving.tion of compound biodegradation is generally based onThe residues of BaA and the 5- ring PAHs (BaPthe autoclaved control. Thus, biodegradation of theBbF, BkF and DBalA) in the sterilized TLP system,compound would be underestimated because the com-after 113 d incubation, are presented in Fig.2. In Con-pound might also be biodegraded in the autoclavedtrast to biodegradation of the lower-ring PAHs (Fig. 1),the biodegradation of BaA and 5-ring PAHs was notTo quantify this underestimation, PAHs degrada-observed in the autoclaved TLP slurries during the 113tion rates in AS and CS were calculated based on thedays incubation, showing similar degradation dynami-HgCla-sterilized control and the autoclaved control, recs to PAHs in the HgCl-sterilized TLP surrie. How-spectively. The degradation rate was only calculatedever, in the control without sterilization, significantfor those compounds which showed biodegradation indegradation of the five PAHs occurred, with about 30%the autoclaved soil slurry (Fig. 1). As shown in Tableto 87% and 10% to 83% of the initial concentrationsII, based on HgCla-sterilized control, about 61% toremaining in AS and CS respectively (Fig.2). The re91% of the 6 PAHs were degraded in the AS and CS soilsults showed that microorganisms able to degrade BaAslurries in 113 days, whereas only 0.3% to 40% were de-and 5-ring PAHs and/or some populations involved ingraded based on the autoclaved control. This indicatedPAHs co metabolism were eliminated from the origi-that PAH degradation would be highly underestimatednal soil by autoclaving, indicating that autoclavingwhen the autoclaving could not eliminate all the bac-changed the structure of the microbial population interia in soils. Based on the HgClx-sterilized control,the soil.degradation rates decreased gradually from the low-Reasessment of biodegradation rates in the TLP soilring PAHs to the high-ring PAHs in both AS and CSsoil slurries and this was in arreement with former reslurrypor中国煤化工al, 2010). However,As aforementioned, dissipation of PAHs occurred indiffeY片CN M H Gation were observedboth the autoclaved and HgCla-sterilized TLP soil slur-in ASaud woul Bult Uastu Uu the autoclaved con-ries (Fig. 1). As the sterilized control was used to dis-trol. Biodegradation rates of FLR and ANT were muchSTERILIATION EFFECT ON PAHS DISSIPATION63higher than those of the other 4 PAHs for both soil slur~Bank, T. L, Kukkeadapu, R. K., Madden, A. S.,. Ginder Vogel,ries, showing an opposite result to the common obser-M. A., Baldwin, M. E. and Jardine, P. M. 2008. Effects ofgamma-sterilization on the physico-chemical propertics ofvation that PYR is more dificult to degrade than ANTnatural sediments. Chem. Geol. 251(1- 4)4):1-7.and FLR (Bossert and Bartha, 1986; Thiele-Bruhn andBastiaens, L., Springael, D., Wattian, P., Harrms, H, Dewachter,Brimmer, 2005). Therefore an ineffective control, likeR, Verachtert, H. and Diels, L. 2000. Isolation of adherentthe autoclaved TLP soil slurry in this study, mightpolycyclic aromatic hydrocarbon (PAH)- degrading bacteriausing PAH sorbing carriers. Appl. Environ. Microbiol.lead to a misunderstanding of a compound's degrada-66(5): 1834-1843.tion behavior.Benjamnin, L. 1997. Molecular structures and dynamics at liquid-liquid interfaces. Annu. Rev. Phys. Chem. 48: 407- -451.TABLE IIBenjamin, I. 2009. Solute dynamics at aqueous interfaces. Chem.Phys. Lett. 469(4-6): 229 -241.Degradation rates of polycyclic aromatic bhydrocarbons (PAHs)in the soils from an agricultural site (AS) and a contaminatedBerns, A. E, Philipp, H, Narres, H. -D., Burauel, P, Vereecken,H. and Tappe, W. 2008. Fffect of gammarsterilization andaite (CS) calculated based on HgClr-sterilized and autoclavedcontrolautoclaving on soil organic matter structure as studied bysolid state NMR, UV and fuorescence spectroecopy. Eur.PAHBased on HgCl2-Based on auto-J. Soil Sci 59(3); 540- -50.sterilized control、claved controlBossert, I. D. and Bartha, R. 1986. Structure-biodegradabilityrelationshipes of polycyclic aromatic hydrocarbons in soil.SASBull. Environ. Contam. Tox. 37(1): 490- 495.二%Chen, J., Wong, M. H., Wong, Y. S. and Tamn, N. F. Y.2008.Naphthalene (NAP)63.51 60.980.632.02Multi-factors on biodegradation kinetics of polycyclie aro-Fluorene (FLR)81.4878.9840.1417.74matic hydrocarbons (PAHs) by Sphingormonas sp. a bacte-Phenanthrene (PHE) 76.6272.830.280.46rial strain isolated from mangrove sediment. Mar. Pollut.Anthracene (ANT)81.99 80.19 38.4831.09Bull. 57(6- 12): 695 -702.Fluoranthene (FLN)90.85 88.56 .3.170.26Deziel, E, Comneau, Y. and Vllenur, R. 199 Two liquid-phasePyrene (PYR)91.4985.791.541.0bioreactors for enhanced degradation of hydrophobic/toxiccompounds. Biodegradation. 10(3): 219 233.Gauthier, E., Deziel, E, Villemur, R, Juteau, P., Lepine, F. andCONCLUSIONSBeaudet, R. 2003. Initial characterization of new bacteriadegrading high-molecular weight polyeyelic aromatic bhydro-No bacteria were detected and no significantcarbons isolated from a 2-year enrichment in a two-liquid-phase culture system. J. Appl. Microbiol. 94(2): 301-311.biodegradation of PAHs occurred in the soil slurriesGuieysse, B., Cirne, M. d. D. T. G. and Mattiasson, B. 2001.with HgCla-sterilization, showing that HgCl2 is anMicrobial degradation of phenanthrene and pyrene in a two-effective and efficient bactericide for sterilization ofliquid phase partitioning bioreactor. Appl. Microbiol. Biot.water -soil/sediment slurry. On the other hand, auto-56(5): 796- -802.claving of soil slurry at 121。C for 45 min did notJuhasz, A. L., Stanley, G. A. and Britz, M. L. 2000. Degrada-tion of high molecular weight PAHs in contaminated soil bycompletely eliminate the microorganisms capable ofa bacterial consortium: Efects on microtox and mutagenic-degrading PAHs in soil. It is necessary to prolong theity bioassays. Bioremediation J. 4(4): 271 283.autoclaving time and/or repeat autoclaving more timesKruger, E. L, Rice, P. J., Anhalt, J. C, Anderson, T. A. andCoats,J. R.1997. Comparative fates of atrazine and deethy-on consecutive days to ensure complete sterilization.latrazine in sterile and nonsterile soils. J. Environ. Qual.The autoclaved soil/sediment should be analyzed for26(1): 95-101. .microbial growth before a biodegradation experimentLehto, K-M., Vuorimaa, E. and Lernmetyinen, H. 2000. Phois performed to confirm the effectiveness of the ster-tolysis of polycyclic aromatic hydrocarbons (PAHs) in di-lute aqucous solutions detected by fuoreecence. J. Photoch.ilization. This would help to avoid wrong evaluationPhotobio. A. 136(1-2): 53 60.of biodegradation of PAHs in the TLP system and toLemic, J, TomaJevicCanovic, M, Adamovic, M., KovaCevic,enable better understanding of the biodegradation be-D. and Milicevic, s. 2007. 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