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Benzo[a]pyrene degradation using simultaneously combined chemical oxidation, biotreatment with Fusarium solani and cyclodextrins
Bioresource Technology 100 (2009) 3157–3160
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Benzo[a]pyrene degradation using simultaneously combined chemical oxidation, biotreatment with Fusarium solani and cyclodextrins Catherine Rafin *, Etienne Veignie, Antoine Fayeulle, Gheorghe Surpateanu Laboratoire de Synthèse Organique et Environnement (EA2599), Université du Littoral Côte d’Opale, 145 Avenue Maurice Schumann, 59140 Dunkerque, France
a r t i c l e
i n f o
Article history: Received 27 November 2008 Received in revised form 8 January 2009 Accepted 11 January 2009 Available online 25 February 2009 Keywords: Polycyclic aromatic hydrocarbons Fusarium solani, biodegradation Fenton’s reaction Cyclodextrins, benzo[a]pyrene
a b s t r a c t The interest of simultaneously combining chemical (Fenton’s reaction) and biological treatments for the degradation of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) has been studied in laboratory tests. An optimal concentration of 1.5 10 3 M H2O2 as Fenton’s reagent was firstly determined as being compatible with the growth of Fusarium solani, the Deuteromycete fungus used in the biodegradation process. For the enhancement of BaP solubilisation, cyclodextrins were also used in the performed tests. The best degradation performance was achieved through the use of 5 10 3 M hydroxypropyl-b-cyclodextrin (HPBCD) in comparison with randomly methylated-b-cyclodextrin (RAMEB). When Fenton’s treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%) in the presence of HPBCD for 12 days of incubation. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) containing five fused benzene rings, is considered to be one of the 16 PAHs defined as priority pollutants by the US Environmental Protection Agency (EPA) due to its carcinogenicity, teratogenicity and acute toxicity. This pollutant tends to persist in the environment partly because of its very low water solubility and its strong adsorption onto the soil organic matter. Among methodologies developed to extract PAHs from solid matrixes, cyclodextrins (CDs) are commonly used as alternative non-toxic extracting agents (Rivas, 2006). CDs are cyclic oligosaccharides composed of a-1,4-linked glucopyranose subunits that easily form inclusion complexes with a wide variety of organic molecules. These inclusion complexes show an apparent solubility higher than those of the compound alone in aqueous phase. The degradation of PAH needs an initial oxidation stage, that could be produced by either a chemical or a biological process. Fenton-based technology is by far the most studied chemical oxidation technology in soil remediation. Fenton’s reaction can also be applied in combination with bioremediation, each method possessing its own advantages and drawbacks (Kulik et al., 2006). However, bioremediation of PAH-polluted soil is severely hampered by the low rate degradation of high molecular weight PAHs due to limited mass transfers and pollutant bioavailability and to the scarcity of
adequate microorganisms. Therefore, substantial investigation has focused attention on microorganisms able to degrade high molecular weight PAHs for cleaning up contaminated sites (Juhasz and Naidu, 2000; Rafin et al., 2006). In our previous studies, we isolated a Deuteromycete fungus F. solani (Mart.) Sacc. (1881) [teleomorph: Haematonectria haematococca (Berk. and Broome) Samuels and Rossman, Ascomycota, Hypocreales, Nectriaceae] that was able to mineralise [7,10-14C]benzo[a]pyrene (BaP) rapidly (Rafin et al., 2000). Nevertheless, the BaP degradation rate obtained in treatment inoculated with the fungus remains quite low. To enhance bioremediation process efficiency, the present work was conducted to study, at laboratory scale, the interest to combine simultaneously chemical oxidation (by Fenton’s reaction) with biological treatment (in presence of F. solani) for BaP degradation. BaP was selected as a high molecular weight PAH model, composed of five aromatic rings, well known for its very weak solubility (Kow 6.06, Juhasz and Naidu, 2000) and therefore weak bioavailability. Various CDs were studied in order to evaluate how their complex-forming capacity with the pollutant could influence BaP degradation rate in combined chemical–biological treatment.
2. Methods 2.1. Chemicals
* Corresponding author. Tel.: +33 3 28 65 82 78; fax: +33 3 28 65 82 31. E-mail address: rafi[email protected] (C. Rafin). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.01.012
Benzo[a]pyrene (BaP) and hydrogen peroxide (H2O2) 30% were purchased from Acros Organics (Noisy-Le-Grand, France).
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Dichloromethane (DCM) and methanol (MeOH) were obtained in the highest purity grade available from Merck (Darmstadt, Germany). Distilled deionised water was used throughout this work. b-cyclodextrin (b-CD) and hydroxypropyl-b-cyclodextrin (HPBCD) were kindly donated from Roquette Frères (Lestrem, France). Randomly methylated-b-cyclodextrin (RAMEB) was purchased from Wacker-Chemie (Lyon, France). 2.2. Media and microorganism Malt yeast extract agar (MYEA) medium was used for routine growth. The standard mineral salts medium (MM) consisted of (g l 1): KCl, 0.25; NaH2PO4 2H2O, 3.235; Na2HPO4 2H2O, 5.205; MgSO4, 0.244; NH4NO3, 1; and trace-element solution consisting of (mg l 1): ZnSO4 7H2O, 1; MnCl2 4H2O, 0.1; FeSO4 7H2O, 1; CuSO4 5H2O, 0.5; CaCl2 2H2O, 0.1; MoO3, 0.2. The culture medium was adjusted to pH 7.0. The study was carried out using F. solani called F33 previously isolated from petroleum-contaminated soil. Stock cultures were maintained on MYEA slants at 18 ± 1 °C and subcultured every 3 months. 2.3. H2O2 toxicity to F. solani Cultures were conducted in 25 ml MM containing 10 g l 1 of glucose in Erlenmeyer flasks. After sterilisation (121 °C for 20 min), inoculation was performed by adding a spore suspension of F. solani (aged 7 days), prepared as described previously (Rafin et al., 2000), so as to obtain a final concentration of 104 spores ml 1. After inoculation, flasks were stirred for 24 h at room temperature on a reciprocating shaker (Infors, Massy, France, 150 min 1) so as to induce spore germination. Then, H2O2 was introduced every day in order to obtain final nominal concentrations of 0, 2.3 10 4, 4.6 10 4, 1.16 10 3, 2.3 10 3, 4.6 10 3, 1.16 10 2 and 2.3 10 2 M. All treatments were stirred for 5 days on a reciprocating shaker. Three replicates were run for each H2O2 concentration. Fungal growth was estimated by biomass produced over a total period of 5 days. Mycelium was filtered through pre-dried cellulose filters in a vacuum filtration apparatus, then lyophilised and weighted. Results were expressed as mean value (M) ± standard error (SE) for three replicates. 2.4. Cyclodextrins degradation by F. solani In a first set of experiments, the fungus was grown in MM-medium (50 ml) per 100-ml Erlenmeyer flask with either glucose as reference or one CD (b-CD, HPBCD, RAMEB) as sole carbon source (5.82 carbon equivalent g l 1). In a second set of experiments, glucose (1.42 carbon equivalent g l 1) was added in each CD treatment in order to induce fungal growth. After sterilisation, spores’ inoculation (104 spores ml 1) was performed. Incubation was conducted over 5 days at room temperature on a reciprocating shaker. Three replicates were run for each treatment. Biomass (M ± SE) was estimated as previously described.
tion of 5 10 3 M. For evaluating biological degradation by F. solani, inoculation was performed by adding a spore suspension (final concentration of 104 spores ml 1). Penicillin flasks were then sealed with silicone septa and aluminium crimps. Oxygenation of the cultures was performed by a sterile syringe needle. Non-inoculated treatments were set up similarly for chemical oxidation assessment. Fenton’s reaction was performed by introducing, through the sterile syringe needle, H2O2 at a final daily concentration of 1.5 10 3 M. Addition of H2O2 was done from the 1st day to the 12th day of culture. Fenton’s reaction was conducted at near neutral pH. In chemical–biological combined treatment, inoculation and H2O2 addition were performed as described previously. All treatments were incubated at room temperature in the dark for 12 days on a reciprocating shaker. All the treatments were effected in triplicates. 2.6. BaP extraction and analytical procedure At the end of incubation, cultures were lyophilised for 3 days. Flasks containing total lyophilised cultures were introduced into a Soxhlet apparatus and extracted for 16 h with DCM. Organic fractions were concentrated in 20 ml DCM/MeOH (50:50, v/v). An aliquot of the extract diluted 1/2000 was used for BaP measurement. BaP fluorescence in DCM extract was directly read on a Perkin–Elmer LS B50 spectrofluorimeter (excitation 295 nm, emission 406 nm, time integration 10 s). Three runs were realised per treatment. The percentage of BaP degradation was given by the formula: [(mEc mT)/mEc] 100, where mEc was the quantity of BaP obtained in extraction controls and mT was the quantity of BaP obtained in each treatment. In extraction controls, 98% of initial BaP was recovered. Statistical analysis was performed by a two-sample t-test comparing treatments expressed as M ± SE (at 99% confidence). 3. Results and discussion 3.1. H2O2 toxicity to F. solani The dose–response curve of F. solani to H2O2 concentrations after 5 days of incubation in MM-medium is a typical sigmoid dose–response curve (Fig. 1). The lowest observed effect concentration (LOEC) and the effective concentration for 50% of inhibition (EC50) determined graphically are, respectively, 2.33 10 4 M and 4 10 3 M. Hydrogen peroxide is a widely used biocide for
2.5. BaP degradation studies Cultures for BaP degradation were conducted in a micro-method established as follows: BaP (40 mg) was dissolved in 10 ml acetone and 0.250 ml was delivered into 22-ml penicillin flasks so as to give a final BaP quantity of 1 mg per flask. Subsequently, the solvent was evaporated. Nine milliliters of MM-medium supplemented with FeSO4 7H2O (working concentration 5 10 4 M) were added into the flasks and sterilised. Stock solutions of carbon sources were prepared, sterilised by filtration and added into the autoclaved BaP MM-medium: glucose (at a final concentration of 10 g l 1) and CDs solutions (HPBCD, RAMEB) at working concentra-
Fig. 1. Dose–response curve of Fusarium solani to H2O2 concentrations after 5 days incubation in MM-medium. Bars indicate standard errors.
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disinfection, sterilisation and antisepsis in various fields. However, microorganisms differ in their sensitivity to H2O2. Relevant data concerning Fusarium species are scarce. In vitro studies showed that two commercially available H2O2-based compounds, ZeroTolÒ (2.0% peroxyacetic acid and 27.0% hydrogen peroxide) and SaniDateÒ (12.0% peroxyacetic acid and 18.5% hydrogen peroxide) were effective in causing 100% spores mortality of Fusarium foetens (LD50 = 258 and 66.7 ll l 1, respectively) at rates that would allow their use in irrigation water in greenhouses (Elmer, 2008). However, due to the compounds’ formulation, data is difficult to compare to our results. In a study conducted on the adaptation of Fusarium decemcellulare to oxidative stress, Medentsev et al. (2001) found that H2O2 completely inhibited the growth of exponential phase fungal cells at concentrations higher than 10 3 M. As observed in our experiments, a concentration higher than 4.6 10 3 M is necessary to completely inhibit the growth of F. solani. That could be due to a sensitivity difference either in the strain or in the growth phase. In fact, Medentsev et al. (2001) underlined that the stationary phase (obtained after 36 h of growth) was more resistant to the oxidative stress than the exponential phase cells. In the following experiments, we chose to work at 1.5 10 3 M, a balance concentration efficient for Fenton’s oxidation and biocompatible to fungal growth. 3.2. Biodegradation of cyclodextrins by F. solani Table 1 shows the fungal biomass accumulated in presence of reference (glucose) or test carbon source (CD) in MM liquid culture. In a first set of experiments, the fungus was grown either with glucose or with one of the studied CDs (b-CD, HPBCD, and RAMEB) as the sole carbon source. The reference glucose reached high fungal growth with 166 mg biomass produced after 5 days of incubation. In these conditions, only the native b-CD allowed fungal growth with a biomass slightly smaller (136 mg) than with glucose. Fungal growth did not occur in the presence of CD derivatives (RAMEB and HPBCD). As F. solani metabolises b-CD as sole carbon source, this telluric fungus produces the CD-degrading enzymes responsible for the cleavage of the CD ring belonging probably to glucoamylases (Bhatti et al., 2007) Even though these enzymes might be produced in all treatments, we might hypothesise that degradation of RAMEB or HPBCD also requires the enzymatic activity involved in the cleavage of ether links (such as methyl- or hydroxypropylgroups, respectively). These enzymes might not be induced due to insufficient carbon sources. Indeed, carbon source available in spores might be rapidly exhausted during the spore germination. Thereby, a second set of experiments was conducted in which glucose (1.42 carbon equivalent g l 1) was added as a supplementary carbon source in each CD treatment. In these conditions, only the native b-CD was again biodegraded by the fungus. In RAMEB or HPBCD treatments, only the added glucose was metabolised by F. solani as these treatments permitted to produce the same biomass as in the presence of glucose (about 31 mg). Similar results on biodegradability of CDs were obtained in studies most often conducted in soils. It is commonly reported that the native CDs (a-, b- and c-CD) are completely and readily biode-
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gradable (Verstichel et al., 2004) while the derivative ones are more resistant. For b-CDs, Oros et al. (1990, 2001) found several plant-associated bacteria (Agrobacterium, Bradyrhizobium, Xanthomonas and Corynebacterium) as well as soil fungi (Trichoderma species) metabolising CDs as sole carbon source with the following biodegradability order: unsubstituted > carboxymethyl > hydroxypropyl > polymethyl. Nevertheless, in more complex environments such as formerly polluted soils, Fava et al. (1998) reported that HPBCD was metabolised by the indigenous soil microorganisms. Even RAMEB, known as the most resistant CD to the microbial attack, could be slowly depleted in soils under favourable conditions (Fenyvesi et al., 2005). In studies conducted with RAMEB as the sole carbon and energy source in mineral medium, this cyclodextrin was found to markedly sustain the growth of inoculated specialised bacteria and also to enhance the frequency of occurrence of genes involved in polychlorinated biphenyls degradation by bacteria (Fava et al., 2003). Unfortunately, until now, little data is available on CDs biodegradation by fungi. 3.3. Benzo[a]pyrene degradation studies The results on benzo[a]pyrene degradation conducted in liquid medium with or without cyclodextrin are exhibited in Fig. 2. Three treatments were compared: Fenton chemical oxidation (F), biodegradation (B) and a combined chemical–biological treatment (FB). In the absence of cyclodextrin, the loss of BaP during the Fenton’s reaction (at a H2O2 concentration of 1.5 10 3 M) was negligible. It is well known that the efficiency of such an oxidation process strongly depends both on the PAH availability and on the applied H2O2/ferrous ions (Kulik et al., 2006). Only the solubilised BaP could be oxidised by hydroxyl radicals (Veignie et al., accepted for publication). Without cyclodextrin, the very low aqueous solubility of BaP limits the quantity of BaP soluble and therefore the efficiency of Fenton’s reaction. Biological treatment with F. solani led to a BaP degradation rate of 4% after 12 days of incubation. This result is in agreement with our previous studies (Rafin et al., 2006). In treatment FB, BaP degradation was higher and reached 11%. This enhancement of BaP degradation might be explained by the alternative BaP metabolic pathway of F. solani suggested in Veignie et al. (2004). This BaP metabolic pathway involves reactive oxygen species, such as hydroxyl radicals (OH), produced by the fungus itself. This free radical generating system which is stimulated by a high concentration of H2O2 in fungal cultures could be a key component in BaP biodegradation by F. solani.
Table 1 Accumulation of dry mass (mg) of Fusarium solani after 5 days incubation in MMmedium. Cyclodextrin
Alone (5.82 CEqu)
+ glucose (1.42 CEqu)
CD RAMEB HPBCD
136 ± 6 <6 <6
118 ± 12 31 ± 2 32 ± 1
F. solani accumulated 166 ± 2 and 32 ± 2 mg dry mass when growing on 5.82 and 1.42 carbon equivalent g l 1 (CEqu) in the medium, respectively.
Fig. 2. Degradation of benzo[a]pyrene after 12 days incubation in MM-medium (bars indicate standard errors). F: Fenton’s reaction (1.5 10 3 M H2O2/ 0.5 10 3 M FeSO4 7H2O), pH 7.0, H2O2 was added from the 1st day to the 12th day. B: Biological degradation by Fusarium solani. FB: Combined Fenton’s reaction and biological degradation.
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In the presence of RAMEB, the loss of BaP during the Fenton’s reaction was 20%, an increase of fivefold compared to the control treatment without cyclodextrin. That confirms once that the efficiency of the Fenton’s treatment is strongly dependent on the capacity of CDs to solubilise BaP. However, this enhancement of BaP solubility was not correlated to an enhancement of biodegradation by F. solani. F and FB treatments present the same percentage of BaP degradation. Thus, the enhancement of BaP solubilisation, which is a prerequisite for the first steps of oxidation (in both chemical and biological processes) seems not to be sufficient to enhance degradation in a combined process conducted with RAMEB. In the presence of HPBCD, when Fenton’s treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%). The combination of Fenton’s oxidation with biotreatment achieved the highest degradation rate of BaP obtained in the present study. This result is particularly interesting as the degradation study was conducted for 12 days at neutral pH, which is a pH compatible with fungal growth but not the best pH for Fenton’s reaction. The efficiency of FB treatment seems to be strongly dependent on the capacity of CDs to solubilise BaP, with one important parameter: the quantity of solubilisation. In our previous study (Veignie et al., accepted for publication), we showed that the quantity of solubilised BaP differed between both CDs, with HPBCD more efficient than RAMEB. HPBCD appears to be an excellent choice as it allows not only a rapid supply of BaP but also in higher quantity than RAMEB, when the fungus is competent to degrade it, at the early stages of cultures (Rafin et al., 2000). In literature, most of the results obtained in integrated treatments combining Fenton with biodegradation confirmed the positive effect of a such technology on PAH degradation. Nevertheless, most of the investigations were conducted by applying a chemical oxidation prior to condition organic contaminants for biodegradation (Goi and Trapido, 2004; Kulik et al., 2006). In the present study, we have chosen to develop a simultaneous chemical and biological treatment that might have great advantages over a remediation strategy based on a sequential application. It required optimal low doses of H2O2 to achieve first steps of oxidation of recalcitrant compounds as maintaining maximum of microbial integrity for biodegradation. Such a process would be more costeffective as well as more compatible with soil integrity and especially indigenous microorganisms’ activity in polluted soils, instead of introducing microorganisms into chemically treated-soil. 4. Conclusion BaP degradation in simultaneously combined Fenton’s oxidation and microbial degradation by F. solani in MM-medium was found to be superior when carried out with HPCD in comparison with RAMEB. This indicated firstly high efficiency of HPBCD to solubilise high molecular weight PAHs like BaP when the fungus is
competent to degrade it. Secondly, it permits also Fenton’s degradation at low H2O2 concentrations compatible with fungal growth. This result is especially interesting as the degradation study was conducted for 12 days. The overall results obtained in this study revealed the interest in developing a new technology of soil remediation, based on a strategy of two simultaneous complementary remediation approaches. Acknowledgements The authors thank S. Bones for help in English revision of this article. References Bhatti, H.N., Rashid, M.H., Nawaz, R., Asgher, M., Perveen, R., Jabbar, A., 2007. Purification and characterization of a novel glucoamylase from Fusarium solani. Food Chem. 103, 338–343. Elmer, W.H., 2008. Preventing spread of Fusarium wilt of Hiemalis begonias in the greenhouse. Crop Prot. 27, 1078–1083. Fava, F., Di Gioia, D., Marchetti, L., 1998. Cyclodextrin effects on the ex-situ bioremediation of a chronically polychlorobiphenyl-contaminated soil. Biotechnol. Bioeng. 58, 345–355. Fava, F., Bertin, L., Fedi, S., Zannoni, D., 2003. Methyl-b-cyclodextrin-enhanced solubilization and aerobic biodegradation of polychlorinated biphenyls in two aged-contaminated soils. Biotechnol. Bioeng. 81, 381–390. Fenyvesi, E., Gruiz, K., Verstichel, S., De Wilde, B., Leitgib, L., Csabai, K., Szaniszlo, N., 2005. Biodegradation of cyclodextrins in soil. Chemosphere 60, 1001–1008. Goi, A., Trapido, M., 2004. Degradation of polycyclic aromatic hydrocarbons in soil: the fenton reaction versus ozonation. Environ. Technol. 25, 155–164. Juhasz, A.L., Naidu, R., 2000. Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degradation of benzo[a]pyrene. Int. Biodeterior. Biodegrad. 45, 57–88. Kulik, N., Goi, A., Trapido, M., Tuhkanen, T., 2006. Degradation of polycyclic aromatic hydrocarbons by combined chemical pre-oxidation and bioremediation in creosote contaminated soil. J. Environ. Manage. 78, 382–391. Medentsev, A.G., Arinbasarova, A.Y., Akimenko, V.K., 2001. Adaptation to the phytopathogenic fungus Fusarium decemcellulare to oxidative stress. Microbiology 70, 26–30. Oros, G., Cserhati, T., Fenyvesi, É., Szejtli, J., 1990. Microbial decomposition of some cyclodextrin derivatives by bacteria associated with plants. Int. Biodeterior. Biodegrad. 26, 33–42. Oros, G., Cserhati, T., Forgacs, E., 2001. Decomposition of native cyclodextrins and cyclodextrin derivatives by various Trichoderma species. Biol. J. Armenia 53, 237–244. Rafin, C., Potin, O., Veignie, E., Lounes-Hadj Sahraoui, A., Sancholle, M., 2000. Degradation of benzo[a]pyrene as sole carbon source by a non white rot fungus Fusarium solani. Polycyclic Aromat. Compd. 21, 311–329. Rafin, C., Veignie, E., Woisel, P., Cazier, F., Surpateanu, G., 2006. New potential of a Deuteromycete fungus Fusarium solani in benzo[a]pyrene degradation: an ecophysiological hypothesis? In: Glazer, M.P. (Ed.), New Frontiers in Environmental Research. Nova Science Publishers Inc., New York, pp. 165–179. Rivas, F.J., 2006. Polycyclic aromatic hydrocarbons sorbed on soils: a short review of chemical oxidation based treatments. J. Hazard. Mater. 138, 234–251. Veignie, E., Rafin, C., Woisel, P., Cazier, F., 2004. Preliminary evidence of the role of hydrogen peroxide in the degradation of benzo[a]pyrene by a non-white rot fungus Fusarium solani. Environ. Pollut. 129, 1–4. Veignie, E., Rafin, C., Landy, D., Fourmentin, S., Surpateanu, G. Fenton degradation assisted by cyclodextrins of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene. J. Hazard. Mater., accepted for publication. Verstichel, S., De Wilde, B., Fenyvesi, É., Szejtli, J., 2004. Investigation of the aerobic biodegradability of several types of cyclodextrins in a laboratory-controlled composting test. J. Polym. Environ. 12, 47–55.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Benzo[a]pyrene degradation using simultaneously combined chemical oxidation, biotreatment with Fusarium solani and cyclodextrins Catherine Rafin *, Etienne Veignie, Antoine Fayeulle, Gheorghe Surpateanu Laboratoire de Synthèse Organique et Environnement (EA2599), Université du Littoral Côte d’Opale, 145 Avenue Maurice Schumann, 59140 Dunkerque, France
a r t i c l e
i n f o
Article history: Received 27 November 2008 Received in revised form 8 January 2009 Accepted 11 January 2009 Available online 25 February 2009 Keywords: Polycyclic aromatic hydrocarbons Fusarium solani, biodegradation Fenton’s reaction Cyclodextrins, benzo[a]pyrene
a b s t r a c t The interest of simultaneously combining chemical (Fenton’s reaction) and biological treatments for the degradation of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) has been studied in laboratory tests. An optimal concentration of 1.5 10 3 M H2O2 as Fenton’s reagent was firstly determined as being compatible with the growth of Fusarium solani, the Deuteromycete fungus used in the biodegradation process. For the enhancement of BaP solubilisation, cyclodextrins were also used in the performed tests. The best degradation performance was achieved through the use of 5 10 3 M hydroxypropyl-b-cyclodextrin (HPBCD) in comparison with randomly methylated-b-cyclodextrin (RAMEB). When Fenton’s treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%) in the presence of HPBCD for 12 days of incubation. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) containing five fused benzene rings, is considered to be one of the 16 PAHs defined as priority pollutants by the US Environmental Protection Agency (EPA) due to its carcinogenicity, teratogenicity and acute toxicity. This pollutant tends to persist in the environment partly because of its very low water solubility and its strong adsorption onto the soil organic matter. Among methodologies developed to extract PAHs from solid matrixes, cyclodextrins (CDs) are commonly used as alternative non-toxic extracting agents (Rivas, 2006). CDs are cyclic oligosaccharides composed of a-1,4-linked glucopyranose subunits that easily form inclusion complexes with a wide variety of organic molecules. These inclusion complexes show an apparent solubility higher than those of the compound alone in aqueous phase. The degradation of PAH needs an initial oxidation stage, that could be produced by either a chemical or a biological process. Fenton-based technology is by far the most studied chemical oxidation technology in soil remediation. Fenton’s reaction can also be applied in combination with bioremediation, each method possessing its own advantages and drawbacks (Kulik et al., 2006). However, bioremediation of PAH-polluted soil is severely hampered by the low rate degradation of high molecular weight PAHs due to limited mass transfers and pollutant bioavailability and to the scarcity of
adequate microorganisms. Therefore, substantial investigation has focused attention on microorganisms able to degrade high molecular weight PAHs for cleaning up contaminated sites (Juhasz and Naidu, 2000; Rafin et al., 2006). In our previous studies, we isolated a Deuteromycete fungus F. solani (Mart.) Sacc. (1881) [teleomorph: Haematonectria haematococca (Berk. and Broome) Samuels and Rossman, Ascomycota, Hypocreales, Nectriaceae] that was able to mineralise [7,10-14C]benzo[a]pyrene (BaP) rapidly (Rafin et al., 2000). Nevertheless, the BaP degradation rate obtained in treatment inoculated with the fungus remains quite low. To enhance bioremediation process efficiency, the present work was conducted to study, at laboratory scale, the interest to combine simultaneously chemical oxidation (by Fenton’s reaction) with biological treatment (in presence of F. solani) for BaP degradation. BaP was selected as a high molecular weight PAH model, composed of five aromatic rings, well known for its very weak solubility (Kow 6.06, Juhasz and Naidu, 2000) and therefore weak bioavailability. Various CDs were studied in order to evaluate how their complex-forming capacity with the pollutant could influence BaP degradation rate in combined chemical–biological treatment.
2. Methods 2.1. Chemicals
* Corresponding author. Tel.: +33 3 28 65 82 78; fax: +33 3 28 65 82 31. E-mail address: rafi[email protected] (C. Rafin). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.01.012
Benzo[a]pyrene (BaP) and hydrogen peroxide (H2O2) 30% were purchased from Acros Organics (Noisy-Le-Grand, France).
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Dichloromethane (DCM) and methanol (MeOH) were obtained in the highest purity grade available from Merck (Darmstadt, Germany). Distilled deionised water was used throughout this work. b-cyclodextrin (b-CD) and hydroxypropyl-b-cyclodextrin (HPBCD) were kindly donated from Roquette Frères (Lestrem, France). Randomly methylated-b-cyclodextrin (RAMEB) was purchased from Wacker-Chemie (Lyon, France). 2.2. Media and microorganism Malt yeast extract agar (MYEA) medium was used for routine growth. The standard mineral salts medium (MM) consisted of (g l 1): KCl, 0.25; NaH2PO4 2H2O, 3.235; Na2HPO4 2H2O, 5.205; MgSO4, 0.244; NH4NO3, 1; and trace-element solution consisting of (mg l 1): ZnSO4 7H2O, 1; MnCl2 4H2O, 0.1; FeSO4 7H2O, 1; CuSO4 5H2O, 0.5; CaCl2 2H2O, 0.1; MoO3, 0.2. The culture medium was adjusted to pH 7.0. The study was carried out using F. solani called F33 previously isolated from petroleum-contaminated soil. Stock cultures were maintained on MYEA slants at 18 ± 1 °C and subcultured every 3 months. 2.3. H2O2 toxicity to F. solani Cultures were conducted in 25 ml MM containing 10 g l 1 of glucose in Erlenmeyer flasks. After sterilisation (121 °C for 20 min), inoculation was performed by adding a spore suspension of F. solani (aged 7 days), prepared as described previously (Rafin et al., 2000), so as to obtain a final concentration of 104 spores ml 1. After inoculation, flasks were stirred for 24 h at room temperature on a reciprocating shaker (Infors, Massy, France, 150 min 1) so as to induce spore germination. Then, H2O2 was introduced every day in order to obtain final nominal concentrations of 0, 2.3 10 4, 4.6 10 4, 1.16 10 3, 2.3 10 3, 4.6 10 3, 1.16 10 2 and 2.3 10 2 M. All treatments were stirred for 5 days on a reciprocating shaker. Three replicates were run for each H2O2 concentration. Fungal growth was estimated by biomass produced over a total period of 5 days. Mycelium was filtered through pre-dried cellulose filters in a vacuum filtration apparatus, then lyophilised and weighted. Results were expressed as mean value (M) ± standard error (SE) for three replicates. 2.4. Cyclodextrins degradation by F. solani In a first set of experiments, the fungus was grown in MM-medium (50 ml) per 100-ml Erlenmeyer flask with either glucose as reference or one CD (b-CD, HPBCD, RAMEB) as sole carbon source (5.82 carbon equivalent g l 1). In a second set of experiments, glucose (1.42 carbon equivalent g l 1) was added in each CD treatment in order to induce fungal growth. After sterilisation, spores’ inoculation (104 spores ml 1) was performed. Incubation was conducted over 5 days at room temperature on a reciprocating shaker. Three replicates were run for each treatment. Biomass (M ± SE) was estimated as previously described.
tion of 5 10 3 M. For evaluating biological degradation by F. solani, inoculation was performed by adding a spore suspension (final concentration of 104 spores ml 1). Penicillin flasks were then sealed with silicone septa and aluminium crimps. Oxygenation of the cultures was performed by a sterile syringe needle. Non-inoculated treatments were set up similarly for chemical oxidation assessment. Fenton’s reaction was performed by introducing, through the sterile syringe needle, H2O2 at a final daily concentration of 1.5 10 3 M. Addition of H2O2 was done from the 1st day to the 12th day of culture. Fenton’s reaction was conducted at near neutral pH. In chemical–biological combined treatment, inoculation and H2O2 addition were performed as described previously. All treatments were incubated at room temperature in the dark for 12 days on a reciprocating shaker. All the treatments were effected in triplicates. 2.6. BaP extraction and analytical procedure At the end of incubation, cultures were lyophilised for 3 days. Flasks containing total lyophilised cultures were introduced into a Soxhlet apparatus and extracted for 16 h with DCM. Organic fractions were concentrated in 20 ml DCM/MeOH (50:50, v/v). An aliquot of the extract diluted 1/2000 was used for BaP measurement. BaP fluorescence in DCM extract was directly read on a Perkin–Elmer LS B50 spectrofluorimeter (excitation 295 nm, emission 406 nm, time integration 10 s). Three runs were realised per treatment. The percentage of BaP degradation was given by the formula: [(mEc mT)/mEc] 100, where mEc was the quantity of BaP obtained in extraction controls and mT was the quantity of BaP obtained in each treatment. In extraction controls, 98% of initial BaP was recovered. Statistical analysis was performed by a two-sample t-test comparing treatments expressed as M ± SE (at 99% confidence). 3. Results and discussion 3.1. H2O2 toxicity to F. solani The dose–response curve of F. solani to H2O2 concentrations after 5 days of incubation in MM-medium is a typical sigmoid dose–response curve (Fig. 1). The lowest observed effect concentration (LOEC) and the effective concentration for 50% of inhibition (EC50) determined graphically are, respectively, 2.33 10 4 M and 4 10 3 M. Hydrogen peroxide is a widely used biocide for
2.5. BaP degradation studies Cultures for BaP degradation were conducted in a micro-method established as follows: BaP (40 mg) was dissolved in 10 ml acetone and 0.250 ml was delivered into 22-ml penicillin flasks so as to give a final BaP quantity of 1 mg per flask. Subsequently, the solvent was evaporated. Nine milliliters of MM-medium supplemented with FeSO4 7H2O (working concentration 5 10 4 M) were added into the flasks and sterilised. Stock solutions of carbon sources were prepared, sterilised by filtration and added into the autoclaved BaP MM-medium: glucose (at a final concentration of 10 g l 1) and CDs solutions (HPBCD, RAMEB) at working concentra-
Fig. 1. Dose–response curve of Fusarium solani to H2O2 concentrations after 5 days incubation in MM-medium. Bars indicate standard errors.
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disinfection, sterilisation and antisepsis in various fields. However, microorganisms differ in their sensitivity to H2O2. Relevant data concerning Fusarium species are scarce. In vitro studies showed that two commercially available H2O2-based compounds, ZeroTolÒ (2.0% peroxyacetic acid and 27.0% hydrogen peroxide) and SaniDateÒ (12.0% peroxyacetic acid and 18.5% hydrogen peroxide) were effective in causing 100% spores mortality of Fusarium foetens (LD50 = 258 and 66.7 ll l 1, respectively) at rates that would allow their use in irrigation water in greenhouses (Elmer, 2008). However, due to the compounds’ formulation, data is difficult to compare to our results. In a study conducted on the adaptation of Fusarium decemcellulare to oxidative stress, Medentsev et al. (2001) found that H2O2 completely inhibited the growth of exponential phase fungal cells at concentrations higher than 10 3 M. As observed in our experiments, a concentration higher than 4.6 10 3 M is necessary to completely inhibit the growth of F. solani. That could be due to a sensitivity difference either in the strain or in the growth phase. In fact, Medentsev et al. (2001) underlined that the stationary phase (obtained after 36 h of growth) was more resistant to the oxidative stress than the exponential phase cells. In the following experiments, we chose to work at 1.5 10 3 M, a balance concentration efficient for Fenton’s oxidation and biocompatible to fungal growth. 3.2. Biodegradation of cyclodextrins by F. solani Table 1 shows the fungal biomass accumulated in presence of reference (glucose) or test carbon source (CD) in MM liquid culture. In a first set of experiments, the fungus was grown either with glucose or with one of the studied CDs (b-CD, HPBCD, and RAMEB) as the sole carbon source. The reference glucose reached high fungal growth with 166 mg biomass produced after 5 days of incubation. In these conditions, only the native b-CD allowed fungal growth with a biomass slightly smaller (136 mg) than with glucose. Fungal growth did not occur in the presence of CD derivatives (RAMEB and HPBCD). As F. solani metabolises b-CD as sole carbon source, this telluric fungus produces the CD-degrading enzymes responsible for the cleavage of the CD ring belonging probably to glucoamylases (Bhatti et al., 2007) Even though these enzymes might be produced in all treatments, we might hypothesise that degradation of RAMEB or HPBCD also requires the enzymatic activity involved in the cleavage of ether links (such as methyl- or hydroxypropylgroups, respectively). These enzymes might not be induced due to insufficient carbon sources. Indeed, carbon source available in spores might be rapidly exhausted during the spore germination. Thereby, a second set of experiments was conducted in which glucose (1.42 carbon equivalent g l 1) was added as a supplementary carbon source in each CD treatment. In these conditions, only the native b-CD was again biodegraded by the fungus. In RAMEB or HPBCD treatments, only the added glucose was metabolised by F. solani as these treatments permitted to produce the same biomass as in the presence of glucose (about 31 mg). Similar results on biodegradability of CDs were obtained in studies most often conducted in soils. It is commonly reported that the native CDs (a-, b- and c-CD) are completely and readily biode-
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gradable (Verstichel et al., 2004) while the derivative ones are more resistant. For b-CDs, Oros et al. (1990, 2001) found several plant-associated bacteria (Agrobacterium, Bradyrhizobium, Xanthomonas and Corynebacterium) as well as soil fungi (Trichoderma species) metabolising CDs as sole carbon source with the following biodegradability order: unsubstituted > carboxymethyl > hydroxypropyl > polymethyl. Nevertheless, in more complex environments such as formerly polluted soils, Fava et al. (1998) reported that HPBCD was metabolised by the indigenous soil microorganisms. Even RAMEB, known as the most resistant CD to the microbial attack, could be slowly depleted in soils under favourable conditions (Fenyvesi et al., 2005). In studies conducted with RAMEB as the sole carbon and energy source in mineral medium, this cyclodextrin was found to markedly sustain the growth of inoculated specialised bacteria and also to enhance the frequency of occurrence of genes involved in polychlorinated biphenyls degradation by bacteria (Fava et al., 2003). Unfortunately, until now, little data is available on CDs biodegradation by fungi. 3.3. Benzo[a]pyrene degradation studies The results on benzo[a]pyrene degradation conducted in liquid medium with or without cyclodextrin are exhibited in Fig. 2. Three treatments were compared: Fenton chemical oxidation (F), biodegradation (B) and a combined chemical–biological treatment (FB). In the absence of cyclodextrin, the loss of BaP during the Fenton’s reaction (at a H2O2 concentration of 1.5 10 3 M) was negligible. It is well known that the efficiency of such an oxidation process strongly depends both on the PAH availability and on the applied H2O2/ferrous ions (Kulik et al., 2006). Only the solubilised BaP could be oxidised by hydroxyl radicals (Veignie et al., accepted for publication). Without cyclodextrin, the very low aqueous solubility of BaP limits the quantity of BaP soluble and therefore the efficiency of Fenton’s reaction. Biological treatment with F. solani led to a BaP degradation rate of 4% after 12 days of incubation. This result is in agreement with our previous studies (Rafin et al., 2006). In treatment FB, BaP degradation was higher and reached 11%. This enhancement of BaP degradation might be explained by the alternative BaP metabolic pathway of F. solani suggested in Veignie et al. (2004). This BaP metabolic pathway involves reactive oxygen species, such as hydroxyl radicals (OH), produced by the fungus itself. This free radical generating system which is stimulated by a high concentration of H2O2 in fungal cultures could be a key component in BaP biodegradation by F. solani.
Table 1 Accumulation of dry mass (mg) of Fusarium solani after 5 days incubation in MMmedium. Cyclodextrin
Alone (5.82 CEqu)
+ glucose (1.42 CEqu)
CD RAMEB HPBCD
136 ± 6 <6 <6
118 ± 12 31 ± 2 32 ± 1
F. solani accumulated 166 ± 2 and 32 ± 2 mg dry mass when growing on 5.82 and 1.42 carbon equivalent g l 1 (CEqu) in the medium, respectively.
Fig. 2. Degradation of benzo[a]pyrene after 12 days incubation in MM-medium (bars indicate standard errors). F: Fenton’s reaction (1.5 10 3 M H2O2/ 0.5 10 3 M FeSO4 7H2O), pH 7.0, H2O2 was added from the 1st day to the 12th day. B: Biological degradation by Fusarium solani. FB: Combined Fenton’s reaction and biological degradation.
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In the presence of RAMEB, the loss of BaP during the Fenton’s reaction was 20%, an increase of fivefold compared to the control treatment without cyclodextrin. That confirms once that the efficiency of the Fenton’s treatment is strongly dependent on the capacity of CDs to solubilise BaP. However, this enhancement of BaP solubility was not correlated to an enhancement of biodegradation by F. solani. F and FB treatments present the same percentage of BaP degradation. Thus, the enhancement of BaP solubilisation, which is a prerequisite for the first steps of oxidation (in both chemical and biological processes) seems not to be sufficient to enhance degradation in a combined process conducted with RAMEB. In the presence of HPBCD, when Fenton’s treatment was combined with biodegradation, a beneficial effect on BaP degradation (25%) was obtained in comparison with biodegradation alone (8%) or with chemical oxidation alone (16%). The combination of Fenton’s oxidation with biotreatment achieved the highest degradation rate of BaP obtained in the present study. This result is particularly interesting as the degradation study was conducted for 12 days at neutral pH, which is a pH compatible with fungal growth but not the best pH for Fenton’s reaction. The efficiency of FB treatment seems to be strongly dependent on the capacity of CDs to solubilise BaP, with one important parameter: the quantity of solubilisation. In our previous study (Veignie et al., accepted for publication), we showed that the quantity of solubilised BaP differed between both CDs, with HPBCD more efficient than RAMEB. HPBCD appears to be an excellent choice as it allows not only a rapid supply of BaP but also in higher quantity than RAMEB, when the fungus is competent to degrade it, at the early stages of cultures (Rafin et al., 2000). In literature, most of the results obtained in integrated treatments combining Fenton with biodegradation confirmed the positive effect of a such technology on PAH degradation. Nevertheless, most of the investigations were conducted by applying a chemical oxidation prior to condition organic contaminants for biodegradation (Goi and Trapido, 2004; Kulik et al., 2006). In the present study, we have chosen to develop a simultaneous chemical and biological treatment that might have great advantages over a remediation strategy based on a sequential application. It required optimal low doses of H2O2 to achieve first steps of oxidation of recalcitrant compounds as maintaining maximum of microbial integrity for biodegradation. Such a process would be more costeffective as well as more compatible with soil integrity and especially indigenous microorganisms’ activity in polluted soils, instead of introducing microorganisms into chemically treated-soil. 4. Conclusion BaP degradation in simultaneously combined Fenton’s oxidation and microbial degradation by F. solani in MM-medium was found to be superior when carried out with HPCD in comparison with RAMEB. This indicated firstly high efficiency of HPBCD to solubilise high molecular weight PAHs like BaP when the fungus is
competent to degrade it. Secondly, it permits also Fenton’s degradation at low H2O2 concentrations compatible with fungal growth. This result is especially interesting as the degradation study was conducted for 12 days. The overall results obtained in this study revealed the interest in developing a new technology of soil remediation, based on a strategy of two simultaneous complementary remediation approaches. Acknowledgements The authors thank S. Bones for help in English revision of this article. References Bhatti, H.N., Rashid, M.H., Nawaz, R., Asgher, M., Perveen, R., Jabbar, A., 2007. Purification and characterization of a novel glucoamylase from Fusarium solani. Food Chem. 103, 338–343. Elmer, W.H., 2008. Preventing spread of Fusarium wilt of Hiemalis begonias in the greenhouse. Crop Prot. 27, 1078–1083. Fava, F., Di Gioia, D., Marchetti, L., 1998. Cyclodextrin effects on the ex-situ bioremediation of a chronically polychlorobiphenyl-contaminated soil. Biotechnol. Bioeng. 58, 345–355. Fava, F., Bertin, L., Fedi, S., Zannoni, D., 2003. Methyl-b-cyclodextrin-enhanced solubilization and aerobic biodegradation of polychlorinated biphenyls in two aged-contaminated soils. Biotechnol. Bioeng. 81, 381–390. Fenyvesi, E., Gruiz, K., Verstichel, S., De Wilde, B., Leitgib, L., Csabai, K., Szaniszlo, N., 2005. Biodegradation of cyclodextrins in soil. Chemosphere 60, 1001–1008. Goi, A., Trapido, M., 2004. Degradation of polycyclic aromatic hydrocarbons in soil: the fenton reaction versus ozonation. Environ. Technol. 25, 155–164. Juhasz, A.L., Naidu, R., 2000. Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degradation of benzo[a]pyrene. Int. Biodeterior. Biodegrad. 45, 57–88. Kulik, N., Goi, A., Trapido, M., Tuhkanen, T., 2006. Degradation of polycyclic aromatic hydrocarbons by combined chemical pre-oxidation and bioremediation in creosote contaminated soil. J. Environ. Manage. 78, 382–391. Medentsev, A.G., Arinbasarova, A.Y., Akimenko, V.K., 2001. Adaptation to the phytopathogenic fungus Fusarium decemcellulare to oxidative stress. Microbiology 70, 26–30. Oros, G., Cserhati, T., Fenyvesi, É., Szejtli, J., 1990. Microbial decomposition of some cyclodextrin derivatives by bacteria associated with plants. Int. Biodeterior. Biodegrad. 26, 33–42. Oros, G., Cserhati, T., Forgacs, E., 2001. Decomposition of native cyclodextrins and cyclodextrin derivatives by various Trichoderma species. Biol. J. Armenia 53, 237–244. Rafin, C., Potin, O., Veignie, E., Lounes-Hadj Sahraoui, A., Sancholle, M., 2000. Degradation of benzo[a]pyrene as sole carbon source by a non white rot fungus Fusarium solani. Polycyclic Aromat. Compd. 21, 311–329. Rafin, C., Veignie, E., Woisel, P., Cazier, F., Surpateanu, G., 2006. New potential of a Deuteromycete fungus Fusarium solani in benzo[a]pyrene degradation: an ecophysiological hypothesis? In: Glazer, M.P. (Ed.), New Frontiers in Environmental Research. Nova Science Publishers Inc., New York, pp. 165–179. Rivas, F.J., 2006. Polycyclic aromatic hydrocarbons sorbed on soils: a short review of chemical oxidation based treatments. J. Hazard. Mater. 138, 234–251. Veignie, E., Rafin, C., Woisel, P., Cazier, F., 2004. Preliminary evidence of the role of hydrogen peroxide in the degradation of benzo[a]pyrene by a non-white rot fungus Fusarium solani. Environ. Pollut. 129, 1–4. Veignie, E., Rafin, C., Landy, D., Fourmentin, S., Surpateanu, G. Fenton degradation assisted by cyclodextrins of a high molecular weight polycyclic aromatic hydrocarbon benzo[a]pyrene. J. Hazard. Mater., accepted for publication. Verstichel, S., De Wilde, B., Fenyvesi, É., Szejtli, J., 2004. Investigation of the aerobic biodegradability of several types of cyclodextrins in a laboratory-controlled composting test. J. Polym. Environ. 12, 47–55.