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Bioremediation potential of basidiomycetes isolated from compost
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 6626–6630
Short Communication
Bioremediation potential of basidiomycetes isolated from compost Antonella Anastasi a, Giovanna C. Varese a,*, Francesca Bosco b, Fabiana Chimirri b, Valeria Filipello Marchisio a b
a Dipartimento di Biologia Vegetale, Universita` degli Studi di Torino, Viale Mattioli 25, 10125 Torino, Italy Dipartimento di Scienza dei Materiali ed Ingegneria Chimica, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy
Received 24 April 2007; received in revised form 6 December 2007; accepted 12 December 2007 Available online 31 January 2008
Abstract The potential of a consortium of three basidiomycete mycelia isolated from compost to degrade polycyclic aromatic hydrocarbons (PAH) was first evaluated using a test based on decolorization of Poly R-478 dye. When pre-grown on straw, the consortium decolorized the dye by 83% in 7 days and generated a laccase activity of 663 IU l1. Its ability to degrade naphthalene was investigated in soil microcosms specially suited for this volatile PAH. The kinetic study was conducted at a maximal naphthalene concentration of 500 mg kg1 of soil. Naphthalene concentration, CO2 evolution and phytotoxicity (germination index, GI%) on Lepidium sativum seeds were monitored. The naphthalene concentration decreased by about 70% in three weeks in the presence of metabolic activity, while the GI% increased indicating reduced phytotoxicity. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Fungi; Naphthalene; PAH degradation; Phytotoxicity tests; Poly R-478
1. Introduction Polycyclic aromatic hydrocarbons (PAH) are common, persistent and recalcitrant contaminants in soil. Their distribution and fate within the environment are a matter of growing concern, especially since many are cytotoxic, mutagenic and carcinogenic and have been classified as priority pollutants by the EPA (Aitken and Long, 2004). PAH can be eliminated from polluted soil by incineration, thermal desorption, soil washing etc. These methods, however, are expensive and have an adverse impact on the environment (Canet et al., 2001). Bioremediation is a competitive alternative. Composts have an enormous potential for bioremediation through their support of mesophilic and thermophilic bacteria and ligninolytic fungi endowed with the ability to degrade aromatic pollutants (Semple et al., 2001). However, a better understanding of the compost microflora *
Corresponding author. Tel.: +39 0116705984; fax: +39 0116705962. E-mail address: [email protected] (G.C. Varese).
0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.12.036
involved in PAH degradation is still needed, with particular emphasis on the impact of ligninolytic fungi (AntizarLadislao et al., 2004). Lignin degrading basidiomycetes normally do not assimilate PAH as the sole carbon source, and hence require cometabolites (e.g. glucose) to degrade them (Singh, 2006). Their degradation results in the formation of PAH-quinones catalysed by extracellular ligninolytic enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP), laccases (Lcc) and H2O2-generating enzymes (Singh, 2006). These enzymes are non-specific, non-stereoselective and effective against a broad spectrum of aromatic compounds including anthracene, benzo[a]pyrene, naphthalene and pyrene (Muncnerova` and Augustin, 1994; Novotny et al., 1999). However, basidiomycetes are rarely isolated from compost, both because many of them cannot withstand the temperatures of more than 50 °C generated during the thermophilic stage (Ryckeboer et al., 2003), and because their isolation and morphological identification are impeded by the fact that they are often found as sterile mycelia, while their molecular detection is hampered by the
A. Anastasi et al. / Bioresource Technology 99 (2008) 6626–6630
incompleteness of gene databases and the low taxonomic resolution of DNA sequences. The genera observed in compost include Armillaria, Clitopilus, Coprinus, Fomes, Lentinus, Lenzites, Pleurotus and Trametes. The degradative capability of basidiomycetes from compost, however, has not been investigated. We have previously used a test based on the decolorization of Poly R-478 dye in solid culture to assess the ligninolytic activity of 266 fungal isolates (33 were basidiomycetes) from different kinds of composts (Anastasi et al., 2005). Decolorization ability and ligninolytic enzymes production of the three most promising basidiomycetes were then investigated in greater detail in liquid culture (Anastasi et al., 2006) containing Poly R-478, since the ability to decolorize this dye is also regarded as indicative of a PAH degradation potential (Alcade et al., 2002; Canet et al., 2001). The aim of the present work was first to investigate the ability to decolorize Poly R-478 of the same three basidiomycetes in a consortium and determine its inhibitory/synergic effects on decolorization and enzyme activities, and then to evaluate its PAH degradation capability in soil microcosms polluted with naphthalene. Phytotoxicity due to naphthalene and/or any of its metabolites was monitored by calculating the germination index (GI%) of Lepidium sativum seeds during the incubation period. 2. Methods
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Ltd.) at a final concentration of 0.02%. Inoculum consisted of 0.2 g straw cultures for each strain. A heat-killed control (straw cultures autoclaved at 121 °C for 20 min) and an abiotic (consortium-free) control were set up to check for absorption of the dye on the mycelium or a decrease in absorbance not due to fungal presence. Triplicate cultures were incubated statically at 27 °C and flushed about every 2 days with O2 in sterile conditions. Decolorization was monitored daily for 7 days on 0.3 ml cell free culture medium diluted 10-fold in water and expressed as the percentage decrease of absorbance at 514 nm, i.e. the maximum visible wavelength of absorbance of Poly R-478, with a UV–visible spectrophotometer (Ultrospec 3300 pro; Amersham). LiP activity was evaluated spectrophotometrically via the oxidation of veratryl alcohol at the wavelength of 310 nm in 250 mM sodium tartrate buffer, pH 5, at 39 °C (Tien and Kirk, 1988). MnP activity was evaluated spectrophotometrically via the oxidation of curcumin at the wavelength of 430 nm in 0.5 M sodium tartrate buffer, pH 5, at 22 °C (Paszczynski et al., 1988). Lcc activity was evaluated spectrophotometrically by following the oxidation of 2,20 -azino-bis-(3ethylbenzothiazoline-6 sulphonic acid) (ABTS) at the wavelength of 420 nm in 100 mM sodium acetate buffer, pH 5, at 22 °C (Johannes and Majcherczyk, 2000). Activities were expressed as international units per litre (IU l1). The Spearman correlation test was used to evaluate correlations between decolorization and enzyme activity.
2.1. Microorganisms and inoculum preparation 2.3. Soil microcosms The three basidiomycetes were patented and deposited in the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) collection (DSM 15214, DSM 15215, DSM 15216); they were isolated from compost as sterile mycelia and identified as basidiomycetes morphophysiologically (Anastasi, 2003); rDNA sequencing (Internal Transcribed Spacer – ITS 1, 2 – and 18S) and comparison with sequences in gene databases showed that strains DSM 15214 and DSM 15215 had 100% similarity to Trametes versicolor (L.) Pila´t, while strain DSM 15216 had 99% similarity to different species of basidiomycetes [Bjerkandera adusta (Willd.) P. Karst., Bjerkandera fumosa (Pers.) P. Karst., Lopharia spadicea (Pers.) Boidin] (GenBank, NCBI). All three strains had been shown to display Lcc and/or MnP activity during their decolorization of Poly R-478 in single cultures (Anastasi et al., 2006). Equal portions of straw cultures of each strain, prepared as previously described (Anastasi et al., 2006), were used to inoculate flasks for Poly R-478 decolorization and naphthalene degradation studies. 2.2. Decolorization tests and enzyme assays in liquid cultures Decolorization tests were performed in 500 ml, hermetically sealed Erlenmeyer flasks containing 30 ml of a synthetic glucose medium (Gold et al., 1988) supplemented with an aqueous Poly R-478 solution (Sigma Aldrich Co.,
The static microcosms were prepared, as previously described by Mollea et al. (2005), by placing in hermetically sealed, 500 ml Erlenmeyer flasks 1 g of straw cultures of each strain, 20 ml of a 20 g/l glucose:water solution and 10 g of an uncontaminated, natural soil (pH 6.63, total moisture 12%) then artificially spiked with naphthalene (500 mg kg1) dissolved in ethyl acetate (EA) (Fluka). Heat-killed controls were obtained by preparing microcosms as described above, but with straw cultures pre-autoclaved at 121 °C for 20 min. Microcosms were incubated statically at 27 °C and periodically flushed with wetted pure oxygen at a constant 27 °C, since the results of preliminary experiments (not shown) had made it clear that temperature control during oxygenation was essential to limit the increase in the volatility of the pollutant. However, checking PAH losses during oxygenation was necessary; for this purpose the flowing gaseous phase was absorbed in 40 ml N,N-dimethylformamide (DMF) (Fluka) at the end of the oxygenation system. Ten milliliters of the 20 g/l glucose:water solution were added on days 14 and 28 to reestablish the system’s C source and humidity. 2.4. Respirometric analysis To determine metabolic activity in each microcosm we periodically performed a respirometric analysis by moni-
toring CO2 emission according to the modified Isermeyer method (Alef, 1995). 2.5. Naphthalene degradation Naphthalene was extracted by adding 100 ml EA and agitating the flasks at 150 rpm for 72 h at 27 °C. The filtered extracts and the DMF used to capture naphthalene were diluted and analysed with a Kontron HPLC equipped with a 432 UV–visible detector operating at the wavelength of 254 nm and a ChromoSpher 5 b PAH (4.6 150 mm) C18 reversed-phase column (Chrompack) maintained at 25 °C with a Peltier thermostat. The mobile phase was a 65:35 (v/v) acetonitrile:water solution and the flow rate was 0.9 ml/min. Residual naphthalene was calculated as the sum of the amounts extracted with EA and captured with DMF. The analyses were carried out in triplicate. 2.6. Phytotoxicity tests Toxicity was evaluated by means of a phytotoxicity test on L. sativum seeds applied directly in biotic and killed microcosms, with and without naphthalene, at the start of incubation and after 21 and 42 days. The microcosms were kept in the same conditions as those utilized for the naphthalene degradations experiments. The germination index (GI%) was calculated from the number of germinated seeds and the root length values in the control and in the samples according to the formula: GI% = (Gs Ls)/ Gc Lc)100, where Gs is the mean number of germinated seeds in the sample, Ls is the mean root length of the sample, Gc is the mean number of germinated seeds in the control, Lc is the mean root length of the control. The analyses were duplicated. 3. Results 3.1. Decolorization tests and enzymatic assays in liquid cultures Decolorization of Poly R-478 by the consortium led to an approximately 83% decrease of its initial absorbance
% decolorization
100 80 60 40 20 0 -20 0
1
2
3
4
5
6
7
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Fig. 1. Percentage of decolorization of 0.02% Poly R-478 by the fungal consortium and by the abiotic and killed controls.
700
900
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800 700
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400 300
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A. Anastasi et al. / Bioresource Technology 99 (2008) 6626–6630
naphthalene concentration (ppm)
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40
time (days) biotic line
killed line
CO2 (mg)
Fig. 2. Residual naphthalene in the biotic microcosms containing the fungal consortium and in the killed controls and CO2 emission in the biotic microcosms (arrows show the additions of 10 ml of a 2% w/v glucose:water solution).
value in 7 days. The values of the killed and abiotic controls were unchanged (Fig. 1). LiP activity was absent. MnP and Lcc activities reached 63 and 663 IU l1, respectively, but were not significantly correlated with the decolorization percentage (r = 0.476, p = 0.233; r = 0.310, p = 0.456). 3.2. Naphthalene degradation and detoxification The CO2 produced by the biotic microcosms never fell below 395 mg from the 2nd day onwards (Fig. 2) and rose when glucose was added on the 14th and 28th days. This showed that the consortium was able to survive and efficiently colonize soil in the microcosms. In Fig. 2 the time course of naphthalene concentration (extracted with EA and captured in DMF) for the killed and biotic lines is reported. A distinction between biotic and killed line was evident from the first days of incubation; between the 3rd and the 20th day, the biotic line had the maximum gradient, then naphthalene decrement shut down and remained constant till the end of the experiment, despite the fact that the CO2 pattern showed that the consortium was still metabolically active after the final glucose supplement on the 28th day. The mean residual naphthalene in the biotic microcosms on the 42nd day was 83 mg kg1, corresponding to a 80.24% reduction during the incubation period. In the killed controls the mean residual naphthalene was 272 mg kg1 corresponding to a 42% reduction. The ratio between the two residues shows that the degradation attributable to the consortium alone was 69.49%. Capture of naphthalene by DMF recovered up to 14.52% in the biotic microcosms and 12.6% in the killed controls (data not shown). This gave a more accurate estimate of the fungus-induced degradation. Without this recovery, the naphthalene degradation would have been 90%, in other words well above the real degradative power of the consortium. The GI% was zero at the initial naphthalene concentration (500 mg kg1). It then rose to 188.77% and 205.55% at
A. Anastasi et al. / Bioresource Technology 99 (2008) 6626–6630
days 21 and 42 when the residual concentrations were 103 mg kg1 and 83 mg kg1, respectively. 4. Discussion The fungal consortium displayed a good degradative capability against the complex aromatic molecule Poly R478 (83% decolorization in 7 days), though this was much the same as the 77–87% found for the individual strains (Anastasi et al., 2006). However Lcc activity expressed by the consortium increased up to 5 times, perhaps due to the concurrent presence and synergic activity of the isolates ascribable to T. versicolor, whose ability to decolorize Poly R-478 and degrade PAH, mainly by means of Lcc, is well known (Alcade et al., 2002). Though the ability to decolorize Poly R-478 is regarded as predictive of a PAH degradation potential (Alcade et al., 2002; Canet et al., 2001), the degradative capacity and the production of enzymes in a liquid culture are not enough to posit the use of organisms in the bioremediation of polluted soils. The setting up of microcosms closely simulates PAH biodegradation in soil without prejudice to the maintenance of controlled culture conditions (artificial pollution, constant incubation temperature, insufflation of pure oxygen, absence of competitors, periodic addition of nutrients and water, and monitoring of metabolic activity). Under these conditions, the consortium displayed a good ability to efficiently colonize the soil and reduce a high naphthalene concentration (500 mg kg1): about 70% degradation was achieved after three weeks and good metabolic activity was maintained. Comparison of our results with those obtained in similar microcosms by Mollea et al. (2005) with Phanerochaete chrysosporium, considered a model organism in PAH degradation studies, shows that the consortium removed naphthalene more rapidly; starting from an initial concentration of 600 mg kg1, P. chrysosporium produced a degradation of about 20% after 3 weeks and a maximum of about 55% at the 50th day (values calculated by comparing the biotic line to the killed controls). Another significant outcome of this study was the gradual disappearance of phytotoxicity following reduction of the pollutant. In some cases, in fact, PAH metabolites have proved more toxic than their parent compounds (Sasek et al., 2003). In our study, the seeds did not germinate at first, whereas at the halfway point (naphthalene concentration of 103 mg kg1) and at the end of the test they all germinated and the GI% rose to more than 100%, showing that growth was actually stimulated compared to the controls without naphthalene, as noted in other fuel and diesel oil bioremediation systems by Wang and Bartha (1990). These results, although limited only to naphthalene, are promising of a more general potentiality of the consortium of the three basidiomycetes to degrade and detoxify also more recalcitrant PAH; they also demonstrate that composts, as substrates rich in lignocellulosic materials, can be important sources of microorganisms, including lignin-
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olytic fungi, able to degrade pollutants. Consortium ability to compete with any autochthonous microflora and degrade more recalcitrant PAH are certainly subjects that require further investigation. Acknowledgements This work was supported by CEBIOVEM (Centro di Eccellenza per la Biosensoristica Vegetale e Micobica) and by Marcopolo Engineering S.p.a. (Borgo San Dalmazzo, Italy). References Aitken, M.D., Long, T.C., 2004. Biotransformation, biodegradation, and bioremediation of polycyclic aromatic hydrocarbons. In: Singh, A., Ward, O.P. (Eds.), Biodegradation and Bioremediation. SpringerVerlag, Berlin, Heidelbberg, pp. 83–124. Alcade, M., Bulter, T., Arnold, F.H., 2002. Colorimetric assays for biodegradation of polycyclic aromatic hydrocarbons by fungal laccases. J. Biomol. Screen. 7 (6), 547–553. Alef, K., 1995. Soil respiration. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London, England, pp. 214–216. Anastasi, A., 2003. Analisi floristica e funzionale della micoflora di un compost e un vermicompost: aspetti igienici e possibili applicazioni in campo ambientale. PhD Thesis. University of Turin, Italy, 195 p. Anastasi, A., Varese, G.C., Filipello Marchisio, V., 2005. Isolation and identification of fungal communities in compost and vermicompost. Mycologia 97 (1), 33–34. Anastasi, A., Varese, G.C., Casieri, L., Filipello Marchisio, V., 2006. Basidiomycetes from compost and their dye degradation and enzyme activities. Compost Sci. Util. 14 (4), 284–289. Antizar-Ladislao, B., Lopez-Real, J.M., Beck, A.J., 2004. Bioremediation of polycyclic aromatic hydrocarbon (PAH)-contaminated waste using composting approaches. Crit. Rev. Environ. Sci. Technol. 34, 249– 289. Canet, R., Birnstingl, J.G., Malcolm, D.G., Lopez-Real, J.M., Beck, A.J., 2001. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by native microflora and combinations of white-rot fungi in a coal-tar contaminated soil. Bioresour. Technol. 76, 113–117. Gold, M.H., Glenn, J.K., Alic, M., 1988. Use of polymeric dyes in lignin biodegradation assays. Method. Enzymol. B 161, 74–78. Johannes, C., Majcherczyk, A., 2000. Laccase activity tests and laccase inhibitors. J. Biotechnol. 78, 193–199. Mollea, C., Bosco, F., Ruggieri, B., 2005. Fungal biodegradation of naphthalene: microcosms studies. Chemosphere 60, 636–643. Muncnerova`, D., Augustin, J., 1994. Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons: a review. Bioresour. Technol. 48, 97–106. Novotny, C., Erbanova, P., Sasek, V., Kubatova, A., Cajthaml, T., Lang, E., Krahl, J., Zadrazil, F., 1999. Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi. Biodegradation 10, 159–168. Paszczynski, A., Crawford, R.L., Huynh, V.B., 1988. Manganese peroxidase of Phanerochaete chrysosporium: purification. Method. Enzymol. 161 (B), 264–270. Ryckeboer, J., Mergaert, J., Vaes, K., Klammer, S., De Clercq, D., Coosemans, J., Insam, H., Swings, J., 2003. A survey of bacteria and fungi occurring during composting and self-heating processes. Ann. Microbiol. 53 (4), 349–410. Sasek, V., Bhatt, M., Cajthaml, T., Malachova, K., Lednicka, D., 2003. Compost-mediated removal of polycyclic aromatic hydrocarbons from contaminated soil. Arch. Environ. Con. Tox. 44, 336–342.
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Semple, K.T., Reid, B.J., Fermor, T.R., 2001. Impact of composting strategies on the treatment of soils contaminated with organic pollutants. Environ. Pollut. 112, 269–283. Singh, H., 2006. Fungal metabolism of polycyclic aromatic hydrocarbons. In: Singh, H. (Ed.), Mycoremediationm, Fungal Bioremediation. John Wiley & Sons, Inc., Hoboken, New Jersey, pp. 283–356.
Tien, M., Kirk, T.K., 1988. Lignin peroxidase of Phanerochaete chrysosporium. Method. Enzymol. 161, 238–248. Wang, X., Bartha, R., 1990. Effects of bioremediation on residues, activity and toxicity in soil contaminated by fuel spills. Soil Biol. Biochem. 22, 501–505.
Bioresource Technology 99 (2008) 6626–6630
Short Communication
Bioremediation potential of basidiomycetes isolated from compost Antonella Anastasi a, Giovanna C. Varese a,*, Francesca Bosco b, Fabiana Chimirri b, Valeria Filipello Marchisio a b
a Dipartimento di Biologia Vegetale, Universita` degli Studi di Torino, Viale Mattioli 25, 10125 Torino, Italy Dipartimento di Scienza dei Materiali ed Ingegneria Chimica, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy
Received 24 April 2007; received in revised form 6 December 2007; accepted 12 December 2007 Available online 31 January 2008
Abstract The potential of a consortium of three basidiomycete mycelia isolated from compost to degrade polycyclic aromatic hydrocarbons (PAH) was first evaluated using a test based on decolorization of Poly R-478 dye. When pre-grown on straw, the consortium decolorized the dye by 83% in 7 days and generated a laccase activity of 663 IU l1. Its ability to degrade naphthalene was investigated in soil microcosms specially suited for this volatile PAH. The kinetic study was conducted at a maximal naphthalene concentration of 500 mg kg1 of soil. Naphthalene concentration, CO2 evolution and phytotoxicity (germination index, GI%) on Lepidium sativum seeds were monitored. The naphthalene concentration decreased by about 70% in three weeks in the presence of metabolic activity, while the GI% increased indicating reduced phytotoxicity. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Fungi; Naphthalene; PAH degradation; Phytotoxicity tests; Poly R-478
1. Introduction Polycyclic aromatic hydrocarbons (PAH) are common, persistent and recalcitrant contaminants in soil. Their distribution and fate within the environment are a matter of growing concern, especially since many are cytotoxic, mutagenic and carcinogenic and have been classified as priority pollutants by the EPA (Aitken and Long, 2004). PAH can be eliminated from polluted soil by incineration, thermal desorption, soil washing etc. These methods, however, are expensive and have an adverse impact on the environment (Canet et al., 2001). Bioremediation is a competitive alternative. Composts have an enormous potential for bioremediation through their support of mesophilic and thermophilic bacteria and ligninolytic fungi endowed with the ability to degrade aromatic pollutants (Semple et al., 2001). However, a better understanding of the compost microflora *
Corresponding author. Tel.: +39 0116705984; fax: +39 0116705962. E-mail address: [email protected] (G.C. Varese).
0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.12.036
involved in PAH degradation is still needed, with particular emphasis on the impact of ligninolytic fungi (AntizarLadislao et al., 2004). Lignin degrading basidiomycetes normally do not assimilate PAH as the sole carbon source, and hence require cometabolites (e.g. glucose) to degrade them (Singh, 2006). Their degradation results in the formation of PAH-quinones catalysed by extracellular ligninolytic enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP), laccases (Lcc) and H2O2-generating enzymes (Singh, 2006). These enzymes are non-specific, non-stereoselective and effective against a broad spectrum of aromatic compounds including anthracene, benzo[a]pyrene, naphthalene and pyrene (Muncnerova` and Augustin, 1994; Novotny et al., 1999). However, basidiomycetes are rarely isolated from compost, both because many of them cannot withstand the temperatures of more than 50 °C generated during the thermophilic stage (Ryckeboer et al., 2003), and because their isolation and morphological identification are impeded by the fact that they are often found as sterile mycelia, while their molecular detection is hampered by the
A. Anastasi et al. / Bioresource Technology 99 (2008) 6626–6630
incompleteness of gene databases and the low taxonomic resolution of DNA sequences. The genera observed in compost include Armillaria, Clitopilus, Coprinus, Fomes, Lentinus, Lenzites, Pleurotus and Trametes. The degradative capability of basidiomycetes from compost, however, has not been investigated. We have previously used a test based on the decolorization of Poly R-478 dye in solid culture to assess the ligninolytic activity of 266 fungal isolates (33 were basidiomycetes) from different kinds of composts (Anastasi et al., 2005). Decolorization ability and ligninolytic enzymes production of the three most promising basidiomycetes were then investigated in greater detail in liquid culture (Anastasi et al., 2006) containing Poly R-478, since the ability to decolorize this dye is also regarded as indicative of a PAH degradation potential (Alcade et al., 2002; Canet et al., 2001). The aim of the present work was first to investigate the ability to decolorize Poly R-478 of the same three basidiomycetes in a consortium and determine its inhibitory/synergic effects on decolorization and enzyme activities, and then to evaluate its PAH degradation capability in soil microcosms polluted with naphthalene. Phytotoxicity due to naphthalene and/or any of its metabolites was monitored by calculating the germination index (GI%) of Lepidium sativum seeds during the incubation period. 2. Methods
6627
Ltd.) at a final concentration of 0.02%. Inoculum consisted of 0.2 g straw cultures for each strain. A heat-killed control (straw cultures autoclaved at 121 °C for 20 min) and an abiotic (consortium-free) control were set up to check for absorption of the dye on the mycelium or a decrease in absorbance not due to fungal presence. Triplicate cultures were incubated statically at 27 °C and flushed about every 2 days with O2 in sterile conditions. Decolorization was monitored daily for 7 days on 0.3 ml cell free culture medium diluted 10-fold in water and expressed as the percentage decrease of absorbance at 514 nm, i.e. the maximum visible wavelength of absorbance of Poly R-478, with a UV–visible spectrophotometer (Ultrospec 3300 pro; Amersham). LiP activity was evaluated spectrophotometrically via the oxidation of veratryl alcohol at the wavelength of 310 nm in 250 mM sodium tartrate buffer, pH 5, at 39 °C (Tien and Kirk, 1988). MnP activity was evaluated spectrophotometrically via the oxidation of curcumin at the wavelength of 430 nm in 0.5 M sodium tartrate buffer, pH 5, at 22 °C (Paszczynski et al., 1988). Lcc activity was evaluated spectrophotometrically by following the oxidation of 2,20 -azino-bis-(3ethylbenzothiazoline-6 sulphonic acid) (ABTS) at the wavelength of 420 nm in 100 mM sodium acetate buffer, pH 5, at 22 °C (Johannes and Majcherczyk, 2000). Activities were expressed as international units per litre (IU l1). The Spearman correlation test was used to evaluate correlations between decolorization and enzyme activity.
2.1. Microorganisms and inoculum preparation 2.3. Soil microcosms The three basidiomycetes were patented and deposited in the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) collection (DSM 15214, DSM 15215, DSM 15216); they were isolated from compost as sterile mycelia and identified as basidiomycetes morphophysiologically (Anastasi, 2003); rDNA sequencing (Internal Transcribed Spacer – ITS 1, 2 – and 18S) and comparison with sequences in gene databases showed that strains DSM 15214 and DSM 15215 had 100% similarity to Trametes versicolor (L.) Pila´t, while strain DSM 15216 had 99% similarity to different species of basidiomycetes [Bjerkandera adusta (Willd.) P. Karst., Bjerkandera fumosa (Pers.) P. Karst., Lopharia spadicea (Pers.) Boidin] (GenBank, NCBI). All three strains had been shown to display Lcc and/or MnP activity during their decolorization of Poly R-478 in single cultures (Anastasi et al., 2006). Equal portions of straw cultures of each strain, prepared as previously described (Anastasi et al., 2006), were used to inoculate flasks for Poly R-478 decolorization and naphthalene degradation studies. 2.2. Decolorization tests and enzyme assays in liquid cultures Decolorization tests were performed in 500 ml, hermetically sealed Erlenmeyer flasks containing 30 ml of a synthetic glucose medium (Gold et al., 1988) supplemented with an aqueous Poly R-478 solution (Sigma Aldrich Co.,
The static microcosms were prepared, as previously described by Mollea et al. (2005), by placing in hermetically sealed, 500 ml Erlenmeyer flasks 1 g of straw cultures of each strain, 20 ml of a 20 g/l glucose:water solution and 10 g of an uncontaminated, natural soil (pH 6.63, total moisture 12%) then artificially spiked with naphthalene (500 mg kg1) dissolved in ethyl acetate (EA) (Fluka). Heat-killed controls were obtained by preparing microcosms as described above, but with straw cultures pre-autoclaved at 121 °C for 20 min. Microcosms were incubated statically at 27 °C and periodically flushed with wetted pure oxygen at a constant 27 °C, since the results of preliminary experiments (not shown) had made it clear that temperature control during oxygenation was essential to limit the increase in the volatility of the pollutant. However, checking PAH losses during oxygenation was necessary; for this purpose the flowing gaseous phase was absorbed in 40 ml N,N-dimethylformamide (DMF) (Fluka) at the end of the oxygenation system. Ten milliliters of the 20 g/l glucose:water solution were added on days 14 and 28 to reestablish the system’s C source and humidity. 2.4. Respirometric analysis To determine metabolic activity in each microcosm we periodically performed a respirometric analysis by moni-
toring CO2 emission according to the modified Isermeyer method (Alef, 1995). 2.5. Naphthalene degradation Naphthalene was extracted by adding 100 ml EA and agitating the flasks at 150 rpm for 72 h at 27 °C. The filtered extracts and the DMF used to capture naphthalene were diluted and analysed with a Kontron HPLC equipped with a 432 UV–visible detector operating at the wavelength of 254 nm and a ChromoSpher 5 b PAH (4.6 150 mm) C18 reversed-phase column (Chrompack) maintained at 25 °C with a Peltier thermostat. The mobile phase was a 65:35 (v/v) acetonitrile:water solution and the flow rate was 0.9 ml/min. Residual naphthalene was calculated as the sum of the amounts extracted with EA and captured with DMF. The analyses were carried out in triplicate. 2.6. Phytotoxicity tests Toxicity was evaluated by means of a phytotoxicity test on L. sativum seeds applied directly in biotic and killed microcosms, with and without naphthalene, at the start of incubation and after 21 and 42 days. The microcosms were kept in the same conditions as those utilized for the naphthalene degradations experiments. The germination index (GI%) was calculated from the number of germinated seeds and the root length values in the control and in the samples according to the formula: GI% = (Gs Ls)/ Gc Lc)100, where Gs is the mean number of germinated seeds in the sample, Ls is the mean root length of the sample, Gc is the mean number of germinated seeds in the control, Lc is the mean root length of the control. The analyses were duplicated. 3. Results 3.1. Decolorization tests and enzymatic assays in liquid cultures Decolorization of Poly R-478 by the consortium led to an approximately 83% decrease of its initial absorbance
% decolorization
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Fig. 1. Percentage of decolorization of 0.02% Poly R-478 by the fungal consortium and by the abiotic and killed controls.
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naphthalene concentration (ppm)
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Fig. 2. Residual naphthalene in the biotic microcosms containing the fungal consortium and in the killed controls and CO2 emission in the biotic microcosms (arrows show the additions of 10 ml of a 2% w/v glucose:water solution).
value in 7 days. The values of the killed and abiotic controls were unchanged (Fig. 1). LiP activity was absent. MnP and Lcc activities reached 63 and 663 IU l1, respectively, but were not significantly correlated with the decolorization percentage (r = 0.476, p = 0.233; r = 0.310, p = 0.456). 3.2. Naphthalene degradation and detoxification The CO2 produced by the biotic microcosms never fell below 395 mg from the 2nd day onwards (Fig. 2) and rose when glucose was added on the 14th and 28th days. This showed that the consortium was able to survive and efficiently colonize soil in the microcosms. In Fig. 2 the time course of naphthalene concentration (extracted with EA and captured in DMF) for the killed and biotic lines is reported. A distinction between biotic and killed line was evident from the first days of incubation; between the 3rd and the 20th day, the biotic line had the maximum gradient, then naphthalene decrement shut down and remained constant till the end of the experiment, despite the fact that the CO2 pattern showed that the consortium was still metabolically active after the final glucose supplement on the 28th day. The mean residual naphthalene in the biotic microcosms on the 42nd day was 83 mg kg1, corresponding to a 80.24% reduction during the incubation period. In the killed controls the mean residual naphthalene was 272 mg kg1 corresponding to a 42% reduction. The ratio between the two residues shows that the degradation attributable to the consortium alone was 69.49%. Capture of naphthalene by DMF recovered up to 14.52% in the biotic microcosms and 12.6% in the killed controls (data not shown). This gave a more accurate estimate of the fungus-induced degradation. Without this recovery, the naphthalene degradation would have been 90%, in other words well above the real degradative power of the consortium. The GI% was zero at the initial naphthalene concentration (500 mg kg1). It then rose to 188.77% and 205.55% at
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days 21 and 42 when the residual concentrations were 103 mg kg1 and 83 mg kg1, respectively. 4. Discussion The fungal consortium displayed a good degradative capability against the complex aromatic molecule Poly R478 (83% decolorization in 7 days), though this was much the same as the 77–87% found for the individual strains (Anastasi et al., 2006). However Lcc activity expressed by the consortium increased up to 5 times, perhaps due to the concurrent presence and synergic activity of the isolates ascribable to T. versicolor, whose ability to decolorize Poly R-478 and degrade PAH, mainly by means of Lcc, is well known (Alcade et al., 2002). Though the ability to decolorize Poly R-478 is regarded as predictive of a PAH degradation potential (Alcade et al., 2002; Canet et al., 2001), the degradative capacity and the production of enzymes in a liquid culture are not enough to posit the use of organisms in the bioremediation of polluted soils. The setting up of microcosms closely simulates PAH biodegradation in soil without prejudice to the maintenance of controlled culture conditions (artificial pollution, constant incubation temperature, insufflation of pure oxygen, absence of competitors, periodic addition of nutrients and water, and monitoring of metabolic activity). Under these conditions, the consortium displayed a good ability to efficiently colonize the soil and reduce a high naphthalene concentration (500 mg kg1): about 70% degradation was achieved after three weeks and good metabolic activity was maintained. Comparison of our results with those obtained in similar microcosms by Mollea et al. (2005) with Phanerochaete chrysosporium, considered a model organism in PAH degradation studies, shows that the consortium removed naphthalene more rapidly; starting from an initial concentration of 600 mg kg1, P. chrysosporium produced a degradation of about 20% after 3 weeks and a maximum of about 55% at the 50th day (values calculated by comparing the biotic line to the killed controls). Another significant outcome of this study was the gradual disappearance of phytotoxicity following reduction of the pollutant. In some cases, in fact, PAH metabolites have proved more toxic than their parent compounds (Sasek et al., 2003). In our study, the seeds did not germinate at first, whereas at the halfway point (naphthalene concentration of 103 mg kg1) and at the end of the test they all germinated and the GI% rose to more than 100%, showing that growth was actually stimulated compared to the controls without naphthalene, as noted in other fuel and diesel oil bioremediation systems by Wang and Bartha (1990). These results, although limited only to naphthalene, are promising of a more general potentiality of the consortium of the three basidiomycetes to degrade and detoxify also more recalcitrant PAH; they also demonstrate that composts, as substrates rich in lignocellulosic materials, can be important sources of microorganisms, including lignin-
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olytic fungi, able to degrade pollutants. Consortium ability to compete with any autochthonous microflora and degrade more recalcitrant PAH are certainly subjects that require further investigation. Acknowledgements This work was supported by CEBIOVEM (Centro di Eccellenza per la Biosensoristica Vegetale e Micobica) and by Marcopolo Engineering S.p.a. (Borgo San Dalmazzo, Italy). References Aitken, M.D., Long, T.C., 2004. Biotransformation, biodegradation, and bioremediation of polycyclic aromatic hydrocarbons. In: Singh, A., Ward, O.P. (Eds.), Biodegradation and Bioremediation. SpringerVerlag, Berlin, Heidelbberg, pp. 83–124. Alcade, M., Bulter, T., Arnold, F.H., 2002. Colorimetric assays for biodegradation of polycyclic aromatic hydrocarbons by fungal laccases. J. Biomol. Screen. 7 (6), 547–553. Alef, K., 1995. Soil respiration. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London, England, pp. 214–216. Anastasi, A., 2003. Analisi floristica e funzionale della micoflora di un compost e un vermicompost: aspetti igienici e possibili applicazioni in campo ambientale. PhD Thesis. University of Turin, Italy, 195 p. Anastasi, A., Varese, G.C., Filipello Marchisio, V., 2005. Isolation and identification of fungal communities in compost and vermicompost. Mycologia 97 (1), 33–34. Anastasi, A., Varese, G.C., Casieri, L., Filipello Marchisio, V., 2006. Basidiomycetes from compost and their dye degradation and enzyme activities. Compost Sci. Util. 14 (4), 284–289. Antizar-Ladislao, B., Lopez-Real, J.M., Beck, A.J., 2004. Bioremediation of polycyclic aromatic hydrocarbon (PAH)-contaminated waste using composting approaches. Crit. Rev. Environ. Sci. Technol. 34, 249– 289. Canet, R., Birnstingl, J.G., Malcolm, D.G., Lopez-Real, J.M., Beck, A.J., 2001. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by native microflora and combinations of white-rot fungi in a coal-tar contaminated soil. Bioresour. Technol. 76, 113–117. Gold, M.H., Glenn, J.K., Alic, M., 1988. Use of polymeric dyes in lignin biodegradation assays. Method. Enzymol. B 161, 74–78. Johannes, C., Majcherczyk, A., 2000. Laccase activity tests and laccase inhibitors. J. Biotechnol. 78, 193–199. Mollea, C., Bosco, F., Ruggieri, B., 2005. Fungal biodegradation of naphthalene: microcosms studies. Chemosphere 60, 636–643. Muncnerova`, D., Augustin, J., 1994. Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons: a review. Bioresour. Technol. 48, 97–106. Novotny, C., Erbanova, P., Sasek, V., Kubatova, A., Cajthaml, T., Lang, E., Krahl, J., Zadrazil, F., 1999. Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi. Biodegradation 10, 159–168. Paszczynski, A., Crawford, R.L., Huynh, V.B., 1988. Manganese peroxidase of Phanerochaete chrysosporium: purification. Method. Enzymol. 161 (B), 264–270. Ryckeboer, J., Mergaert, J., Vaes, K., Klammer, S., De Clercq, D., Coosemans, J., Insam, H., Swings, J., 2003. A survey of bacteria and fungi occurring during composting and self-heating processes. Ann. Microbiol. 53 (4), 349–410. Sasek, V., Bhatt, M., Cajthaml, T., Malachova, K., Lednicka, D., 2003. Compost-mediated removal of polycyclic aromatic hydrocarbons from contaminated soil. Arch. Environ. Con. Tox. 44, 336–342.
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