Complete degradation of anthracene by Manganese Peroxidase in organic solvent mixtures

1 Complete degradation of anthracene by Manganese Peroxidase in organic solvent mixtures G. Eibes, T. Lú-Chau, G. Feijoo, M.T. Moreira* and J.M. Lema Dept. of Chemical Engineering. School of Engineering, University of Santiago de Compostela. Santiago de Compostela. E-15782, Spain. *Author for correspondence (Fax: 34-981-528050; [email protected]) 2 Abstract The goal of this study is the development of a system based on the use of the ligninolytic enzyme Manganese Peroxidase (MnP) for the degradation of polycyclic aromatic hydrocarbons (PAHs), of which anthracene was selected as an example. A main problem of these compounds is their poor solubility in aqueous media. Therefore, the addition of different water miscible organic solvents (acetone, methyl-ethyl-ketone, methanol and ethanol) was considered as a previous step to increase the bioavailability of anthracene. Due to the maximal solubilisation of anthracene and the minimum loss of MnP activity, acetone was selected as the optimal cosolvent, allowing to enhance 140- fold the anthracene solubility for an acetone concentration of 36% (v/v). The in vitro degradation of anthracene by MnP was investigated for different concentrations of the main cofactors and substrates that affect the catalytic cycle of MnP (Mn2+, H2O2 and organic acids) as well as for other environmental parameters (temperature, air/oxygen atmosphere and light source). The system attained a nearly complete degradation of anthracene, around 100%, after 6 hours of operation under optimal conditions. Key words: anthracene, biodegradation, Manganese Peroxidase, miscible organic solvents, solubility, stability 3 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are pollutants produced via natural and anthropogenic sources, generated during the incomplete combustion of solid and liquid fuels or derived from industrial activities. These compounds are hydrophobic with low water solubility, thus they are easily adsorbed onto organic matter as soils and sediments. Besides, their recalcitrant behaviour greatly hampers their naturally biological degradation [1, 2]. Among other possibilities, an environmentally friendly approach for PAHs degradation could be based on the use of white rot fungi, which are known to degrade a great variety of complex compounds due to their complex enzymatic system [3]. Lignin Peroxidase (LiP) and Manganese Peroxidase (MnP) are extracellular peroxidases produced by white rot fungi and the onset of their production is associated to secondary metabolism conditions in response to nutrient depletion [4]. In particular, manganese and nutrient nitrogen have been shown to have strong regulating effects [5]. The ligninolytic system is nonselective, consequently other aromatic substrates, such as PAHs are potentially oxidized and biodegraded by white rot fungi [6, 7, 8]. The catalytic action of these enzymes generates more polar and water-soluble metabolites, such as quinones, which are more susceptible to further degradation by indigenous bacteria present in soils and sediments [9, 10]. However, a wider application of these enzymes is hindered by the fact that enzymes work properly in aqueous media, where nonpolar compounds as polyaromatics present a very low solubility. An increased solubilisation of polyaromatics in aqueous media would have beneficial effects on the potential degradation of these compounds [11, 12, 13]. A good 4 approach to enhance PAHs solubility in several orders of magnitude is the addition of water-miscible cosolvents or surfactants [14, 15, 16]. These latter compounds may present a low solubilisation of PAHs and partial inhibition of the ligninolytic activity [17]. Although enzymatic catalysis in organic solvents is considered a promising approach for solving environmental problems, most of the available work is related to hydrolytic enzymes, applied for synthesis of organic compounds [18, 19]. The potential of using more complex enzymes such as ligninolytic enzymes produced by white rot fungi, which require specific environmental conditions for the activation of their catalytic cycle, is almost untapped [8]. The goal of this work is the evaluation of a system based on the use of MnP for the degradation of a PAH model compound, anthracene, in water-miscible organic solvents. Anthracene, a three-ring PAH, was chosen due to its low aqueous solubility (0.07 mg/L [20]) and this compound has been proved to be a substrate of ligninolytic peroxidases [6]. Enzymatic degradation was selected as an alternative to bacterial processes because the biological degradation usually requires long periods of treatment (from 2 to 4 weeks) and presents lag phases (2 days) till the degradation begins [21, 22]. The initial stage of the process was the selection of the most appropriate cosolvent from a list of four relatively safe, easily available and fairly inexpensive chemicals: acetone, methyl-ethyl-ketone (MEK), methanol and ethanol. The influence of the solvent on MnP activity was used as a criterion for this selection. In a second stage, the optimisation of the degradation process was conducted taking into account specific physico-chemical factors which may directly affect the activation of the MnP catalytic cycle and the degradation rate of anthracene: (a) the concentration of cofactors and substrates required for the action of MnP (Mn2+, H2O2, organic acids) [23, 24] and 5 (b) operating parameters such as temperature, light source and maintenance of air or oxygen atmosphere [25]. 6 2. Materials and methods 2.1. Chemicals Anthracene and anthraquinone were obtained from Janssen Chimica (99% purity). Acetone, methanol and ethanol were purchased from Panreac (chemical purity); methyl-ethyl-ketone was supplied by Sigma-Aldrich (99.5% purity). 2.2. MnP production MnP was obtained from two metabolically distinct white-rot fungi, Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) and Bjerkandera sp. BOS55 (ATCC 90440), with some different catalytic properties, the latter presenting a superior resistance against high concentrations of H2O2 [26]. P. chrysosporium was cultured in 250-mL Erlenmeyer flasks on the N-limited BIII medium [27]. B. sp. BOS55 was grown in a 10-L fermenter (Braun-Biotech International) on skimmed cheese whey medium [28]. Once the peak production of MnP was detected, the fermentation was stopped. Crude enzyme was concentrated by ultrafiltration using a 10- kDa cut-off type YM-10 membrane (Amicon), and then it was centrifuged for 10 min at 20,000 × g. 2.3. MnP activity assays MnP activity was determined by monitoring the oxidation of 2,6- dimethoxyphenol (DMP) spectrophotometrically at 30ºC (Cecil CE 7200, UK). The reaction mixture contained 50 mM sodium malonate (pH 4.5), 1 mM DMP, 1 mM MnSO4, and up to 600 μL of supernatant in a total volume of 1 mL. The reaction was initiated by adding 0.4 mM H2O2. One MnP activity unit was defined as the amount of 7 enzyme transforming 1 μmol DMP per minute [29]. 2.4. Anthracene solubility assays The solubility of anthracene was determined in 20-mL aliquots containing 25 mg anthracene (final concentration of 1.25 mg/L) with different concentrations of solvent ranging from 1% to 100%. The aliquots were placed in 100-mL Erlenmeyer flasks sealed with teflon plugs, in triplicate, equilibrated for 24 h on a shaker (150 rpm) at 20ºC or 30ºC. Afterwards, the 20-mL assays were filtered through a Millex-LCR13 cartridge (Millipore Corp.), with a pore diameter of 0.45 μm and analysed by highpressure liquid chromatography (HPLC). 2.5. Effect of water: solvent mixtures on MnP stability The stability of crude MnP from cultures of B. sp. BOS55 and P. chrysosporium was evaluated in water: solvent mixtures by the determination of the peroxidase titers of MnP at periodic intervals. The assay conditions evaluated included: two temperatures (20ºC and 30ºC), a solvent concentration attaining the solubilisation of 10 mg/L of anthracene according to the results from the anthracene solubility assays and 10 mM sodium malonate (pH 4.5) in a total volume of 10 mL. 2.6. Anthracene biodegradation assays Oxidation of anthracene was carried out in 100-mL Erlenmeyer flasks, sealed with teflon plugs, with magnetic stirring at room temperature, i.e. 23ºC (except when indicated). The reaction mixture (50 mL) consisted of acetone 36% (v:v), anthracene (5 mg/L) and MnP (200 U/L) with different concentrations of the main cofactors and 8 substrates reported for MnP (Mn2+, H2O2 and organic acid: malonic, oxalic, citric and tartaric acid). Mn2+ concentration was assayed at 20 μM and 100 μM, while H2O2 was added continuously at three addition rates: 1, 5 and 25 µmol/L·min. In the experiments where the effect of the organic acid was considered, malonic acid concentration ranged from 1 mM to 30 mM. Experiments with oxalic, citric and tartaric acid at similar concentrations were also conducted. The possible effects of other environmental parameters, such as temperature, light and oxygen atmosphere, on the degradation of anthracene were also investigated. The influence of temperature was evaluated in assays performed at 23ºC, 30ºC and 40ºC. An oxygen atmosphere was also investigated by flushing industrial oxygen at periodic intervals (3 min every 30 min). Samples were withdrawn periodically to determine anthracene and anthraquinone concentrations by HPLC as described above, and the evolution of MnP activity was spectrophotometrically determined. To verify that degradation took place only due to an enzymatic oxidation, controls were run in parallel using boiled MnP. No change in anthracene concentration after 6-8 h of incubation was observed in any controls (data not shown). 2.7. Analytical determinations A HP 1090 HPLC, equipped with a diode array detector monitoring the absorbance at 253 nm, a 4.6 × 200 mm Spherisorb ODS2 reverse phase column (5 μm; Waters) and a HP ChemStation data processor were used for determining the anthracene concentration. The injection volume was set at 10 μL and the isocratic eluent (80% acetonitrile and 20% water) was pumped at a rate of 0.4 mL/min. 9 3. Results 3.1. Solubility of anthracene in water: solvent mixtures The solubility of anthracene in four water miscible solvents: acetone, methylethyl-ketone (MEK), ethanol and methanol, was determined at 20ºC (Figure 1) and 30ºC. Concentrations higher than 70% acetone dissolved anthracene completely (1.25 g/L) while the alcohols attained lower solubilities, only being equivalent at 100% solvent. The addition of MEK provided the highest anthracene solubilities in a concentration range between 10 and 30% (v:v) in comparison with the other solvents. However, higher concentrations of MEK resulted in the formation of two differentiated phases: aqueous and non-aqueous, which impeded the utilisation of MEK as a water miscible solvent. Table 1 shows the solvent concentrations for the solubilisation of 1, 10 and 100 mg/L of anthracene at 20 and 30ºC. The solubilisation at 30ºC was slightly more beneficial for all cosolvents since it implied a reduction between 7-12% of the total addition of the organic solvent in comparison with that required for 20ºC. The minimum concentrations of the organic solvents required to attain a solubility of 10 mg/L, which is a 140-fold increase of the anthracene solubility in water at 20ºC (0.07 mg/L [20]), were: 27% MEK, 36% acetone, 44% ethanol and 55% methanol (Table 1). 3.2. Stability of MnP in solvent: water mixtures The effect of solvent: water mixtures on the activity and stability of crude MnP from B. sp. BOS55 and P. chrysosporium were evaluated. MnP activity was determined instantaneously after mixing the solvent mixtures with MnP. Acetone: water mixtures 16 action of MnP depends on the combined action of several compounds, referred as substrates, cofactors and mediators, which initiate, participate and allow the completion of the catalytic cycle. Therefore, for optimizing the catalytic action of the enzyme, special attention was paid to study the influence of the following main factors: H2O2 and Mn2+ concentrations, organic acids and other operating parameters such as temperature and oxygen atmosphere . The continuous addition of H2O2 at a controlled flow (5 μmol/L·min) permitted the progressive participation of H2O2 in the catalytic cycle through a suitable regeneration of the oxidised form of the enzyme, minimising the peroxide-dependent inactivation of the peroxidase [29]. Organic acids are required in the catalytic cycle of MnP because they facilitate the release of Mn3+ from the active site and also stabilize this species in aqueous solution [24, 46]. In addition, Kuan et al. [30] reported that complexed Mn2+ is the preferred substrate for the oxidised form of MnP compound II. Our results confirm that the concentration of the organic acid (e.g. malonic) is decisive on the action of the enzyme: on the one hand, degradation extent is improved, but on the other hand, activity loss also increases. Oxalic and malonic acids have been shown to be oxidatively decarboxylated by Mn3+ [47], generating a carbon dioxide anion radical which permits the endogenous formation of H2O2 via Mn2+ and a superoxide radical. The resulting accumulation of H2O2 may explain the greatest activity loss for both acids at high concentrations, specially oxalate which produces higher H2O2 concentrations [48]. Regarding to the other organic acids, it is possible that the binding of tartaric and citric acid (C4 and C6, respectively) to the enzyme is sterically hindered, being, therefore, the extent of degradation even lower than the corresponding to the reaction without exogenous 17 organic acid. The crude MnP contains lactic acid in a concentration of 1 mM from the fermentation medium, which would be enough to permit a low degradation extent. Oxygen atmosphere increases the anthracene oxidation. This fact which has been observed in degradation of azo dyes in water may be attributed to the catalase-type activity of MnP [49]. MnP releases atomic oxygen which could be directly used for the degradation of anthracene. In this case, it is interesting to see that the maximum degradation rate is coincident with the highest dissolved oxygen concentration in the medium (27.9 mg/L). The degradation of anthracene outcomes into its total decomposition to the dead-end product: the anthraquinone [3, 50]. The degradation mechanism, probably arising via one-electron oxidative pathway, has a large complexity with the generation of intermediate compounds such as anthrol and anthrone [51]. The apparent discrepancy between the expected ratio 1:1 of anthraquinone and anthracene and that obtained in this experimental work, around 1:2, indicates the presence of relative amounts or these or other intermediate compounds. In fact, the final step to anthraquinone is likely to be limiting the overall reaction rate of the process, as we determined an increase of the anthraquinone concentration around 10% in samples measured after 24 h. In this sense, ongoing research has as an objective the deeper knowledge of the degradation mechanism and kinetics and the way to enhance the rate of the whole process. Moreover, future work will be also focused on the biological degradation of anthraquinone by bacterial populations. Therefore, the overall process will be considered as a combination of an in vitro enzymatic system in the initial stage of degradation and a bacterial biological treatment to complete the process. 18 5. Conclusions The proposal of the enzyme-based oxidation for the initial degradation of anthracene, as a model compound representative of recalcitrant polyaromatics, is the main focus of this work. 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Solvent concentration required for the solubilisation of 1, 10 and 100 mg/L of anthracene Solvent concentration (%) Solvent T (ºC) 1 mg/L 10 mg/L 100 mg/L MEK 20 17 27* ND 30 14 24 ND Acetone 20 21 36 53 30 19 33 49 Ethanol 20 31 44 64 30 28 41 60 Methanol 20 37 55 76 30 32 51 67 ND: not determined *Bold type values represent the values selected for the following experiments 32 Figure 3 B A 33 Figure 4 B A 34 Figure 5