Enzymatic Degradation of Low Soluble Compounds in Monophasic Water:Solvent Reactors. Kinetics and Modeling of Anthracene Degradation by MnP

1 Enzymatic degradation of low soluble compounds in monophasic water:solvent reactors. Kinetics and modelling of anthracene degradation by MnP G. Eibes*, M. T. Moreira, G. Feijoo and J. M. Lema Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, E-15782. Santiago de Compostela, Spain Phone: +34-981563100 Ext.16740; e-mail: [email protected] 2 Abstract Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds presenting low water solubility and high hydrophobicity, which greatly hampers their natural biodegradation. The enzymatic degradation of a model compound, anthracene, was evaluated in presence of a miscible solvent for an increased solubility. Manganese peroxidase, a ligninolytic enzyme from white-rot fungi, was used as biocatalyst in a medium containing acetone. The kinetic parameters of the enzymatic degradation of anthracene, obtained from fed-batch experiments, were applied to model the operation of a continuous reactor. Kinetics comprised a Michaelis-Menten equation, modified with an autocatalytic term, assumed to the effect of quinones acting as electron carriers, and a logistic function related to enzyme activity. The continuous reactor has been operated for 108 h, attaining a 90% of anthracene degradation, which demonstrated the feasibility of the system for its application in the removal of poorly-soluble compounds. The model of this reactor permitted to predict accurately anthracene degradation in different conditions, such as external addition of anthraquinone and different enzymatic activities. Keywords: Enzyme bioreactors; Manganese peroxidase; Anthracene; Miscible solvent; Modelling 3 Introduction Recalcitrant compounds are a major threat for the environment and in many cases they can constitute a health risk to humans and animals. In recent years there has been an increasing interest in the search of biological systems capable of degrading persistent pollutants. Among diverse recalcitrant compounds, polycyclic aromatic hydrocarbons (PAHs) present low solubility and great affinity to be adsorbed on the soil matrix and thus, their desorption from soil limits their availability for microbial biodegradation. Enzymatic degradation is an attractive approach for the removal of recalcitrant compounds since it presents a series of advantages with respect to microbial treatment: shorter operational times, easier control and operation, non-sterile conditions, etc. The extracellular degrading system of white-rot fungi has permitted in vitro PAH biodegradation, this process is usually limited by the low solubility of the compounds in aqueous media (Field et al. 1995). The use of an organic solvent to desorb PAHs from the soil matrix can assist the degradative action of the enzymes (Field et al. 1996). The catalytic action of the ligninolytic enzyme manganese peroxidase (MnP) generates polar and water-soluble metabolites, such as quinones, which are susceptible for more readily biodegradation than the parent compounds (Meulenberg et al. 1997). The economic feasibility of an enzymatic process is mostly influenced by the optimisation of the enzymatic reactor design and operation (Illanes and Wilson 2003), and particularly by the enzyme stability and its potential inactivation (Buchanan et al. 1998). Therefore, a comprehensive knowledge of enzyme kinetics is essential. Although Michaelis-Menten is the most widely used model to predict enzymatic reaction kinetics, in many processes enzymatic reactions do not follow the sequence of events, where free enzyme forms a single enzyme-substrate complex and further dissociates into free enzyme and product (Bailey and Ollis 1986; Segel 1993). That is the case of enzymatic 4 reactions carried out by MnP, where two different substrates are used during the catalytic cycle. Furthermore, enzyme deactivation may be significant and especially evident for an operation during an extended period of reaction, as may occur in the case of oxidation of barely biodegradable compounds by MnP (Eibes et al. 2005; Mielgo et al. 2003). The objective of this work is to study the feasibility of enzymatic treatment of poorly-soluble and recalcitrant compounds in monophasic reactors. Anthracene, a tricyclic PAH, was selected as a model compound and acetone was added at 36% (v:v), which increased 140-fold anthracene solubility in aqueous media. Acetone at concentrations below 10% does not substantially inhibit aerobic or anaerobic sludges (Eibes, 2007). Regarding the biodegradability, there are studies with several different strains bacteria (aerobic and anaerobic) which demonstrated that acetone is readily biodegradable (Platen and Schink 1989; Price et al. 1974). Different configurations of the reactor were also taken into account to determine the influence of the different factors affecting process kinetics. The work will also consider the development of a model describing substrate conversion, product generation and enzyme deactivation. Materials and methods Chemicals Anthracene and anthraquinone were obtained from Janssen Chimica (95-99% purity). Acetone was obtained from Panreac (chemical purity). H2O2 (30% v:v), sodium malonate and manganese sulphate were from Sigma-Aldrich. MnP production MnP was obtained from Bjerkandera sp. BOS55 (ATCC 90940). The fungus was grown in a 10-L fermenter BIOSTATE B. Braun-Biotech International (Melsungen, 5 Germany), on skimmed cheese whey medium (Feijoo et al. 1999; Moreira et al. 2001). Once the peak production of MnP was detected, the fermentation was stopped by decreasing temperature and the extracellular liquid was vacuum filtered. High molecular weight polysaccharides were precipitated by freeze-thawing and removed by filtration. Crude enzyme was concentrated by ultrafiltration using a 10 kDa cut-off type YM-10 membrane (Amicon), and was then centrifuged for 10 min at 20,000×g. Neither Lignin peroxidase nor Laccase activities were detected in any of the separated fractions during the procedure. Analytical techniques MnP activity was determined spectrophotometrically (Cecil CE 7200, UK) at 30ºC and 468 nm as described by Field et al (1992). The method is based on the oxidation of 2,6- dimethoxyphenol (2,6-DMP) by MnP system to form a quinone dimer. The molar extinction coefficient is 49600 M-1 cm-1. MnP assays were carried out with 200 µL sodium malonate (250 mM, pH 4.5), 50 μL 2,6-DMP (20 mM), 50 μL MnSO4 (20 mM), 50 μL of the sample whose MnP activity is to be determined and 550 μL water. The reaction was started by the addition of 100 μL H2O2 (4 mM), ending up with a volume of 1 mL. One unit of activity was considered as the amount of enzyme which oxidizes one µmol per min. Anthracene and anthraquinone were analyzed by a HP 1090 HPLC, equipped with a diode array detector monitoring the absorbance at 254 nm, a 4.6 x 200 mm Spherisorb ODS2 reverse phase column (5 μm; Waters) and a HP ChemStation data processor. The injection volume was set at 10 μL and the isocratic eluent (80% acetonitrile and 20% water) was pumped at a rate of 1 mL min-1. Fed-batch reactors Oxidation of anthracene was carried out in 100-mL Erlenmeyer flasks sealed with 6 Teflon plugs, with magnetic stirring at room temperature (about 22ºC). The reaction mixture (50 mL) consisted of acetone 36% (v:v), anthracene (28 μM), MnP (200 U L-1), Mn2+ (20 μM), malonic acid (20 mM) at pH 4.5 and anthraquinone (14 μM) when indicated. The reaction started with the continuous addition of 5 μmol L-1 min-1 of H2O2 supplied by a peristaltic pump running at 20 μL min-1. The dilution effect caused by H2O2 addition was considered to calculate accurate concentrations in the reactor. MnP (2.5 mL of enzymatic crude) was periodically added in the reactor. Samples were withdrawn at regular intervals to determine anthracene and anthraquinone concentrations by HPLC and the evolution of MnP activity was spectrophotometrically determined. Experiments were run in duplicate. To verify that degradation took place only due to 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). Semi-continuous reactor Oxidation of anthracene was carried out in 250-mL Erlenmeyer flasks sealed with Teflon plugs, with magnetic stirring at room temperature (about 22ºC). The volume of the reactor was 150 mL and the hydraulic retention time (HRT) was 11.5 h. All reagents were added continuously but for MnP, which was added once enzymatic activity reached zero in the reactor. Concentrations of the different compounds in the reactor at the beginning of the reaction were as follows: anthracene 28 μM; acetone 36% (v:v); sodium malonate 20 mM; Mn2+ 20 μM and MnP 200 U L-1. The process was initiated by the addition of two solutions: i) A solution containing anthracene 34 μM, acetone 42%, Mn2+ 23.4 μM and sodium malonate 23.4 mM (pH 4.5), was pumped at 10 mL h-1 using a high precision pump P-500 (Pharmacia) with Teflon tubes to avoid anthracene adsorption; and ii) A solution of H2O2: 5 μmol L-1·min-1 was pumped at 2 mL h-1 using 7 a Masterflex peristaltic pump (Cole Palmer). This H2O2 solution was stored in a cool box and periodically changed. During the time course of the experiments, MnP pulses were regularly added to restore enzyme concentrations to levels around 200 U L-1. A third pump was used for the discharge of the effluent. Samples were obtained directly from the Erlenmeyer flask. Continuous reactor Continuous oxidation of anthracene was carried out in a reactor working under identical conditions to those of semi-continuous reactor. In this case, three solutions were added: i) A solution containing anthracene 41 μM, acetone 52%, Mn2+ 28.9 μM and sodium malonate 28.9 mM (pH 4.5) was pumped at 9 mL h-1 using a high precision pump P-500 (Pharmacia) with Teflon tubes to avoid anthracene adsorption; ii) A solution of H2O2 5 μmol L-1·min-1 was pumped at 2.5 mL h-1 through a Masterflex peristaltic pump (Cole Palmer) and iii) A solution containing different MnP activities: 3350, 4630 and 7500 U L-1 was used to pump MnP at different addition rates: 36, 50 and 75 U L-1·h-1, respectively. MnP crude was stored in a cool box to minimise thermal inactivation. A fourth pump was used for the discharge of the effluent. Samples were obtained directly from the Erlenmeyer flask. Method of numerical integration A software package using an algorithm based on a Runge-Kutta formula (the Dormand- Prince pair) was used to solve the set of nonlinear ordinary differential equations. It is a one-step solver: in computing y(tn), it needs only the solution at the immediately preceding time point, y(tn-1). 8 Results and discussion Batch experiments: The kinetic model As previously stated, the oxidation mechanism of MnP is a multi-step process, comprising different reactions at different rates. Although the enzyme’s first substrate is usually considered the substrate of the reaction, the slowest step of the degradation will limit the apparent kinetics of the whole process. In the present work, the real oxidizing agent of anthracene is the Mn3+-malonate complex. The formation of this complex is followed by oxidation of MnP to MnPI by H2O2 and reduction by Mn2+ of MnPI and MnPII to the initial state (See catalytic cycle in Figure 1). An excess of H2O2 may lead to partial or total irreversible inactivation of the enzyme. The first oxidation, reductions to produce Mn3+ and formation of the malonate complex are rapid reactions, as shown by their kinetic constants (Wariishi et al. 1989; Wariishi et al. 1992). When the enzymatic reaction is faster than the oxidation of the final recalcitrant compounds, the latter reaction turns out to be the limiting step in the degradation process. Therefore, the overall kinetics will match up with the degradation kinetics of the final substrate, in this case, anthracene. A first experiment was carried out maintaining MnP activity in the range around 100 to 200 U L-1, because lower enzymatic activities have been shown to greatly limit degradation extent (Mielgo et al. 2003). Figure 2A shows the time course profile of an experiment performed during 7 h with enzyme pulses added after 1.5 h and 5.5 h. As a preliminary approach, first-order and Michaelis-Menten kinetics were considered; the former was taken into account due to the very low concentration of the final substrate (around 28 µM of anthracene). Significant discrepancies relative to experimental data are evident in Figure 2B, where experimental, first-order and Michaelis Menten degradation rates were plotted. The kinetics constants considered to 9 plot the models were those obtained from the integrated first-order equation (kcat=0.39 h-1 and r2=0.97) and the Eadie-Hotstee linearisation (rm=7.02 μM h-1, KM=8.47 μM and r2=0.62). In the case of first-order kinetics the decrease of oxidation rates would be very rapid during the first hours whereas for Michaelis-Menten kinetics lower degradation rates than first order would be observed. The experimental degradation rates followed a different pattern for the first 2 h, where a bell-shaped curve was observed. The first increase and further stabilisation of the degradation rate observed in the experiment could be explained by the presence of an activating agent, which may first increase degradation rates and subsequently balance the decrease of anthracene concentration. Quinones have been described to play a role as electron carriers in redox processes (Kudlich et al. 1997; Méndez-Paz et al. 2005; van der Zee et al. 2000). Considering that anthraquinone is the main metabolite produced in the anthracene degradation by MnP (Eibes et al. 2006), a possible explanation of the reaction behaviour could arise from the autocatalytic effect of the products formed, as described in Figure 3. Therefore a modified Michaelis-Menten equation including an auto-catalytic effect caused by the reaction products was considered as the model basis (equation 1): ( ·) m S M rS r a bP KS ⋅ =− ⋅+ + (eq 1) Considering that products refer not only to anthraquinone but also other intermediates formed during degradation (anthrone, dihydroxyanthrone), the rate of substrate formation in a batch experiment is given by: ( ) 00 · m M rS dS a bP S S dt K S ⋅ =− ⋅+ +−   + (eq 2) In order to validate this approach, an experiment where anthraquinone was added at the beginning of the reaction (14 μM), was carried out. Degradation rates were higher in this experiment, since 90% of anthracene had been degraded after 5 h whereas 16 Feijoo G, Moreira MT, Roca E, Lema JM. 1999. Use of cheese whey as a substrate to produce manganese peroxidase by Bjerkandera sp BOS55. J Ind Microbiol Biotechnol 23:86-90. Field JA, Baten H, Boelsma F, Rulkens WH. 1996. Biological elimination of polycyclic aromatic hydrocarbons in solvent extracts of polluted soil by the white rot fungus, Bjerkandera sp strain BOS55. Environ Technol 17:317-323. Field JA, Boelsma F, Baten H, Rulkens WH. 1995. Oxidation of anthracene in water/solvent mixtures by the whiterot fungus, Bjerkandera sp strain BOS55. Appl Microbiol Biotechnol 44:234-240. Field JA, de Jong E, Feijoo G, de Bont JAM. 1992. Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white-rot fungi. Appl Environ Microbiol 58:2219-2226. Illanes A, Wilson L. 2003. Enzyme reactor design under thermal inactivation. Crit Rev Biotechnol 23:61-93. Kudlich M, Keck A, Klein J, Stolz A. 1997. Localization of the enzyme system involved in anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 and effect of artificial redox mediators on the rate of azo dye reduction. Appl Environ Microbiol 63:3691-3694. Méndez-Paz D, Omil F, Lema JM. 2005. Anaerobic treatment of azo dye Acid Orange 7 under batch conditions. Enzyme Microb Technol 36:264-272. Meulenberg R, Rijnaarts HHM, Doddema HJ, Field JA. 1997. Partially oxidized polycyclic aromatic hydrocarbons show an increased bioavailability and biodegradability. FEMS Microbiol Lett 152:45-49. Mielgo I, López C, Moreira MT, Feijoo G, Lema JM. 2003. Oxidative degradation of azo dyes by manganese peroxidase under optimized conditions. Biotechnol Prog 19:325-331. Moreira MT, Palma C, Mielgo I, Feijoo G, Lema JM. 2001. In vitro degradation of a polymeric dye (Poly R-478) by manganese peroxidase. Biotechnol Bioeng 75:362-368. 17 Platen H, Schink B 1989. Anaerobic degradation of acetone and higher ketones via carboxylation by newly isolated denitrifying bacteria. J Gen Microbiol 135:883- 891. Price KS, Waggy GT, Conway RA 1974. Brine shrimp bioassay and seawater BOD of petrochemicals. J Water Pollut Control Fed 46:63-77. Reading NS, Aust SD. 2000. Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability. Biotechnol Prog 16:326-333. Segel IH. 1993. Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. New York: Wiley. 984 p. Sutherland GRJ, Aust SD 1996. The effects of calcium on the thermal stability and activity of manganese peroxidase. Arch Biochem Biophys 332:128-134. van der Zee FP, Lettinga G, Field JA. 2000. The role of (auto)catalysis in the mechanism of an anaerobic azo reduction. Water Sci Technol 42:301-308. Wariishi H, Dunford HB, MacDonald ID, Gold MH. 1989. Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism. J Biol Chem 264:3335-3340. Wariishi H, Valli K, Gold MH. 1992. Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium - kinetic mechanism and role of chelators. J Biol Chem 267:23688-23695. 18 Table I. Stationary values of the enzymatic function at the different stages of the continuous reactor operation Stage E* (U L-1) S* (μM) P† (μM) Y† (E) I 30 11.0 17.34 0.311 III 49 8.5 19.84 0.396 IV 76 4.0 24.34 0.753 * Experimental data (Figure 7) † Calculated from the set of equations 3 and 4 at steady state conditions 19 Table II. Values of inactivation decay constants in fed-batch reactors in absence (FB 1) and presence (FB 2) of external anthraquinone, continuous reactor (CR) and semicontinuous reactor (SCR) kd1 kd2 r2 FB 1 0.66 ± 0.17 0.42 ± 0.08 0.97 FB 2 0.86 ± 0.36 0.62 ± 0.11 0.99 CR 1.00 0.73 SCR 0.25 0.73 20 Table III. Values of kinetic parameters (modified Michaelis-Menten r2 0.998; enzymatic function r2 0.996) rm (μM h-1) kM (μM) a b (μM-1) c d e 13.30 10.99 0.572 0.009 1.07 54.42 -1.82 21 CAPTIONS TO FIGURES Figure 1 Catalytic cycle of MnP Figure 2 (A) Time course of anthracene (), anthraquinone (▲) and MnP enzymatic activity () during MnP fed-batch experiment (B) Experimental degradation rates () compared to those obtained using first-order (─ · ─) and Michaelis-Menten (─) models Figure 3 Autocatalytic mechanism of the anthracene oxidation by MnP Figure 4 Modelling of anthracene using Michaelis-Menten and autocatalytic kinetics in the MnP fed-batch reactors in absence (□) and in presence of anthraquinone (○) in the initiation of the reaction. Kinetic parameters: rm: 13.574 μM h-1; KM=14.72 μM; a=0.549; b=0.008 μM-1 (r2=0.995) Figure 5 Sequence scheme for obtaining kinetic parameters Figure 6 Fitted (—) and experimental data of anthracene () and MnP enzymatic activity () at different addition rates of MnP: I) 36 U L-1 h-1, II) no addition of MnP, III) 50 U L-1 h-1 and IV) 75 U L-1 h-1 Figure 7 Function defining the effect of enzymatic activity on the degradation of anthracene (eq. 6). Kinetic parameters are shown in Table III Figure 8 Modelling of first-order inactivation of enzymatic activity () and anthracene degradation (○) using Michaelis-Menten, autocatalytic kinetics and an enzymatic function in the MnP fed-batch reactors in absence (A) and in presence of external anthraquinone (B). Kinetic parameters are shown in Table III Figure 9 Fitted (—) and experimental data of: A) anthracene disappearance () and B) MnP enzymatic activity () during semi-continuous experiment 22 Figure 1 MnP MnP I H 2 O 2 H 2 O MnP II Mn2+ Mn3+ Mn2+ Mn3+ MnP III (inactive) H2O2 H2O Anthracene Oxidised products Anthracene Oxidised products 23 0 5 10 15 20 25 30 02468 Anthracene (µM) Anthraquinone (µM) 0 50 100 150 200 250 MnP activity (U/L) 0 2 4 6 8 10 0 2 4 6 8 time (h) r (µM/h) Figure 2 A B 24 Figure 3 Mn+3 Mn+2 QUINONES Anthracene Oxidized products MnPII MnP MnPI MnPII 25 0 5 10 15 20 25 30 012345678 time (h) Anthracene (µM) Figure 4