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Author's personal copyA.I. Rodarte-Morales et al. / Biochemical Engineering Journal 66 (2012) 38–45 39restricts the growth of the fungus to the external and inner surfaceof the support. However, the use of a support for the degradationof certain compounds has to take into consideration the potentialadsorption of these compounds onto the support surface, whichmay render for limited biodegradation and the need of an extractionstage to desorb them.The aim of this study was to assess which is the best type ofculture as free or immobilized mycelia as well as the bioreactorconfiguration: STR and FBR to carry out the continuous degradationof five pharmaceutical compounds belonging to different therapeuticgroups: anti-inflammatory drugs (diclofenac, ibuprofenand naproxen), anti-epileptics (carbamazepine) and tranquilizers(diazepam). Furthermore, the influence of gas supply either as acontinuous flow of air or as pulses of oxygen was evaluated in theoperation of the FBR. Additionally, the identification of the majordegradation products of the three anti-inflammatory drugs wasconducted.2. Materials and methods2.1. Microorganism and pre-inoculum preparationThe white-rot fungus Phanerochaete chrysosporium (ATTC24725) was obtained from the culture collection of the Universityof Santiago de Compostela (Spain). The pre-inoculum was culturedin static using Fernsbach flasks (1 L) with 100 mL of modified Kirkmedium [21] and 6 plugs of malt extract agar (glucose, 10 g/L;agar, 15 g/L; malt extract, 3.5 g/L) with active fungus. Concerningthe fungal immobilization, the support consisted of polyurethanefoam cubes (volume, 0.5 cm 3 ; density, 20 kg/m 3 ; superficial area,414 ± 10 m 2 /m 3 ) obtained from Copo Ibérica (Vigo, Spain) [11].A support/liquid volume ratio of 0.015 g/mL was used. From thehomogenized mycelium, 9 mL were added to Erlenmeyer flasks(250 mL) containing 90 mL of modified Kirk culture medium inorder to form pellets or immobilize the fungus. Flasks were incubatedat 30 ◦ C and agitated at 150 rpm in an orbital shaker (C24Incubator Shaker, New Brunswick Scientific, USA) for 3–5 days.Grown cultures of pellets or immobilized fungus were withdrawnby filtration using sterile gauze and used as inoculum for the bioreactorsin a proportion of 10%.2.2. Pharmaceutical compounds and chemicalsThe target compounds considered were diclofenac (DCF),ibuprofen (IBP), naproxen (NPX) and carbamazepine (CBZ), all ofthem purchased from Sigma–Aldrich as pure grade. Diazepam(DZP) from Roche Pharma as pure grade was used. A stock solutionprepared at three different concentrations: 1 mg/L (DCF, IBPand NPX), 0.5 mg/L (CBZ) and 0.25 mg/L (DZP) was used. A range ofsolvents were used for the extractions and for the preparation ofthe pharmaceutical mixtures: acetone, ethyl acetate, acetonitrile,methanol and n-hexane; all of them purchased from J.T. Baker (allof them >99.5% pure).2.3. Comparison of a continuous stirred tank reactor (STR) usingP. chrysosporium pellets and immobilized fungus for thedegradation of DCF, IBP, NPX, CBZ and DZPDegradation experiments were performed in a 2-L stirred tankfermenter Biostat B plus (Sartorius, Melsungen, Germany). Theculture vessel consisted of a jacketed glass vessel designed fortemperature control via a thermostated system. Dissolved oxygenconcentration and pH were continuously monitored using pO 2 andpH electrodes and data were processed by software MFCS/DA 3.0(Module operator service program, Sartorius sedimentation Systems,Germany). The bioreactor vessel was sterilized and filledwith 1.5 L of modified Kirk medium (pH 4.5) previously sterilized[21] and pellets or immobilized fungus from Erlenmeyer flasks. Thecontinuous operation was conducted for 50 days with a hydraulicresidence time (HRT) of 24 h, feeding addition rates of glucose(125–300 mg/L h), ammonium tartrate (0.6–2.08 mg/L h) and continuousair flow (0.5–3 L/min) to maintain significant levels ofdissolved oxygen and temperature of 30 ◦ C. The feed medium wassterilized and spiked with the pharmaceutical compounds at differentconcentrations: 1 mg/L (DCF, IBP, NPX), 0.5 mg/L (CBZ) and0.25–0.5 mg/L (DZP) were added in the upper port of the reactorvessel while the effluent was withdrawn from the bottom of thereactor. Samples were withdrawn daily for monitoring the mainoperational variables and twice per week to determine the residualconcentration of the pharmaceutical compounds.2.4. Operation of a continuous fixed-bed reactor (FBR) using P.chrysosporium immobilized in polyurethane foam for thedegradation of DCF, IBP, NPX, CBZ and DZPThe continuous operation of the FBR was conducted for 100 dayswith a 24 h HRT, feeding addition rates of glucose (125–250 mg/L h)and ammonium tartrate (0.6–2.08 mg/L h) and temperature of30 ◦ C. The bioreactor configuration consisted of a glass jacketedcolumn with an internal diameter of 4.5 cm and a height of 20 cmwith two gas supply systems: continuous air flow (1 L/min) andpulsation of oxygen by means of a pulsing device consisting ofan electrovalve located at the end of a flexible membrane tube(oxygen pulsation frequency of 0.062 s −1 ) [22]. Dissolved oxygenwas measured off-line by means of an electrode (Oxi 340i fromWissenschaftlich Technische Werk, Germany). Samples were withdrawnfrom the middle (every 10 days) and upper ports (twice perweek) of the reactor.2.5. Adsorption of the pharmaceutical compounds on the fungalbiomass and on the polyurethane foam supportAt the end of the operation of the STRs, a final extraction wascarried out to determine the concentration of pharmaceuticalsthat could be adsorbed on the biomass and/or the support. Forthis purpose, 50 mL of blended mycelium (for pellets) or immobilizedfungus was placed in an Erlenmeyer flask, supplementedwith 50 mL of acetonitrile and agitated for 2 h at 150 rpm. In thecase of the immobilized fungus from the FBR, samples were takenfrom the upper, the middle and the lower section of the column.After the agitation of the samples with acetonitrile, 4 mL of eachsample were withdrawn and diluted in 100 mL of distilled water,to proceed with the solid phase extraction (SPE), stage requiredprior to gas chromatography-mass spectrometry.2.6. Determination of the concentration of residualpharmaceutical compounds by gas chromatography-massspectrometry (GC–MS)Residual concentrations of DCF, IBP, NPX, CBZ and DZP weremeasured by gas chromatography-mass spectrometry (GC–MS).Prior to determination, a solid phase extraction (SPE) of the sampleswas carried out with 60 mg OASIS HLB cartridges (Waters closet,Milford, MA, USA) previously supplemented with 3 mL of ethylacetate, 3 mL of methanol and 3 mL of distilled water (pH 2) to allowthe determination of the soluble fraction of these compounds [23].2.7. Identification of degradation productsThe identification of the major degradation products ofDCF, IBP and NPX was performed by GC–MS, using N-methyl-N-(tert.-butyldimehtylsilyl)trifluoroacetamide (MTBSTFA) as the
Author's personal copy40 A.I. Rodarte-Morales et al. / Biochemical Engineering Journal 66 (2012) 38–45derivatized agent. From the derivatized structures of each antiinflammatorydrug as well as their metabolites, these moleculeswere identified to suffer a fragmentation process. Then, the GC–MSchromatograms were analyzed in order to find those peaks withsimilar mass spectrum (MS) to the ones expected after the fragmentationof the parent compound.2.8. Analytical techniquesThe concentration of glucose was analyzed with the dinitrosalicylicacid (DNS) method using D-glucose as a standard [24]. Totalorganic carbon (TOC) concentration was measured in a Shimadzuanalyzer (TOC 5000). The concentration of total nitrogen (NT) wasdetermined using a Dohrmann nitrogen analyzer. The pH value wasmeasured using a pH meter GLP 21 from CRISON (Barcelona, Spain).Peroxide concentration was measured by a colorimetric methodwith Peroxide-Test strips (Merck, Darmstadt, Germany). Biomassconcentration was determined as dry weight with 0.45 m poresizefilters. Enzymatic activity of manganese peroxidase (MnP) wasmeasured by the oxidation of dimethoxyphenol (DMP) in a spectrophotometer(Shimadzu UV-1603) as described by Field et al. [7].3. Results3.1. Degradation of DCF, IBP, NPX, CBZ and DZP by free pellets ofP. chrysosporium in a STRThe removal results of the three anti-inflammatories by fungalpellets of P. chrysosporium in the STR are shown in Figs. 1–3. DCF andIBP were efficiently degraded during the experiment; only limitedremoval percentages were detected at the start of the bioreactoroperation (Figs. 1a and 2a). Removal of NPX was negligible duringthe first days of experiment and steadily increased up to maximumvalues of 92% (Fig. 3a). The removal of CBZ, however, was negligibleduring the first two weeks of the experiment (Fig. 4a). From day 20to day 50 the removal of CBZ ranged from 24% to 63%. Finally, itwas not possible to detect DZP during the first days of the experiment,which may have been due to an analytical error. Therefore,the feeding rate of this compound was increased to 0.6 mg/L d up today 40; thereafter, the feeding rate was reduced to 0.2 mg/L d. Thisstrategy did not improve the degradation of DZP, but at least thecompound was detected by GC–MS (data not shown). The quantificationof the adsorption of the pharmaceutical compounds ontothe fungal biomass indicated that the three anti-inflammatorieswere hardly adsorbed on the biomass while DZP showed the highestconcentration per gram of biomass. The analysis of the potentialadsorption on the vessel surface reported negligible concentrationsfor all compounds (Table 1).The main parameters of the fermentation profile of the fungalbioreactor showed constant glucose consumption rate (Fig. 6a), dissolvedoxygen concentration between 2 and 8 mg O 2 /L (Fig. 7a),constant pH and low MnP activity (data not shown). On day28, approximately 500 mL of medium (including pellets and freemycelium) were removed from the STR and replaced with freshmedium due to an excessive mycelium growth. Biomass content atthis time of operation was 7.3 g/L, similar to the concentration atthe end of the experiment (8 g/L). Pellets showed a slight formationof hyphal branches (9.3 mm) up to day 10; thereafter, fragments offungal mycelia formed pellets of smaller size: 7 mm (days 30 and50).3.2. Degradation of DCF, IBP, NPX, CBZ and DZP by immobilized P.chrysosporium in a STRIn accordance to the previous experiment, DCF and IBP wereefficiently degraded, with removal percentages higher than 93%for both compounds (Figs. 1b and 2b). A high removal yield forNPX was detected during the first 3 days (higher than 90%); however,a decrease in the efficiency of elimination until percentagesbetween 65% and 77% was observed for the rest of the operation(Fig. 3b). Regarding the removal of the antiepileptic and the tranquilizer,these drugs, characterized by their recalcitrant behavior,were only partially removed with values up to 50% during certainperiods (Figs. 4b and 5b). Similar fermentation profiles to those ofthe free pellets reactor were found in this experiment (Figs. 6b and7b); however, a significant lower concentration of biomass (2 g/L)was measured at the end of the experiment. The determinationof the residual concentration of the pharmaceutical compoundsadsorbed onto the polyurethane foam was conducted (Table 1). Aremarkably high content of all the compounds was found in thesupport. Diclofenac showed the highest amount (15.9 mg of drugper gram of polyurethane foam with biomass) followed by NPX. IBP,on the contrary, was the one with the lowest amount adsorbed.3.3. Degradation of DCF, IBP, NPX, CBZ and DZP by immobilized P.chrysosporium in a FBR using air and oxygen supplyThe removal of the target compounds by immobilized fungusin a FBR is shown in Figs. 1–5. DCF, IBP and NPX were largelyremoved throughout the experiment under both aeration conditions(Figs. 1–3); while CBZ and DZP presented partial degradation(Figs. 4 and 5). The measurement of the concentration of CBZ inthe middle and upper ports evidenced gradients of concentrationthrough the reactor column (Fig. 4c). Comparing the results fromthe experiments with different gas supply, lower amounts of DCF,IBP and CBZ were detected in the FBR with oxygen pulsation whilenegligible effect of the gas supply was found for the removal of DZP.In contrast, NPX was detected in higher amounts in the oxygen reactor(Table 1). Low concentrations of the considered pharmaceuticalcompounds (between 0.005 and 0.125 g per gram of biomass) weredetected in the polyurethane foam of the FBR at the end of theexperiment.P. chrysosporium was able to consume most of the glucose addedin the air FBR (Fig. 5c). In contrast, almost negligible values of glucosewere detected after 36 days of operation in the oxygen FBR.Hence, feed was added also through the medium port (Fig. 5d).Enzymatic activity was detected below 30 U/L in both FBRs (airand oxygen). Dissolved oxygen concentration was maintained ina range between 6 and 8 mg O 2 /L when air was the gas supply(Fig. 7c) and 15 and 23 mg O 2 /L when oxygen was supplied (Fig. 7d).The final concentration of biomass in the air and oxygen FBRs was11.3 g/L in the air reactor and 6.4 g/L in the oxygen reactor. Preferentialpathways due to the fungal growth on the support surfacewere observed in the FBR after two weeks. A slight darkening of thesupport through the column was observed on day 35 in the air reactor;meanwhile a complete darkening was observed on day 20 inthe oxygen reactor, possibly due to the precipitation of manganeseoxides.3.4. Identification of the major degradation productsThe identification of the main degradation products of theanti-inflammatory drugs was conducted using the GC–MS chromatogramsobtained during the FBRs experiments. When thesedrugs are derivatizated, the hydroxyl group from each moleculereacts with the MTBSTFA resulting in the loss of a hydrogenatom and the formation of methyl-tertbutyl-sylil derivatives [17].According to literature, the main degradation products of DCF, IBPand NPX are 4-hydroxy-DCF, 1-hydroxy-IBP and 6-O-desmethyl-NPX [25–27]. The estimation of the amount of these compoundswas carried out from the relative area of the peaks correspondingto the degradation products on days 20, 40, 68 and 100 and the
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