J. Microbiol. Biotechnol. (2015), 25(6), 803–813 http://dx.doi.org/10.4014/jmb.1410.10054 Research Article Review jmb

Fungal Growth and Manganese Production in a Deep Tray Solid-State Bioreactor, and In Vitro Decolorization of Poly R-478 by MnP Xinshan Zhao, Xianjun Huang, JuntaoYao, Yue Zhou, and Rong Jia*

College of Life Science, Anhui University, Hefei 230601, P.R. China

Received: October 23, 2014 Revised: December 11, 2014 The growth of Irpex lacteus F17 and manganese peroxidase (MnP) production in a self- Accepted: December 13, 2014 designed tray bioreactor, operating in solid-state conditions at a laboratory scale, were studied. The bioreactor was divided into three layers by three perforated trays. Agro- industrial residues were used both as the carrier of bound mycelia and as a nutrient medium

First published online for the growth of I. lacteus F17. The maximum biomass production in the bioreactor was December 24, 2014 detected at 60 h of fermentation, which was consistent with the CO2 releasing rate by the

*Corresponding author fungus. During the stationary phase of fungal growth, the maximum MnP activity was Phone: +86-551-6386-1005; observed, reaching 950 U/l at 84 h. Scanning electron microscopy images clearly showed the Fax: +86-551-6386-1281; growth situation of mycelia on the support matrix. Furthermore, the MnP produced by I. E-mail: [email protected] lacteus F17 in the bioreactor was isolated and purified, and the internal peptide sequences were also identified with mass spectrometry. The optimal activity of the was detected at pH 7 and 25°C, with a long half-life time of 9 days. In addition, the MnP exhibited significant stability within a broad pH range of 4-7 and at temperature up to 55°C. Besides this, the MnP showed the ability to decolorize the polymeric model dye Poly R-478 in vitro. pISSN 1017-7825, eISSN 1738-8872

Copyright© 2015 by Keywords: Irpex lacteus F17, tray bioreactor, solid-state fermentation, manganese peroxidase, The Korean Society for Microbiology polymeric dye Poly R-478, decolorization and Biotechnology

Introduction white-rot fungi, including chrysosporium, Trametes versicolor, Pleurotus ostreatus, Bjerkandera sp. BOS55, and White-rot fungi produce extracellular ligninolytic , others, have been reported to produce MnP [10]. such as manganese peroxidase (MnP, E.C. 1.11.1.13), Owing to the potential applications of MnP in treating peroxidase (LiP, E.C. 1.11.1.14), and laccase (Lac, E.C. environmental pollutants, there is general interest in 1.10.3.2) that can efficiently degrade lignin as well as many producing the enzyme biotechnologically. However, large- recalcitrant organic pollutants. Among these enzymes, MnP scale enzyme production from white-rot fungi is limited is one of the most frequently used for the transformation of mainly owing to the lack of suitable bioreactor configurations environmental pollutants, such as synthetic dye decolorization for fungal growth and their metabolism [27]. Although as well as polycyclic aromatic hydrocarbons and tetracycline several studies on the development of innovative bioreactor degradation [7, 11, 33]. MnP is a glycosylated - systems have already been carried out [7, 25, 32], an ideal containing enzyme and can catalyze the oxidation of Mn2+ bioreactor for ligninolytic enzyme production from white-rot 3+ 3+ to Mn in a H2O2-dependent reaction. Mn is stabilized by fungi has not yet been shown [6, 8]. Furthermore, the high organic , and the Mn3+-acid complex acting as the production cost is another barrier to the commercialization active mediator can attack phenolic lignin structures, of these enzymes. resulting in the formation of unstable free radicals that Solid-state fermentation (SSF), which is carried out without tend to disintegrate spontaneously. A large number of the use of free-flowing water, is the most economic process

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for ligninolytic enzyme production owing to the use of 121°C for 20 min. The grown mycelial mat collected from several agro-industrial residues as the nutrient medium for white- slants was washed with sterile water and homogenized by using a rot fungi. This solid matrix can also be employed as a Waring blender. Then, 500 ml flasks containing 150 ml of liquid medium (potato extract 200 g/l, glucose 20 g/l, KH PO 3.0 g/l, natural support for the development of filamentous fungi 2 4 and MgSO ·7H O 1.5 g/l) were inoculated with 20 ml of the in SSF. In addition, these agro-industrial residues are rich 4 2 mycelial suspension and incubated at 28°C on a rotary shaker at in lignin or cellulose, which are inducers for ligninolytic 120 rpm for 3 days for the formation of mycelial pellets. enzyme production, thus stimulating much higher enzyme The homogenized mycelia obtained by crushing the mycelial production in SSF processes. In fact, SSF has become an pellets in a Waring blender were inoculated into a 7.23 L bioreactor alternative large-scale production system for enzymes and containing 300 g of solid and 300 ml of water (the water secondary metabolites [9, 30]. holding capacity was 1 ml/g (1 ml water per gram dry medium)). I. lacteus F17 (CCTCC AF 2014020) — a local white-rot Fermentations were performed at 28°C for 7 days. basidiomycete isolated by our laboratory — has been found to be a producer of MnP. The fungus I. lacteus has Bioreactor Configuration and Operating Conditions been reported to be very efficient in degrading a wide The designed tray bioreactor used in this study contains a variety of recalcitrant organic pollutants, including various cylindrical glass vessel with 16.0 cm diameter, 36.0 cm height, and types of dyes and polycyclic aromatic hydrocarbons (PAH) 7 L working volume (Fig. 1). The outer frame of the bioreactor and [2, 13, 19, 26, 28]. MnP proved to be the main enzyme the air-intake casing were made of glass. The bioreactor was vertically divided into three layers: top (10 cm height), middle (10 cm during the decolorization and PAH degradation by I. lacteus [2, 13, 29]. These features suggest this organism to be a good candidate for further applications at a larger scale and make it an interesting objective to scale up ligninolytic enzymes production. In the present study, a laboratory-scale bioreactor was designed to examine the feasibility of utilizing agro-industrial residues as the nutrient and support for the growth of the white-rot fungus I. lacteus F17, as well as for scale-up of MnP production. Furthermore, the MnP produced by I. lacteus F17 in the bioreactor was purified and partially characterized to gain more information on its stability for application. Finally, the ability of the MnP to degrade aromatic pollutants was also assessed by monitoring the decolorization of the polymeric model dye Poly R-478.

Materials and Methods

Microorganism and Growth Medium I. lacteus F17 was isolated from a decayed wood chip pile in the vicinity of Hefei, China, and was stored in the China Center for Type Culture Collection (CCTCC AF 2014020). The fungus was cultured on potato dextrose agar (PDA) slants for 7 days at 28°C and then preserved at 4°C. The PDA medium contains 200 g/l of potato extract, 20 g/l of glucose, and 20 g/l of agar.

Solid-State Fermentation SSF substrate preparation was based on results of our previous research. Dried, ground, and sifted pine sawdust, rice straw, and Fig. 1. The solid-state deep tray bioreactor. soybean powder mixed at the ratio of 0.52:0.15:0.33 in weight were (A) Schematic diagram of external circulation of air. 1, power; 2, air used both as an attachment place and to supply nutrients to the pump; 3, air filter; 4, NaOH solution; 5, bioreactor. (B) Schematic I. lacteus F17. diagram of the right and front views of the solid-state reactor. 6, gas Prior to use, these solid materials were mixed and autoclaved at exhaust; 7, cover; 8, air inlet; 9, sampling port.

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height), and bottom (10 cm height). Each layer was composed of a phosphate buffer (pH 7.4) with 5% (v/v) glutaraldehyde for 2 h at removable perforated tray, where 100 g initial dry weight of the 4°C. Then, the samples were dehydrated sequentially using 20%, mixed solid matrix was placed, forming a layer of about 3.5 cm 40%, 60%, 80%, and 100% ethanol [10]. The samples were coated thickness, respectively. Each perforated tray, made of stainless with gold before examination under a scanning electron microscope steel, had 34 circular holes with a diameter of 0.5 mm. The 10 cm (Hitachi S-4800 SEM, Japan). The substrate without fungus was height between each tray in the deep-bed bioreactor was designed considered as the blank. All the images were taken at a magnification for better temperature control and metabolic heat removal. Three of 500×. sampling ports (inner diameter 2.4cm) were arranged above each tray. The bioreactor had two air inlets and a gas exhaust from the Enzyme Activity Assays outside to ensure effective circulation of air. The air pumped into To obtain enzyme extracts, 5 ml/g dry matrix of 10 mM sodium the bioreactor was separated through five vents at each layer, acetate buffer (pH 4.5) was added to the sample in an Erlenmeyer which kept the atmosphere near saturation and uniform distribution. flask, and the flask was incubated on a rotary shaker at 120 rpm A ground glass covered the top of the bioreactor, and three rubber for 30 min. Then, the supernatants were used for enzyme activity plugs were used to seal the sampling ports. A constant flow assays. ventilation was achieved in the bioreactor through an air pump The MnP activity in the supernatant was measured with Mn2+ in that moved the air through a solution of NaOH, which absorbed 0.11 M sodium lactate buffer. Oxidation of Mn2+ to Mn3+ was

CO2. During the process of SSF, the entire bioreactor was kept determined by the increase in absorbance at 240 nm [14]. One unit inside a constant-temperature incubator maintained at 28°C, (U) of MnP activity was defined as the oxidization of 1 µmol of and humidified air was supplied continuously at airflow rate of Mn2+ per minute. The enzyme activities were expressed in U/l. 0.4l/min. The samples were removed from three sampling ports The (LiP) assay was based on the oxidation of at regular intervals of 12 or 24h and were analyzed for enzyme veratryl alcohol to veratraldehyde, determined by the increase in activity and biomass. The experiments were conducted in triplicate absorbance at 310nm [12]. One unit (U) of LiP activity was and the results described are the mean of three replicates. defined as the oxidation of 1 µmol of veratryl alcohol per minute. The laccase (Lac) assay was based on the oxidation of ABTS, Biomass Analysis determined by the increase in absorbance at 420 nm [35]. One unit In the present study, the biomass produced during SSF was (U) of Lac activity was defined as the oxidation of 1 µmol of ABTS quantitatively analyzed each day by measuring the nucleic per minute. All the experiments were performed in triplicate, and present in the fermentation medium [20]. Samples of the medium all the enzyme activity assays were carried out at 25°C. (0.4g) from each layer were collected every 24h, except that the first sample was taken at 12 h after the start of SSF. After Enzyme Purification pulverizing the sample in a white porcelain mortar, 25 ml of 5% In order to investigate the enzyme production by I. lacteus F17 trichloroacetic acid was added and the mixture was constantly during SSF in the bioreactor, enzyme activities were analyzed stirred for 25 min at 80°C. Subsequently, the mixture was cooled daily. The enzyme extracts containing the highest MnP activity in an external ice bath. After centrifugation of the mixture (for were concentrated by ammonium sulfate (70%) and dialyzed 15 min at 5,653 ×g and 4°C), the absorbance of the supernatant at against 10 mM of sodium acetate buffer (pH 4.5). After this, the 260 nm was measured, and the medium without inoculation crude enzyme was loaded onto a DEAE-cellulose column (Whatman through the same treating process was used as the blank control. DE52, England) (2.6 × 30 cm), which had been equilibrated with Different amounts of mycelia were weighted precisely and dealt 10 mM of sodium acetate buffer (pH 4.5) at 4°C. Elution of the with the method as mentioned above. The linear relationship was achieved with a flow rate of 100 ml/h by establishing between the weight of mycelia and the absorbance of the a linear gradient first with 10 mM of sodium acetate buffer supernatant at 260 nm was calculated. The biomass was expressed (pH 4.5) and then with 0–0.5 M of NaCl at 4°C. Fractions with as grams per gram matrix. Furthermore, CO2 emission during SSF MnP activity were pooled, concentrated by a Stirred Ultra was also tested by carrying out acid–base titration at every 12 h to filtration Cell Model 8010 system (10 kDa cut-off polyethersulfone analyze the fungal growth condition. All the experiments were membranes; Millipore Corporation, USA) to approx. 1 ml, and performed in triplicate, and the results were expressed as the then loaded to a Sephadex G-75 gel filtration column (Fluka, USA) mean values. Deviation values are standard deviations based on (1.7 × 100 cm). Proteins from this column were eluted with 10 mM triplicate determinations. of sodium acetate buffer (pH 4.5) at a flow rate of 20 ml/h and temperature of 4°C. The peak containing MnP was collected, Scanning Electron Microscopy concentrated, and then applied to FPLC (FPLC with Superdex 75 The status of I. lacteus F17 mycelium growth in the tray 10/300 column (Pharmacia)), which was previously equilibrated bioreactor was observed by scanning electron microscopy (SEM). with 10 mM of sodium acetate buffer (pH 4.5). Elution of proteins The mycelium samples collected from the middle layer of the from this column was detected by continuous recordings of bioreactor at different fermentation periods were fixed in 0.1 M absorbance at 280 and 406 nm. The enzyme solution was stored at

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4°C for the characterization and decolorization experiments. The decolorization efficiency was calculated according to the following Eq. (1): Enzyme Characterization A –A Enzyme electrophoresis and mass spectrometry. Electrophoresis Decolorization () % = ------0 -t ×100% (1) A0 on polyacrylamide gel in denatured conditions (SDS-PAGE) was carried out using a 10% Tris/HCl separation gel and 3% Tris/HCl where A0 is the initial absorbance at 520 nm and At refers to the stacking gel. After SDS-PAGE, the gels were stained with Coomassie absorbance at 520 nm at reaction time t. The data were the mean Brilliant Blue R-250. values of triplicates. The was excised from the gel and digested with trypsin. Then the resulting peptide mixture was analyzed by LC/MS (liquid Results chromatography-mass spectrometry) with a mass spectrometer, ProteomeX-LTQ (ThermoFisher Scientific, USA). All complete or Production of Biomass in the Deep Tray Bioreactor partial sequences of this protein were searched against those of the In SSF, the mycelium cannot be directly quantified proteins from BLAST similarity search to obtain the identifications because of its strong adherence to the solid matrix; hence, with higher confidence. indirect methods such as analyses of ATP, enzymatic activity, respiration rate, nutrient consumption, and cell Determination of Kinetic Parameters of MnP constituents are employed to evaluate the biomass. Nucleic The K and k values of MnP were calculated by the m cat acids evolution could be used as an indirect measurement hyperbolic non-linear least-square method, using MnSO as the 4 of the growth of the white-rot fungus. The biomass substrate at varying concentrations (0.025-0.2 mM). The reactions production profile of I. lacteus F17 in the tray bioreactor is were carried out in 0.11 M of sodium lactate buffer (pH 4.5) with depicted in Fig. 2A. In SSF using agricultural waste as the 0.1 mM of H2O2. substrate, the biomass of the fungus increased rapidly in Effects of Temperature and pH on MnP Activity and Stability the bioreactor at the beginning of fermentation, peaked at Determination of the optimum temperature for MnP activity 60 h, and then reached a plateau until 156 h. The maximum was carried out in 0.11 M of sodium lactate buffer over temperatures biomass production at the top, middle, and bottom layers from 15°C to 65°C at pH 4.5. To evaluate the thermal stability of of the bioreactor was 0.25, 0.23, and 0.24 g per gram matrix, the purified MnP, the enzyme was incubated at different respectively. The rate of CO2 released by I. lacteus F17 temperatures ranging from 25°C to 65°C for 1-3 days and aliquots under SSF is shown in Fig. 2A, which indicated the metabolic of samples were removed at every 24 h for analysis. activity of the fungus in the tray bioreactor. The release rate The effect of pH on MnP was determined at 25°C using citrate– rapidly increased for 36 h, reached to the highest value at phosphate buffer with a pH range of 2.2–8. The pH stability was 60 h, and then reduced significantly. When compared with assayed by incubating the enzyme at different pH values (pH 3–7, biomass production, the fungus grew rapidly by consuming citrate–phosphate buffer) at 25°C for 0-35 h. The remaining activities carbon sources at the beginning of the 60 h fermentation, were measured using the enzyme activity assays mentioned above. leading to high rate of CO2 release. After 60 h, the biomass production remained almost constant and the CO release In Vitro Decolorization of Poly R-478 by MnP 2 rate decreased, indicating that the respiratory metabolism Dye decolorization was measured spectrophotometrically at 520 nm (WFV UV-2100 Spectrophotometer; Shanghai, China), of fungus was becoming increasingly stressed. which is the maximum visible absorbance of the dye Poly R-478. The reaction mixture contained citrate–phosphate buffer (62.8 mM), Production of MnP in the Deep Tray Bioreactor

MnSO4 (1mM), H2O2 (0.2 mM), Poly R-478 dye (0.001%), and Fig. 2B presents the MnP production by I. lacteus F17 200 µl of the purified MnP or 100 µl of crude MnP in a total during SSF in tray bioreactor. MnP appeared after 36 h of volume of 1 ml. The MnP activity employed was 900 U/l. The fermentation, and then started to increase rapidly to a high reaction was initiated by the addition of H2O2 and the mixture was level. The activity of MnP was approx 950 U/l, with maximum incubated at different temperatures and pH conditions for 1 h. activity observed at the bottom layer of the bed at 84 h, The decolorization of Poly R-478 represents the percentage decrease along with LiP and Lac activities of about 31 U/l and 17 U/l, in the peak of absorbance at 520 nm. Control samples, without respectively. After that, the MnP activity decreased drastically enzyme or H O , were done in parallel under identical conditions. 2 2 until the end of cultivation. When compared with the The optimal temperature and pH conditions for dye decolorization biomass, the MnP activity started to increase and gradually were carried out in vitro with purified MnP and crude enzyme preparations. reached the maximum value while the biomass remained

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Fig. 3. SEM of the carriers in the bioreactor at different times. (A) Substrate without fungus at a magnification of 500×. (B) Samples after 24 h of inoculation at a magnification of 500×, exhibiting the beginning of mycelium production. (C) Micrograph showing lush growth of mycelium after 48 h of inoculation at a magnification of 500×. (D) Micrograph showing mycelium closely entangled in the support matrix after 60 h of inoculation.

environment for the fungus.

Fermentation Kinetics Model Fitting Fig. 2. Tray bioreactor data determined at different times. A logistic model can be used as the mathematical model (A) The total CO release rate and biomass at the three levels of the 2 to reveal the fungal growth condition [7, 36], which can be tray bioreactor measured from 12 to 156 h of SSF. (B) The MnP defined as follows: activity at the three levels of the bioreactor measured every 12 h of SSF. Deviation values are standard deviations based on triplicate N N = ------m - (2) determinations. t –µt 1N+ ()m ⁄ N0 – 1 exp The Luedeking–Piret equation has also been used for the stable, indicating the production of a secondary metabolite kinetic analysis of extracellular enzyme production. According by the fungus when its growth rate was zero. to this model, the formation rate depends on both the instantaneous biomass concentration N and the growth SEM Observation rate dN/dt: The morphology of I. lacteus F17 grown on support matrix was examined by SEM. Fig. 3 shows the SEM ------dP = α------dN- + βN (3) dt dt images of the mycelium growth status after 0, 24, 48, and 60 h of SSF. The mycelia could be easily observed on the By substituting Eq. (2) into Eq. (3) and integrating it, the surface of the medium mixed with pine sawdust, rice following equation could be obtained: straw, and soybean powder. They were densely entangled expµt in the support matrix after 60 h of SSF (Fig. 3D). These P = N ------α –------α + --β-ln⎛⎞ 1+ ------t m –µt ⎝⎠ ()N ⁄N –1 exp +1 Nm ⁄N0 µ Nm ⁄N0 –1 results were consistent with the data regarding I. lacteus m 0 (4) F17 biomass production in the bioreactor, indicating that the local tray bioreactor provided an appropriate growth where Nt is the biomass synthesized during the time

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interval considered (g per gram matrix), N0 is the initial biomass of the inoculum (g per gram matrix), Nm is the maximum biomass during fermentation (g per gram matrix), µ is the maximum specific growth rate (h−1), t is the differential time interval (h), α is the growth-associated product formation, and β is the non-growth-associated product formation coefficient and other variables. The average biomass and MnP production evaluated at the three layers can be used as a parameter to measure the ability of the entire bioreactor to enhance fungal growth and MnP production. Eqs. (2) and (4) were solved by a data processing software, Origion 8.0. Through the nonlinear least-squares fitting technique, the program provided the result as the output. The fitted equation can be given as Fig. 4. SDS-PAGE of samples containing MnP from various follows: purification steps. Molecular mass markers are shown on the left of the gel; lane 1: N = ------0.23575 - (5) purified MnP; lane 2: samples after Sephadex G-75 gel filtration; lane t –0.06681t 1+ 3.37869exp 3: samples after DEAE-Cellulose DE52 column; lane 4: crude enzyme.

P = 0.23575 ------–4569.29145 - + 1043.52993 sequences of four peptides showed low levels t –0.06681t 3.37869exp + 1 of identity with MnP from P. chrysosporium (57%, 30%, 67%, exp0.06681t + 1737.20581ln⎛⎞ 1 + ------12h≤≤ t 84h (6) and 71%, respectively). It also showed relatively high levels ⎝⎠3.37869 of identity with MnPs from P. ostreatus and T. versicolor, The Adj. R2 of the nonlinear regression of the biomass ranging from 42% to 100%. Besides this, the amino acid and MnP production was 0.95 and 0.96, respectively, sequences of these four peptides were in full accordance indicating that Eq. (5) can be used to determine the with those of the MnP of I. lacteus F17 (AGO86670.2), which relationship between production of biomass and time of was predicted by the full-length cDNA sequence obtained fermentation. Eq. (6) represents the relationship between by our laboratory. Namely, the purified MnP in this work MnP production and time of fermentation. may be the deduced MnP (AGO86670.2) or its isozymes from I. lacteus F17. Purification and Identification of MnP The MnP was isolated and purified for further examination Kinetic Parameters of the Purified MnP of its enzyme properties. Crude enzyme was extracted The kinetic constants of the purified MnP were determined based on typical Michaelis–Menten behavior. The K and from SSF culture of I. lacteus F17 in the tray bioreactor at m k values of MnP for Mn2+ were found to be 80 µmol/l and 84 h, corresponding to the maximum MnP activity, and cat -1 concentrated by ammonium sulfate precipitation. Fractionation 273.9 s , respectively. Furthermore, the catalytic efficiency (specificity constant, k /K ) of the purified enzyme was of MnP was performed by DEAE-cellulose anion-exchange cat m 6 -1 -1 chromatography, followed by Sephadex G-75 gel filtration 3.4 × 10 mol l s . chromatography. SDS-PAGE (Fig. 4) revealed that the molecular mass of the purified MnP was about 43.2 kDa, Effects of Temperature and pH on the Activity and which agreed well with the molecular mass range of the Stability of Purified MnP MnP family. As illustrated in Fig. 5A, the purified MnP showed its Four peptides with m/z of 689.33, 979.52, 1,054.53, and 1,625.74 maximal activity at 25°C, and had a relatively high activity corresponding to the amino acid sequences VACPDGK, at 25–55°C. Furthermore, the enzyme was stable at low and PNPVAAAPDK, LQSDHDLAR, and TACEWQSFVNNQAK, moderate temperatures, maintaining more than 90% of its were obtained by LC-MS. Then, they were searched against maximal activity after incubation at temperature ranging a nonredundant protein database using BLASTp in NCBI. from 25°C to 45°C for 24 h. However, a rapid loss of Table 1 shows the homology of the internal sequences of stability occurred at temperatures higher than 55°C. The I. lacteus F17 MnP with those of representative MnPs. The half-lives of MnP at pH 4.5 were 19, 18, 18, and 5 days, and

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Table 1. Sequence homology for internal peptides of I. lacteus F17 MnP by NCBI BLAST short sequence search. [M+H]+ Amino acid sequences Similar sequences in the database of the NCBI (Accession No.) Identity (%) 689.33 VACPDGK 27VACPDGK(AGO86670.2) 100 26ATCADGR(BAA33009.2) 42 26VACPDGV(CAA83148.1) 86 358IAHCPDG(AAA33744.1) 57

979.52 PNPVAAAPDK 153PNPVAAAPDK(AGO86670.2) 100 154PPATAASPNG(BAA33009.2) 40 160PDATQPAPDL(CAA83148.1) 44 18AAPTAVCPD (AAA33744.1) 30

1054.53 LQSDHDLAR 255LQSDHDLAR(AGO86670.2) 100 255IQSDHDLAR(BAA33009.2) 89 262LQSDSELAR(CAA83148.1) 78 259LQSDFALAH (AAA33744.1) 67

1625.74 TACEWQSFVNNQAK 267TACEWQSFVNNQAK(AGO86670.2) 100 267TACEWQSFVNNQAK(BAA33009.2) 100 274TACEWQSFVNNQAK(CAA83148.1) 100 271TACIWQGFVNEQAF(AAA33744.1) 71 AGO86670.2: MnP of I. lacteus; BAA33009.2: MnP of Pleurotus ostreatus; CAA83148.1: MnP of Trametes versicolor; AAA33744.1: MnP of Phanerochaete chrysosporium.

Fig. 5. Effects of temperature and pH on the activity and stability of purified MnP from I. lacteus F17. (A) The optimum temperature and thermostability of purified MnP. For thermal stability, the enzyme was incubated for 24 h at various temperatures. The residual MnP activity was assayed under standard assay conditions (maximum activity = 100%). (B) The optimum pH and pH stability of purified MnP. For pH stability, the purified MnP was incubated for 24 h at 25°C in various pHs in citrate–phosphate buffer. The residual MnP activity was assayed under standard assay conditions (maximum activity=100%). Deviation values are standard deviations based on triplicate determinations.

2.1 h at 25°C, 35°C, 45°C, 55°C, and 65°C, respectively. enzyme was stable in the pH range of 4-7, exhibited an In addition, the effect of varying the pH on the activity of optimum pH of about 7, and retained more than 90% of the the purified MnP was also examined. Fig. 5B shows the maximal activity after 24 h incubation at 25°C in citrate– influence of pH in the range of 2.2–8 on MnP activity. The phosphate buffer with different pH values. In addition, the

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half-lives of MnP at 25°C were 3, 14, 28, 28, and 9 days at pH 3, 4, 5, 6, and 7, respectively. Therefore, the purified MnP exhibited great stability in these conditions of the temperature ranging from 25°C to 55°C and the pH range of 3-7.

In Vitro Decolorization of Poly R-478 Poly R-478 is a charged, sulfonated, anthraquinone polymeric dye, which has been shown to be useful for the screening of fungi to degrade lignin. This dye was found to be one of the recalcitrant organic compounds and could remain colored for a long time [18]. Thus, it was chosen to examine the degrading capability of both crude and purified MnP. The optimal temperature and pH for crude MnP to decolorize Poly R-478 was first evaluated (Fig. 6A). Efficient decolorization was only observed at pH 4.0 and decreased rapidly at other values. Thus, for practical application, an acidic pH system is required for the enzymatic decolorization. The optimal temperature for decolorization was around 45°C. However, the effective range of temperature was broader compared with pH. Furthermore, the effects of temperature and pH on decolorization efficiency by the purified MnP were evaluated, and optimum values of 35°C and 4.0 were detected (Fig. 6B). The corresponding maximum decolorization percentages were 21% for crude enzyme and 14% for purified enzyme after 1 h of reaction (Fig. 6). Then, the decolorization of Poly R-487 was enhanced slowly along with the reaction time extension. The decolorization for prolonged times is in accordance with the thermostability of this enzyme. Fig. 6. Poly R-478 decolorization by crude MnP and purified MnP at different pH values and temperatures. Discussion (A) The effect of pH (black triangle) and temperature (black square) on Poly R-478 decolorization by crude MnP. The reaction mixture For the cultivation of I. lacteus F 17 on a solid substrate, a contained citrate–phosphate buffer (62.8 mM), MnSO4 (1 mM), H2O2 cylindrical static bed bioreactor, aerated from the two sides (0.2 mM), Poly R-478 dye (0.001%), and 100 µl of crude MnP (900 U/l) with moist air, was set up in this study for batch SSF at a in a total volume of 1 ml. (B) The effect of pH (black triangle) and laboratory scale (see Fig. 1). The design of this deep tray temperature (black square) on Poly R-478 decolorization by purified bioreactor combined ideas of both the solid-state tray MnP. A 200 µl aliquot of purified MnP (900 U/l) was used to decolorize the Poly R-478 dye (0.001%) in a total volume of 1 ml, and reactor and packed bed reactor to overcome some problems, other conditions are the same as in (A). Deviation values are standard such as the requirement of large areas and the occurrence deviations based on triplicate determinations. of a dry bed, as well as mycelium damage. Three perforated trays in the bioreactor formed a thin layer, allowing good attachment of the fungus to the support composed of agro- its surface was covered with a layer of fungal hyphae (see industrial residues, as well as facilitating appropriate Fig. 3D). The major components of rice straw are cellulose oxygen and nutrient diffusion. The support material for the and hemicelluloses, which are macromolecules composed attachment and growth of I. lacteus F 17 was a mixture of of different sugar monomers. Pine sawdust is not only a pine sawdust, rice straw, and soybean powder. After 60 h carbon source but also an important inducer for ligninolytic of incubation, the support was completely penetrated and enzymes production by white-rot fungi. Thus, the support

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material used in this study is not only a good immobilization period in the deep tray bioreactor. Furthermore, CO2 matrix with very low cost but also provides all the nutrients measurements also provided valuable information about needed for fungal growth and enzyme production. Taken the progress of mycelium growth and metabolism on the together, the main advantage of this bioreactor is the solid medium tested in the bioreactor. The profile of CO2 reduction of MnP production costs as well as the ability to emission is shown in Fig. 2A, which was noted to be in mimic the natural environment for rapid mycelium growth. agreement with the amount of I. lacteus F17 biomass In addition, this bioreactor is simple and has less stringent produced. During early growth, the nucleic acid content was control of operative conditions. higher and there was a strong increase in CO2 production Moilanen et al. [16] reported that the maximum MnP before 60 h of fermentation, indicating aerobic metabolism in activity (340 nkat/g DM) occurred after 14 days of fermentation the tray bioreactor. During the late growth phase, the in a solid-state reactor with an internal conveyor, but their nucleic acid content remained almost constant, and the MnP activity stayed at almost the same level at 100 g-scale MnP activity of I. lacteus F17 grown on the solid medium in plastic jars and 4 kg-scale in bioreactor cultivation. A was high, while the CO2 production decreased consistently similar study was carried out by Vivekanand et al. [32] through the end of fermentation, indicating that the using a bioreactor that contained four circular aluminum digestion of carbon sources present in the growth medium trays, and the maximum MnP activity (1,339.0 ± 131.23 U/l) gradually became more difficult and that secondary was obtained after 6 days of SSF by Aspergillus fumigatus metabolism was initiated. The causes of deceleration of VkJ2.4.5. Phanerochaete chrysosporium BKM-F-1767 had also growth may be the accumulation of inhibitory metabolites produced 1,293U/l of MnP activity, in a fixed-bed bioreactor in the medium, exhaustion of readily utilizable nutrients, or on the 7th day [17]. Compared with these reports, 950 U/l the onset of oxygen limitation. To analyze the bioreactor of MnP activity was achieved after 3.5 days of SSF in our performance, logistic and Luedeking–Piret equations were bioreactor, indicating a higher MnP activity obtained from applied to determine the different kinetic parameters related our bioreactor in a relatively short time. In addition, the to fungal biomass production and MnP synthesis. The MnP activity was 1.68-fold higher than that observed in results calculated using the models were comparable with flask cultures under the same conditions. In fact, the tray the experimental data, and the model equations could bioreactors have been proposed as an appropriate type of actually reflect the enzymatic fermentation process and bioreactor for enzyme production through solid-state kinetic mechanism. fermentation [31, 24]. Couto et al. [4, 22] demonstrated that the The purified MnP showed an optimum activity at 25°C, tray bioreactor is an ideal configuration for the production which is slightly lower than that of MnP obtained from of laccase by Trametes versicolor and Trametes hirsuta under other organisms such as Pleurotus ostreatus (30°C) [1], solid-state conditions when compared with other types of I. lacteus (35°C) [26], and Lentinula edodes (40°C) [3]. Moreover, configurations. Rosales et al. [23] focused on obtaining high the enzyme was quite stable up to 55°C, and did not lose laccase activity levels by T. hirsuta in a 1.8 L static tray activity for at least 24 h. Furthermore, it is worth noting bioreactor under solid-state conditions, and they exhibited that this MnP had stronger half-lives within a broad a very high laccase activity of around 12,000 U/l. From temperature range of 25-55°C among MnPs reported so far these research results, it can be indicated that the tray [3, 15, 28]. In fact, good thermostability is a desirable bioreactor is a successful scale-up for the production of feature of an enzyme for enzymatic industrial applications. enzymes from filamentous fungi under SSF conditions and The purified MnP obtained in the present study was stable it is economical and accessible. in the pH range of 3-7, with an optimum pH of 7 at 25°C, Nucleic acid, ergosterol, and glucosamine have been whereas most of the MnPs reported previously exhibited used to evaluate the biomass of fungal Aspergillus terreus in maximum activity at an acidic pH ranging from 4.5 to 5.0 SSF [34]. Nucleic acid proved to be a good biomass [3, 21, 28]. indicator because its content changed only with mycelial In the present study, Poly R-478 was selected as the growth during the cultivation period, whereas the amount lignin model compound, whose strong structure is especially of ergosterol and glucosamine could not accurately represent difficult to degrade through most of the available technologies the biomass changes. In the present study, the nucleic acid [5]. The effects of temperature and pH on the decolorization content of the mycelium was chosen as the indicator to of Poly R-478 by both crude and purified MnP produced estimate the biomass of I. lacteus F17 during the cultivation from I. lacteus F17 were analyzed. It was noticed that the

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optimal temperature for crude and purified MnP to References decolorize was 45°C and 35°C, respectively, greatly different from the optimum temperature of 25°C for the MnP 1. Aslam S, Asgher M. 2011. Partial purification and characterization activity. It is probably attributed to the strong thermal of ligninolytic enzymes produced by Pleurotus ostreatus during stability of the MnP at a broad temperature range of 25- solid state fermentation. Afr. J. Biotechnol. 10: 17875-17883. 45°C, and the enzyme retained more than 90% of its 2. Baborová P, Möder M, Baldrian P, Cajthamlová K, Cajthaml original activity for at least 3 days, and exhibited a half-life T. 2006. Purification of a new manganese peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic of 18 days at 45°C. However, the crude enzyme lost its aromatic hydrocarbons by the enzyme. Res. Microbiol. 157: ability of decolorization with further increases of the reaction 248-253. temperature to 55°C. The purified MnP was effective for 3. Boer CG, Obici L, Souza CGM, Peralta RM. 2006. Purification decolorization of the polymeric dye, but the decolorization and some properties of Mn peroxidase from Lentinula edodes. rate was a little low when compared with the crude MnP Process Biochem. 41: 1203-1207. (Fig. 6), even though the concentration of the purified MnP 4. Couto SR, Moldes D, Liébanas A, Sanromán A. 2003. was 2-fold higher than that of the crude enzyme. From a Investigation of several bioreactor configurations for laccase practical point of view, crude enzyme is preferred owing to production by Trametes versicolor operating in solid-state its low cost. Therefore, the use of crude enzyme for in vitro conditions. Biochem. Eng. J. 15: 21-26. decolorization may be a viable technology for the degradation 5. Couto SR, Moldes D, Sanromán MA. 2006. Optimum stability of some recalcitrant organic compounds. Furthermore, conditions of pH and temperature for ligninase and manganese- the decolorization of Poly R-478 was found to be strictly dependent peroxidase from Phanerochaete chrysosporium. Application to in vitro decolorization of Poly R-478 by MnP. dependent on the pH of the reaction system, and the World J. Microbiol. Biotechnol. 22: 607-612. optimum pH for the decolorization by both types of MnP 6. Couto SR, Sanromán MA. 2005. Application of solid-state from I. lacteus F17 was detected at pH 4. The acidic fermentation to ligninolytic enzyme production. Biochem. Eng. optimum pH in dye oxidation by MnP has already been J. 22: 211-219. reported and was suggested to be due to the increased 7. Eibes G, Cajthaml T, Moreira MT, Feijoo G, Lema JM. 2006. redox potential of the oxidized heme at low pH [15]. Enzymatic degradation of anthracene, dibenzothiophene and A systematic investigation on the growth of I. lacteus F17 pyrene by manganese peroxidase in media containing acetone. and MnP production in the designed bioreactor was Chemosphere 64: 408-414. presented, and MnP purification as well as its kinetic 8. Govender S, Pillay VL, Odhav B. 2010. Nutrient manipulation properties and application in decolorization of the polymeric as a basis for enzyme production in a gradostat bioreactor. model dye were reported in this study. The results indicated Enzyme Microb. Technol. 46: 603-609. that the proposed bioreactor is an effective system for MnP 9. Graminha EBN, Gonçalves AZL, Pirota RDPB, Balsalobre MAA, Da Silva R, Gomes E. 2008. Enzyme production by production by I. lacteus F17 at laboratory scale. The solid-state fermentation: application to animal nutrition. experiments of in vitro decolorization of Poly R-478 by MnP Anim. Feed Sci. Technol. 144: 1-22. indicate this enzymatic degrading ability. Thus, if the 10. Hofrichter M. 2002. Review: lignin conversion by manganese conditions permit the scale-up of enzyme production peroxidase (MnP). Enzyme Microb. Technol. 30: 454-466. through an efficient production system without operational 11. Ibrahim CO. 2008. Development of applications of industrial problems, the enzymatic biodegradation can be selected as enzymes from Malaysian indigenous microbial sources. a viable option for treatment of dye wastewater at an Bioresource Technol. 99: 4572-4582. industrial scale. Therefore, further studies are needed to 12. Jia R, Tang B, Zhang X, He Y. 2004. Effect of veratryl optimize the bioreactor configuration and environmental alcohol and Tween 80 on ligninase production and its roles factors, as well as to further enhance the enzyme product in decolorization of azo dyes by white-rot basidiomycete

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