Decolorization of Congo Red by Phanerochaete

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Environmental Technology

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Decolorization of Congo Red by Phanerochaete chrysosporium: the role of biosorption and biodegradation Francesca Bosco, Chiara Mollea & Bernardo Ruggeri To cite this article: Francesca Bosco, Chiara Mollea & Bernardo Ruggeri (2016): Decolorization of Congo Red by Phanerochaete chrysosporium: the role of biosorption and biodegradation, Environmental Technology To link to this article: http://dx.doi.org/10.1080/09593330.2016.1271019

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Date: 22 December 2016, At: 17:45

Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis Group Journal: Environmental Technology DOI: 10.1080/09593330.2016.1271019

Decolorization of Congo Red by Phanerochaete chrysosporium: the role of biosorption and biodegradation

Francesca Boscoa, Chiara Molleaa* and Bernardo Ruggeria

a

Department of Applied Science and Technology, Politecnico di Torino, Corso Duca

degli Abruzzi 24, 10129 Torino, Italy

*Corresponding Author: Chiara Mollea, [email protected] , Department of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy. Telephone: +390115644714.

Francesca Bosco, [email protected], Telephone: +390115644696.

Bernardo Ruggeri, [email protected], Telephone: +390115644647.

Decolorization of Congo Red by Phanerochaete chrysosporium: the role of biosorption and biodegradation Abstract: The degradation of Congo Red by means of P. chrysosporium BKM-F1767 is reported in this work. Solid and liquid cultures have been prepared to evaluate in vivo biodegradation as well as the role of biosorption phenomena on mycelium. Moreover, in vitro tests have been performed to define the influence of MnP on dye decolorization. P. chrysosporium, cultivated on MEA in the presence of Congo Red 0.005% (w/v), has shown good growth and the ability to decolorize the dye in the 25-39°C temperature range. It has also been cultivated in a low NMM liquid medium with the aforementioned dye concentration in immobilized stationary cultures inducted for LiP and MnP production. Congo Red was absorbed on the biomass and then decolorized (93% and 85% for the LiP and MnP cultures, respectively). The cultures with added Congo Red have shown a higher MnP synthesis rate than a control without the dye. The enzymatic degradation of Congo Red has also been investigated by means of the extracellular fluid for different MnP activities (0-300 IU/l); the decolorization percentage has been found to be clearly related to the enzyme concentration up to a value of about 200 IU/l. Keywords: P. chrysosporium; Congo Red; absorption; biodegradation; Manganese Peroxidase.

1. Introduction Several thousands of synthetic dyes are available on the market: over 7*105 tons and approximately 10,000 different dyes and pigments are produced annually throughout world [1]. Among these, the dyes commonly used in industry are divided into azo, triphenylmethane or heterocyclic/polymeric dyes [2]. Dyes are used extensively in the biomedical, food, plastic and textile industries, and for this reason large amounts are released into the environment: it is estimated that 10-14% of dye is lost in effluents during the dyeing process [3, 4]. A common feature of synthetic dyes is their low biodegradability: therefore, when spilled into the environment, they are persistent and many are also toxic [5, 6]. The treatment of dye-containing effluents is based above all on physical and chemical operations, such as adsorption, concentration, chemical oxidation and incineration. These methods are expensive and sometimes produce hazardous substances, and for this reason biodegradation is considered an attractive option. Since dyes are not usually removed by means of conventional wastewater treatment processes, because of their resistance to microbial attack, different biological treatment approaches have been tested [6, 7]. Among the different classes of aerobic microorganisms that are able to decolorize industrial effluents, many fungal genera have been employed either in the living or dead form. The research studies can be grouped together on the basis of the adopted mechanism, that is, bioaccumulation, biosorption or biodegradation. The bioaccumulation mechanism pertains to yeasts [8, 9]. Biosorption studies have mostly been conducted on fungi different from wood-rot ones which have been applied to heavy metal adsorption [10]. Most of the works related to the biodegradation of textile

dyes have concentrated on the use of wood-rot fungi, which produce lignin degrading enzymes [11]. White Rot Fungi (WRF) have been shown to possess a remarkable potential in the field of dye removal from effluents [12, 13], because they use an oxidative decolourization mechanism that offers the advantage of producing less toxic metabolites than the initial dye [14]. In nature, these microorganisms are able to degrade lignin, and their biodegradation activity is related to the production of Lignin-Modifying Enzymes (LMEs) composed of one or more extracellular enzyme classes: Lignin Peroxidase (ligninase, LiP, EC 1.11.1.14), Manganese dependant Peroxidase (MnP, EC 1.11.1.13) and Cu containing Laccase (Lac, EC 1.10.3.2) [15]. A new group of Ligninolytic hemecontaining peroxidases, which combine the structural and functional properties of LiPs and MnPs, are the versatile peroxidases (VPs). In addition, the enzymes involved in hydrogen peroxide production, such as glyoxal oxidase (GLOX) and aryl alcohol oxidase (AAO, EC 1.1.3.7), are considered to belong to the ligninolytic system [16]. Different authors have described the dye degradation capability of WRF with different LME producing characteristics [17-19], although most studies have used the “standard” Phanerochaete chrysosporium microorganism. P. chrysosporium ligninolytic cultures have been shown to decolorize azo, triphenylmethane and heterocyclic dyes in the presence of veratryl alcohol and H2O2 [17, 20]. Most studies deal with the degradation of azo dyes, which are the most frequently used ones. In the work of Tatarko and Bumpus [21], decolourization was verified in both liquid and solid cultures and related to LiP enzymes. According to Pasti-Grigsby et al. [22], different LMEs, and in particular LiP and MnP, are able to decolorize different azo-dyes [23]. The total decolorization of amaranth, orange G and new coccine has been demonstrated in liquid cultures of P. chrysosporium in the presence of MnP alone [24]. The role of biosorption

in azo dye decolorization (the removal of dye from a solution through binding to the mycelium) was reported to be very important in the work of Cripps et al. [17]; on the contrary, in Ligninolytic cultures of P. sanguineus, biosorption was shown to account for less than 3% of azo and triphenylmethane dye removal [25]. In a study on dye decolorization by means of P. chrysosporium and Trametes versicolor [26], it was observed that, in addition to enzymatic degradation, the biosorption of dye on fungal hyphae also contributed to the total removal of the colour from an aqueous solution. The fate of biosorbed dye may include permanent binding to fungal hyphae, physical desorption and/or enzymatic degradation by LMEs or intracellular enzymes. Dye biodegradation processes, using immobilized growing cells, seem to be more promising than those with free cells, since the immobilization allows the microbial cells to be used repeatedly and in a continuous way. Many examples of dye biodegradation, carried out with immobilized fungi, have been reported in the review by Rodriguez-Couto [16]: P. chrysosporium was grown on cubes of polyurethane foam, wheat straw or wood chips; T. versicolor was encapsulated in polyvinyl alcohol (PVAL)-hydrogel and alginate beads and immobilized on stainless steel sponge and pine wood chips. Although many studies regarding the metabolic pathway of azo-dye degradation by WRF and their ligninolytic enzymes have been performed [27, 28], to the best of the authors’ knowledge, the specific contribution of biosorption and biodegradation to the degradation mechanisms of Congo Red by means of P. chrysosporium has not yet been clarified. In this context, the main aim of the present work was to establish the role of biosorption phenomena on living and dead mycelia. Moreover, the in vivo and in vitro biodegradation of living mycelia have been evaluated and commented on.

2. Materials and methods 2.1 Microorganism Phanerochaete chrysosporium BKM-F-1767 was purchased from Mycothéque de l'Université Catholique de Louvain (MUCL, Belgium). The fungus was maintained on 2% Malt Extract Agar (MEA) plates at 4°C. Mycelial growth and spore production on agar plates usually requires 3-4 days at 39°C.

2.2 Chemicals Congo Red diazo dye was used in this study (C.I. number 22120; C.I. name Direct Red 28). All the utilized reagents were of analytical grade and were purchased from SigmaAldrich Co. Mycological peptone was purchased from Oxoid Limited.

2.3 Congo Red decolorization on agar plates The capability of Phanerochaete chrysosporium to grow and sporulate on a solid medium (2% agar) was tested at 39°C by means of MEA and on a low Nitrogen Mineral Medium (NMM) [29]. The MEA medium contained (per litre): agar-agar 20 g, malt extract 20 g, mycological peptone 2 g, and glucose 20 g. The NMM medium contained: agar-agar 20 gl-1, glucose 1% w/v, dimethyl succinic buffer pH 5.5 0.01 M, ammonium tartrate dibasic 0.2 gl-1, and soybean asolectin 0.75 gl-1. The NMM medium was also supplemented with a Basal III medium (10% v/v) and Vitamine B1 (1 gl-1), both of which were sterilised by means of filtration. The Basal III medium had the following composition (per litre): Trace element solution (10% v/v), KH2PO4 20 g, MgSO4*7H2O 10.24 g, and CaCl2 1 g. The Trace elements solution contained (per litre): MgSO4*7H2O 3 g, MnSO4 0.56 g, NaCl 1 g, FeSO4*7H2O 0.1 g, CoCl2*6H2O 0.1 g, ZnSO4*7H2O 0.1 g, CuSO4*5H2O 0.1 g, AlK(SO4)2*12H2O 0.01 g, H3BO3 0.01 g,

NaMoO4*5H2O 0.01 g, and nitrilotriacetic acid 1.5 g. The dye degradation tests on the solid medium were performed on MEA [29], supplemented with a Congo Red solution sterilised by means of filtration (sterile filters MFS-25 cat. n° 25CS020AS, pore diameter 0.20 μm). In order to obtain the desired final concentration (0.005% w/v) on the MEA, 200 μL of dye stock solution (0.5%) was added to the agar medium before it solidified. MEA plates without dye were used as a control of the basal growth rate. A mycelium plug (8 mm diameter), cut from the edge of a 96 hour old colony grown on a MEA, was inoculated at the centre of each plate. The plates were incubated at 25°C, 30°C or 39°C in a humidified chamber, to prevent desiccation of the agar, until complete mycelial growth and sporulation had occurred. The radial growth of P. chrysosporium was evaluated daily on the plates, with and without Congo Red; two perpendicular diameters were measured for each plate. The growth rate was expressed as cm/h of the colony diameter over time; the results were the mean of six replicates.

2.4 Congo Red decolorization in liquid cultures Low NMM was used in liquid cultures [29]. Immobilized stationary cultures of P. chrysosporium were developed in 500 mL Erlenmeyer flasks containing a monolayer of Rasching ceramic rings of ¼ inch diameter and 30 mL of low NMM. The bottom of each flask was covered with Rasching rings which were dried at 105°C for 24 hours and sterilised by autoclaving at 121°C for 20 min. A conidia suspension in distilled water (5*106 conidia/mL), obtained from 4 day old plates, was used as the inoculum (10% v/v). The cultures were maintained at 39°C in a thermostatic room until ammonia depletion had occurred (about 48 hours). When the ammonia was exhausted, MnP induction was carried out by adding MnSO4 * H2O (2050.9 ppm),

while LiP production was induced by adding veratryl alcohol (2.5 mM) and asolectin (0.1 gl-1) [30] [31]. At the same time, the temperature was shifted to 30°C, and Congo Red (0.005% w/v) was added. The cultures were flushed with wet pure-oxygen (4 minutes, 150 NL/h) at the inoculation and induction times, and whenever a sample was taken to measure the MnP activity and/or the medium decolorization. Biotic, abiotic and killed controls were conducted for each experiment, along with biotic tests: i.

a biotic reference test, without Congo Red, to verify the biomass growth and ligninolytic enzyme production;

ii.

a biotic test, with Congo Red, to evaluate the overall biodegradation.

iii.

a biotic control, with Congo Red, to verify the biomass contribution. The biomass was separated from the Extracellular Fluid (EF), washed with sterile distilled water, and maintained in the DMS buffer at a pH 5.50 or 4.80;

iv.

an abiotic uninoculated control, with Congo Red, to evaluate all the nonbiological decolorization phenomena, carried out at different pH values (4.8 and 5.5);

v.

a killed control, with Congo Red, to quantify the dye biosorption on the biomass. A 2-day old culture was inactivated by means of thermal sterilization and then supplemented with the dye;

2.5 Enzymatic decolorization of Congo Red in vitro MnP (293 IU/L), derived from low NMM cultures, without dye addition, was used for the enzymatic decolorization experiments. Two different kinds of test were performed: Congo Red as a substrate instead of 2,6-dimethoxyphenol (2,6-DMP) in the reaction

mixture, according to the method of Paszczynski et al. [32]; Congo Red in the EF, diluted with H2O or the permeate, to obtain the same enzyme activity as the previous test. The permeate derived from the ultrafiltration of EF (cut-off 10 KDa). All the experiments were performed under the following conditions: 0.3 IU of enzyme activity, 0.005% of Congo Red and at 30°C; exogenous peroxide was not added to the second type of test.

2.6 Influence of MnP activity on decolorization EF derived from low NMM cultures and added with Congo Red (0.005% w/v), was used to measure the influence of MnP activity on dye decolorization. Different values of MnP activity were tested in the range 0-300 IU/L. The samples were incubated at 30°C for up to 166 hours. Samples were taken to evaluate the disappearance of the dye; the initial absorbance value of each sample was used as a reference to establish the dye decolorization.

2.7 Ligninolytic enzyme assays The extracellular fluid of the low NMM cultures of P. chrysosporium was used for the enzymatic assays. LiP activity was determined using the Tien and Kirk method [29]. A 2 mM solution of veratryl alcohol (VA) was used as the substrate, and the assay was performed at pH 3.0 using a sodium tartrate buffer (50 mM). The reaction was started by adding hydrogen peroxide (0.4 mM), and the increase in absorbance at 310 nm was recorded over time (ε= 9300 M-1 cm-1). The MnP activity was determined using the Paszczynski et al. method [32]. The enzymatic activity was measured by spectrophotometrically monitoring the oxidation of

2,6-DMP at 568 nm (ε = 23100 M-1 cm-1) and at 22°C. A Hewlett Packard 8452A Diode Array spectrophotometer was used. The reaction mixture contained the culture fluid, sodium tartarate buffer (100 mM, pH 5.0), MnSO4 (0.1 mM), 2,6-DMP (1 mM) and H2O2 (0.1 mM). The reaction was initiated by adding hydrogen peroxide. The hydrogen peroxide solution was prepared daily in order to prevent any loss of activity. One unit of both of the enzymatic activities was defined as one μmol of product formed per minute.

2.8 Glucose and ammonium assays Glucose and ammonium concentrations were determined spectrophotometrically in the extracellular fluid, using enzymatic kits (Boehringer Mannheim numbers 716251, 1112732, respectively).

2.9 Decolourization assays The decolourization of Congo Red in the liquid medium was recorded by measuring the absorbance of the filtered culture samples or of the enzyme mixture. The absorbance decrease at 490 nm (maximum absorption wavelength) was used to establish the decolourization ability of the fungus or the enzyme. Samples were diluted until an absorbance value below 1 was reached. The results refer to the % of absorbance decrease or the residual dye concentration (mg/L).

2.10

Sorption of dye by the fungal mycelium

Dye adsorption by the mycelia was tested using killed biomass. After 48 hours of fungal growth, the biomass was inactivated by means of thermal sterilisation (see v. paragraph 2.4). Congo Red (0.005% w/v) was added to these cultures.

3. Results and discussion 3.1 Decolorization of Congo Red on solid cultures The ability of P. chrysosporium to decolorize Congo Red was first tested on a solid medium. In order to evaluate the best solid medium for the microorganism growth and sporulation, two different agar media were tested without Congo Red, at the optimal growth temperature of 39°C: MEA and low NMM. In the first case, the fungal growth rate was higher and sporulation was more homogeneous; for this reason, MEA was chosen for the subsequent degradation tests with dye. The experimental tests on the MEA plates, in the presence of Congo Red 0.005%, were performed at three different temperatures: 25°, 30° and 39°C. A significant decrease in the growth rate, due to the presence of the dye, was observed for each temperature. The behaviour of the sample compared with that of the control (30°C), is reported in Figure 1 as an example. The fungus was able to decolorize the dye at all the examined temperatures, but the decolourization was not complete. Any attempt to quantify the % of the Congo Red degradation on the solid culture proved useless, because the decolourization was not homogeneous, as can be observed in Figure 2. The incomplete decolourization was probably related to the non-homogeneous distribution of LMEs inside the agar medium.

3.2 Decolorization of Congo Red in liquid cultures The study on decolorization capability of P. chrysosporium was performed in the low NMM containing 0.005% of dye (w/v) in immobilized stationary cultures. In order to identify the role of extracellular enzyme activity (LiP and MnP) in Congo Red degradation, two different types of induction were applied at the end of the trophophase, as described in the “Materials and Methods” section. The results obtained in the cultures that were inducted for LiP or MnP production are reported in Figure 3; 0.033g/ml

(decolourization % =66) and 0.036g/ml (decolourization % =73) of dye were degraded, respectively, 13 hours after the addition of the dye. The trend of the degradation curves was found to be very similar after 140 hours; Congo Red was decolorized by 93% for the LiP inducted cultures, and 85% for the MnP inducted ones. The decolourization % obtained in the presence of LiP induction is in agreement with the results of Tatarko and Bumpus [21], while the percentage of MnP induction agrees with that of Noreen et al. (2011) [23]. In the biological sorption control (heat killed mycelium) after 50 hours, a total sorption of the dye on the biomass was evident for both of the inducted lines (Figure 4). The adsorbed dye was 96% in the LiP inducted line and 95% in the MnP one at 140 hours after the induction. The biomass and the EF colour of both a biotic and a killed line were compared at the end of a biodegradation experiment, that is, after a 140 hours, (Figures 5a and 5b). The biomass colour of the killed control was very different from that of the biotic lines; biodegradation of the dye was carried out in the latter, while biosorption of the dye occurred on the biomass without degradation in the killed control. A comparison of the EFs (Figure 5b) confirmed the different behaviour of the decolorization process in the killed (biosorption) and biotic lines (biosorption and enzymatic degradation); a yellow coloured extracellular fluid appeared in the latter instead of a colourless one. The decolorization of Congo Red by P. chrysosporium was clearly related to the biosorption on the biomass and LME biodegradation, as described for Trametes versicolor in the work by Wang and Yu [33], and for Alternaria alternata CMER F6 by Chakraborty et al. [34]. The contribution of different enzyme classes was evaluated by means of in vivo and in vitro experimental tests: Tatarko and Bumpus [21] described the

role of the LiP enzyme. The role of MnP has been evaluated and reported in the present work. MnP activity has been measured in the cultures to establish the effect of dye on enzyme synthesis and secretion (Figure 6). In comparison to the biotic control line (no added dye), the culture with added Congo Red showed a higher MnP synthesis rate, while the maximum MnP values reached in the dye culture and biotic control were very similar around 270-300 UI/l. The positive effect of Congo Red on the MnP synthesis rate has also been described in the work by Harazono et al. [35] in liquid cultures with P. sordida YK 624.

3.3 Biomass contribution to dye decolorization The biomass contribution to Congo Red decolorization was also been studied (Figure 7). The fungal mycelium was washed with sterile distilled water, and then kept in a DMS buffer at pH 5.50 or 4.80 (pH 5.50 is the same as that of the buffer used to set up the cultures, while pH 4.80 is usually found at the beginning of the idio-phase when dye is added). Congo Red was added after the removal of the medium and its substitution with the DMS buffer. The abiotic uninoculated control, containing the dye, was prepared at pH 4.80 and at pH 5.50. As far as the abiotic controls are concerned, no effect of pH was pointed out on the Abs variations for a pH value of 5.50. On the contrary, spectral variations (hypochromic and bathochromic effects) were observed at pH 4.80. As far as the biotic controls are concerned, a new MnP production (32.75 IU/L) was registered for the biomass maintained at pH 4.80, after 46 hours after the addition of the dye, while no new activity was observed for that maintained at pH 5.50. On the basis of these results, it has been hypothesized that Congo Red had been able to induce

MnP production in the biotic control at pH 4.80. Even though there was no enzyme activity in the cultures at pH 5.50 after 46 hours, a dye concentration decrease of 86.67% was registered. This decrease was comparable with that obtained in the biotic control at pH 4.80 in which MnP activity was observed. The decolorization observed at pH 5.50 is due to a mycelium biosorption phenomenon, and probably also to biomass activity, due to the presence of membrane oxidative enzymes, such as cytochrome P450; this contribution has also been reported by Rodriguez et al. [36] and Trupkin et al. [37].

3.4 Enzymatic decolorization of Congo Red in vitro The degradation tests in the liquid medium have shown that both sorption on the biomass and enzymatic degradation of the dye (LMEs) were involved in the dye removal process, due to the presence of P. chrysosporium. In order to obtain experimental evidence on the involvement of LME activity in dye decolorization, in vitro tests were performed in the presence of EF derived from MnP inducted cultures. Experimental tests were carried out at 30°C with 0.3 IU of the enzyme in a kinetic mixture or in the EF, as described in the “Materials and Methods” section. Two series of tests were performed in the EF: in the first one, the EF was diluted (1:3) with distilled water, while in the second one, the dilution was obtained using a permeate from the EF ultrafiltration process, to maintain the same chemical environment as in the in vivo degradation experiment. Decolorization was not observed in the presence of EF diluted with distilled water. The results related to the kinetic mixture and the EF diluted with the permeate are shown and compared in Figure 8 with the decolorization obtained in the permeate control. Dye decolorization was incomplete in both tests. Nevertheless, a higher decolorization (58%) was reached in the presence of EF diluted with permeate

after 30 hours. Both the MnP activity and the permeate chemical composition had a positive effect on Congo Red degradation. The same observation about the influence of EF on the decolorization of Remazol Brilliant Blue R (RBBR) has been reported by other authors (see, for example, Novotny et al. [38]).

3.5 Influence of MnP activity on decolorization The decolorization of Congo Red was also been investigated by means of the EF at different MnP activities (0-300 IU/L), obtained from low NMM cultures without Congo Red. The dye was added at the same concentration as that used in the in vivo and in vitro tests (0.005% w/v). The decolorization percentage was evaluated after 166 hours of incubation, at the same time as the in vivo experiments. The obtained results are shown in Figure 9. The decolourization percentage is clearly related to the enzyme concentration up to a value of about 200 IU/L. No remarkable differences in the decolourization percentage can be observed in the presence of higher enzyme activity (200-300 IU/L) (medium value 70%). A decrease in the absorbance values, corresponding to a decolorization percentage of 20-30%, was also obtained without MnP enzymes (permeate derived from EF ultrafiltration). The active role of EF in the decolorization of Congo Red, observed in the previously described in vitro tests, and confirmed in this experiment, has also been registered in the presence of Congo Red in EF diluted with the permeate (Figure 8).

4. Conclusions The present study has demonstrated that the examined Congo Red degradation was due to the biosorption on the biomass, the activity of the membrane oxidative enzymes and the LME biodegradation in the liquid phase. It has been observed that the role of the LiP and MnP enzymes in the dye degradation was very similar. Moreover, it has been hypothesized that Congo Red can induce MnP production. The obtained results point-out the advantage of in vivo biodegradation. In fact, the synergistic effect of biosorption, membrane enzyme and LME activity, as well as the extracellular fluid chemical composition, allowed a higher percentage of dye degradation to be obtained than in the in vitro experiments.

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Figure 1. Decrease of the growth rate for P. chrysosporium in the presence of Congo Red on MEA at 30°C.

Figure 2. Decolorization of Congo Red by P. chrysosporium on MEA at 30°C.

Figure 3. Decolorization of Congo Red by P. chrysosporium cultures inducted for LiP and MnP production.

Figure 4. Sorption of Congo Red by P. chrysosporium in the biological sorption control.

Figure 5. Biomass colour (5 a) and extracellular fluid colour (5 b) of biotic (left) and killed (right) lines of P. chrysosporium at the end of biodegradation experiment.

Figure 6. MnP activity of a biotic line.

Figure 7. Biomass contribution to Congo Red decolorization in the abiotic and biotic controls at two different pH values.

Figure 8. Residual color % comparison among the kinetic mixture, the EF diluted with the permeate and the permeate control.

Figure 9. Decolorization % of Congo Red in the EF at different MnP activity values.