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Induction of laccase activity in the white rot fungus Pleurotus ostreatus using water polluted with wheat straw extracts
Bioresource Technology 133 (2013) 142–149
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Induction of laccase activity in the white rot fungus Pleurotus ostreatus using water polluted with wheat straw extracts Alejandra Parenti a, Elaia Muguerza a, Amaia Redin Iroz a, Alejandra Omarini a, Enma Conde b, Manuel Alfaro a, Raúl Castanera a, Francisco Santoyo a, Lucía Ramírez a, Antonio G. Pisabarro a,⇑ a b
Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Spain Department of Chemical Engineering, Faculty of Science, University of Vigo (Campus Ourense), Ourense, Spain
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Wheat straw water extracts induce
efficiently laccase activity in fungal cultures. " The wheat straw water extract inducer effect is accumulative. " The isoenzyme Lacc10 is the main responsible for the laccase induced activity. " Wheat industry polluted water effluents can be used to induce ligninolytic enzymes.
a r t i c l e
i n f o
Article history: Received 7 November 2012 Received in revised form 11 January 2013 Accepted 13 January 2013 Available online 30 January 2013 Keywords: Laccase activity Pleurotus ostreatus Wheat straw Inducer Wastewater effluent
a b s t r a c t The purpose of this work was to explore the use of polluted water effluents from wheat straw using industries as inducers of lignocellulolytic enzymatic activities in cultures of white rot basidiomycetes. For this purpose, we studied the effect of a wheat straw water extract on the evolution of the laccase activity recovered from submerged cultures of Pleurotus ostreatus made in different media and under various culture conditions. Our results demonstrated an accumulative induction effect in all the cultures and conditions tested. This induction is parallel to changes in the laccase electrophoretic profiles recovered from the culture supernatants. The isoenzyme that appeared to be mainly responsible for the laccase activity under these conditions was laccase 10, as confirmed by sequencing the induced protein. These results support the idea of using wheat straw effluents as inducers in liquid cultures of P. ostreatus mycelia for the production of ligninolytic enzymatic cocktails. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose is the major reservoir of organic carbon on Earth. It is made up of three major polymers present in plant cell walls: cellulose, hemicellulose, and lignin. Cellulose is the predominant polysaccharide in plant residues, where it forms the main component of cell walls and is responsible for plant support. ⇑ Corresponding author. Tel.: +34 948169107; fax: +34 948169732. E-mail address: [email protected] (A.G. Pisabarro). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.072
Hemicellulose is a heteropolysaccharide composed of frequently branched hexoses, pentoses, uronic acid, and lower sugars held together by b1 ? 4 glycosidic bonds. Hemicellulose is easily hydrolyzable, in contrast with the more resistant cellulose moiety. Finally, lignin is a complex, amorphous, and optically inactive hydrophobic polymer. Lignin and hemicellulose surround and cement the cellulose fibers to strengthen the cell walls (Salmén and Burgert, 2009). White-rot fungi are unique organisms that are able to degrade the lignin moiety of lignocellulose, making cellulose available for
A. Parenti et al. / Bioresource Technology 133 (2013) 142–149
other organisms and for industrial processes involved in the production of biofuels. There are two fungal strategies for degrading lignocellulose: white rot fungi primarily attack the lignin and leave the cellulose accessible, whereas brown rot fungi attack the cellulose without significantly degrading the lignin. White rot fungi have been extensively studied because of this ability to selectively degrade lignin (Kirk and Cullen, 1998). Pleurotus ostreatus (Jacq.: Fr.) Kumm. (Dikaryia, Basidiomycota, Agaricomicotina, Agaricales) is a white rot basidiomycete industrially cultivated on a variety of lignocellulosic agricultural wastes as an edible mushroom of high nutritional value (Mattila et al., 2001; Reis et al., 2012). This basidiomycete may be considered a model organism of the Pleurotus genus, which includes other species that are capable of growing on a variety of agricultural lignocellulosic wastes and that are able to degrade dyes and aromatic compounds. These fungi are useful for decontaminating wastes with a high content of this type of toxic compounds (Faraco et al., 2009; Martínez et al., 2005). P. ostreatus produces a large number of extracellular enzymes and can be used as a source of lignin-degrading enzymes for the biotreatment of wastes and effluents (Ruiz-Duenas et al., 2009), as well as for bio-pretreatment of lignocellulose for the production of second-generation biofuels (Talebnia et al., 2009). Lignin degradation or modification is a key step in the deconstruction of the lignocellulosic material (Vinoth Kumar et al., 2012). In P. ostreatus, this process involves enzymes such as laccases (Phenol oxidases, Lac, EC 1.10.3.2), manganese peroxidase (MnP, EC 1.11.1.13), and versatile peroxidases (VP, EC 1.11.1.16), whose activity is complemented by that of a number of accessory enzymes that include glyoxal oxidase (GLOX, EC 1.1.3) and arylalcohol oxidase (AAO, EC 1.1.3.7) (Hammel and Cullen, 2008). The biotechnological relevance of the lignin-degrading enzymes and, especially, the use of laccases in processes that are aimed at the degradation of water pollutants and involved in the pulp and paper industry has prompted research on methods for their efficient recombinant production (Theerachat et al., 2012) and for their stabilization in industrial processes (Vinoth Kumar et al., 2012). In vivo, the source of carbon and nitrogen in various culture media plays an important role in the production of ligninolytic enzymes (Elisashvili and Kachlishvili, 2009; Kaal et al., 1995). Laccases are induced by the presence of certain aromatic compounds or lignin-related phenolic derivatives such as ferulic acid, 2.5-xylidine, p-anisidine or veratryl alcohol (de Souza et al., 2004). Numerous studies have utilized agro-industry-derived products in the production of ligninolytic enzymes to be applied as alternatives for bioremediation (Karp et al., 2012; Kurt and Buyukalaca, 2012). Industries that use straw (i.e., industrial mushroom production) produce large volumes of wastewater that is highly polluted with straw extractives. To study the use of these effluents in applied processes, we investigated the effect of an aqueous straw extract as an inducer of ligninolytic enzymes in P. ostreatus cultures produced in different media and under various culture conditions. The present study is of relevance for establishing the culture conditions for the production of ligninolytic enzyme cocktails using natural wastes as feedstock.
2. Methods 2.1. Organisms and culture conditions Two P. ostreatus strains were used in this work: the dikaryotic N001 and the monokaryotic PC9 (Larraya et al., 1999, 2000). Both strains were maintained and grown in Petri dishes containing agar-malt (20 g/L malt extract, 15 g/L agar) (Eger et al., 1976), incubated at 25 °C in the dark and subcultured every 8 days.
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Two liquid culture media were used: SMY (10 g/L sucrose, 10 g/ L malt extract and 4 g/L yeast extract), and M7GY (10 g/L glucose, 2 g/L ammonium tartrate, 0.5 g/L MgSO47H2O, 1 g/L KH2PO4, 0.5 g/L KCl, 1 g/L yeast extract, and 1 mL/L of trace elements solution). The solution of trace elements contains the following: 0.1 g/L Na2B4O7H2O, 0.07 g/L ZnSO47H2O, 0.01 g/L CuSO45H2O, 0.01 g/L MnSO44H2O, 0.05 g/L FeSO47H2O, and 0.01 g/L (NH4)6Mo7O24H2O. The mycelium grown on the agar surface was used to inoculate 90 mL of liquid medium (either SMY or M7GY) in 250 mL Erlenmeyer flasks. These cultures were incubated for 24 h with orbital shaking (150 rpm) at 25 °C in the dark to produce the pre-inoculum I0. The 90 mL I0 pre-inocula were homogenized and added to 500 mL Erlenmeyer flasks containing 135 mL of the corresponding liquid culture medium to produce the inocula I1. These inocula were incubated for 48 h with orbital shaking (150 rpm) at 25 °C in the dark before being homogenized and used to start the experimental cultures. The experimental cultures were performed using 500 mL Erlenmeyer flasks containing 135 mL of culture medium inoculated with 15 mL of the corresponding I1 inoculum. All the cultures were incubated at 25 °C in the dark. The shaken cultures were placed in orbital shakers that were set at 150 rpm. Each experiment was performed using three biological replicas for the biomass and three independent measures of each replica for the enzymatic measurements. At the corresponding sampling times (days 3, 7, 12, 14, 19, 21 and 24 of culture), a portion of the culture’s supernatant was aseptically removed for measuring the enzymatic activities and the reducing sugar content. 2.2. Aqueous extract of wheat straw The wheat straw extract used as inducer was prepared as follows: 150 g of dry wheat straw was soaked in 1500 mL of distilled water and shaken in an orbital shaker at 150 rpm for 5 h at 25 °C. After this extraction time, the straw was eliminated by filtration through a Miracloth (Merck Millipore International), and the filtrate was centrifuged at 4500 rpm for 10 minutes at 20 °C. The supernatant was then sterilized at 121 °C for 20 min. The extract was kept frozen until use. For the induction experiments, 50 mL of the inducer extract was added to the experimental cultures described above. An equivalent volume of distilled water was added to the control cultures. The induction treatment was performed at culture days 12 and 21. 2.3. HPLC identification and quantification The samples were analyzed using an Agilent HPLC 1100 instrument equipped with a Waters Spherisorb ODS2 column (5 lm, 250 mm 4.6 mm) and diode-array detector (DAD detector), operating at 30 °C. The injection volume was 20 lL, and the flow rate was 1 mL/min. A non-linear gradient of solvent A (acetonitrile/5% [v/v] formic acid in water, 10:90) and solvent B (acetonitrile/5% [v/v] formic acid in water, 90:10) was used as follows: 0 min, 100% A; 40 min, 85% A, 15% B; 45 min, 100% B; 55 min, 100% B; 60 min, 100% A; and 65 min, 100% A. The samples were analyzed in triplicate. Phenolic compounds were identified by comparisons of the retention time and UV–visible spectral data with those of authentic compounds. Quantification was performed from calibration curves that were obtained using model compounds (Conde et al., 2011). 2.4. Evaluation of biomass and pH For the determination of the biomass, parallel cultures were prepared in 100 mL Erlenmeyer flasks containing 20 mL of medium, 2.2 mL of I1 inoculum and 7.4 mL of wheat straw extract for
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induced or distilled water for the control cultures. At the corresponding sampling times, three randomly selected flasks (biological replicas) were removed from the incubator, filtrated through a 10 lm nylon using a vacuum pump, and dried in an oven at 50 °C to a constant weight. The culture filtrate was used to monitor the pH of the culture. 2.5. Enzyme activity assays The laccase and manganese peroxidase enzymatic activities were determined colorimetrically as described by (Santoyo et al., 2008). The laccase activity was determined by detecting the product of the oxidation of 2.6-dimetoxiphenol (DMP, e468 = 49,600 M 1 cm 1). The reaction mixture contained 450 lL of the culture filtrate described above and 500 lL of 10 mM DMP in 100 mM acetate buffer (pH 5.0). The reaction was performed for 1 min at room temperature before the variation in the absorbance at 468 nm was recorded. After measuring the laccase activity, 50 lL of MnSO4 was added to the sample and incubated for 1 min before adding 30 lL of an H2O2 solution containing 22.5 lL of 30% (w/v) H2O2 in 10 mL of Milli-Q water, and the reaction was allowed to proceed for an additional minute before the A468 was measured. The manganese peroxidase (MnP) activity was estimated as the difference between the two activity measurements. One unit of enzyme activity was defined as the formation of 1 lmol of product per min. All the assays were performed in triplicate using a Shimadzu UV-1800 spectrophotometer. Samples were taken at days 3, 5, 7, 10, 12, 14, 17, 19, 21 and 24 of culture for both treatments.
(Tris 25 mM, glycine 192 mM and methanol 20%) at 100 V for 75 min at 4 °C. Afterwards, the membranes were stained using Coomassie Brilliant Blue G-250 (Sigma) to visualize the protein bands. Proteins bands migrating to 40 kDa were excised, washed twice with Milli-Q water for 10 min and stored at 20 °C in an Eppendorf tube containing Milli-Q water until further analysis. 2.9. Protein identification by peptide fingerprint and mass spectrometry (MALDI-TOF/TOF) These analyses were performed at the Proteomics Unit of the Genomics and Proteomics Centre (http://www.ucm.es/info/gyp/ proteomica/presentacion.htm) at the Complutense University of Madrid. The SDS–PAGE gel fragment was digested using trypsin. Peptide analyses were performed in duplicate by mass spectrometry MALDI-TOF to obtain the peptide fingerprint. Automatic fragmentation of the peptides was performed to obtain one to three peptides, which were analyzed by MS/MS. The protein identification was performed via peptide fingerprints (MS data), peptide sequences (MS/MS data) or a combination of both techniques, as well as by an automatic identification search by the GPS (Global Protein Server) attached to the TOF–TOF data. The identified peptides were blasted to the P. ostreatus PC15 v2.0 database (http://genome.jgipsf.org/PleosPC15_2/PleosPC15_2.home.html) for identifying the protein. 3. Results and discussion
2.6. Measurement of reducing sugars
3.1. Description of the growth of the induced and uninduced cultures
The reducing sugar content of the liquid culture medium in each of the conditions and treatments was determined with dinitrosalicylic acid reagent (DNS), using the colorimetric method described by Miller (1959). The reaction mixture containing 250 lL of distilled water, 250 lL of sample solution or glucose (1% w/v) and 1 mL of DNS reagent was heated for 5 min and then cooled to 4 °C. Subsequently, 9 mL of distilled water was added, the mixture was stirred, and the absorbance at 540 nm was recorded.
The industrial production of edible mushrooms produces large volumes of waste water that is highly polluted with compounds derived from the rinsing and wetting of the cereal straw used as the culture substrate. In this work, we investigated the use of this spent water as an inducer of ligninolytic enzymatic activity by the white rot edible basidiomycete P. ostreatus using liquid cultures of the fungus and a laboratory-produced wheat straw extract as inducer. The wheat extraction process was performed at 25 °C as a model for the extraction that occurs naturally while the mushrooms are being cultured under industrial conditions. As a preliminary step, we analyzed the composition of this extract using HPLC (Table 1). The principal extract was gallic acid, which represented 57.2% of the total mass of the extractable phenols. The dryweight concentration of the extract was 11.05 mg/L (SD 0.5 mg/L), and its pH was 6.3. To determine the effect of the wheat straw extract as an inducer of lignin-degrading activities in P. ostreatus, it was necessary first to determine the effect of the wheat straw extract on mycelial growth under the culture conditions of the assay. For this purpose, we monitored the accumulation of biomass in submerged cultures using the dikaryotic N001 and the monokaryotic PC9 P. ostreatus strains. The study was conducted using two different culture media (rich SMY and minimal M7GY), two culture conditions (shaking and static), and the presence or absence of added inducer (Fig. 1). Other recent studies have focused on the effect of the culture type (solid fermentation vs. submerged culture) (Castanera et al., 2012) of the solid substrate (Elisashvili et al., 2008; Karp et al., 2012; Kurt and Buyukalaca, 2012; Membrillo et al., 2008) on laccase production by P. ostreatus. By contrast, the use of microbial combinations obtained from spent mushroom cultivation substrates in the decolorization of textile effluents has been recently studied (Singh et al., 2012). We focused the present study on submerged cultures with the long-range goal of producing enzyme cocktails useful for the biological pretreatment of lignocellulosic wastes to be used for bioethanol production (Salvachúa et al., 2011).
2.7. Non-denaturing electrophoresis analysis Non-denaturing polyacrylamide gel electrophoresis (PAGE) was performed as described by Sambrook (1989), using 5% acrylamide in 0.5 M Tris (pH 6.8) and 10% acrylamide in 1.5 M Tris (pH 8.8) for the stacking and resolving gels, respectively. The running buffer solution used contained 25 mM Tris and 190 mM glycine (pH 8.3). Samples (15 lL) were mixed with the loading buffer, as described below. Electrophoresis was performed at 120 V for 105 min, and the gels were then washed three times for 20 min in sodium acetate buffer (50 mM, pH 5.0) and stained with 10 mM DMP until the activity bands attained the appropriate intensity to be photographed. The loading buffer contained 50 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, and 10% (v/v) glycerol. The samples were not heated before loading. 2.8. Protein identification Samples of supernatant from N001-induced cultures were concentrated using the Amicon Ultra Ultracel-3000 MWCO Centrifugal Filter Device (Millipore, Billerica, MA). The extracellular proteins were separated by SDS–PAGE gel electrophoresis and electroblotted onto ProBlott polyvinylidene difluoride membranes (Applied Biosystems). Electroblotting was performed in a transfer buffer
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A. Parenti et al. / Bioresource Technology 133 (2013) 142–149 Table 1 Simple phenol composition of the wheat straw extract. Concentration (lM)
RT (min)
Compound
mg/L
SD
%
3.63 8.98 9.84 10.40 13.24 14.59 18.37
Gallic acid Vanillic acid 4-Hydroxybenzaldehyde Syringic acid Vanillin p-Coumaric acid Ferulic acid
28.71 7.19 1.24 3.81 3.08 3.82 2.31
0.79 0.17 0.05 0.05 0.16 0.10 0.08
57.2 14.3 2.5 7.6 6.1 7.6 4.6
Biomass (g/l)
1.00
A
0.80
0.80
0.60
0.60
0.40
0.40
0.20
0.20
0.00
42.19 10.69 2.54 4.81 5.06 5.82 2.97
B
0.00 0
3
6
9
12
15
18
21
24
27
C
1.00 Biomass (g/l)
Culture
168.76 42.76 10.15 19.23 20.24 23.27 11.90
Shaking
Static 1.00
Extract
0
0.80
0.60
0.60
0.40
0.40
0.20
0.20
0.00
6
9
3
6
9
12
15
18
21
24
27
12 15 18 Time (days)
21
24
27
D
1.00
0.80
3
0.00 0
3
6
9
12
15
18
21
24
27
Time (days)
0
Fig. 1. Biomass (g/L) evolution in submerged cultures made in SMY (A and B) and M7GY (C and D) of the N001 (circles) and PC9 (triangles) strains cultivated in static (A and C) or shaking (B and D) conditions. The filled symbols represent the controls and the open symbols the induced cultures. The arrows indicate the induction times.
The biomass values attained in the control (i.e., without inductor) cultures were similar in the static and shaking conditions for both strains that were cultivated in the same medium, although the kinetics were different in the two culture types. In the shaking cultures, the stationary phase was entered after three to 6 days of culture, whereas in the static cultures, the onset of the stationary phase could be observed only after 12 (SMY) or 20 (M7GY) days of culture. The culture yield was higher in the rich SMY medium than in the minimal M7GY broth (0.22–0.26 g/L in M7GY and 0.45–0.62 g/L in SMY). A different and unexpected behavior was observed, however, upon addition of the inducer. Whereas no effect was observed when the inducer was added to shaking cultures in any of the two culture media (Fig. 1B and D), growth stimulation was observed when the inducer was added to the static cultures growing in both types of media. This effect nearly doubled the biomass accumulation in the N001 cultures performed in SMY. In fact, the PC9 and N001 induced cultures performed in SMY were those from which a higher biomass yield was recovered in all the experiments. To further characterize the macroscopic effect of the inducer on the growth, we monitored the variation of the pH and of the reducing sugar concentration in the supernatant of the induced and uninduced cultures. There was a trend toward an increase in the pH of the culture supernatant in all the cultures (i.e., different media and shaking vs. static conditions), as the pH changed from 6.0 to
8.0 (as limit values, data not shown). This variation was unaffected by the addition of inducer. An unexpected observation, however, was made in the shaken SMY cultures of the two strains; a sharp pH oscillation of two pH units (from 6.0 to 8.0 and back to 6.0) was observed during the first 6 days of culture that corresponded to the exponential growth of the culture. After the oscillation, when the cultures had already entered the stationary phase, the pH rose again to the final values of 8.0 within 6–10 days. The evolution of the reducing sugars detected in the supernatant was also monitored in the induced and control cultures (Fig. 2) and revealed different kinetics depending on the culture conditions, as was observed in the case of biomass accumulation. The reducing sugar concentration in the supernatant decreased steadily in the static cultures and precipitously in the shaking cultures. In fact, the timepoint at which a nearly undetectable reducing sugar level was attained in the shaking cultures matched the onset of the stationary phase, suggesting that sugar limitation was one of the main contributing factors in these cultures. However, in the case of the static cultures, the stationary phase was reached when reducing sugars were still detected in the cultures, suggesting that carbon source exhaustion was not the main reason for the growth arrest in these conditions. Although the addition of the wheat straw extract produced an increase in the biomass accumulated in the static but not in the shaking cultures (Figs. 1 and 2), this accumulation was
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A. Parenti et al. / Bioresource Technology 133 (2013) 142–149
Shaking
Static
A
Reducing Sugars (g/l)
8.00 6.00
6.00
4.00
4.00
2.00
2.00
0.00
0.00 0
3
6
9
12
15
18
21
24
27
C
12.00 Reducing Sugars (g/l)
B
8.00
0
10.00
8.00
8.00
6.00
6.00
4.00
4.00
2.00
2.00
0.00
0.00 0
3
6
9
12 15 18 Time (days)
21
24
27
6
9
3
6
9
12
15
18
21
24
27
12 15 18 Time (days)
21
24
27
D
12.00
10.00
3
0
Fig. 2. Free reducing sugar concentration (g/L) evolution in the supernatant of submerged cultures made in SMY (A and B) and M7GY (C and D) of the N001 (circles) and PC9 (triangles) strains cultivated in static (A and C) or shaking (B and D) conditions. The filled symbols represent the controls and the open symbols the induced cultures. The arrows indicate the induction times.
not solely the consequence of the sugars added to the extract because this addition did not produce an increase in the level of reducing sugars present in the culture (Fig. 2, panels 2B and D). Moreover, addition of the inducer produced a decrease, rather than an increase, in the levels of free reducing sugars in the static cultures (panels 2A and C), and there was no biomass increase after addition of the inducer to the shaking cultures (Fig. 1B and D)—which is important because sugar exhaustion may be a major cause for entering the stationary phase in this type of culture. Therefore, what could the effect of the inducer on mycelial growth be? In the static cultures, the mycelium floats on the culture surface, forming a cake that becomes more compact over time. By contrast, in shaking cultures, the mycelium forms flocks (pellets) that grow until reaching a certain size and then divide into new, smaller flocks. In the static cultures, incipient differentiation occurs, in which an aerial mycelium is formed and is fed by a basal mycelium that is in close contact with the culture medium. In the shaking cultures, this type of differentiation does not occur. Mycelium differentiation can be triggered by environmental inducer signals (Arjona et al., 2009). We hypothesize here that wheat straw extract triggers a type of differentiation process in the static cultures but not in the shaking cultures. In fact, the compactness of the mycelium cake increases after addition of the inducer, and the cultures tend to produce droplets of secondary metabolites and aromas under these conditions (data not shown). All of these observations indicate that the stationary phase was produced by different factors in the two culture media and conditions, that both types of stationary phase differ physiologically, and that the production of lignocellulolytic enzymes should be different, depending on the physiological conditions of the mushroom (Elisashvili and Kachlishvili, 2009; Soden and Dobson, 2001). 3.2. Ligninolytic activities in induced and uninduced cultures The kinetics of the laccase activity in the supernatant of the different cultures studied is presented in Fig. 3. The levels of phenol
oxidases and manganese peroxidases in the control cultures were very low, as reported previously (Castanera et al., 2012). The addition of the inducer, however, had a major effect on the production of laccase because it was followed by strong induction of the laccase activity recovered in the supernatant. Similar increases in laccase production upon the use of complex culture media lignocellulose compounds has been reported by other authors (Kahraman and Gurdal, 2002). This induction effect appeared to be cumulative, as well as culture and medium dependent. The largest increase was observed in the shaking cultures of N001 produced in SMY, although levels higher than 200 U/L were attained in many other cases. In the rich SMY medium, the induction effect was predominant in the dikaryotic strain N001; by contrast, in the minimal M7GY medium, the effect was similar in both strains or slightly higher in the case of the PC9 monokaryons. An unexpected result was obtained when the induction was studied using the monokaryotic strain PC9 in shaking SMY. Under these conditions, there was very little laccase induction in PC9, whereas a strong induction was observed in the dikaryotic strain N001. The laccase activity was inducible in PC9 when cultured in SMY, as a moderate but clearly noticeable laccase induction was observed for this strain under static growth. Furthermore, the laccase activity was similarly induced in N001 and PC9 cultures under shaking conditions when M7GY was used as the culture broth. Consequently, the inducer effect was dependent on the strain, culture medium and growing conditions. N001 is a dikaryon containing the genome of PC9 plus the genome of PC15. The differential behavior of the two strains in SMY could indicate the presence of genetic elements in PC15 that could relieve the inhibitory effect that compounds present in the SMY medium could have in the laccase production. Finally, the production of laccase activity upon the influence of the straw inducer cannot be correlated with the possible differentiation effect of this inducer because the larger laccase induction effects are produced in cultures where no differentiation should occur (shaking cultures performed in SMY).
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Shaking
Static
A
Laccase (U/l)
700 600
600
500
500
400
400
300
300
200
200
100
100 0
0 0
3
6
9
12
15
18
21
24
0
27
C
700 Laccase (U/l)
B
700
600
500
500
400
400
300
300
200
200
100
100
0
6
9
3
6
9
12
15
18
21
24
27
12 15 18 Time (days)
21
24
27
D
700
600
3
0 0
3
6
9
12 15 18 Time (days)
21
24
27
0
Fig. 3. Evolution of the laccase activity (U/L) recovered from the supernatant of submerged cultures made in SMY (A and B) and M7GY (C and D) of the N001 (circles) and PC9 (triangles) strains cultivated in static (A and C) or shaking (B and D) conditions. The filled symbols represent the controls and the open symbols the induced cultures. The arrows indicate the induction times.
The manganese peroxidase (MnP) activity recovered from the supernatant was low in all the tested experimental conditions compared with the laccase activity recovered under the same conditions (data not shown). No MnP activity was recovered from the M7GY cultures. In the SMY cultures, a 4-day MnP activity peak (>430 U/L) was observed in the N001 static cultures 6 days after the inducer addition (at culture day 18). In the rest of cultures and conditions, the MnP activity was low (<50 U/L), and there was no induction signal for this enzymatic activity. Moreover, in all cases, the MnP activity values were lower in the induced cultures than in the uninduced cultures. It is well known that laccase activity is induced by organic compounds (de Souza et al., 2004; Elisashvili et al., 2008; Santoyo et al., 2008) and metal ions (Palmieri et al., 2000); furthermore, both xenobiotic and metal-responsive elements in the laccase gene promoters have been described (Piscitelli et al., 2011). In most of the studies conducted with the addition of laccase inducers, the concentrations used have far exceeded those present in natural extracts. For example, Cu2+ concentrations of 150 lM (Palmieri et al., 2000) and phenolic compound concentrations of 250 lM (de Souza et al., 2004) were found to induce the enzymatic activity in different Pleurotus species. By contrast, the conditions tested in the present study were much milder. The copper concentration in M7GY is 0.04 lM, and the concentrations of ferulic acid and vanillin—the major laccase inducers present in the wheat straw according to de Souza et al. (2004)—in the culture medium after the addition of the straw were 2.97 and 5.06 lM, respectively. These results suggest that efficient laccase induction may be achieved solely with the addition of the products present in the water waste. 3.3. Laccase isoenzyme profiles The laccase isoenzymes recovered from the culture supernatants were studied by non-denaturing gel electrophoresis and in situ staining of the activity using DMP (Fig. 4). Three laccase isoenzyme bands with apparent molecular weights of 36.8, 52.2 and
77.9 kDa were observed, the patterns of which were more influenced by the culture conditions (shaking vs. static) than by the culture medium (SMY vs. M7GY). Two isoforms (36.8 and 52.2 kDa) appeared to be basally produced in the control static cultures, which confirmed the results presented in Fig. 3A and C. In this type of culture, addition of the inducer produced an increase in the signal and the appearance of smaller signals of higher molecular mass (77.9 kDa). In the case of the shaking cultures, on the contrary, the picture was quite different. There was no laccase signal in any of the uninduced samples, confirming the low enzymatic activity that was detected in these samples (Fig. 3B and D). Upon addition of the inducer, a strong signal was observed in the SMY cultures corresponding to the 36.8 kDa band, and a secondary band of 52.2 kDa was observed in the N001 M7GY cultures. The appearance of these bands was also consistent with the activity data presented in Fig. 3B and D. In the case of PC9 cultivated with shaking in M7GY without an inducer, a 52.2 kDa band was clearly observed in the zymograms (Fig. 4). This band disappeared when the inducer was added and was replaced by the aforementioned band of 36.8 kDa. Carefully analyzing the enzyme activity data, we observed a low but noticeable basal activity of laccase in these PC9 cultures (up to 20 U/L in uninduced cultures). Therefore, we concluded that this basal activity was performed by the 52.2 kDa isoenzyme that was later replaced by the 36.8 kDa isoenzyme. In summary, after addition of the inducer, a marked increase in the low-molecular-weight laccase activity was observed in the static cultures and a moderate increase in the activity was observed in the shaking cultures. These changes were qualitatively independent of the culture medium. Comparison of all these experiments revealed that the laccase isoenzyme profile is more complex in the static than in the shaking cultures. 3.4. Identification of the induced isoenzyme of 36.8 kDa The laccase isoenzyme with an apparent molecular mass of 36.8 kDa appeared to be constitutive in the uninduced static cul-
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SMY
M7GY
N001 12 M C I
14 C I
21 C
N001
PC9 24 I C
14
12 I
C
I
C
21 I
C
12
24 I
C
I
C
14 C
PC9
21 I
C
24 I
C
12 I
C
14 C
21 I
C
24 I
C
I
STA 77.9 52.2 36.8
SHK 77.9 52.2 36.8
Fig. 4. Zymograms of the laccase activity revealed with 2,6 DMP recovered from the supernatant of submerged cultures made in SMY and M7GY of the N001 and PC9 strains cultivated in static (upper panels) or shaking (lower panels) conditions. M indicates a molecular size marker, C lanes are the controls and I lines are the induced. The culture times at which the samples were harvested are indicated.
Fig. 5. Aminoacid sequence of the P. ostreatus laccase 10 where the peptides detected by peptidic footprint and peptide fragmentation of the 36.8 kDa laccase isoenzyme are highlighted within the boxes.
tures and inducible by the wheat straw extract in both static and shaking cultures. In the shaking cultures, the 36.8 KDa protein was the sole isoenzyme detectable after induction. To determine the corresponding laccase gene, the isoenzyme was extracted from non-denaturing gels, and the protein was identified by peptide fingerprinting and mass-spectrometry. Three peptides were identified by the combined analysis (Fig. 5) and blasted against SwissProt, yielding the best hit for a protein of 57 kDa with accession number Q12739. This protein was blasted to the P. ostreatus PC15 v2.0 database and corresponded unequivocally to Lacc10 (Evalue 0.0) (P. ostreatus v2.0 gene ID 1089723). This combined analysis allowed us to identify this isoform as the product of the lacc10 gene at a confidence level of p < 0.05 (sequence coverage of 10%). This gene, along with lacc2, is the most highly expressed gene and is the main source of laccase activity in submerged cultures performed in minimal medium (Castanera et al., 2012; Piscitelli et al., 2011). The electrophoretic mobility of this isoform did not correspond with that predicted for Lacc10 in P. ostreatus (56.8 kDa). This discrepancy possibly indicates that this enzyme is processed after its translation. In fact, we could not detect any peptide mapping to the first 200 monoacids of this protein. Alternatively, it is possible that the protein migrated to a lower apparent molecular weight under non-denaturing conditions, as has been described in the laccases of Schizophyllum commune and Armillaria mellea (Rehman and Thurston, 1992; Thurston, 1994) and explained by the higher molecule’s compactness and nonspherical shape.
4. Conclusion The effect of the addition of a wheat straw water extract (as a model for the wastewater effluents of straw industries) on the production of laccase activity by various types of submerged cultures of the white rot basidiomycete P. ostreatus was investigated. In all the conditions and strains tested, the inducer had a major and additive effect on the enzymatic activity recovered from the culture supernatants. The induction depended on the culture medium and conditions and was especially relevant to the activity of the Lacc10 isoenzyme. These results suggest the utility of these effluents for the production of ligninolytic enzyme cocktails.
Acknowledgements This work was supported by funds from the projects AGL201130495 of the Spanish National Research Plan; Bioethanol-Euroinnova, from the Autonomous Government of Navarre; and by additional institutional support from the Public University of Navarre. AP, EM, AR and AO carried out the enzymatic determinations. EC made the HPLC analyses. AO set up the straw-extraction protocol. MA and RC, carried out the protein analyses. AGP revised and edited the manuscript. FS supervised the statistics. LR led and coordinated the project. The manuscript was written by AP and AGP.
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Martínez, A.T., Speranza, M., Ruiz-Dueñas, F.J., Ferreira, P., Camarero, S., Guillén, F., Martínez, M.J., Gutiérrez, A., del Río, J.C., 2005. Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin. Int. Microbiol. 8, 195–204. Mattila, P., Könkö, K., Eurola, M., Pihlava, J.-M., Astola, J., Vahteristo, L., Hietaniemi, V., Kumpulainen, J., Valtonen, M., Piironen, V., 2001. Contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms. J. Agric. Food Chem. 49, 2343–2348. Membrillo, I., Sánchez, C., Meneses, M., Favela, E., Loera, O., 2008. Effect of substrate particle size and additional nitrogen source on production of lignocellulolytic enzymes by Pleurotus ostreatus strains. Bioresour. Technol. 99, 7842–7847. Palmieri, G., Giardina, P., Bianco, C., Fontanella, B., Sannia, G., 2000. Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Appl. Environ. Microbiol. 66, 920–924. Piscitelli, A., Giardina, P., Lettera, V., Pezzella, C., Sannia, G., Faraco, V., 2011. Induction and transcriptional regulation of laccases in fungi. Curr. Genomics 12, 104–112. Rehman, A.U., Thurston, C.F., 1992. Purification of laccase I from Armillaria mellea. J. Gen. Microbiol. 138, 1251–1257. Reis, F.S., Barros, L., Martins, A., Ferreira, I.C.F.R., 2012. Chemical composition and nutritional value of the most widely appreciated cultivated mushrooms: an inter-species comparative study. Food Chem. Toxicol. 50, 191–197. Ruiz-Duenas, F.J., Morales, M., Garcia, E., Miki, Y., Martinez, M.J., Martinez, A.T., 2009. Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases. J. Exp. Bot. 60, 441–452. Salmén, L., Burgert, I., 2009. Cell wall features with regard to mechanical performance. A review. Holzforschung 63, 121–129. Salvachúa, D., Prieto, A., López-Abelairas, M., Lu-Chau, T., Martínez, A.T., Martínez, M.J., 2011. Fungal pretreatment: an alternative in second-generation ethanol from wheat straw. Bioresour. Technol. 102, 7500–7506. Santoyo, F., González, A.E., Terrón, M.C., Ramírez, L., Pisabarro, A.G., 2008. Quantitative linkage mapping of lignin-degrading enzymatic activities in Pleurotus ostreatus. Enzyme Microb. Technol. 43, 137–143. Singh, R., Ahlawat, O.P., Rajor, A., 2012. Identification of the potential of microbial combinations obtained from spent mushroom cultivation substrates for use in textile effluent decolorization. Bioresour. Technol. 125, 217–225. Soden, D.M., Dobson, A.D., 2001. Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147, 1755–1763. Talebnia, F., Karakashev, D., Angelidaki, I., 2009. Production of bioethanol from wheat straw: an overview on pretreatment, hydrolysis and fermentation. Bioresour. Technol. 101, 4744–4753. Theerachat, M., Emond, S., Cambon, E., Bordes, F., Marty, A., Nicaud, J.M., Chulalaksananukul, W., Guieysse, D., Remaud-Simeon, M., Morel, S., 2012. Engineering and production of laccase from Trametes versicolor in the yeast Yarrowia lipolytica. Bioresour. Technol. 125, 267–274. Thurston, C.F., 1994. The structure and function of fungal laccases. Microbiology 140, 19–26. Vinoth Kumar, V., Prem Kumar, M.P., Thiruvenkadaravi, K.V., Baskaralingam, P., Senthil Kumar, P., Sivanesan, S., 2012. Preparation and characterization of porous cross linked laccase aggregates for the decolorization of triphenyl methane and reactive dyes. Bioresour. Technol. 119, 28–34.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Induction of laccase activity in the white rot fungus Pleurotus ostreatus using water polluted with wheat straw extracts Alejandra Parenti a, Elaia Muguerza a, Amaia Redin Iroz a, Alejandra Omarini a, Enma Conde b, Manuel Alfaro a, Raúl Castanera a, Francisco Santoyo a, Lucía Ramírez a, Antonio G. Pisabarro a,⇑ a b
Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Spain Department of Chemical Engineering, Faculty of Science, University of Vigo (Campus Ourense), Ourense, Spain
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Wheat straw water extracts induce
efficiently laccase activity in fungal cultures. " The wheat straw water extract inducer effect is accumulative. " The isoenzyme Lacc10 is the main responsible for the laccase induced activity. " Wheat industry polluted water effluents can be used to induce ligninolytic enzymes.
a r t i c l e
i n f o
Article history: Received 7 November 2012 Received in revised form 11 January 2013 Accepted 13 January 2013 Available online 30 January 2013 Keywords: Laccase activity Pleurotus ostreatus Wheat straw Inducer Wastewater effluent
a b s t r a c t The purpose of this work was to explore the use of polluted water effluents from wheat straw using industries as inducers of lignocellulolytic enzymatic activities in cultures of white rot basidiomycetes. For this purpose, we studied the effect of a wheat straw water extract on the evolution of the laccase activity recovered from submerged cultures of Pleurotus ostreatus made in different media and under various culture conditions. Our results demonstrated an accumulative induction effect in all the cultures and conditions tested. This induction is parallel to changes in the laccase electrophoretic profiles recovered from the culture supernatants. The isoenzyme that appeared to be mainly responsible for the laccase activity under these conditions was laccase 10, as confirmed by sequencing the induced protein. These results support the idea of using wheat straw effluents as inducers in liquid cultures of P. ostreatus mycelia for the production of ligninolytic enzymatic cocktails. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulose is the major reservoir of organic carbon on Earth. It is made up of three major polymers present in plant cell walls: cellulose, hemicellulose, and lignin. Cellulose is the predominant polysaccharide in plant residues, where it forms the main component of cell walls and is responsible for plant support. ⇑ Corresponding author. Tel.: +34 948169107; fax: +34 948169732. E-mail address: [email protected] (A.G. Pisabarro). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.072
Hemicellulose is a heteropolysaccharide composed of frequently branched hexoses, pentoses, uronic acid, and lower sugars held together by b1 ? 4 glycosidic bonds. Hemicellulose is easily hydrolyzable, in contrast with the more resistant cellulose moiety. Finally, lignin is a complex, amorphous, and optically inactive hydrophobic polymer. Lignin and hemicellulose surround and cement the cellulose fibers to strengthen the cell walls (Salmén and Burgert, 2009). White-rot fungi are unique organisms that are able to degrade the lignin moiety of lignocellulose, making cellulose available for
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other organisms and for industrial processes involved in the production of biofuels. There are two fungal strategies for degrading lignocellulose: white rot fungi primarily attack the lignin and leave the cellulose accessible, whereas brown rot fungi attack the cellulose without significantly degrading the lignin. White rot fungi have been extensively studied because of this ability to selectively degrade lignin (Kirk and Cullen, 1998). Pleurotus ostreatus (Jacq.: Fr.) Kumm. (Dikaryia, Basidiomycota, Agaricomicotina, Agaricales) is a white rot basidiomycete industrially cultivated on a variety of lignocellulosic agricultural wastes as an edible mushroom of high nutritional value (Mattila et al., 2001; Reis et al., 2012). This basidiomycete may be considered a model organism of the Pleurotus genus, which includes other species that are capable of growing on a variety of agricultural lignocellulosic wastes and that are able to degrade dyes and aromatic compounds. These fungi are useful for decontaminating wastes with a high content of this type of toxic compounds (Faraco et al., 2009; Martínez et al., 2005). P. ostreatus produces a large number of extracellular enzymes and can be used as a source of lignin-degrading enzymes for the biotreatment of wastes and effluents (Ruiz-Duenas et al., 2009), as well as for bio-pretreatment of lignocellulose for the production of second-generation biofuels (Talebnia et al., 2009). Lignin degradation or modification is a key step in the deconstruction of the lignocellulosic material (Vinoth Kumar et al., 2012). In P. ostreatus, this process involves enzymes such as laccases (Phenol oxidases, Lac, EC 1.10.3.2), manganese peroxidase (MnP, EC 1.11.1.13), and versatile peroxidases (VP, EC 1.11.1.16), whose activity is complemented by that of a number of accessory enzymes that include glyoxal oxidase (GLOX, EC 1.1.3) and arylalcohol oxidase (AAO, EC 1.1.3.7) (Hammel and Cullen, 2008). The biotechnological relevance of the lignin-degrading enzymes and, especially, the use of laccases in processes that are aimed at the degradation of water pollutants and involved in the pulp and paper industry has prompted research on methods for their efficient recombinant production (Theerachat et al., 2012) and for their stabilization in industrial processes (Vinoth Kumar et al., 2012). In vivo, the source of carbon and nitrogen in various culture media plays an important role in the production of ligninolytic enzymes (Elisashvili and Kachlishvili, 2009; Kaal et al., 1995). Laccases are induced by the presence of certain aromatic compounds or lignin-related phenolic derivatives such as ferulic acid, 2.5-xylidine, p-anisidine or veratryl alcohol (de Souza et al., 2004). Numerous studies have utilized agro-industry-derived products in the production of ligninolytic enzymes to be applied as alternatives for bioremediation (Karp et al., 2012; Kurt and Buyukalaca, 2012). Industries that use straw (i.e., industrial mushroom production) produce large volumes of wastewater that is highly polluted with straw extractives. To study the use of these effluents in applied processes, we investigated the effect of an aqueous straw extract as an inducer of ligninolytic enzymes in P. ostreatus cultures produced in different media and under various culture conditions. The present study is of relevance for establishing the culture conditions for the production of ligninolytic enzyme cocktails using natural wastes as feedstock.
2. Methods 2.1. Organisms and culture conditions Two P. ostreatus strains were used in this work: the dikaryotic N001 and the monokaryotic PC9 (Larraya et al., 1999, 2000). Both strains were maintained and grown in Petri dishes containing agar-malt (20 g/L malt extract, 15 g/L agar) (Eger et al., 1976), incubated at 25 °C in the dark and subcultured every 8 days.
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Two liquid culture media were used: SMY (10 g/L sucrose, 10 g/ L malt extract and 4 g/L yeast extract), and M7GY (10 g/L glucose, 2 g/L ammonium tartrate, 0.5 g/L MgSO47H2O, 1 g/L KH2PO4, 0.5 g/L KCl, 1 g/L yeast extract, and 1 mL/L of trace elements solution). The solution of trace elements contains the following: 0.1 g/L Na2B4O7H2O, 0.07 g/L ZnSO47H2O, 0.01 g/L CuSO45H2O, 0.01 g/L MnSO44H2O, 0.05 g/L FeSO47H2O, and 0.01 g/L (NH4)6Mo7O24H2O. The mycelium grown on the agar surface was used to inoculate 90 mL of liquid medium (either SMY or M7GY) in 250 mL Erlenmeyer flasks. These cultures were incubated for 24 h with orbital shaking (150 rpm) at 25 °C in the dark to produce the pre-inoculum I0. The 90 mL I0 pre-inocula were homogenized and added to 500 mL Erlenmeyer flasks containing 135 mL of the corresponding liquid culture medium to produce the inocula I1. These inocula were incubated for 48 h with orbital shaking (150 rpm) at 25 °C in the dark before being homogenized and used to start the experimental cultures. The experimental cultures were performed using 500 mL Erlenmeyer flasks containing 135 mL of culture medium inoculated with 15 mL of the corresponding I1 inoculum. All the cultures were incubated at 25 °C in the dark. The shaken cultures were placed in orbital shakers that were set at 150 rpm. Each experiment was performed using three biological replicas for the biomass and three independent measures of each replica for the enzymatic measurements. At the corresponding sampling times (days 3, 7, 12, 14, 19, 21 and 24 of culture), a portion of the culture’s supernatant was aseptically removed for measuring the enzymatic activities and the reducing sugar content. 2.2. Aqueous extract of wheat straw The wheat straw extract used as inducer was prepared as follows: 150 g of dry wheat straw was soaked in 1500 mL of distilled water and shaken in an orbital shaker at 150 rpm for 5 h at 25 °C. After this extraction time, the straw was eliminated by filtration through a Miracloth (Merck Millipore International), and the filtrate was centrifuged at 4500 rpm for 10 minutes at 20 °C. The supernatant was then sterilized at 121 °C for 20 min. The extract was kept frozen until use. For the induction experiments, 50 mL of the inducer extract was added to the experimental cultures described above. An equivalent volume of distilled water was added to the control cultures. The induction treatment was performed at culture days 12 and 21. 2.3. HPLC identification and quantification The samples were analyzed using an Agilent HPLC 1100 instrument equipped with a Waters Spherisorb ODS2 column (5 lm, 250 mm 4.6 mm) and diode-array detector (DAD detector), operating at 30 °C. The injection volume was 20 lL, and the flow rate was 1 mL/min. A non-linear gradient of solvent A (acetonitrile/5% [v/v] formic acid in water, 10:90) and solvent B (acetonitrile/5% [v/v] formic acid in water, 90:10) was used as follows: 0 min, 100% A; 40 min, 85% A, 15% B; 45 min, 100% B; 55 min, 100% B; 60 min, 100% A; and 65 min, 100% A. The samples were analyzed in triplicate. Phenolic compounds were identified by comparisons of the retention time and UV–visible spectral data with those of authentic compounds. Quantification was performed from calibration curves that were obtained using model compounds (Conde et al., 2011). 2.4. Evaluation of biomass and pH For the determination of the biomass, parallel cultures were prepared in 100 mL Erlenmeyer flasks containing 20 mL of medium, 2.2 mL of I1 inoculum and 7.4 mL of wheat straw extract for
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induced or distilled water for the control cultures. At the corresponding sampling times, three randomly selected flasks (biological replicas) were removed from the incubator, filtrated through a 10 lm nylon using a vacuum pump, and dried in an oven at 50 °C to a constant weight. The culture filtrate was used to monitor the pH of the culture. 2.5. Enzyme activity assays The laccase and manganese peroxidase enzymatic activities were determined colorimetrically as described by (Santoyo et al., 2008). The laccase activity was determined by detecting the product of the oxidation of 2.6-dimetoxiphenol (DMP, e468 = 49,600 M 1 cm 1). The reaction mixture contained 450 lL of the culture filtrate described above and 500 lL of 10 mM DMP in 100 mM acetate buffer (pH 5.0). The reaction was performed for 1 min at room temperature before the variation in the absorbance at 468 nm was recorded. After measuring the laccase activity, 50 lL of MnSO4 was added to the sample and incubated for 1 min before adding 30 lL of an H2O2 solution containing 22.5 lL of 30% (w/v) H2O2 in 10 mL of Milli-Q water, and the reaction was allowed to proceed for an additional minute before the A468 was measured. The manganese peroxidase (MnP) activity was estimated as the difference between the two activity measurements. One unit of enzyme activity was defined as the formation of 1 lmol of product per min. All the assays were performed in triplicate using a Shimadzu UV-1800 spectrophotometer. Samples were taken at days 3, 5, 7, 10, 12, 14, 17, 19, 21 and 24 of culture for both treatments.
(Tris 25 mM, glycine 192 mM and methanol 20%) at 100 V for 75 min at 4 °C. Afterwards, the membranes were stained using Coomassie Brilliant Blue G-250 (Sigma) to visualize the protein bands. Proteins bands migrating to 40 kDa were excised, washed twice with Milli-Q water for 10 min and stored at 20 °C in an Eppendorf tube containing Milli-Q water until further analysis. 2.9. Protein identification by peptide fingerprint and mass spectrometry (MALDI-TOF/TOF) These analyses were performed at the Proteomics Unit of the Genomics and Proteomics Centre (http://www.ucm.es/info/gyp/ proteomica/presentacion.htm) at the Complutense University of Madrid. The SDS–PAGE gel fragment was digested using trypsin. Peptide analyses were performed in duplicate by mass spectrometry MALDI-TOF to obtain the peptide fingerprint. Automatic fragmentation of the peptides was performed to obtain one to three peptides, which were analyzed by MS/MS. The protein identification was performed via peptide fingerprints (MS data), peptide sequences (MS/MS data) or a combination of both techniques, as well as by an automatic identification search by the GPS (Global Protein Server) attached to the TOF–TOF data. The identified peptides were blasted to the P. ostreatus PC15 v2.0 database (http://genome.jgipsf.org/PleosPC15_2/PleosPC15_2.home.html) for identifying the protein. 3. Results and discussion
2.6. Measurement of reducing sugars
3.1. Description of the growth of the induced and uninduced cultures
The reducing sugar content of the liquid culture medium in each of the conditions and treatments was determined with dinitrosalicylic acid reagent (DNS), using the colorimetric method described by Miller (1959). The reaction mixture containing 250 lL of distilled water, 250 lL of sample solution or glucose (1% w/v) and 1 mL of DNS reagent was heated for 5 min and then cooled to 4 °C. Subsequently, 9 mL of distilled water was added, the mixture was stirred, and the absorbance at 540 nm was recorded.
The industrial production of edible mushrooms produces large volumes of waste water that is highly polluted with compounds derived from the rinsing and wetting of the cereal straw used as the culture substrate. In this work, we investigated the use of this spent water as an inducer of ligninolytic enzymatic activity by the white rot edible basidiomycete P. ostreatus using liquid cultures of the fungus and a laboratory-produced wheat straw extract as inducer. The wheat extraction process was performed at 25 °C as a model for the extraction that occurs naturally while the mushrooms are being cultured under industrial conditions. As a preliminary step, we analyzed the composition of this extract using HPLC (Table 1). The principal extract was gallic acid, which represented 57.2% of the total mass of the extractable phenols. The dryweight concentration of the extract was 11.05 mg/L (SD 0.5 mg/L), and its pH was 6.3. To determine the effect of the wheat straw extract as an inducer of lignin-degrading activities in P. ostreatus, it was necessary first to determine the effect of the wheat straw extract on mycelial growth under the culture conditions of the assay. For this purpose, we monitored the accumulation of biomass in submerged cultures using the dikaryotic N001 and the monokaryotic PC9 P. ostreatus strains. The study was conducted using two different culture media (rich SMY and minimal M7GY), two culture conditions (shaking and static), and the presence or absence of added inducer (Fig. 1). Other recent studies have focused on the effect of the culture type (solid fermentation vs. submerged culture) (Castanera et al., 2012) of the solid substrate (Elisashvili et al., 2008; Karp et al., 2012; Kurt and Buyukalaca, 2012; Membrillo et al., 2008) on laccase production by P. ostreatus. By contrast, the use of microbial combinations obtained from spent mushroom cultivation substrates in the decolorization of textile effluents has been recently studied (Singh et al., 2012). We focused the present study on submerged cultures with the long-range goal of producing enzyme cocktails useful for the biological pretreatment of lignocellulosic wastes to be used for bioethanol production (Salvachúa et al., 2011).
2.7. Non-denaturing electrophoresis analysis Non-denaturing polyacrylamide gel electrophoresis (PAGE) was performed as described by Sambrook (1989), using 5% acrylamide in 0.5 M Tris (pH 6.8) and 10% acrylamide in 1.5 M Tris (pH 8.8) for the stacking and resolving gels, respectively. The running buffer solution used contained 25 mM Tris and 190 mM glycine (pH 8.3). Samples (15 lL) were mixed with the loading buffer, as described below. Electrophoresis was performed at 120 V for 105 min, and the gels were then washed three times for 20 min in sodium acetate buffer (50 mM, pH 5.0) and stained with 10 mM DMP until the activity bands attained the appropriate intensity to be photographed. The loading buffer contained 50 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, and 10% (v/v) glycerol. The samples were not heated before loading. 2.8. Protein identification Samples of supernatant from N001-induced cultures were concentrated using the Amicon Ultra Ultracel-3000 MWCO Centrifugal Filter Device (Millipore, Billerica, MA). The extracellular proteins were separated by SDS–PAGE gel electrophoresis and electroblotted onto ProBlott polyvinylidene difluoride membranes (Applied Biosystems). Electroblotting was performed in a transfer buffer
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A. Parenti et al. / Bioresource Technology 133 (2013) 142–149 Table 1 Simple phenol composition of the wheat straw extract. Concentration (lM)
RT (min)
Compound
mg/L
SD
%
3.63 8.98 9.84 10.40 13.24 14.59 18.37
Gallic acid Vanillic acid 4-Hydroxybenzaldehyde Syringic acid Vanillin p-Coumaric acid Ferulic acid
28.71 7.19 1.24 3.81 3.08 3.82 2.31
0.79 0.17 0.05 0.05 0.16 0.10 0.08
57.2 14.3 2.5 7.6 6.1 7.6 4.6
Biomass (g/l)
1.00
A
0.80
0.80
0.60
0.60
0.40
0.40
0.20
0.20
0.00
42.19 10.69 2.54 4.81 5.06 5.82 2.97
B
0.00 0
3
6
9
12
15
18
21
24
27
C
1.00 Biomass (g/l)
Culture
168.76 42.76 10.15 19.23 20.24 23.27 11.90
Shaking
Static 1.00
Extract
0
0.80
0.60
0.60
0.40
0.40
0.20
0.20
0.00
6
9
3
6
9
12
15
18
21
24
27
12 15 18 Time (days)
21
24
27
D
1.00
0.80
3
0.00 0
3
6
9
12
15
18
21
24
27
Time (days)
0
Fig. 1. Biomass (g/L) evolution in submerged cultures made in SMY (A and B) and M7GY (C and D) of the N001 (circles) and PC9 (triangles) strains cultivated in static (A and C) or shaking (B and D) conditions. The filled symbols represent the controls and the open symbols the induced cultures. The arrows indicate the induction times.
The biomass values attained in the control (i.e., without inductor) cultures were similar in the static and shaking conditions for both strains that were cultivated in the same medium, although the kinetics were different in the two culture types. In the shaking cultures, the stationary phase was entered after three to 6 days of culture, whereas in the static cultures, the onset of the stationary phase could be observed only after 12 (SMY) or 20 (M7GY) days of culture. The culture yield was higher in the rich SMY medium than in the minimal M7GY broth (0.22–0.26 g/L in M7GY and 0.45–0.62 g/L in SMY). A different and unexpected behavior was observed, however, upon addition of the inducer. Whereas no effect was observed when the inducer was added to shaking cultures in any of the two culture media (Fig. 1B and D), growth stimulation was observed when the inducer was added to the static cultures growing in both types of media. This effect nearly doubled the biomass accumulation in the N001 cultures performed in SMY. In fact, the PC9 and N001 induced cultures performed in SMY were those from which a higher biomass yield was recovered in all the experiments. To further characterize the macroscopic effect of the inducer on the growth, we monitored the variation of the pH and of the reducing sugar concentration in the supernatant of the induced and uninduced cultures. There was a trend toward an increase in the pH of the culture supernatant in all the cultures (i.e., different media and shaking vs. static conditions), as the pH changed from 6.0 to
8.0 (as limit values, data not shown). This variation was unaffected by the addition of inducer. An unexpected observation, however, was made in the shaken SMY cultures of the two strains; a sharp pH oscillation of two pH units (from 6.0 to 8.0 and back to 6.0) was observed during the first 6 days of culture that corresponded to the exponential growth of the culture. After the oscillation, when the cultures had already entered the stationary phase, the pH rose again to the final values of 8.0 within 6–10 days. The evolution of the reducing sugars detected in the supernatant was also monitored in the induced and control cultures (Fig. 2) and revealed different kinetics depending on the culture conditions, as was observed in the case of biomass accumulation. The reducing sugar concentration in the supernatant decreased steadily in the static cultures and precipitously in the shaking cultures. In fact, the timepoint at which a nearly undetectable reducing sugar level was attained in the shaking cultures matched the onset of the stationary phase, suggesting that sugar limitation was one of the main contributing factors in these cultures. However, in the case of the static cultures, the stationary phase was reached when reducing sugars were still detected in the cultures, suggesting that carbon source exhaustion was not the main reason for the growth arrest in these conditions. Although the addition of the wheat straw extract produced an increase in the biomass accumulated in the static but not in the shaking cultures (Figs. 1 and 2), this accumulation was
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Shaking
Static
A
Reducing Sugars (g/l)
8.00 6.00
6.00
4.00
4.00
2.00
2.00
0.00
0.00 0
3
6
9
12
15
18
21
24
27
C
12.00 Reducing Sugars (g/l)
B
8.00
0
10.00
8.00
8.00
6.00
6.00
4.00
4.00
2.00
2.00
0.00
0.00 0
3
6
9
12 15 18 Time (days)
21
24
27
6
9
3
6
9
12
15
18
21
24
27
12 15 18 Time (days)
21
24
27
D
12.00
10.00
3
0
Fig. 2. Free reducing sugar concentration (g/L) evolution in the supernatant of submerged cultures made in SMY (A and B) and M7GY (C and D) of the N001 (circles) and PC9 (triangles) strains cultivated in static (A and C) or shaking (B and D) conditions. The filled symbols represent the controls and the open symbols the induced cultures. The arrows indicate the induction times.
not solely the consequence of the sugars added to the extract because this addition did not produce an increase in the level of reducing sugars present in the culture (Fig. 2, panels 2B and D). Moreover, addition of the inducer produced a decrease, rather than an increase, in the levels of free reducing sugars in the static cultures (panels 2A and C), and there was no biomass increase after addition of the inducer to the shaking cultures (Fig. 1B and D)—which is important because sugar exhaustion may be a major cause for entering the stationary phase in this type of culture. Therefore, what could the effect of the inducer on mycelial growth be? In the static cultures, the mycelium floats on the culture surface, forming a cake that becomes more compact over time. By contrast, in shaking cultures, the mycelium forms flocks (pellets) that grow until reaching a certain size and then divide into new, smaller flocks. In the static cultures, incipient differentiation occurs, in which an aerial mycelium is formed and is fed by a basal mycelium that is in close contact with the culture medium. In the shaking cultures, this type of differentiation does not occur. Mycelium differentiation can be triggered by environmental inducer signals (Arjona et al., 2009). We hypothesize here that wheat straw extract triggers a type of differentiation process in the static cultures but not in the shaking cultures. In fact, the compactness of the mycelium cake increases after addition of the inducer, and the cultures tend to produce droplets of secondary metabolites and aromas under these conditions (data not shown). All of these observations indicate that the stationary phase was produced by different factors in the two culture media and conditions, that both types of stationary phase differ physiologically, and that the production of lignocellulolytic enzymes should be different, depending on the physiological conditions of the mushroom (Elisashvili and Kachlishvili, 2009; Soden and Dobson, 2001). 3.2. Ligninolytic activities in induced and uninduced cultures The kinetics of the laccase activity in the supernatant of the different cultures studied is presented in Fig. 3. The levels of phenol
oxidases and manganese peroxidases in the control cultures were very low, as reported previously (Castanera et al., 2012). The addition of the inducer, however, had a major effect on the production of laccase because it was followed by strong induction of the laccase activity recovered in the supernatant. Similar increases in laccase production upon the use of complex culture media lignocellulose compounds has been reported by other authors (Kahraman and Gurdal, 2002). This induction effect appeared to be cumulative, as well as culture and medium dependent. The largest increase was observed in the shaking cultures of N001 produced in SMY, although levels higher than 200 U/L were attained in many other cases. In the rich SMY medium, the induction effect was predominant in the dikaryotic strain N001; by contrast, in the minimal M7GY medium, the effect was similar in both strains or slightly higher in the case of the PC9 monokaryons. An unexpected result was obtained when the induction was studied using the monokaryotic strain PC9 in shaking SMY. Under these conditions, there was very little laccase induction in PC9, whereas a strong induction was observed in the dikaryotic strain N001. The laccase activity was inducible in PC9 when cultured in SMY, as a moderate but clearly noticeable laccase induction was observed for this strain under static growth. Furthermore, the laccase activity was similarly induced in N001 and PC9 cultures under shaking conditions when M7GY was used as the culture broth. Consequently, the inducer effect was dependent on the strain, culture medium and growing conditions. N001 is a dikaryon containing the genome of PC9 plus the genome of PC15. The differential behavior of the two strains in SMY could indicate the presence of genetic elements in PC15 that could relieve the inhibitory effect that compounds present in the SMY medium could have in the laccase production. Finally, the production of laccase activity upon the influence of the straw inducer cannot be correlated with the possible differentiation effect of this inducer because the larger laccase induction effects are produced in cultures where no differentiation should occur (shaking cultures performed in SMY).
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Shaking
Static
A
Laccase (U/l)
700 600
600
500
500
400
400
300
300
200
200
100
100 0
0 0
3
6
9
12
15
18
21
24
0
27
C
700 Laccase (U/l)
B
700
600
500
500
400
400
300
300
200
200
100
100
0
6
9
3
6
9
12
15
18
21
24
27
12 15 18 Time (days)
21
24
27
D
700
600
3
0 0
3
6
9
12 15 18 Time (days)
21
24
27
0
Fig. 3. Evolution of the laccase activity (U/L) recovered from the supernatant of submerged cultures made in SMY (A and B) and M7GY (C and D) of the N001 (circles) and PC9 (triangles) strains cultivated in static (A and C) or shaking (B and D) conditions. The filled symbols represent the controls and the open symbols the induced cultures. The arrows indicate the induction times.
The manganese peroxidase (MnP) activity recovered from the supernatant was low in all the tested experimental conditions compared with the laccase activity recovered under the same conditions (data not shown). No MnP activity was recovered from the M7GY cultures. In the SMY cultures, a 4-day MnP activity peak (>430 U/L) was observed in the N001 static cultures 6 days after the inducer addition (at culture day 18). In the rest of cultures and conditions, the MnP activity was low (<50 U/L), and there was no induction signal for this enzymatic activity. Moreover, in all cases, the MnP activity values were lower in the induced cultures than in the uninduced cultures. It is well known that laccase activity is induced by organic compounds (de Souza et al., 2004; Elisashvili et al., 2008; Santoyo et al., 2008) and metal ions (Palmieri et al., 2000); furthermore, both xenobiotic and metal-responsive elements in the laccase gene promoters have been described (Piscitelli et al., 2011). In most of the studies conducted with the addition of laccase inducers, the concentrations used have far exceeded those present in natural extracts. For example, Cu2+ concentrations of 150 lM (Palmieri et al., 2000) and phenolic compound concentrations of 250 lM (de Souza et al., 2004) were found to induce the enzymatic activity in different Pleurotus species. By contrast, the conditions tested in the present study were much milder. The copper concentration in M7GY is 0.04 lM, and the concentrations of ferulic acid and vanillin—the major laccase inducers present in the wheat straw according to de Souza et al. (2004)—in the culture medium after the addition of the straw were 2.97 and 5.06 lM, respectively. These results suggest that efficient laccase induction may be achieved solely with the addition of the products present in the water waste. 3.3. Laccase isoenzyme profiles The laccase isoenzymes recovered from the culture supernatants were studied by non-denaturing gel electrophoresis and in situ staining of the activity using DMP (Fig. 4). Three laccase isoenzyme bands with apparent molecular weights of 36.8, 52.2 and
77.9 kDa were observed, the patterns of which were more influenced by the culture conditions (shaking vs. static) than by the culture medium (SMY vs. M7GY). Two isoforms (36.8 and 52.2 kDa) appeared to be basally produced in the control static cultures, which confirmed the results presented in Fig. 3A and C. In this type of culture, addition of the inducer produced an increase in the signal and the appearance of smaller signals of higher molecular mass (77.9 kDa). In the case of the shaking cultures, on the contrary, the picture was quite different. There was no laccase signal in any of the uninduced samples, confirming the low enzymatic activity that was detected in these samples (Fig. 3B and D). Upon addition of the inducer, a strong signal was observed in the SMY cultures corresponding to the 36.8 kDa band, and a secondary band of 52.2 kDa was observed in the N001 M7GY cultures. The appearance of these bands was also consistent with the activity data presented in Fig. 3B and D. In the case of PC9 cultivated with shaking in M7GY without an inducer, a 52.2 kDa band was clearly observed in the zymograms (Fig. 4). This band disappeared when the inducer was added and was replaced by the aforementioned band of 36.8 kDa. Carefully analyzing the enzyme activity data, we observed a low but noticeable basal activity of laccase in these PC9 cultures (up to 20 U/L in uninduced cultures). Therefore, we concluded that this basal activity was performed by the 52.2 kDa isoenzyme that was later replaced by the 36.8 kDa isoenzyme. In summary, after addition of the inducer, a marked increase in the low-molecular-weight laccase activity was observed in the static cultures and a moderate increase in the activity was observed in the shaking cultures. These changes were qualitatively independent of the culture medium. Comparison of all these experiments revealed that the laccase isoenzyme profile is more complex in the static than in the shaking cultures. 3.4. Identification of the induced isoenzyme of 36.8 kDa The laccase isoenzyme with an apparent molecular mass of 36.8 kDa appeared to be constitutive in the uninduced static cul-
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SMY
M7GY
N001 12 M C I
14 C I
21 C
N001
PC9 24 I C
14
12 I
C
I
C
21 I
C
12
24 I
C
I
C
14 C
PC9
21 I
C
24 I
C
12 I
C
14 C
21 I
C
24 I
C
I
STA 77.9 52.2 36.8
SHK 77.9 52.2 36.8
Fig. 4. Zymograms of the laccase activity revealed with 2,6 DMP recovered from the supernatant of submerged cultures made in SMY and M7GY of the N001 and PC9 strains cultivated in static (upper panels) or shaking (lower panels) conditions. M indicates a molecular size marker, C lanes are the controls and I lines are the induced. The culture times at which the samples were harvested are indicated.
Fig. 5. Aminoacid sequence of the P. ostreatus laccase 10 where the peptides detected by peptidic footprint and peptide fragmentation of the 36.8 kDa laccase isoenzyme are highlighted within the boxes.
tures and inducible by the wheat straw extract in both static and shaking cultures. In the shaking cultures, the 36.8 KDa protein was the sole isoenzyme detectable after induction. To determine the corresponding laccase gene, the isoenzyme was extracted from non-denaturing gels, and the protein was identified by peptide fingerprinting and mass-spectrometry. Three peptides were identified by the combined analysis (Fig. 5) and blasted against SwissProt, yielding the best hit for a protein of 57 kDa with accession number Q12739. This protein was blasted to the P. ostreatus PC15 v2.0 database and corresponded unequivocally to Lacc10 (Evalue 0.0) (P. ostreatus v2.0 gene ID 1089723). This combined analysis allowed us to identify this isoform as the product of the lacc10 gene at a confidence level of p < 0.05 (sequence coverage of 10%). This gene, along with lacc2, is the most highly expressed gene and is the main source of laccase activity in submerged cultures performed in minimal medium (Castanera et al., 2012; Piscitelli et al., 2011). The electrophoretic mobility of this isoform did not correspond with that predicted for Lacc10 in P. ostreatus (56.8 kDa). This discrepancy possibly indicates that this enzyme is processed after its translation. In fact, we could not detect any peptide mapping to the first 200 monoacids of this protein. Alternatively, it is possible that the protein migrated to a lower apparent molecular weight under non-denaturing conditions, as has been described in the laccases of Schizophyllum commune and Armillaria mellea (Rehman and Thurston, 1992; Thurston, 1994) and explained by the higher molecule’s compactness and nonspherical shape.
4. Conclusion The effect of the addition of a wheat straw water extract (as a model for the wastewater effluents of straw industries) on the production of laccase activity by various types of submerged cultures of the white rot basidiomycete P. ostreatus was investigated. In all the conditions and strains tested, the inducer had a major and additive effect on the enzymatic activity recovered from the culture supernatants. The induction depended on the culture medium and conditions and was especially relevant to the activity of the Lacc10 isoenzyme. These results suggest the utility of these effluents for the production of ligninolytic enzyme cocktails.
Acknowledgements This work was supported by funds from the projects AGL201130495 of the Spanish National Research Plan; Bioethanol-Euroinnova, from the Autonomous Government of Navarre; and by additional institutional support from the Public University of Navarre. AP, EM, AR and AO carried out the enzymatic determinations. EC made the HPLC analyses. AO set up the straw-extraction protocol. MA and RC, carried out the protein analyses. AGP revised and edited the manuscript. FS supervised the statistics. LR led and coordinated the project. The manuscript was written by AP and AGP.
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