Production of lignocellulolytic enzymes from floriculture residues using Pleurotus ostreatus

1. Grupo de Biotecnología Ambiental e Industrial (GBAI), Laboratorio de Biotecnología Aplicada, Departamento de Microbiología, Pontificia Universidad Javeriana. Bogotá, Colombia. 2. Grupo de Biotecnología Ambiental e Industrial (GBAI), Laboratorio de Microbiología ambiental, Departamento de Microbiología, Pontificia Universidad Javeriana. 3. Grupo de Procesos Químicos y Bioquímicos, Laboratorio de Ingeniería Química, Departamento de Ingeniería Química y Ambiental, Universidad Nacional de Colombia. Bogotá, Colombia. 4 Grupo de Procesos Químicos y Bioquímicos, Laboratorio de Ingeniería Química, Departamento de Ingeniería Química y Ambiental, Universidad Nacional de Colombia. Bogotá, Colombia.


Introduction
Forestry and agricultural activities such as the production of paper, lumber and many other agro industries generate large amounts of waste.These residues are considered an environmental contamination issue.A high percentage of these residues are indiscriminately burned, generating greenhouse gases, and their outdoor decomposition generates leaching into water bodies and promotes the proliferation of pests (Asocolflores 2002).
Lignocellulose is the major structural component of woody and non-woody plants and represents an important source of renewable organic matter.Lignocellulose contains lignin, hemicellulose and cellulose, compounds with chemical properties Production of lignocellulolytic enzymes from floriculture residues using Pleurotus ostreatus original article that increase the biotechnological potential of the lignocellulosic substrate (Malherbe & Cloete 2002).
To exploit the potential of lignocellulose as feedstock to produce high added-value products, a physical, chemical or biological pretreatment process is always necessary to modify its three-dimensional structure.Some biological pretreatments have been studied, among these; degradation using enzymes produced by fungi (Stajić et al. 2006, Membrillo et al. 2008, Bettin et al. 2011, Větrovský et al. 2013), including the lignocellulosic degradation using P. ostreatus (Isikhuemhen & Mikiashvilli 2009, Quevedo-Hidalgo 2012, Thakur et al. 2013).
Cellulases and other enzymes with similar or complementary enzymatic pathways are used in the food, beer, wine, animal feed, textile, laundry and paper industries, as well as in agriculture research (Bhat 2000).Laccase and manganese peroxidase are enzymes that have potential industrial applications because these enzymes remove oxidized phenolic compounds and a variety of aromatic compounds.The conversion of toxic compounds and textile dyes present in wastewater and the bleaching and removal of lignin from wood fibers and non-wood are two examples of industrial applications of these enzymes (Lundell et al. 2010).
Floriculture is one of the main activities of the agricultural sector in Colombia, with a 13 % share of the world trade of flowers, ranking second after the Netherlands, which has a 42 % market share.According to Asocolflores (2010), more than 50 types of flowers are produced over an area of 7,266 hectares in Colombia.The main types of flowers are Rosa (33 %), carnations (12.7 %), mini carnations (6.7 %), Chrysanthemum and pompons (8 %) (Asocolflores 2010).The floriculture sector generates approximately 1.5 m 3 /ha/day or between 0.8 and 1 t/ha/week of vegetable residues, depending on the type of flower and the production cycle.Approximately 9,591 t/month of residues are generated by Rosa production while 2,325 t/month are generated by Chrysanthemum cultures.Residues result from the cutting, postharvesting and planting startup steps.
Considering the volume of floriculture residues generated in Colombia and the fact that enzyme production from floriculture wastes has not been reported previously, in this article, we evaluate enzyme production from the degradation of lignocellulose residues of Rosa and Chrysanthemum using P. ostreatus.The production of laccase, manganese peroxidase, endoglucanase, exoglucanase and β-glucosidase was evaluated after a submerged fermentation process using flower residues and P. ostreatus.Enzyme extracts containing all the enzymes mentioned previously were obtained from the two assessed residues.The process using the Chrysanthemum residues produced the extracts with the highest enzymatic activities.

Materials and methods
Cultivos del Norte (Tocancipá, Colombia) supplied the Chrysanthemum and Rosa residues (stems and leaves).The residues were analyzed for lignin, cellulose and hemicellulose contents using the neutral detergent fiber method (NDF) (Van Soest et al. 1991).Phosphorus, calcium, potassium, zinc, magnesium, copper and manganese content was assessed using flame atomic absorption spectrophotometry (Thermo Scientific ICE3000) following the procedure described in ASTM D1971-11 Practice B for digestion and ASTM D4691-11 for measuring the elements.Nitrogen content was determined by the procedure described in ASTM D5373-08 (Table 1).Before degradation, the residues were dried at 104 °C for 24 h and milled to a particle size smaller than 1 mm.All reagents used in this study were of analytical grade.P. ostreatus (HPB/P3) were obtained from the Biotechnology Laboratory of the Pontificia Universidad Javeriana (Bogotá, Colombia).Stock cultures of this fungus were maintained on wheat bran agar at 4 °C.

Enzyme production from Chrysanthemum and Rosa
Two assays were performed for each residue, one with and one without inductors addition (in total four cultures, each one run by triplicate) to evaluate enzyme production from Chrysanthemum and Rosa.
Fungal inoculum were grown on a static culture at 30 ± 2 °C in 250 mL Erlenmeyer flasks containing 50 mL of the following (per liter): 50 g of floriculture wastes, 2 g of yeast extract, 5 g of peptone, 0.075 g of MnSO4.H2O, 1 g of KH2PO4, 0.5 g of MgSO4.7H20, and 175 g of wheat bran.This medium was inoculated with ten fungal disks (5 mm diameter) taken from the edge of the mycelium grown on bran extract agar in petri dishes (7-day old).After seven days, mycelial biomass was obtained and was inoculated (5 % of dry mycelium based on substrate volume) to the degradation stage.
The degradation of Chrysanthemum and Rosa residues was performed in a 1.5 L bioreactor (TECFERM, Procesos Biotecnológicos y Medioambientales, Bogotá, Colombia) with an agitation speed of 240 ± 5 rpm and aeration rate of 2 vvm for 24 h, with P. ostreatus in submerged fermentation under the optimum conditions as determined by Quevedo-Hidalgo et al. (2012) for reducing sugars.The optimum conditions for Chrysanthemum and Rosa residues were 6.3 % (w/v) concentration and pH 5.6, and 11.8 % (w/v) concentration and pH 5.6, respectively (Quevedo-Hidalgo et al. 2012).For the assays without inductors, Cu +2 and Mn +2 concentrations were the same as those originally present in each residue.For the assays with inductors, copper sulfate and manganese sulfate were added simultaneously; each concentration was 7.5 mM.
The culture was separated from the fungal biomass and insoluble components of the culture medium by centrifugation (at 22,000 g for 15 min) at 4 °C, followed by filtration using a Millipore membrane (0.45 µm).The extract was stored at -20 °C and it was used to determine the presence of the following enzymes: laccase, manganese peroxidase (MnP), endoglucanase, exoglucanase, and β-glucosidase.
Finally, because Chrysanthemum and Rosa residues contain Zn +2 and Cu +2 , we added Zn +2 or Cu +2 to final concentrations of 20, 40 and 80 ppm to assess laccase were compared to the activities obtained without the addition of these metals.

Evaluation of the solid fraction obtained from the degradation process of Chrysanthemum and Rosa residues
Because the solid fraction of the residues can contain adsorbed enzymes, they were washed 5 times by centrifugation with distilled water (at 1,500 g for 10 min at 4 °C).The remaining solids were re-suspended with 50 mM sodium citrate buffer pH 5.0 and stirred at 150 rpm for two hours at 4 °C.The supernatant was separated by centrifugation at 10,800 g for 10 min at 4 °C, then filtered through a 0.45 µm Millipore membrane and used to measure the enzymatic activities.The enzymatic activity of the solid fraction was related with the extract to determine the percentage of adsorption.The process was evaluated with and without the addition of these inductors to establish the effects of manganese and copper sulfate on the enzymatic activity.
The activity of endoglucanase (EC 3.2.1.4)was measured with carboxymethylcellulose (CMC) (low viscosity, Sigma, St. Louis, MO, USA).The reaction mixture contained 1 mL of 2 % (w/v) substrate in 50 mM sodium citrate buffer (pH 5.0), and 1 mL sample (Ghose 1987).The reaction mixture was incubated at 40 °C for 60 min; the reaction was stopped by boiling the sample for 5 minutes.The sample was then cooled for 5 minutes and centrifuged at 2,500 g for 15 min.The amount of reducing sugars released was determined using the Somogy-Nelson method (Somogy 1952).A standard curve was obtained by using glucose.One unit of enzyme activity was defined as the amount of enzyme releasing 1 µmol of reducing sugars as glucose per min.
The activity of exoglucanase (EC 3.2.1.91)was determined using p-nitrophenyl-β-D-cellobioside (PNPC, Sigma, St. Louis, MO, USA).The reaction mixture contained 160 µL of 5 mM PNPC in a 50 mM sodium acetate buffer (pH 5.0) and a 40 µL sample.Reaction mixtures were incubated at 40 °C for 60 min.The reaction was stopped by adding 100 µL of 0.5 M sodium carbonate, and the absorbance was read at 400 nm.Enzyme activity was calculated by using a standard curve of p-nitrophenol.One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of p-nitrophenol per min (Valášková & Baldrian 2006).

Statistical analysis
To establish significant differences between Chrysanthemum and Rosa residues as substrates and with and without inductors, we performed an ANOVA with Tukey test using a confidence interval of 95 % (α = 0.05).Data were tested for normality (Shapiro-Wilk) and subjected to the Levene test for homogeneity in the group variances.In all the cases, differences of p < 0.05 were regarded as statistically significant.SAS 9.0 for Windows was used.

Enzyme production from Chrysanthemum and Rosa
According to the ANOVA, statistically significant differences were observed for the enzymatic activity of laccase, exoglucanase and β-glucosidase (p<0.0001), and for endoglucanase (p=0.0007), between the different cultures completed.
The enzymatic activities of Chrysanthemum residues were higher than those of Rosa residues, except for endoglucanase (Figure 1).Regarding the enzymes involved in the degradation of lignin, laccase and MnP, the maximum enzyme activity for both residues was obtained by adding inductors.The behavior of endoglucanase and β-glucosidase was different and the maximum enzyme activity for both residues was obtained without inductors (Figure 1).The addition of inductors did not increase exoglucananse enzymatic activity.
Enzyme activities for the degradation of Chrysanthemum residues were 4,693.4U/L and 673.8 U/L for laccase and for MnP, respectively (Table 2).Laccase activity was 4.8 times higher when the inductors Cu +2 and Mn +2 were added, while the MnP activity was 62 % higher.On the other hand, the behavior for endoglucanase and β-glucosidase was different, and the presence of inductors decreased their activity.The addition of inductors reduced the

Enzyme
With
Regarding the enzymes involved in the degradation of lignin from Rosa residues, the maximum enzyme activity was 2,640 U/L for laccase and 439.2 U/L for MnP when inductors were used (Table 3).When Cu +2 and Mn +2 were added, the activities of these enzymes increased six times and 36 %, respectively.As described for Chrysanthemum residues, the addition of inductors reduced the enzymatic activity of endoglucanase and β-glucosidase.Their activities were reduced about 14 % and 28 %, respectively.Unlike the behavior observed with Chrysanthemum residues, exoglucanase activity augmented 7 % when inductors were added.
Cu +2 addition increased laccase activity in all cases (Table 4), whereas the addition of Zn +2 decreased it in both Chrysanthemum and Rosa extracts (Table 4).The maximum reduction in activity was found in Rosa extract.

Evaluation of the solid fraction obtained from the degradation process of Chrysanthemum and Rosa residues
The presence of enzymes in the solid fraction was assessed to check whether they were adsorbed in the residue.For both residues, in all cases, the enzyme activity was higher in the liquid fraction than in the solid (Table 5).The percentage of adsorbed laccase enzymes was low for both residues.Approximately 30 % of the endoglucanase was adsorbed for both residues.

Enzyme production from Chrysanthemum and Rosa
The laccase activity obtained in this study was higher than the activity reported by other researchers using P. ostreatus and measuring the enzymatic activity with ABTS.For example Isikhuemhen & Mikiashvilli (2009) obtained 164.6 U/L for laccase from wheat straw mixed with solid waste obtained from the anaerobic digestion of litter from the commercial production of broiler chickens.Another example is the study developed by Větrovský et al. (2013); these authors reported 16.7 mU/ml when laccase was produced from a synthetic CLN medium with cellulose as the source of carbon and high C/N ratio typical of wood.The highest laccase activity obtained by these authors was 206.8 mU/ml using Fomes fomentarius.
As previously mentioned, the addition of inductors increased the laccase activity 4.8 times with Chrysanthemum and 6 times with Rosa.Other authors have reported the effect of the inductors, copper and manganese, in improving laccase activity depending on the substrate, organism, crop, and the concentrations of these metals (Giardina et al. 2000, Baldrian et al. 2005, Stajić et al. 2006, Lu & Ding 2010).For example, Baldrian et al. (2005) reported that average laccase activity increases 79 % with Cu addition in cultures of P. ostreatus on wheat straw.Stajić et al. (2006) reported peaks of laccase activity, grown on dry mandarin peels, which depended on the concentration of Cu +2 , the time of cultivation and the P. ostreatus strain.
According to the characterization of the waste (Table 1), the copper contents in the residues of Rosa and Chrysanthemum were 8.99 ppm and 24.6 ppm, respectively.This difference explains why laccase activity was higher when Chrysanthemum residues were used as a substrate.Copper induces laccase activity, as reported in cultures of Trametes versicolor (Collins 1996), P. ostreatus (Palmieri 2000), Trametes pubescens (Galhaup & Haltrich 2001), and Botryosphaeria sp.(Dekker & Barbosa 2001), and P. ostreatus (Tinoco et al. 2011).However, some studies have showed that high copper concentrations can be inhibitory (Murugesan et al. 2009).The lignin content is another factor that may influence the difference found in the laccase activity of these residues.The lignin content was 17.5 % for Chrysanthemum residues and 14.8 % for Rosa residues (Table 1).Tinoco et al. (2011) observed that the addition of lignin to the culture medium had a strong positive influence on the production of laccase by P. ostreatus.When copper was added simultaneously, the activity of that enzyme was higher than the sum of the values determined using each inductor separately; this shows a synergistic effect on the production of the enzyme (Tinoco et al. 2011).The lignin and copper contents in Chrysanthemum residues are higher than those in Rosa residues (Table 1), explaining why laccase activities were higher in the former residue.
The MnP activity increased 62 % and 36 % with the addition of inductors for Chrysanthemum and Rosa residues, respectively.The addition of Mn +2 can have positive or negative effects on the MnP activity; as a result, this metal must be evaluated for each process.For example, Giardina et al. (2000) produced MnP using P. ostreatus in a solid culture of poplar sawdust; they reported an increase in the MnP activity as a consequence of the addition of Mn +2 to the culture.On the other hand, Baldrian et al. (2005), reported that the MnP activity was negatively affected by the presence of Mn +2 , Cu +2 and Pb +2 and that the MnP activity was lower in the presence of Mn +2 than in the absence of this metal when P. ostreatus was cultured on lignocellulose.
Concerning the evaluation of the enzymes involved in the degradation of cellulose, the results show that the enzyme complex necessary to hydrolyze cellulose was present in the obtained extract.According to the results of this work, the activity of β-glucosidase stresses the production from Chrysanthemum and Rosa residues, with or without inductors, because the activities are higher than the values reported by other studies.For instance, Větrovský et al. ( 2013) obtained an activity of 3.88 U/ml using a synthetic medium while the value obtained in our work was 9,513 U/L using Chrysanthemum residue without inductors.The activities of endo-and exo-glucanases were different with and without inductors, so we can affirm that copper and manganese have effect on the activity of these enzymes, although this is less important than the effect of these inductors on laccase and MnP.
The activity of β-glucosidase was 14 % lower when the process was performed with the addition of copper and manganese.That reduction can be explained because copper is an inhibitor as reported by Lin et al. (2010).These authors found that the presence of Cu +2 (0.1 mM) inhibited the activity of β-glucosidase, and this activity was not increased by the addition of Mn +2 (0.1 mM).
Enzymes that exhibited activity after the degradation of the Chrysanthemum and Rosa residues have several industrial uses.For example, laccase is used in industrial processes such as bleaching of stains and the degradation of polycyclic aromatic hydrocarbons; β-glucosidase also plays an important role in the degradation of lignocellulosic substrates.The addition of β-glucosidase speeds up sugar production, which is the end product of the degradation of lignocellulosic substrates.This enzyme catalyzes cellobiose hydrolysis, avoiding inhibition of endoglucanase and exoglucanase synthesis (Kumar et al. 2008).Therefore, establishing the production conditions to obtain β-glucosidase with high activity (9,513 U/L) and productivity (396.4U/L/h) is an essential contribution to the study of the hydrolysis of cellulose.
Because the effects of copper and zinc on laccase activity can be either positive or negative, we conducted experiments adding these metals to laccasecatalyzed reactions.Results showed that laccase activity increased with the addition of Cu +2 and diminished with the addition of Zn +2 .The effect of zinc can be explained by the results of Murugesan et al. (2009).Their results indicated that laccase activity increased when Ca +2 , Cu +2 , Co +2 and Zn +2 ions were added to the culture medium at low concentrations (up to 1 mM), and decreased if the concentrations of Cu +2 and Zn +2 were increased to 5 mM.It is then possible that the initial concentration of Zn +2 in the residues evaluated in this work might be higher than the limit inhibitory concentration and that the addition of zinc reduced laccase activity.Although the inhibitory limit concentration was not determined in this work, the initial concentration of Zn +2 was higher in Rosa than in Chrysanthemum (Table 1).The previous explains why the reduction in laccase activity is higher in the former residue with the same final Zn +2 concentration in the enzymatic reaction.

Evaluation of the solid fraction obtained from the degradation process of Chrysanthemum and Rosa residues
The adsorption percentages for exoglucanase and MnP depend largely on the type of waste.In general, more enzymes were adsorbed on Rosa than on Chrysanthemum residues (Table 5); other researchers observed similar behavior.For example, Valášková & Baldrian (2006) reported the presence of laccase, MnP, exoglucanase, and β-glucosidase in the solid fraction of a liquid culture of P. ostreatus in a synthetic medium; the percentages of adsorption of these enzymes were 38.8 %, 5.4 %, 87 % and 65 %, respectively.As in this study, the percentage of adsorption of exoglucanase was the highest.This behavior can be explained by the irreversible adsorption of cellulases by the residue, specifically by cellulose, and the addition of surfactants is recommended.Surfactants change the surface tension between the liquid and solid phases and minimize the binding of cellulases to cellulose (Sun & Cheng 2002).

Conclusion
In this study, ligninases and cellulases were produced from Chrysanthemum and Rosa residues using P. ostreautus in a submerged culture.Under the conditions evaluated here, the best substrate was the Chrysanthemum residue; the obtained extract had higher enzymatic activity than extracts from the Rosa residue.The highest enzymatic activities for both residues were obtained for laccase and β-glucosidase and the lowest for endoglucanase.The activity of β-glucosidase was higher without the presence of inductors, whereas laccase activity was enhanced by the presence of inductors.

Fig. 1 .
Fig. 1.Enzymatic activity as a function of the residue used as substrate and the presence of inductors.Culture 1: Chrysanthemum residues with inductors.Culture 2: Chrysanthemum residues without inductors.Culture 3: Rosa residues with inductors.Culture 4: Rosa residues without inductors.Laccase MnP Endoglucanase β-glucosidase Exoglucanase .Letters represent Tukey homogeneous subsets.a* corresponds to the culture with the best activity for each enzyme, followed in order by a, b, c and d.

Table 1 .
Characterization of the Chrysanthemum and Rosa residues.activity in the extracts.Each assay was performed by triplicate.Laccase activities with added Zn +2 or Cu +2

Table 2 .
Enzyme activity in the liquid extract after the degradation of the Chrysanthemum residue using P. ostreatus.

Table 3 .
Enzyme activities in the liquid extract after the degradation of the Rosa residue using P. ostreatus.

Table 4 .
Enzyme activities in the liquid extract after the degradation of the Rosa residue using P. ostreatus.

Table 5 .
Enzyme activities in the solid fraction after the degradation of the Chrysanthemum and Rosa residues using P. ostreatus.Data are the average of three measurements ±SD.