Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune

Accepted Manuscript Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune Eka Metreveli, Eva Kachlishvili, Steven W. Singer, Vladimir Elisashvili PII: DOI: Reference:

S0960-8524(17)30816-7 http://dx.doi.org/10.1016/j.biortech.2017.05.148 BITE 18174

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

23 March 2017 21 May 2017 23 May 2017

Please cite this article as: Metreveli, E., Kachlishvili, E., Singer, S.W., Elisashvili, V., Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune, Bioresource Technology (2017), doi: http://dx.doi.org/ 10.1016/j.biortech.2017.05.148

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Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune

Eka Metreveli a, Eva Kachlishvili a, Steven W. Singer b,c, Vladimir Elisashvili a,


a

Agricultural University of Georgia, 240 David Agmashenebeli alley, 0159 Tbilisi,

Georgia b

c

Joint BioEnergy Institute, Emeryville, California 94608, USA

Biological Systems and Engineering Division, Lawrence Berkeley National

Laboratory, Berkeley, CA 94720

_______________


Corresponding author at: Agricultural University of Georgia, Kakha Bendukidze

University campus, 240 David Agmashenebeli alley, 0159 Tbilisi, Georgia E-mail address: [email protected] (V. Elisashvili)

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ABSTRACT

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnococcus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7 U/mL total cellulase, 142 U/mL endoglucanase, 104 U/mL xylanase, and 5.2 U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus.

Keywords: White-rot basidiomycetes Co-cultivation Cellulase Xylanase Submerged fermentation

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1. Introduction

The saprotrophic white-rot basidiomycetes (WRB) are the major agents of wood bio-deterioration in natural ecosystems, producing hydrolytic and lignin-modifying enzymes for wood deconstruction (Baldrian and Valaškova, 2008; Hatakka and Hammel, 2010). Unlike the soil fungi, the WRB have capacity to completely mineralize native lignocellulosic biomass without association with other microbes. Plant polysaccharide-degrading enzymes, such as endo-1,4-ß-D-glucanase (EC 3.2.1.4), exo1,4-ß-D-glucanase (EC 3.2.1.91), and xylanase (EC 3.2.1.8), are of fundamental importance for efficient depolymerization of plant carbohydrate polymers to fermentable sugars and they are essential for the food, textile, pulp and paper industries, agriculture, and lignocellulosic bio-refineries (Elisashvili, 1993; Sukumaran et al., 2005; Phitsuwan et al., 2013; Juturu and Wu, 2014). Recently, several species of WRB have been studied in submerged and solid-state fermentation of lignocellulose, some of which have shown high potential for the production of individual groups of hydrolytic enzymes under appropriate cultivation conditions (Elisashvili et al., 2009; Agnihotri et al., 2010; Jagtap et al., 2014; Juturu and Wu, 2014). Thus, Coprinellus disseminatus produced 469 U/mL of alkali-thermo-tolerant xylanase along with negligible cellulase activity (Agnihotri et al., 2010). Armillaria gemina secreted up to 146 U endoglucanase/mL, 15 U β-glucosidase/mL, and 1.72 U FPA/mL (Jagtap et al., 2014). Saccharification of the pre-treated poplar biomass with Celluclast 1.5L and Novozyme 188 supplemented with the A. gemina enzyme preparation was 20% more efficient in the production of reducing sugars compared to the commercial enzymes alone. Moreover, Jagtap et al. (2013) achieved very high β-glucosidase activity (45.2 U/mL) in

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submerged cultivation of Pholiota adiposa in optimized medium containing rice straw and corn steep powder. However, physiological studies of glycoside hydrolases production by WRB are still limited. Various approaches and strategies have been widely explored to accelerate the cellulase and xylanase synthesis and to increase their yield (Elisashvili, 1993; Sukumaran et al., 2005; Agnihotri et al., 2010; Jagtap et al., 2014; Juturu and Wu, 2014; Rytioja et al., 2014; Yoon et al., 2014). However, in these studies, WRB were cultivated as mono-cultures. Little is known about the production of hydrolases by dual basidiomycetes cultures or in co-cultivation with other microorganisms. Recently, modulation of lignocellulose-deconstructing enzyme activity during co-cultivation of WRB with other basidiomycetes fungi and soil microfungi. The experimental results indicated that harnessing interspecies interactions is a promising strategy to increase fungal protein production. Hu et al. (2011) reported that high activities of β-glucosidase, α-cellobiohydrolase, β-galactosidase and laccase were produced by co-cultivation of ascomycete Aspergillus oryzae with WRB Phanerochaete chrysosporium. Co-culturing of P. chrysosporium with Trichoderma reesei QM6a and T. reesei Rut C30 induced the production of cellulolytic proteins and stimulated expression of hemicellulolytic enzymes (Adav et al., 2012). However, cellulase and xylanase production in submerged co-cultivation of two WRB has not been addressed, except in two publications (Kalyani et al., 2013; Metreveli et al., 2014). In particular, the synergistic interaction of Agaricus arvensis and Sistotrema brinkmannii led to 2.3-3-fold higher total cellulase activity than that produced by either single culture (Kalyani et al., 2013). The aim of this study was to investigate how the interactions between two WRB affect the production of cellulases and xylanases to identify a fungal pair that efficiently

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secretes both enzymes, and to establish the co-cultivation conditions ensuring an enhanced production of the target enzymes. Cellulase and xylanase production by mono and dual cultures of four WRB was evaluated under submerged fermentation conditions. Differential regulation of cellulase and xylanase activity was revealed upon interaction of individual fungi. Co-cultivation conditions were optimized for high hydrolase accumulation by Irpex lacteus and Schizophyllum commune, the best producers of cellulase and xylanase, respectively. Moreover, the enzyme complex derived from the mixed culture showed complementary interactions between cellulases, xylanases and βglucosidases, ensuring increased hydrolysis of wheat straw polysaccharides.

2. Materials and methods

2.1. Organisms and inoculum preparation

The following WRB deposited in the Agricultural University of Georgia basidiomycetes culture collection have been used in this study: Irpex lacteus BCC 104, Pycnoporus coccineus BCC 310, Schizophyllum commune BCC 632, and Trametes hirsuta BCC 17. Fungal inocula were prepared by growing the mycelia on a rotary shaker at 150 rpm and 27 oC in 250 mL flasks containing 100 mL of standard medium containing per liter: 15 g glucose, 1 g KH2PO4, 0.5 g MgSO4·7H2O, 3 g peptone, 2 g yeast extract, pH 6.0. After 7 days of fungal cultivation mycelial pellets were harvested and homogenized with a Waring laboratory blender.

2.2. Paired interactions on agar plates

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For compatibility testing, interspecies interaction of the tested fungi was performed in 9 cm Petri dishes containing 15 ml of above-mentioned agarized medium containing 20 g/L dried wheat-based distillers’ grains with solubles (DDGS) instead glucose. The DDGS contained 35.1% crude protein, 4.7% starch, 6.4% cellulose, 17.3% hemicellulose. Agar pieces with pre-grown mycelia of individual I. lacteus, P. coccineus, and T. hirsuta were inoculated aseptically on the same agar plate (2 cm from the edge of the plate) and paired with S. commune at opposite sides (40-45 mm apart) to allow interaction of the WRB at the middle of the Petri dish (see Fig. 1). The plates were sealed with parafilm and incubated at 27 oC. Once the mycelial fronts had contacted each other, the cultivation was continued for 2 additional days and then different regions of paired cultures were analyzed for enzyme activity. The mycelial fronts with the interspecies interaction zone as well as incubation zones within the individual fungal mycelium (30–40 mm from the interaction zone) were removed from paired cultures by using a sterile scalpel. The total wet weight of mycelial agar from each region was 200 mg. The materials were transferred in 5 mL 50 mM citrate buffer (pH 5.0), cut and shaken gently for 30 min. The extracts were clarified by centrifugation at 4 oC (10,000 x g, 10 min) and clear supernatants were used for enzyme activity measurements.

2.3. Cultivation conditions in the shake-flasks experiments

Submerged cultivation was carried out on the rotary shaker Innova 44 (New Brunswick Scientific, USA) at 150 rpm and 27 oC in 250 mL flasks containing 50 mL

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of medium containing per liter: 1 g KH2PO4, 0.5 g MgSO4·7H2O, 5 g peptone, 5 g yeast extract, crystalline cellulose or/and lignocellulosic materials at various concentrations as a growth substrate. Avicel was used at the concentration of 10 g/L, banana and mandarin peels, wheat bran and wheat straw were used at a concentration of 40 g/L were used to study the growth substrate effect on enzyme production by I. lacteus 104 and S. commune 632 co-culture. When the effect of nitrogen sources on the enzyme production was studied, inorganic and organic nitrogen compounds were used at the final concentration of 20 mM. The medium pH was adjusted to 6.0 before sterilization. The flasks were inoculated with 5 mL of mycelial homogenate. After 5, 8, 11, and 14 days of growth, samples (1 mL) were taken from the flasks and solids were separated by centrifugation (Eppendorf 5417R, Germany) at 10,000 x g for 10 min at 4 oC. The supernatants were analyzed for pH, reducing sugars and enzyme activities.

2.4. Cultivation in fermenter

To scale up the cellulase and xylanase production by the dual culture of I. lacteus BCC 104 and S. commune BCC 632, their cultivation was performed in the 7 l fermenter LiFlus GX (South Korea) with three Rushton impellers. The fermenter was filled with 5 L of the optimized medium containing per liter: 20 g mandarin peels, 10 g Avicel, 1 g KH2PO4, 0.5 g MgSO4, 5 g yeast extract, 1 g (NH4)2SO4. Polypropylene glycol 2000 (3 mL) was added as an antifoam agent and the medium pH was adjusted to 5.0. The fermenter equipped with pH, temperature and pO2 probes was sterilized (121 o

C, 40 min) and inoculated with homogenized mycelia of individual fungi (250 mL

each). Fermentation was performed without baffles at 27 oC and at the constant airflow

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rate of 1 v/v/min. During the fermentation process samples were collected daily and analyzed for enzyme activity. After 7 days of fermentation, the fungal biomass was separated from the culture liquid by successive filtration and centrifugation at 6,000 rpm for 20 min at 4 oC. The enzyme preparation was isolated from the culture liquid by precipitation with (NH4)2SO4 at 70% saturation and the precipitate was dissolved in 0.05 M acetate buffer (pH 5.5).

2.5. Wheat straw saccharification

The wheat straw (WS) enzymatic hydrolysis experiments were conducted in standard conditions (0.1 mM citrate buffer, pH 5.0, 50°C) with a gentle agitation at 150 rpm for 48 h. Base-pretreated (1.5% NaOH) wheat straw with a composition 64.6% cellulose, 17.3% hemicellulose, and 9.4% lignin was used as the biomass substrate. Accelerase 1500 (Genencor, Finland) and crude enzyme preparations isolated (as described above) from the supernatants of individual I. lacteus cultures and co-cultures of I. lacteus with S. commune in fermenter were used as enzyme sources. The reaction mixture (10 mL) contained 200 mg substrate, 10 or 20 FPU/g substrate, as well as 300 µg tetracycline and 200 µg cycloheximide to prevent microbial contamination. Mixtures with heat inactivated enzymes served as controls. Samples were taken from the reaction mixture after 24 and 48 h of saccharification, heated in a boiling water bath for 2 min and then centrifuged at 10,000 x g for 5 min at 4 oC. The supernatants were analyzed for reducing sugars using the dinitrosalicylic acid reagent method (Miller, 1959).

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2.6. Analytical methods

After fermentation, the separated biomass was dried to constant weight at 60 oC. To determine protein concentration, the fungal biomass was first treated with 0.5% trichloroacetic acid for 15 min in a boiling water bath and then centrifuged at 10000 g for 10 min, washed with 96% ethanol, and dried at 60 oC to constant mass. The nitrogen content in fungal biomass was estimated by the Kjeldahl procedure with a Nessler reagent using the coefficient of 4.38. The supernatants obtained after removal of biomass were analyzed for protein concentration and enzymatic activity. Protein concentration was determined using the Bradford Reagent (Serva, Heidelberg, Germany) according to the manufacturer’s instructions. The total cellulase activity (filter paper activity, FPA) was measured with Whatman filter paper No. 1 according to IUPAC recommendations (Ghose, 1987). Endoglucanase (CMCase) activity was assayed using 1% low-viscosity carboxymethyl cellulose in 50 mM citrate buffer (pH 5.0) at 50 oC for five minutes (Ghose, 1987). Xylanase activity was determined using 1% birch wood xylan (Roth 7500) in 50 mM citrate buffer (pH 5.0) at 50 oC for 10 min (Bailey et al., 1992). Glucose and xylose standard curves were used to calculate the cellulase and xylanase activities. In all assays, the release of reducing sugars was measured using the dinitrosalicylic acid reagent method (Miller, 1959). One unit of enzyme activity was defined as the amount of enzyme, releasing 1 µmol of reducing sugars per minute.

2.7. Statistical analysis

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All experiments were performed twice using three replicates each time. The results are expressed as the mean ± SD. The mean values as well as standard deviations were calculated by the Excel program (Microsoft Office 2010 package) and only values of p ≤ 0.05 were considered as statistically significant.

Fig. 1

3. Results and discussion

3.1. Effect of co-cultivation

Preliminary screening of recently isolated WRB from various Georgian ecosystems revealed several promising producers of hydrolases in their submerged cultivation in crystalline cellulose-containing medium. Among these isolates, I. lacteus, P. coccineus, and T. hirsuta were distinguished with high endoglucanase and filter paper (FP) activities, whereas S. commune expressed the highest xylanase and β-glucosidase activities. Hydrolysis of the polysaccharides in lignocellulosic biomass can be improved by creating a cocktail of hydrolytic enzymes derived from different fungi. Therefore, production of cellulase and xylanase by dual cultures of three cellulase producers with the best xylanase and β-glucosidase producer was compared with the monocultures. The paired growth and compatibility of the three cellulase producers with S. commune and their enzymatic activity were compared on the agar plates containing DDGS as a carbon source. All fungi grew rapidly and their mycelial fronts met within

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5-6 days followed by the formation of a mycelial barrage across the center line of the plate. T. hirsuta and I. lacteus formed dense mycelia in the interface zone with S. commune while an inhibition zone of 1 mm between fronts of P. coccineus and S. commune was visible. Moreover, some thickening of P. coccineus mycelia was also observed nearby the contact zone. After formation of a barrage, the fungal cultivations were continued for two days and then the secreted enzymes were extracted. The measurement of endoglucanase and xylanase activities revealed distinct differences in enzymatic activity among individual WRB (Fig. 1). The CMCase activity varied from 0.11 U/mL to 0.18 U/mL while the xylanase activity ranged from 0.19 U/mL to 1.65 U/mL. The CMCase activity at the interaction zone between I. lacteus and S. commune increased more than 2-3-fold as compared with those from the individual fungi regions. No significant differences were observed in activity between regions and interaction zone of P. coccineus with S. commune, while endoglucanase activity in interaction zone of T. hirsuta with S. commune appeared to be significantly lower than that of individual fungi. For xylanase, enzymatic activity in the interaction zone was 2-4-fold higher compared with those detected in the regions of S. commune partner fungi.

Table 1

Subsequently, the mono- and dual cultures were assessed for their potential to produce hydrolases in submerged cultivation in the synthetic medium containing 1.5% crystalline cellulose. The fungi showed good growth in the form of pellets in this medium and they were capable of synthesizing the main hydrolytic enzymes

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participating in lignocellulosic polysaccharide degradation (Table 1). Among the individual fungi, P. coccineus 310 accumulated the highest endoglucanase (65.6 U/mL,) and FP (5.6 U/mL) activities. S. commune 632 appeared to be the weakest producer of these enzymes but this strain secreted the highest xylanase activity. The enzymatic activity measurements obtained from each culture were compared to those from the mixed cultures. They indicated that co-cultivation of the tested fungi in the cellulose-based medium generally was unfavorable for the target enzymes production. The mixed cultivation of S. commune 632 with P. coccineus 310 dramatically reduced CMCase (14-fold) and FPase (11-fold) production. In contrast, the co-culturing of S. commune 632 with I. lacteus 104 resulted in less than a two-fold decrease in CMCase and FPase activity. It is worth noting that the levels of xylanase activities achieved in co-cultivation of three fungi with S. commune 632 were comparable with those in their monocultures, although significantly lower than that in the xylanase producer monoculture. Supplementation of the Avicel-based medium with milled mandarin peels promoted accumulation of all hydrolases activities by mono- and mixed cultures (Table 1). In particular, cellulase and xylanase activities of T. hirsuta 17 increased more than twofold as compared with those in the cellulose-containing medium. A significant increase in enzymatic activity was observed in the cultivation of S. commune 632 in response to mandarin peels supplementation. Of the dual cultures, the highest cellulase activity was again observed in the co-cultivation of S. commune 632 with I. lacteus 104 while the best xylanase activity was detected in the dual culture of S. commune 632 and P. coccineus 310. It is interesting that even in the co-cultivation of P. coccineus 310 or T. hirsuta 17 with S. commune 632 cellulases and xylanase levels increased compared with

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those in the only cellulose-containing medium. Nevertheless, cultivation of these fungal pairs gave lower cellulase activity as compared to single cultures. Co-cultivation of I. lacteus 104 with S. commune 632 provided the highest levels of CMCase and FP activities while the dual culture’ xylanase activity was higher than that in the monoculture of I. lacteus 104. Analysis of the data received revealed several distinctive features of mixed cultures. The fungal response to co-culture was species-dependent and enzymatic activity of the dual cultures depended on the combination of fungi. Specifically, a significant decrease of cellulase activity was observed upon interaction of S. commune 632 with P. coccineus 310 or T. hirsuta 17, while an increase in enzymatic activity was observed in the mixed cultivation of I. lacteus 104 with S. commune 632 in the 1.5% Avicel + 4% MP-containing medium. We can assume that S. commune was antagonistic to P. coccineus 310 or T. hirsuta 17 in co-cultivation, but this fungus was compatible with I. lacteus 104 and their synergistic interaction led to a higher cellulase activity as compared with the activity produced by either monoculture. This finding is in agreement with several reports showing changes in cellulase production during interspecies interactions by different fungi (Kalyani et al., 2013; Metreveli et al., 2014). In particular, Metreveli et al. (2014) described co-cultivation of two strains of the same species (I. lacteus 98 and 104) in Avicel containing medium produced an additive effect on the cellulase and xylanase activities of monocultures. At the same time, cocultivation of I. lacteus 104 with Trametes versicolor decreased enzymatic activity whereas mixed cultivation of I. lacteus 104 with P. coccineus 310 increased enzymatic activity. Other studies have demonstrated a species-specific change in lignin-modifying

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enzymatic activity in response to WRB co-cultivation (Chi et al., 2007; Dong et al., 2012). The WRB co-culturing effect is somewhat enzyme-specific. In the co-cultivation of P. coccineus 310 and T. hirsuta 17 with S. commune 632 in the Avicel-based medium, the CMCase and FP activities decreased 5-14-fold as compared with their monocultures whereas the xylanase activity of paired P. coccineus 310 decreased only by 24% while that of T. hirsuta 17 increased by 32% (Table 1). Moreover, in the 1.5% Avicel + 4% mandarin peels-containing medium production of cellulase by dual cultures of P. coccineus 310 or T. hirsuta 17 with S. commune 632 was still inhibited whereas xylanase secretion was increased. Other authors demonstrated that the solid-state cofermentation of corn stover by I. lacteus CD2 and Auricularia polytricha AP only slightly increased cellulase activity compared with monocultures, while the xylanase activity increased 5.0- and 3.0-fold compared with that of A. polytricha AP and I. lacteus CD2 monocultures, respectively (Ma et al., 2011). Ma and Ruan (2015) demonstrated that the fungal–fungal interaction in co-fermentation of agricultural wastes significantly enhanced the secretion of Coprinus comatus laccase, however, the production of CMCase and xylanase by Trichoderma reesei was decreased. The mechanism of this phenomenon is unclear, although it is possible that the co-cultivation of these fungi resulted in appearance of specific compounds accelerating the individual enzymes synthesis. The extent of interspecies interaction also depended on the availability of carbon source. As shown in Table 1, co-cultivation of S. commune 632 with P. coccineus 310 or T. hirsuta 17 in the 1.5% Avicel-based medium resulted in a dramatic decrease of CMCase and FPase activity. One possible explanation is that in the presence of

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recalcitrant crystalline cellulose a severe antagonistic competition for this unique growth substrate between the fungi took place. To prevent this competition and to create conditions favoring the both fungi growth at the initial stage of co-cultivation, the Avicel containing medium was enriched with the mandarin peels (40 g/L) since this material contains 34% total sugars and organic acids and favors cellulase and xylanase production by various groups of WRB in both submerged and solid-state fermentation (Elisashvili, 1993; Elisashvili et al., 2009; Metreveli et al., 2014). Indeed, this medium ensured an abundant growth of four WRB and significantly increased the enzymatic activity of all monocultures, especially in the cultivation of S. commune and T. hirsuta 17 (Table 1). Moreover, the presence of mandarin peels in the Avicel-containing medium favored hydrolase accumulation by all dual cultures although enzyme production in this medium was delayed during 3 days of S. commune 632 co-cultivation with other fungi, probably, because of catabolite repression by easily metabolizable sugars. By this time, the CMCase and xylanase activities, for example, in dual culture of I. lacteus 104 and S. commune 632 were 1.3 and 0.5 U/mL (data not shown), respectively, while the same enzymes activity of I. lacteus 104 + S. commune 632 in medium containing only Avicel achieved 13.2 and 6.8 U/mL, respectively.

Fig. 2 3.2. Effect of growth substrate

The dual culture of I. lacteus 104 and S. commune 632 was selected for the subsequent co-cultivation experiments as this pair provided the highest levels of

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CMCase and FP activities while their xylanase activity was higher than that in the monoculture of I. lacteus 104. The supplementation of nutrient medium with agroindustrial wastes/by-products to accelerate the cellulase and xylanase synthesis by individual fungi has been widely explored (Elisashvili, 1993; Agnihotri et al., 2010; Jagtap et al., 2014; Juturu and Wu, 2014; Rytioja et al., 2014; Yoon et al., 2014). The positive impact of mandarin peels on cellulase and xylanase production by the dual culture suggested that it would be interesting to elucidate the effect of other cheap lignocellulosic materials on enzyme accumulation. As shown in the Fig. 2, the enzymatic activity of the I. lacteus 104 and S. commune 632 co-culture strongly depended on the type of growth substrate in the medium. Among the growth substrates tested, mandarin peels followed by the banana peels provided the highest cellulase activity of the dual culture. However, neither mandarin peels nor other lignocellulosic materials generated cellulase and xylanase activities of the dual culture as high as the activities produced by cultivation on crystalline cellulose. In particular, the levels of CMCase and FPA accumulated in the presence of the lignocellulosic growth substrates appeared to be 2-8-fold lower than those in the Avicelbased medium. Wheat straw was the poorest growth substrate for cellulase production by these fungi, but it provided the highest xylanase activity of the dual culture, which slightly exceeded that in the cellulose-containing medium. It is worth noting that the dual culture growth in the presence of lignocellulosic materials was accompanied by a rapid elevation of the medium pH to 6.8-7.0 after 4 days of cultivation to pH 7.4-7.9 on the day 14 while in the Avicel-containing medium it varied between 5.1-5.6, near the optimal pH for cellulase and xylanase activity.

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Table 2

Since the mixture of mandarin peels and crystalline cellulose ensured an efficient secretion of both cellulase and xylanase by I. lacteus 104 and S. commune 632, the concentrations and ratio of these materials in the nutrient medium was varied to identify optimal conditions. Comparatively high activities of cellulase and xylanase were observed in medium containing 1% Avicel (Table 2). Supplementation of this medium with 2% mandarin peels favored accumulation of the target enzymes, increasing endoglucanase and xylanase activities more than two- and 3-fold, respectively. It should be noted that co-cultivation of the dual culture in the 1.0% Avicel + 2% MP-containing medium maintained pH range between 5.0 (4 days) and 5.3 (11 days), ensuring that coculture grew abundantly with a high level of protein production. Moreover, an optimal and abundant growth of I. lacteus 104 and S. commune 632 excluded an accumulation of sugars in the nutrient medium (their concentration did not exceed 0.3 mg/mL during entire period of co-fermentation) and prevented catabolite repression of the synthesis of the target enzymes. However, a further increase of the mandarin peels concentration to 4% significantly reduced activities of both enzymes, although they still were higher than those activities observed in the Avicel-containing medium. Unexpectedly, the elevation of the cellulose concentration from 1 to 2% negatively affected the hydrolase accumulation by the dual culture. The xylanase activity was lowered considerably in this medium. As shown in Table 2, supplementation of 2% Avicel-based medium with 2% mandarin peels significantly improved the cellulase and xylanase secretion by I. lacteus and S. commune. However, the levels of both enzymes activity were lower than those in the 1% Avicel + 2% mandarin peels-containing

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medium. Finally, a further increase in the concentration of mandarin peels to 4% in addition to 2% Avicel significantly inhibited enzyme secretion by the dual culture. The data received indicate that elevated concentrations of the cellulose and mandarin peels negatively affect the hydrolase synthesis by the dual culture. In this respect, the response of I. lacteus 104 and S. commune 632 to the high concentration of both substrates differed from that of I. lacteus 104 and P. coccineus 310 pair, which expressed the highest cellulase and xylanase activities at elevated concentrations of cellulose and mandarin peels (Metreveli et al. 2014). Moreover, addition of the supplemental growth substrate (mandarin peels) correlated with an increase in the enzymatic activity. These observations emphasize the diversity and complexity of the physiological processes involved in co-cultivation of individual WRB and hydrolase production. A synergistic effect on cellulase and xylanase secretion by I. lacteus 104 and S. commune 632 was observed by combining crystalline cellulose with mandarin peels at the optimal concentration and ratio as described above. Subsequent optimization of cellulase and xylanase production by this paired culture was performed using medium containing 1% Avicel and 2% mandarin peels.

Table 3 3.3. Effect of nitrogen source

The physiology of cellulase production by individual and co-cultivated WRB is still poorly investigated. A recent study has shown that cellulase and xylanase production by the paired I. lacteus 104 and P. coccineus 310 is highly influenced by various culture

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parameters (Metreveli et al., 2014). To further optimize the target enzymes production by I. lacteus 104 + S. commune 632, the effect of nitrogen sources on the fungal coculture growth and cellulase and xylanase production was studied. The data presented in the Table 3 demonstrated that the fungal biomass and cellulase activity increased in the presence of all tested nitrogen source as compared with the control without additional nitrogen. Of these sources, peptone, potassium and ammonium nitrate ensured a 46-48% increase in the fungal biomass yield. All the tested nitrogen sources increased the cellulase activity of the mixed culture of I. lacteus 104 and S. commune 632 as compared with the control medium, but differently affected the xylanase activity. Ammonium sulphate among inorganic compounds and casein hydrolysate among organic nitrogen sources provided the highest endoglucanase (128 U/mL and 132 U/mL, respectively) and total cellulase (5.3 and 5.0 U/mL, respectively) activities of the dual culture. The xylanase activity was slightly increased only in media supplemented with (NH4)2SO4 or casein hydrolysate, other nitrogen sources had no affect or lowered the enzymatic activity. However, the mixed culture productivity (enzyme activity/fungal biomass), for example, in the control and in the best ammonium sulphate or casein hydrolysate-containing media showed comparable values of CMCase (202, 189, 197 U/mg biomass protein, respectively) and FP (7.3, 7.8, 7.5 U/mg biomass protein, respectively) activities. These results suggest that an increase in cellulase activity correlates with increased fungal biomass accumulation due to addition of a nitrogen source.

Table 4

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Subsequently, the effect of nitrogen source concentration on the enzyme accumulation was studied at varying (NH4)2SO4 and casein hydrolysate concentrations in the nutrient medium (0 to 80 mM nitrogen). The data in Table 4 show that both nitrogen sources equally favored fungal growth and their biomass gradually increased with increasing nitrogen in the medium. Supplementation of the control medium with nitrogen sources at concentration of 10, 20, 40, and 80 mM as nitrogen resulted in the fungal biomass protein gains of 12-14%, 36-40%, 52-56%, and 56-60%, respectively, after 14 days of submerged fermentation. Between these two nitrogen sources, ammonium sulphate was preferable for the hydrolase production. Both CMCase and FP activities of the dual culture also gradually increased from 93.9 to 138.8-141.4 U/ml and from 3.9 to 5.9-6.0 U/ml, respectively, with an elevation of (NH4)2SO4 concentration from 0 to 40-80 mM nitrogen while the maximum xylanase activity was achieved at the 20 mM of salt concentration. When I. lacteus 104 and S. commune 632 were cocultivated in the medium containing casein hydrolysate the highest enzymatic activities were achieved at the nitrogen concentration of 20 mM. However, the enzymatic activity values in the medium appeared to be lower than those in the (NH4)2SO4-containing medium. Therefore, in the further experiments, ammonium sulphate at concentration of 40 mM was used as a nitrogen source.

Fig. 3

3.4. Effect of inoculum ratio, time and order of fungi pairing

21

Fig. 3 shows the results of enzyme production by mono and dual cultures of I. lacteus 104 and S. commune 632 in response to the pairing time of the fungi and the order of fungal inoculation. The enzymatic activity of both monocultures gradually increased and reached maximum after 11 days of fermentation (CMCase - 76 and 33 U/mL, xylanase – 70 and 381 U/mL, respectively) while the FPA (4.7 and 3.2 U/mL) reached a maximum at day 5. When both fungi were inoculated simultaneously at zero time and equal rate, endoglucanase and total cellulase activities of mixed culture was enhanced by 75 and 36%, respectively, as compared with those in the monoculture of I. lacteus 104 and the maximum of both CMCase and FP activities (133 U/mL and 5.6 U/mL, respectively) was observed after 8 days of co-fermentation. The dual culture xylanase activity appeared to be 4 times lower than that in S. commune but it was higher by 34% as compared with that of I. lacteus monoculture. Analysis of the enzyme accumulation kinetics suggests that both fungi developed independently and I. lacteus 104 delivered cellulases to the medium whereas S. commune 632 was mainly responsible for the accumulation of xylanase activity. Almost the same behavior of co-culture with a rather lower enzyme activities was revealed when the medium was inoculated with I. lacteus 104 and S. commune 632 in the ratio 4:1. However, inoculation of I. lacteus 104 and S. commune 632 in the ratio 1:4 caused a 2-fold decrease in the dual culture CMCase activity and 3-fold decrease of FPA as compared with those of the control I. lacteus monoculture. It should be noted that in this case, the xylanase activity two times exceeded that in the control culture, obviously owing to domination in the co-culture of S. commune 632. Finally, the results obtained show that cellulase and xylanase production by I. lacteus and S. commune was affected by the time and order of individual fungal inoculation

22

(Fig. 3). When the inoculation time of I. lacteus 104 with S. commune 632 was delayed for 3 days it displayed a negative effect on cellulase activity although the xylanase activity increased by 40% (to 98 U/mL as compared with 70 U/mL in the I. lacteus 104 monoculture). Evidently, in this case, S. commune inhibited growth of I. lacteus and cellulase accumulation, since the activities of CMCase and FP achieved their maximum on the day 5th and then gradually decreased whereas xylanase activity of the paired culture continued to increase till the day 8th. However, when conversely S. commune 632 was inoculated by I. lacteus 104 with 3 days’ delay two-fold decrease of CMCase activity and a significant decrease of FPA was observed during the co-fermentation process. It is worth noting that the maximum activity of both enzymes was achieved on the day 5 (two days after pairing) and subsequent co-cultivation of fungi did not accompany with cellulase secretion. In contrast, as depicted in Fig. 3, the dual culture xylanase activity greatly increased and achieved 312 U/mL after 8 days of co-cultivation. Despite the ability of I. lacteus 104 to grow rapidly, this fungus did not overcome an antagonistic effect of the already grown biomass of S. commune 632. Results received in the study of the effect of individual fungi inoculum ratio confirm this conclusion. In particular, when the I. lacteus 104 and S. commune 632 inoculum ratio was 1:4, the cellulase activity appeared to be low and very close to those in the S. commune monoculture. Although the xylanase activity in the co-culture was 2.7 times lower than that in S. commune, this enzyme activity was 2-fold higher as compared with that in I. lacteus monoculture. Inoculation of I. lacteus 104 and S. commune 632 in ratio 4:1 favored cellulase accumulation but decreased xylanase activity of the co-culture. It is interesting that in the co-cultivation of T. reesei RUT-30 with Phanerochaete chrysosporium, the

23

maximum endoglucanase, cellobiohydrolase, and β-glucosidase activities were observed when the inoculation time of P. chrysosporium was delayed for 1.5 day, whereas inoculation delayed for 3 d had a negative effect on these activities and 0 d delay gave intermediate values (Yang et al., 2013).

Fig. 4

3.5. Enzyme production in fungi co-cultivation in fermenter

Based on the experiments described above, the co-cultivation of I. lacteus 104 and S. commune 632 was performed in a 7 l laboratory fermenter using an optimized medium containing 1% Avicel in combination with 2% milled mandarin peels as carbon sources, and 1% (NH4)2SO4/0.5% yeast extract as nitrogen sources. During the first stage of fermentation, conditions favoring the polysaccharide hydrolysis of the substrates to supply growing mycelia were generated, while during the second stage conditions beneficial for cellulase and xylanase production were established. During the initial three days of fermentation at pH 5.0, the fungal biomass density rapidly increased while the dissolved oxygen gradually decreased to 4%. The reducing sugar content in the culture liquid after 1 and 2 days of fermentation was 2.4 and 1.0 mg/mL, respectively, and low enzymatic activity was detected (Fig. 4). After three days of cultivation the sugar content decreased to 0.2 mg/mL, whereas endoglucanase, xylanase, and FP activities increased to 23 U/mL, 18 U/mL, and 0.6 U/mL, respectively. At this time, the medium pH was changed and controlled at pH 6 until the end of fermentation to slow the polysaccharide hydrolysis in substrates. The data showed that

24

the CMCase and xylanase activities increased and achieved their maximum on day 6 and 7 of 142 U/mL and 102 U/mL, respectively. The total cellulase activity correlated with the endoglucanase activity secretion and attained 7 U/mL on the day 6. Moreover, the measurement of extracellular protein content correlated with the enzymatic activity. The crude enzyme preparation (total volume 175 mL) obtained from the culture liquid by precipitation with ammonium sulfate contained 2940 U/mL CMCase, 2234 U/mL xylanase, 169 U/mL FPA, and 176 U/mL β-glucosidase activities, respectively, and 21.6 mg protein/mL.

Table 5

3.6. Enzymatic saccharification of wheat straw

Fungal cellulases are the most effective enzymes for biomass saccharification (Mathew et al., 2008; Juturu and Wu, 2014). In this study, the hydrolysis of pretreated wheat straw was compared using: a commercial enzyme preparation (Accellerase 1500), the crude enzymes obtained after I. lacteus monoculture cultivation in the flasks, and the I. lacteus and S. commune co-cultivation in fermenter. At a FPA load of 10 U/mL, the substrate saccharification after 24 h resulted in a total reducing sugar yield of 5 mg/mL and 3.8-4.2 mg/mL, respectively (Table 5). Saccharification for 48 h resulted in an increase of total reducing sugars to 6.4 mg/mL and 4.7-5.6 mg/mL, respectively. Doubling the concentration of the commercial and in-house enzymes accelerated the hydrolysis rate and led to an increase of reducing sugar content up to 7.8 mg/mL and 6.5-7.2 mg/mL, respectively, after 48 h incubation. These concentration represented

25

yields from wheat straw hydrolysis of 52.9% and 44.1-48.8%, respectively. The degree and duration of hydrolysis of lignocellulosic substrates and sugar yield is largely dependent on the availability of a mixture of different enzymes and their activity (Mathew et al., 2008). Therefore, the enzymatic mixture derived from the mixed fermentation showed a higher cellulose hydrolysis potential than the enzymes from the I. lacteus monoculture. This positive effect may result from the complementary interactions of cellulases, xylanases and β-glucosidases of the dual culture ensuring a more complete polysaccharide hydrolysis. Moreover, the presence of elevated concentrations of β-glucosidase diminished the inhibitory effect of cellobiose. Thus, as compared with the monocultures of I. lacteus and S. commune, their mixed cultivation provided a more balanced enzymatic mixture compared to the monocultures, improving the hydrolysis of lignocellulosic material.

4. Conclusions

The results presented here highlight that accumulation of cellulase and xylanase in co-cultures of WRB depends on combinations of individual fungi. Comparison of a number of combinations of WRB identified I. lacteus and S. commune as a particularly favorable co-culture for enzyme accumulation The extent of enzyme accumulation by I. lacteus and S. commune was strongly dependent on the type and concentration of growth substrate and nitrogen source. The time and order of fungal inoculation also was critical for optimizing protein production. The co-cultivation of I. lacteus and S. commune resulted in an enzymatic mixture that was better balanced than what could be obtained from a single monoculture and it performed comparably to a commercial

26

enzymatic mixture in the saccharification of pretreated wheat straw. Therefore, understanding mechanistic details of interspecies interactions may lead to improved fungal enzyme production for biomass saccharification.

Conflict of interests The authors have no conflicts of interest to declare.

Acknowledgements

This work was financially supported by the Shota Rustaveli National Science Foundation (grant No. 31/62).

References

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4. Baldrian, P., Valaškova, V., 2008. Degradation of cellulose by basidiomycetous fungi, FEMS Microbiol. Rev. 32, 501–521. 5. Chi, Y., Hatakka, A., Maijala, P., 2007. Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes? Int. Biodeterior. Biodegrad. 59, 32–39. 6. Dong, Y.C., Wang, W., Hu, Z.C., Fu, M.L., Chen, Q.H., 2012. The synergistic effect on production of lignin-modifying enzymes through submerged co-cultivation of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora using agricultural residues. Bioprocess Biosyst. Eng. 35, 751–760. 7. Elisashvili, V., 1993. Biosynthesis and properties of cellulases and xylanases of higher basidiomycetes (Review). Appl. Biochem. Microbiol. 29, 257-266. 8. Elisashvili, V., Kachlishvili, E., Tsiklauri, N., Metreveli, E., Khardziani, T., Agathos, S.N., 2009. Lignocellulose-degrading enzyme production by white-rot basidiomycetes isolated from the forests of Georgia. World J. Microbiol. Biotechnol. 25, 331-339. 9. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257– 268. 10. Hatakka, A., Hammel, K.E., 2010. Fungal biodegradation of lignocelluloses, in: M. Hofrichter (Ed.), The Mycota X. Industrial Applications. Springer, Berlin, Heidelberg, pp. 319–340. 11. Hu, H.L., van den Brink, J., Gruben, B.S., Wösten, H.A.B., Gu, J.D., de Vries, R.P., 2011. Improved enzyme production by co-cultivation of Aspergillus niger and Aspergillus oryzae and with other fungi. Int. Biodeterior. Biodegrad. 65, 248–252.

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12. Jagtap, S.S., Dhiman, S.S., Kim, T.S., Li, J., Kang, Y.C., Lee, J.K., 2013. Characterization of a β-1,4-glucosidase from a newly isolated strain of Pholiota adiposa and its application to the hydrolysis of biomass. Biomass Bioenergy. 54, 181-190. 13. Jagtap, S.S., Dhiman, S.S., Kim, T.S., Kim, I.W., Lee, J.K., 2014. Characterization of a novel endo-β-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis. Appl. Microbiol. Biotechnol. 98, 661-669. 14. Juturu, V., Wu J.C., 2014. Microbial cellulases: engineering, production and applications. Renew. Sustain. Energy Rev. 33, 188–203. 15. Kalyani, D., Lee, K.M., Kim, T.S., Li, J., Dhiman, S.S., Kang, Y.C., Lee, J.K., 2013. Microbial consortia for saccharification of woody biomass and ethanol fermentation. Fuel. 107, 815–822. 16. Ma, K., Ruan, Z., 2015. Production of a lignocellulolytic enzyme system for simultaneous bio-delignification and saccharification of corn stover employing coculture of fungi. Bioresour. Technol. 175, 586–593. 17. Ma, F., Wang, J., Zeng, Y., Yu, H., Yang, Y., Zhang, X., 2011. Influence of the cofungal treatment with two white rot fungi on the lignocellulosic degradation and thermogravimetry of corn stover. Process Biochem. 46, 1767–1773. 18. Mathew, G.M., Sukumaran, R.K., Singhania, R.R., Pandey, A. 2008. Progress in research of fungal cellulases for lignocellulose degradation. J. Sci. Ind. Res. 67, 898907. 19. Metreveli, E., Kachlishvili, E., Denchev, D., Elisashvili, V., 2014. Improved cellulase and xylanase production by co-cultivation of white-rot basidiomycetes. Ecol. Eng. Environ. Prot. №1, 5-11.

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Figure Captions

Fig. 1. Cellulase and xylanase activity of mono- and dual cultures of basidiomycetes grown on the EPR-containing agar medium. Il – I. lacteus, Pc – P. coccineus, Sc – S. commune, Th – T. hirsita. Fig. 2. Effect of growth substrates on the I. lacteus + S. comune enzyme activity.

Fig. 3. Effect of individual I. lacteus (Il) and S. commune (Sc) inoculum ratio, time and order of second fungus inoculation on the dual culture enzyme activity. Fig. 4. Profiles of hydrolases activity and extracellular protein content in I. lacteus + S. commune co-cultivation in a laboratory fermenter.

31

1.8 CMCase Xylanase

Enzyme activity (U/mL)

1.5 1.2 0.9 0.6 0.3 0 Il

Fig. 1.

Pc

Sc

Th

Il + Sc

Pc + Sc

Sc + Th

32

CMCase

Xylanase

4

FPA

40 3 30 2 20 1 10

0

0 Avicel

Fig. 2.

BP

MP

WB

WS

FPA (U/mL)

CMCase, Xylanase (U/mL)

50

Fig. 3.

5 mL Sc + 5 mL Il on day 3

5 mL Il + 5 mL Sc on day 3

4 mL Il + 1 mL Sc

FPA

400

300 4

200 3

100 2

1

0 0

FPA (U/mL)

Xylanase

2.5 mL Il +2. 5 mL Sc

CMCase

1 mL Il + 4 mL Sc

500

5 mL S. commune (Sc)

5 mL I. lacteus (Il)

CMCase, Xylanase (U/mL)

33

7

6

5

34

CMCase

8

Xylanase 7

CMCase, Xylanase (U/mL)

FPA 120

β-Glucosidase

6

Protein

5

90

4 60

3 2

30 1 0

0 0

1

2

3

4

5

Fermentation days Fig. 4.

6

7

FPA, β-Glucosidase (U/mL); Protein (mg/mL)

150

35

Table 1 Cellulase and xylanase activity of mono- and dual cultures of basidiomycetes. Fungi

Final pH

CMCase

FPA

Xylanase

(U/mL)

(U/mL)

(U/mL)

1.5% Avicel-containing medium I. lacteus 104

6.3 ± 0.1

51.7 ± 4.3

4.8 ± 0.7

57.9 ± 4.6

P. coccineus 310

6.5 ± 0.1

65.6 ± 7.3

5.6 ± 0.7

63.5 ± 10.3

S. commune 632

6.1 ± 0.1

28.0 ± 3.9

1.4 ± 0.2

194.1 ± 28.6

T. hirsuta 17

5.8 ± 0.1

34.3 ± 3.1

2.5 ± 0.3

48.8 ± 5.0

I. lacteus 104 + S. commune 632

5.4 ± 0.2

30.7 ± 6.5

2.8 ± 0.4

44.7 ± 6.1

P. coccineus 310 + S. commune 632

7.0 ± 0.1

4.8 ± 0.7

0.5 ± 0.3

51.1 ± 8.0

T. hirsuta 17 + S. commune 632

7.1 ± 0.1

7.8 ± 0.9

0.6 ± 0.4

64.2 ± 10.7

1.5% Avicel + 4% MP-containing medium I. lacteus 104

6.5 ± 0.1

73.2 ± 10.3

5.1 ± 0.7

67.2 ± 7.0

P. coccineus 310

6.4 ± 0.1

71.9 ± 6.7

6.0 ± 0.5

65.6 ± 5.8

S. commune 632

6.7 ± 0.1

48.0 ± 6.3

3.7 ± 0.4

334.1 ± 52.1

T. hirsuta 17

6.0 ± 0.1

64.6 ± 11.8

5.3 ± 0.8

73.0 ± 12.1

I. lacteus 104 + S. commune 632

6.8 ± 0.2

91.6 ± 13.0

5.7 ± 0.9

96.0 ± 13.0

P. coccineus 310 + S. commune 632

7.1 ± 0.2

24.2 ± 3.5

2.2 ± 0.4

214.2 ± 25.8

T. hirsuta 17 + S. commune 632

7.2 ± 0.1

30.1 ± 3.4

2.5 ± 0.3

140.3 ± 18.5

36

Table 2 Effect of growth substrates concentration and ratio on the paired I. lacteus + S. comune enzyme activity. Growth

Final

CMCase

FPA

Xylanase

substrates

pH

(U/mL)

(U/mL)

(U/mL)

1.0% Avicel

5.8 ± 0.1

40.2 ± 5.6

3.9 ± 0.3

45.2 ± 6.8

1.0% Avicel + 2% MP

6.5 ± 0.2

118.0 ± 13.2

5.2 ± 0.6

121.0 ± 22.5

1.0% Avicel + 4% MP

6.1 ± 0.1

72.4 ± 10.5

2.8 ± 0.5

68.5 ± 8.7

2.0% Avicel

5.5 ± 0.1

36.8 ± 5.7

3.1 ± 0.3

27.5 ± 5.0

2.0% Avicel + 2% MP

6.0 ± 0.1

68.0 ± 9.6

4.4 ± 0.5

98.5 ± 13.5

2.0% Avicel + 4% MP

5.1 ± 0.1

16.9 ± 2.2

1.9 ± 0.3

29.0 ± 4.1

37

Table 3 Effect of nitrogen sources (20 mM as nitrogen) on the S. commune + I. lacteus growth and enzyme activity.

Nitrogen sources

Biomass

CMCase

FPA

Xylanase

(U/mL)

(U/mL)

(U/mL)

protein (mg/mL)

Control

0.48 ± 0.04

97.0 ± 7.9

3.5 ± 0.4

105.2 ± 11.0

KNO3

0.70 ± 0.06

127.2 ± 11.0

4.0 ± 0.5

89.7 ± 10.2

(NH4)2SO4

0.68 ± 0.04

128.4 ± 14.3

5.3 ± 0.4

115.3 ± 11.2

NH4NO3

0.71 ± 0.05

113.8 ± 12.7

3.7 ± 0.5

95.3 ± 9.1

Casein hydrolysate

0.67 ± 0.04

132.1 ± 12.1

5.0 ± 0.3

112.9 ± 12.7

Peptone

0.70 ± 0.04

122.4 ± 11.8

3.9 ± 0.5

104.0 ± 10.6

Yeast extract

0.57 ± 0.03

121.1 ± 10.2

3.6 ± 0.4

92.3 ± 10.1

38

Table 4 Effect of nitrogen source concentration on the paired S. commune and I. lacteus enzyme activity.

Nitrogen

Biomass

concentration

protein

CMCase

FPA

Xylanase

(U/mL)

(U/mL)

(U/mL)

(mg/mL) (mM)

0.50 ± 0.03

93.9 ± 6.8

4.1 ± 0.4

102.4 ± 11.5

10

0.56 ± 0.03

115.3 ± 10.4

5.1 ± 0.4

112.5 ± 9.2

20

0.70 ± 0.04

127.6 ± 9.3

5.3 ± 0.4

120.2 ± 11.8

40

0.76 ± 0.05

138.8 ± 15.0

6.0 ± 0.5

116.0 ± 15.0

80

0.78 ± 0.06

141.4 ± 17.2

5.9 ± 0.6

105.1 ± 13.4

10

0.57 ± 0.03

109.8 ± 8.1

3.9 ± 0.3

104.3 ± 8.3

20

0.68 ± 0.04

127.0 ± 12.1

4.3 ± 0.3

120.2 ± 10.3

40

0.78 ± 0.05

121.0 ± 11.7

4.3 ± 0.5

118.3 ± 12.4

80

0.80 ± 0.05

117.3 ± 13.2

3.9 ± 0.5

113.5 ± 11.2

Control (NH4)2SO4

Casein hydrolysate

40

Table 5 Pretreated wheat straw saccharification by in-house and commercial enzymes.

Enzyme load

Reducing sugars (mg/mL) Duration of hydrolysis

24 h

48 h

I. lacteus enzyme, 10 FPU/g

3.8 ± 0.10

4.7 ± 0.14

I. lacteus enzyme, 20 FPU/g

5.5 ± 0.13

6.5 ± 0.15

Co-culture enzyme, 10 FPU/g

4.2 ± 0.11

5.6 ± 0.12

Co-culture enzyme, 20 FPU/g

5.7 ± 0.10

7.2 ± 0.16

Accellerase 1500, 10 FPU/g

5.0 ± 0.11

6.4 ± 0.14

Accellerase 1500, 20 FPU/g

6.4 ± 0.12

7.8 ± 0.13

41

HIGHLIGHTS White-rot basidiomycetes are promising sources of cellulases and xylanases. Irpex lacteus and Schizophyllum commune showed compatible interaction. Availability of carbon source determined outcomes of interspecific interaction. Co-culture conditions optimization improved hydrolases production. Synergistic enzyme preparation exhibited 11% higher hydrolysis of biomass.