Enzymatic Hydrolysis of Lignocellulosic Agricultural Wastes to Fermentable Glucose

Thermophilic micromycetes from the collection of microscopic fungi active strain producers of extracellular cellulases has been selected. Hydrolytic potential of cellulase preparations isolated from the selected strains has been investigated according to hydrolysis of cellulose in agricultural wastes. The wastes have been pretreated biologically (by basidial fungi) and thermo-mechanically (2 atm, at 140C, for1 hour). During 10-days of basidial fungi cultivation more than 50% of lignin was utilized from wheat straw, cornstubble, rice straw and potato straw. The following enzymatic treatment of biologically fermented substrates was converted from 54 to 85% of cellulose to glucose. These data are comparable and sometimes even exceed the analogous data of previously used thermo-mechanical pretreatment of substrates. © 2016 Bull. Georg. Natl. Acad. Sci.

Enzymatic hydrolysis of cellulose in lignocellulosic biomassesto fermentable glucose isthe most important technological processes among all possible enzyme technologies. The cost of ethanol production based on current technologies from lignocellulosic materials is relatively high, and the main challenges are the low yield and high price of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis process of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the deepness of cellulose hydrolysis. The lignin acts as a barrier to enzyme and microbial penetration in lignocellolose substrates significantly decreasing the yields of fermentable sugars; it negatively affects the overall process of hydrolysis most often making it uneconomical [1].
The multi enzymatic lignicellulose degradation is quite complicated process. There are several enzymes acting simultaneously, such as: laccase, oxidizing phenol ring containing compounds by forming phenoxy radicals and quinones; cellulases and xylanase perfoming -endo and -exo type hydrolysis of cellulose and xylan. Laccase, actively participating in lignocellulose hydrolyses process, is one of the most versatile enzymes, which can find application in many Bull. Georg. Natl. Acad. Sci., vol. 10, no. 2,2016 different industrial sectors [2,3].
To overcome this limitation, some physical, chemical and biological pretreatment of the lignocellulose are used for effective cellulose hydrolysis [4,5]. Enzymes hydrolyzing and oxidizing these biopolymers are found in plants [6][7][8] but the equilibrium of their hydrolysis and/or oxidative degradation is so strongly shifted toward their synthesis that hydrolysis becomes negligible process and should not be taken into consideration.
For successful industrial realization of cellulose enzymatic hydrolysis in addition to high specific activity of enzymes from mycelyal fungi, up to now recognized as the best producers of enzymes (cellulases/xylanases/laccase), some other characteristics of enzymes are required: heat-resistance under the high temperature regimen (60-65°C), resistance against inhibition by terminal products of cellulose hydrolysis, deep and effective hydrolysis of different lignocellulose substrates. Enzymatic hydrolysis of cellulose, which is the main component of plant mass (makes above 60% of all plant mass) from the point of view of fermentable glucose and biofuel production in lagre scale, becomes the most important technological process among all possible enzyme technologies [9][10][11][12].

Materials and Methods
Thermo-mechanical pretreatment. Lignocellulosic substrates: wheat straw corn straw, rice straw, potato straw. All residues were dried at 60 o C and milled to dust extent (<1 mm  MgSO 4 -0.5; Initial pH was adjusted to 5.7 prior to sterilization. The nutrient medium was sterilized at 121 º C for 20 min. After 7-10 days of fungi cultivation, mycelium was inoculated to conduct the solid-state fermentation (SSF) of lignocellulose containing materials [13].
Cultivation conditions. Solid-state fermentation (SSF) of selected plants residues was carried out at 27°C in 250-ml flasks containing 5g of lignocel-lulosic substrates moistened with 18 ml of the nutrient medium (g/l): NaNO 3 -2.0; yeast extract-3.0; KH 2 PO 4 -0.9; K 2 HPO 4 -0.3; MgSO 4 ×7H 2 O-0.5; 0.2m M CuSO 4 ×5H 2 O; pH 5.8. The flasks were inoculated with 5ml of mycelial homogenate. To determine enzyme activity, after 10 and 15days of cultivation, the extracellular enzymes were extracted from the 5g of cell biomass, which previously was washed twice with 10 ml of distilled water (total volume 20 ml). The extract was centrifuged at 10 000 g for 15 min at 4 º C. The final filtrate was used for determination of enzyme activities. Remained wet biomass was dried at 60 0 C and used for hydrolysis.
Enzyme activities assay. Viscosimetric activity was determined as a result of enzyme action on soluble Na-CMC according to the method modified by Rodionova et al. [14]. Aliquots of appropriately diluted culture filtrate as enzyme source was added to Whatman No.1 filter paper strip (1×6 cm; 50 mg) immersed in one milliliter of 0.05 M acetate buffer, pH 4.5. After incubation at 50°C for 1 h, the reducing sugar release was estimated by dinitrosalicylic acid (DNS) method [15.16]. One unit of filter paper (FPU) activity was defined as the amount of enzyme releasing 1mole of reducing sugar from filter paper per ml per min. To obtain enzyme preparation, culture liquid was precipitated by ethanol in the ratio of 1 volume liquid to 4 volumes of cold ethanol (+2-4°C). Xylanase activity was determined by mixing 70 µl appropri ately diluted samples with 630 µl of birch wood xylan (Roth 7500) (1% w/v) in 50 mM citrate buffer (pH 5.0) at 50 o Cfor 10 min [17]. Glucose and xylose standard curves were used to calculate cellulase and xylanase activities. In all assays, the release of reducing sugars was measured using the dinitrosalicylic acid reagent method [18]. Laccase activity was determined by monitoring the A420 change related to the rate of oxidation of 1 mM 2,2-azino-bis-[3-ethylthiazoline-6sulfonate] (ABTS) in 100 mmol sodium tartrate buffer (pH 4.5). Assays were performed in 1 ml spectrophotometric cuvette at 30 ± 1 o C with adequately diluted culture liquid. One unit of laccase activity was defined as the amount of enzyme, which leads to the oxidation of 1 mmol of ABTS per minute [19].
Determination of glucose. The amount of glucose was determined by glucosooxidase-peroxidase method. 3 ml of glucosooxidase-peroxidase reagent was added to 0.2 ml of analyzing solution. After delaying for 30 minutes, the intensity of formed color was measured on spectrophotometer at 420 nm of wavelength. The amount of glucose was estimated by preliminarily diagrammed calibration curve. To determine the biosynthesis potential of the selected strains enzymes (hydrolyses: CMCase, xylanase, FPA and laccase) the basidial fungi strains of different genera and families differing in lignin utilization ability were cultivated on wheat straw by solid state fermentation (SSF). Data of these experiments are presented in Table 1. As seen from the Table,  is interesting to underline that the most active laccase producer strains in above experimental conditions exposed a low activities of cellulose and xylan de-grading enzymes.

Results and Discussion
To determine the efficiency of mycelial fungi strains individual potential of lignin utilization in agro wastes, such complicated compound as wheat straw was chosen. After basidial fungi solid-state fermentation on wheat straw in final solid-state fermented biomasses, the following components were determined: soluble compounds, cellulose, lignin, and xylan. The final amounts of these components indicate on the deepness of microbial transformation of wheat straw. As it was revealed, qualitatively the amounts of these components differed in final biomasses (Table 2).
First, it should be stated that basidial strains are performing the utilization of lignin in different extent. Lignin was effectively degraded by Ganoderma strains, the most active producers of laccase. The final amount of this biopolymer was in both cases less than 10% (8.8 and 9,4). In the biomass trans-

Mycelia Fungi Strains Extracellular Cellulase Activities
For the hydrolysis of cellulose in agro-wastes fifty producers of cellulases with different extracellular activities were selected from the collection of mycelial fungi collection (3500 strains). As optimal conditions, for the cultivation of selected strains were 40-45 o C, they were considered as thermotolerants producing comparatively heat-stable forms of cellulases.
According to our results to carry out the hydrolysis of cellulose to fermentable glucose in partially delignified wheat straw or other agricultural wastes, more promising seems to be cellulases from Penicillium canescence TK-2 and Trichoderma viride 16-3 (Table 3). In this Table typical extracellular activities of the most active producers are given. From the above presented strains (Table 3) due to the highest activity and previously estimated reasonable heat stability of cellulases the enzyme preparation from Penicillium canescens TK-2 was cho-sen. The optimal temperature of cellulases action isolated from this strain was 55°C. The enzymatic hydrolysis of agricultural wastes was carried out in a reactor, during 24 h, ambient: 0.05 M acetate buffer, pH 4,5. Concentration of the substrate was 50g/l, the correlation of enzyme activity units and substrate was 60 CMC units per 1g of substrate. Results of enzymatic hydrolysis of thermo mechanically and biologically (by basidial fungi) pretreated substrates are presented in Table 4.
As it was previously proved by the authors, thermo-mechanical pretreatment of the above listed agricultural wastes is one of the most effective methods for their following enzymatic hydrolysis, sometimes allowing to reach 90-100% of substrates cellulose conversion to glucose. Therefore, this method has been chosen for the comparison with biological treatment performed by basidial fungi. According to the data presented in Table 4, 5 and Figure 2, pretreatment biotechnology by basidial fungi is comparable and sometimes (wheat straw and rice straw) even exceeds thermo-mechanical method in efficiency.