Saccharification and hydrolytic enzyme production of alkali pre-treated wheat bran by Trichoderma virens under solid state fermentation
© El-Shishtawy et al.; licensee BioMed Central. 2015
Received: 25 November 2014
Accepted: 1 May 2015
Published: 28 May 2015
In continuation of our previously interest in the saccharification of agriculture wastes by Bacillus megatherium in solid state fermentation (SSF), we wish to report an investigation and comparative evaluation among Trichoderma sp. for the saccharification of four alkali-pretreated agricultural residues and production of hydrolytic enzymes, carboxymethyl cellulase (CMCase), filter paperase (FPase), pectinase (PGase) and xylanase (Xylase) in SSF. The optimization of the physiological conditions of production of hydrolytic enzymes and saccharification content from Trichoderma virens using alkali-pretreated wheat bran was the last goal.
The physico-chemical parameters of SSF include incubation time, incubation temperature, moisture content of the substrate, incubation pH, supplementation with carbon and nitrogen sources were optimized.
Saccharification of different solid state fermentation sources wheat bran, date's seeds, grass and palm leaves, were tested for the production of fermentable sugar by Trichoderma sp. The maximum production of hydrolytic enzymes CMCase, FPase, PGase and Xylase and saccharification content were obtained on wheat bran. Time course, moisture content, optimum temperature, optimum pH, supplementation with carbon and nitrogen sources were optimized to achieve the maximum production of the hydrolytic enzymes, protein and total carbohydrate of T. virens using alkali pre-treated wheat bran. The maximum production of CMCase, FPase, PGase, Xylase, protein and carbohydrate content was recorded at 72 h of incubation, 50-70 % moisture, temperature 25-35 °C and pH 5. The influence of supplementary carbon and nitrogen sources was studied. While lactose and sucrose enhanced the activity of PGase from 79.2 to 582.9 and 632.6 U/g, starch inhibited all other enzymes. This was confirmed by maximum saccharification content. Among the nitrogen sources, yeast extract and urea enhanced the saccharification content and CMCase, PGase and Xylase.
The results of this study indicated that alkali pre-treated wheat bran was a better substrate for saccharification and production of hydrolytic enzymes CMCase, FPase, PGase and xylase by T. virens compared to other alkali-pretreated agricultural residues tested.
Agricultural residues, forests and agro industrial practices generally accumulated in the environment and caused pollution problem. Active efforts were being made to convert these organic waste resources into either glucose or alcohol, and use this either as fuel or as a valuable starting material for chemical synthesis . Saccharification of polysaccharides to glucose by microbial hydrolytic enzymes which had attracted the attention of the researchers, as this was the first step of bioconversion of organic material into valuable products such as sugar, fine chemicals and biofuels . As the cost of cellulosic substrates play the central role in determining the economy of the saccharification process, lot of emphasis had been given to the usage of low price substrates and therefore screening of the agricultural wastes for release of sugars as organic wastes from renewable forest and agricultural residues . The saccharification of different agro-wastes had been reported by other workers employing enzymes from different organisms [4–6].
Recently, a significant interest raised in using solid state fermentation (SSF) instead of submerged fermentation (SmF). The advantages of SSF in comparison to traditional SmF were better yields, easier recovery of products, the absence of foam formation and smaller reactor volumes. Moreover, contamination risks were significantly reduced due to the low water contents and, consequently, the volume of effluents decreases . Another very important advantage was that, it permits the use of agricultural and agro-industrial residues as substrates which were converted into products with high commercial value like secondary metabolites, organic acids, pesticides, aromatic compounds, fuels and enzymes . Furthermore, the utilization of these compounds helps in solving pollution, which otherwise caused their disposal . For enzyme production, the costs of these techniques were lower and the production higher than submerged cultures [10,11].
Structural properties of cellulose such as the degree of crystallinity, the degree of polymerization and the surface area, limit accessibility of substrate to enzyme and had been demonstrated  to affect the rate of enzymatic hydrolysis of cellulose. Pretreatment methods, which disrupted the highly-ordered cellulose structure and the lignin-carbohydrate complex, remove lignin, and increase the surface area accessible to enzymes, promoted the hydrolysis, and increased the rate and extent of hydrolysis of cellulose in various lignocellulosic residues. The enzymatic hydrolysis of cellulosic materials correlated with the level of cellulose crystallinity  complete enzymatic hydrolysis of the polysaccharides of lignocelluloses required a concerted action of a complex array of hydrolases including cellulase, xylanase, pectinase, and other side-group cleavage enzymes .
Several cell-decomposing microorganisms produce cellulases which were the most economic and available sources for fermentable sugar production, because these microorganisms could grow on inexpensive media. The genus Trichoderma, filamentous ascomycetes were widely used in industrial applications because of high secretary capacity and inducible promoting characteristics . The structural complexity were often easily degraded by xylanases, mannanases etc. which were present in some cellulase preparations, so that their presence may actually lead to increased production of reducing sugars and greater susceptibility of the residual cellulose [16–18].
In continuation of our interest in the saccharification of agriculture wastes by SSF , we wished to report an investigation and comparative evaluation among Trichoderma sp., T. reesei, T. viride, T. harzianum and T. virens for the saccharification of four alkali-pretreated agricultural residues, wheat bran, date’s seeds, wild grass and palm’s leaves under solid state fermentation for the production of hydrolytic enzymes, carboxymethyl cellulase (CMCase), filter paperase (FPase), pectinase (PGase) and xylanase (Xylase). The polysaccharide composition of these agricultural residues included different concentrations from cellulose, hemicelluloses and lignin [20–22]. The optimization of the physiological conditions of production of hydrolytic enzymes and saccharification content from T. virens using alkali-pretreated wheat bran was the last goal.
T. reesei, T. viride, T. harzianum and T. virens were obtained from National Research Centre, Cairo, Egypt and maintained on potato dextrose agar. The slants were grown at 28 °C for seven days and stored at 4 °C.
Pretreatment of agricultural wastes
Wheat bran, date's seeds, grass and palm leaves were chosen as the sole nutrient source for solid-state fermentation (SSF). They dried in an oven at 80 °C for 24 h. The dried substrates were then milled in a commercial mill and sieved. The mean diameter of the dried substrates was 1.0 mm. The substrates were pretreated with 1.0 M NaOH at 121 °C and 15 psi pressure for 1 hr at the ratio of 1:10 (w/v) [23,24]. The pretreated materials were washed with tap water until the pH of the filtrate reached 7.0. The washed materials were dried at 60 °C overnight to constant weight and stored at room temperature for further use.
The medium used for inoculum of Trichoderma sp. preparation contained (g l−1): KH2PO4, 28; (NH4)2SO4, 19.6; Urea, 4.2; MgSO4. 7H20, 4.2; CoCl2, 4.2; FeSO4. 7H20, 0.07; MnSO4. 7H20, 0.021; ZnSO4 7H20, 0.019; CaC12, 0.028; yeast extract, 7; and glucose, 15; pH 5.0 ± 0.2. The media were sterilized by autoclaving at 121 °C pressure of 15 psi for 15 min. The culture was incubated and shaken at 30 °C for 48 hr in an orbital shaking incubator at 150 rpm before transferring to the production medium .
Solid state fermentation
SSF was performed to study the effect of various physicochemical parameters required for the optimum production of enzymes and saccharification content by Trichoderm sp. prior to inoculation, the agriculture waste was sterilized in an autoclave for 20 min at 121 °C and 1.2 atmospheres. To each 50 ml Erlenmeyer flask, 5 g of sterilized agriculture waste, 5 × 105 spores/g, and appropriate amount of water (10 % moisture) were added. The physico-chemical parameters included incubation time, incubation temperature (20, 30, 35, 40, 45 °C), moisture content of the substrate (10 %, 20 %, 40 %, 60 %, 100 %) and incubation pH (4 to 8) were optimized. The pH was adjusted using 0.1 M NaOH or HCl. Studies were also performed to evaluate the influence of different carbon sources (glucose, maltose, starch, sucrose, lactose at 1 % w/v) and nitrogen sources (yeast extract, urea, sodium nitrate, ammonium sulphate, ammonium chloride at 1 % w/v) when added to the fermentation medium contained agriculture waste. Each experiment is done in 3 sets.
Crude enzyme was extracted by mixing a 5 g of fermented matter with 50 ml distilled water on a rotary shaker (180 rpm/min) overnight. The suspension was then centrifuged at 12000 rpm for 10 min and the supernatant was designated as a crude extract.
Carboxymethylcellulase (CMCase), filter paperase (FPase), pectinase (PGase) and xylanase (Xylase) activities were assayed by determining the liberated reducing end products using glucose, glucose, galacturonic acid and xylose as standards, respectively . Substrates used were CM-cellulose, filter paper, polygalacturonic acid and birchwood xylan for CMCase, FPase, PGase and Xylase, respectively. The reaction mixture (0.5 ml) contained 1 % substrate, 0.05 M sodium acetate buffer pH 5.5 and 0.1 ml crude extract. Assays were carried out at 37 °C for 1 h. Then 0.5 ml dinitrosalicylic acid reagent was added to each tube. Then the reaction mixture was mixed well, and heated in a boiling water bath for 10 min. After cooling to room temperature, the absorbance was measured at 560 nm. One unit of enzyme activity is defined as the amount of enzyme which liberated one μmol of reducing sugar per min under standard assay conditions. All the experimental work was run in triplicates.
Protein concentration was determined according to the dye binding method of Bradford  with bovine serum albumin as standard.
Determination of total reducing sugars
Total reducing sugars were determined by the method of Miller . The reaction mixture contained 0.5 ml of crude extract and 0.5 ml dinitrosalicylic acid reagent. The tubes were heated in a boiling water bath for 10 min. After cooling to room temperature, the absorbance was measured at 560 nm. Glucose served as the calibration standard for total reducing sugar determination.
Determination of total soluble carbohydrates
Total soluble carbohydrates were determined by the method of Dubois et al. . The reaction mixture contained 25 μl of a 4:1 mixture of phenol and water, 0.8 ml of crude extract and 2 ml of concentrated sulfuric acid. Then mixed well, and heated in a boiling water bath for 30 min. The absorbance was determined at 480 nm. Glucose served as the calibration standard for total carbohydrate determination.
The obtained data were statistically analyzed as a randomized complete block design with three replicates by analysis of variance (ANOVA) using the statistical package software SAS (SAS Institute Inc., 2000, Cary, NC., USA). Comparisons between means were made by F-test and the least significant differences (LSD) at level P = 0.05. Correlations coefficient among the different parameters were also calculated by SAS.
Results and discussion
Sacchrification enzyme activities of different fungal isolates grown on lignocellulosic substrates under solid state fermentation
Enzyme activities (U/g dry substrate)
Shamala and Sreekantiah 
Ustok et al. 
Botella et al. 
Aspergillus terreus M11
Gao et al. 
Aspergillus niger KK2
Kang et al. 
Debing et al. 
Rodriguez-fernandez et al. 
Patil and Dayanand 
Myceliophthora sp. IMI 387099
Badhan et al. 
Kalogeris et al. 
Trichoderma harzianum and Trichoderma virens
cantaloupe and watermelon
Mohamed et al. 
Trichoderma harzianum SNRS3
Rahnama et al. 
Trichoderma reesei MCG77
Latifan et al. 
The present study revealed the saccharification potential of T. virens on alkali pre-treated wheat bran as an agricultural waste in SSF. The optimal conditions for production of CMCase, FPase, PGase and xylase and sccharification content utilizing alkali pre-treated wheat bran as the solid substrate in SSF included incubation for 72 h, temperature at 25-35 °C, substrate moisture content of 50-70 % and pH at 5.0.
This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH) – King Abdulaziz City for Science and Technology - the Kingdom of Saudi Arabia – award number (11-ENE1527-03). The authors also, acknowledge with thanks Science and Technology Unit, King Abdulaziz University for technical support.
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