Research Article | Open Access
Marli Camassola, Aldo J. P. Dillon, "Effect of Different Pretreatment of Sugar Cane Bagasse on Cellulase and Xylanases Production by the Mutant Penicillium echinulatum 9A02S1 Grown in Submerged Culture", BioMed Research International, vol. 2014, Article ID 720740, 9 pages, 2014. https://doi.org/10.1155/2014/720740
Effect of Different Pretreatment of Sugar Cane Bagasse on Cellulase and Xylanases Production by the Mutant Penicillium echinulatum 9A02S1 Grown in Submerged Culture
The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum 9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.253 ± 0.147 U·mL−1) was detected in the medium on the sixth day of cultivation when bagasse samples were pretreated with sodium hydroxide, hydrogen peroxide, and anthraquinone. Endoglucanase enzyme production was also enhanced by pretreatment of the bagasse. Nine cultures grown with bagasse possessed higher β-glucosidase activities on the sixth day than the culture grown with cellulose. The highest xylanase activity was observed in cultures with cellulose and with untreated sugar cane bagasse. These results indicate that pretreated sugar cane bagasse may be able to serve as a partial or total replacement for cellulose in submerged fermentation for cellulase production using P. echinulatum, which could potentially reduce future production costs of enzymatic complexes capable of hydrolyzing lignocellulosic residues to form fermented syrups.
Lignocellulosic biomass is the most abundant organic material on earth and could provide a suitable low-cost feedstock for the production of ethanol fuel and other chemicals in the future . These materials generally consist of up to 75% of cellulose and hemicellulose, which are compounds that cannot be easily converted to simple monomeric sugars due to their recalcitrant nature . Sugar cane bagasse is a waste product of the sugar extraction process. Currently, the sugar cane bagasse generated in the plant is consumed for energy production through cogeneration, becoming self-sustainable energy plant, and in some cases spares energy to electricity sales. This is an abundant, low-cost lignocellulosic material that could be a promising feedstock for the use as a carbon source in the fermentation medium for the production of cellulase enzymes . Additionally, it contains xylan, an inducer of xylanase production .
Xylanases are produced on an industrial scale for use as a food additive for poultry to increase feed efficiency and in wheat flour to improve dough handling and the quality of baked products. The interest in xylanases has markedly increased recently due to the other potential industrial uses, particularly in pulping and bleaching processes, using cellulase-free preparations . Cellulolytic enzymes are used in a large number of processes including supplementation of animal feeds, extraction of fruit and vegetable juices, pulp and paper manufacturing, starch processing, textile processing, and ethanol production [6–10]. Furthermore, growing concerns regarding the potential consequences of a worldwide shortage of fossil fuels, the emission of greenhouse gases, and air pollution caused by incomplete combustion of fossil fuels have resulted in an increased focus on the production of bioethanol from lignocellulosics materials [11, 12] and use of cellulases and hemicellulases in the enzymatic hydrolysis of lignocellulosic material .
The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production . The challenges are to obtain cellulase-producing mutant microorganisms [14, 15] and economic cellulase production processes in combination with inexpensive sources of inducers along with total or partial recycling of enzymes.
This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum strain 9A02S1  using sugar cane bagasse pretreated with different chemicals as an inducer of these enzymes. Mutants of P. echinulatum are potential producers of cellulases for cellulose hydrolysis because they offer relatively good thermal stability of filter paper activity (FPA) and β-glucosidase enzymes at 50°C, maintaining the activity for more than 80 hours , high levels of enzymatic production , and good proportions of FPA and β-glucosidase for the efficient hydrolysis of cellulose as compared to the cellulases of T. reesei . Therefore, this strain has potential value for the enzymatic hydrolysis of cellulose and lignocelluloses to produce glucose syrup.
2. Materials and Methods
2.1. Strain and Culture Media
The cellulolytic mutant P. echinulatum strain 9A02S1 (DMS 18942) was used in this study. The strain was obtained by exposing wild-type P. echinulatum strain 2HH to ultraviolet (UV) light and hydrogen peroxide (H2O2) . These strains are stored in the culture collection of the Division of Process Biotechnology, Institute of Biotechnology, Caxias do Sul, Rio Grande do Sul, Brazil.
The media used were based on 10x concentrated Mandels and Reese solution (MS)  containing (in g·L−1) KH2PO4, 20; (NH4)2SO4, 13; CO(NH2)2, 3; MgSO4·7H20, 3; CaCl2, 3; FeSO4·7H2O, 0.050; MnSO4·H2O, 0.0156; ZnSO4·7H2O, 0.014; and CoCl2, 0.0020. It was used swollen cellulose to prepare the strain maintaining medium. Swollen cellulose gel was produced by placing 5 g of Cellufloc 200 cellulose (Celulose e Amido Ltda, Suzano, São Paulo, Brazil), 60 mL of distilled water, and 20 glass beads ( mm) in a 500 mL Erlenmeyer flask, which was then sealed and autoclaved at 120°C for 30 min, after which the flask and its contents were shaken for 48 h at 180 rpm, and 140 mL of distilled water was added. The swollen cellulose was stored at 4°C until use.
The strain 9A02S1 was grown and maintained on cellulose agar (C-agar) consisting of distilled water containing 1% (v/v) swollen cellulose, 10% (v/v) 10x MS, 0.1% (w/v) proteose peptone (Oxoid L85), and 2% (w/v) agar and the pH was adjusted to 6.0. Cultures were grown on C-agar slants for up to 7 days at 28°C until conidia formed and then stored at 4°C until use.
2.2. Obtention of Delignified Bagasse Samples
The delignification of bagasse was generally carried out using different quantities of sodium hydroxide, hydrogen peroxide, anthraquinone (AQ), and ethylenediaminetetraacetic acid (EDTA) solutions at 120°C for 20 minutes. Liquor ratios and reagent concentrations for the mixtures are shown in Table 1. The samples were ground into fragments of size between 0.5 and 1 cm.
2.2.1. Enzyme Production
Shake-flask experiments were carried out in 500 mL Erlenmeyer flasks with 100 mL of production medium containing 10% (v/v) 10x MS, 1% (w/v) microcrystalline cellulose-200 or pretreated sugar cane bagasse samples from different alkaline pretreatments (Table 1), 0.2% (w/v) soy meal, and 0.1% (v/v) Tween 80. The flasks were inoculated with sufficient conidial suspension to give a final concentration of conidia per mL and shaken at 28°C, 180 rpm for 6 days. Samples were removed at various times and centrifuged at 2800 ×g for 10 minutes. The supernatant was analyzed for extracellular enzyme activity. Experiments were carried out in triplicate and twice.
2.3. Enzymatic Assays
Total cellulase activity assay was analyzed on filter paper (filter paper activity—FPA), according to Ghose . β-glycosidase activity was measured using salicin as the substrate, according to Chahal . Endoglucanase activity was determined according to Ghose  using 2% (w/v) carboxymethylcellulose solution in citrate buffer, pH 4.8. Xylanase activity was determined in the same way as endoglucanase activity, except that 1% xylan from oat spelt solution was used as the substrate in place of carboxymethylcellulose. The concentration of reducing sugar was estimated as either xylose or glucose equivalents by the dinitrosalicylic acid (DNS) method according to Miller .
One unit (U) of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of reducing sugar from the appropriate substrate per minute per mL of crude filtrate under the assay conditions.
2.4. Analytical Methods
The mycelial biomass was estimated by the quantity of N-acetylglucosamine according to the method described in Reissig et al. . The Klason lignin of the samples was determined by the method described by Templeton and Ehrman (1995) .
2.5. Statistical Tests
The results were statistically analyzed using the PrismGraphPad program (GraphPad Software, Inc., CA, USA) to perform analysis of variance with Tukey’s post hoc test for a .
3. Results and Discussion
Different pretreatments of sugar cane bagasse were tested to determine which were optimal for using bagasse as a component of the medium for cellulase production by P. echinulatum. The efficiency of the pretreatments was measured by determining Klason lignin (Table 1). It was found that the use of greater reagent concentrations in the pretreatments resulted in higher delignification indices, measured as the amount of lignin present in the substrate after the different pretreatments.
According to Kim and Holtzapple (2006) , an effective lignocellulose treatment process should remove all of the acetyl groups and reduce the lignin content to about 10% in the treated biomass; this reduction was obtained in the samples of this work. Additionally, Kong et al. (1992)  reported that alkalis remove acetyl groups from hemicellulose, thereby reducing the steric hindrance of hydrolytic enzymes and greatly enhancing carbohydrate digestibility. This reduction of crystallinity may be because H2O2 pretreatment can swell and dissolve cellulose, while NaOH can even penetrate into the amorphous area of cellulose and destruct the neighboring crystalline regions  (Wang et al. 2008). EDTA is used in the process to prevent the decomposition of peroxide . The anthraquinone also has important function during the pretreatment, since it is agent which reduces lignin intermediates formed during pulping and thereby prevents the lignin intermediates from condensing during pulping .
Enzyme production experiments were carried out using samples of sugar cane bagasse from different treatments to verify secretion of cellulases and xylanases by P. echinulatum, strain 9A02S1. As controls, cultures were grown with microcrystalline cellulose and untreated sugar cane bagasse (Tables 2, 3, 4, and 5).
|Values (means) with the same letters for the same day did not differ significantly by Tukey’s test ().|
|Values (means) with the same letters for the same day did not differ significantly by Tukey’s test ().|
|Values (averages) with the same letters for the same day do not differ significantly by Tukey’s test ().|