alexa Optimization of Cellulase Enzyme Production from Sugarcane Pressmud Using Oyster Mushroom - Pleurotus Sajor-Caju by Solid State Fermentation | OMICS International
ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Optimization of Cellulase Enzyme Production from Sugarcane Pressmud Using Oyster Mushroom - Pleurotus Sajor-Caju by Solid State Fermentation

Nitin Prakash Pandit* and Sanjiv Kumar Maheshwari
School of Biotechnology, IFTM University, Lodhipur, Delhi Road, Moradabad 244 102, Uttar Pradesh, India
Corresponding Author : Nitin Prakash Pandit
School of Biotechnology, IFTM University
Lodhipur, Delhi Road, Moradabad 244 102, Uttar Pradesh, India
E-mail: [email protected]
Received January 03, 2012; Accepted March 09, 2012; Published March 11, 2012
Citation: Pandit NP, Maheshwari SK (2012) Optimization of Cellulase Enzyme Production from Sugarcane Pressmud Using Oyster Mushroom - Pleurotus Sajor- Caju by Solid State Fermentation. J Bioremed Biodegrad 3:140. doi: 10.4172/2155-6199.1000140
Copyright: © 2012 Pandit NP, et al. This is an open-a ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Cellulose is often a major component of different waste streams from various agriculture and sugarcane industries. Bioconversion of cellulosic components into fermentable sugars has currently been accomplished with the help of the microbial enzyme, cellulase (Endo-β-1, 4-glucanase, Exo-β-1, 4-glucanase and β-glucosidase). In this study, Pleurotus sajor-caju was tested for cellulase production by solid state fermentation (SSF) using sugarcane pressmud as substrate. Production rate of enzyme were optimized with different parameters like pretreatment of substrate with NaOH, HCl, H 2 O 2 and NaOH + H 2 O 2 (1-5%) solutions, thickness of the substrate (0.4-2.0 cm), incubation times (1-16 days), optimum pH (2.0-8.0) and temperature (15 ÌŠC-40 ÌŠC). The highest activities of Endo-β-1, 4-glucanase, Exo-β-1, 4-glucanase and β-glucosidase were obtained as follows: 13.94, 8.84 and 12.29 Units (μmole of glucose released/min/g substrate), respectively, for the pretreatment of pressmud with 2% (H 2 O 2 +NaOH); 18.37, 12.09 and 16.47 Units (μmole of glucose released/min/g substrate), respectively, when the thickness of the substrate was 0.8 cm (10 g); 15.51, 11.09 and 13.99 Units (μmole of glucose released/min/g substrate), respectively, on 10 th day of incubation; 17.65, 13.49 and 14.62 Units (μmole of glucose released/min/g substrate), respectively, at 5 pH and 18.98, 13.63 and 18.54 Units (μmole of glucose released/min/g substrate), respectively, at 25 ÌŠC. The optimum culture conditions for the production of cellulases by Pleurotus sajor-caju were 10 days of incubation time at 25 ÌŠC and pH 5.0 when presmud was pretreated with H 2 O 2 +NaOH (2%) and the thickness of the substrate was 0.8 cm (10 g) under solid state fermentation.

Keywords
Pressmud; Cellulase; Endo-β-1, 4-glucanase; Exo-β-1, 4-glucanase; β-glucosidase; Solid-state fermentation; Pleurotus sajorcaju
Introduction
Sugarcane is one of the important cash crops in India and plays pivotal role in both agricultural and industrial economy of the country. In India approximately 5.2 million tones of sugarcane pressmud are generated as agrowaste from sugarcane industries. An agricultural waste is a cheap source of cellulose for the production of different useful products all over the world [1]. The most abundant renewable organic compound in the biosphere is cellulose, which accounts for 40-50% of plant composition and its production is expected to be 1010 tones from cell wall of plants per year [2]. Cellulase production from agrowastes is economical as compared to production from pure cellulose [3]. Three major structural polymers combined to make up lignocellulose are called cellulose (a homopolymer of β-D-glucosyl units), hemicellulose (a cluster of heteropolymers which contain xylans, arabinans, mannans, galactans), and lignin (an intricate polyphenolic polymer) [4].
Cellulases are a group of enzymes that break down cellulose into glucose monomers [5]. Bacterial and fungal cellulases are traditionally separated into three classes: Endoglucanases (EGs) (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21) [6] based on the ability to degrade carboxymethylated cellulose (CMC), whereas EGs being the most efficient [7]. The Endo- β -glucanase is responsible for the scission of the inner bonds in the cellulose chains yielding glucose and cell-oligosaccharides. Exo-β-glucanase (cellobiohydrolases) cleaves non-reducing end of cellulose with cellobiose as the main structure [8,9]. The β - glucosidase (cellobiase) hydrolyses cellobiose to glucose [10].
Cellulase enzyme, has its importance due to major role in industrial applications [11]. It is used for bioremediation, waste water treatment and also for single cell protein [12]. It has also importance in food sciences like food processing in coffee, drying of beans by for efficient purification of juices when used mixed with pectinases, paper and pulp industry and as a supplement in animal feed industry. This enzyme helpful for plant protoplast isolation, plant viruses investigations, metabolic and genetic modification studies [11,13,14]. This enzyme have also pharmaceutical importance, treatment of phytobezons (a type of bezoar cellulose existing in humans stomach) and a key role in textile industry especially as its detergent applications to recover properties of cellulose related textiles and biofuels production from cellulosic biomass [1].
Some important bacteria and fungi are the most prominent sources of cellulase enzymes, which participate in the conversion of cellulose to glucose [15]. Among these organisms, fungi have been studied extensively because their elongated hyphae create mechanical pressure on the cellulose structure, causing them to produce large amounts of cellulose [16]. Most fungal cellulases have the capability of decomposing cellulose, hemicelluloses and lignin in plants by secreting multifarious set of hydrolytic and oxidative enzymes [17]. Cellulases producing fungi include genra Aspergilli [1] Aspergillus niger and Aspergillus terreus, Rhizopus stolonifer [18] Trichoderma, Penicillium, Botrytis Neurospora etc. [19]. Sodium hydroxide (NaOH), Hydrochloric acid (HCl) and Hydrogen peroxide (H2O2) pretreatment of sugarcane bagassi is one of the best-known methods of increasing the digestibility of cellulosic materials [20]. The prime requirement for production of any enzyme is a high yielding organism for which the selection of a cultivation technique and composition of media and culture conditions of supporting media should be properly formulated [21,22]. Hydrolytic enzymes used in industries can be produced by solid state fermentation (SSF) [23].
Solid State Fermentation (SSF) is a way of fermenting substrate in the presence of excessive moisture in growth medium in spite of large amount of water being provided. SSF is an environmental friendly (less waste water production), low energy required and economical technology in synthesizing cellulase enzyme in response to submerged fermentation [24]. SSF from last decade has made its importance in the production of value added products i.e., secondary metabolites, alkaloids, enzymes, organic acids, bio-pesticides (mycopesticides and bio-herbicides), biosurfactants, biofuels, aroma compounds, biopulping, degradation of toxic compounds, biotransformation, nutritional improvement of crops, biopharmaceuticals and bioconversion of agricultural waste [25]. The present study aimed to obtain a high yield of cheap cellulase by using Pleurotus Sajor-Caju (Oyster mushroom) through solid state fermentation and also exploiting local sugarcane-waste like pressmud. This study will help in proper disposal of sugarcane-waste resulting in resolution of the environmental problems.
Materials and Methods
Substrate and microorganism selection
The sugarcane waste especially Pressmud (PM) was used as Lignocellulosic substrate and it was collected from the Simbhaouli Sugar Mill, Simbhaoli, Ghaziabad, Uttar Pradesh, India. The substrate was dried in oven at 70°C and grinded mechanically with electric grinder to make it in powdered form and sieve to 40 meshes. Pleurotus sajor-caju was selected for production of cellulase enzyme. The pure culture of said strain was obtained from IMTECH, Chandigarh, India.
Maintanence of Pleurotus Sajor-Caju and preparation of vegetative inoculum
Potato dextrose agar (PDA) medium with 0.1 μg/L L-arginine added, was prepared in petri plates and formed into slants in test tubes, which were stored at 4°C. P. sajor-caju was grown on PDA at temperatures of 15, 20, 25, 30, 35, and 40°C and at pH levels of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. Every 24 h, growth of colonies was recorded. The maximum colony diameter was 7.5 cm. A loop full of conidia from 10-12 days old slant culture of P. sajor-caju aseptically transferred to a cotton wool plugged Erlenmeyer conical flask containing 200 ml of sterilized Czapek-Dox medium with following contents (3.0g NaNO3; 1.0g H2PO4; 0.01g FeSO4.7H2O; 0.01g KCl; 0.5g MgSO4.7H2O and 4g Sucrose/Lit.). The flask was incubated at 25°C on a rotary shaking incubator for 10-14 days and the freshly grown mycelial suspension was used as vegetative inoculum.
Pretreatment of the substrate
As alkali treatment on lignocellulosic substrates results in a disruption of the lignin seal, hydration and swelling of the cellulose, and a decrease in crystallinity [26,27], pretreated lignocellulosic substrates were used as the sole source of carbon to detect the cellulolytic activity of the selected fungal strains. The substrate (sugarcane pressmud) powder were soaked individually in 1% to 5% NaOH, HCl, H2O2 and H2O2 + NaOH (1:1) solutions at a ratio of 1:15 (substrate:solution), heated in a boiling water bath for 1 hr and then cooled. All chemically treated substrates were separated from the solution by filtering with muslin. Excess chemicals were removed by repeated washing with tap water until the pH was neutral. The substrates were then squeezed to remove excess water, spread over newspaper and allowed to dry overnight in an oven at 90°C [28,29].
Composition of fermentation medium
Five gram of lignocellulosic substrate was taken individually in 250 ml conical flasks. To give a final moisture content of 80%, 20 ml of distilled water was added individually to each flask. The flasks were then cotton plugged and autoclaved at 121°C for 20 min. Then, 3 ml of vegetative inoculums of Pleurotus Sajor-Caju was used to inoculate 1 flask. The inoculations were done under aseptic conditions in a laminar airflow cabinet. Substrate (5g), moisture level (20 ml) and fungal inoculum (3 ml) were kept constant for all optimizing steps.
Selection of optimum conditions for cellulase production under SSF
The strategy was adopted for optimizing the engaged parameters enhancing cellulase yield was to optimize one specific parameter and process it at the optimized level in the next experiment [30].
Optimization of pretreatment condition of substrate
The substrate (sugarcane pressmud) powder were soaked individually in 1% to 5% NaOH, HCl, H2O2 and H2O2+NaOH (1:1) solutions at a ratio of 1:15 (substrate:solution) and inoculated with P. sajor-caju respectively to determine optimum pretreatment condition at which said strain would express high cellulase activity was to select.
Optimization of thickness of substrate in fermented medium
Thickness of substrate was optimized from 0.4-2.0 cm (5-25 g) to select optimum thickness of substrate at which P. sajor-caju would exhibit hyper cellulase activity was selected.
Optimization of incubation period
Duplicate Erlenmeyer flasks using pressmud inoculated with P. sajor-caju were incubated for a period of 1-16 days to select the optimum incubation period for the production of cellulases. The growth was assessed every 24 hrs and the best incubation period at which employed strain would give maximum cellulase activity was selected.
Optimization of pH
pH was optimized from 2.0-8.0 (50 mM) to select optimum pH at which P. sajor-caju would exhibit hyper cellulase activity was selected.
Optimization of temperature
Duplicate flasks inoculated with P. pleurotus were kept at 15°C, 20°C, 25°C, 30°C, 35°C and 40°C, respectively to determine the optimum temperature at which said strain would express high cellulase activity was to select.
Enzyme extraction by solid state fermentation
After incubation, in each fermented cultures, 50 ml of 0.05 M citrate buffer (pH 5.0) was added to each flask (except in case of pH optimization where used 50 ml of DW for each flask) and left for shaking on a rotary shaker at 150 rpm for 1 h. The fermented samples were then filtered through muslin. Because the cellulase was strongly absorbed by the cellulose [31-33], 50 mL of 0.05 M citrate buffer was added in 2 successive washings to recover the enzyme successfully. The filtrates were centrifuged at 1000 × g for 30 min. The supernatants were taken as crude enzyme extracts and stored at 4°C.
Enzyme assay
There are 3 main types of enzymes present in the cellulase system: endo-β-1,4-glucanase, exo-β-1,4-glucanase, and β-glucosidase. ‘μmole of glucose released/min/g substrate’ was taken as an enzyme unit. The enzymes were analyzed as follows: (1) Combined assay for endo- and exoglucanase and β-glucosidase: For the endoglucanase assay, 1% carboxymethylcellulose (CMC) and for the exoglucanase assay, 1% cellulose solution was prepared. For the combined assay of endo- and exo- glucanase, Strips (1 × 6 cm) of Whatman No. 1 filter paper were used as substrate. Standard cellulase enzyme was used as the positive control and distilled water was used as the negative control. For the β-glucosidase assay, p-nitrophenyl-β-D, glucopyranoside (PNPG) solution (0.3 g PNPG dissolved in 40 mL distilled water) was used as substrate (pH was maintained neutral: 7.0). Endoglucanase hydrolyzed carboxymethylcellulose was used to produce free carboxymethyl glucose units. The free carboxymethyl glucose units reacted with 3, 5-dinitrosalicylic acid (DNS) reagent to form a colored complex, which was detected spectrophotometrically at 540 nm [34]. Exoglucanase removed glucose from nonreducing ends of the major chain or shorter chains of cellulose. The carbonyl group of this glucose and other reducing sugars reacted with 3, 5-dinitrosalicylic acid to form a colored complex which was detected spectrophotometrically at 540 nm [34]. β-glucosidase hydrolyzed p-nitrophenyl-β-D-glucopyranoside and released p-nitrophenol, which was determined by spectrophotometer at 410 nm [34]. (2) Filter paper assay (FPase) method: 1 mL of appropriately diluted enzyme solution was added in test tubes and 1 mL of 0.05 M citrate buff er (pH 4.8) and 50 mg (1 × 6 cm strip) of Whatman No. 1 filter paper that had been curled around a glass rod was added. The test tubes were incubated for 60 min at 50°C. After incubation, 3 ml of DNS reagent was added. Then the tubes were boiled for 15 min in a boiling water bath and 1 mL of 40% sodium potassium tartarate was added, while the tubes were still warm. After cooling to room temperature, absorbance was measured at 540 nm [34]. Standard cellulase enzyme was used as the positive control and distilled water was used as the negative control.
Results and Discussion
Optimization of pretreatment condition of substrate
Pressmud was pretreated with different concentrations of NaOH, HCl, H2O2 and H2O2+NaOH (1-5%). It was observed that there were no significant changes in cellulases activity when the substrate was pretreated with NaOH, HCl and H2O2 (1-5%) but best results were obtained, for 2.0% H2O2+NaOH treatment, which gave higher yield of Endo-β-1, 4-glucanase, Exo-β-1, 4-glucanase and β-glucosidase as 13.94, 8.84 and 12.29 Units (μmole of glucose released/min/g substrate), respectively (Figure 1). It may be due to increase in the crystallinity and decrease in the lignin contents after treatment, so the substrate becomes more acceptable for the microorganisms.
Optimization of thickness of substrate in fermented medium
Effect of varying thickness of substrate (pressmud) ranging from 0.4-2.0 cm (5.0-25 g per flask) was investigated for the production of cellulases (Figure 2). It was found that the highest activities of Endo-β-1, 4-glucanase, Exo-β-1, 4-glucanase and β-glucosidase were obtained as 18.37, 12.09 and 16.47 Units (μmole of glucose released/ min/g substrate), respectively, when the thickness of the substrate was 0.8 cm (10 g). As the depth was increased further, a significant loss in enzyme activity was observed. P. sajor-caju an aerobic fungus and requires adequate supply of aeration. As the depth of the substrate was increased the metabolic pathway of the fungus effected, which reduce its extracellular cellulolytic efficiency [35].
Optimization of incubation period
In the present study, the fermentation medium was incubated for different time intervals (2-16 days). The highest activities of Endo-β-1, 4-glucanase, Exo-β-1, 4-glucanase and β-glucosidase were obtained as 15.51, 11.09 and 13.99 Units (μmole of glucose released/min/g substrate), respectively, on 10th day of incubation (Figure 3). Further increase in the incubation period effects lethally. It might be due to the depletion of nutrients in substrate, which resulted in the inactivation of enzyme synthesis with the passage of time. Another reason is that initially the substance was more susceptible, which made rapid rise in enzyme synthesis. With the lapse of time, the susceptible portion was completely hydrolyzed to glucose, which severely inhibited the biosynthesis of cellulolytic enzymes [36-38].
Optimization of pH
The production of cellulases by P. Sajor-kaju at different pH (2.0- 8.0) of fermentation medium was also studied (Figure 4). Results showed that the highest activities of Endo-β-1, 4-glucanase, Exo-β-1, 4-glucanase and β-glucosidase were obtained as 17.65, 13.49 and 14.62 Units (μmole of glucose released/min/g substrate), respectively, at pH 5. Further increase or decrease in pH results in the reduction of enzyme activity, which shows that the acidic pH is more favorable for the growth of P. sajor-kaju strain utilizing cellulosic biomass.
Optimization of temperature
Fungal strain P. sajor-caju was also grown under all the optimized conditions at different temperatures (15-40°C) in order to optimize the fermentation temperature for enhanced cellulase production (Figure 5). It was found that the highest activities of Endo-β-1, 4-glucanase, Exo-β-1, 4-glucanase and β-glucosidase were obtained as 18.98, 13.63 and 18.54 Units (μmole of glucose released/min/g substrate), respectively, at 25°C. Secretion rate from the strain was better at 25°C. Negative effects were observed on the production by increase or decrease in the temperature.
Conclusion
Pressmud is a sugarcane processing byproduct; enriched with cellulosic biomass and is a preferable solid-substrate for the stimulation of cellulase enzymes by a number of fungal strains. The oyster mushroom Pleurotus Sajor-Caju is more potent species for the production of cellulases. Cellulase production is directly proportional to the crystallinity of biomass from which it is produced i.e., higher the crystallinity, better will be the yield of cellulases. Pretreatment of solid substrate with different chemicals act as scouring, sequestering and bleaching agent to enhance the crystallinity. It enhances the production rate of cellulases as it breaks lignin and carbohydrate bonds for successful degradation of cellulosic biomass by microorganisms [39]. Pretreatment of pressmud is an important parameter for the hyper production of cellulolytic enzymes as by this, lignin and carbohydrate bonds are broken down and the pressmud becomes easily susceptible to the microorganisms and ultimately the production rate enhances. The exploitation of this process on industrial scale can be a regular and better source of cellulases for a number of different applications.
Acknowledgements
This work was supported by School of Biotechnology, IFTM University, Moradabad, Uttar Pradesh, India. The authors wish to record his sincere thanks to Dr. Rajiv Dutta, Director, Department of Biotechnology; Prof. R. M. Dubey, Vice Chancellor and Prof. Anupam Srivastav, Pro Vice Chancellor, IFTM University, Moradabad, for their valuable suggestions and encouragement during the course of this study.
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