Studies on Cellulase Production by Solid state Fermentation using Sweet Sorghum bagasse

Author Name: *Lavudi Saida, Harinder Singh Oberoi, M.Laxmi Narasu
Author Email: lavudisaida@jntuh.ac.in

Abstract

The present study involves production of cellulolytic enzymes by a newly isolated strain of Aspergillus niger isolated from decaying sugarcane bagasse. Solid-state-fermentation was employed using sweet sorghum bagasse as the substrate for the production of enzyme. One factor at a time approach was used to optimize the process variables like moisture content, pH, temperature, and incubation period. A maximum of 19.2 FPU/g-ds of cellulose and 384.5 IU/g-ds of β-glucosidase activity of were obtained at 70% moisture content, a temperature of 30 °C, pH of 5 and incubation time of 96 h. This study showed an effective utilization of sweet sorghum bagasse for the production of cellulolytic enzymes which could be effectively used for hydrolysis to simple sugars for production of bioethanol.

Keywords

Aspergillus niger, sweet sorghum bagasse, wheat bran, Solid state fermentation, cellulase, β-glucosidase.

Introduction

Lignocellulosic waste residues obtained from energy crops, wood and agricultural residues represent the most abundant sources of renewable biomass [1]. It is estimated that the photosynthetic process produces about 150 billion tons of dry material annually with respect to carbon, of which about 50% is cellulose [2]. Cellulase enzymes are the key drivers for biomass utilization through the bioconversion of the most abundant cellulosic wastes into its monomeric forms [3]. Researchers have strong interests in cellulases because of their applications in industries such as, starch processing, grain alcohol fermentation, malting and brewing, extraction of fruit and vegetable juices, pulp and paper industry, and textile industry [4]. A potential challenging area where cellulases would have a central role is the bioconversion of renewable cellulosic biomass to commodity chemicals [5]. In recent years, some important agro-industrial residues such as sugarcane bagasse, kinnow waste, wheat bran, rice straw, leaves, corn stover etc. have been used as substrates by solid state fermentation (SSF) for the production of enzymes [4,7,8]. Cost reduction of the process can be achieved by the use of high enzyme producers and use of cheaper substrates [9]. Reduction in the production cost and improvement in cellulase yield could also be achieved using appropriate and low cost carbon and nitrogen sources in the formulation of fermentation medium [10, 11]. Thus, the use of rice straw for production of cellulases which have the potential of bioconversion of cellulosic biomass to sugars seems to be a good alternative. Microbial degradation of lignocellulosic wastes and the downstream products resulting from it is due to the concerted action of several enzymes, the most prominent of which are cellulases.
Cellulase is a multienzyme system composed of three main enzymes viz., Endoglucanase, Exoglucanase and β-Glucosidase which act synergistically and catalyze the hydrolysis of cellulose and cello-oligosaccharide derivatives. Cellulolytic enzymes comprise of endoglucanase (EG; 1,4-β-D-glucan-4-glucano-hydrolases; EC3.2.1.74), exoglucanase (EC 3.2.1.91 and EC 3.2.1.74) and β-glucosidase (BGL; EC 3.2.1.21). Endoglucanases cleave glycosidic bonds randomly within the interior of cellulose polymer chain. Exoglucanase acts progressively on the reducing and non-reducing ends of cellulose chains, releasing cellobiose and glucose respectively as major products. The β-glucosidase hydrolyzes soluble cellodextrins and cellobiose to glucose [14]. In addition, auxiliary enzymes, such as, glucuronidase, acetylesterase, feruloyl esterase, xylanase, β-xylosidase, galactomannanase and glucomannanase attack hemicelluloses, thereby releasing pentose sugars, such as xylose and arabinose. Media for cellulase enzyme production with components like cellulose, glucose, yeast extract, peptone, urea, KH2PO4, (NH4)2SO4, MgSO4, FeSO4, MnSO4, CoCl2, CaCl2 etc have been reported previously [16, 17]. As large number of basal ingredients are screened and optimized in fermentation processes, experiments based on multi-factorial design are difficult, thus statistics-based experimental methods have been designed for media optimization [18, 19]. Solid-state fermentation (SSF) holds tremendous potential for production of commercially important enzymes because of several advantages, such as higher fermentation productivity, higher concentration of end product, higher product stability, lower catabolic repression and cultivation of microorganisms specialized for water-insoluble substrates [15]. The present study reports the optimization and validation of process parameters using one factor at a time method in SSF.

Conclusion

In the present study optimal process parameters for enzyme production by Aspergillus niger ,were found to be pH of 5.0, moisture content of 70%, a Temperature of 30 °C, and a substrate ratio (SSB:WB)- 4:1 and Incubation time of 96h. Similar kind of work has been done by [4], on cellulase enzyme production. Work was performed on 2 levels of bagasse (2 and 5%) and it was reported that the optimal variables as mixed substrate (RB: WB 1:4, 3:2), temperature 30°C, initial culture pH 5.5. Optimal conditions for the endogluconase production by T. viridae strain was reported 97 U/ml and 95 U/ml [28].
It is reported in the literature that solutions with higher desirability gave optimum temperature of 30°C, pH of 5 and moisture content of 70% [8]. Our results are also in agreement with the observations recorded in the previous studies. In the present investigation enzyme activities under the optimized conditions for both the F-pase and β-glucosidase, and Protein content were maximized.

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