CELLULOSE CHEMISTRY AND TECHNOLOGY BIODIVERSITY OF CELLULASE PRODUCING BACTERIA AND THEIR APPLICATIONS NADEEM AKHTAR, * AANCHAL, * DINESH GOYAL * and ARUN GOYAL ** *Department of Biotechnology, Thapar University, Patiala, 147 004, Punjab, India ** Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781 039, Assam, India ✉Corresponding author: Dinesh Goyal, [email protected] Received August 30, 2014 Bioconversion of cellulose, mainly by bacteria and fungi, plays a key role in the global carbon cycle. The biodiversity of these microorganisms in nature is due to diverse cellulosic substrates and geoclimatic conditions, but despite their abundance, only a few are efficiently involved in the biodegradation process. Bacterial cellulase is preferred over fungal one, owing to the higher bacterial growth rate, broad range of tolerance, synergy of the complex enzyme system, higher compatibility and feasibility towards genetic engineering. This paper reviews the diversity of aerobic and anaerobic cellulolytic bacteria, their cellulase activity, stability and application in biodegradation of lignocelluloses in various industries. Most of the cellulolytic bacteria belong to phyla Actinobacteria, Bacteroidetes, Fibrobacteres, Firmicutes and Proteobacteria. Among aerobic bacteria, members of Bacillaceae and Paenibacillaceae families mainly inhabit soil, cowdung and compost. However, anaerobic species of Clostridia have mostly been reported to originate from sediments of hot springs, soil and compost, whereas Butyrivibrio , Fibrobacter , Halocella and Ruminococcus are found in rumen. Purified bacterial cellulases generally have wide pH and temperature tolerance and a molecular mass in the range of 30-250 kDa. Efforts have been made to increase cellulase activity, all with limited success. Extremophilic bacteria, such as Acidothermus cellulolyticus , Clostridium straminisolvens , Geobacillus pallidus , Sporotrichum thermophile , Thermobifida fusca , Thermomonospora curvata and Rhodothermus marinus , have proved to be a better choice for cellulase production to improve the process economics for various industrial applications. Keywords : Bacillus sp., biodiversity, cellulase, hemicellulose INTRODUCTION Cellulose, the most abundant, widely have great potential in biofuel production over the available, low cost, polymeric, renewable past few decades. Many types of organisms resource on Earth, has attracted recent worldwide produce cellulases, which move through secretory concern in the search of alternative ways to meet pathways to reach the extracellular space, where future energy demands. Efforts have been made to enzymatic reactions take place.4 Both fungi and reduce international dependence on conventional bacteria have been widely used for their abilities petroleum resources, and to minimize the impact to produce a wide variety of cellulases. More of rising energy and feedstock material costs. 1-3 emphasis has been placed on the use of fungi due Complete and efficient hydrolysis of cellulose is to their capability to produce copious amounts of the result of synergistic actions between different cellulases, which can be easily extracted and enzymes, which include exo-glucanases, endo- purified, and are less complex than bacterial glucanases and β-glucosidase. glycoside hydrolases. However, due to certain Cellulases belong to the glycoside hydrolase advantages, such as the higher growth rate of family of enzymes and act by cleaving β,1-4 bacteria, synergy of their complex enzyme glycosidic bonds of polysaccharides and system, higher compatibility and feasibility oligosaccharides. Due to the ability of these towards genetic engineering, bacteria are enzyme systems to convert cellulosic materials preferred over fungi. In addition, bacteria inhabit into useful sugars, they have been considered to a variety of environmental and industrial niches, Cellulose Chem. Technol., 50 (9-10), 983-995(2016) NADEEM AKHTAR et al . and their cellulolytic properties are extremely chemical treatment.14 The dominant sugars in resistant to environmental stress.5 hemicelluloses are mannose in softwoods and Over the years, culturable, cellulase producing xylose in hardwoods and agricultural residues.15-17 bacteria have been isolated from diverse habitats, These heteropolymers contain galactose, glucose, such as composting heaps, decaying plant arabinose and a small amount of rhamnose, materials, feces of ruminants and hot springs.6 glucuronic acid, methyl glucuronic acid and Bacteria show higher growth rates as compared to galacturonic acid.12,14 In contrast to cellulose, fungi, and have a good potential for use in hemicelluloses have a random, amorphous and cellulase production.7 Among bacteria, recent and branched structure with little resistance to widespread concern regarding the need of an hydrolysis and they are more easily hydrolyzed by efficient cellulase producer targets extremophiles, acids to their monomer components.18-22 Lignin is which may evade harsh conditions during a very complex, recalcitrant molecule constructed bioconversion processes. Cellulases provide a key of phenylpropane units linked in a three- opportunity for achieving tremendous benefits dimensional structure, which encapsulates from biomass utilization,8 contributing to cellulose and hemicelluloses, which, in turn, satisfying the global industrial demand for reduces the efficiency of hydrolysis. enzymes. The cellulase market is expected to expand dramatically due to their applications in Cellulase: The enzyme complex the hydrolysis of pretreated cellulosic materials to Cellulases are inducible enzymes, which are sugars and further to bioethanol and other bio- synthesized by a large number of microorganisms, based products.9 either cell-bound or extracellular during their The search is on for efficient cellulase growth on cellulosic materials. 23 Historically, producing bacteria, with high cellulase activity, cellulases have been divided into three major which could be effective over a broad range of groups: endoglucanase (EC 3.2.1.4), tolerance in order to achieve optimum utilization exoglucanase or cellobiohydrolase (EC 3.2.1.91) of these enzymes for use in biofuel and and β-glucosidase (EC 3.2.1.21) and the bioproduct industries. The current focus is now on synergistic actions of these enzymes are a widely screening and developing novel cellulose accepted mechanism for cellulose hydrolysis.10,24- degrading enzymes with special characteristics.10 27 Figure 1 presents a simplified schematic The exploitation of bacteria in the search for diagram of the enzymatic actions of cellulases, improved enzymes provides a means to upgrade involving exoglucanase, endoglucanase and β- the feasibility of lignocellulosic biomass glucosidase on cellulose. Endoglucanase conversion, ultimately providing the means to a randomly cleaves the internal bonds in the ‘greener’ technology. 5 amorphous region; exoglucanase cleaves the The current review envisages characterization exposed chains produced by endoglucanases, of different cellulolytic aerobic and anaerobic whereas β-glucosidase hydrolyses the cellobiose bacteria and their biodiversity in nature . The unit produced by the action of exoglucanase. paper discusses the benefits of bacteria over fungi These enzymes can either be free in aerobic and the trends in utilizing cellulosic substrates to microorganisms, or grouped in a multicomponent produce cellulases in making the bioconversion enzyme complex, cellulosome, in anaerobic process more efficient and compatible. cellulolytic bacteria. 24 Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria Lignocellulose and its composition and protozoans, 28 which acts as a biocatalyst for Cellulose, or β,1-4-glucan, is the most the conversion of cellulosic substrates to abundant and ubiquitous linear polymer of fermentable sugar; this process incurs major costs glucose with widespread industrial use. Cellulose in a biorefinery. 29 microfibrils, roughly one third of the total mass of Cellulose has attracted attention as a many plants, contain 500 to 14,000 glucose units renewable resource that can be converted into linked by β,1-4 glycosidic bonds.11,12 The bioenergy and value-added bio-based products. 30 cellulose fibrils are associated in the form of Enormous amounts of agricultural, industrial and bundles 13 and attached to each other by municipal cellulosic wastes have accumulated or hemicelluloses, amorphous polymers of different been used inefficiently due to the high cost of sugars, pectins and covered by lignin, which their utilization processes. 31,32 Therefore, it would makes cellulose resistant to both biological and be of considerable economic interest to develop 984 Cellulase processes for effective treatment and utilization of agricultural processes, some of these are: citrus cellulosic wastes as a cheap carbon source. These peel waste, coconut biomass, sawdust, paper pulp, biomasses comprise leaves, stems and stalks from industrial waste, municipal cellulosic solid waste sources such as corncob, corn stover, sugarcane and paper mill sludge. 33 bagasse, rice, rice hulls, woody crops and forest residues. Besides, there are multiple sources of lignocellulosic waste from industrial and Figure 1: A simplified schematic representation of the enzymatic action of cellulase, involving exoglucanase, endoglucanase and β-glucosidase, on cellulose In the last two decades, research has been impressive collection of more than 14,000 fungi aimed at developing