Isolation of Cellulose-Degrading Actinomycetes and Evaluation of their Cellulolytic Potential

Nine isolates of cellulose-degrading Actinomycetes were isolated from different sediment samples from the Bhitarkanika Mangrove Forest. Cellulase and hydrolytic activity was confirmed with clear zone around the colony on basal salt agar medium using Congo Red. The enzyme assays for two enzymes, filter paper cellulase (FPCase), and carboxymethylcellulase (CMCase), were examined by methods recommended by the International Union of Pure and Applied Chemistry (IUPAC). The extracellular cellulase activities ranged from 0.266±0.001 to 0.734±0.001 IU/mL for FPC and 0.501±0.014 to 1.381±0.024 IU/mL for CMCase or endoglucanase assay.


Introduction
Cellulose is the most abundant renewable carbon source available on the earth for production of commercial value added feedstock chemicals, when converted efficiently to monomeric D-glucose units [1]. The availability of cellulose makes it an attractive and useful raw material for producing many industrially important commodity products [2]. From the starting, both chemical and enzymatic hydrolysis has been employed for effective conversion of crude cellulosic biomass into fermentable sugar; make it to be simple to use in different industrial purposes. The old method is currently being employed as most common means for producing mono-saccharides from cellulose. Apart from it, chemical hydrolysis often leads to the accumulation of undesired non-sugar byproducts that poses big problems in recovery of resultant products and use as a cost effective product [3]. On the other hand, enzymatic hydrolysis overrides all such problems and can be used effectively over chemical hydrolysis. It has a great potential of the lower solubility in the aqueous medium [4]. Cellulase enzyme system which completely controls the enzymatic hydrolysis process comprises three classes of soluble extracellular enzymes: (1) 1, 4-β-endoglucanase, (2) 1, 4-β-exoglucanase, and (3) β-glucosidase (β-D-glucoside glucohydrolase or cellobiase). Generally endoglucanase is responsible for random cleavage of internal β-1, 4-glycosidic bonds a long a cellulose chain. Exoglucanase is mostly required for cleavage of the non-reducing end of a cellulose chain and splitting of the elementary fibrils from the crystalline cellulose and β-1, 4-glucosidase hydrolyses cellobiose and water-soluble cello-dextrin to glucose [5,6]. Synergetic activities of all these three enzymes provide the best result of completely hydrolysis of raw cellulose to glucose [7][8][9]. Microorganisms are giving an outstanding contribution regarding the synthesis of extracellular cellulase enzymes and are effectively used for the conversion of the cellulosic biomass to simple sugar molecules. Several microorganisms are reported as potential of cellulose productions viz. Actinomycetes, Bacteroides succinogenes, Butyrivibrio fibrisolvens, Clostridium species, Ruminococcus albus, Aspergillus species, Chaetomium species, Fusarium species, Methanobrevibacter ruminantium, Myrothecium species, Penicillium species, Trichoderma species etc. [10,11]. These microorganisms plays vital role as mineralizes of organic matter and thereby influencing the productivity of the marine environment. Cellulase is widely used, due to its potential applicability in various industrial processes such as production of bioethanol [12,13], triphasic biomethanation [14], agricultural and plant waste management [15,16]; chiral separation and ligand binding studies [17] etc.
Present work focuses on the isolation of cellulose degrading actinomycetes from a mangrove forest area (sediments and plant decomposed soils) and assessment of its cellulolytic potential.

Sample Collections
An extensive collection of various sediment samples were set up in Bhitarkanika National Park (20 0 44.387"N, 2 Isolation of Cellulose-Degrading Actinomycetes and Evaluation of their Cellulolytic Potential 086 0 52.042") during the Monsoon season (2013).The sediment samples were kept in sterile falcon tubes and after reaching the lab all are kept in -20 0 C till further processing.

Isolation and Screening of Cellulose Degrading Actinomycetes
Sediment and plant decomposed soil were collected from Bhitarkanika National Park. Soil samples were kept in 70 0 C to eradicate the moisture. One gram of each soil sample was transferred to aliquot of 9 mL sterile distilled water in sterile test tube. The solution was vigorously haken by vortex mixture at constant speed 4000 rpm for 15min. The soil suspension was then subjected to serial dilutions up to 10 -5 . Each dilution was inoculated in ISP-2 Medium (Yeast extract: 4g/L, Malt extract powder: 10g/L, Glucose: 4g/L, Agar: 18g/L) in appropriate plate in duplicate. The plates were incubated for 7 days at 28 o C. Pure culture actinomycetes strains are stored in ISP-2 medium at 4 0 C and Glycerol stock was prepared for future use.
Actinomycetes strains were screened for their ability to produce the hydrolytic enzymes: cellulase in a plate assay method using 1% carboxymethyl cellulose in a basal salt media, respectively, according to [18] at the incubation period, 0.1% Congo red solution was added and counterstained with 1M NaCl for 15-20min. The zone of cellulose hydrolysis was appeared as a clear area around the colony.

Cellulase Enzyme Production
The selected BAY isolates were cultured at 37 o C at 150 rpm in orbital shaker for enzyme production in basal salt media composed of KH 2 PO 4 0.5 g, MgSO 4 0.25 g, and gelatin 2 g, distilled water 1 L and containing Whatman filter paper No.1 ( 0.05 g / 20mL) and at pH 6.8-7.2. Broth culture after six days of incubation period was subjected to centrifugation at 5000 rpm for 15 min at 4 o C. The cells and other debris are settled down at the bottom and form the pellet.The cell free supernatant after centrifugation was collected and stored as crude enzyme preparation at 4 o C for further enzyme assays.

Cellulase Enzyme Assays
Total cellulase activity (FPCase and CMcase) was determined by standard method [18]. Endoglucanase (β, 1-4 endoglucanase-EC 3.2.1.4) activity was assayed by measuring the amount of reducing sugar from carboxymethyl cellulose (CMC). The cellulase activity was determined according to the methods recommended by the International Union of Pure and Applied Chemistry (IUPAC) commission on biotechnology [19]. Endoglucanase (CMCase) activity was determined by incubating 0.5mL of crude enzyme supernatant with 0.5mL of 2% carboxy methyl cellulose in 0.05M sodium citrate buffer (pH 4.8) at 55 o C for 30 minute. FPCase activity was also determined by incubating 0.5mL of crude enzyme supernatant with 1.0mL of 0.05M sodium citrate buffer (pH4.8) containing Whatman no.1 filter paper (50 mg). After incubation for an hour at 55 o C, the reaction was terminated by adding 3mL of 3, 5-dinitrosalicylic acid (DNS) reagent to 1mL of reaction mixture.
Reducing sugars were estimated spectrophotometrically with 3, 5-dinitrosalicylic acid [20] using glucose as standard. The enzymatic activity of total FPCase and endoglucanase (CMCase) were defined in international units (IU). One unit of enzymatic activity is defined as the amount of enzyme that releases 1 μmol reducing sugars (measured as glucose) per mL per minute.

Statistical Analysis
All data of results are analyzed by the one way ANOVA by SPSS and presented with (±S.D.). Each experiments were carried out independently by taking three replicates. The average mean of results of the three replicates are represented with standard deviations.

Results and Discussion
Cellulases actively convert cellulose to simple fermentable sugars readily accessible for human consumption and genus Streptomyces is known as the largest producer of cellulases [21]. Extensive research revealed that cellulases produced from Streptomyces sp. are of optimum alkaline pH and highly thermostable [22]. Besides Streptomyces, several other genera like Micromonospora and Thermobifida are known to be produced recombinant cellulases [23]. A recombinant cellulase with thermal and pH stability is also reported from Streptomyces thermoviolaceus. This enzyme is highly stable and active in the presence of commercial detergents and other ionic liquids, proves its superiority to the existing commercial cellulases [24]. Cellulase from Thermomonospora fusca has been used for degradation of avicel and cotton [25].

Isolation and Screening of Cellulose Degrading Actinomycetes
Cellulase activity of actinomycetes was carried out by preliminary screening method using the hydrolysis of substrate incorporating in the basal salt medium. The present study is a preliminary characterization of cellulase activities of the strains among the 50 actinomycetes isolated from soil samples of different locations. A total of nine actinomycetes isolates (BAY 41,03,04,40, 22,33,23,08 & 21) found to be positive on screening media (cellulose Congo-Red agar) producing clear zone (Figure 1) indicated the cellulase enzyme activity.

Cellulolytic Potential of Actinomycetes
Actinomycetes are one of the important microbial communities greatly responsible for cellulose degradation of the plant sources. The effect of major environmental factors such as temperature, salinity and pH are found to be important parameters that influence enzyme production and activities [26]. All nine actinomycetes (BAY 41,03,04,40,22,33,23,08 & 21) isolates are selected for the production of cellulase enzymes and their cellulose degrading potential is estimated. The total cellulase activity ranges from 0.501±0.014 to 1 [27]. Till date fungal strains are reported to be the highest cellulase activity among the microorganisms and the fungal species Trichoderma reesei has the highest cellulase activity [28]. Actinomycetes which are also known as filamentous bacteria are very close to fungal characteristics and are showing quite more cellulase activity in comparison to the pure bacterial species. Recently, Gupta et al. [2] studied the bacterial cellulase activities ranged from 0.012 to 0.196 IU/mL for FPC and 0.162 to 0.400 IU/mL for endoglucanase assay. The CMCase value of actinomycetes is higher than activity of CMCase produced from some known natural isolates (expressed per mL of cell-free culture broth), for example, Cellulomonas sp.  [29][30][31][32][33]. This result was supported by the findings of Howell and Mangat (1978) [34]. The cellulase activities of all isolates are shown in the Figure 2 and Figure 3.

Conclusion
The present study highlighted the potential cellulase activity of strains (BAY 03, 21 & 22) which can be used for production of biofuel and other industrial products. These cellulase enzymes from actinomycetes can strengthen other microbial cellulase sources from fungal and bacterial cellulase. Further investigations are required to make use of the full potential of these organisms for cellulase production by modern protein engineering technology.