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International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume- 4, Issue-3, Jul.-2016 ISOLATION OF CELLULASE PRODUCING THERMOPHILIC BACTERIAL FROM HOT SPRING 1 BALAKRISHNAN KUNASUNDARI, 2YI PENG TEOH, 3IBRAHIM ROSHITA 1,2 Faculty of Engineering Technology, Universiti Malaysia Perlis (UniMAP), P.O Box 77, D/A Pejabat Pos Besar Kangar, Perlis, 01000, Malaysia.. E-mail: [email protected], [email protected], [email protected] Abstract— This study focuses on the isolation of thermophiliccellulase producing bacteria from hot spring, HutanLipur Kuala Woh, Tapah, Perak, Malaysia. The cellulose degrading bacteria were isolated based on their halo zone formation on carboxymethylcelluloseagar medium stained with congo red indicator.Total 4 isolates were obtained from the primary screening technique. Only one isolate showed the highest celullase activity with the maximum carboxymethycellulose hydrolysis capacities in the range of 1.5 to 1.8 cm. From the 16S rDNA analysis, the isolate was identified as MeiothermusSilvanus strain UniMAP06. Keywords— Cellulase, Cellulose, Isolation, Thermophile, Meiothermus. of anaerobic cellulolytic bacteria are Bacteroidescellulosolvensand Clostridium thermocellum (Sun and Cheng, 2002; Sukumaran et al, 2005; Kuhad et al., 2010; Saranraj et al., 2012). Most emphasis has been placed on the use of fungi because of their capability to produce copious amounts of cellulolytic enzymes and often less complex than bacterial cellulase allowing for easy extraction and purification. It can therefore be more readily cloned and produced via recombination in a rapidly growing bacterial host. However, the isolation and characterization of novel cellulase from bacteria are now becoming widely exploited. There are several reasons for these shifts (Miranda et al., 2009). The higher growth rate of bacteria together with increased function and synergy of multi enzyme complexes of bacterial cellulases. Besides, abilities of bacteria to inhabit various environmental conditions resulting in production of cellulolytic enzymes that able to tolerate harsh conditions with improved bioconversion rate. Researchers are now focusing on the efficient utilization of these enzymes for production of various value-added products. The present study aimed to isolate and screen for potential thermophilic cellulolytic bacteria for hot spring. I. INTRODUCTION Malaysia is a country located at Southeast Asia, which has tropical climate and is blessed with agriculture resources such as oil palm and rubber. Over the past four decades, agriculture industry has significantly contributed to the economy (Goh et al, 2010).Lignocellulosic waste materials from agricultural and forest residues represent the largest renewable reservoir in Malaysia with potential generation of more than 104.55 million tonne of biomass waste annually (Shahabuddin et al, 2012). In accordance to the concept of waste-to-wealth which has been widely popular, lignocellulosic waste materials are potentially viable feedstock for conversion into valuable chemical compounds which can lead to the sustainability and advancement of energy and chemicals industries (Goh et al, 2010). Lignocellulosic biomass generally consists of cellulose which is an ample natural biopolymer on earth and the most dominating agricultural waste. It is a branched glucose polymer made of hundreds to thousands of β (1→4) linked D-glucose units (Sharadha et al., 2013).Lignocellulose is, unfortunately, a heterogeneous and recalcitrant material which is highly resistant to depolymerization. In order to exploit lignocellulosic materials for production of renewable products, it must first be broken down into constituent compounds, such as sugars, that can be more easily converted using chemical and biological processes (Kumar et al, 2008; Wackett, 2008; Matt et al, 2012). A variety of microorganisms take part in cellulose hydrolysis with an aid of a multienzyme system. Among the best-characterized cellulase from fungi systems are as follows: Trichodermakoningii, Trichodermareesei, Fusariumsolani, Aspergillusniger and Penicillumfuniculosum. Some of the aerobic cellulolytic bacteria which are having bestcharacterized cellulase systems are Cellulomonasspp., Cellvibrio spp. and Thermomonospora spp. Examples II. DETAILS EXPERIMENTAL 2.1.Enrichment, Isolation and Screening Cellulases Producing Thermophilic Bacteria of The water samples were collected aerobically in sterile Schott bottle (500 mL) from the hot spring, HutanLipur Kuala Woh, Tapah, Perak, Malaysia and stored at -20ºC until used.The collected water samples were serially diluted in 10ml of sterile distilled water. 100 µL of each dilution and water samples (without dilution) were spread on mineral agar media with the following composition [KH2PO4, 1.36 g/L; (NH4)2SO4, 1.0 g/L; MgSO4.7H2O, 0.2 g/L; Isolation of Cellulase Producing Thermophilic Bacterial From Hot Spring 155 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 FeSO4, 10.0 mg/L; NaCl, 2.0 g/L; yeast extract, 1.0 g/L supplemented with 0.3% carboxymethylcellulose (CMC)] (Patel et al., 2006) and then incubated at 55ºC for 72 h.After growth, colonies were analyzed for morphological differences and repeated streaking was done at 55ºC for 72 h on respective agar plates until pure colonieswas obtained. The plateswere flooded with 1% Congo red and 1 M NaCl in order to observe for cellulolytic activity of isolated strain. Theformation of a clear zone of hydrolysis indicatedthe cellulose degradation. The ratio of the clearzone diameter to colony diameter was measuredin order to select for the highest cellulase producer (Ariffin et al., 2006). The largest ratio was assumed to contain the highest activity. Volume- 4, Issue-3, Jul.-2016 identification as it showed highest cellulolytic activity compared to all other isolates. Analysis of 16S rDNAgene sequence The genomic DNA of strain 191 was extracted following the manufacturer’s instruction of Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) and used as the template for PCR amplification of 16S rDNA. A partial 16S rDNA region was amplified using the two primers F27 (50AGAGTTTGATCCTGGCTCAG-30) and 1492R (50-GGTTACCTTGTTACGACTT-30). The PCR amplification was performed for 30 cycles using T100 Thermal Cycler (Bio-Rad Laboratories Inc., USA). Each cycle consisted of denaturation at 94° C for 1 min, annealing at 64°C for 30 s and extension at 72°C for 1 min. The amplified DNA fragments were sequenced at First Base Sdn. Bhd. (Malaysia) and analyzed by a similarity search in the GenBank database using the BLAST. 3.2 Genotypic Identification of the Isolate Analysis of 16S rDNA gene sequence revealed that this isolate showed 99% identity to those of several Meiothermussilvanus strains. This isolate was designated as UniMAP06. The species of the genus Meiothermus comprises strictly aerobic, nonhalophilic and moderately thermophilic heterotrophs with growth temperature ranges between approximately 35 and 68°C. Up to date, there are five species of bacteria belonging to the genus Meiothermushave been described which are Meiothermusruber, Meiothermuschliarophilus, Meiothermussilvanus, Meiothermuscerbereus, and MeiothermustaiwanensisThese bacteria are generally red-pigmented, except for the yellow-pigmented strains of M. timidus and M. chliarophilus (Luciana et al., 2010; Mori et al., 2012). Detailed microscopic observations revealed that the strain UniMAP06 formed Gram negative with rod-shaped cells of variable length. Besides, when the strain was grown on broth media, large clump of cells formed which indicate the features of the genus Meiothermus(Sikorski et al., 2010). This strain showed highest cell density at 55°C in broth media even though growth was observed from the range of 40 to 58°C. So far to our knowledge, there is no information available on cellulase production from M. silvanus. III. RESULTS AND DISCUSSION 3.1 Screening for cellulolytic activity The present study focused on the isolation of thermophilic cellulolytic bacteria considering the fact that they are ideal biocatalyst for modern biotechnology applications in various industries due to thermostability and better yields under extreme operational conditions (Acharya and Chaudhary, 2012). Fourisolates which were morphologically distinct were obtained and maintained as pure culture in CMC agar plates. One of the isolates were determined to be red-pigmented as shown in Figure 1(A). The other three isolates appeared as opaque and pale yellow colonies and revealed to be morphologically different under microscopic observation. The clearing zone size and colony diameter of these isolates were measured when incubated aerobically at 55°C. The results showed that the maximum clearing zone of 2.1 cm [Figure 1(B)] was detected with the redpigmented isolate. Other isolates produced clearing zone of 0.5 to 1.1 cm demonstrating all the isolates exhibited cellulolytic activity. However, only the redpigmented isolate was selected for further genotypic CONCLUSIONS A new source of thermophilic cellulolytic bacterium has been identified from hot spring which is Isolation of Cellulase Producing Thermophilic Bacterial From Hot Spring 156 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 designated as M. silvanus strain UniMAP06. However, further research on the optimization of the cellulase activity is required to prove the efficiency of this bacterium in the bioconversion of lignocellulosic wastes. [6] ACKNOWLEDGMENTS [7] This study was supported by Fundamental Research Grant Scheme (FRGS), Grant No: 900300530[FRGS/1/2015/SG05/UNIMAP/02/4] entitled: Identification of New Cellulolytic Bacterial Strains from Tropical Mangrove Soil. [8] [9] REFERENCES [1] [2] [3] [4] [5] [10] C.S Goh, K.T. Tan, K.T Lee and S. 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