BIODIVERSITAS ISSN: 1412-033X Volume 20, Number 11, November 2019 E-ISSN: 2085-4722 Pages: 3134-3141 DOI: 10.13057/biodiv/d201105 Cellulolytic enzyme-producing thermophilic Actinobacteria isolated from the soil of Cisolok Geysers, West Java, Indonesia PUTRI PRATIWI SETYANINGSIH 1,* , FITRIA NINGSIH 1,2,* , MAZYTHA KINANTI RACHMANIA 1 , WINDA AYU SYAFITRI 1 , DHIAN CHITRA AYU FITRIA SARI 1,2 , SHUHEI YABE 3,4 , AKIRA YOKOTA 3 , ARIYANTI OETARI 1,2 , WELLYZAR SJAMSURIDZAL 1,2,♥ 1 Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia. Jl. Prof. DR. Sudjono D. Pusponegoro, Kampus UI Depok, Depok 16424, West Java, Indonesia 2 Center of Excellence for Indigenous Biological Resources-Genome Studies, Faculty of Mathematics and Natural Sciences, Universitas Indonesia. Jl. Prof. DR. Sudjono D. Pusponegoro, Kampus UI Depok, Depok 16424, West Java, Indonesia. Tel.: +62-21-7270163, Fax.: +62-21-78849010, ♥ email: sjwelly@hotmail.com; sjwelly@sci.ui.ac.id 3 Department of Microbial Resources, Graduate School of Agricultural Sciences, Tohoku University, 468-1 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-0845, Japan 4 Hazaka Plant Research Center, Kennan Eisei Kogyo Co., Ltd., 44 Aza-Inariyama, Oaza-Ashitate, Murata-cho, Shibata-gun, Miyagi 989-1311, Japan *These authors contributed equally to the work Manuscript received: 8 August 2019. Revision accepted: 8 October 2019. Abstract. Setyaningsih PP, Ningsih F, Rachmania MK, Syafitri WA, Sari DCAF, Yabe S, Yokota A, Oetari A, Sjamsuridzal W. 2019. Cellulolytic enzyme-producing thermophilic Actinobacteria isolated from the soil of Cisolok Geysers, West Java, Indonesia. Biodiversitas 20: 3134-3141. This study investigated 17 thermophilic Actinobacteria isolated from the soil of geysers in the Cisolok geothermal area, West Java, as potential producers of cellulase. Screening for cellulase was performed on minimal (Mm) agar medium with and without the addition of 1% (w/v) carboxymethylcellulose (CMC) and microcrystalline cellulose (MCC), then incubated at 45, 50, 55 and 60°C for up to 7 days. Formation of clear zones around colonies indicated cellulose hydrolysis. The results showed that 15, 14, 4, and 3 isolates showed cellulolytic activity on CMC agar medium at 45, 50, 55, and 60°C, respectively, after 7 days of incubation. Three potential isolates showed cellulolytic activity on MCC agar medium after being incubated for 7 days at 45°C. Molecular identification based on the 16S rRNA gene was performed for three isolates with positive cellulolytic activity at 60°C. The results showed that the three isolates are closely related to Actinomadura keratinilytica WCC-2265 T with 99.93-100% sequence similarities. A phylogenetic tree based on 16S rRNA gene sequences confirmed that the three isolates were clustered together with Actinomadura keratinilytica WCC-2265 T with 100% bootstrap value. The tree also showed that cellulase producers and non-cellulase producers in Thermomonosporaceae are grouped into different clades. Keywords: Cellulase, Cisolok geothermal area, thermophilic Actinobacteria, soil INTRODUCTION Actinobacteria is one of the largest phyla within the domain Bacteria which comprises Gram-positive bacteria with high G+C content in their DNA. They are frequently found in soil due to their abilities in carbon cycling (Edwards 1993), and are mostly mesophilic (Satyanarayana et al. 2013). Actinobacteria can also be found in high- temperature habitats such as geothermal areas, deserts, and deep-sea hydrothermal vents (Satyanarayana et al. 2013), with growth temperatures, range from 40 to 80°C (Shivlata and Satyanarayana 2015). These thermophilic Actinobacteria has gained interest and are largely studied for their metabolites production, such as various antibiotics and industrial important thermostable enzyme (Hamedi and Wink 2017; Lewin et al. 2017). Thermophilic Actinobacteria were reported to produced many important enzymes, such as amylases, cellulases, lipases, proteases, and xylanases. Studies on extracellular enzymes produced by thermophilic Actinobacteria have garnered interest for application in various industries due to their thermostability (Satyanarayana et al. 2013). The advantages of thermostable enzymes from thermophiles include great stability, and enhanced activity in the presence of common protein denaturants (Matsui et al. 2000). They also reduce the risk of contamination (Haki and Rakshit 2003). Cellulases, as one of important enzymes, are widely used in several industries, such as textiles, pulps, and biofuels (Satyanarayana et al. 2013). Cellulases act upon cellulose by breaking the polysaccharide down into oligosaccharide and glucose (Rani et al. 2016). Cellulose is one of the most abundant polysaccharides and is a major component of plants, composed of glucose units linked by β-1,4-glycosidic bonds (Sharma and Yazdani 2016). Thermostable cellulases are required to break the crystalline celluloses, such as biopolishing of cotton fabric in the textile industry because it is a more efficient process at high temperature (Ando et al. 2002). Cellulase-producing bacteria have more advantages than fungi e.g. better stability, shorter generation time, easy to cultivate using inexpensive carbon and nitrogen sources, and larger amount of enzymes secreted (Li et al. 2008). Additionally, several thermophilic Actinobacteria have been reported as cellulase producers, for example,