Jurnal Biologi Indonesia diterbitkan oleh Perhimpunan Biologi Indonesia. Jurnal ini memuat hasil penelitian ataupun kajian yang berkaitan dengan masalah biologi yang diterbitkan secara berkala dua kali setahun (Juni dan Desember). Editor Ketua Prof. Dr. Ibnu Maryanto Anggota Prof. Dr. I Made Sudiana Dr. Deby Arifiani Dr. Izu Andry Fijridiyanto Dewan Editor Ilmiah Dr. Abinawanto, F MIPA UI Dr. Achmad Farajalah, FMIPA IPB Prof. Dr. Ambariyanto, F. Perikanan dan Kelautan UNDIP Dr. Didik Widiyatmoko, Pusat Konservasi Tumbuhan Kebun Raya-LIPI Dr. Dwi Nugroho Wibowo, F. Biologi UNSOED Dr. Gatot Ciptadi F. Peternakan Universitas Brawijaya Dr. Parikesit, F. MIPA UNPAD Dr. Faisal Anwari Khan, Universiti Malaysia Sarawak Malaysia Assoc. Prof. Monica Suleiman, Universiti Malaysia Sabah, Malaysia Dr. Srihadi Agungpriyono, PAVet(K), F. Kedokteran Hewan IPB Y. Surjadi MSc, Pusat Penelitian ICABIOGRAD Drs. Suharjono, Pusat Penelitian Biologi-LIPI Dr. Tri Widianto, Pusat Penelitian Limnologi-LIPI Dr. Witjaksono Pusat Penelitian Biologi-LIPI
Sekretariat Eko Sulistyadi M.Si, Dewi Citra Murniati M.Si, Hetty Irawati PU, S.Kom Alamat d/a Pusat Penelitian Biologi - LIPI Jl. Ir. H. Juanda No. 18, Bogor 16002 , Telp. (021) 8765056 Fax. (021) 8765068 Email :
[email protected];
[email protected];
[email protected];
[email protected] Website : http://biologi.or.id
Jurnal Biologi Indonesia : Akreditasi: No. 657/AU3/P2MI-LIPI/07/2015.
JURNAL BIOLOGI INDONESIA
Diterbitkan Oleh: Perhimpunan Biologi Indonesia Bekerja sama dengan PUSLIT BIOLOGI-LIPI
OBITUARI Redaksi Jurnal Biologi Indonesia telah kehilangan seorang editor penelaah Dr. Ir Sri Sulandari, M.Sc. yang telah berpulang kerahmat Allah SWT pada tanggal 18 Agustus 2015 Jam 16.10 di RSCM, Jakarta. Jabatan terakhir almarhumah sebagai Peneliti Madya/IVc di Pusat Penelitian Biologi-LIPI sebagai ahli DNA Molekuler yang menekuni kajian DNA pada ayam lokal Indonesia dan berbagai hidupan liar khususnya pada burung. Tiga tahun terakhir sangat aktif berusaha menyelamatkan populasi kambing Gembrong di Kabupaten Karanganyar, Bali. Almarhumah meninggalkan seorang suami Prof. Dr. Muladno, MSA yang bekerja sebagai guru besar di Fakultas Peternakan, Institut Pertanian bogor dan saat ini juga sebagai Direktur Jendral Peternakan dan Kesehatan Hewan, Kementerian Pertanian, serta dua anak laki-laki Aussie Andry Vermarchnanto M. dan Endyea
Mendelian.
Jurnal Biologi Indonesia yang diterbitkan oleh PERHIMPUNAN BIOLOGI INDONESIA bekerjasama dengan PUSLIT BIOLOGI-LIPI. Edisi volume 11 No. 2 tahun 2015 memuat 15 artikel lengkap dan satu artikel tulisan pendek. Penulis pada edisi ini sangat beragam yaitu dari Balai Besar Penelitian Veteriner-Deptan, Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian, Bogor, Balai Penelitian Tanaman Sayuran Lembang, Bandung, Departemen Konservasi Sumberdaya Hutan dan Ekowisata, Fakultas Kehutanan-IPB, Dept. Biokimia FMIPA-IPB, Institut Sains dan Teknologi Nasional Jakarta, Pusat Penelitian dan Pengembangan Sumberdaya Pesisir & Laut, Balitbang Kelautan & Perikanan, Kementerian Kelautan & Perikanan, Departemen Manajemen Sumberdaya Perairan, Fakultas Perikanan dan Ilmu Kelautan, IPB. Program Studi Manajemen Sumberdaya Perairan, Fakultas Ilmu Kelautan dan Perikanan-Universitas Maritim Raja Ali HajiTual, Pusat Konservasi Tumbuhan Kebun Raya–LIPI, Puslit Biologi-LIPI, Puslit Bioteknologi-LIPI.
Jurnal Biologi Indonesia mengucapkan terima kasih dan penghargaan kepada para pakar yang telah turut sebagai penelaah dalam Volume 11 No 2, Desember 2015: Dr. Niken Tunjung Murti Pratiwi, Fakultas Perikanan dan Ilmu Kelautan IPB Dr. Agus Prijono Kartono, Fakultas Kehutanan IPB Ir. Drs. Eko Harsono MSi, Puslit Limnologi-LIPI Dra. Donowati Tjokrokusumo M.Phil, Pusat Teknologi Bioindustri, BPPT Ir. M. Syamsul Arifin Zein MSi, Puslit Biologi LIPI Drh. Anang S. Achmadi MSc, Puslit Biologi LIPI Dr. Yuyu S. Poerba, Puslit Biologi LIPI Ir. Dwi Agustiyani MSc, Puslit Biologi LIPI Dr. Apon Zaenal Mustopa, Puslit Bioteknologi LIPI Dr. Yopi Puslit Bioteknologi LIPI Dr. Joeni S. Rahajoe, Puslit Biologi LIPI Dr. Wartka Rosa Farida, Puslit Biologi LIPI
BIOLOGI
Halaman Efikasi Vaksin Inaktif Bivalen Avian Influenza Virus Subtipe H5N1 (Clade 2.1.3. dan Clade 2.3.2) di Indonesia NLP. Indi Dharmayanti & Risa Indriani
169
Klon-klon Kentang Transgenik Hasil Persilangan Terseleksi Tahan terhadap Penyakit Hawar Daun Phytophthora infestans Tanpa Penyemprotan Fungisida di Empat Lapangan Uji Terbatas Alberta Dinar Ambarwati, Kusmana, & Edy Listanto
177
Penambahan Inokulan Mikroba Selulolitik pada Pengomposan Jerami Padi untuk Media Tanam Jamur Tiram Putih (Pleurotus ostreatus) Iwan Saskiawan
187
Identifikasi Molekular dan Karakterisasi Morfo-Fisiologi Actinomycetes Penghasil Senyawa Antimikroba Arif Nurkanto & Andria Agusta
195
Populasi dan Kesesuaian Habitat Langkap (Arenga obtusifolia Mart.) di Cagar Alam Leuweung Sancang, Jawa Barat Didi Usmadi, Agus Hikmat, Joko Ridho Witono, & Lilik Budi Prasetyo
205
Optimasi Produksi Enzim Amilase dari Bakteri Laut Jakarta (Arthrobacter arilaitensis ) Awan Purnawan, Y. Capriyanti, PA. Kurniatin, N. Rahmani, & Yopi
215
Pengaruh Antioksidan Eksopolisakarida dari Tiga Galur Bakteri Asam Laktat pada Sel Darah Domba Terinduksi tert-Butil Hidroperoksida (t-BHP) Fifi Afiati, Nina Ainul Widad, & Kusmiati
225
Ekosistem Lamun sebagai Bioindikator Lingkungan di P. Lembeh, Bitung, Sulawesi Utara Agustin Rustam, Terry L. Kepel, Mariska A. Kusumaningtyas, Restu Nur Afi Ati, August Daulat, Devi D. Suryono, Nasir Sudirman, Yusmiana P. Rahayu, Peter Mangindaan, Aida Heriati, & Andreas A. Hutahaean
233
Identification of Bioactive Compound from Microalga BTM 11 as Hepatitis C Virus RNA Helicase Inhibitor Apon Zaenal Mustopa, Rifqiyah Nur Umami, Prabawati Hyunita Putri, Dwi susilaningsih, & Hilda Farida
243
Kemampuan Cerna Protein dan Energi Metabolisme Perkici Pelangi (Trichoglossus haematodus ) Rini Rachmatika & Andri Permata Sari
253
Optimasi Enzim α-Amilase dari Bacillus amyloliquefaciens O1 yang Diinduksi Substrat Dedak Padi dan Karboksimetilselulosa Yati Sudaryati Soeka, Maman Rahmansyah, & Sulistiani
259
Kajian Aspek Ekologis dan Daya Dukung Perairan Situ Cilala Niken T.M. Pratiwi, Sigid Hariyadi, Inna Puspa Ayu, Aliati Iswantari, Novita MZ, & Tri Apriadi
267
Halaman Penanda Genetik Tarsius (Tarsius spp.) dengan Menggunakan Gen Cytochrome Oxidase I 275 (COI) DNA Mitokondria (mtDNA) Melalui Metode Sekuensing Wirdateti, Sri Wijayanti Wulandari, & Paramita Cahyaningrum Kuswandi Carboxymethyl Cellulose Hydrolyzing Yeast Isolated from South East Sulawesi, Indonesia Atit Kanti
285
Uji Bakteri Simbiotik dan Nonsimbiotik Pelarutan Ca vs. P dan Efek Inokulasi Bakteri pada Anakan Turi (Sesbania grandiflora L. Pers.) Sri Widawati
295
TULISAN PENDEK Mating behavior of Slow Loris (Nycticebus coucang ) at Captivity Wartika Rosa Farida & Andri Permata Sari
309
Carboxymethyl Cellulose Hydrolyzing Yeast Isolated from South East Sulawesi, Indonesia Khamir Penghidrolisa Selulosa Karboxymethil yang Diisolasi dari Sulawesi Tenggara Indonesia Atit Kanti
Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong Science Centre, Jl.Raya BogorJakarta Km. 46, Cibinong 16911, Indonesia
[email protected] Received: May 2015, Accepted: May 2015 ABSTRACT The objective of study was to isolate, identify and characterize the CMC-ase producing yeast from South East Sulawesi, Indonesia. We isolated 142 strains and obtain 53 strains (37.32%) were CMC-ase producer consist of 26 species residing within 10 genera. Candida was the most diverse genus consisting of 15 species. It is important to note that several strains residing within this genus could be candidate for new taxa, among others Candida aff. cylindracea PL2W1, Candida aff. insectorum PL3W6, Candida aff. friedrichii MKL7W3, Candida aff. lessepsii, Candida aff. tenuis. Five new candidates for novel species of cellulolytic yeast close to Yamadazyma mexicana: were Yamadazyma aff. mexicana (5 strains). Pichia, Pseudozyma, Sporodiobolus, and Sporobolomyces were other cellulolytic yeasts found in South East Sulawesi. It is obvious, that leaf litter was a good source for cellulolytic yeasts. This CMC-ase producing yeasts dominate this biome, and production of extracellular cellulase is critical strategy for such yeast to survive in cellulose rich ecosystem such as leaf-litter. This finding would suggest that yeasts play key role on hydrolyzes of cellulose and important resources for sustainable energy research. Keywords: cellulolytic yeasts, secondary forest, South East Sulawesi, leaf-litter ABSTRAK Penelitian bertujuan untuk melakukan proses isolasi, identifikasi, dan karakterisasi khamir yang mempunyai kemampuan untuk memproduksi CMC-ase dari Sulawesi Tenggara, Indonesia. Sebanyak 142 strain telah diseleksi dan 53 strain merupakan penghasil CMC-ase (37,32%), yang terdiri dari 26 jenis yang tergolong dalam 10 marga. Candida merupakan marga yang paling beragam, terdiri dari 15 jenis. Hal penting yang perlu dicatat adalah beberapa strains yang termasuk marga tersebut merupakan kandidat taksa baru, diantaranya Candida aff. cylindracea PL2W1, Candida aff. insectorum PL3W6, Candida aff. friedrichii MKL7W3, Candida aff. lessepsii, Candida aff. tenuis. Lima kandidat jenis baru yang merupakan khamir selulolitik yang berkerabat dekat dengan Yamadazyma mexicana: adalah Yamadazyma aff. mexicana (5 strains). Pichia, Pseudozyma, Sporodiobolus, dan Sporobolomyces adalah khamir selulolitik lain yang ditemukan di Sulawesi Tenggara. Berdasarkan dari hasil yang didapat, sangat jelas bahwa serasah merupakan sumber yang potensial untuk mendapatkan khamir selulolitik. Khamir penghasil CMC-ase ini mendominasi bioma tersebut, dan produksi dari selulase ekstraseluler merupakan strategi kunci agar dapat bertahan hidup di ekosistem yang kaya selulosa seperti serasah. Dari penemuan ini maka dapat diambil kesimpulan bahwa khamir memegang peranan penting pada proses hidrolisis selulosa dan merupakan sumber penting bagi penelitian energi terbarukan. Kata Kunci: khamir selulolitik, hutan sekunder, Sulawesi Tenggara, serasah
INTRODUCTION Up to date ecological studies of yeast from natural habitats have been conducted extensively mostly in temperate regions (Wang & Bai 2004; Butinar et al. 2005). Taxonomy and ecology data indicate a need for additional studies in tropical ecosystems, particularly in Asia (Nakase et al. 2006; Takashima et al.
2012). Indonesia is a tropical nation comprised of over 17,000 islands is rich in biodiversity, having unique flora and fauna (Allend 2009), and presumably microbes as well. Rifai 1995 estimated Indonesia has more than 200,000 species of fungi. However, little information on species diversity of Indonesia indigenous yeast and yeast-like fungi has been published. Studies of Indonesian yeasts primarily related
Atit Kanti
to their role in fermented foods (Abe at al. 2004; Kuriyama et al.1997). Early studies of yeast from natural environment in Indonesia include Deinema in 1961, who found Candida bogoriensis from the surface of leaves of the flowering shrubs Randia melleifera (Rubiaceae) in Bogor. In recent years, more studies have been performed to explore yeast diversity in Indonesia (Sjamsuridzal et al. 2010; Sudiana & Rahmansyah 2002). While microorganisms are very important sources for bioindustry however few study conducted to assess the important of forest as genetic resources for many interest. Cellulase are primarily obtained from cellulolytic fungi: Trichoderma reseei, T. viride, T. lignorum (Fahrurrozi et al. 2010), T. koningi, Chrysosporium lignorum, C. pruinosum, Fusarium solani, Neusrospora crassa, Aspergillus (Golddbeck et al. 2013a), Penicelllium, Acremonium strictum (Golddbeck et al. 2013b), including that from several group of cellulolytic bacteria: Cellulomonas, Cellulosimicrobium cellulans (Lo et al. 2009) Clostridium clariflavum, Bacillus group, Flavobacterium, and Paenibacillus (Zhanga et al. 2013), and Fermicutes and Actinomycetes group (Zainudin et al. 2013). On the other hand the role of yeast on biodegration of cellulose is few explored, particularly using yeast from tropical forest (Jimenez et al. 2007; Kanti et al. 2014). Sulawesi has high biodiversity, and reported to have high biodiversity of flora (Cannon 2007) and fauna (Koch 2011). At the microbial diversity level, further study is needed to verify the species richness of this area, particularly cellulolytic yeast. We evaluated two areas in South East Sulawesi that have different covering vegetation. Leaf litter, soil and leaf surfaces are common habitats for yeast (Takashima et al. 2002; Chang et al. 2012). This paper is concerned with the isolation of cellulolytic yeast from natural habitats in Southeast Sulawesi, Indonesia and their phylogenetic affiliation based on partial 26S rDNA and ITS1/5.8S/ITS2 region. MATERIALS AND METHODS Leaf and leaf litter samples were aseptically collected from two sites that differ in elevation, forest type, and land use type.
286
Papalia Protected Forest is a tropical lowland monsoon wet forest dominated by evergreen tree, with a high humidity and high rain fall. Papalia, located in South Konawe, GPS S 04◦ 13’ 526”, E 122 44 301”, having altitude <1000 m asl. Whereas Mekongga Protected Forest covers lowland and upland rain forest sites included hill forest, montane forest with elevation gradient ranges from 100 m to 2500 m asl. Land use type is dominated by cocoa plantations. Mekongga rain forest is located near Tinukari, North Kolaka, GPS S 03◦ 38’ 085”, E 121 04 311”. Leaf and leaf litter sampling were conducted in 2009 and 2010. Six leaves and 6 leaf litter samples were collected in Papalia, and six leaves and 10 leaf litter were collected from Mekongga Protected Forest. Yeasts were isolated from leaf, leaf litter, and soil. One g of soil or leaf litter was added to 25 mL of saline/Tween (0.85% NaCl, 0.01% Tween 80, v/v) buffer in a 7 oz Whirl-Pak filter bag (Cat. B01385WA, Nasco, Salida, CA, USA) and shaken to suspend the microbes. The bag had two separated compartments which allowed separation of suspension from debris. Two hundreds μL of suspension were plated on Rose Bengal Chlorampenicol Agar (RBCA) plates supplemented with chloramphenicol. Leaves were plated using two methods, washing and direct plating. For washing, leaves were added to 10 mL of saline/Tween buffer in a 7 oz. Whirl-Pak filter bag and processed as detailed previously. Aliquots of 200 μL and 50 μL of these samples were plated on RBCA, which prevents growth of bacteria and slows down the spread of molds. For direct plating, the leaf and leaf litter were weighed and cut into small pieces of about 2cm2. The leaf and leaf litter were washed with 30 ml of sterile distilled water and vortexed for 5 min. Washed materials were placed directly onto RBCA plates. Ballistospore-producing yeasts were isolated from leaves using the ballistospore-fall method. Briefly, aseptically collected segments of leaves were attached to the underside of a Petri dish lid using Vaseline, and the plate was incubated lid-side up. Ballistospores ejected onto the surface of the agar germinated, and yeasts were cultivated. Incubation of the plates
Carboxymethyl Cellulose Hydrolyzing Yeast Isolated From South East Sulawesi
was done for 5 days at room temperature. Strain purification was done at least twice by selecting the different type of yeast colony and plated on potato dextrose agar (PDA, CM0139, OXOID) at room temperature. Yeast DNA template was prepared from freshly-grown cells on the PDA plate and used for colony PCR. Five uL of lysed yeast cell suspension was used for PCR amplification of the partial 26S rDNA sub unit with primers NL1 and NL4 (O’Donnel, 1992), using GoTaq master mix (Promega, M7122). PCR products were visualized on 2% agarose and sequenced with both primers using Big Dye terminator v3.1. Cycle Sequencing Ready Reaction Kit (Applied Biosystems) following the manufacturer’s instructions. The partial 26S sequences determined in this study were compared to those in the EMBL/ GenBank/DDBJ databases using the nucleotide Basic Local Alignment Search Tool (BLASTn). The ITS1/5.8S/ITS2 region of selected strains was also amplified with primers ITS1 and ITS4 Kurztman and Robnett, 1998 when species identifications were ambiguous. Sequences were aligned using CLUSTALX (Altschul et al. 1997). The distance matrix for the aligned sequence was calculated using the two-parameter methods of Kimura, 1980. The neighbor-joining (NJ) method & Nei (1987) was used to construct all phylogenetic trees. Cellulolytic yeast is defined as yeasts grow on CMC as the sole carbon sources, and produce CMC-ase. To obtain the cellulolytic each yeast was grown on CMC agar containing 3 g L-1 yeast extract, 5 g L-1 peptone ,10 g L-1 CMC, K2HPO4 5 g L-1,(NH4)2SO4 0,5 g L1 ,MgSO4.H2O 0,2 L-1, FeCl3.6H2O 0,01 g L-1, MnSO4.H2O 0.001 g L-1. ,20 g L-1 agar (pH 6.2 ± 0.2) and incubated for 5 days at room temperature. Cellulolytic yeast was determined by pouring 2 mL congo red 1 M solution into grown colonies, and kept for 10 minutes. Observing clear zone was done by pouring the congo red solution and replaced with NaCl 0.1N. Cellulolytic yeast was indicated by formation of clear zone surrounding colonies and the ratio between clear zone divided by colony’s size indicating cellulolytic capacity Yeast isolates were preserved by two methods, in 20 % glycerol solution at -80°C,
and by lyophilization. Yeasts were deposited in the Indonesian Culture Collection (InaCC, www.biologi.lipi.go.id) at the Indonesian Institute of Sciences, Research Center for Biology; the Forest Microbes Collection (FORDACC, http://fordamof.org/) at the Forestry Research and Development Agency, Indonesian Ministry of Forestry; and the Phaff Yeast Culture Collection, Department of Food Science and Technology, University of California Davis (UCDFST, phaffcollection.ucdavis.edu). RESULTS Diversity of CMC-ase producing yeast Numerous of yeasts were isolated from two sites (Mekongga and Papalia) in South East Sulawesi. The cellulolytic yeast was defined as yeast grow on CMC used as the sole carbon sources and produce CMC-ase (Endoglucanases EG1, EC. 3.2.1.4) hydrolyze soluble, substituted celluloses such as Carboxymethyl Cellulose (CMC) by attacking the carbohydrate chain (14,β glucosidic bond) internally and randomly. This can be visualized by culturing yeast both on liquid and solid media contain CMC. Formation of clearing zone on CMC-media poured with 1 % congo red and production of CMC-ase were used to screen cellulolytic yeast. We obtained numerous species of yeasts (Figure 1). Of 142 strains tested 43 strains were cellulolytic yeasts consist of 10 genera and 26 species, of whose Candida was the most diverse genus consisting of 15 species. Sporidiobolus runiaes were most prevalent CMC-ase producing yeast from South East Sulawesi represented by 10 isolates (Figure.1). Candida intermedia, Pseudozyma aphidis and Asterotremella humicola were second predominating CMC-ase producing yeast. The most frequently isolated yeast genus from Mekongga and Papalia was Candida. Candida was isolated from both locations and sample types. Candida is a polyphyletic genus, with species placed in 14 families within the class Saccharomycotina. In fact, over 400 of the 1600 known species of yeasts have been placed in the genus Candida (Fell & Boekhout 2011). Due to its taxonomic diversity, it is not surprising that Candida is ecologically diverse also, occupying niches including human infections,
287
Atit Kanti
Figure 1. Diversity of CMC-ase producing yeast from South East Sulawesi
Figure 2. Phylogenetic placement of yeast from South East Sulawesi within Yamadazyma clade based on D1/ D2 region of the nLSU rDNA sequences
288
Carboxymethyl Cellulose Hydrolyzing Yeast Isolated From South East Sulawesi
soil (Amprayna et al. 2012), insect frass, fruit (Wilson & Chalutz 1989), and many other habitats. Some yeasts isolated in this study are ecologically important for sustainable agriculture (Fracchia et al. 2003). Sporiodiobolus pararoseus shows promise for biocontrol of the fungal plant pathogen Botrytis cinerea (Huang et al. 2012). Novel taxa of CMC-ase producing yeast Comparison of the D1/D2 region of LSU rDNA data showed 14 strains belonging to 11 different species had homology values less than 99%, indicating that they may be novel species. A number of potential novel species were collected in this study, as indicated by 1% or higher sequence divergence from previously known species in the D1D2 domain. The results of ITS sequence analysis of these isolates confirmed that they are likely novel species. Our molecular analysis revealed that these yeast isolates are phylogenetically diverse and distributed
within the phyla Ascomycota (Yamadazyma clade and Metschnikowia clade) (Figure 2 and Figure 3). The novel species candidates were mostly residing within genus Candida (13 species: Candida aff. cylindracea PL2W1, Candida aff. insectorum PL3W6, Candida aff. friedrichii MKL7W3, Candida aff. lessepsii PLE3W1, MKL7W4, Candida aff. tenuis PL3DP3, MKL6W4. We isolated 5 strains of cellulolytic yeast close to Yamadazyma mexicana: Yamadazyma aff. mexicana MKL6DP1, MKL6DP2, MKL8W2, MKL6W2, MKL6W1), and our study reveal detection of many undescribed yeast from Indonesia (Figure.4). Further study is needed to describe the novel taxa found in this study. Phylogeography of CMC-ase producing yeast Sample sources were collected from secondary forest in Mekongga and Papalia, South East Sulawesi. Both places harbor numerous species of Cellulolytic yeast (Figure. 4 and 5). There were 15 species including 6 candidates for novel species (3
Figure 3. Phylogenetic placement of yeast from South East Sulawesi within Metschnikowia clade based on D1/ D2 region of the nLSU rDNA sequences
289
Atit Kanti
Figure 4. Candidate for novel species of CMC-ase producing yeast
Figure 5. CMC-ase producing yeast isolated from Mekongga
Figure 6. Cellulolytic yeast isolated from Papalia
strains of Yamadazyma aff. scolyti, Y. aff. mexicana , Candida aff. tenuis, C. aff. lessepsii, C. aff. friedrichii, and C. aff. endomychidarum ) Figure. 4. Eleven species including 3 candidates for novel species of cellulolytic yeast (Candida aff. tenuis, C. aff. insectorium, C. aff. cylindracea) were found in Papalia (Figure. 6). Yamadazyma were only isolated
from Mekongga, and Asterotremella and Sporodiobolus were only cultivated from Papalia.
290
CMC-ase yeast producer on Litter Litter are good sources for cellulolytic yeast, as indicated by numerous cellulolytic yeasts obtained from litter collected both from
Carboxymethyl Cellulose Hydrolyzing Yeast Isolated From South East Sulawesi
Mekongga (Figure. 5) and Papalia ( Figure. 6). This study supports other studies that concluded that leaf litter and plant leaf surfaces are good sources of a diversity of yeast cultures for research (Wang et al. 2004, Nasase et al. 2002, Fungsin et al. 2002). CMC-ase yeast producer on leaf Seven species of cellulolytic yeasts were isolated from Papalia leaf, and none was from Mekongga. Sporodiobolus were common genera, and Sporodiobolus ruineneniae and Candida intermedia were dominant in leaf (Figure. 7 ,8 and 9).
Sporodiobolus ruineneniae seem to be a tropical yeast because only found in tropical region. It was first found in Indonesia by Ruinen in (1963) then it was also isolated in Thailand but not in the temperate zone (Nakase et al. 2006). DISCUSSION Little information was previously available about yeasts on the island of Sulawesi, Indonesia, one of the five largest islands that makes up this richly biodiversity and biogeographically significant region (Sihotang et al. 2012). We found a broad taxonomic diversity of cellulolytic yeast species from this exploratory survey. Because plant surfaces and leaf litter have been sampled extensively (Amprayna et al. 2012; Wilson & Chalutz, 1989), novel taxa were not expected. However, numerous potentially novel species of cellulolytic yeast were obtained. Novel strains of known species were obtained, expanding the known geographic and habitat range of these known species. A variety of ascomycetous and basidiomycetous yeasts were cultivated. Basidiomycetous yeasts are more likely to utilize a broader range of carbon sources than ascomycetes, and have been cultivated more frequently from lownutrient habitats such as leaf surfaces (Joo et al. 2008). The most frequently cellulolytic yeast isolated from South East Sulawesi were Yamadazyma, Pseudozyma and Candida. Candida was isolated from both locations and
Figure 7. Mekongga CMC-ase producing yeast from litter
Figure 8. Papalia CMC-ase producing yeast from litter
Figure 9. CMC-ase producing yeasts obtained from Papalia leaf
291
Atit Kanti
ubiquitous on litter. Candida is a polyphyletic genus, with species placed in 14 families within the class Saccharomycotina. In fact, over 400 of the 1600 known species of yeasts have been placed in the genus Candida (Junyapate et al. 2013). Due to its taxonomic diversity, it is not surprising that Candida is ecologically diverse also, occupying niches including human infections, soil, insect frass, fruit. Important applications using Candida species include agent for bioremediation, Candida catenulata, and biofertilizer, Candida tropicalis HY. Yamadazyma, Pseudozyma and Candida, isolated repeatedly in this study, is a well known species having wide distribution and having high xylose transport capacity. This study supports other studies that concluded leaf litter and plant leaf surfaces are good sources of a diversity of yeast cultures for research (Wang & Bai. 2004; Altschul et al. 1997). We found leaf litters are good sources for cellulolytic yeast. The strains isolated in this study were deposited in Indonesian Culture Collection (InaCC). Isolation of numerous species of yeasts include novel taxa reaffirm that South East Sulawesi are rich in biodiversity of flora, fauna and microorganism, and potential genetic resources for sustainable development. ACKNOWLEDGMENTS Yeasts used in this study were isolated and identified as part of a collaborative project between the University of California Davis and the Government of the Republic of Indonesia, funded by Grant Number U01TW008160 from the US National Institutes of Health Fogarty International Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health, the Office of Dietary Supplements, the National Science Foundation, the Department of Energy, or the Department of Agriculture. The author acknowledge Kyria Boundy-Mills, Ph.D and Dr. Irnayuli Sitepu for manuscritps editing. Yeni Yuliani for laboratory work.
292
REFERENCES Abe, A., N. Sujaya, T. Sano & K. Asano. 2004. Microflora and Selected Metabolites of Potato Pulp Fermented with an Indonesian Starter Ragi Tapé, Food Technol.Biotechno. 42 (3): 169-173. Allend, GR. & MV. Erdmann. 2009. Reef fishes of the Bird’s Head Peninsula, West Papua, Indonesia,” Check List. 5: 587-628. Altschul, SF., TL. Madden, AA. Schäffer, J. Zhang, Z. Zhang, W. Miller & DJ. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 25: 3389 -3402. Amprayna, K., MT. Rosea, M. Kecskésa, L. Pereg, HT. Nguyend, & IR. Kennedya. 2012. Plant growth promoting characteristics of soil yeast Candida tropicalis HY and its effectiveness for promoting rice growth. Applied Soil Ecology. 6: 295-299. Butinar, L., S. Santos, I. Spencer-Martin, A. Oren, & N. Gunde-Cimerman. 2005. Yeast diversity in hypersaline habitats. FEMS Microbiology Letter, vol. 244: 229234. Cannon, C. 2007. Interim Report. The Study on Arterial Road Network Development Plan for Sulawesi Island and Feasibility Study on Priority Arterial Development for South Sulawesi Province. Chang, F.C., Y.R. Liu, S.F. Chen, G.I. Naumov, E.S. Naumova & C.F. Lee. 2012. Five novel species of the anamorphic genus Candida in the Cyberlindnera clade isolated from natural substrates in Taiwan. Antonie van Leeuwenhoek. 102: 9-21. Dewi K. & Hasegawa. H. 2012. Two new species of Syphacia (Nematoda: Oxyuridae) in endemic murid rodents from Sulawesi, Indonesia. J. Helminthology. 2 (12): 1-9. Fahrurrozi, S. Ratnakomala, T. Anindyawati, P. Lisdiyanti, & E. Sukara. 2010. Rapid Assessment of Diverse Trichodermal Isolates of Indonesian Origin for Cellulase Production, Annales Bogorienses. 14 (1): 39-43. Fungsin, B., M. Hamamoto, V. Arunpairojana, V.,Sukhumavasi, J.,Atthasampunna, P., &
Carboxymethyl Cellulose Hydrolyzing Yeast Isolated From South East Sulawesi
Nakase, T. 2002. Kockovaella barringtoniae sp. nov., a new basidiomycetous yeast species isolated from a plant leaf collected in a tropical rain forest in Thailand. International Journal Systematic Evolutionary Microbiology. 52 : 281-4. Goldbeck, R., M.M. Ramos, G.A.G Pereira & F. Maugeri-Filho. 2013. Production of cellulase by a newly isolated strain of Aspergillus sydowii and its optimization under submerged fermentation. International Biodeterioration & Biodegradation. 78: 2433. Goldbeck, R., M.M. Ramos, G.A.G. Pereira, & F. Maugeri-Filho. 2013. Cellulase production from a new strain Acremonium strictum isolated from the Brazilian Biome using different substrates, Bioresource Technology. 128: 797-803. Jiménez, M., A.E Gonzáles, M.J. Martínez, A.T. Martínez & B.E. Dale. 2002. Screening of yeasts isolated from decayed wood for lignocellulose-degrading enzyme activities, Mycol. Res. 9 (11): 1299-1302. Joo, H.S., P.M. Ndegwa, M. Shoda, & C.G. Phae. 2008. Bioremediation of oil-contaminated soil using Candida catenulate and food waste. Environmental Pollution. 156: 891- 896. Junyapate, K., S. Jindamorakot, & S Limtong S. 2013. Yamadazyma ubonensis f.a., sp. nov., a novel xylitol-producing yeast species isolated in Thailand. Antonie Van Leeuwenhoek. DOI 10.1007/s10482-013-0098-8. Kang, S. W., Y. S. Park, J.S. Lee, S.I. Hong and S.W. Kim. 2004. Production of Cellulases and Hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresource Technology. 91: 153-156. Kanti, A. , E. Sukara, K. Latifah, N. Sukarno, & K. Boundy-Mills. 2013. Indonesian oleaginous yeasts isolated from Piper betle and P. nigrum. Mycosphere. 4 (5): 1015-1026. Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal Mollecular and Evolution. 16: 111-120. Koch, A. 2011. The Amphibians and Reptiles of Sulawesi : Underestimated Diversity in
a Dynamic Environment. Biodiversity Hotspots 5: 383-404. Kuriyama, H., D. Sastraatmadja, Y. Igosaki, K. Watabane, A. Kanti, and T. Fukatsu. 1997. Identification and Characterization of Yeast isolated from Indonesian fermented food. Mycoscience 38: 441-445. Kurztman, C.P. & C.J. Robnett. 1998. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Leeuwenhoek. 73: 331-71. Lo, Y-C., G.D. Saratale, W-M. Chen, M-D. Bai & J -S Changa. 2009. Isolation of cellulosehydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production, Enzyme and Microbial Technology. 44: 417-419. Nakase, T., S. Jindamorakot, S. Am-In, S., W. Potachararoen, & M. Tanticharoen. 2006. Yeast biodiversity in tropical forest of Asia, In Biodiversity and ecophysiology of Yeast, Rosa, C., Peter., G,. Berlin Heidelberg, Ed. Springer-Verlag: 441-60. Nakase, T., S. Suzuki & M. Takashima. 2002. Bullera taiwanensis sp nov and Bullera formosensis sp nov, two new ballisto conidium-forming yeast species isolated from plant leaves in Taiwan. Journal Genetic Applied. Microbiology. 48: 345-355. Nakase, T., S. Suzuki, M. Takashima, M. 2002. Bullera taiwanensis sp nov and Bullera formosensis sp nov, two new ballistoconidium-forming yeast species isolated from plant leaves in Taiwan. J. Gen. Appl. Microbiol., 48: 345-355. O’Donnel, K. 1992. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris). Current. Genetic. 22: 213-220. Rifai, MA. 1995. The biodiversity of Indonesian microorganisms. in Proc. of UNESCO Regional Workshop on Culture Collection of Microorganisms in Southeast Asia, Yogyakarta. Sihotang, VBL., EA. Widjaja & D. Potter. 2012. Medicinal plant knowledge of Tolaki and Toraja inTinukari Village and its surrounding in : Proc. Strategies and
293
Atit Kanti
Challenges on Bamboo and Potential Non Timber Forest Products (NTFPs) Management and Utilization, Bogor. Sjamsuridzal, W., A. Oetari, A. Kanti, R. Saraswati, C. Nakashima, Y. Widyastuti & A. Katsuhiko. 2010. Ecological and Taxonomical Perspective of Yeast in Indonesia. Micro biology Indonesia. 4: 49-57. Sudiana, IM. & M. Rahmansyah. 2002. Species and functional diversity of soil microflora at Gunung Halimun National Park. Jakarta : BCP-JICA. LIPI Press. Takashima, M., T. Sugita, BH. Van, M. Nakamura, M. Endoh & M. Ohkuma. 2012. Taxonomic richness of yeast in Japan within subtropical and cool temperate areas. Plos ONE [Online]. 7(11). Available: e50784, 2012. doi: 10.1371/ journal.pone.0050784. Wang, QM. & FY. Bai. 2004. Four new
294
species of the genus Sporobolomyces from plant leaves. FEMS Yeast Research. 4: 579-586. Wilson, CL., & E. Chalutz. 1989. Postharvest biological control of Penicillium rots of citrus with antagonistic yeasts and bacteria. Sc. Hortic. 40: 105-112. Zainudin, MHM., MA. Hassan, M. Tokura & Y. Shirai. 2013. Indigenous cellulolytic and hemicellulolytic bacteria enhanced rapid cocomposting of lignocellulose oil palm empty fruit bunch with palm oil mill effluent anaerobic sludge. Bioresource Technology. 147: 632-635. Zhanga, Q., M. Tian, L. Tang, H. Li, W. Li , J. Zhang, H. Zhang & Z. Mao. 2013. Exploration of the key microbes involved in the cellulolytic activity of a microbial consortium by serial dilution. Bioresource Technology. 132: 395-400.
PANDUAN PENULIS Naskah dapat ditulis dalam bahasa Indonesia atau bahasa Inggris. Naskah disusun dengan urutan: JUDUL (bahasa Indonesia dan Inggris), NAMA PENULIS (yang disertai dengan alamat Lembaga/ Instansi), ABSTRAK (bahasa Inggris, dan Indonesia maksimal 250 kata), KATA KUNCI (maksimal 6 kata), PENDAHULUAN, BAHAN DAN CARA KERJA, HASIL, PEMBAHASAN, UCAPAN TERIMA KASIH (jika diperlukan) dan DAFTAR PUSTAKA. Penulisan Tabel dan Gambar ditulis di lembar terpisah dari teks. Naskah diketik dengan spasi ganda pada kertas HVS A4 maksimum 15 halaman termasuk gambar, foto, dan tabel disertai CD. Batas dari tepi kiri 3 cm, kanan, atas, dan bawah masing-masing 2,5 cm dengan program pengolah kata Microsoft Word dan tipe huruf Times New Roman berukuran 12 point. Setiap halaman diberi nomor halaman secara berurutan. Gambar dalam bentuk grafik/diagram harus asli (bukan fotokopi) dan foto (dicetak di kertas licin atau di scan). Gambar dan Tabel di tulis dan ditempatkan di halaman terpisah di akhir naskah. Penulisan simbol a, b, c, dan lain-lain dimasukkan melalui fasilitas insert, tanpa mengubah jenis huruf. Kata dalam bahasa asing dicetak miring. Naskah dikirimkan ke alamat Redaksi sebanyak 3 eksemplar (2 eksemplar tanpa nama dan lembaga penulis). Penggunaan nama suatu tumbuhan atau hewan dalam bahasa Indonesia/Daerah harus diikuti nama ilmiahnya (cetak miring) beserta Authornya pada pengungkapan pertama kali. Pustaka didalam teks ditulis secara abjad. Contoh penulisan Daftar Pustaka sebagai berikut : Jurnal : Achmadi, AS., JA. Esselstyn, KC. Rowe, I. Maryanto & MT. Abdullah. 2013. Phylogeny, divesity , and biogeography of Southeast Asian Spiny rats (Maxomys). Journal of mammalogy 94 (6):1412-123. Buku : Chaplin, MF. & C. Bucke. 1990. Enzyme Technology. Cambridge University Press. Cambridge. Bab dalam Buku : Gerhart, P. & SW. Drew. 1994. Liquid culture. Dalam : Gerhart, P., R.G.E. Murray, W.A. Wood, & N.R. Krieg (eds.). Methods for General and Molecular Bacteriology. ASM., Washington. 248-277. Abstrak : Suryajaya, D. 1982. Perkembangan tanaman polong-polongan utama di Indonesia. Abstrak Pertemuan Ilmiah Mikrobiologi. Jakarta . 15 –18 Oktober 1982. 42. Prosiding : Mubarik, NR., A. Suwanto, & MT. Suhartono. 2000. Isolasi dan karakterisasi protease ekstrasellular dari bakteri isolat termofilik ekstrim. Prosiding Seminar nasional Industri Enzim dan Bioteknologi II. Jakarta, 15-16 Februari 2000. 151-158. Skripsi, Tesis, Disertasi : Kemala, S. 1987. Pola Pertanian, Industri Perdagangan Kelapa dan Kelapa Sawit di Indonesia.[Disertasi]. Bogor : Institut Pertanian Bogor. Informasi dari Internet : Schulze, H. 1999. Detection and Identification of Lories and Pottos in The Wild; Information for surveys/ Estimated of population density. http//www.species.net/primates/loris/lorCp.1.html.