EISSN: 1998-1066 (Online) ISSN: 1995-0756 (Print)

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Ad va nces in Environmental Biology

Country: Jordan

Subject Area: Environmental Science I Agricultural and Biological Sciences

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Category

Agricultural and

Biological Sciences

(miscellaneous)

Environmental Science

[miscellaneou~)

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

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'Jal~ Publisher: American·E~ias1an Network for Scientific Information. Publication type: Journals. ISSN: 19981066, 19950756

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48 129 788 1.211

0,08 0, 39 0,23 0,21

0,08 0,39 0,23 0,20

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45 95 657 1.037

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Search terms: ENVIRONMENTAL Total journals found: 177

t. ADVANCES IN ENVIRONMENTAL BIOLOGY Monthly ISSN: 1995-0756

AM-EURASIAN NETWORK SCI INFORMATION-AENSI, AL HUSSEIN BIN TALAL UNIV, BIOL DEPT, C/O DR ABDEL RAHMAN MOHAMMAD SAID AL-TA W AHA, PO BOX 20, MA AN, JORDAN, 00000

1. Zoological Record

2. AMERICAN-EURASIAN JOURNAL OF AGRICULTURAL & ENVIRONMENTAL SCIENCES Monthly ISSN: 1818-6769

INT DIGITAL ORGANIZATION SCIENTIFIC INFORMATION-IDOSI, PO BOX 40468, DEIRA, U ARAB EMIRATES, DUBAI, 00000

1. Zoological Record

3. ANNALS OF AGRICULTURAL AND ENVIRONMENTAL MEDICINE Semiannual ISSN: 1232-1966

INST AGRICULTURAL MEDICINE, JACZEWSKIEGO 2, PO BOX 185, LUBLIN, POLAND, 20-950

1. Science Citation lndex Expanded 2. Current Contents - Agricu1ture. Biology & Environmental Sciences 3. Z9ologiool Record

4. APPLIED AND ENVIRONMENT AL MICROBIOLOGY Semimonthly ISSN: 0099-2240

AMER SOC MICROBIOLOGY, J 752 N ST NW, WASHINGTON, USA, DC, 20036-2904

I . ·Science Citation Index 2. Science ,Citation Index Expanded 3. Current Contents - Agriculture. Biology & Environmental Sciences 4. Current Contents - Life Sciences 5. Zoological Record 6. BIOSIS Previews

5. APPLIED AND ENVIRONMENTAL SOIL SCIENCE Irregular ISSN: 1687-7667

HINDA WI PUBLISHING CORPORATION, 410 PARK.AVENUE, 15THFLOOR, #287 PMB, NEW YORK, USA, NY, I 0022

1. Zoological Record

6. APPLIED CATALYSIS B-ENVIRONMENTAL Semimonthly ISSN: 0926-3373

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AEB Volume 8, Number 14: Special7, 2014 -~-------------

Preliminary Study of Laotian Black Crested Gibbon Activity Budget in Ban Toup, Nam Kan National Protected Area, Lao PDR

SingphoneLuangleuxay, Kham Youanechuexian and Pongthep Suwanwaree

1-6

The Status of Laotian Black Crested Gibbon Nomascus concolor lu in Nam Kan National Protected Area, Lao POR

Kham Youanechuexian, Phaivanh Phiapalath and Pongthep Suwanwaree

7-13

Relation of Ganoderma Ergosterol Content to Basal Stem Rot Disease Severity Index

Chong, K.P .• Eldaa, P.A. and Jedol Dayou

14-19

Increasing the Energy Output from Living-plants Fuel Celts with Natural Photosynthesis.

Choo Ying Ying and Jedol Dayou

20-23

Chemical Composition of Bio-Oil Obtained from Biomass via Thermal Controlled inside The Continuous Pyrolysis Reactor

Kittiphop Promdee, Omrumpha Soubsawwong, Chintana Sanvong, Tharapong Vitidsant

24-29

-----------Some Interpretations on FTIR Results for the Detection of Ganoderma Boninense in Oil Palm Tissue

Amnyitte Alexander, Jedol Dayou, Coswald Stephen Sipaut, Chong Khim Phfn and Lee Ping Chin

30-32

Fed-batch Production of Valuable Biosurfactant, Rhamnolipid, from Waste Cooking Oil by Indigenously Isolate Pseudomonas aeruginosa USM-AR2

Zainatul ·Asyiqin Samsu, Zulail<ha Yusof, Mohd Syafiq Awang, Nurasshifa Md Noh, Ahmad Ramli Mohd Yahya

33-38

Effects of Varied Amount of Carbon Dioxide and Pressure on Asphaltene Stability

Josefina Barnachea Janier, Mohamad Afzal B. Jalil, Radzuan B. Razali, Afza Bt. Shafie, Samsul Ariffin B. Abdul Karim

39-43

Room Temperature Synthesis of Water-Soluble Starch-Stabilized CdSe Quantum Dots for Latent Fingerprints Detection

Tanutkuh Palakawong Na Ayudhaya, Panida Viwattana, Kheamrutai Thamaphat, Khemika Lomthaisong

44-49

Community Succession of Methanotrophic Bacteria Based on PMOA GENE In Rice Fields

Hendri Sutanto, 'Iman Rusmana, Nisa Rachmania Mubarik

50-56

Blomolecular React ion and Heat Controlled In the Reactor for Synthesis of Charcoal and Blo-011 Derived from Mixed Grass

Kittiphop Promdee, Chintana Sanvong, Somruedee Satitkune, Tharapong Vitidsant

57-62

Diversity of Nitrogen Fixing Bacteria Based on nilH Gene in Rice Fields

Randi Hadiar.ta, Iman Rusmana, Nisa Rachmania Mubarik

,

I

63-69

--------------------------·-----------------·----------------------------Improving Power Output Prediction from Ocean Salinity and Temperature Energy Converter using Viscosity Model

Fuei Pien Chee, Shu Kim Lee, Jedol Dayou, Ejria Saleh and H.L.H. Chong

70-77

_ ....... ------------------------------·-----------------------------

Initial Investigation on Using Copperas By-Product to Remove Colour from Domestic Wastewater by Coagulation and Flocculation

Hamidi Abdul Aziz and Wan lzatul Saadiah Wan Kamar

78-82

----------------------·------------------------------------·------·---Removal of COO and Colour from Landfill Leachate using Ferric Chloride by Coagulation.flocculation Treatment

Hamidi Abd Aziz and Sifi Fatihah Ramli

83-90

Selected Heavy Metals and Polycyclic Aromatic Hydrocarbon in Commercial Fishes Caught From UMT Enclosed Lagoon, Terengganu, Malaysia

Ong M.C., Yong J.C. , Khoo X. Y., Tan Y.F. and Joseph B

91-98

Effect of Rainfall on the Transmission Model of Conjunctivitis

Jantraporn Suksawat and Surapol Naowarat

99-104

. -------·-------·--------------------------·-----·------------------·-----------·------

Microbiological Risk Assessment of Fresh Water Aquaculture Fish: From Farm to Table

Ibrahim, A.B., Mohd Khan, A., Norrakiah, A.S.

105-1 11

School's Indoor Air Quality and Respiratory Health Implications among Children

Chua Poh Choo, Juliana Jalaludin, Zailina Hashim

112-121

The Impact from Free Flow of 11 Logistics Services Provider to Performance

Efficiency in Beverage Manufacturing Firm in Thailand

Jarun Bootdachi and Sirirak Khaochaimaha

122-130

In-vivo Blood Glucose Control by Phenolic Compound from Vernonia amygdalina, Andrographls panlculata, and Plthecellob/um jlrlnga in Streptozotoxin-lnduced Diabetic Rats

Asna Mohd Zain and Amirul Abu

131-134

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, Prof. Dr. Majid Monanemi Prof. of Physical Chemistry, Science & Research Campus, Islamic Azad University, Tehran P.O. Box:14155/775 Iran.

Prof. Dr. Wenju Liang Professor, Institute of Applied Ecology, Chinese Academy of Sciences, P.O.Box 417, Shenyang 110016 hina.

• Prof. Or. Abd Al-kareem Al-Sallal Professor of Applied Microbiology, Biotechnology and Genetic Eng., Jordan University of science and Technology, Jordan. ·

• Dr. Abeer Abbass EL-Saharty Associate Professor, Marine Chemistry Lab., Marine Environment Division, National Institute of Oceanography & Fisheries (NIOF), Kayat Bay, El-Anfoushy, Alex., Egypt

• Dr. lgnacy Kitowski Department of Nature Conservation, Institute of Biology, University of Maria-Curie Sklodowska, Akademicka 19, PL-20-033 Lublin, Poland.

• Dr. Andrzej Komosa Department of Radiochemistry and Colloid Chemistry, Maria Curie Sklodowska University, Lublin, Poland.

• Benott SCHOEFS Professeur de Biologie et Physiologie Vegetales Directeur du champs disciplinaire •physiologie" Directeur du M1 "Sciences du vegetal" Directeur du M2P "Plantes Productions Biotechnologies UMR CNRS (5184)/INRA (1088)/Universite de Bourgogne - Plante- Microbe-Environnement Bat CMSE 17, Rue Sully, BP 86510. 21065 DIJON CEDEX,France

• Dr. Robin DUPONNOIS Directeur de Recherche a l'IRD, Laboratoire Commun de Microbiologie, IRD/ISRNUCAD, Centre de

Recherche de Bel-Air, BP 1386 CP 18524 Dakar-Senegal

, or. Hidetaka HORI Ph.D. Laboratories of Applied Bioscience, Graduate School of Science and Technology. Niigata University, Niigata 950-2181, Japan.

, or. Panos S. ECONOMIOIS Professor Emeritus at the Aristotle University, Karakasi str. 79. GR-544 53 Thessaloniki, Greece

l . Or. Christopher (Kitt} E. Bagwell, Ph.D. Savannah River National Laboratory, SRNL Environmental Sciences & Biotechnology

, or. Yuexia Wang College of Bioscience and Biotechnology, Yangzhou University, NO. 12 East Wenhui Road, Yangzhou City, Jiangsu Province, China

, or. Rashed Al-Sa'ed (Dr. Eng.) Associate Professor in Environmental Sciences & Engineering, Institute of Environmental and water Studies (IEWS), Birzeit University , P.O. Box 14 , Birzeit, West Bank.Palestine

, Or. Rais Ahmad Department of Applied Chemistry, F/O Engg. & Technology, AMU Aligarh, India

, Or. Marius Ciprian Branzila Technical University of lasi, Faculty of Electrical Engineering, Department of Electrical Measurements and Materials, Bd. Dimitrie Mangeron 53 lasi, 700050 Romania

Associate Editors::

, Dr. Mohammad Wedyan Biological Department, Al Hussein Bin Talal University, Ma'an, P.O. Box 20, Jordan .

• Or. Cai zhiquan Xishuangbanna Tropical Botanical Garden, The Chinese Academy of Sciences, Menglun, Yunnan 666303, P.R. China .

• Dr. S.S. Dudeja Department of Microbiology, CCS Haryana agricultural University, Hisar 125 004, India .

• Dr. 8.K. Tyagi Officer in-Charge, Centre for Research in MedicaJ Entomology (Indian Council of Medical Research), 4-Sarojini Street,Chinna Chokkikulam,Madurai - 625 002,lndia .

• Dr. Fa Yuan Wang Department of Resources and Environmental Science, Agricultural College, Henan University of Science and Technology, 70 Tianjin Road, Luoyang, Henan Province 471003, P.R. China.

• Dr. Nishi Mathur Head of Department, Department Of Biotechnology, Mahila P.G. Mahavidyalaya, Jodhpur-342001, Rajasthan, India.

• Dr. F.M. Aminuzzaman Department of Plant Pathology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh.

• DR. DHRUVA KUMAR JHA Gauhati University, Campus Guwahati-781 014, Assam.India.

• Dr SSS Sarma Professor & National Researcher.National Autonomous University of Mexico, Campus lztacala AP 314, CP 54090 Los Reyes, lztacala, Tlalnepantla Edo. de Mexico Mexico.

• Prof Dr. SVS Rana Head Dept. of Zoology, Cooordinator Dept. of Env. Science CCS University, Meerut.

l• Dr. Murat Dem1r Istanbul University, Faculty of Forestry, Department of Forest Construction and Transportation, 34473 Bahcekoy I Sariyer / Istanbul, Turkey .

. . • Dr. Zafer OLMEZ Artvin Coruh University, Faculty of Forestry, 08000 Artvin, Turkey.

1• Dr. YASIR HASAN SIDDIQUE FIBR Human Genetics and Toxicology Lab, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh - 202002 (UP) India.

• Dr. P.R. Salve Scientist, Environmental Impact and Risk Assessment Division. National Environmental Engineering Research Institute (NEERI). Nehru Marg, Nagpur-440 020(M.S.), India

• Dr. S. KARTHIKEYAN Lecturer in Physics, St. Joseph's Collge of Engineering, Chennai-600 119 Tamil Nadu, India.

• Dr. Nuray MISIR Karadeniz Technical University, Faculty of Forestry, 61080, Trabzon,Turkey.

• Dr. Bragadeeswaran CAS in Marine Bilogy, Annamalai University, Parangipettai 608 502 Cuddalore, Tamil Nadu, India

• Dr. Sevil TOROGLU Biology Department, Faculty of Arts and Sciences, University of KSU, 46100 Avsar Campus, Kahramanmaras, Turkey.

• Dr. Taiga Akpovughaye Department Of Biological Sciences, K.S.U. , P.M.B. 1008, Anyigba, Kogi State.

• Dr. Jitendra Panwar Biological Sciences Group, Birla Institute of Technology & Science (BITS), Pilani-333 031 (Rajasthan) INDIA.

• Or. Ezekiel Olatunji Department of Fisheries, Cross River University ofTechnology, Calabar, P.M.B. 102, Obubra, Nigeria .

• or. Ali Gazanchian Department of Genetic and Physiology, gricultural and Natural Resources Research Center of Khorassan, Addresses: Mashhad, Razavi Khorassan Province, Iran Box P.O.: 91735-1148, Mashhad, Iran .

• Dr. Shahid A. Soomro Paul-Hindemith-Allee4/Apt. 312, 0-80939 Munchen, Germany.

• Dr. YOUGASPHREE NAIDOO Senior Lecturer, School of Biological and Conservation Sciences, UKZN -Westville campus .

• Or. Ranya Aly Helmy Amer Department of Environmental Biotechnology, Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research and Technology Application, New Burg 8-Arab City, Universities and Research Institutes Zone, 21934 Alexandria.Egypt.

• Dr. A. Karthikeyan Division of Forest Protection, Institute of Forest Genetics and Tree Breeding, P.O. Box: 1061; R.S. Puram, Coimbatore- 641 002, India .

• Assist. Prof. Dr. NOket SIVRI Istanbul University, Faculty of Engineering, Dept. of Environmental Engineering, Avcilar Kampus 34320. Istanbul TURKIYE .

• Or. Kamelia Mahmoud Osman Ahmed Department Microbiology, Faculty Veterinary Medicine, University Cairo, Egypt.

• Dr. Slavomlr Ceri'lansk Comenius University in Bratislava, Faculty of Natural Sciences, Department of Ecosozology and Physiotactics, Mlynska dolina 1, 842 15 Bratislava, Slovakia.

, Prof. Dr. Renato G. Reyes College of Arts and Sciences, Central Luzon State University, Science City of Muf\oz, Nueva Ecija.

• Dr. A.O.Togun Deptartment of Crop Protection & Environmental Biology, Faculty of Agriculture & Forestry, University of Ibadan, Ibadan, Nigeria.

• Dr. Abdelwahid Saeed Ali Department of Veterinary Preventive Medicine and Public Health, Faculty of Veterinary Medicine, University of Khartoum, Khartoum North, Postal code: 11115, P.O. Box: 321, SUDAN

• Dr. Ama~esh Chandra Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi - 284003 India.

• Or. KALIMUTHU KANOASAMY Department of Botany, Government Arts College. Coimbatore - 641 018. India.

• Or. Abdul Latief A. Al--Ghzawi Department of Biology and Biotechnology, Faculty of Science, The Hashemite University, Zarqa­Jordan.

• Prof. Magdy Tawfik Khalil Zoology Dept, Fae. Science, Ain Shams Univ., Cairo, Egypt. Aquatic Ecology, Biodiversity, Management & Conservation

• Dr. Halil Erhan EROGLU Department of Biology, 66200 YOZGAT, Bozok University, Faculty of Science and Arts, Turkey.

• Prof. Shikui DONG chool ofE11vironmental Sciences, eijing Normal University, No. 19, Xingjiekou Waidajie, aidian District, Beijing, 100875, .R. China

J, Dr. Naveed Ahmed Khan Senior Lecturer in Microbiology, School of Biological & Chemical Sciences, Birkbeck College, University of London, Malet Street, London WC1 E 7HX, England, U.K.

• DR. DHRUVA KUMAR JHA Department of Botany, Gauhati University, Guwahati-781 014, Assam, India.

• Dr. lgnacy Kitowski Department of Nature Conservation, Institute of Biology, Maria Curie-Sklodowska University, Akademicka 19, PL-20-033 Lublin, Poland

• Dr. Nida Mohammad Ismail Salem Industrial Chemistry Center, Royal Scientific Society P.O. Box: 1438 Aljubaiha, Amman 11941 , Jordan.

• Dr. MUHAMMAD MSIM Department of Field Crops, Faculty of Agriculture, University of Ankara, Ankara , Turkey.

• Dr. Song he Zhang College of Environment, Hohai University, Xi Kang road N0.1. Gulou district, Nanjing 210098, Jlangsu

Regional Editors::

• Dr. Oscar Martinez AlvarezStation de Biologie Marine du Museum National d'Histoire, Naturelle et du College de France. BP 225, 29182 Concarneau Cedex, France.

• Dr. Piotr Tryjanowski Department of Behavioural Ecology, Adam Mickiewicz University, Umultowska 89, PL 61-614 Poznan, Poland.

• Dr. Rafael Caballero Garcia de Arevalo Centro de Ciencias Medioambientales, C/ Serrano 115 bis, Madrid 28006, Spain.

, Dr. OmOr BAYSAL Turkish Ministry of Agriculture and Rural Affairs, West Meditereanean Agricultural Research Institute (BATEM), Plant Pathology Department, P.B. 35, 07100 Anlalya/Turkey.

, Or. Aamir Nazir, Ph.D. Medical College of Georgia, USA .

• Or. Li, Feng-Rui Department of Ecology and Agriculture, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences.China .

• Md. Amin Uddin Mridha Department of Botany, University of Chittagong, Bangladesh.

, Dr. D.J. BAGYARAJ INSA Senior Scientist & Chairman, NBRCD # 41 RBI Colony, Anand Nagar, Bangalore, India.

, Dr. Shyam Singh Yadav PhD, FISGPB,FISPRO,FISPPB, MSIGMA Xi (U.S.A.), Technical Expert, United Nations Development Programme, Yemen .

• Or. Gamal M. Fahmy Professor of Plant Ecology, Department of Botany, Faculty of Science, University of Cairo, Giza 12613, Egypt.

• Dr. Sarwoko Mangkoedihardjo Department of Environmental Engineering, Sepuluh Nopember lnsttute of Technology (ITS), Campus ITS Sukolilo Surabaya 60111, Indonesia .

• Or. Bob Redden Curator, Australian Temperate Field Crops Collection, DPl-Vlc, Private mail bag 260, Horsham Vic 3401 , Australia

Editors::

• Dr. Ahmad K. Hegazy Head of Ecology Division, Prof. of Conservation & Applied Ecology, Botany Department, Faculty of Science, Cairo University, Giza 12613, Egypt

• Prof. Or. Marcelo Enrique Conti SPES - Development Studies Research Centre, Universit~ di Roma 'La Sapienza" Via Del Castro Laurenziano 9, 00161 Rome, Italy

• Prof. Cesar G. Demayo, Professor of Genetics and Entomology, Department of Biological Sciences MSU-lligan Institute of Technology ,9200 lligan City, Philippines

Contact Us

• J. Editor in Cheif - Dr. Abdel Rahman Tawaha • ~Address: Amman-Jordan

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Advances In Enllironmental Biology (AEB) Instruction for Aulhors

The instructions for authors include informabon about preparing a manuset1pt for submlSSion to the Journal of interest. er 1ter1a for pubticabon and the onbne submission process Ori\Jinal research pap8'S, review articles, technical reports and short communications in all aspects of Agricuhure. Biological. lnformat10n. Health & L~e Sciences, Zoology, Humanity, Social and Applied Sciences etc .. can be submitted on the understanding that the 'Mll'k is not prev10usly published or undef C011S1deration f()( pubficatvn elsewhere.

SUBMISSION PROCESS

Microsoft Word formats of Manuscript (MS) should be submitted 1n electronic f()(m (e-ma~ attachment) to the Editorial Office of Advances in Enllironmental Biology {AEB) 1ournal.

Note For Mieles with muttple authors, we would expect a 'corresponding auth()(' lo be responsible for the arttele during the refereemg process aftei stA:ilnission

COVER LETTER

The covei letter should be a separate 'MXd file alongside the manuscript sent as an ernad message and should contain,

• Full names of aulhor(s) and affdialions AffiHation must be included Name of Univl!fSity, Name ol Deparlmen~ Name of Facutty, Box 3030 City. Counry

• A short biography of author (s)

• Corresponding Author: Clearly indicate who Will handle correspondence at all stages of refereeing and publication process. Ensure that Name of Univ«sily, Name of Department. Name of Faculty, Box.3030. City. Country. Phone numbers (with country and area code) are provided in addition to the e-mail address and the complete postal address.

• The name of the corresponding author should be marked With an aslerisk (') for 1denhficahon

TYPES OF PAPERS

Standard full·lengtll pape• These should desa1be new and carefully confirmed findings including experimental procedures A paper of this nature should conta111 the Abstract. Key v.ords. Ablllevtahons. Introduction, Materials and Melhods, Results, Discussion (or Resuhs af\d Discussion). Acknowtedgemenls, References. Tables, and L~ends to figures A typical slandard paper contains 10-15 manuscnpl pages {W!lh figures)

Short Communica1ion: It must not exceed 6-12 manuscnpt pages (wlh figures) and must conlam lhe Abstracl, Key words. Abbrev1alions. the core of the pap8' Acknowledgements. References. Tables. and Legends lo figures II should report a C<JOl)leled 'Mll'k but not preliminaty findings.

Review article: This should give an overview Of a lopcal held of interest for a Wide spectrum of readers af\d should conlain lhe absb'acl. topical sectoos and subsecbans. and references Review article should be concise ancf no longer lttan Hl-15 manuscnpt pages

FORMAT DETAILS

Manuscript Style

An manuscript should be clearly >M~ten in a concise grammatical corr eel English manner. The aulh()( must full the papei 1n template iournal form and Re-w-de lhe abslract like the formal f()( AJBAS Journal

Manuscrl!)ls \hal do nol conform to lhese requirements and manuscnpl formal may be returned 10 lhe aulhor fOI cOl'recbon

SubmiS$ion of Manuscript Send your manuscr rpts IMlh allachment lo

abs:!el al-lawaha@mail 111cg11 J

abdellawaha@yaho9 Cl.Jill

along v.ith covering lettei

Prep~ring Manuscripts

Tex1 Font Trnesnewroman F onl Size. 10 No Columns

Miiin Headings rolloW1119 mall headings should be provided 11 the manusaipt 'Miiie prepartng Main headings should not be nurrbered 111 the manusaipt

Introduction Materials and Methods Results OISCUSSlOll Conclusion Ack~ement (opbonal) Reeences

Sequence ol Preparation Arlx:le Ttle Aulhors and thet' fut adcresses Abstracl (300 lo 500 v.«ds) Key 'Mll'ds lntroducbon Materials and Methods Results Discussion ConclUSIOn Acknov.tedgement References

Abs1rKt .ebstrad of ~ w:irds should be provided summenmg tr iel lntroduc:bon, methods llSed. results and concluslon of the stud/. No Sib headngs should be given in tins secbon.

Figures Figures should be of good quality and cleasly readable Graphs and same like figures should be drawn m corelciawor M1crosoh Excel Scienbfic Pictures should have at least a resolution or 300 DPI (Dots Per Inch) for goodpnntmg quality.

Referenc:es Rererences m the text should be Ill ful if they have one ex two authols {e g AJ-Tawaha 2004 AJ-Tawaha and Segum, 2006} m the case of mult"4e authcxs they should be c~ed as Turk et al. 2004 Full References should be provided" the REFERENCES soct10!'

References:

A Examp!esc'crtahon :i teYI The use of an'l!utha's name (wllhout 1mtials} followed by a dale ex year of pubhcallon is used lor references I01Jnd 111 !ext

Peiry (2003} early proved thal This is 111 agreement ~lh the resulls obtained by severnl authors {Brown 1999, Kramer, 2004, Smllh. 2008)

Zhang and C!teng (2001) repcxted that This was later round to be incor•cct (KurtlJf and Ahmed. 2000)

When there •e more than two authors. only the irsl author's name should be menboned loUo"'9d by et :.r as seeo below

Prince et al (1990} staled that S1rnlar results 'M!re reported recenriy (Smilh et al. 2003)

In the event that an author crted has had two or mcxe W01ks published during the same yeai !he relerence. bolh 1n the '.ext and in lhe reference hst. should be ldenhhed by a tower case lel!er hke 'a and 'b after the date lo disllngu1sh the wOiks Fortnstance(Slephen, 2001a,b}

Journal Artrcies Ouyang, D J Bartholic and J Selegeao. 2005 Assessing Sedtmenl Loading from Agricultural O'oplands 1n !he Great Lakes Basin J<unal ol American Science. 1(2) 14-21

A Book. Durbin, R . S R Edcty A Krogh and G Mltchison. 1999 81ologicaJ Sequence Analysis Probabi1sbc Models ol Prolerns and Nuc:lelc Acids Canillqje lJfwersdy Press

A Chapter in a Book Leach. J . 1993 lrqiacts of lhe zetwa mussel (OlelSSena polymcrpha} on waler quatrty and hsh spawning reefs of Western Lake Erie In Zetra mussels btology, mpacts and conrot Eds . Nalepa T and D Schloesser Ann Albor Ml Lewis Publisheis. pp 381-397

A Report MakarelMcz, J.C., T. Lev.is and P. Be11ram, 1995 Epttimnetic phy1oplanklon and zooplanktonbiomass and species composition in Lake Michigan. 1983-1992 U.S. EPA Great Lakes National Program, Chicago, IL. EPA 90&-R.es-009

Conference Proceedngs: Slock, A, 2004. S19nal Transduction in Bacteria. In the Proceedings of the 2004 Maik.ey Scholars Conference. pp 80-89,

A Thesis· Strunk, J.L .. 1991. The ex1raction of mercury iom sediment and the geochemical partilioning of mercu.ry in secimenls from Lake Superia, M. S thesis, Michigan Slate Univ., East Lansing, Ml

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Advances ia Eaviroamcntal BioloJO-, ~(14) Special 2014, Pages: 63-69

AE'NSJ Journal$ .

Advances in Environmental Bioiogy

Diversity of Nitrogen Fixing Bacteria Based on nijH Gene in Rice Fields

l Randi Hadianta, 21man Rusmana, 2 Nisa Rachmania Mubarik

1Gruciua/i: Schwl. Bugur .,grimlt11ra/ Uniw:rsily. Darmugu Cumpuv, Bugur, West .!<Nu, lnJunesia 166/JIJ 1D 1purtrm:nl of Bi<>lugy. F<1t11lty u{Mu1ltt!l11u1icy um! Nuturul Scirm r, Bugw Ag11t-ulturul UmvcrsllV. Dunnusu Cumpu>, Bugor, We.11 Juvu, Indonesia 166.'SU

ARTICLE INFO Ardd' history: Reuned 15 Jwtt 1014 Recm-ed m rensl!ii form 8 Julv W/4 Aatpted 14 Stptember 2014 A.vail<1bl~ on/int 27 September :llu

Key,.VJrtls: B1vfe11i/izcr. DGGJ:;. mfH gen.•, ni1~gcn,tlxi11g hat1eri11

ABSTRACT Bjological nnrogen fixation is ao important proc~s in changing Nl gas in the lllmQlphe-c into ammcn1um "llilich pcrfonncd by nitrogen fixing baaeriL The molecular analysis of nifil g.:ne is coonnooJy u~ aod pro'·'d more a.ccurate for ddccung die dm:n1ty of niirogcn fixing bacteria coowncd in !he nee ficld soil. The di,·mi1y of n11rogen-fixing bacteria based on n11ll !!..:nc can be determined by molo:.."1lbr approM;hcs, LC. DGGE (Dcnallll'alll OnM.hcnt Gel El..'CU'Ophorcsis). This r~h were cooducted to study the d1\'ersny of n1l(Ogen fuiog bacteria in rice fields soil and also to mea<nlre the plant gro'Mll. The plant gn,.,,.1h parameters oi rice shuwed the trcat111ct11 whi~b soaked by biofcrtilizcr were better than comm! tn.'af.lllCUt aod treallUent w1ucb ~prcad by biofcnilaer. However, tho; divc"1ty of nitrogco fixing l>aqcria was '11<)re vary tn control treabUcut bused on l)CJGE re;;uh~. There were lhre.: of eight b3.o.ds which always appears in .:very month. The ,.;qucn1:1ng n.-sult$ sbow.:d all se<lucnccs wcrc cw~1fied u uncultured bacterium clone nitrogcna<e i.ron prOtein (niflI) gcoc, patttal .:ds.

() 2014 1\1':1'.SI Publisher All rk>h1 s resened To Cite Thi$ Artiri.-: Rllld1 Had1anta. Iman Rusmana. Nisa !Uchnwna Mubarik. Diversity of Nnrogcn Ft'llD" Bactcna S.,,,.,d on rutH Gene in Rice F1el<b .. fdv. F.nl'irntl. JJWJ., 8(14), 113-119. WU

INTRODUCTION

Nitrogen is a macro clement required by plants but the compo~i tion of78% ni trogen in the atmosphcr.: cannot be used directly by plan~. Plants can only abso1b soluble nitrogen in the soil by using root. Supplying the nitrogen in the soil can be done by fertili2a1ion or naturally with the help of m1croorganim1S. B1ological 11.itrogeu fixation i!> an unpotulllt prm:ess in nature which performed by nHrogen-fix.ing bactena in changing free nitrogen gases in the: atnio,phere into 11r1moruum Ammonmn 1• nr 1mr "'..l!' rJtrog~·t source: in th:: c:cosysrc:ru_ l.iit.'"C'gen fixing bactena 1s able to hvc freely (free living) and can also hvc in symbiosis with plant<>. Riological nitrogen fixation is limited to prokaryotes while there is no report for eukaryotes as uiirogen fixer

The ability of rulrogcn fixing bacter1c1 to convert nitrogen ia the auno~pherc 1nw ammoruum due 10 11;

nitrogenasc cnqme. Nicrogenase enzyme consists ol l\~o subunits that dinitrogcnase and dmitrogenase reducrase. Dinitrogenase enzyme is encoded by 11i}D and nrjK gene, while dmitrogeoasc: reductase enzyme: encoded by nijH gene. The diversity of nitrogen-fixing b:icteria can be identified by molecular upproac~s. One of the m<llecular te<'hniques that can be u:.cd is DGGE (Denimu ing Gradienl Gel Electrophoresis).

DGGE can distinguish species based on differences in the GC composition of tbi: analy.(Cd DNA ~cquenccs [ 10]. This is caused hy the uc;e of denaturant gradienl gel uc;cd. The diYer<;i1y of nitrogen-fixing hacteri:1 sn1died by nijH gene. The use of 11(/l I gene in DGGE is commonly used and proven to be more accurate for the detection of diversity nitrogen fixing bacteria contained in the soil [21,-1,5.11 ].

Accordmg 10 report<; by Pingak [l::!], the use of bio-tertih7ers in rice fields cao mcrc3~c nee protlucti\ ity. Rice producliv1ry can be supported by a various factors, such as nitrogen. Nitrogen elemem itself can be acquired from ferti li;ration or nitrogen fixing bacteria. Research related to the relalionsh1p between the u~e ol bio-lertihzcr and nee proJuctJ"ity associated wid1 the di,·ersiry of nitrogen fixing bacteria in nee fieldc; hac; not hc.:n rcpo11eJ in Sukabumi. This re!>carch aimed to study the diversity of nitrogcn-tixmg bactena in rice fields h~ OGG[ technique.

Corresponding Author: 1111.in R.usmana, Bogor Agricultural University, Depanment ofBiolog,y, Faculty of Mathematics and Natural Science, Bogor 16680, West Java, Indonesia. T,..I• +~1-'">C:. 1-2~'>")R"t'l t:' ... mttil· ; ,.. •• ...,...nJn\tn1'..,.. ;_A

64 Jman Rosmana ~' 111, 2014 AdnMH ia EaviroamHIU Biology, 8(14) Special 2014, Pagl!' b3-69

MATERIAL AND M ETllODS

This study was conducted in June 2013 - May 2014. Field research was performed in rice fields at Cidahu village, Cicurug, Sukabumi, West Java. The analysis was performed at the Laboratory of Microbiology and Laboratory oflntegrated, Department of Biology, Bogor Agricultural Unive~ity. ·1 he treaunent used in this study were control, soaked and spread. The control treatment using 300 kg/ha of NPK fertilizer. Soaked and spread treatment using 200 kg/ha of NPK fertilizer and biofertilizcrs. Rice clump in the soaked treatment was soaked with biofertilizer while in the spread treatment, biofertilizer was spread directly into rice fields.

Soil sampling. Rice field soil in the control, soaked and spread treatment were taken by using a 10 ml syringe. 2

repJjcatioos were used per treatment. Control treatment has an area of 150 m2 while soak and spread plots ha\·e an area of 600 m2. Soil sampling is done every 30 days during the rice planting namely 30 DAP (days after plantiug), 60 DAP and 90 DJ\P. A total of± 1 kg of soil in each treaonent was taken in the beginning and end of the growing season then scot and analyzed a1 lhe Soil Research institute, Bogor to determine the physical and chemical characteristics of the soil.

Plalll Growth Meas11rement: Plant growth measurement was carried out by measuring the plant height and number of tillers. There were

I 0 clumps that had been used in this study. Measurement of wet weight and dry weight of the clump, roots and seeds are also carried Ollt in the end of the planting.

DNA £ttmrno11. DNA extraction was done by using the Power Soil DNA Isolation Kit (Mob10 laboratories, Carlsbad, CA.

USA). Extrncaon was done according to the procedures of the company. The quality of DNA then checked using Nano Drop 2000 (Thennu Scientific, Wilmmgton, DE, USA).

Po(1 muase Cham Reaction DNA was amplified by USIJlg PolF I PolR primer sets f 13 ]. 40 CIC sequences were attached to th~ end of the

forward pnmer. PCR was performed by using KAPA Hot Stan Read}mix (KAPA Biosystems. Wilmington, MA. USA). Each PCR reaction contained 12.5 µL KAPA Hotstart Readymix, 1.25 µL forward primer (0.5 uM), 1.25 µL reverse primer (0.5 uM), 3 µL template (-100 ng) and 7 µL nuclease free water. PCR was performed using T­Gral,lient Thcrfnocycler (Biomelra GmbH. Goeuingcn. Gerrnanr). The PCR thermal cycling coodilions were as follo"v-;: m111al denaturation at 95 °C for I min; 30 cycles of dena1ura1ion at 95 °C for I 5 s. annca1mg at 55 °C for 15 sand elongation at 72 C for 15 s; and a final elongation step at72 °C for 5 mm. Products were r'Jll at 1.5 % agarose gd for checked correct ~i7e, and stored at -20 °C until analyzed on OGGF

DGGE Analysi.f OGGE wa-: ~rfornml using the D Code Univer<>al Muuition .Detecuon Sy-;um lBio-Rad. llen:ules, CA.

USA). 'the DGGf geb were prepared and nm under the following conditions 2S uT of the PCR p1oduct was loaded omo I mm thick IS"'o (w/\') polyacrylam1de gels (acrylamide-b1sacrylam1de (37.5.1)] in I TAE buffer (40mM Tris, 20mM acetic acid, aml lmM EDTA) using urc:a as the denaturing agent with a denaturing gradient from 35% 10 60% (100% denaturant corre::.ponded to 7M urea and 40% (v/v) deionized fonnamide). Electrophoresis was perfonncd at 60 °C and IJO V for 6 h. After clectrophores1s. the gel was stained for 60 min in SYBR Safe {l11vi1Togcn- \itolecular Probe~. Carlsbad, CA, USA) and visualized by G:BOX (Syngene, Frederick, MD. USA). Band-: appearing on the gels were excised and translerred to micro tubes containing 50 ul ddH20. l'hc bands were incuba1ed at 4 °C' ovcnught Band analy7ed using Phorctix ID (Touil Lab) LO estimate total band that appeared.

Se'luencing tmd Phyloge11111ic 7i·ce Co11s1ruc1io11: Supernatant from each band was checked usmg Nano Drop 2000 to dc1cnmnc the quanuty of DNA from

excised band. A 10 µL (-SO ng) supematalll w11~ re-amplified under the PCR conduions described aoove using non GC pnmcr. The PCR productS were sent to I stBASE Malaysia. Sequencing rc-;ults will be analy7ed using BLAST program \\ith NIRI database and then the phylogenclic tree created using MEGA 5.2 [J 7].

RF.SULT A ~D OISCUSS101'1

Snil Charac1eris1ics: Soil in 1he control lreatmem contained of 10% sand, 43% silt and 47% clay Soil io the soaked treatment

contained of 16% sand, 33% silt and 5 i % clay. Soil on spread treatment contained of 18% sand. 38°-o silt and

65 Iman Rusm:ina et al, 2014

AcfnnttS ill Enviro•111tntal Biology, 8(14) Special 2014, Pages· "'·69

48% clay. Soil in I.he control treatment, soak and spread was tbe same pH range, ic pH 5. Carbon content in the control treatment, soak and spread is 1.97%, 1.81 %, and 1.91 %, respectively. Nitrogen content of the control treatment, soak and spread is 0: 17%, 0: 17% aoJ 0: 14%, respecLivdy. CIN ratio of each trcaullent was 12, 11 aod 14, respec1ivcly. Based on 1he criteria of the USDA [19], 1he soil in the control treatment classified as Silty Clay, while soaked treatment classified as Clay and spread treatment classified as Clay. Based on the criteria of BA.LIT ANAH [2], those soil'! was classified as acidic soil (pH 4.5 - S.5) with carbon and nitrogen was low while the CIN ratio of the 1hree soil was classified as moderate. According AGRlSNET [I), rice plants can grow well in Clay soil, Silty Clay and Silly Clay Loam. The opumum pH condition which required to grow rice well ranged 5-7. So. the 'iOil characteristics in this study fairly well for rice planting.

Rice growth: Height measurement results indicated that rice plants at soaked treatment showed muimum height than

other treatments. Control treatment occupied the lowest position among others. The number of tillers in soaked treatment also showed compared to the other two treatments. Spread treatment place at second place followed by control treatment (Figure l ). Soaked treatment had the highest wet weight and dry weight compared to the other treatments in all parametm measured (Figure 2). These results were supported with reportS [12] that the $Qaked treatment resulted in the best growth response than the other two treatments.

He Wit and Number of Tille11

100

Ill so

6,)

-J 26 26

lO ~llR 0

He~tl Tilltr

l'I CCIM'O' . \Q.\.f'(J Q C\p~.•d

Fig. 1: I lc:1ght and number of tillers tn the control. soaked and spread treatment.

WelWelgbl 180 160 H9.S

140

111 ~·20 ~ JOO 74.75 ·r 80 15t?S ii i 3: 60

38 I It)

20 ~ u ......

B (u.irul '•c.:i.'J''j! Spi-~

(a)

DryWf'ight

lllO

3 ISO

l: .'!!! 1ro ..

l~.~~ ~·iii 3: so 11 'lb l 'l.6.S6

0 =-• Fnl (ump Root G,a,.,

P•rt of Plant

" c..c~1 10 • \1 •4"•f el ~r>'~dd

(b) Fig. 2: Weight of wet (a) and dry lb) of the clump. roo1s and grain of rice in the control. \Oakcd and spread

treatment.

66 1nun Rusmana Dal, 2014

Ad\loett in E•~U-o•mflllll Bioloc. 8(14) Spec1al 2014, Page'!: 63-69

DNA Extraction: DNA was extracted from soil showed various results ranged arouod 24 - 28 ng/uL Tbc purity of the DNA

obtained from the extraclioo is quite good wilh an average A260/A280 parameter ranges 1.9 (Table 1). Re\.'Ommcnded value of A260/A280 ratio is ranged 1.8 - 2.0 (14). Good quality of DNA template was important for downstream process hke PCR.

DNA Amplificatio11: Nine samples were successfully amplified by PolF-GC/PolR primer with fragment size obtained as expected,

around 360 bp (Fig 3). PolF I PolR primer was designed to amplify nijH genes possessed by nitrogen fixing bacteria and has been shown to successfully ampltfy 19 imponant species of oitrogen-fixmg bacteria such as Azospirillum. Agrobacterium, Burkholderia, Pseudomonas, Rhizobium, Streptomyc~. and Xamhomonas (13].

Table I : T DNA be No I 2 3 4 s 6 7 8 9

p S I DN bol cooccntrallnn us111sr: ower 01 A atJon L

Treatment DNA Concentration lnot .. 1 )

Control 3-0 DAP 26 Soaked. 30 DAP 24.4 Soread, J() DA P 24.5 Control, 60 DAP 28 Soaked, 60 OAP 24.4 S1>rcild 60 DAP 27.9 Control 90 DAP 27.4 <;oakcd. 90 OAP 26.6 Sorcad. 90 D . .\P 27.2

Mabcdefg:h

3000 bp

1000 bp

500 bp

A260/A280 1.96 I 97 1.9

1.94 l.95 l.9R 199 1.'ll l.9S

360 bp

Fig. 3: The ni/H gene amphlica1ion resul1 wi1h PolF-GCfPolR rinmer on 1.5% agarose gel. \Veth from left 10 nght 100 bp marker (a) control 30 OAP. (b) ~oaked 30 OAP, (c) spread 30 DA.P, (d) •'Mtrol ~O DAl>, tc) ~aked 60 OAP. (f) spread 60 OAP, (g) control 90 OAP, (h) soaked 90 DAP, (i) spread 90 OAP.

DGG£ Profile: DGGE results showed all analyzed s.imple were separated into several separate bands. f.ach separate bands

represent one of us own species. The results showed there were three bands that always appeared in lht: treatment every month. Control treatmenl showed the highest number of band~ with different vanattons of each month. Snaked t:reaunem tended 10 be stable every month with 4 bands. Spread trearmcnt showed incrcasms number of bands from 4 bands in firs1 month into 6 bands in the 1hird month (Figure 4).

fhc control u·eatmenl showed 1.he highest diYersity compared than other treatment~. ll was probably because there was no biofcrtilizcr applicauon then there is no dominance dfec1 at this tn:atmcn1. Btofcrt1hzcr contain metanotroph bacterial isolates capable of enhancing the growth of rke planis. However, the 1mrnduct1on of biofcrtilizers decreased nitrogen-fixing bacteria communities. Eventhough the highest d1vermy of nitrogen­fixing bactc;:ria v.:as obtamcd in the control treatment but it had lmle role to encourage the gro"'1h of nee plan1s. Jt can be shown by the less growth of rice plunls in conirol treatment than soaked and spn::ad lrcacmenl. There wen: three reason to CJ<platn this pbcnomc:non. First, the diversity of nitrogen-fixing bacteria found in the control treatment may be high bu! not necessarily active. The activity nijH genes can be more accurately known when it analyzed by RNA approach [I I). Sl><:ond, bacterial isolates were used as a hiological fertilizer could encourngc the growth of rice because i1 has been tested accordrng to Piogak [12). Third, some bacterial isolates metanotroph used as a biological fertili7.er also bas the capabil11y of nitrogen fixation (3).

67

7

-.~ '-- ..__ .....__ ! b c

.: _,...,.

ActuocH ill Eniro1mtntal Biology, 8(14) Special 2014, Pagct· 63-69

! b c d e f

- -- - --2

3 I 4 5 I - .... -6 7

8 ---

g h

- - - I

6

8

Fig. 4: DGGE profile analysis of nijH gene, Left: Photo ofG: BOX and Righi: Interpretation of Pboretix lD software. The number showed the bands which cut to be rcamplified. Wells from left to right: (a) control 30 OAP, (b) soaked 30 OAP, (c) spread 30 DAP, (d) control 60 OAP, (e) soaked 60 DAP, (f) spread 60 OAP, (g) control 90 OAP, (h) soaked 90 DAP. (i) spread 90 DAP.

Seque11ci11g a11d Phylogenetic Tree· BLAST results showed that all sequence!> obtained were uncultured bactena dinito~cnase (111jH) gene from

various bactena (Table '.!). Thi:. mtfa:atcJ tJ1at the sequences obtained bdung to unculrurable bacteria. It nl~o showed that the sequence found was relatively new a$ a percentage identity of the results of the bl~t sequence databases under 95% of the - 360bp sequences of bases. Band I. 6. and 8 were showed on DGGt gel on each treatment every month. The three of bands were suspected to dominant bacteria in nee field due to appear m each tn:atment 1n every month The resulL~ showed that hand I hatl closene~~ to U11c11/t11red bai:terwm c:/011e MDE_amb_35jl d111i1rogenase reductase (nijH) gene. partial eds for 89%. Baud 6 bud closeness to Uncultured bactel'i11m clone clo.4-42 11itroge11ase iron protein (nifli) gene, partial eds for 88% and band 8 had closeness to Uncultured bacterium clone Sipa-L24 mtrogcnase iro11 protein (mjl I) gene partial cd~ for 92%.

Table 2: Results of Bl AS'l seQuencc of1he 11iJI I 11cne Blmd Des~ripnon Query E Value Ba...e/H;i,c llknmy Acc:.i.100

Cover i'OumbC!' I Un.:ulruml ba..1cnum done 100% Se-114 JOR W! 89'!. Kf'84Mll9.l

~IDE_amb_35f2 dmnro~enase re<luc1asc (.(11/ll) 11.ene, oMial ~d~

2 Uncultured b•~erium clone Sipa-10 97°'t 5e-1)4 JO'JJJI 93•. JX'.!6!4'.17.l lllllO!ICna<c 1100 J>fOICln tn1/H) gene,

-~cd'

' Uncuh111ed bal:tcrium cl"ne Sir"-'" '19'% So-149 1~\' '1 .f .. !)<•. .rx'.!t1~H< I ni1rogcn:hc 11on prou:rn tnifH} gene.

n;uuaJ c,t, 4 l}ocuhwcd baclcnum clooc Sipa-14 97"/o 3e-t26 JO' Hl 92•. JX26X413J

nilrol!et13~ iron 1uutcm (11i/H) gene. nartial eds

5 Unculrurtd bancrium clone JSR~-:! 9~"-~ 9c-107 -29.: 111) :Sil"• HM750439.I JUllll'Ogcna.se l'NucUl.5e (111/l l) !!el'le pnrual

eds 6 Uncultured bae1enum doll<! do.\-42 97~'. 2e-102 2SS ~1i\ R~"• JX26~272 I

nicrogcnnsc iron prolcm tnr/H) gene. nanial eds

7 Un.;ul1urctl ti.cicrium CIC111e ac;2 ) 97°• 3c- I Iii !9) 1' 7 ~. JX07%20 I dn1itrog.:iia~ •TOO pf\\ICIR (nijH) g.:nc ,

oarual cd~ ~ Uocuhurcd bdcleriuni d<lnc S1pa-L24 JOO% 5,~ 1w 33 I 1(,1) 92°. Kf032J72 I

ni1rogc11a<e iron pru1ci11 111ijH) gene. oarual ~J,

From the phylogeny tree (fig. 5) 11 can be seen that the bands I, o ..1.nd ):( hatl closeness 10 Pse11do111011as WITZl!l'I, Anaeromywbacter sp Halorhodu.~pira haloµhila with compan:il 1estricted to - 360 bp range only Pseudomcmas stutzeri is a bacteria which has broad habitat and relati,·el) resistant to changes in envcronmental conditions [9]. Thi~ ability makes P. sruturi \ery tolerant of changes in management and planting methods on a depressing place. Perez found 20 DGGE sequences related to P s1t11~1•ri on agricultural land cultiv:ited org::nically and conventionally [ I I]. Diallo also found P. stutzeri of ~cveral sequences which were found

68 Iman Rusmana D al, 2014

Aduoct:s ia Eaviroameotal Biology, 8(14) Special 2014, Page<;· 63-69

predominantly in the soil [7). IT. ha/op/11/a was classified into gamma prmeobactcria, purple sulfphur bactena, Gram-negati\'e and photorotroph. H. halophila is belong to potential nitrogen fixing bacteria and djspersed quite widely. Some research and studies have shown that H. halophila bas 11!/H gene and capable of fixing nitrogen [18] [4] (161. Anaeromyxobacter sp. is belong lO the detta proteobacteria bacteria, rod-shaped, Gram-negath·e, capable of fanning spores, and natural habitat in soil (15]. Anaeromyxobacter sp. also capable of fixing atmosphenc nitrogen and from several studies has been shown that it has nifH gene (8) l16] (20].

·-· Urctlfured beel•num clone cloA-42 IV~ llUn protOln (,,.n '' (lef)tt f"tJtfUJI t:"9- JX268212 1

•""""' ..... _,, ................ llG23...._,_ ... r-o--•-.JX079d». Uncu1!<""'1 _,_ c-~ M-•Jron "'°'""' ~llfff) gMi. f*1Rt1..i. l(FIJ32t12.1

A~)')Obctw $f>.. CP{)()()llJA I

• b-.d5

.,,

,.....,,,,,..., 11J. FJ&e7'n I

Ps~ $p F.Ja1,T• 1

U'llCiJltUl'ed b-..J.,i..vn ~ S•.fO nit~•"'°" Pf°'..,. (n tH} ~ l*fW cm JXU&f31 t

Lklctll~ b1JK.l•n"'n c.((;VlO S~:Jf: ndr~~· H<N• OfOI•:'' (1Hftt) gon. "''"'Ill ct# JX2684-.1S T

UlCt.itul8d bll(.lerun c/onit S~.'U M~e NOl'l ,Orof~ (nt/HJ l.k"MI l*'r-' Cd$ Jl!"'4'M.1111

-- 1' A b-2

A b-14

.Az~ .. "-' hydrty:Jlulus EF62C~ r

~---------- ------ J.ltf,._..«OCC,... ~AV1?113Co r

Fig. 5: Phylogenetic tree of8 nijH gene :.equences obtained from DGGI" anaJ)'sis Phylogeny tree wa~ constrvcted with the Neighbour Joining method wich IOOOX boo1srrap Yaluc.

Eight bands were successfully sequenced didn '1 have closeness wi1h merano1roph bacteria which W&S u~cd

as b1ofertililer and added to the rice fields. Some metanotroph bactena ilia.I added as a biofenih:zcr has been tested an<l analyzed the ability of nitrogen fixation f3]. However, this study found no sequence which simjJar 10 metanotroph bacleria. It was because the different primer used in thi~ research instead to recognize the mcthanotroph bactena PolF PolR primer designed to amplify the nitrogen fixing bacteria m w1dcl:,. coverage while the primers used by Bintarti [3) \V:l.S a special primer designed b} Dedysh [6] for m1.:tanotroph bactcna !h:ll had nhility of nnrngen fixation. The target amplicuns from cwo pnmcr alsG J1ffor.:nt. f'olF PolR 1 .• im.:r h .. J a target of360 bp. \\hi le primers designed Dedysh had a target of 453 bp (6).

Co11ciusio11: ' The dfrcrsity of nitrogen-fixing bacteria obtained from rice fields Cicurug village, Sukabumi tend to vary.

Control treatment had the most dhersit} in comparison \\1th the soaked and ,,,read trea1men1. Sequencing results showed that all th-: bands were belong into uncultured bacteria dinitogcnase (n(fJI) gene.

REFERENCES

I 11 AGRJSNET, 2014.' Rice Soil Requirement. flntcnm). (download 18 Ma) '.!014]. http: ''1kkimagrisnet.org/Gencral 1en/R ice _Soil_ Requirement.aspx

(2) BALITA.\IAII Balai Penelitian Tanah, 2005.' 1nalisis K1mta Tanah. Tanaman .. lir dan P11puk Bogor (IO)· Balai Pcnelitian Tanah.

Pl Bintarti. A.F., 2011\. Amonium scrta karakterisasi gen nyH dan 11ijD bakteri mctanoLrol a>al saw;ih (thesis) Bogor ( ID) lnstitut Pertaman Bogor

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69 Iman Rusmana n 111, 2014 Aduncts ia Eavin11unent1tl Biology, 8{14) Special 2014, Pages: 63-69

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