Naskah Cosat Revisi FINAL 22-12-2014

Organized by Sponsored by Supported by
JAIF
Japan-ASEAN Cooperation
Proceeding
ASEAN COSaT 2014
This proceeding and the individual contributions contained in it are protected under copyright
by LIPI Press and the following terms and condition apply to their use:
Photocopying
Single photocopies of single articles may be made for personal use as allowed by copyright laws.
Permission of the Publisher and payment of a fee is required for all other photocopying, including
multiple or systematic copying, copying for advertising or promotional purposes, resale, and all
forms of document delivery. Special rates are available for educational institutions that wish to
make photocopies for non-profit educational classroom use.
Derivative Works
Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for
internal circulation with their permissions. Permission of the Publisher is required for resale or
distribution outside the institution. Permission of the Publisher is required for all other derivative
works, including compilations and translations.
Electronic Storage or Usage

Permission of the Publisher is required to store or use electronically any material contained in
this journal, including any article or part of an article. Please consult
Except as outlined above, no part of this publication may be reproduced, stored in a retrieval
system or transmitted in any form or by any means, electronic, mechanical, photocopying,
recording or otherwise, without prior written permission of the Publisher.
Notice
No responsibility is assumed by the Publisher for any injury and/or damage to persons or property
as a matter of products liability, negligence or otherwise, or from any use or operation of any
methods, products, instructions or ideas contained in the material herein. Because of rapid
advances in the medical sciences, in particular, independent verification of diagnoses and drug
dosages should be made.
Although all advertising material is expected to conform to ethical (medical) standards, inclusion
in this publication does not constitute a guarantee or endorsement of the quality or value of such
product or of the claims made of it by its manufacturer.
Proceeding
ASEAN COSaT 2014
The Proceeding of
ASEAN Conference on Science and Technology 2014
– 9th ASEAN Science and Technology Week (ASTW-9)
Innovation for better ASEAN Community:

Science and Technology Innovation in Food, Energy,
Water and Related Topics for ASEAN Development.
Editor in Chief
Prof. Dr. Estiko Rijanto
LIPI Press
© 2014 Indonesian Institute of Sciences (LIPI)
Cataloging in Publication Data

Proceeding of ASEAN Conference on Science and Technology 2014/Estiko Rijanto (Ed.). –
Jakarta: LIPI Press, 2014.
pp. xvi + pp. 562; 17,6 x 25 cm
ISBN 978-979-799-812-7
1. ASEAN COSAT 2. Science and Technology
600
Copyeditor : Sri Suratmini Nugroho

Annisa Waharyudisti
Proofreader : Martinus Helmiawan
Layouter : Ariadni
Siti Qomariyah
Cover Designer : Junaedi Mulawardana
First Edition : December 2014

Abstracts available online at penerbit.lipi.go.id
Published by:
LIPI Press, member of Ikapi
Jln. Gondangdia Lama 39, Menteng,
Jakarta 10350
Phone: (021) 314 0228, 314 6942
Fax.: (021) 314 4591
E-mail: press@mail.lipi.go.id
JAIF
Japan-ASEAN Cooperation
iv
Contents
Editorial Foreword............................................................................................................. xi
The Remarks by the Indonesia National ASEAN COST Chair.........................................xiii
Preface by Minister of Research, Technology and Higher Education, the Republic of
Indonesia............................................................................................................................ xv
SCB (14)
Gamma Radiation Induced Changes of Molecular and Phytochemi-
cal Profiles on Mutants of Andrographis paniculata (Burm.F.) Wal-
lich Ex Ness
(J. I. Royani, et al.)............................................................................................................3
The Chemical Constituent and Antioxidant Activity of the (-)-Epi-
cathecin From AnEndophytic Fungus Mycoleptodiscus indicus
(P. C. Mawarda, T. Ernawati and Y. Srikandace)..............................................................19
Potency of Hibiscus (Hibiscus Rosa-Sinensis L.) As Antioxidant To Re-
duce Carbon Tetra Chloride Compounds In Red Blood Cells
(D. Priadi and Kusmiati).................................................................................................29
Purification of Bioactive Peptide with Proteases Inhibitory Activi-
ties from Streptomyces misionensis
(J. M. Yusoff, K. Simaran and Z. Alias)...........................................................................37
Isolation, Identification, and Screening of Locally Isolated Xan-
thomonas sp.
(N. I. S. Bokhari, K. Simarani and M. S. M. Annuar).....................................................43
Ultraviolet Irradiation Effect of Penicillium chrysogenum on Peni-
cillin Production
(D. Hardianto, et al.).......................................................................................................49
Isolation and Cloning of Partial Her-2 Gene from Indonesian
Breast Cancer Patients for DNA Vaccine Development
(Desriani and L. Triratna)................................................................................................57
Heterologous Expression of Recombinant Plantaricin Ws34 in Esch-
erichia coli
(A. S. Putri, et al.)............................................................................................................65
Evaluation of Low Temperature Induced Mutant of Soybean Mosaic
Virus for Cross Protection in Soybean
(W. R. Andayanie and P. G. Adinurani.)..........................................................................73
Process Design of Peptone Production from Peanut Meal as By-
Product of Peanut Oil Industry Using Crude Papain
(M. Rahayuningsih and N.G. Wiranti)............................................................................85
v
Potency Of IAA Hormone Produced By Endophytes Bacteria Isolated
From Shorea Selanica On Supporting The Growth Of Paraserinthes
Falcataria
(T. Widowati, et al.)........................................................................................................93
Production And Characterization Of The Biosurfactant By The
Formation Of Glycolipid Isolated From Pseudozyma Hubeiensis
Y10bs025
(M. Sari, F. Afiati and W. Kusharyoto)...........................................................................103
Application Of Marker Assisted Selection And Sensory Test For
Selecting Aroma On F2 Progeny Of Rice Derived From Crossing Be-
tween Ciherang X Basmati
(S. Sari, et al.)................................................................................................................111
Development Of Rice Lines Resistant To Brown Planthopper With
Aromatic Traits: Selection Based On Molecular Marker
(A. P. Asri, N. Carsono and S. Amien)...........................................................................122
SCSER (12)
Assessment of E-Waste Recovery Facilities in Selangor and Kuala
Lumpur, Malaysia
(N. A. M. Nordin and P. Agamuthu).............................................................................129
Public Perception on Current Waste Management System: A Malay-
sian Case Study
(S. H. Fauziah and S. F. Ser)..........................................................................................135
Biomass Flow and Carbon Sequestration in An Organic Farm
(L. H. Yeng and P. Agamuthu).......................................................................................147
Biomass Gasification for Power Generation Using Dual Chamber
Circulating Fluidized Bed Reactor
(H. Wahyu, et al.)..........................................................................................................153
Roof Mounted Micro-Wind Turbine For Power Generation In Coast-
al Housing In Semarang, Indonesia
(D. P. Sari).....................................................................................................................161
Performance Of A Radial Turbine For Small Organic Rankine Cycle
Power Generation System
(M. Arifin, B. Wahono and A. D. Pasek).......................................................................169
Effect Of Reaction Time And Cellulase Loading On Dilute Alkali
Pretreatment Of Sugarcane Bagasse To Produce Fermentable Sug-
ars For Bioethanol Production
(T. Fajriutami dan Rizky Rissa Bella).............................................................................177
Performance Of Microbes Consortium On Single-Chamber Micro-
bial Fuel Cell As Electricity Generation
(D. Rahayuningwulan, D. Permana dan H. E. Putra)....................................................187
vi
Heat Release Analysis Of A Two Cylinders in Diesel Engine Fuelled
With Ethanol-Diesel Blends
(Y. Putrasari, et al.)........................................................................................................199
An Evaluation For Enzymatic Saccharification of Fast-Growing
Tree Species From Secondary Forest in West Kalimantan
(L. Risanto, et al.)..........................................................................................................209
A Novel Microwave-Biological Pretreatment Effect On Cellulose
And Lignin Changes Of Betung Bamboo (Dendrocalamus Asper)
(W. Fatriasari, et al.)......................................................................................................219
The Emergence of Biogas Technology For Reducing Rural Poverty:
Empirical Studies In Java Island
(L. Ariana).....................................................................................................................231
SCMG (9)
Assessment Of Erosion Potentials On Various Cropping Patterns Us-
ing Usle: Case Of Subang Region, West Java
(R. I. Sholihah, et al.)....................................................................................................243
New Stage Of International Collaboration On Climatological Ob-
servation
(M. D. Yamanaka) .......................................................................................................253
Simultaneous Correlation Analysis Of Australian Summer Monsoon
Index (Ausmi) Against Rainfall In Bali Region
(S. Mujiasih and I. G. A. Purbawa)................................................................................261
Developing Strategy For Monitoring And Decision Support System
For Smoke Haze Trans-Boundary Problem Within Asean Region
(S. D. A. Kusumaningtyas)............................................................................................275
The Potential Impact Of Carbon Monoxide Emission To The Commu-
nity Health In The Vicinity Of Baranangsiang Toll Gates
(Y. V. Paramitadevi, A. S. Yuwono, and M. Widyarti)....................................................289
The Mechanism Of Dry Mid-Atmosphere In The Western Maritime
Continent During Rainy Season IN 2014
(Supari, et al.)................................................................................................................299
Design Of Automatic Measurement Instrument For Water Dis-
charge On Drainage Monitoring System
(R. T. Wahyuni).............................................................................................................311
Acehseis, A Local Seismic Experiment In Bener Meriah And Central
Aceh
(M. Muzli, et al.)...........................................................................................................319
Selection Of Global Gmpes Models For Seismic Hazards Assessments
In Indonesia (Case Study Sumatra-Java Area)
(A. Rudyanto, et al.)......................................................................................................329
vii
SCMIT (7)
Led-Based Spectrometer For Advanced Chemistry Laboratory Ex-
periments
(M. A. Alagao, et al.).....................................................................................................343
Introduction Investigation: Executive Information System For
University
(S. Warnars, Sasmoko and N. Susianna) ......................................................................353
Design Of Implementation Delay Tolerant At Wireless Mesh Net-
works Using IBR-DTN and Batman-Adv
(H. Yuliandoko, S. Sukaridhoto and M. U. H. A. Rasyid).............................................365
Development Of A Programmable Multipurpose Forced Convection
Type Dryer
(E. C. Guevarra)............................................................................................................377
A Study Of Network Speech Recognition Using Tcp
(A. Jarin, K. Ramli and Suryadi)....................................................................................389
Aural And Photonic Spectrum Based Digital Pest Controller For
Oryza Sativa (Rice)
(I. A. P. Banlawe)...........................................................................................................401
Development Of Programmable Logic Controller (Plc)-Based Cof-
fee Pulper For Wet Process
(M. R. Perena)...............................................................................................................413
SCMST (13)
Effect Of Sintering Temperature Rate On Physical Properties Of Po-
rous Tricalcium Phosphate (Tcp) Ceramics
(A. Fadli, A. Rasyid and R. Firmansyah)........................................................................427
Study Of Kinetics And Thermodynamics As Well As The Effect Of
The Presence Of Co-Ions In Influencing Adsorption Cu2+ Ion By Coal
Fly Ash Adsorbent
(A. Zakaria, et al.)..........................................................................................................433
Organophosphorus (Ops) In The Environment: Effects Of Repeated
Application Of Chlorpyrifos On Agricultural Soil
(C. Carol and S. H. Fauziah).........................................................................................443
Bonded Prfeb Magnet: Fabrication And Characterization
(D. Aryanto, et al.)........................................................................................................449
Effect Of Sintering Temperature On Dielectric Constant Of Silica
Prepared From Rice Husk Ash
(Qudratun, et al.)..........................................................................................................457
Synthesis And Characterization Of Al-Doped Lithium Titanate Li-
4
ti5o12 As Anode Material For Li-Ion Battery
(S. Priyono, et al.)..........................................................................................................463
viii
Cellulose Fibers From Oil Palm Fronds Reinforced Polylactic Acidb
Composite
(F. A. Syamani, Y. D. Kurniawan and L. Suryanegara)...................................................473
Isolation And Characterization Of Lignin From Alkaline Pretreat-
ment Black Liquor Of Oil Palm Empty Fruit Bunch And Sugarcane
Bagasse
(M. A. R. Lubis, et al.)...................................................................................................483
Changes In The Cellulose Crystallinity During Different Phases Of
Distilled Vetiver Root Cellulose Fibers Development
(F. A. Syamani, et al.).....................................................................................................493
Bioethanol Production Using Saccharomyces Cerevisiae Immobil-
ised On Fresh And Modified Sugarcane Bagasse
(S. H. Anita, et al.)........................................................................................................503
Effect Of Ph On Extraction Efficiency And Distribution In Nickel
Ion Separation Using Solvent Extraction
(A. Maimulyanti and A. R. Prihadi)...............................................................................515
Ethoxylated Glycerol Monooleate: Palm Oil Based Nonionic Sur-
factant For Oil-In-Water Emulsion Systems
(I. B. Adilina, et al.).......................................................................................................523
Conversion Of Citronella Oil And Its Derivatives To Menthol Over
Bifunctional Nickel Zeolite Catalysts
(I. B. Adilina, et al)........................................................................................................531
APPENDIX
Editorial Board................................................................................................................ 541
Authors Index................................................................................................................... 543
ix
Editorial Foreword
The ASEAN Conference on Science and Technology 2014 was held in Bogor -
Indonesia on August 18th–19th, 2014. The conference was a part of activities of
the 9th ASEAN Science and Technology Week (ASTW) event under the theme
“Innovations from the Most Dynamic Region on Earth” which was held on August
18th–28th 2014. It is a triennial event of the ASEAN Committee on Science and
Technology (COST). Innovation for better ASEAN Community was selected as
the sub-theme for the Conference. The Conference covered all subcommittees
and flagships in a focused topic, i.e. Science and Technology Innovation in food,
energy, water and related topics for ASEAN development.
The Proceeding of ASEAN Conference on Science and Technology 2014
– 9th ASEAN Science and Technology Week (ASTW-9) contains papers which
had been presented and discussed in the parallel sessions during the Conference
and have undergone review process. The authors come from Indonesia, Malaysia,
Thailand, Philippines and Japan.
This proceeding is an excellent result of collaborative work. We would like to
express our gratitude to international editorial board members, reviewers, authors
and the publisher for the quality of their works. We would also convey our sincere
appreciation to ASEAN Secretariat, The Ministry of Research, Technology and
Higher Education of the Republic of Indonesia and JAIF for their continued
support.
We hope this proceeding could be a constructive contribution in development
of science and technology, especially among ASEAN member states.
Jakarta, 20 November 2014

Editor in Chief
xi
The Remarks by the Indonesia National ASEAN
COST Chair
The 9th ASEAN Science and Technology Week (the 9th ASTW) was successfully
organized by the Ministry of Research and Technology, Republic of Indonesia at
Bogor, West Java from 18th to 28th August 2014. ASTW event is a flagship project
of the ASEAN Committee on Science and Technology (ASEAN COST) which
is aimed at demonstrating major achievements made and exploring potential of
S&T generated both in ASEAN Member Countries and Dialogue Partners. The
ASTW is conducted triennially on a rotational basis amongst ASEAN Member
Countries. This forum is expected to be used as a media for opening windows of
opportunities for the triple helix plus societal communities (ABG-S) to promote
networking and to expand their S&T co-operations, not only amongst the
ASEANs, but also with ASEAN Dialogue Partners. As the ASTW is deemed as
an important event; therefore, Indonesia has been very pleased to host the ASTW
twice, in 2005 (7th ASTW) and in 2014 (9th ASTW). As a reference other member
countries holding the previous ASTW are: Malaysia (1st, 1986), Philippine (2nd
in 1989 and 8th in 2008), Singapore (3rd in 1992), Thailand (4th in 1995), Viet
Nam (5th in 1998), and Brunei Darussalam (6th in 2001).
The 9th ASTW was proceeded by the 4th ASEAN Science Congress and
Sub Committee Conferences or known as the ASEAN Science and Technology
Conference 2014 (ASEAN COSAT 2014). It was opened by former Minister
of Research and Technology the Republic of Indonesia, Prof. Dr. Ir. Gusti
Muhammad Hatta, M.Si on August 18th 2014. The ASEAN COSAT 2014 was
organized in two days with the following agendas: (1) Convener by Organizing
Committee Chairman, Prof. Dr. Estiko Rijanto; (2) Opening Remarks by His
Excellency Prof. Dr. Ir. Gusti Muhammad Hatta; (3) Plenary session Day 1 by
four keynote speakers: Innovation and Entrepreneurship in Sweden by Dr. Sven
Thore Holm (Sweden), Innovation and Entrepreneurship in ASEAN: Challenge
and Opportunities by Dr. Warsito P. Taruno (Indonesia), ASEAN as a driving force
of global innovation by Dr. Haruo Takeda from Hitachi Ltd (Japan), Science and
Technology Innovation for Food Security by Prof. Ram Rajasekhran (India); (4)
Oral Parallel session Day 1 (7 Sub Committees in 7 Rooms) and Poster session
xiii
Day 1; (5)Plenary session Day 2 by five keynote speakers: Blue Economy for Small
Island Developing State by Mr. Nico Barito (Ambassador, Republic of Seychelles),
Human environmental security in ASEAN: Water –Energy-Food Nexus by Prof.
Robert Delinom (LIPI, Indonesia), Basic material research for renewable energy
applications by Dr. Nicola Seriani (The Abdus Salam ICTP, Italy), An Application
of Synchrotron-based X-ray Absorption Spectroscopy on Materials by Dr. Pinit
Kidkhunthod (Thailand), and China-ASEAN Technology Transfer Center: a Great
Boost to China-ASEAN Science and Technology Cooperation and Technology
Innovation by Dr. Ye Bo (People Republic of China); (6)Oral Parallel sessions
Day 2 (7 Sub Committees in 7 Rooms) and Poster session Day 2.
We are pleased to see the completion of The Proceeding of ASEAN Conference
on Science and Technology 2014 – 9th ASEAN Science and Technology Week
(ASTW-9). As a scientific documentation it will contribute to the development
of science and technology especially among ASEAN member states.
Finally we would like to convey our gratitude to the international edito-
rial board and reviewers who have dedicated up to the end in completing this
proceeding.

Indonesia National ASEAN COST Chair
Dr. Agus R. Hoetman
xiv
Preface
The Minister of Research, Technology and
Higher Education,
The Republic of Indonesia
First, I would like to congratulate all of the ASEAN Science, Technology and
Innovation (STI) policy makers and scientists, for the success of the 9th ASEAN
Science and Technology Week (9th ASTW) spectacular event, which was conducted
in Bogor, 18–27 August 2014. I acknowledged that out of 15 activities, the 4th
ASEAN Science and Technology Congress and Conferences (18–19 August
2014), is one of a very important activities, which aims to expose all of S&T
achievements in the past 6 years, since the 8th ASTW in Manila (2008). Therefore,
special gratitude was also delivered to the ASEAN joint committe for this congress
and conference.
Secondly, I would like to take this opportunity to inform you that as of
October 20, 2014, the former Indonesian Ministry of Research and Technology
(RISTEK) has been transformed into the Ministry of Reserach, Technology and
Higher Education (RISTEK-DIKTI). With regard to this development, I am
convinced that Indonesian commitment in the ASEAN Committe on Science and
Technology (ASEAN COST) would be more firmed and continued, especially in
supporting the implementation of ASEAN Plan of Action on Science, Technology
and Innovation (APASTI) 2015–2020, with its vision on “A Science, Technology
and Innovation-enabled ASEAN which is innovative, vibrant, sustainable and
economically integrated”.
APASTI 2015–2020 has several goals, among others are to encourage active
collaboration between public and private sectors, and to improve human resources,
through capacity building and talent mobility. All of these goals are in line with
RISTEK-DIKTI Ministry in the next five years. Therefore, I am expecting that
most of ‘research results presented in this ASEAN congtess and conference’
could be ‘leveraged up’ into prototypes (lab and industrial scales), which then the
‘useful and valuable products’ could be massively produced to fulfill the ASEAN
xv
and world markets. In addition, I also believe that both of the academic papers
and their commercial transformations into the valuable reserach products would
encourage more numbers of techno-entrepreneurs in ASEAN countries, as one
solution to the global challanges that we should face together.
Ending my remarks, I would like to emphasize that developing a proceeding
after the congress and conference is surely very useful to empower Indonesia as
the ‘knowledge-based society’country, as well as to expose the scientific results
and achievements to the users, industries and societies. Regarding this, I would
like to express my appreciation to the editorial board and reviewers for the efforts
and commitments of the International editorial boards and reviewers, as well as
the publishers, who have been working hard to the completion of the 4th ASEAN
Conference on Science and Technology (ASEAN COSAT 2014). I am sure this
proceeding would lead to STI product commercializations in the future, which
benefit all of the ASEAN and world community.

Minister of Research, Technology and Higher Education,
The Republic of Indonesia
Prof. Dr. M. Nasir
xvi
SCB
1
GAMMA RADIATION INDUCED CHANGES OF
MOLECULAR AND PHYTOCHEMICAL PROFILES ON
MUTANTS OF Andrographis paniculata (Burm.f.)
Wallich Ex Ness
J. I. Royania,*, A. Purwitob, W. Sumaryonoa, D. Hardiantoa and A. Mahsunaha
The Agency for the Assessment and Application of Technology
a
630 Bld PUSPIPTEK Area, Serpong, Tangerang

b
Departement of Agronomy and Horticulture-Faculty of Agriculture, Bogor Agriculture
University,
Darmaga, Bogor
Abstract
Sambiloto (Andrographis paniculata (Burm.f.) Wallich Ex Ness is a medicinal plant that became
the pre-eminent national and prospective to be developed in Indonesia. Self pollination and
habitual inbreeder from sambiloto affected the low of genetic variation. Quality improvement
of the plants is one strategy that can be used to increase the genetic diversity and improve the
content of the active compounds in medicinal plants. One method to improve genetic diversity
and the content of active compounds is by using radiation. The aim of this research was to find
out changes in mutant characters of sambiloto plants irradiated with gamma rays on molecular
and phytochemicals profiles. Sambiloto seeds were irradiated using gamma rays Cobalt 60 and
grown vegetatively with ex vitro propagation from M1V1 to M1V4 generations. Molecular analysis
of mutant by 10 primers of ISSR marker was used to obtain DNA profiles. Determination of
phytochemical profiles of mutant at M1V4 generation was done by using HPLC method. The
results showed that 5 primers out of 10 primers could distinguish changes of DNA profile, 4
primers showed the same number and size bands and 1 primer could not amplified. Analysis
of genetic similarity obtained 6 groups with genetic distance 0.79–1.00. HPLC analysis with a
wavelength of 230 nm showed the variation of the profiles and contents of phytochemical mutants
of sambiloto. The contents of andrographolide varied in the range between 6.5%–10.9%. Highest
content of andrographolide was found in DK300 mutant, while the lowest one was found in
control. In DB60 mutant, andrographolide content had the same content as control; however,
chromatogram revealed the presence of 4 peaks compared to the control that just had 2 peaks.
Key words: Andrographis paniculata, Gamma radiation, DNA profiles, Phytochemical profiles
i. Introduction
Andrographis paniculata (Burm.f.) Wallich Ex Ness, which is also called “King
Bitter”, is an annual herbaceous plant that comes from Peninsular India and Sri
Langka (Lattoo et al., 2006, Mishra et al., 2007; Jarukamjorn & Nemoto, 2008).
* Corresponding author. Email: idhajr@yahoo.com
3
4 Proceeding ASEAN COSAT 2014
These plants grow naturally in Southeast Asia (India and Srilanka), Pakistan and
Indonesia, but extensively cultivated in China and Thailand, eastern and western
of India, and Mauritius (Mishra et al., 2007). In Indonesia, A. paniculata is
well known as sambiloto, and eventhough A. Paniculata is not originally from
Indonesia, this plant became the pre-eminent national and prospective to be
developed in Indonesia. Subramanian et al. (2012) also said that A. paniculata
is a bitter plant with a sweet future to describe how important this plant is for
medicinal.
Lattoo et al. (2006) reports that A. paniculata is hermaphroditic, self-
compatible, habitual inbreeder and obligate autonomous selfing in the species.
These reasons were affected by the low genetic variation of A. paniculata. Research
generated by Pandey and Mandal (2010) found that there is no variation of A.
paniculata genotype at the phenotypic level from 5 different location in India.
Wijarat et al. (2012) also reinforce this research that could not detect genetic
variation in 58 A. paniculata accessions from Thailand which were evaluated by
SSR, AFLP and RAPD markers. On the other side, the level of active compound
of A. paniculata, namely andrographolide, without any treatment were very low
too. Many researcher reported that the level of andrographolide is between 0.1–2%
(Sabu et al., 2001; Raina et al., 2007; Sharma et al., 2009).
Improvement of quality of the plants is one strategy that can be used to
increase the genetic diversity and improve the content of the active compounds
in medicinal plants. Mutation breeding is a breeding technique that creates
variability in the mutated population through heritable changes in the genotypic
and phenotypic utilized for effective selection of particular traits (Tah et al., 2008).
Induced mutation is the easier method to create genetic variability compared
with other breeding methods (Minn et al., 2008). Mutation by using ionizing
irradiation is one of the most widely used method to generate mutant. Gamma
rays is a mutagen that has a high energy irradiation that can cause damage to
the covalent bonding or hydrogen bonding molecules/biomolecules in cells that
result in damage to chromosomes, genes and ends on cell death (Xiang et al.,
2002). The biological effect of gamma-rays is based on the interaction with atoms
or molecules in the cell, particularly water, to produce free radicals (Borzouie et
al., 2010). These radicals can damage or modify important components of plant
cells and have been reported to affect differentially the morphology, anatomy,
biochemistry and physiology of plants including changes in the plant cellular
structure and metabolism depending on the radiation dose.
Gene mutation without phenotypic expression is usually unrecognized. To
recognize gene mutation in mutants, various methods has been applied to detect
the effect of mutagen in plants. Different methods are available to investigate the
Innovation for Better ASEAN Community 5
effect of mutagens on plants. In medicinal plants, the change of mutant characters

could be detected in morphology, physiology, molecular and phytochemical level.
The aim of this research was to find out changes in mutant characters of A.
paniculata plants that irradiated with gamma rays on molecular and pytochemical
profiles.
ii. MATERIAL AND METHODS

Materials used for this research were A. paniculata seeds from Kanigoro Karan-
ganyar accession. The seeds were obtained from Indonesia Research Institute
for Medicinal and Aromatic Plants (BALITTRO).
A. Irradiation treatments, plantation and subculture of mutants

For the exposure of gamma irradiation on the seeds, 60Co had been used. Seeds
of A. paniculata in clean plastic were irradiated in Cobalt 60 Gamma Chamber
machine at National Nuclear Energy Agency with treatment dose of: 0, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275 and 300 Gy.
After irradiation, seeds were planted in pot trays and germination for 6 weeks.
Viability of seed germination from each treatments were calculated to determine
the LD50 value with CurveExpert 1.3 software.
After 8 weeks of planting, shoots from M1V0 generation were cut approxi-
mately 10 cm to subculture to M1V1 until M1V4 generation is obtained with
ex vitro propagation method.
B. Determination of molecular profiles of mutants

1) Extraction of DNA genome
Total genomic DNA of A. paniculata in M1V2 generation was isolated using
CTAB modification method. Fresh leaves (200 mg) were well grounded in
extraction buffer solution with mortar and pestle to fine paste thoroughly.
DNA pellet obtained was dried, then 200 ul TE buffer solution was added
and kept at freezer (-20°C) for subsequent use. DNA quality was checked
visually using electrophoresis machine on 0,8% agarose at 100 volt for 30
minutes. DNA bands are exposed by UV-ray illuminator and documented
using GELDOC. Nanodrop machine was used to determine the isolated
DNA quantity.
2) Amplification of DNA using ISSR primers
Ten primers of ISSR marker were used to amplify 31 genomic DNAs of
A. paniculata mutants. ISSR primers used were: SBLT2 {(AG)8T}, SBLT3
{(AG)8C}, SBLT5 {(GA)8C}, SBLT8 {(CT)8G}, SBLT13 {(GAA)6}, SBLT14
{(GACA)4}, SBLT15 {(GA)8}, SBLT17 {(TG)8G}, SBLT18 {(CCCT)4} and
SBLT19 {(GATA)2(GACA)2}. ISSR amplification was carried out with 10

primers using 200-300 ng of genomic DNA. PCR reactions consisted of DNA
template (1 µl extract of DNA genome added by DNA free water), 1.5 mM
MgCl2, 0.2 mM ISSR primer, 0.2 mM dNTP, 1x PCR buffer, 1 U Taq DNA
polymerase (dream taq-fermentas) and add free DNA/RNAse water up to total
volume of 20 µl. The initial condition was: Pre-PCR heating at 94 °C for 5
minutes, then followed by subsequent steps as followed: 40 cycles consisting
of denaturation at 94 °C for 45 seconds, annealing at 50 °C up to 58°C for
45 seconds and extension at 72 °C for 2 minutes. After 40 cycles finished,
post-PCR extension was at 72 °C for 5 minutes. Modification of annealing
temperature of the PCR was conducted to optimize the ISSR primer reaction.
3) Visualization of amplified DNA and Molecular analysis
Amplified DNA products were analyzed through electrophoresis on 1.5%
agarose gel containing 1xTBE (tris borate EDTA) and syber safe to stain the
DNA. Gel running at 100 volts for 30 minutes and DNA bands were exposed
under UV illuminator and documented using GELDOC.
Polymorphic products from ISSR analyses were scored qualitatively for
presence or absence of the amplified products for each primer. If a product was
present as bands, it was designated as 1, and the absence of it was designated
as 0. The cluster analysis was done using NTSYS software version 2.02 and
Dice similarity coefficient was utilized to generate dendrogram by means of
Unweighted Pair Group Method of Arithmetic Means (UPGMA) employing
the Sequential, Agglomerative, Hierarchical and Nested Clustering (SAHN)
from NTSYS program. The dendrogram obtained was used to evaluate the
genetic distance of mutants compared with control.
C. Determination of pytochemical profiles

1) Extraction of A. paniculata leaves
Freshly collected plant leaves from 4 mutants of A. paniculata that exposed
to gamma radiation and 1 leaves from control were washed thoroughly and
dried in the oven at 60 °C for 4 hour to obtain dried material. Dried materials
were weighed for 0.05 g and grounded to fine powder. The fine powder of
A. paniculata leaves were placed into 15 mL falcon tube and then extracted
with 2.5 mL methanol solution as solvent extraction. Dried materials with
methanol into falcon were shaken horizontally at 300 rpm for 30 minutes.
Further extracts were centrifuged at 5,000 rpm for 10 minutes. Superna-
tant was collected and re-extracted crude similar as in the above manner.
Afterwards, supernatant collected from 2 times extraction was concentrated
using centrifugal concentrator for 2 hours until dried. Then dry extracts were
dissolved in 1 mL methanol solution and prepared to inject into the HPLC

instrument.
2) Analysis of pytochemical profiles by HPLC
Determination of changes of phytochemical profiles from A. paniculata mu-
tants performed by HPLC using Photodiode Array Detector. For comparison
to phytochemical profiles, andrographolide (Sigma-Aldrich) was used as
standard. Calibration curve for linear regression was made based on peaks
area using standard solutions of andrographolide. Stationary phase using a
Symmetry C18 column (150 x 4.6 mm) size 5 μm. The mobile phase was
water with pH 3.0 (formic acid): acetonitrile in the ratio (70:30 v/v). Column
equilibrated with the mobile phase for an hour. Isocratic elution was carried
out at room temperature and at the pump at a flow rate of 1 mL/min. The
detection was made at a wavelength of 230 nm. The solution was injected as
much as 10 mL. The evaluation was based on the retention time and peak area
formed by linear regression. Chromatogram of standard was compared with
chromatograms from mutants and control of A. paniculata plant. Calculated
levels of andrographolide were formed from each mutant.
III. RESULTS AND DISCUSSIONS

1) The effect of irradiation on seeds germination and LD50
Sensitivity of radiation on A. paniculata seeds can be seen from germination
of seeds in the range of irradiation doses as compared to control. The result
for irradiation sensitivity based on survival percentage of irradiated and
control seeds demonstrated that significant reduction in survival percentage
was observed with increasing gamma dosage.
Dhakshanamoorthy et al. (2011) states that the decrease in germination
at higher doses of the mutagens may be attributed to disturbances at cellular
level (caused either at physiological or physical level) including chromosomal
damages. Based on germination rate, CurveExpert 1.3 analysis was obtained
LD50 from the seeds of irradiation with dose at 140.363 Gy (Figure 1). In
this study, the function of the Gaussian model was the best model to describe
the population mortality of A. paniculata with r = 0.954.
2) Molecular profiles of A. paniculata mutants in M1V2 generation
The PCR amplification using 10 primers of ISSR gave rise to reproducible
amplification products. ISSR primers produced different numbers of DNA
bands, depending upon their simple sequence repeat motifs. The result showed
that 31 mutants based on different morphology were observed in M1V2
generation. These 31 mutants and 1 control were then analyzed using ISSR
molecular marker.
S = 7.12167613
r = 0.95458304
0
90.0
0 y=78.4*exp((-(-22.14-x)^2)/(2*171.3^2))
80.0
Seeds germination (%)
benih berkecambah (%)
0
70.0
0
60.0
140.363 Gy
0
50.0
0
40.0
0
30.0
0
20.0
0
10.0
0.0 30.0 60.0 90.0 120.0 150.0 180.0 210.0 240.0 270.0 300.0 330.0
Dosis (Gy)
Dosoge (Gy)
Figure 1. LD50 of seeds A. paniculata irradiated with Cobalt 60 Gamma rays
Molecular analysis showed that genomic DNA of A. paniculata mutants

produced multiple bands using 10 primers of ISSR. Of 10 primers used,
9 primers were amplifying the genomic DNA and 1 primer could not
be amplified. Based on electroforegram, 5 primers, i.e.: SBLT2, SBLT3,
SBLT13, SBLT15 and SBLT18 can distinguish changes in the DNA profiles
of mutants compared with control and 4 primers, i.e.: SBLT5, SBLT8,
SBLT14 and SBLT19 shows the same number and size bands with control.
The total number of amplifield DNA bands yields was 1.450 bands, out of
which 152 bands (10.48%) were polymorphic and 1.298 (89.52%) were
monomorphic. Fragments ranged in size from 1.000–8.000 bp (Table 1).
The number of fragments produced by various ISSR primers ranged from
2–12 with an average of 161 bands per primer. The largest number of bands
was identified in primer SBLT3 and SBLT18 (11 bands) and the smallest in
primers SBLT15 (1 band).
The qualitative data from electroforegram showed that there were 3 types
of DNA profiles of A. paniculata mutant amplified by 10 primers of ISSR.
The first type was DNA profile of mutants with the same DNA bands with
wild type. It can be seen in 4 primers that were used, i.e.: SBLT5, SBLT8,
SBLT14 and SBLT19. DNA profile with this type was monomorphic. The
second type was DNA profile with reduced DNA bands compared with wild
type plant. These type were shown in 5 primers, i.e. : SBLT2, SBLT3, SBLT13,
dan SBLT15. While the third type was DNA profile that had additional
bands compared with wild type. In this research, primer of SBLT18 had this
characteristic. The second and third type of DNA profiles were polymorphic
bands. Copra (2005) states that polymorphism of amplified bands is caused
by: (i) base substitutions or deletion in the priming sites, (ii) insertions that
render priming sites too distant to support amplification or (iii) insertions
or deletions that change the size of the amplified fragment. Appearance of
new bands in this research can be explained as the results of different DNA
structure (breaks, transpositions, deletion, etc). The polymorphism revealed
by ISSR due to deletion and or addition may be caused by variation in DNA
binding patterns by gamma rays.
Based on these polymorphic bands, a similarity coefficient matrix was
calculated and a similarity dendrogram was obtained using a UPGMA cluster
analysis (Figure 2). A cophenetic correlation of r = 0.89 was obtained, which
indicates a good fit between the original similarity matrix and the resulting
clustering analysis (Rohlf, 1997). On the basis of the similarity dendrogram,
from 32 irradiated A. paniculata plants, they could be classified into six major
clusters at a Nei’s genetic distance with each group divided into sub-clusters.
This grouping tendency does not refer to a single character or single dose only,
but spreads on each character and the dose. Jaccard’s similarity coefficient
showed genetic distance of A. paniculata ranged from 0.79 to 1.00. Furthest
genetic distance obtained in the DK300, DG275 and DG70 compared with
controls (DN). These coefficient similarities showed that A. paniculata, even
through treatment with gamma rays mutagen, has genetic variation, still
remains low.
The genetic alterations are produced by ionizing radiation due to ionization
and excitations of the DNA molecule. There are two effects of ionizing irradia-
tion on the heredity material: gene mutations and chromosome breaks (Atak
et al., 2004). Irradiation by this physical mutagenic agent leads DNA break
formation via direct and indirect detrimental effects. In direct interactions, the
radiation energy is transferred to the targets and in indirect interactions energy
is absorbed by the water present in the external medium. After hydrolysis of
water, secondary messenger molecules affect the biomolecules (Esnault et
al., 2010). They react with most of the biomolecules including DNA and
scavenger electrons from them. The oxidation of biomolecules by the radicles
damage their structure and biological activity. It is worth, genetic alterations
occur on the DNA molecules. This is the cause of mutations that depends
on radiation (Selvi et al., 2007).
According to Dhakshanamoorthy et al. (2011), the disappearance of normal
bands (loss of bands) maybe related to the occurrence of DNA damage (e.g.
single and double-strand breaks, modified bases, abasic sits, oxidized bases,
bulky adducts), DNA-protein cross links, point mutation and/or complex
DN
DK250
DB50
DG200
D3125
DB70
DG60
DG125
D390
D3200
DK90
DB90
DK175
DG40
DKR275
D3250
D3300
DB60
DK150
D4275
DK275
DC3150
DK200
DG150
DB275
DK225
D3100
DG300
D3150
DK300
DG275
DG70
0.79 0.84 0.89 0.95 1.00
Koefisien Kemiripan
Figure 2. Dendogram constructed based on UPGMA model calculated from genetic distance of 32
irradiated A. paniculata by ISSR marker
chromosomal rearrangements induced by gamma radiation. While the addi-

tion of the DNA bands may reveal a change on some oligonucleotide priming
sites due to mutations (new annealing event), large deletions (bringing to
preexisting annealing site closer) and homologous recombination. Atienzar et
al. (2006) reported that mutations can only be responsible for the appearance
of new bands if they occur at same locus in a sufficient number of cells to be
amplified by PCR. The new bands could be attributed to mutation, while the
disappearance of bands could be attributed to DNA damage.
3) Phytochemical profiles of A. paniculata mutants in M1V4 generation
We used 4 leaves of mutants and 1 from control to determine the changes
of pytochemical profiles. The reason for 4 mutants used for comparison of
this pytochemical profiles based of dendrogram data (Figure 2). We used
mutans plant with farthest of genetic distance from control (DG70, DG275
and DK300 mutants) and plant from the middle of the dendrogram (DB60
mutants) in M1V4 generation. Andrographolide standard used for this
research in the range of retention time of 4.98 ± 0.01 with equation of a
linear regression of standard was Y = 18606X +76026 and the correlation
coefficient was R2 = 0.993, which indicated a good linear regression for

analysis of andrographolide.
Analysis using HPLC with a wavelength of 230 nm resulted in the variation
of the profiles and contents of phytochemical mutants of A. paniculata. The
contents of andrographolide varied in the range between 6.5%–10.9% (Figure
5). Highest content of andrographolide found in DK300 mutant while the
lowest content found in control (DN). The higher dose of irradiation given
made the increasing levels of andrographolide produced. There were increased
levels of andrographolide up to 1.66% in DK300 mutant compared to control.
In control, the normal of andrographolide content amounted to 2.01%
(Data from BALITTRO, not shown), with the condition if A. paniculata
grown using seeds until harvest in 120 days. In this research, propagation
of A. paniculata was done by ex vitro method and the result showed that
andrographolide content increased up to 4% with 60 days of harvest period.
This can be explained that on conditions of propagation by cutting, parts of
such plants were subjected to stress. Reported by Datta et al. (2011), stress
of plants caused by external factors such as radiation not only causes chro-
mosomal changes but also the pathway disruption of some chemical reaction
linked to abnormal of plant growth and mechanisms of defense of the cell.
This stress will stimulated the endogenous hormone to produce plant growth
regulators such as cytokinin or GA3 to maintain metabolic and physiological
systems and stimulate the growth of shoots. It is known that the effect of
GA3 also change the number of metabolites in cells by regulating the activity
of key enzymes such as DXS and HMGR. Both of these enzymes play a role
in terpenoid biosynthesis pathway either through MVA (HMGR enzyme)
or DXP (DXS enzyme) pathways that produces IPP and DMAPP, which are
precursors for the synthesis of diterpene (Jha et al., 2011). The increase of
andrographolide content on ex vitro plants of A. paniculata and irradiated
plants on M1V4 generation indicated also caused by this. These contents of
andrographolide were very high compared to the contents of andrographolide
isolated from several different locations in Indonesia, with the average ranges
between 0.95%–2% dry weight (Royani et al., 2012).
Research conducted by Koobkokkruad et al. (2008), with mutant of
Artemisia annua that irradiated by gamma rays at 8 Gy, have been able to
increase the levels of artemisinin content from 0.18% to 0.70% or an increase
by approximately 3.89%. Aryanti (2011) also found artemisin in Artemisia
cina mutant was increased from 40 mg/100g to 2103 mg/100g than parent
or an increase of approximately 52%. Likewise, research by Moghaddam et
al. (2011) found content of flavonoid was increased from 2.64 ± 0.02 mg/g
to 8.94 ± 0.04 mg/g or an increase of 3.38% in Centella asiatica mutant. In
Lithospermum erythrorhizon mutant (Chung et al., 2006), shikonin production

increased (71.0 mg/l) at 16 Gy was almost 4-fold amounts to that of control
cultures (17.7 mg/l).
Chromatograms peaks generated by HPLC in control plant obtained two
peaks with retention times of 2.823 dan 4.818, the last digit was the peak of
andrographolide. In DB60 mutant, andrographolide content had almost the
same content as control but in chromatogram, it had four peaks. They were
2.837, 5.014, 6.806 and 9.379 with andrographolide peak obtained in 5.014.
The addition of the peak in 6.806 and 9.379 were indicated the addition of
different phytochemical profiles than control as a result of irradiation.
In DG70 mutant, profiles of chromatogram obtained 3 peaks in the
range of 5.011, 6.703 and 9.331. These profiles were also different from those
seen in the control where the peak with 2.823 value was lost in the mutant
DG70, while the value of 6.703 and 9.331 were indicated the same profile
with DB60 peak. Andrographolide content on DG70 mutant was also higher
than the control, it was 7.8%.
10.9
12 9.9
Andrographolide content (%)
10 7.8
6.5 6.8
8
6
4
2
0 Figure 3. The contents of androgra-
DN DB60 DG70 DG275 DK300 pholide from A. paniculata mutants
In DG275 mutant, profiles of chromatogram were identified to also have 3

peaks, namely 2.863, 5.017 and 6.875. These profiles were different from control
because there was an additional peak in 6.875 and peak of 5.017 indicated the
peak of andrographolide. The addition of 6.875 peak is similar to that found
in DB60 and DG70 mutants. Andrographolide levels of mutant DG275 also
increased 9.9% compared to the control with 6.5%.
In DK300 mutant, chromatogram peaks indicated the presence of 2 peaks
at 2.882 and 5.023. Phytochemical profiles in DK300 mutant generated had
the same profile as control but the level of andrographolide was higher than 3
mutants and control, which was at 10.9% level.
Of the five chromatograms that formed by gamma rays, the effects of

phytochemical profiles were clearly visible on the different mutants than control.
These differences of phytochemical profiles from DB60, DG70 and DG275
mutants possibility generating new compounds when its compared to control.
It can be explained that irradiation by gamma rays causes oxidative stress and
the impact on the biomolecules that cause conformational changes, oxidative,
breaking of covalent bonds and the formation of free radicals (Lee et al 2005).
The hydroxyl radical (HO-) and superoxide anion (O2-) formed by the radiation
can modify the properties of the protein and lipid molecules that cause oxidative
modification of peroksidase protein and lipid.
The location of the new peaks that formed from phytochemical profiles is
likely that the compound was not much different from the andrographolide
compound in basic structure but in a different kind like neo-andrographolide,
deo-xyandrographolide, or dehydroandrographolide. This possibility was because
the standard used for phytochemical profile was just andrographolide so the other
peaks would not be ascertained.
Gamma rays can cause different levels of damage to the cells. Biological
damage occurs most direct and mediated by reactive oxygen species (ROS) such
as hydrogen radical (HO), superoxide radical (O2), hydrogen peroxide (H2O2),
and singlet oxygen formed from the radiolysis of water (El-Beltagi et al., 2011).
The presence of water radiolysis by gamma radiation causes changes of
chemical from protein that caused by fragmentation, cross-linking, aggregation
and oxidative caused by oxygen radicals were formed (Lee et al., 2005). ROS are
highly reactive in membram lipids, proteins and DNA. ROS was known by its
ability to activate signal nitrogenmonoksida (NO) and NADPH oxidase (Zhang
& Bjorn, 2009). ROS was also believed to be the main cause of stress injuries
and rapid cellular damage (El-Beltagi et al., 2011). Added by Kume and Matsuda
(1995) that the radiation causes behind changes in protein conformation at the
molecular level by means of break covalent bonds from polipetida chains.
In DK300 mutant, phytochemical profile of the mutant with the control
obtained two peaks with the same retention time but contains andrographolide
higher levels than control. This can be explained hypothetically that the role of
these enzymes in the biocatalyst of biosynthesis of the other compounds non
andrographolide were blocked and resulting the optimization of the biosynthesis
of the andrographolide or an increase enzymes that play important role in the
synthesis of the andrographolide.
Allegedly, an increase in the enzyme 3-hydroxy-3-methylglutaryl-coenzyme
A reductase (hmgr) which is one of the key enzymes that play a role in the
accumulation and chlorophyll content of andrographolide on A. paniculata
1.00
andrographolide
2.823
0.50
AU
4.818
0.00
2.00 4.00 6.00 8.00 10.00
Minutes
1.00
andrographolide
6.807
2.837
9.379
0.50
AU
0.00
5.014
2.00 4.00 6.00 8.00 10.00
Minutes
1.00
andrographolide
6.704
9.331
0.50
5.011
AU
0.00
2.00 4.00 6.00 8.00 10.00
Minutes
1.00 andrographolide
5.017
2.864
6.876
0.50
AU
0.00
2.00 4.00 6.00 8.00 10.00
Minutes
1.00 andrographolide
5.024
2.882
0.50
AU
0.00
2.00 4.00 6.00 8.00 10.00
Minutes
Figure 4. The results of HPLC chromatograms in control and A. paniculata mutants
(Jha et al., 2011). Increased of shikonin compounds by gamma-rays on the

plant Lithospermum erythrorhizon was also due to the increase of enzyme activity
geranyltransferase PHB, as a precursor of shikonin, which resulted in increased
accumulation of shikonin (Chung et al., 2006). Another possibility is that the
expression of genes that involved in the synthesis of andrographolide to increase.
The study conducted by Jha et al. (2011) about the profile of expression of A.
paniculata plants with elicitor GA3 and jasmonic acid can alter expression of
HMGR gene. In this study, the gamma rays were also thought to be elicitor to
increase expression of HMGR gene in A. paniculata plants. Similar research was
also reported in maize plants exposed by UV-B rays, where the expression of genes
that associated with photosynthesis in maize decreased and genes that associated
with antioxidant increased (Casati & Walbot, 2003).
The similarity of the number of peaks at the phytochemical profiles of control
and DK300 mutant compared with the other mutants (DB60, DG70 and
DG275) that have more peaks can be explained that mutation by irradiation was
random. So that the possibility of the mutant had the addition peak of more than
control due to a modulation in the pattern of proteins that induce the presence
or loss of some protein bands (Hegazi & Hamideldin, 2010) which resulted in a
conformational protein change in the making up of the compound.
IV. Conclusion
Genetic distance of A. paniculata ranged from 0.79 to 1.00 indicating that the
genetic variation was still remained low even though it was treated with gamma
radiation mutagen. Futher study such as the structure elucidation of coumpounds
need to be done in order to see what will come up in the A. paniculata mutant
DB60, DG70 and DG275.
V. R eferences
1) Lattoo, S. K., Khan, S., Dhar, A. K., Choudhary, D. K., Gupta, K. K. and
P. R. Sharma. (2006). Genetics and mechanism of induced male sterility in
Andrographis paniculata (Burm. f.) Nees and its significance. Current Science,
91 (12), 515–519.
2) Mishra, S. K., Sangwan, N. S. and R. S. Sangwan. (2007). Phcog Rev.: Plant
Review Andrographis paniculata (Kalmegh): A Review. Pharmacognosy Reviews,
1 (2), 283–298.
3) Jarukamjorn, K. and N. Nemoto. (2008). Pharmacological Aspects of
Andrographis paniculata on Health and its Major Diterpenoid Constituent
Andrographolide. Journal of Health Science, 54 (4), 370–381.
4) Subramanian, R., Asmawi, M. Z. and A. Sadikun. (2012). A bitter plant

with a sweet future? A comprehensive review of an oriental medicinal plant:
Andrographis paniculata. Phytochemistry Reviews, 11, 39–75.
5) Pandey, A. K. and A. K. Mandal. (2010). Variation in morphological
characteristics and andrographolide content in Andrographis paniculata
(Burm.f.) Nees of Central India. Iranica Journal of Energy & Environment, 1
(2), 165–169.
6) Wijarat, P., Keeratinijakal, V., Toojinda, T., Vanavichit, A. and S. Tragoonrung.
(2012). Genetic evaluation of Andrographis paniculata (Burm. f.) Nees revealed
by SSR, AFLP and RAPD markers. Journal of Medicinal Plants Research, 6
(14), 2777–2788.
7) Sabu, K. K., Padmesh, P. and S. Seeni. (2001). Intraspecific variation in active
principle content and isozymes of Andrographis paniculata Nees (Kalmegh):
a traditional hepatoprotective medicinal herb of India. Journal of Medicinal
and Aromatic Plant Sciences, 23, 637–647.
8) Raina, A. P., Kumar, A. and S. K. Pareek. (2007). HPTLC analysis of
hepatoprotetive diterpenoid andrographolide from Andrographis paniculata
Nees (Kalmegh). Indian Journal of Pharmaceutical Sciences, 69 (3), 473–475.
9) Sharma, S. N., Sinha, R. K., Sharma, D. K. and Z. Jha. (2009). Assessment
of intra-specific variability at morphological, molecular and biochemical level
of Andrographis paniculata (Kalmegh). Current Science, 96 (3), 402–408.
10) Tah, P. R. (2008). Studies of leaflet mutants in Mungbean (Vigna radiate (L)
Wilczek). International Journal of Plant Breeding and Genetics, 2 (2), 75–84.
11) Minn, M., Khai, A. A. and K. M. Lwin. (2008). Study on the Effect of
Gamma Radiation on Rice Sin Thwe Latt (IR53936). GMSARN International
Conference on Sustainable Development: Issues and Prospects for the GMS. 12–14
Nov. 2008.
12) Xiang, T. H., Yang, J. B., Zhu, Q. S., Li, L., Ni, D. H., Wang, X. F. and
D. N. Hang. (2002). Molecular biological effect of (CO)-C-60 gamma-ray
irradiation on rice genome DNA. Progress in Biochemistry and Biophysics, 29,
754–759.
13) Borzouei, A., Kafi, M., Khazae, H., Naseriyan, B. and A. Majdabadi. (2010).
Effects of Gamma Radiation on Germination and Physiological Aspects of
Wheat (Triticum Aestivum L.) Seedlings. Pakistan Journal of Botany, 42 (4),
2281–2290.
14) Erdem, G. and S. Oldacay. (2004). Employment of RAPD technique to assess
the genetic stability of Helianthus annuus treated with different mutagenic
agents. Journal of Applied Sciences, 4 (2), 277–281.
15) Abd El-Twab, M. H. and F. A. Zahran. (2010). RAPS, ISSR and RFLP
analysis of phylogenetic relationhips among congeneric species (Anthemideae,
Asteraceae) in Egypt. International Journal of Botany, 6 (1), 1–10.
16) Dhakshanamoorthy, D., Selvaraj, R. and A. L. A. Chidambaram. (2011).
Induced mutagenesis in Jatropha curcas L. using gamma rays and detection
of DNA polymorphism through RAPD marker. Comptes Rendus Biologies,
334, 24–30.
17) Chopra, V. L. (2005). Mutagenesis: Investigating the process and processing
the outcome for crop improvement, Current Science, 89 (2), 353–359.
18) Atak, C., Sema, A., Leyla, A. and C. Yasemin. (2004). Induced of plastid
mutations in soyben plant (Glycine max L. Merril) with gamma radiation
and determination with RAPD. Mutation Research, 556, 35–44.
19) Selvi, B. S., Ponnuswami, V. and T. Sumathi. (2007). Identification of DNA
polymorphism induced by gamma irradiation in amla (Emblica officinalis
Gaertn.) grafts of V1M1 and V2M1 generation. Journal of Apllied Sciences
Research, 3 (12), 1933-1935.
20) Atienzar, F. A. and N. J. Awadhesh. (2006). The random amplified polymor-
phic DNA (RAPD) assay and related techniques applied to genotoxicity and
carcinogenesis studies: a critical review. Mutation Research, 613, 76–102.
The Chemical Constituent and Antioxidant
Activity of The (-)-Epicathecin from an
Endophytic Fungus Mycoleptodiscus indicus
Panji Cahya Mawardaa,*, Teni Ernawatib and Yoice Srikandacea

a
Research Center for Chemistry, Indonesian Institute of Sciences, Jalan Cisitu Sangkuriang-
Bandung, Indonesia
b
Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan Puspitek Serpong-
Tangerang, Indonesia
Abstract
The current study was to investigate the chemical constituent and antioxidant activity of
(-)-epicathecin from an endophytic fungus Mycoleptodiscus indicus. The fungus was isolated from
Indonesian medicinal plant Thyponium flageliiforme. The endophytic fungus was fermented in
Potato Dextrose Broth and extracted with ethyl acetate. Ethyl acetate extract was fractionated using
column chromatography techniques to obtain flavones. The structure of flavones was elucidated
through spectral data based on 1H-NMR, 13C-NMR, and LC-MS spectrometer. The compound
was identified as (-)-epicatechin with molecular weight of 290.11 g/mol. The compound was
tested with DPPH radical scavenging method and it showed strong antioxidative activity with
IC50 0.6 µg/mL. At the first time, (-)-epicathecin from M. indicus Indonesia was reported.
Key words: Mycoleptodiscus indicus, (-)-epicathecin, Antioxidant activity
I. Introduction
Oxidant and free radical are continuously produced as by-product in normal cel-
lular metabolism. The uncontrollable production of free radical leads to oxidative
stress and becomes a main trigger of human degenerative diseases development
[11,21,18]. The harm characteristics of free radical can be well controlled by
antioxidant because it inhibits the oxidation processes on substrates. Therefore, it
is essentially needed to develop a drug that overcomes free radical problems from
organisms which have antioxidant compound. As one of the richest countries
at biodiversity, Indonesia has medical herbs and microbes that can be utilized as
drugs resources [22]. It potentially could be explored for bioactive compound
discovery that serves as antioxidant.
One of the microbe that is promising to be developed as a new source for
bioactive antioxidant compound is endophytic microbes. These microbes live
symbiotically in plant tissue at certain period and survive by forming a colony
on that particular place. Every medicinal plant could possibly have some differ-
* Corresponding author. Phone: +6285711749663. Email: panji.cahya@gmail.com
19
ent kind of endophytics that are capable at producing bioactive compound or

secondary metabolites which are hypothesized as a result of coevolution or genetic
transfer from its host plant in to endophytic microbes [17].
The ability of endophytics for producing the same secondary metabolites
with its host plant is a great and reliable. It could be used to produce the same
secondary metabolites that are isolated from its host plant. Then, if the endophytic
microbes from medical herbs are able to produce flavones or the same secondary
metabolites with its host, we do not have to chop the plant down for obtaining
its symplisia which possibly needs a dozen of years to be harvested. Some of the
endophytics have been successfully isolated and cultivated in a proper media,
likewise, its secondary metabolites which have been successfully isolated, purified,
and structurally elucidated [13]. More than 300,000 plants species spreading
around the globe are associated with one or more endophytic microbes [16].
The endophytic microbes usually live in medicinal plants. Typhonium flagelli-
forme is one of medicinal plant which has endophytic microbes. The plant has been
known to contain Ribosome Inacting Protein (RIP) and antioxidant compound.
According to previous study, one of endophytic fungi that has been isolated from
Typhonium flagelliforme is Mycoleptodiscus indicus. It potentially is able to produce
various active secondary metabolites as antioxidant. This endophytic fungus is
remarkably believed to produce the same secondary metabolites with Typhonium
flagelliforme. Mycoleptodicin A and Mycoleptodicin B have been reportedly pro-
duced by Mycoleptodiscus indicus that lives in Desmotesin comparabilis [7]. Whereas,
Mycoleptone A,B,C, austdiol, eugnitin, 6-metoksieugenin, 9-hydroxyieugenin
have been reportedly produced by Mycoleptodiscus indicus who lives in Borreria
verticilata [19].
There are also a significant major compound of flavonoids in other endophytic
fungi. The fungal endophytics which associated with a medicinal plant, Nerium
oleander L. (Apocynaceae) showed flavonoids, phenolic acids and aliphatic com-
pounds [23]. The objective of this research was to isolate the chemical compound
and to evaluate its antioxidant activity from Mycoleptodiscus indicus who lives in
rodent tuber. The antioxidant activity from the isolated compound was determined
by 1,1-diphenyl-2-picrilhydrazil (DPPH-radical) method.
II. MATERIAL AND METHOD

A. Chemicals and Materials
Mycoleptodiscus indicus isolate, PDA, PDB, eluent (100% Hexane, ethyl acetate
100%, 100% methanol), MeOH for DPPH Analysis p.a grade, DPPH for sigma.
B. Instruments
Silica gel with a column length of 35 cm; diameter of 4.5 cm and a length of 55
cm; diameter of 2.5 cm; Liquid-Mass Spectrometry chromatography Mariner
Biospectrometry: UVJVis. Simadzu Detector (Perkin Elmer Series 200), LC MS
Mariner, Phenomenex column 2 x 150 mm containing C18; NMR spectrometer
Jeol Inova Unity Plus (500 MHz and 250 MHz), Incubator.
C. Method
1) Fermentation and Extraction
The endophytic fungus M.indicus was cultivated on Potato Dextrose Agar
plates at 25 °C for 7–14 days. Three pieces (0.5 X 0.5 cm) of mycelia agar
plugs were inoculated into 10L PDB and incubated at room temperature
under agitation 150 rpm for 20 days. After this, the media of the fungus
was separated from mycelia by vacuum filtration. The media was extracted
with ethyl acetate and evaporated using rotary vaccum evaporator at 40°C
to obtain the crude extract.
2) Isolation and Identification
Isolation of the crude extract used modification of standard purification
methods for natural products. Initial fractionation by dry flask chromatog-
raphy techniques and distribution components differences in of a mixture
between the two phases. Stationary phase silica gel 60 (E. Merck 7734) and
the mobile phase n-hexane; n-hexane: ethyl acetate (4:1, 3:1, 2:1, 1:1); ethyl
acetate; ethyl acetate: methanol (4:1, 3:1, 2:1, 1:1); and methanol, using
a variety of comparison with a range of 0–100%. The first fractionation
produced 140 fractions. The analysis process of chromatogram for each frac-
tion used silica gel aluminum plates GF254. Spots were observed under UV
light with a wavelength of 254 nm and 366 nm. The fractions that showed
similar chromatogram then combined into 26 fractions (A-Z). The fraction
R (75–79) showed a specific spot and a large number of isolates, so it’s carried
for the purification process using preparative TLC techniques. The weight of
fraction R was 125.7 mg.
Isolation of the fraction R (75–79) was performed by preparative TLC
techniques with ethyl acetate mobile phase. TLC results were observed
and marked in UV light with a wavelength of 366 nm. The marked part
was extracted, filtered, and evaporated to obtain 20.3 mg pure fractions.
Spectroscopic Analysis was conducted by LC-MS, ‘H-NMR and 13C-NMR
spectrometer, using NMR (500 MHz and 250 MHz, CD30D) with CD3OD
solvent.
3) Inhibition Assay for DPPH Radical Scavenging Activity
Heading the DPPH radical scavenging activity was performed using estab-
lished procedure [24]. The 1.5 mg DPPH was dissolved in 50 mL methanol.
The mixture was vigorously shaken and incubated in the dark for 30 minutes
at room temperature. The absorbance was measured at 517 nm. Ascorbic acid
was taken as a positive control, DPPH was taken as negative control, and
methanol was taken as blanko. The scavenging ability was calculated as: DPPH
radical scavenging activity (% inhibition) = [1-(A1-A2)/A0] x 100, where A0 is
the absorbance in the lack of the test compound, A1 is the absorbance in the
presence of the test compound and DPPH, and A2 is the absorbance in the
lack of DPPH. The IC50 values (the concentration of the antioxidant required
to scavenge 50% of DPPH present in the test solution) were obtained from
linear regression analysis of the concentration-response curves plotted for
tested compound.
III. R esult and Discussion

The ability of endophytic microbes at producing the same secondary metabolites
as its host plant is a fundamental reason why we did the research. It was a great
and reliable opportunity to produce secondary metabolites from Typhonium flagel-
liforme without chopping the plant down since the endophytic fungus that live on
that plant could possibly produce the same secondary metabolites. The isolated
compound that has been successfully purified and identified is (-)-epicathecin.
The ethyl acetate extract of Mycoleptodiscus indicus fermentation was fractioned
by column chromatography and was purified by preparative TLC. The used
isolation method was the modification method of standard purification for natural
products and chromatogram analysis [10, 14, and 15]. This method has been
efficiently proved at clustering compound group that has similar chromatogram
pattern from each fraction. The fractionation yielded 140 fractions which were
combined in to 26 fractions based on similarity of chromatogram pattern.
The purified compound that has been isolated from 26 fractions was from
Fraction R1 (75–79). The mass of the purified fraction, a brown powder, was 20.3
mg and has molecular formula C15H14O6 from LC-MS spectrum with [M+] ion
at 291.11. The rendement of this compound was 1.01%. It was then analysed by
NMR Spectrometer. The structure of the purified fraction was elucidated on the
basis of its 1H NMR and 13C NMR analyses (Table 1) and closely similar to those
of (-)-epicatechin. Based on characteristic signals of δ H 4.82 (1H, br s, H-2), δ
H 4.17 (1H, m, H-3), δ H 2.86 (1H, dd, J = 4.8, 16.8 Hz, Ha-4) and δ H 2.73
(1H, dd, J = 2.8, 16.8 Hz, Hb-4), it showed C-ring protons of flavan-3-ol. The
data of δ H 5.93 (1H, d, J = 2.4 Hz, H-6) and δ H 5.91 (1H, d, J = 2.4 Hz,
H-8) showed the meta-coupled aromatic proton signals which also revealed the
presence of a flavan-3-ol skeleton. The ABX-type aromatic signals at δ H 6.97
Figure 1. (-)-epicatechin molecular structure
Table 1 1H and 13C NMR spectral data of fraction R1

Position δH δC
2 4.82 (1H, br s) 79.97
3 4.17 (1H, m) 67.58
4 2.86 (1H, dd, J = 4.8, 16.8 Hz) 29.37
2.73 (1H, dd, J = 2.8, 16.8 Hz)
5 157.77
6 5.93 (1H, d, J = 2.4 Hz) 96.44
7 158.11
8 5.91 (1H, d, J = 2.4 Hz) 95.95
9 157.46
10 100.14
11 132.38
12 6.97 (1H, d, J = 1.6 Hz) 115.40
13 6.75 (1H, d, J = 8.0 Hz) 146.03
14 145.87
15 115.96
16 6.79 (1H, dd, J = 2.0, 8.4 Hz) 119.47
1
H and 13C NMR spectra were recorded in methanol-d4 at 400 MHz and 100 MHz, respectively. Chemi-
cal shifts (δ) were given in ppm versus TMS.
(1H, d, J = 1.6 Hz, H-12), δ H 6.75 (1H, d, J = 8.0 Hz, H-13) and δ H 6.79
(1H, dd, J = 2.0, 8.4 Hz, H-16) corresponding to a B-ring were recognized.
The basic structure was therefore deduced as (-)-epicatechin. Furthermore, the
stereochemistry of (-)-epicatechin was suggested by the proton signal of H-2,
which appeared as a broad singlet at δ H 4.82 [3,12].
The data of 13C-NMR showed 15 signals for 15 carbons (Table 1). Those
signals were δC 79.97 (C-2), 67.58 (C-3), 29.37 (C-4), 157.77 (C-5), 96.44
(C-6), 158.11 (C-7), 95.95 (C-8), 157.46 (C-9), 100.14 (C-10), 132.38 (C-11),
115.40 (C-12), 146.03 (C-13), 145.87 (C-14), 115.96 (C-15) and 119.47 (C-16).
The 1H NMR and 13C NMR data of this fraction were similar with those reported
in the literatures for (-)-epicatechin [4,5,9]. Epicatechin has various biological
activities as follows antimicrobial [2], antipasmodic, broncodilator, vasodilator,
and effectively is used for gingitivis patient. This compound is also popular for
cosmetic and has been tested as antiaging, antiacne, and help to maintain the
weight loss [6].
The antioxidant activity of this compound was evaluated according to the
method previously described. The DPPH is free radical compound that gives
purple colour solution in methanol. Its purple colour is reduced to yellowish
solution in methanol by presence of antioxidant compound. The principle of
this method is the ability of a molecule at donating a hydrogen atom to a radical.
The critical factor in free radical scavenging is the propensity of the hydrogen
donation [1]. The DPPH radical scavenging activities of the purified compound
are presented in Figure 2. The compound was made in to 5 final concentrations
as follows: 3.125 ppm, 1.563 ppm, 0.781 ppm, 0.391 ppm, and 0.195 ppm.
Each concentration exhibits different percentage of inhibition.This compound
exhibited significant inhibition activity with IC50 0.6 µg/mL.
Figure 2. Antioxidant activity of (-)-epictechin

IV. Conclusion
Based on 1H NMR and 13C NMR spectra and compared to previous spectral data
that has been reported, the compound of Fraction R1 from Mycoleptodiscus indicus
fermentation extract was identified as (-)-epicatechin. This compound showed
significant activity in the DPPH radical scavenging with IC500.6 µg/mL. To our
knowledge, this is the first report on antioxidative activity of (-)-epicatechin from
Mycoleptodiscus indicus. It was indicated that this endophytic fungus could be
considered as potential source for antioxidant bioactive compound.
V. Acknowledgement
This research has been funded by the Indonesian Government Budget for
Indonesian Institute of Sciences (LIPI). We would also like to thank Dr. Linar
Zalinarudin dan Dra. Puspa Dewi N. Lotulung, M.Eng.
IV. R eferences
1) Bondet, V., Williams, W. B. and C. Berset. (1997). Kinetics and mechanisms
of antioxidant activity using the DPPH free radical method. Lebensmittel-
Wissenschaft und-Technologie, 30, 609–615.
2) Dogra, S, C. (1987). Antimikrobial Agents Used in Ancient India. Indian
Journal of History of Science, 22, 164–169.
3) Fan, P. H., Lou, H. X., Yu, W. T., Ren, D. M., Ma, B. and M. Ji. (2004).
Novel flavanol derivatives from grape seeds. Tetrahedron Letters, 45,
3163–3166.
4) Foo, L. Y., Lu, Y., Howell, A. B. and N. Vorsa. (2000). The structure of
cranberry proanthocyanidins which inhibit adherence of uropathogenic
P-fimbriated Escherichia coli in vitro. Phytochemistry, 54, 173–181.
5) Foo, L. Y., Lu, Y., Mcnabb, W. C., Waghorn, G. and M. J. Ulyatt. (1997).
Proanthocyanidins from Lotus pedunculatus. Phytochemistry, 43, 1689–1696.
6) Ghayur, M, N., Khan, H. and A. H. Gilani. (2007). Antispasmodic,
Bronchodilator and Vasodilator Activities of (+)-Catechin, a Naturally
Occurring Flavonoid. Archives of Pharmacal Research, 30 (8), 970–975.
7) Ortega, H. E., Graupner, P. R., Asai, Y., Dyke, K. T., Qiu, D., Shen, Y. Y.,
Rios, N., Arnold, A. E., Coley, P. D., Kursar, T. A., Gerwick, W. H. and
L. Cubilla-Rios. (2013). Mycoleptodiscins A and B, Cytotoxic Alkaloids
from the Endophytic Fungus Mycoleptodiscus sp F0194. Journal of Natural
Products, 76, 741–744.
8) Mao, S. C., Gu, Q. Q., Cui, C. B., Han, B., Cai, B. and H. B. Liu. (2004).
Phenolic compounds from Sargentodoxa cuneata (Olive) Rehd. Et Wils
and their antitumor activities. Chinese Journal of Medicinal Chemistry, 14,
326–330.
9) Ohuchi, K. and L. Levine. (1980). Alpha-Tocopherol inhibits 12-0-tet-
radecanoyl-phorbol-13-acetate-stimulated deacylation of cellular lipids,
prostaglandin production, and changes in cell morphology of Madin-Darby
canine kidney cells. Biochimica et Biophysica Acta, 619 (1), 9–11.
10) Pham-Huy, L., He, H. and C. Pham-huy. (2008). Free radicals antioxidants
in disease and health. International Journal of Biomedical Science, 4, 89–96.
11) Pizzolatti, M. G., Venson, A. F., Smania, A. J., Smania, E. F. and R. Braz-
Filho. (2002). Two Epimeric Flavalignans from Trichiliacatigua (Meliaceae)
with Antimicrobial Activity. Zeitschriftfurr Naturforschung, 57, 483–488.
12) Maksum, R. (2005). Peranan Bioteknologi dan Mikroba Endofit dalam
Pengembangan Obat Herbal. Majalah Ilmu Kefarmasian, 2 (3), 113–126.
13) Sewell, P. A. and B. Clarke. (1980). Chromatographic Separations, Analytical
Chemistry by Open stimulated deacylation of cellular lipids, prostaglandin
production, and changes in cell morphology of Madin-Darby canine kidney
cells. Biochimica et Biophysica Acta, 619 (1), 9–11.
14) Stahl, E. (1985). Analisis Obat Secara Kromatografi dan Mikroskopi (K.
Padmawinata and I. Soediro, Trans.) (pp. 1-10). Bandung, Indonesia: ITB.
15) Strobel, G. A. and B. Daisy. (2003). Bioprospecting for Microbial Endo-
phytics and Their Natural Products. Microbiology and Molecular Biology
Reviews, 67 (4), 491–502.
16) Tan, R. X. and W. X. Zou. (2001). Endophytes: a rich source of functional
metabolites. Natural Product Reports, 18, 448–459.
17) Valko, M., Leibfritiz, D., Mancola, J., Cronin, M. T. and M. Mazur. (2007).
Free radicals and antioxidant in normal physiological functions and human
disease. International Journal of Biochemistry and Cell Biology, 39, 44–84.
18) Andrioli, W. J., Conti, R., Araújo, M. J., Zanasi, R., Cavalcanti, B. C.,
Manfrim, V., Toledo, J. S., Tedesco, D., de Moraes, M. O., Pessoa, C., Cruz,
A. K., Bertucci, C., Sabino, J., Nanayakkara, D. N. P., Pupo, M. T. and J.
K. Bastos. (2014). Mycoleptones A−C and Polyketides from the Endophytic
Mycoleptodiscus indicus”. Journal of Natural Products, 77, 70–78.
19) Young, I. S. and J. V. Woodside. (2001). Antioxidant in health and disease.
Journal of Clinical Pathology, 35, 176–186.
20) Zuhud, E. A. M. (2009). Potensi Hutan Tropika Indonesia sebagai Penyangga

Bahan Obat Alam untuk Kesehatan Bangsa. Jurnal Bahan Alam Indonesia,
6 (6), 27–23.
21) Huang, W-Y., Cai, Y-Z., Hyde, K. D., Corke, H. and M. Sun. (2007).
Endophytic fungi from Nerium oleander L (Apocynaceae): main constituents
and antioxidant activity. World Journal of Microbiology and Biotechnology,
23, 1.253–1.263.
22) Sekhon-Loodu, S., Warnakulasuriya, S. N., Rupasinghe, H. P. and F. Shahidi.
(2013). Antioxidant ability of fractionated pple peel phenolics to inhibit
fish oil oxidation. Food Chemistry, 140, 189–186.
Potency of Hibiscus (Hibiscus rosa-sinensis
L.) as Antioxidant to Reduce Carbon
Tetrachloride Compounds in Red Blood
Cells
Dody Priadi and Kusmiati*
Research Centre for Biotehnology-LIPI
Jl. Raya Bogor Km.46, Cibinong 16911, Indonesia
Abstract
Hibiscus (Hibiscus rosa-sinensis L.) is a species of flowering plant that is easily found and used for
traditional medicine. Hibiscus flower decoction is used to treat various diseases, such as urinary
tract infections, coughing, whooping cough, dysentery, bronchitis, tuberculosis and various
degenerative diseases. These properties are related to the content of compounds as antioxidants
such as anomuricin and polyphenols. Antioxidant compounds may reduce the risk due to free
radical oxidation process to prevent degenerative diseases. Hibiscus flowers were extracted by
maceration method with ethanol and ethyl acetate solvents. The extract containing antioxidant
of 10, 20, 30, and 40 ppm was tested against sheep red blood cell that induced with CCl4 as
oxidant. Parameters measured were malondialdehyde (MDA) content and activity of superoxide
dismutase (SOD) and catalase enzymes. Data was analyzed using Kruskal-Wallis test. Results
showed that the highest reduction in MDA content (0.7679 nmol/ml) was obtained by the
treatment of ethyl acetate extract at the concentration of 10 ppm. The highest activities of the
SOD and catalase enzymes were 0.8874 U/ml and 460.655 U/ml respectively, obtained by the
treatment of 40 ppm of the extract containing antioxidants.
Key words: Hibiscus (Hibiscus rosa-sinensis L.), Antioxidants, MDA, SOD, Catalase
I. Introduction
An excessive oxidation reactions in the body can trigger the formation of a
highly active free radicals which damage the structure and function of cells, and
unconsciously, the body is constantly form free radicals, either through normal
cellular metabolism, inflammation, malnutrition, and due to the response against
influences from outside the body, such as environmental pollution, ultraviolet
(UV) radiation, cigarette smoke and others [1].
Antioxidants are substances that inhibit oxidative stress in the molecule.
Antioxidants can be categorized into enzymatic antioxidants (enzymes) such as
superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase.
Meanwhile, non-enzymatic antioxidant (extra-cellular) includes vitamin E,
* Corresponding author. Phone: +62-21-8754587. Email: kusmiati02@yahoo.com
29
vitamin C, β-carotene, glutathione, ceruloplasmin, albumin, uric acid and

selenium [2].
Hibiscus (Hibiscus rosa-sinensis L.) is one of the ornamental plants that are
widely used as traditional medicine in Indonesia in the form of fresh flowers or a
stew. Herb or decoction of hibiscus flowers is proven to be able to cope with various
diseases, such as gonorrhea, bronchitis and tuberculosis due to chemical efficacy
of antioxidants containing polyphenols such as flavonoids anomuricin reducing
the risk oxidation process by free radicals causing several of degenerative diseases
[3]. Hibiscus rosa-sinensis flower extract exhibited antioxidant, antihyperglycemic
and antihyperlipidemic activities mainly at the dose of 250 mg/kg bwt without
showing any toxic effects on experimental rats [4]. Leaves of this plant species
inhibited the process of spermatogenesis of ddy strain mice. Therefore, it can be
developed into a male contraception [5].
The objective of the study was to identify antioxidant activity of Hibiscus
flower extracts in vitro in sheep red blood cells induced by carbon tetrachloride,
determining the optimal solvent concentration to reduce MDA levels and to
increase the activity of SOD and catalase, and determining the optimal concentra-
tion of hibiscus extract containing antioxidants.
II. Method/M aterial

Preparation hibiscus flower extract
Hibiscus (Hibiscus rosa-sinensis L.) was obtained from Research Institute for
Medicinal and Aromatic Plants, Bogor. Petals of the approximately 2 days-
blooming flowers were cleaned and dehydrated in room temperature for 3 days
and were then powdered using a blender. The powder was stored in a clean and
sealed container. A total of 20 grams of powdered hibiscus flower were macerated
with ethanol andethyl acetate (200 ml) separately, and soaked for 24 hours at
room temperature while stirring using an orbital shaker. The filtrate was separated
using a filter paper.
Figure 1. Hibiscus flower

Phytochemical screening of the powdered flowers of hibiscus was conducted

at the Laboratory of Bioprocess 3 Research Centre for Biotechnology-LIPI
Cibinong. Sheep blood was obtained from the Microbiology Division of Faculty
of Medicine, University of Indonesia. The red blood of sheep was separated into
blood plasma and cells by centrifugation at 3.000 rpm at 5oC for 5 minutes.
Blood plasma was used for analysis of MDA (malondialdehyde) content. MDA
content was analyzed according to [6], a volume of each 0.5 ml ethanol and ethyl
acetate extract of hibiscus flower (10, 20, 30 and 40 ppm) were added into a half
milliliter of plasma and then incubated at room temperature for 15 minutes. The
solution was induced with carbon tetrachloride (CCl4) solution and incubated
at room temperature for 15 minutes, centrifuged at 3000 rpm for 5 minutes. A
number of the supernatant was treated with trichloroacetic acid (TCA) 20% and
thiobarbituric acid (TBA) 0.67%, and homogenized. The mixture was heated for
30 minutes in a waterbath at 100 °C and then cooled. Absorption of the solution
was measured at λ 532 nm. Tetra ethoxy propane (TEP) was used as a standard.
The blood cell was washed 3 times with phosphate buffered saline solution
(PBS) and centrifuged at 3.000 rpm at 5 °C for 5 minutes. The red blood cells
(RBC) were used for the study of superoxide dismutase (SOD) and catalase
enzymes activity. The extract of hibiscus flower was added into red blood cells
and incubated at room temperature for 15 minutes. Oxidant of CCl4 was then
added prior to incubation at room temperature for 15 minutes. The mixture was
diluted with distilled water and centrifuged for 5 minutes at 3.000 rpm. Details
of the treatment are presented in Table 1.
Activity of SOD enzyme was analyzed according to [7]; one volume of RBC
supernatant was diluted with distilled water and extracted with one volume
of chloroform-ethanol (3:5), was shaken and centrifuged at 2.500 rpm for 10
minutes. A volume at 50 µl the aqueous phase was added into carbonate buffer
pH 10.2 and 50 µl, of 0.02 M epinephrine solution. Absorption was measured
at λ 480 nm at 30 °C.
Activity of catalase enzyme was analyzed according to [8]; a volume of 100 µl
RBC supernatant was added into H2O2 0.059 M and 1.9 ml 0.05 M phosphate
buffer pH 7. Absorption of enzymatic reaction was measured at λ 240 nm.
Data were analyzed using a non-parametric method of Kruskal-Wallis and
Mann-Whitney for those not normal and not homogeneous. The data were
processed with SPSS 16 statistical software.
Outline of the method above is described in Figure 2.
Table 1. Group of treatments

Group Treatments
I Normal Control (RBC)
II Negative Control (RBC + CCl4)
III Positive Control (RBC + CCl4 + Vit C 20 ppm)
IV RBC + CCl4 + Ethanol extract of Hibiscus flower (10 ppm)
V RBC + CCl4 + Ethanol extract of Hibiscus flower (20 ppm)
VI RBC + CCl4 + Ethanol extract of Hibiscus flower (30 ppm)
VII RBC + CCl4 + Ethanol extract of Hibiscus flower (40 ppm)
VIII RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (10 ppm)
IX RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (20 ppm)
X RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (30 ppm)
XI RBC + CCl4 + Ethyl acetate extract of Hibiscus flower (40 ppm)
Figure 2. Outline of the method of study of hibiscus flower as antioxidant


A. Phytochemical Screening Test
Results of phytochemical screening showed that hibiscus flower powder extracted
with ethanol solvent contains alkaloids, flavonoids, saponins, tannins and trit-
erpenoids, whereas those extracted with ethylacetate solvent contains alkaloids,
flavonoids, and tannins. Flavonoid was obtained in considerable number from
hibiscus flower. Flavonoids test using magnesium and hydrochloric acid gives a
positive result indicated with formation of orange color in amil alcohol solution
(Table 2).
Table 2. Result of fitochemical test of hibiscus flower
No. Compound Powder Ethanol extract Ethyl acetate extract
1 Alkaloid + + +
2 Flavonoid + + +
3 Saponin + + -
4 Tannin + + +
5 Triterpenoid + + -
6 Steroid - - -
Note: + = available; - = not available
B. MDA Content
Free radicals were detected by the presence of lipid peroxidation, which
will produce secondary products, such as malondialdehyde (MDA).
Levels of MDA were detected by UV-VIS spectrophotometer at λ max 532 nm.
MDA content in group II (negative control) was 1.2591 nmol/ml that
increased by 23.33% of the normal control. This suggests that under normal
conditions, the content of lipid peroxidation in the body is low. Increasing of
free radicals in the blood caused the lipid peroxidation process which increase
formation of MDA. This was associated with oxidation increase of unsaturated
fats as a result of the addition of carbon tetrachloride. Group III, IV, V, VI, VII,
VIII, IX, X and XI showed a decrease in MDA levels of 50.38%, 25.75%, 11.52%,
7.60%, 4.11%, 39.01%, 20.05%, 14.35%, and 6.63% respectively compared
to the negative control. The treatments of hibiscus flower extract and vitamin C
(positive control) showed that it potentially inhibits free radicals. It seems that
the ethanol extract of hibiscus flower was more effective as antioxidants (Figure
3). Result of statistical analysis showed that there was a significantly different
result (p<0.05) between the groups of analysis.
Figure 3. MDA content in red blood

cells of sheep induced by carbon
tetrachloride.
C. SOD Enzyme Activity

Table 3 showed that SOD enzyme activity was increased with the addition of
vitamin C (Group III) as well as group VIII and XI compared with group I and
II. SOD enzyme activity in group III, IV, V, VI, VII, VIII, IX, X, XI i.e. 66.75%,
17.67%, 41.95%, 51.51%, 60.37%, 6.18%, 26.04%, 45.75% and 53.95%
respectively from the negative control (Figure 4).
Ethanol is the best solvent for hibiscus flower extraction in enhancing activity
of SOD compared with ethylacetate. The low concentration of SOD in the blood
leading to high reactive oxygen (O2*-) plays an important role in inflammation.
Low concentration of SOD caused the release of prostaglandins as an important
factor of inflammation. There is a possibility of the ethanol extract of hibiscus
flowers acted as anti-inflammatory, because it can prevent the decrease in SOD
enzyme activity in the blood [9].
Study conducted by [10] showed that the activity of SOD and the concentra-
tion of MDA were highest in the group of patients with the lowest success of the
hypo-osmotic swelling (HOS) test. The assessment of the antioxidant enzymes
and MDA in addition to the semen analysis and the HOS test may be greatly
useful in diagnosing infertility in men having oxidative stress in their etiology.
Figure 4. SOD enzyme activity in

red blood cells of sheep induced by
carbon tetrachloride.
D. Catalase Enzyme Activity

Figure 2 showed that catalase enzyme activity in group II was lower than group I
due to the red blood cells in group II already induced by carbon tetrachloride. In
group II, the catalase enzyme in red blood cells has been used in part to neutralize
free radicals of carbon tetrachloride. This caused reduction of catalase enzyme
activity in red blood cells with the result that the ability to neutralize peroxidation
of hydrogen peroxide was decreased.
The addition of carbon tetrachloride and vitamin C in group III as well as
group IV, V, VI, VII, X and XI with that addition of hibiscus flower extract caused
a higher activity of catalase enzyme compared to group I and II. An increase in
catalase activity in group III and VII shows the antioxidant effects of the ethanol
extract of the hibiscus flower. Meanwhile, group VIII and IX did not show any
antioxidant activity of ethylacetate. Ethanol extract at a concentration of 40 ppm
was the most effective dose to reduce carbon tetrachloride oxidation for up to
38.06%. Increased activity of catalase enzyme in group III, IV, V, VI, X, and IX
were 86.29%, 7.43%, 23.69%, 4.82% and 6.36% respectively from the negative
control (Figure 5).
Results of statistical analysis showed that there were significantly different
(p<0.05) result between the groups of analysis.
Figure 5. Catalase enzyme activity

in red blood cells of sheep induced
by carbon tetrachloride.
IV. Conclusion
Treatments of ethanol and ethyl acetate extract of Hibicus flower reduced MDA
content in blood plasma as well as increased activity of SOD and catalase enzyme
in red blood cells induced by carbon tetrachloride.
The highest reduction of MDA content (0.7679 nmol/ml) in red blood plasma
was obtained by ethyl acetate extract of Hibiscus flower (10 ppm). The highest
activities of SOD and catalase enzyme were 0.8874 U/ml and 460.655 U/ml
respectively, obtained by ethanol extract of hibiscus flower (40 ppm). Ethanol is

a polar compound so that the secondary metabolites in the hibiscus flower can
be extracted perfectly. Further study has to be done using experimental animal
to obtain more informative result.
V. R eferences
1) Ahmad, S. A., Hakim, E. H. and L. Makmur. (2009). Ilmu Kimia dan
Kegunaan Tumbuh-tumbuhan Obat Indonesia (p. 120). Bandung: ITB.
2) Halliwel, B. and J. M. C. Gutteridge. (1989). Free Radicals in Biology and
Medicine (pp. 196–200) (2nd ed.). New York: Oxford University Press.
3) Mursito, B. (2002). Tanaman Hias Berkhasiat Obat (p. 33). Jakarta: Penebar
Swadaya.
4) Sankaran, M. and A. Vadivel. (2011). Antioxidant and antidiabetic effect of
hibiscus rosasinensis flower extract on streptozotocin induced experimental
rats-a dose response study. Notulae Scientia Biologicae, 3 (4), 13–21.
5) Mawuntu, M. (2008). The extract of “shoe flower” (Hibiscus rosasinensis
L.) leaves inhibit the spermatogenesis of ddy strain mice. Medical Journal of
Indonesia, 17, 157–62.
6) Tüközkan, N., Erdamar, H. and I. Seven. (2006). Measurement of total
malondialdehyde in plasma and tissues by high-performance liquid chroma-
tography and thiobarbituric acid assay. Fırat Tıp Dergisi, 11 (2), 88–92.
7) Achuba, I. Fidelis. (2005). Effect of vitamins C and E intake on blood lipid
concentration, lippid peroxidation, superoxide dismutase and catalase activi-
ties in petroleum-contaminated diet fed rabbit. European Journal of Scientific,
12 (1), 1–8.
8) Aebi, H. (2974). Catalase. In Bergmeyer (Ed,), Methods in Enzymatic Analysis
(pp. 674–684). New York: Academic Press.
9) Alfonso, V. and R. Chumpy. (2007). Reactive oxygen spesies and superoxide
dismutases: Role in joint disease, Joint Bone Spine. Science Direct Journal,
74, 324–329.
10) Zelen, I., Mitrović, M., Jurišić-Škevin, A. and S. Arsenijević. (2010). Activity
of superoxide dismutase and catalase and content of malondialdehyde in
seminal plasma of infertile patients. Medicinski pregled, 63 (9-10), 624–629.
PURIFICATION OF BIOACTIVE PEPTIDE WITH
PROTEASES INHIBITORY ACTIVITIES FROM
STREPTOMYCES MISIONENSIS
Juwaini Mohd Yusoff, Khanom Simarani* and Zazali Alias
Institute of Biological Sciences, Faculty of Science, University of Malaya,
50603 Kuala Lumpur, Malaysia
Phone: +603 79675843
Abstract
Actinomycetes are the major source of the biologically active compounds. Extensive bioactive
compounds are yet to be screened from these known vast producers of antimicrobials for
future benefits. During the screening of over 400 soil actinomycete isolates for the production
of epsilon poly-L-lysine (ε-PL), a potentially bioactive protein was discovered. The molecular
weight of the protein was estimated as 6 kDa by using Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis (SDS-PAGE). Cation Exchange Chromatography revealed that the protein
sample was positively charged and thus subjected to proteases activity. The putative protein
exhibited an enhanced activity towards the well-characterized serine proteases; trypsin and
α-chymotrypsin, while inhibiting the activity of α-amylase, suggesting an interesting finding
particularly in enzyme modulation aspect. Furthermore, it showed antagonistic activity against
some pathogens including Gram negative Xanthomonas campestris, Ralstonia and Erwinia, as well
as Gram positive B. cereus. Subsequently, the isolate was identified as Streptomyces misionensis
based on its 16S rRNA gene sequence.
Key words: Actinomycetes, Peptide, Serine Proteases
I. Introduction
Soil microbes represent an important source of biologically active compounds.
Most of the chemically diverse compounds with biological activities were
discovered from actinomycetes. Actinomycetes have provided nearly 80% of the
world’s antibiotics, prominently from the genera Streptomyces and Micromonospora,
and the search for new bioactive compounds continues until today [1].
Enzymes are biomacromolecules (usually proteins), which have important
roles in regulating most of the chemical reactions involved in various biological
processes of living organisms. Besides in vivo functions of enzymes, they are also
widely used in pharmaceutical, medical, food, environmental and industrial fields
as well as in life science studies. With those diverse commercial applications, the
regulation of enzyme activity and stability has always becomes the main issue that
* Corresponding author. Phone: +603 79675843. Email: hanom_ss@um.edu.my
37
attracts great attention. Many enzyme regulators have been discovered, ranging
from proteins, peptides and synthetic organic molecules, and the studies on that
aspect continue until now [2].
II. M aterials and Methods

A. Isolation, Screening and Identification of Bacterial Strain
Actinomycetes used in this study were isolated from various types of soil samples
including forest, agricultural, rhizospheric and polluted soils. The isolation proce-
dures were followed as previously described by Hirohara [3]. Purified colonies were
maintained and subcultured on ISP 2 medium prior to use. Actinomycete strains
selected by appearances on the agar plate were subjected to two-stage cultivation
as described by Hirohara [3] for the production of the bioactive metabolites. The
potential isolate was identified based on its 16S rRNA gene sequence.
B. Protein Separation and Concentration

The production broth of actinomycete cultures was filtered using membrane filter
to remove the cells and was subjected to acetone precipitation and centrifugation
using protein concentrator prior to SDS-PAGE, enzymatic assays and antibacterial
testing. The protein concentration was determined by method of Bradford [4]
using bovine serum albumin (BSA) as a standard.
C. Enzymatic Activity Assay

Serine proteases; trypsin and chymotrypsin activity was performed as described by
Jennings [5] using the substrates sodium-benzoyl-DL-arginine ρ-nitroanilide for
trypsin and, N-benzoyl-L-tyrosine ρ-nitroanilide for chymotrypsin. For α-amylase
inhibitory assay, the method was adapted as outlined by Feng [6].
D. Antimicrobial Activity
The antibacterial activity was determined using agar well diffusion method [1]. B.
cereus, X. campestris, Ralstonia and Erwinia strain obtained from UM Microbiology
Culture Collection were used as antimicrobial test strains.
III. R esults and Discussion

Various types of soil samples, including forest, garden, agricultural, rhizospheric
and polluted soils were collected from different places as to increase the chances
of getting different actinomycete strains.
Pure homogenous preparation of peptide using Tricine SDS-PAGE indicated a
single band of low molecular weight (MW) protein approximately 6 kDa (Figure
1) after protein separation by acetone precipitation method. As the samples was

from crude part, that means they were of homogenous proteins, allowing for the
thick clearly visualized band on SDS-PAGE. This has become the interesting part
of the study; checking whether the protein possesses biological activities. Thus,
we attempted to measure the activity of the isolated peptide towards the selected
enzymes particularly serine proteases. Two well-characterized serine proteases;
trypsin and chymotrypsin which have important biomedical and industrial ap-
plications were opted for the next stage of the study along with α-amylase enzyme.
Trypsin and chymotrypsin activity assays revealed an interesting finding that
the locally isolated peptide has the capability to significantly enhance the activity
of both of the serine proteases (Table 1). To the best of our knowledge, the
improvement of the proteases activity was rarely documented, in fact this is the
first report that described the ability of the potential peptide from an actinomycete
which could enhance the activity of proteases. Similarly, the study done by Jin
[2] reported the specific enhancement in substrate-dependent trypsin digestion
of phosphoproteins by PEGlated GO nanosheets.
Other than having an effect on the protease activities, the isolated peptide
was shown to exhibit antibacterial property against several selected bacteria when
qualitatively determined by the well diffusion method (Table 2). A clear zone of
inhibition was observed towards some Gram negative plant pathogens including
X. campestris, Ralstonia and Erwinia as well as Gram positive B. cereus. To correlate
this data with the obtained MW of the locally isolated protein, we believed that
it might be the antimicrobial peptide (AMP) as its MW falls within the range
for the AMP. Subsequently, the potential isolate was identified as Streptomyces
misionesis by 16S rRNA gene sequencing.
For our best knowledge, this is the first study that reported the isolation of
AMP with biological activities from Streptomyces misionesis, proposing that this
research could be a basis for constructing new natural antimicrobial compounds
with higher specific activity and broader range of action to serve as a solution
for the never-ending antimicrobial resistance issues as well as to reduce the side
effect problems of using synthetic antibiotics.
Figure 1. SDS-PAGE of pure homogenous

preparation of peptide. Lane 1 shows the
benchmark standard marker (Invitrogen).
Lane 2 shows the presence of single band
protein with MW estimated around 6 kDa.
Gel was stained with colloidal Coomassie blue.
Table 1. Enzymatic activity assays

Assay % Activity Activity
α-amylase 78.87 Inhibited
Trypsin 67.18 Enhanced
Chymotrypsin 329.32 Enhanced
Table 2. Antibacterial property of

peptide III. Conclusion
Pathogen Inhibition A locally isolated actinomycete strain,
B. cereus positive identified as Streptomyces misionensis, was
X.campestris positive found to produce a peptide with a molecular
Ralstonia positive weight of 6 kDa which is within a range
Erwinia positive for MW of AMP. The peptide was capable
of enhancing the activity of serine protease
enzymes; trypsin and chymotrypsin, while
inhibiting the activity of α-amylase and exhibited antagonistic action against X.
campestris, Ralstonia, Erwinia and B. cereus. Despite the preliminary data obtained,
this research serves as a fundamental for future works on elucidating enzymes
regulation and stability. Further research on proteases activity using selection of
more substrates can be done to study the effects of substrates on the activity in
details. Besides that, enzyme kinetics of the bioassays should be performed to
particularly elucidate the mode of action as well as the concentration at which
the peptide activity is optimum. To understand the chemical structure of the
newly-isolated peptide, it is best to obtain the amino acid sequence of it.
IV. Acknowledgement
Authors would like to thank University of Malaya for providing the following
research funds: PG057-2013A, MOHE FP020-2013A, and RG048-11BIO.
V. R eferences
1) Pandey, A., Ali, I., Butola, K. S., Chatterji, T. and V. Singh. (2011). Isolation
and characterization of actinomycetes from soil and evaluation of antibacterial
activities of actinomycetes against pathogen. International Journal of Applied
Biology and Pharmaceutical Technology, 2 (4), 384–391.
2) Jin, L., Yang, K., Zhang, S., Tao, H., Lee, S.-T., Liu, Z. and R. Peng. (2012).
Functionalized Graphene Oxide in Enzyme Engineering: A Selective Modula-
tor for Enzyme Activity And Thermostability. ACS Nano, 6, 4864–4875.
3) Hihohara, H., Takehara, M., Saimura, M., Masayuki, A. and M. Miyamoto.
(2006). Biosynthesis of poly (ε-L-lysine)s in two newly isolated strains of
Streptomyces sp. Applied Microbiology and Biotechnology, 73, 321–331.
4) Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye binding.
Analytical Biochemistry, 72, 248–254.
5) Jennings, C., West, J., Walne, C., Cralk, D. and M. Anderson. (2001).
Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted
from Oldenlandia affinis. PNAS, 98, 10614–10619.
6) Feng, G. H., Chen, M. S., Kramer, K. J. and G. R. Reeck. (1991).
Reversed-Phase High Performance Liquid Chromatographic Separation
Wheat Proteinaceous Inhibitors of Insect and Mammalian α-amylase. Cereal
Chemistry, 68, 95–99.
7) Young, G. O. (1964). Synthetic structure. In J. Peters (Ed.), Plastics, 2nd ed.,
vol. 3 (pp. 15–64). New York: McGraw-Hill.
ISOLATION, IDENTIFICATION AND SCREENING OF
LOCALLY ISOLATED Xanthomonas sp.
Nur Izlin Shafinaz Bokhari, Khanom Simarani** and Mohamad Suffian
Mohamad Annuar
Institute of Biological Sciences, University of Malaya
50603, Kuala Lumpur, Malaysia
Phone: +603 79674371
Abstract
Xanthan gum is a water soluble, complex exopolysaccharide which have great commercial values
produced industrially by pytopathogen called Xanthomonas sp. Commercially, because of its
rheological properties, it is widely used as a thickener, viscosifier and stabilizer, as well as emulsifier
in both food and non-food industries including dairy products, bakeries product, beverages,
cosmetics, textiles, paper miling and pharmaceutical product. Samples were isolated from diseased
vegetables, and were subjected to set of simple phenotypic. The strains were further identified
by BIOLOG. All positive isolates were compared by fermention in shake flask, under controlled
conditions, and the production of xanthan gum were compared to Xanthomonas campestris pv.
Campestris strain from ATCC culture collection (ATCC33913). 411 bacteria were isolated, and
55 isolates were presumptively identified as Xanthomonas spp. based on the biochemical test.
Thirteen of the presumptive isolates were identified as Xanthomonas campestris pv. Campestris,
Xanthomonas campestris pv. Raphani, Xanthomonas campestris pv. Begonia A, Rhizobium vitis,
Acinetobacter johnsonii, and Microbacterium maritypicum using BIOLOG. Strain C206 has
productivity (0.1074) compare to others as well as control strain, ATCC.
Key words: Xanthan gum, Xanthomonas campestris, Optimization
I. INTRODUCTION
Xanthan gum is a complex extracellular polysaccharide which have great
commercial significance [6] secreted by Xanthomonas campestris [1] during its
normal life cycle. These plant pathogens caused black rot in family members of
the Brassicaceae (Cruciferae) which include cabbage, broccoli and cauliflower.
The subsequent infection of these bacteria will cause defoliation, resulting in
loss of weight and deterioration of its quality [5]. This disease can be accounted
as the major disease constraint in the tomato production all over the world and
in the cabbages plantation in Africa [5]. X. campestris pv. Campestris is the main
producer of xanthan gum, where it thrives well in warm and humid climates [9].
* Corresponding aAuthor. PhoneTel: : +603 79675843. Email: hanom_ss@um.edu.my
43
The growth of Xanthomonas sp. and the production of the xanthan gum are
influenced by various types of factor that need to be controlled during the process
of xanthan gum synthesis. The factors include the types of bioreactor, medium
composition, type of strain used and also other factors, such as temperature,
pH and dissolved oxygenconcentration [4,8,3]. The synthesis of xanthan gum
involved an aerobic, submerged fermentation process where the bacteria that have
been purified will be cultured in a well-areated medium containing inexpensive
substrates and nutrient including glucose, nitrogen and other trace elements.
Glucose and sucrose are most frequently used as the carbon sources [8]. Other
types of substrates also might be used such as sucrose, barley, hydrolysed rice,
sugar cane molasses and coconut juice [3].
Due to its superior properties, xanthan gum is applied in different area of
industries. In pharmaceutical, cosmetic, paper, paint textile and oil industries,
xanthan gum is mainly used as gelling and suspending agent, as flocculants or
used for viscosity control [1]. For example, the application of xanthan gum in
toothpaste allows easy extrusion from the toothpaste tube. In food industry, it
was widely used as a thickening and suspending agent in dairy product, bakery
products, beverages and also in pet foods. On the other hand, xanthan gum
functions as a stabilizer and thickener in salad dressings [1], and gelling and
emulsifying agent in food industries besides being used as an inhibitor of ice
crystal formation control [1]. In this study, xanthan gum production, recovery
and properties will be studied by a locally isolated culture of Xanthomonas, which
are isolated from cabbages and lettuces.
II. MATERIAL AND METHODS

A. Bacterial isolation and identification
Samples of diseased vegetables that show a typical spot symptom of Xanthomonas
infection was randomly collected. Bacterial isolation was done based on proce-
dures described by [5]. The presumptive colonies were identified according to
morphological, physiological and biochemical and BIOLOG.
B. Screening among selected strains

Ten isolated strains that were identified as Xanthomonas campestris were screened.
Yield and viscosity of the polymer synthesized in conventional medium were
analysed.
C. Cell production
Xanthan production was done in a batch fermentation method, in the orbital
shaker as have been describe by [12]. In this study, Yeast Malt Media (YM) was
used as the inoculums medium that consist of (g/l); glucose (10), malt extract
(3), yeast extract (3) and peptone (5).
D. Xanthan production
The batch fermentations were carried out in a 250 mL conical flask with working
volume of 100 ml containing 10% of inoculums concentration [5]. A complex
medium containing (g/l); glucose (30), yeast extract (3), K2HPO4 (2) and MgSO4.
H2O (0.1) was used as the fermentation medium. Samples were harvested after
72 hours and centrifuged at 13,000 rpm for 30 min to separate the cell mass and
xanthan gum.
E. Viscosity
The viscosity of 3% aqueous solution of the polymer synthesis by the ten strains
were compared with viscosity of xanthan gum synthesized by Xanthomonas
campestris pv. Campestris strain from ATCC culture collection (ATCC33913).
F. Analytical methods
The cell biomass was determined using dry cell weight method. Xanthan gum
was precipitated by adding cell-free supernatant with three times volumes of
acetone.
III. RESULTS AND DISCUSSIONS

A. Isolation and Identification
411 bacteria were isolated from both sampling site. 51 isolates were presumptively
identified as Xanthomonas sp. based on the biochemical test. Table 1 shows the
biochemical characteristic of Xanthomonas sp. Twelve presumptive isolates were
further identified using BIOLOG microbial identification system. Results of
the BIOLOG are as in table 2. According to literature [7], Xanthomonas have
smooth, yellow and mucoid colony. All of twelve isolates have these appearances.
Table 1. Biochemical characteristic of Xanthomonas sp.

Characteristic of Xanthomonas sp.
Gram staining Gram negative
3% KOH Positive
Oxidase Negative
Catalase Positive
Table 2. Results of BIOLOG after 24 hours of incubation

Isolates Identification
C85 Acenetobacter johnsonii
C195 No ID
C205 No ID (xanthomonas campestris pv campestris)
C206 No ID (xanthomonas campestris pv campestris)
C253 Xanthomonas campestris pv raphani
C254 Xanthomonas campestris
C256 No ID ( Stenotrophomonas maltophilia)
C272 Xanthomonas campestris pv raphani
C279 xanthomonas campestris pv campestris
C316 Microbacterium maritypicum
B. Screening among selected strains

Ten isolates that were identified as Xanthomonas campestris were further screened by
evaluating the viscosity of exopolymer produced and its productivity. The analysis
shows that xanthan production was influenced by the strains. The productivity
(g h-1) varies from 0.0747 to 0.1074. Figure 1 shows the productivity among
isolates. Strain C206 has the highest productivity (0.1074) compare to others as
well as control strain, ATCC. Xanthan gum synthesized by strain C194 presented
the highest viscosity. Figure 2 shows the comparison of viscosity of polymer
produced among all isolates. Results of viscosity show that the viscosity produced
was dependent on the strains.
Figure 1. Productivity by different strains of newly

isolated X. campestris.
Figure 2. Viscosity of polymers synthesized by different strains of newly

isolated X. campestris.
IV. CONCLUSION
The production of xanthan gum and viscosity of the polymers was influenced by
strains and culture conditions. From the comparative studies, it was concluded
that isolates C279 has the highest productivity, but the yield obtain by all strains
was not appropriate for industrial production which are 10 to 20gl-1 according
to literature. The culture condition should be optimized and revalued in attempt
to increase the production.
V. Acknowledgement
The author would like to thank University of Malaya for the sponsorship and
research grant (RG048-11B10) and Institute of Research Management and
Consultancy, University of Malaya for providing Postgraduate Research Grant
(PG057-2013A) and also Ministry of High Education (MOHE) for providing
a research grant (FP020-2013A).
VI. REFERENCES
1) Becker, A., Katzen, F., Puhler, A. and L. Ielpi. (1998). Xanthan gum
biosynthesis and application: a biochemical/genetic perspective. Applied
Microbiology and Biotechnology, 50, 145–152.
2) Sharma, B. R., Naresh, L. and N. Dhuldhoya. (2006). Xanthan gum- A Boon
to Food Industry. Food Promotion Cronicle, 5, 27–30.
3) Faria, S., Viera, P., and M. M. Resende. (2010). Application of a model using
the phenomenological approach for detection of growth and xanthan gum
production with sugar cane broth in a batch culture. LWT-Food Science and
Technology, 43, 498–506.
4) Hsu, C. H. and Y. Lo. (2003). Characterization of xanthan gum biosynthesis
in a centrifugal, paxked-bed reactor using metabolic flux analysis. Process
Biochemistry, 38, 1617–1625.
5) Shenge, K. C., Mabagala, R. B. and C. N. Mortensen. (2007). Identification
and characterization of strains of Xanthomonas campestris pv Vesicatoria from
Tanzania by Biolog system and sensitivity to antibiotics. African Journal of
Biotechnology, 6, 15–22.
6) Ochoa, F. G., Santos, V. and Alcon. (1995). Xanthan gum production: an
unstructured kinetic model. Enzyme and Microbial Technology, 17, 206–217.
7) Ochoa, F. G., Santos, V., Casas, J. and E. Gomez. (2000). Xanthan gum:
production, recovery and properties. Biotechnology Advance, 18, 549–579.
8) Rosalam. S., Krishnaiah, D. and A. Bono. (2008). Cell free xanthan gum
using continous recycled packed fibrous-bed bioreactor-membrane. Malaysian
Journal of Microbiology, 4, 1–5.
9) Soudi, M., Alimadadi, N. and P. Ghadam. (2011). Minimal phenotypic test
for simple differentiation of Xanthomonas campestris from other yellow-
pigmented bacteria isolated from soil. Iranian Journal of Microbiology, 84-91.
10) Psomas, S. K. and Liakopoulou-Kyriakides. (2007). Optimization study of
xanthan gum production using response surface methodology. Biochemical
Engineering Journal, 35, 273–280.
11) Massomo, S. M. S., Nielsen, H., Mabagala, R. B., Mansfeld-Giese, K., Hock-
enhull, J. and C. N. Mortensen. (2003). Identification and characterization of
Xanthomonas campestris pv campestris strains from Tanzania by pathogenicity
test, Biolog, rep-PCR and fatty acid methyl ester analysis. European Journal
of Plant Pathology, 109, 775–789.
12) Salah, R. B., Chaari, K., Besbes, S., Ktari, N., Blecker, C., Deroanne, C. and
A. Hammadi. (2010). optimisation of xanthan gum production by palm date
juice by-products using response surface methodology(RSM). Food Chemistry,
121, 627–633.
ULTRAVIOLET IRRADIATION EFFECT OF
Penicillium chrysogenum ON PENICILLIN
PRODUCTION
Dudi Hardianto*, Uli Julia, Eka Siska, Diana Dewi, Suyanto, Erwahyuni E.
Prabandari, Lira Windriawati and Danang Waluyo
Biotech Center, Agency for the Assessment and Application of Technology, Puspiptek Area
Serpong 15314, Indonesia
Abstract
Penicillin is the oldest β-Lactam antibiotic that is produced by the filamentous fungus Penicillium
chrysogenum. Since the discovery of penicillin by Fleming, much effort has been invested to
improve productivity of Penicillium chrysogenum. Strain improvement to increase the penicillin
production can use random mutation with physical and chemical mutagents. In this research,
UV irradiation was used to obtain Penicillium chrysogenum mutant. Penicillin production was
determined by HPLC, and productivity of Penicillium chrysogenum mutants were compared
to wild type. The penicillin yield of mutants are varied, and mutant M12 produced 1.23 fold
compared to wild type.
Key words: Penicillin, Penicillium chrysogenum, Ultraviolet, Mutation
I. Introduction
Penicillin is the first antibiotic discovered and used for treatment of bacterial
infections. The structure of β-lactam penicillin consists of a bicyclic nucleus
formed by a β-lactam ring and a thiazolidine ring containing a sulfur atom
and an acyl side chain bound to the amino group present at C-6 [1]. Although
some bacteria are resistant to penicillin, this antibiotic is still widely used today.
Penicillin is used in treatment, many gram-positive bacterial infections, such as
Staphylococcus pyogenes (strep throat) and Streptococcus pneumoniae (respiratory
tract infection, otitis media) [2,3]. Bacterial cell wall synthesis is inhibited by
penicillin. Penicillin binds the enzyme transpeptidase that links the peptidoglycan
molecules in bacterial cell wall.
Penicillin is industrially produced by the filamentous fungus Penicillium
chrysogenum and commercial production of penicillin began in 1941 [4]. The
production of penicillin by the submerged fed-batch fermentation in stainless
tank reactors of 30,000–100,000 galon capacity is important for several decades
* Corresponding author. Email: dudihd@gmail.com
49
[5] and optimization of penicillin production is very important for the penicillin
companies [6]. Since the discovery of the production of antibiotics by Fleming
in 1929, much effort has been invested in the selection and synthesis of strains
with improved productivity [7]. Classical strain improvement with random
mutation and screening has been used to obtain overproducing strains. The UV
irradiation is one of the techniques for strain improvement and its technique is
very effective for mutation because the bases DNA absorb the UV irradiation.
The effect of the absorbed UV irradiation is the formation of thymine dimers and
cross links in the same strand [8]. Mutants are unstable, and mutagenic events
are random and do not necessarily affect only the genes involved in antibiotic
synthesis. Re-isolation of mutants, since prolonged storage of high producing
strains is needed to know stability of mutants.
The increase in penicillin fermentation productivity and high recovery yield
(>90%) has led to significant cost reduction, despite increasing labor, energy and
raw materials costs. In 1953, the bulk cost for penicillin production was ~$300/
kg. In 1980, the bulk price for penicillin was ~$35/kg. In the late 1990s, bulk
penicillin cost ranged from $10 to 20/kg and bulk marketed costs for 6-APA
have been estimated to range from $35 to 40/kg [5].
The aim of this research was obtaining Penicillium chrysogenum mutant that
produces higher amount of penicillin than the wild type.
II. METHOD/MATERIAL
Microorganism. Strain of Penicillium chrysogenum in this study was obtained from
Biotech Center Culture Collection, Agency for the Assessment and Application
of Technology.
Mutation by UV Irradiation. Czapek Dox Agar was used to select Penicil-
lium chrysogenum mutants. A conidial from 10 days old culture of Penicillium
chrysogenum was suspended in distilled water and adjusted to about 103 conidia/
ml. Conidial suspension was irradiated by short wave UV irradiation of 254 nm
and the time of irradiation varied from 5 to 30 minutes with 5 minutes interval.
50 µl conidial suspension of the growth of Penicillium chrysogenum mutants were
inoculated, spreaded into Czapek Dox Agar plates, and the inoculated plates were
incubated for 10 days at 28oC.
Isolation and Selection Mutants. After 10 days, mutant colonies were separated
by inoculation each mutant colony on Czapek Dox Agar plates. The inoculated
plates were incubated for 10 days at 28oC.
Production of Penicillin. One of the mutant colony was inoculated in seed
medium and incubated in shaker for 36 hours at 28oC. 10% Suspension inoculum
was inoculated in fermentation medium and incubated in shaker for 10 days at

28 oC.
Penicillin Analysis in Broth of Fermentation. Broth of fermentation was
centrifuged at 15,000 g, and supernatant was filtered through 0.45 µm filter.
10 µl sample was injected to HPLC mechine from Waters with C18 column
(Symmetry), mobile phase: 5 mM KH2PO4 and 6 mM H3PO4: Acetonitrile (60
: 40), flow rate 1.0 mL/min, and ultraviolet detector λmax: 210 nm.
III. result and discussion

P. chrysogenum wild type was irradiated by UV from 5–30 minutes. In this
research, effect of UV irradiation on P. chrysogenum was evaluated by survival
curve (Figure 1). The increase in time of UV exposure on P. chrysogenum caused
a gradual decrease of survivals. DNA bases absorb UV light induce the formation
covalent crosslinking of thymine in the same strand DNA or thymine dimers.
The thymine dimers do not have base pairing and when the DNA replicate, the
wrong base may be inserted [9,10]. The lethal of P. chrysogenum may be caused by
disruption of replication, alteration of gene that inhibit vegetation, or alteration
of the biochemical structure of the molecules that are essential to P. chrysogenum’s
survival (membrane or enzymes to metabolism).
Figure 1. Effect of UV exposure time on P. chrysogenum

The survivals of P. chrysogenum mutants were selected and tested for penicil-
lin production. Penicillin yield of mutants varied from 1.635 to 4.594 ppm.
Mutant M12 produced 1.23 fold (4.594 ppm) compared to wild type (3.305
ppm) (Table 1). The hyperproduction of mutant may cause mutation of one
or more biosynthesis of penicillin gene or the disrupsion of the lys2 gene (gene
for lysine biosynthesis). The mutation of biosynthesis of penicillin gene may
increase enzymes activity for penicillin production or disruption of the lys2 gene
increase aminoadipic acid (precursor for penicillin biosynthesis). In Penicillium
chrysogenum, the biosynthesis of penicillin and lysine have several steps in
Figure 2. HPLC chromatogram of lovastatin standard
Figure 3. HPLC chromatogram of lovastatin from fermentation broth of P. chrysogenum wild type
Figure 4. HPLC chromatogram of lovastatin from fermentation broth of P. chrysogenum mutant
Table 1. Penicillin production of mutants

Penicilium chrysogenum Concentration of Penicillin (ppm)
Wild type 3711
M1 2534
M2 3193
M3 2674
M4 2068
M5 2482
M6 2638
M7 3193
M8 2859
M9 2650
M10 1635
M11 2087
M12 4594
M13 3067
M14 2548
M15 4404
M16 4216
M17 3663
M18 4138
M19 4272
Figure 5. Biosynthesis pathway of penicillin and lysine in P. chrysogenum (Casqueiro

et al., 1999)
common (Figure 5). It should be possible to increase penicillin production by

the disrupsion of lys2 gene [11]. The poor result of penicillin of mutants may be
caused by mutation in penicillin biosynthesis genes. The mutation of biosynthesis
of penicillin gene may cause a decrease enzymes activity for penicillin production.
IV. conclusion
The penicillin yield of mutants varied from 1.635 to 4.594 ppm and mutant
M12 from 30 minutes-irradiated produced 1.23 fold compared to wild type.
V. references
1) Liras, P. and J. F. Martin. (2006). Gene cluster for β-lactam antibiotics and
control of their expression: Why have clusters evolved, and from where did
they originate? International Microbiology, 9, 9–19.
2) Christensen, B., Thykaer, J. and J. Nielsen. (2000). Metabolic characteriza-
tion of high- and low-yielding strains of Penicillium chrysogenum. Applied
Microbiology and Biotechnology, 54 (2), 212–217.
3) Rayamajhi, N., Cha, S. B. and H. S. Yoo. (2010). Antibiotics resistances:
Past, present and future. Journal of Biomedical Research, 11 (2), 65–80.
4) Newbert, R. W., Barton, B., Greaves, P., Harper, J. and G. Turner. (1997).
Analysis of a commercially improved Penicillium chrysogenum strain series:
Involvement of recombinogenic regions in amplification and deletion of the
penicillin biosynthesis gene cluster. Journal of Industrial Microbiology and
5) Elander, R. P. (2003). Industrial production of β-lactam antibiotics. Applied
Microbiology and Biotechnology, 61, 385–392.
6) Thykaer, J. and J. Nielsen. (2003). Metabolic engineering of β-lactam
production. Metabolic Engineering, 5 (1), 56–69.
7) Harris, D. M., van der Krogt, Z. A., Klaassen, P., Raamsdonk, L. M., Hage,
S., van den Berg, M. A., Bovenberg, R. A. L., Pronk, J. T. and J. Daran.
(2009). Exploring and dissecting genome-wide gene expression responses of
Penicillium chrysogenum to phenylacetic acid consumption and penicillinG
production. BMC Genomics, 10, 75.
8) Parekh, S., Vinci, V. A. and R. J. Strobel. (2000). Improvement of microbial
strain and fermentation processes. Applied Microbiology and Biotechnology,
54, 287–301.
9) Irum, W. and T. Anjum. (2012). Production enhancement of cyclosporin ‘A’
by Aspergillus terreus through mutation. African Journal of Biotechnology, 11
(7), 1736–1743.
10) Ravalat, J., Douki, T. and J. Cadet. (2001). Direct and indirect effect of
UV radiation on DNA and its components. Journal of Photochemistry and
Photobiology B: Biology, 63, 88–102.
11) Casqueiro, J., Guteirrez, S., Banuelos, O., Hijarrubia, M. J. and J. F. Martin.
(1999). Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene
leads to penicillin overproduction. Journal of Bacteriology, 181 (4), 1181–1188.
Isolation and Cloning of Partial HER-2 Gene
from Indonesian Breast Cancer Patients for
DNA Vaccine Development
Desriani* and Lita Triratna
Research Center for Biotechnology-Indonesian Institute of Sciences
Jl. Raya Bogor KM 46, Cibinong Bogor, Indonesia
Abstract
Over-expression of proto-oncogene HER-2 occurs in 20–30% of the total breast cancer cases
associated with increased metastatic potential and poor prognosis. It consists of a 620 aa
extracellular domain, followed by a 23 aa trans-membrane domain and a 490 aa intracellular
domain with tyrosine kinase activity. Treatment using the monoclonal antibody, trastuzumab, was
an expensive treatment with an incidence rate of resistance in single use reaches a high percentage of
66-88%. Until now, there are continuous efforts for materials or methods improvement for HER-
2 breast cancer treatment. Vaccination is an attractive alternative approach to provide protective
immunity. Several reports show that a DNA vaccine encoding full-length or truncated HER-2
was immunogenic since it may generate protective immunity. Furthermore, HER-2 engineered
epitope can also be used as a vaccine active agent. Here in this study, we were successful in isolating
and cloning partial of HER-2 gene which include extracellular and trans-membrane domain with
total size of 2.127 base pairs (bp) into pGEMT-easy. This gene material will allow us to develop
new vaccine candidate in the future works.
Key words: HER-2, Proto-oncogene, DNA vaccine, Immunogenic, Vaccination
I. Introduction
Breast cancer cases in the world tend to increase. In 1975, there were about
500,000 cases. After 30 years, nearly 1.4 million cases of breast cancer were
diagnosed across the world in 2008. In 2012, nearly 1.7 million new cases were
diagnosed (second most common cancer overall). This represents about 11%
of all new cancer cases and 23% of all female cancers. It is predicted that the
number of cases will rise to 2.1 million by 2030 (World Cancer Research Fund
International, 2013).
There are 3 types of breast cancer: ER/PR positive breast cancer, ER/PR nega-
tive breast cancer, and HER-2/neu positive breast cancer, with the percentage of
65–75%, 5–10%, and 20-25%, respectively. Although HER-2/neu breast cancer
is only one quarter of all breast cancer cases in the world, this breast cancer tends
to be much more aggressive and fast-growing.
* Corresponding author. Phone: +62-21-8754587. Email: gerodes@yahoo.com
57
HER-2/neu, also called HER-2 (human epidermal growth factor receptor 2), is
ERBB2 gene. It is one such gene that can play a role in the development of breast
cancer. The HER-2 gene makes HER-2 proteins. HER-2 proteins are receptors on
breast cells. Normally, HER-2 receptors help control how a healthy breast cells
grows, divides or do proliferation, and repairs itself. But in about 25% of breast
cancers, the HER-2 gene doesn’t work correctly and makes too many copies of
itself (known as HER-2 gene amplification). All these extra HER-2 genes tell
breast cells to make too many HER-2 receptors (HER-2 protein overexpression).
This makes breast cells grow and divide in an uncontrolled way (Clifford and
Hudis, 2007).
The drug trastuzumab is a humanized monoclonal antibody targeted
against the extracellular portion of HER-2. Genentech Company (South San
Francisco, CA) has been developing and producing trastuzumab with the trade
name Herceptin. Since released in 1998, trastuzumab has become an important
treatment for patients with invasive breast cancer (Ross et al., 2004). This is the
first HER-2 targeted agent to be approved by the united States Food and Drug
Administration (FDA) for the treatment of both early stage and metastatic
HER-2 overexpressing. Although HER-2 targeted therapies have had a significant
impact on patient outcomes, resistance to these agents reaches a high percentage
of 66–88% (Pohlman et al., 2009). Furthermore, trastuzumab is also associated
with an increased risk of cardiac dysfunction (Seidman et al., 2002).
Vaccination is an attractive alternative approach to provide protective
immunity against tumor that overexpress HER-2 oncogen product (Hurvitz
et al., 2013). Several reports show that a DNA vaccine encoding full-length or
truncated HER-2 was immunogenic since it may be effective in inducing protective
antitumor immunity (Rovero et al., 2000; Jasnska et al., 2003; Jacob et al., 2006;
Dimitriadis et al., 2009). Chen et al. (1998) reported that co-injection of DNA
vaccine encoding full length or truncated HER-2/neu co-injection together with
a plasmid vector that encoded IL-2 could enhance the ability of HER-2 DNA
vaccine to induce protective antitumor immunity. Norell et al. (2010) reported
that HER-2-pDNA vaccination in conjunction with GM-CSF and IL-2 was well
tolerated and can induce long lasting cellular and humoral immune responses
against HER-2 patients. HER-2 epitope, 597-626 sequence is reported as a
potential vaccine candidate to reduce tumor (Garrett et al., 2006). Until now,
the most effective vaccine has not been found, although there are many studies
to developed DNA Vaccine HER-2.
In this study, we were successful in isolating and cloning partial of HER-2
gene which includes extracellular domain (ECD) and trans-membrane domain
with total size of 2.127 base pairs (bp) into pGEMT-easy. This gene material will
allow us to develop new vaccine candidate in the future works.

2.1. Tissue Collection
Breast cancer tissues from patients in Muhammad Djamil Padang Hospital and
Dharmais Hospital Jakarta were collected and saved at -80°C.
2.2. Total RNA Isolation

The total RNA from breast cancer tissue was isolated by using Purelink RNA
mini kit (Invitrogen/Ambion).The total RNA was confirmed to 1% agarose
electrophoresis with 1X TBE (Tris Boris EDTA) Buffer. Then, these total RNA
can be stored at -80°C for more than a month.
2.3. One Step PCR (Polymerase Chain Reaction)

The pure total RNA was used as a template to synthesis cDNA and to amplify
ECD and trans-membrane domain by using Superscript III One-Step RT-PCR
system with platinum Taq High Fidelity; two specific primers (forward and reverse),
dNTPs, and ddH2O. All these reagent would be transfered into PCR tube (200
microLiter). The PCR product confirmed in 1% Agarose, electrophoresis with
1X TBE buffer.
2.4. Sequencing
PCR product was sequenced with ABI machine and then identified with Basic
Local Alignment Search tool (BLAST) software.
2.5. Cloning
The pure DNA was inserted into the plasmid cloning, pGEM-Teasy, by using
ligase. They were transformed into Escherichia coli DH5alpha by heat shock
technique. This E.coli recombinant was incubated in shaker incubator at 150 rpm,
for 16 hours or overnight, at 37°C. PCR colony and second by using restricted
plasmid isolation were used in order to confirm the success of cloning.

3.1 Tissue collection
The research was conducted using ethical clearance issued by Ministry of Health
Efforts, Dharmais Cancer Hospital National Cancer Center. We have managed
to collect 110 breast cancer tissues. The fresh tissue samples were stored at
-80°C or in liquid nitrogen. Breast cancer tissue identified as HER-2 + through
immunohistochemistry screening then was used as a RNA isolation source.
3.2. Total RNA Isolation

Total RNA from breast cancer tissue have been successfully isolated (picture 1).
Figure 1 shows that there are 28S RNA and 18S RNA. The size were 2.8 kilobase
pairs (kb) and 1.8 kb, respectively. 1 kb DNA ladder was used as the marker (left
side).
3.3. One Step PCR

The extracellular domain (ECD) and trans-membrane domain from HER-2 gene
was successfully isolated with total size of 2,127 base pairs (bp) (Figure 2).
3.4 Cloning
Cloning of the gene encoding the extracellular domain (ECD) and trans-
membrane domain was successful. The size of pGEM-Teasy, as a plasmid or vector
cloning, is 3.015 bp, while the size of the gene is 2,127 base pairs (bp). In total,
the size of plasmid and insert gene is 5.477 bp (Figure 3).
Figure 1. Total RNA Isolation

Result.
(a) (b) (c)

Figure 2. One step PCR Result. (a) and (b) are the unpurified results; (c) is the
purified result.
PCR colonies and enzyme restriction site analysis of targeted plasmid isolation
were done to confirm the cloning results (Figure 4). DNA sequencing methods
was also done in order to confirm DNA bases constituents (data was not shown).
Many approaches for the genetic immunization of tumor have been investi-
gated. Injection of tumor with DNA plasmid designed to promote an immune
response to the tumor resulting the minimal efficiency (only affecting the injected
tumor) and minimal systemic immunologic impact. The most potential part of
DNA, as the candidate of DNA vaccine for immunotheraphy agent, need to be
examined more. (Kirkwood et al., 2012). Not only DNA vaccine, DNA delivery
system and ageing could also affect the effectiveness of immune response (Smorlesi
et al., 2006; Provincialli et al., 2003).
Here in this study, we were succeeded in the isolating and cloning partial of
HER-2 gene which include extracellular and trans-membrane domain with the
total size of 2,127 base pairs (bp) into pGEMT-easy. This gene material will allow
us to develop new vaccine candidate in the future works.
IV. Conclusion
The gene encoding extracellular membrane and trans-membrane HER-2 protein
has been successfully isolated and cloned into plasmid cloning, pGEM-Teasy,
and transformed into E.coli DH5alpha. These genetic materials can be stored for
long term and potentially used in the development of DNA vaccine for HER-2
positive breast cancer.
Figure 3. pGEM-Teasy and insert gene encoding ECD and trans-

membrane domain.
Figure 4. (A) PCR colonies results. (B)

Plasmid isolation results.
V. Acknowledgments
Thank you to the competitive program from Indonesian Institute of Science
(LIPI) as the funder. We would like to thank Dharmais Hospital in Jakarta and
M. Djamil Hospital in Padang, for their cooperation in this research in providing
the breast cancer tissue. To Neneng Hasanah, Rivai, M. Ali Warisman, and Wiwit
Amrinola, thank you very much for making this research possible.
VI. R efference
1) Chen, Y. et al. (1998). DNA vaccines encoding full-length or truncated neu
induce protective immunity against neu-expressing mammary tumors. Cancer
Research, 58, 1965-1971.
2) Clifford, A. and M. D. Hudis. (2007). Trastuzumab-mechanism of action
and use in clinical practice. New England Journal of Medicine, 357, 39-51.
3) Dimitriadis, A. et al. (2009). The mannosylated extracellular domain of Her-2/
neu produced in P. pastoris induces protective antitumor immunity. BMC
Cancer, 1-9.
4) Hurvitz, S. A. et al. (2013). Current approaches and future directions in the
treatment of HER2 positive breast cancer. Cancer Treatments Review, 39,
219-229.
5) Garret, J. T. et al. (2010). Novel engineered trastuzumab conformational
epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/
neu. The Journal of Immunology, 8, 1720-1730.
6) Jacob, J. et al. (2006). Activity of DNA vaccines encoding self or heteroulogous

Her-2/neu in Her-2 or neu transgenic mice. Cellular Immunology, 240,
96-106.
7) Jasnska, J. et al. (2003). Inhibition of tumor cell growth by antibodies induced
after vaccination with peptides derived from the extracellular domain of Her-2/
neu. International Journal of Cancer, 107, 976-983.
8) Kirkwood, J. M. et al. (2012). Immunotherapy of cancer in 2012. CA: A
Cancer Journal for Clinicians, 62, 309-336.
9) Norell, H. et al. (2010). Vaccination with a plasmid DNA encoding HER-2/
neu together with low doses of GM-CSF and IL-2 in patients with metastatic
breast carcinoma: A pilot clinical trial. Journal of Translational Medicine, 8,
53-64.
10) Pohlman, P. R., Mayer, I. A. and R Mernaugh. (2009). Resistance to
trastuzumab in breast cancer. Clinical Cancer Research, 24, 7479-7491.
11) Provincialli, M. et al. (2003). Low effectiveness of DNA vaccination against
HER-2/neu in ageing. Vaccine, 21, 843-848.
12) Ross, J. S., Fletcher, J. A., Bloom, K. J., Linette, G. P., Stec, J., Symmans,
W. F., Pusztai, L. and G. N. Hortobagyi. (2004). Targeted therapy in breast
cancer. Molecular and Cellular Proteomics, 3 (4), 379-398.
13) Rovero, S. et al. (2000). DNA vaccination against rat Her-2/neu p185 more
effectively inhibits carcinogenesis than transplantable carcinomas in transgenis
BALB/c mice. Journal of Immunology, 165, 5133-5142.
14) Seidman, A. et al. (2002). Cardiac dysfunction in the trastuzumab clinical
trials experience. Journal of Clinical Oncology, 20, 1215-1221.
15) Smorlesi, A. et al. (2006). Evaluation of different plasmid DNA delivery
systems for immunization against HER-2/neu in a transgenic murine model
of mammary carcinoma. Vaccine, 24, 1766-1775.
16) Tai, W., Mahato, R. and K. Cheng. (2012). The role of HER-2 in cancer
therapy and targeted drug delivery. Journal of Controlled Release, 146 (3),
264-275.
Heterologous Expression of Recombinant
Plantaricin WS34 in Escherichia coli
Andini Setyanti Putria, Rifqiyah Nur Umamib, Apon Zaenal Mustopab,*

and Hasim Danuria
a
Graduate student of Biochemistry Department, Bogor Agricultural University
Jl. Raya Darmaga Kampus IPB Darmaga, Bogor 16680, Phone/fax: +62-251-8423267
b
Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI)
Jl. Raya Jakarta-Bogor Km 46, Cibinong, Bogor 16911,
Phone: +62 (021) 8754587 Fax: +62 (021) 8754588
Abstract
Plantaricin W is a two peptide lantibiotic produced by Lactobacillus plantarum S34 isolated from
bekasam, a traditional fermented food of meat products from Waykanan, Lampung. Plantaricin
W is a potential antimicrobial peptide. However, the expression of recombinant plantaricin W
has not been reported. To express a recombinant plantaricin WS34, we constructed and expressed
the gene encoding plantaricin W in pET-32a by using the T7 RNA polymerase promoter with
approximately 33 kDa in size in Escherichia coli BL21 (DE3)(pLysS). The result revealed that the
plantaricin WS34 peptide was expressed as a translational fusion protein with thioredoxin and
His-tag. When expressed in E. coli, recombinant plantaricin W was found to be accumulated in
the cell cytoplasm but forming an inclusion body. In this study, two solubilization strategies were
done and showed that inclusion bodies were solubilized at alkaline condition in the presence of
urea solution under mild condition and the expression of plantaricin WS34 has been confirmed
with western blot. The expression of plantaricin WS34-pET32a was succesfully expressed in E. coli.
Key words: Inclusion body, Plantaricin W, Protein expression
I. Introduction
Bacteriocin are bactericidal compounds, act as a suppressor of competitor species.
Bacteriocins kill bacteria by disrupting membrane integrity so it does not induce
resistance [1]. Based on its properties, bacteriocin can be one of the solutions
for antibiotic resistance problems, and it can be used as alternative antibiotic.
Bacteriocin produced by lactic acid bacteria (LAB) was classified into four class [2].
The class I bacteriocin (lantibiotics) defined as ribosomally synthesized peptides
containing the thioether amino acids lanthionine (Lan) and 3-methyl-lanthionine
(meLan) [3]. Plantaricin W is a new family of two-peptide lantibiotics. According
to [3] and [4], plantaricin W was able to inhibit a large number of Gram-positive
and Gram-negative bacteria, such as E. coli, B. cereus, S. typhimurium and S. aureus.
* Corresponding author. Email: azmustopa@yahoo.com
65
Lantibiotics plantaricin W is produced from Lactobacillus plantarum [3]. In

this study, we isolated Lactobacillus plantarum S34 from bekasam, a traditional
fermented food of meat products from Waykanan, Lampung. The use of plan-
taricin W as alternative antibiotic required extraction technique from natural
resources in a large scale that is difficult to purified in high yield. One approach
that can be done is making the recombinant plantaricin. According to [5], this
technique allows to produce the target protein abundantly, not expensive, and
more stable. Bacteriocin production systems are currently utilized in expression
from native strain [6], recombinant expression system by using Escherichia coli,
recombinant expression in LAB [7], and chemical synthesis.
There are several advantages of using tag systems, such as a maltose-binding
protein (MBP), His-tag, or glutation-S-transferase. In this study, Plantaricin
W was expressed as a translational fusion protein with thioredoxin, S-tag and
His-tag. Polyhistidine-tag (his-tag) is a short affinity tag consisting polyhistidine
residues, widely used to purify recombinant protein utiliz immobilized metalaf-
finity chromatography. Immobilized metal affinity chromatography is based on
interaction between a transition metal ion (Co2+, Ni2+, Cu2+, Zn2+) immobilized
on a matrix and specific amino acid chains [8]. Histidine is the amino acid that
show the strongest interaction with immobilized metal ion matrix. Plantaricin W
also contains polypeptide that specifically recognized by enterokinase to remove
the tag from protein [8].
Expression of genetically engineered protein in E. coli often results in the
accumulation of the protein product in inactive insoluble form that deposits
inside the cells, called inclusion bodies [9]. This protein can be expressed as
inclusion bodies because of the protein interest is toxic or lethal to the host cell,
then inclusion body expression may be the best available production method [9].
We used several strategies to recover active protein from inclusion bodies, to take
advantage of the high expression levels of inclusion body protein.
Heterologous expression of bacteriocin has been widely studied in recent
years [10,11,12,13], but the expression of plantaricin W has not been reported.
In this study, we have expressed plantaricin W in E. coli to enhance the yield
with solubilization and produce an active plantaricin W as antimicrobial peptide.
II. M aterials and methods

A. Bacterial Strains and Media
Transformant Escherichia coli BL21(DE3) pLysS were grown in Luria Bertani
broth medium (Oxoid, Hampshire, UK) at 37°C with shaking. Salmonella thypi
P2KIM Collection, Escherichia coli NBRC 14237, and Staphylococcus aureus
ATCC 6538 were obtained from Biotechnology Research Center, Cibinong
collection, grown in nutrient broth medium (Oxoid) at 37 °C with shaking.

When appropriate, 100 mg/ml ampicillin was added for E. coli.
B. Expression Plantaricin WS34

Expression of recombinant plantaricin WS34, furtherly we name it PlnWS34-p32,
was referred to [14] with modification at the amount of starter culture. Bacterial
cultures of E. coli BL21 transformant (DE3) pLysS carrying the gene Plantaricin
W (Pln W) were grown in Luria Bertani medium (LB), 20 mL has been added
with 100 mg/mL of ampicillin. Bacterial cultures were incubated at 37°C for 16
hours, shaked at 150 rpm. The expression of PlnWS34-p32 was performed by
adding 20 mL of culture into 200 mL of LB medium with 100 mg/mL ampicillin.
The cultures were incubated with shaker at 150 rpm, 37°C for 2 hours or until
the OD600 reached 0.6 (mid log phase), and then the culture were inducted with
0.5 mM IPTG (Thermo Scientific). After inducted, the culture were incubated
at 22 °C for 5 hours with shaking at 150 rpm. After 5 hours, the culture was
centrifuged at 8,000 g, 4°C, for 10 minutes. Removed supernatant (media) and
pellet (medium free cell) are stored at -20°C.
C. Protein Solubilization
We studied two solubilization buffers at low urea and high urea. Solubilization
of inclusion bodies reffered to [15] and [16]. After sonication, insoluble fraction
as inclusion bodies was separated from soluble protein by sentrifugation for 30
min with centrifugation speed at 17,000 g. The cells were washed with washing
buffer (50 mM Tris HCl pH 8, 100 mM NaCl, 2 M Urea, 1% Triton X-100)
and centrifuged at 12,000 g for 5 min. The washed pellet was resuspended in
solubilizing buffer. Two solubilizing buffers were studied. Buffer I that contained
high Urea [14] and buffer II that contained lower urea but high pH [15] had
been optimized to solubilize recombinant protein that cloned in E. coli. Buffer I
contained 8 M Urea, 50 mM Tris HCl pH 8.0, 800 mM mercaptoethanol and
buffer II contained 100 mM Tris HCl pH 12.5 and 2 M urea. Pellet were incubated
over night at mild condition with gentle agitation. Subsequently, solubilized pellet
was centrifuged at 17,000 g for 15 min, 4 °C. Protein was analyzed using SDS-
PAGE. We had observed these solubilization buffers to compare solubilization
ability on inclusion bodies and improving the yield of bioactive protein.
D. Protein Refolding
Protein refolding method was referred to [15]. Dialysis was performed to refold
PlnWS34-p32 after solubilization step. Two dialysis step were applied, first step
was to remove denaturing agent using denaturing removal buffer (50 mM Tris
HCl pH 8, 0.2 mM EDTA). The supernatant of solubilization was dialyzed
overnight in dialyzed tube (MWCO 11 463, Sigma) with 500 mL denaturing

removal buffer at 4 °C with gentle agitation. Secondly, the fraction was dialyzed
to renature and to retain native like secondary structure of PlnWS34-p32. To
renature PlnWS34-p32, the protein was dialyzed in 500 mL refolding buffer (0.4
M urea, 0.2 mM EDTA, 50 mM Tris HCl pH 8.0, 0.25 mM mercaptoethanol)
over night at 4 °C with gentle agitation.
E. Western Blot Analysis

Western blot was performed to detect the presence of the target protein by
using imunoaffinity (Invitrogen, R931-25) [17]. The process of western
blot performed several stages, first electrophoresis with SDS-PAGE without
staining, protein transfer to nitrocellulose membrane (blotting), blocking with
5% skim milk on a nitrocellulose membrane to prevent non specific binding
to the antibodies. These protein has his-tag fusion, probing is done by using
antibodies (anti-his-HRP-Cterm) so that the antibody would bind to proteins
that containing his-tag. Detection is done by adding the HRP substrate that is
TMB (3,3’,5,5’-Tetramethylbenzidine) (Invitrogen) that will react with the HRP
and give colour.
F. Inhibition Activity
The antimicrobial activity of each sample was determined by agar diffusion
method [4] with modification in kind of the medium agar used. A 50 mL
samples were applied to the well (5 mm diameter). Agar containing 108 CFU/
mL of indicator microorganisms (Salmonella typhi and S. aureus). Petri stored at
4 °C for 1-2 hours so that the sample could diffuse into agar before incubated at
optimum temperature. Then incubated at 37°C for 18 hours. Then the diameter
of inhibition zones were measured.

The SDS-PAGE profile of the solubilize PlnWS34-p32 by two buffers is shown
in Figure 1. The purity of PlnWS34-p32 that was solubilize by buffer II (100
mM Tris pH 12.5 and 2 M urea) is better than buffer I (50 mM Tris HCl pH 8,
8 M urea, 800 mM 2-mercaptoethanol). Buffer II can solubilize 48.72%±4.44 of
wet pellet when 500 ml buffer solubilization was added for each 0.1 g wet pellet.
The antimicrobial activity of solubilized PlnWS34-p32 is shown in Table 1
and Figure 2. Based on Table 1, solubilization with buffer II has antimicrobial
activity against bacteria Gram positive and negative compare to solubilization
with buffer I.
The best solubilization method that produced PlnWS34-p32 biologically

active was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea.
Solubilization PlnWS34-p32 inclusion bodies in the presence of low concentra-
tion of urea helped in retaining the existing native-like secondary structures of
PlnWS34-p32 [16]. Since PlnWS34 is class I A bacteriocin, their mode of action
is by pore formation to kill target cells [18].
Escherichia coli has an inducible expression system that strongly suppresses
the expression of the cloned gene in the presence of the expression inducer
(isopropyl-β-d-thiogalactopyranoside-IPTG) [21]. We also observed the expres-
sion when induced with IPTG and not induced (Figure 3). Based on Figure 3,
the expression of PlnWS34-p32 is higher in the presence of IPTG. The expression
of PlnWS34-p32 was confirmed with western blot (Figure 4). Based on Figure 3
and Figure 4, PlnWS34-p32 (~33 KDa ) was successfully expressed.
In the present work, we isolated and constructed recombinant PlnWS34 in
pET 32a. The plantaricin of WS34 belong to class I (lantibiotic) of bacteriocin
produced by Lactobacillus plantarum. Lantibiotic is a peptide containing the
unusual post-tranlationally modified residues lanthionine, 3-methyllanthionine,
and other modified residues. Lantibiotics are devided into type A and B. The
type A are elongated peptides with seven net positive charges. These peptides kill
target cells by pore formation to permeabilizing their membranes. The type B
are globular peptide and have either a low net positive charge, no net charge, or
a net negative charge. Their mode of action is inhibition of enzymatic activity
[18]. According to [3], Plantaricin W are indeed lantibiotic class A.
Plantaricin W peptide was expressed as a translational fusion protein with
thioredoxin, S-tag, and His-tag in E. coli. E. coli is common employed as the
host for vectors expressing recombinant proteins, because of its ease of growth
and generally low cost to cultivate. But sometimes, it is quite common that
overproduced recombinant protein is expressed into insoluble inclussion bodies
[19].
His-tags are the most widely used affinity tags. Purification of his-tagged
protein is based on metal affinity, by using chelated metal ions as affinity ligand.
This is possible to purification of the desired protein from the crude extract cell
of the host cells in a single step [20].
S-tag sequence is a fusion-peptide composed of four cationic, three anionic,
three uncharged polar, and five non-polar residues. This composition makes the
S-tag soluble [8]. Thioredoxin (Trx) is hydrophilic tag that can increase solubility
[8]. Altough Pln W has fused with several tags that could increase solubility,
PlnWS34 was found in a insoluble form. According to [9], this protein can be
expressed as inclusion bodies because of the protein is toxic or lethal to the host
cell, then inclusion body expression may be the best available production method.
M 1 2 Figure 1. Solubilization optimization. Lane 1: PlnWS34-p32

210 solubilized with buffer 1 (8 M Urea, 50 mM Tris HCl pH 8,
800 mM mercaptoethanol), lane 2: Solubilization with buffer
78 II (100 mM Tris pH 12.5 and 2 M urea).
55
45
Table 1. Activities of solubilization PlnWS34-p32 towards
34 different indicator strains
Clear zone (mm)
23 Gram
Indicator Strain Buffer Buffer
(+/-)
I II
16
EPEC K.1.1 - - 2 mm
S. aureus ATCC 6539 + - 4 mm
E. coli NBRC 14237 - - 5 mm
7
S. typhi P2KIM col- - - 2 mm
4
lection
1 2 K
Figure 2. Activities Pln WS34 towards S. typhi P2KIM collection. 1. PlnWS34 (buffer I),
2. PlnWS34-p32 (buffer II), K (negative control, buffer without PlnWS34-p32)
M 1 2 Figure 3. Differentiation be-

210 tween expression PlnWS34-p32
78
55 that is induced with IPTG (lane
45 1) and non induced with IPTG
(lane 2).
34
23
Figure 4. Western Blot of

16
PlnWS34. Lane 1: Solubiliza-
tion of PlnWS34-p32 (mem-
7 brane nitrocellulose), lane 2:
4 Solubilization of PlnWS34
(gel SDS-PAGE).
IV. Conclusion
Solubilization of the recombinant Plantaricin WS34 inclusion bodies in the
presence of low urea improving the yield of bioactive protein during refolding.
The biologically active of recombinant Plantaricin WS34 was succesfully expressed
in E. coli with the size of ~33 KDa.
V. Acknowledgement
This research was funded by Competitive Program Indonesian Institute of Science
2014 granted to Apon Zaenal Mustopa.
VI. R eferences
1) Sang, Y. and F. Blecha. (2008). Antimicrobial peptides and bacteriocins:
alternatives to traditional antibiotics (review). Animal Health Research Reviews,
9 (2), 227–235.
2) Cleveland, J., Montville, T. J., Nes, I. F. and M. L. Chikindas. (2001).
Bacteriocins: safe, natural antimicrobial for food preservation [review].
International Journal of Food Microbiology, 71, 1–20.
3) Holo, H., Jeknic, Z., Daeschel, M., Stevanovic, S. and I. F. Nes. (2001).
Plantaricin W from Lactobacillus plantarum belongs to a new family of
two-peptide lantibiotics. Microbiology, 147, 643–651.
4) Arief, I., Jakaria, Suryati, T., Wulandari, Z. and E. Andreas. (2013). Isolation
and characterization of plantaricin produced by Lactobacillus plantarum strains
(IIA-1A5, IIA-1B1, IIA-2B2). Media Peternakan, 36 (2), 91–100.
5) Ma, J., Pascal, M. W. and C. Paul. (2003). The production of recombinant
pharmaceutical proteins in plants [review]. Nature, 4, 794–805.
6) Tiwari, S. K. and S. Srivastava. (2008). Purification and characterization of
plantaricin LR14: a novel bacteriocin produced by Lactobacillus plantarum
LR/14. Applied Microbiology and Biotechnology, 79 (5), 759–767.
7) Sorvig, E., Gronqvist, S., Naterstad, K., Mathiesen, G., Eijsink, V. G. H. and
L. Axelsson. (2003). Construction of vectors for inducible gene expression
in Lactobacillus sakei and L. Plantarum. FEMS Microbiology Letters, 229,
119–126.
8) Terpe, K. (2003). Overview of tag protein fusions: from molecular and
biochemical fundamentals to commercial systems. Applied Microbiology and
9) Clark, E. B. (2001). Protein refolding for industrial processes. Current Opinion
in Biotechnology, 12, 202–207.
10) Pal, G. and S. Srivastava. (2013). Cloning and heterologous expression of plnE,
-F, -J and –K genes derived from soil metagenome and purification of active
plantaricin peptides. Applied Microbiology and Biotechnology, 98, 1441–1446.
11) Heu, S., Oh, J., Kang, Y., Ryu, S., Cho, S. K., Cho, Y. and M. Cho. (2001).
Gly gene cloning and expression and purification of glycenin A, a bacteriocin
produced by Xanthomonas campestris pv. Glycines 8ra. Applied and Environmental
Microbiology, 67, 4105–4110.
12) Richard, C., Drider, D., Elmorjani, K., Marion, D. and H. Prevost. (2004).
Heterologous expression and purification of active divercin V41, a class IIa
bacteriocin encoded by a synthetic gene in Escherichia coli. Journal of Bacteriology,
186 (13), 4276–-4284.
13) Gibbs, G. M., Davidson, B. E. and A. J. Hillier. (2004). Novel expression system
for large-scale production and purification of recombinant class IIa bacteriocins
and its application to piscicolin 126. Applied and Environmental Microbiology,
70 (6), 3292–3297.
14) Utama, A., Shimizu, A., Morikawa, S., Hasebe, F., Morita, K., Igarashi, A., Hatsu,
M., Takamizawa, K. and T. Miyamura. (2000). Identification and characterization
of the RNA helicase activity of Japanese encephalitis virus NS3 protein. FEBS
Letters, 465, 74–78.
15) Ningrum, R. A., Debbie, S. R., Yeyet, C. and R. Heni. (2011). Optimization of
human interferon a2b soluble protein overproduction and primary recovery of
its inclusion bodies. Microbiology Indonesia, 5, 27–32.
16) Patra, A. K., Mukhopadhyay, R., Mukhija, R., Krishnan, A., Garg, L. C. and A.
K. Panda. (2000). Optimization of inclusion body solubilization and renaturation
of recombinant human growth hormone from Escherichia coli. Protein Expression
and Purification, 18 (2), 182–192.
17) Invitrogen. (2001). Anti-His(C-term) antibody; Anti-His (C-term)-HRP
antibody (R930-25, R931-25). Invitrogen. Carlsbad. USA. (Online). Available:
https://www. invitrogen.com.
18) Jorgenrund, B. M. (2009). Construction of a heterologous expression vector for
Plantaricin F, one of the peptides constituining the two-peptide bacteriocin Plantaricin
EF (thesis). Oslo: University of Oslo.
19) Frank, E. G., McDonald, J. P., Karata, K., Huston, D. and R. Woodgate. (2012).
A strategy for the expression of recombinant proteins traditionally hard to purify.
Analytical Biochemistry, 429, 132–139.
20) Arnau, J., Lauritzen, C., Petersen, G. E. and J. Pedersen. (2006). Current strategies
for the use of affinity tags and tag removal for the purification of recombinant
protein. Protein Expression and Purification, 48, 1–13.
21) Kimelman, A. et al. (2012). A vast collection of microbial genes that are toxic
to bacteria. Genome Research, 22, 802–809.
Evaluation of Low Temperature Induced

Mutant of Soybean mosaic virus for Cross
Protection in Soybean
Wuye Ria Andayanie* and Praptiningsih Gamawati Adinurani
Faculty of Agriculture, Merdeka University
Serayu, Madiun, Indonesia
Abstract
Early natural infection by soybean mosaic virus (SMV) can reduce seed production in soybean.
Cross protection enables the production of SMV free seed and is a mechanism which can
signficantly reduce the impact of SMV. We proposed that attenuated isolates of SMV obtained
by cold temperature treatments are able to produce SMV free seed in soybean [Glycine max
(L.)] Merr. plants. We inoculated the cotyledons with SMV infected plants, incubated at low
temperature, mechanically inoculated seedlings with virus were subjected to cold and finally
transplanted into pot and then into the field. We examined plants with different symptoms.
Serological assays, RT-PCR analysis and electron micrography did not distinguish between the
very mild mosaic symptom of attenuated isolate and the original virulent isolate. However, the
mutant of SMV did not give rise to local lesions on Chenopodium amaranticolor. Our results suggest
that attenuated isolate of SMV is potentially useful for reducing the impact of SMV infection.
When plants are inoculated with the attenuated isolate at 8 days after planting, symptoms of the
disease do not develop.
Key words: Attenuated isolate, Cross protection, Induced mutant, soybean, soybean mosaic virus (SMV)
I. Introduction
Soybean is a valuable economic commodity in Indonesia with great potential for
production in the future. Imports of soybean reached 1.6 million of tons in 2010.
Soybean mosaic disease is a major problem caused by soybean mosaic virus
(SMV), a seedborne and aphid-borne disease. This viral disease reduces seed
quality, decreases oil content and nodulation [1]. Soybean mosaic virus is com-
monly transmitted by aphids and through seed. Mottling of soybean seeds does
not indicate that plants grown from such seeds will contain SMV or whether seed
produced by plants grown from mottled seeds will themselves produce mottled
seed [2,3].
The increase of soybean production was absolutely needed to meet national
needs and security of foreign exchange. Mosaic disease caused by cowpea mild
* Corresponding author. Phone: +62-81335401517. Email: wuye_andayanie@yahoo.com

mottle virus (CMMV) and soybean mosaic virus (SMV) are commonly found in
soybean [Glycine max (L.) Merr.] in East Java Province, Indonesia, but there was
no efficient controlling measure to SMV. Etiology of the viral disease of soybean
crops, prevalent in the soybean growing regions of Ngawi District in East Java
Province, clearly indicates the dominance of SMV. Infection by SMV, especially
at an early stage of cropping, has a serious impact on seed production in soybean.
Early natural infection by SMV has been found to reduce seed production by
13–30% in soybean [4,5]. Therefore, a control strategy that delays SMV infection
improves soybean production. This can be achieved by the use of SMV free seeds
or seedlings to minimize the primary inoculum source that is spread by aphids.
However, it is difficult to obtain SMV free seed from field grown soybean plants
under high disease pressure [6].
The induced resistance of plant is that the inducting factors induce the plant
to resist pathogeny microorganism. The cross protection is induced by attenuated
virus to protecting the plant infected by virus. The effectiveness of the use of
attenuated isolates of SMV to protect soybean plants from the seed transmitted
mosaic disease caused SMV.
Cross protection is described as “a specific symptom, a single virus”, that
is the presence of one virus inhibits the accumulation of second and produces
a specific symptomatology. Cross protection is a phenomenon whereby plants
infected with one strain of the virus do not develop additional symptoms when
inoculated with another strain of the same virus. Cross protection interprets as
a symbiotic union between the virulent and avirulent srains and consider it an
important source of physiological immunity. The coat protein of the mild strain
inhibits the uncoating of the challenging virus and the presence of the mild strain
blocks the systemic movement of the challenge virus [7,8].
Cross protection with a mild or avirulent strain could offer an alternative
route to control by protecting soybean plants from subsequent infection by a
virulent strain. Deployment of such a strain could be similar to using virus-free
seed. If a field is virus free, even if the vector population is high, no infection
will take place because the virus is non-persistent and will not be present in the
vector. Attenuated isolates exploited for cross protection have been obtained by
the treatment of virus with heat, nitrous acid and UV light [9,10]. Heat or cold
treatment may also yield mild isolates [11].
The most common limiting factor hindering the acceptance of cross protection
being used commercially is the difficulty involved in large number of plants
with the mild strain, especially when the virus is non-mechanically transmis-
sible. Another unpredictable drawback is that the mild strain might associate
synergistically with an unrelated virus which can then infect hosts under field
conditions [12,13].
Succes in virus control by cross protection depends on whether the avirulent
virus can invade and replace the virulent virus and whether the virulent virus is
prevented from reestablishing. Ideally, a protective strain should induce no or
very mild symptoms that: will not reduce the market value of the crop; will not
inflict disease on other crops that are not targets for cross protection; will be
completely systemic affecting all host tissues; will be genetically stable, will not
revert to the severe form; will be non-transmissible by vectors in order to limit
non-intentional spread to other crops or fields; will provide protection towards
the widest possible range of severe strains. Also, the inoculum should be easy to
produce, to apply and needs to be very stable, during the process of transportation
and utilization [14].
There is a dire need to investigate the effect of this protection on the yield
parameters and also study should be planed to study the behaviour of these isolates
under natural field conditions, especially their effect on other prevalent crops and
their interaction with the prevalent viruses in the area, before allowing the use of
this isolate for commercial control at field level.
We proposed that attenuated isolates of SMV obtained by cold temperature
treatments may be able to produce SMV free seed on soybean plants. The objec-
tives of this study were therefore (i) to investigate the cross protection offered
by a mild mutant isolate of SMV, induced at low temperature, against a severe
strain and (ii) to detect very mild symptoms of the mutant isolate of SMV in
soybean crops.

The SMV was isolated from soybean samples collected at Ngawi, East Java
Province, Indonesia in 2011 and was maintained on mechanically inoculated
plants of C. amaranticolor in insect proof green house at Merdeka Madiun
University, Indonesia.
A. Inoculation of Soybean Seedling Leaves with SMV

The heavily infected SMV isolate originated from soybean samples collected in
Ngawi District. The presence of SMV was confirmed by local lesion assay, serology
assay, RT-PCR and electron micrograph [5,15].
To produce SMV free seeds, we determined the SMV to protect seedling
plants and detect with Enzyme Linked Immnusorbent Assay (ELISA). Healthy
seeds of soybean were germinated in soil contained in 12 plastic pots, each pot
had two seeds. After germination thinning was done to reduce the number of
seedling to one in each pot. The soybean seedlings were mechanically inoculated
with the severe SMV isolate. Mechanical inoculation was done at the first true leaf
stage by grinding 1 g of infected leaf tissue in 50 ml of 0.05 potassium phosphate
buffer (pH 7.3) and rubbing the inoculum on carborundum (600 mesh) dusted
healthy leaves.
B. Incubation Under Low Temperature

Immediately after inoculation, the plants were cut off at the base of the hypocotyl,
and the stems were immersed in distilled water in vials. The cuttings were then
transferred into different growth cabinets, maintained at one of the following
temperatures: 10oC, 15oC, 20oC and 25°C as a control in a 14 hr photoperiode,
respectively. The cuttings were incubated for periods of 8 days, 14 days or 20 days.
The cotyledons of 8 day old seedling were inoculated with leaf extracts from the
cuttings subjected to incubation at different temperatures to evaluate of SMV
mutants obtained by exposure to cold.
C. Testing Cold Induced SMV Mutantsin the Field

To evaluate of cross protection strategy, soybean seedling was inoculated with the
attenuated SMV isolate. The seedlings (7 days after inoculation with attenuated
isolate) were planted into the field under conditions of natural infection.
The experimental design comprised plots with seedlings inoculated with the
cold induced SMV mutant and non-inoculated (control) plots. Inoculated and
non-inoculated seedlings were planted in the plots at intervals of 0.5 m in 2
rows; 40 plants (5 plants in each row and 4 replication in each row), respectively.
D. Examination of Plant Symptoms

Symptoms were observed and diseased plants were counted for 10 days, 20 days
and 30 days after transplanting into the field. Plants were scored for disease
severity according to the following scale as reported earlier by Ilyas et al., (l992)
as: 0 = no visible syptoms (plants apparently healthy); 1 = very mild mosaic
(mild mosaic mild mosaic on few leaves/plants); 2 = moderate mosaic (mosaic
on many leaves/plant and vein clearing); 3 = severe mosaic (severe mosaic and
mild mottling); 4 = severe mosaic and severe mottling); 5 = severe mosaic plus
severe mottling plus necrosis and occasinally death of plants. To check for the
presence of the attenuated SMV isolate, leaf samples with no visible symptoms
or only very mild symptoms were tested for the ability of their extracts to cause
local lesion on Chenopodium amaranticolor Coste et. Reyn.
Virus inoculum was prepared by grinding systemically infected leaves with a
mortar and pestle at an approximate dilution of 1:10 (w/v) with 0.05 M potassium
phosphate buffer, pH 7.2. Inoculations were performed by rubbing the inoculum

onto C. amaranticolor leaves that had been previously dusted with carborundum,
using a pestle dipped in the inoculum (one dip per leaflet, approximately 100
ul inoculum per leaflet) [5] in an attempt to find out any reasonable local lesion
hosts for mild strain seection. Leaf samples were also tested with ELISA, RT-PCR,
and observed under electron microscopy.
E. Total RNA extraction and RT- PCR

The sensitivity of RT-PCR enabled the detection of SMV from plants in which
the number of virus particles was too low to be detected by ELISA. Total RNA
was extracted from leaf samples grown in an insect proof greenhouse using
Nucleon Phytopure plant DNA extraction kit (Amersham pharmacia biotech,
Buckinghamshire, England).
Samples were obtained from plants with no visible symptom or very mild
symptom and was detected by RT-PCR. First Strand cDNAs Synthesis Kit: Am-
ersham Bioscience, Backinghamshire, Uk) and primer 3093R (5΄-ATGCTCTTC-
GCATGTACTCG-3΄;2.5 pmol). A pair of oligonucleotides amplifying a 1385 bp
fragment at posisition from 4,176 to 5,560 including the CI coding region was
designed based on the sequence published by Jayaram et al. (l992). The forward
primer, CÍ (5΄-GCATTCAACTGTGCGCTTAAAGAAT-3΄), was homologous
to nucleotides 4,176 to 4,200 and the reverse primer, CI3΄ (5΄-TTGAGCT-
GCAAAAATTTACTCACTT-3΄), was complementary to nucleotides 5,536
to 5,560 of SMV strain G2. These primers were used for amplification. PCR
was performed in a reaction mixture containing the following: 0.5 µl of cDNA
solution, 2.5 µl of 10 x PCR buffer (KOD plus Neo), 2.5 µl of 2 dNTP mix,
1.5 of 25 mM MgSO4, 0,5 µl of KOD plus Neo, 0.5 of each primer (50 pmol),
of each forward and reverse, 16.5 µl of sterile distilated water.
Thermocycling was programmed as follows: cDNA synthesis at 42°C for 60
min and 95°C for 5 min, and RNA/cDNA/primer denaturation at 94°C for 2
min, followed by 40 cycles for template denaturation at 94°C for 10 s, primer
annealing at 47 °C for 30 s, and extention at 68 °C for 1 min, and a final extention
at 68°C for 1 min. The amplified products were separated by 1.2% agarose gel
electrophoresis.
F. Electron Microscopy
Electron microscopy was also done for the selected samples to confirm the results
of serological assay. Sap from soybean leaf samples infected with attenuated
isolate was coated on carbon-formvar (400 mesh) grids, negatively stained with
2% phosphotungstate (PTA) pH 6.5 and were examined with JEOL 100 S x
electron microscope.
G. Data Analysis
The percentage of diseased plant was calculated from the number of plants
showing leaf roll and rugose symptom characteristic of the virulent SMV infect.
A randomized complete block design (RCBD) was used for agriculture traits.
Each treatment was performed four times. Disease severity scores associated with
SMV mutant were arcsine-transformed prior to analysis of variance. Analysis of
variance were performed by SAS [17]. Mean separation was done by calculating
the least significant difference (LSD) at P ≤ 0.05.

A. Cross Protection Using Attenuated Isolates in the Field
Disease severity of SMV was significantly lower (P ≤ 0.05, by chi-square test of
independence) in the field where seedlings from the SMV mutant obtained by
cold treatment (at 10°C for 8 days or 14 days) were grown (0–5%), compared
to other treatments. None of the symptomatic or very mild symptom plants in
the field when inoculated with the attenuated isolate produced by the treatment
temperature of 10°C for 14 days. In this study, no difference in the percentage
disease plant were found treatment at 10°C for 8 days or 14 days. An isolate of
virulent SMV obtained from field was subjected to low temperatures and mutants
strain induced; mutant isolates of the virus resulting from treatment at 10°C for
8 days or 14 days caused very mild and mild symptom, respectively.
These results showed that plants had the ability to cross-protect plants from
SMV infection in endemic field. Hence, plants were finally selected for the
attenuated isolate selection. The inoculated seedlings were kept in a green house
with they were transplanted to endemic area.
In contrast, SMV in endemic area used as the challenge virus was detected in
soybean plants that had previously been inoculated with other treatments. These
result indicated that the severity of viral pathogenicity to induce symptoms might
be not associated with cross-protective ability.
B. Serological Assay
Preinoculated soybean healthy samples with SMV maintained at 10°C, 15°C,
20°C, and 25 °C for 8 days, 14 days, 20 days, respectively gave a positive reaction
when tested with the antibodies of SMV (Table 2). Although soybean plants
have very mild and mild symptoms with SMV maintained at 10 °C for 8 days
and 14 days.
C. Differentiation of Attenuated Isolates by RT-PCR

Table 1. Disease Severity of SMV in Soybean Plants Inoculated with the SMV Mutant
Treatments SMV maintained at % Disease plants a
Preinoculated 10 °C 8 days 5 f(2)
14 days 0 f (1)
20 days 25 c (1,2)
15 °C 8 days 55 d (1,2,3)
14 days 85 b (2,3)
20 days 100 a (3,4)
20 °C 8 days 80 c(3,4)
14 days 95 a (3,4)
20 days 100a(4)
25 °C 8 days 95 a(4)
14 days 100 a(3,4)
20 days 100 a (4)
Noninoculated 100 a (3,4)
LSD 5.34
Similar letters in each column indicate no significant difference at 5% level

a
Symptom severity on diseases plants at 30 days after the challenge inoculation: 1 = very mild;
2 = mild; 3 = mosaic; 4 = severe mosaic and stunting
Table 2. Detection of SMV Maintained in Soybean Plants by Indirect ELISA
Treatments SMV maintained at Absorbance value (A405 nm) /

ELISA reaction
Preinoculated 10 °C 8 days 0.364/+
14 days 0.388/+
20 days 0.357/+
15 ° C 8 days 0.358/+
14 days 0.356/+
20 days 0.351/+
20 °C 8 days 0.356/+
14 days 0.351/+
20 days 0.354/+
25 °C 8 days 0.359/+
14 days 0.364/+
20 days 0.361/+
Noninoculated 0.357/+
Healthyleaves 0.117/−
Buffer 0.066/−
Remarks: Sample is considered positive if the absorbance value at 405 nm is up to three times
the value of the negative control (healthy leaves)
1 2 3 4
Figure 1. Specific detection of attenuated isolate by RT-PCR analy-

sis of total extracts using primer CI (lower lanes) which amplify a
1385 bp product. Lane 1 = healthy soybean leaves (negative con-
trol); lane 2 = very mild symptom of soybean leaves; lane 3 = mild
symptom of soybean leaves; lane M = 1 kb DNA ladder
RT-PCR analysis using primers CI yielded a DNA fragment of the expected size
1.385 bp from soybean leaves with very mild and mild symptoms.
D. Electron Microscopy
When checked under electron microscope, virus particles could be detected in
six samples from leaf tissue with very mild symptom. Filamentous particles of
ca. 13 x 750 nm were present in the leaf dip preparation examined by electron
microscopy.
The diagnosis of attenuated isolates showed that serological assay, RT-PCR
analysis and electron micrograph showing the results of very mild mosaic symptom
of attenuated isolate similar to original isolate.
E. Local Lesion Host Assay

No satisfactory local lesion host could be found in the assay with the SMV mutant;
however, the isolate SMV that it originated from caused chlorotic lesions on C.
amaranticolor.
Detection of the mild strain of SMV and its differentiation from severe SMV
is very important in order to prove its efficacy as a succesful cross protectant
[11,10]. Protective inoculation with SMV mutant (temperature 10°C for 8 days
or 14 days) has been effective for control of SMV in endemic areas. Artificial
mutants are being used, with no apparent problem for control of SMV. Those
plants not having been infected at the time of protective inoculation with SMV
mutant to a severe strain of SMV. This efforts could be directed to induce mild
strain, to help farmer controlling the severe virus under field conditions or at least
minimizing the detrimental effects to economic benefecial level. There is the real
possibility of the mild strain mutating to a severe form.
Since polyclonal antibody can detect the presence of virus coat protein from
either infectious or non-infectious, positive reaction with polyclonal antibodies
against SMV was caused by low antibody specificity so that these antibodies
still react with other viruses and strain viruses or SMV mutant. Although not
all samples were tested, a reverse transriptase polymerase chain reaction detection
test showed promise and detected both mild and severe SMV detection. Further
molecular studies are required to differentiate the mild strain of SMV from the
severe strain. In our study, variant mutants that may have been obtained with
cold treatment were not investigated but such an approach might give rise to a
more easily identifiable mutant SMV isolate. However, it is known that SMV
came into the region where the seedling were transplanted via infected seed and
vector (Aphis glycines Matsumura) [5,15].
Figure 2. Electron micrograph of soybean

Figure 3. Symptoms on C.
leaf sample infected with attenuated isolate,
amaranticolor plant infected
showing of SMV viral particles. The bar in
with mutant of SMV andorigi-
the electron micrographs is 100 nm
nal isolate of SMV
IV. Conclusion
Soybean plants with very mild mosaic symptoms were found in SMV mutant
seedlings transplanted from pots after cold treatment. The mutant isolate of SMV
obtained by cold treatment provided effective protection against field spread of
the virulent SMV. Although SMV was aphidborne, the attenuated virus was not
aphid transmitted in soybean field growing conditions. Immunity or tolerance is
established by infection of the attenuated virus and plants showed mild symptom.
This may be explained: 1) plants were effectively protected against the virulent
SMV as it was transmitted in the field; 2) plants were not showing symptoms
when observed at an early stage. After the application of cross protection in the
field, there was a trend for soybean yield to increase because early SMV infection
was reduced. The very mild symptom did not give rise to local lesion on C.
amaranticolor.
V. Acknowledgement
The author gratefully acknowledges Directorate General of Higher Education
for providing financial support by grant competition.
VI. R eferences
1) Sudaryanto, T. and D. K. S. Swastika. (2010). Ekonomi kedelai di Indonesia
(pp. 1–30). Badan Penelitian dan Pengembangan Pertanian, Kementerian
Pertanian: PT Balai Pustaka.
2) Li, D., Chen, P., Shi, A., Shakiba, E., Gergerich, R. and Y. Chen. (2009).
Temperature effects expression of symptoms induced by soybean mosaic
virus in homozygous and heterozygous plants. Journal of Heredity, 100 (3),
348–354.
3) Domier, L. L., Hobbs, H. A., Coppin, Mc. N. K., Bowen, C. R., Steinlage, T.
A., Chang, S., Wang Y. and G. L. Hartman. (2011). Multiple loci condition
seed transmission of Soybean mosaic virus (SMV) and SMV induced seed
coat mottling in soybean. Phytopathology, 101, 750–756.
4) Gunduz, I. (2000). Genetic analysis of Soybean mosaic virus resistance in
soybean. In partial fulfillment of the requirements for the degree of Doctor
of Phylosophy in Crop and Soil Enviromental Science, Virginia Polytechnic
Institute and Institute and State University.
5) Andayanie, W. R., Sumardiyono, Y. B., Hartono, S. and P. Yudono. (2011).
Incidence of soybean mosaic disease in East Java Province. Journal of Agrivita
Science, 33 (1), 15–22.[6]Andayanie, W. R., Sumardiyono, Y. B., Hartono, S.
and P. Yudono. (2011). Identification and seed transmission of Soybean mosaic
virus management. In partial fulfillment of the requirements for the degree
of Doctor of Phytopatology Science, Gadjah Mada University. [7]Freitas,
D. M. S. (2008). Protection between strains of Papaya ringspot virus-type
W in Zuchini squash involves competition for viral replicatin sites. Scientia
Agricola, 65 (2), 183-189.
6) Ziebell, H. and P. C. John. (2009). Effects of dicer-like endoribonucleases
2 and 4 on infection of Arabidopsis thaliana by Cucumber mosaic virus and a
mutant virus lacking the 2 b counter-defence protein gene. Journal of General
Virology, 90, 2288–2292.
7) Hooks, C. R. R. and A. Fereres. (2006). Protecting crop from non persistently
aphid transmitted viruses; a review on the use of barrier plants as a manage-
ment tool. Virus Research, 120, 1–16.
8) Nishiguchi, M. and K. Kobayashi. (2011). Attenuated plant viruses: prevent-

ing virus diseases and understanding the molecular mechanism. Journal of
General Plant Pathology, 77 (4), 221–229.
9) Kosaka, Y. and T. Fukunishi. (1994). Application of cross protection to the
control of black soybean mosaic disease. Plant Disease, 78, 339–341.
10) Untiveros, M. and S. Fuentes. (2007). Synergistic interaction of Sweet
potato chlorotic stunt virus (Crinivirus) with Carla-, Cucumo-, Ipomo-, and
Potyvirus infecting sweet potato. Plant Disease, 91, 669–676.
11) Hwang, T. Y. (2011). Intra host competition and interaction between Soybean
mosaic virus (SMV) Strain in mixed infected soybean. Australian Journal of
Crop Science, 5 (11), 1379–1387.
12) Gal-on, A. and Y. H. Shiboleth. (2006). Cross protection. In G. Loebenstein
and J. P. Carr (Eds.), Natural resistance mechanisms of plant to viruses (pp.
261-288). Springer.
13) Andayanie, W. R. (2012). Diagnosis of soybean seed transmission. Journal of
Tropical Plant Pests and Diseases, 12 (2), 185–191.
14) Konig, G. R. (1981). Indirect ELISA methods for the broad specificity
detection of plant viruses. Journal of General Virology, 55, 53–62.
15) SAS. (2003). SAS/STAT User’s Guide. Release 9.1. edn. SAS Institute Inc.
Cary NC, USA.
Process Design of PeptoneProduction from
Peanut Meal as byproduct of Peanut Oil
Industry Using Crude Papain
Mulyorini Rahayuningsih* and Ninuk Gilang Wiranti
Departement of Agroindustrial Technology, Faculty of Agricultural Technology, Bogor
Agricultural University
Jl. Puspa, Kampus IPB Darmaga, Bogor, Indonesia
Contact author: +628128534505
Email: mulyorinir@yahoo.com
Abstract
Peptone is an important component in the microbial growth media which has function as the
source of amino acids. Up until now, Indonesia still imports peptone which reaches $17.84
milion per year in the last five years. That fact is the reason to develop the research in peptone
production by using protein source material that is available in Indonesia, such as peanut meal
that can be obtained as by product of peanut oil industry. The objectives of this research was to
design the production process of peanut peptone using enzymatic hydrolysis by crude papain.
Design process was performed by determination of the best condition (time of hydrolysis, crude
papain concentration and temperature of hydrolysis. Peanut peptone produced was characterized
on its amino acids content and applied to be used as microbial growth media and compared with
a commercial product. Crude papain powder was obtained from the sap of papaya fruit, through
drying and milling process. The activity of crude papain used in this experiment was 5057.47
U/g min. Hydrolysis process was conducted on peanut meal that have been diluted in water
with ratio of 1:2. The research showed that the best hydrolysis condition of peanut peptone was
obtained by using 0.4% of crude papain for 4 h at temperature of 55 °C. The peptone product
has appearance of brownish yellow color in liquid form. The yield peptone produced was 40.8%.
The analysis of amino acids content showed that peanut peptone produced had high glutamic
acid aspartic acid and arginine. Those 3 amino acids are very important for microbial growth.
The result of growth test of Escherichia coli and Staphylococcus aureus by determining optical
density (OD620nm) and Total Plate Count showed that peanut peptone produced has similar
performance with commercial peptone.
Key words: Enzymatic hydrolysis, Peanut meal, Crude papain, Peanut peptone
I. Introduction
Peptone is a protein hydrolisate product which is common to be used as nitrogen
source in growth media of microorganisms. Up until now, Indonesia still imports
peptone to fulfill its demand due to there is no peptone industry available in
* Corresponding author. Email: mulyorinir@yahoo.com
85
Indonesia. Imported peptone within the last two years increase by 3,296 tonnes
with the value of US $12,15 million in 2012 and become 5,102 tonnes with the
value of US $20,76 million in 2013 [1]. This facts lead to the need of preliminary
research of development of peptone industry in Indonesia using raw materials
which are available such as protein from plants.
Peanut is one of the protein source plants with the potential to be used as
peptone raw material. Usually, peanut is used as materials for snacks, peanut
butter and peanut oil. On the production of peanut oil, peanut meal will be
produced as by products which contain 45–50% crude protein that can be
utilized as raw material of peptone. Usually, by products of peanut oil industry
is used for feed or traditional food, such as oncom which have low added value.
By conducting this research, peanut meal is utilized as peptone raw material and
applied to substitute imported peptone as microbial growth media. This research
was aimed to design the production process of peanut peptone using enzymatic
hydrolysis by crude papain. Design process was performed by determination of
hydrolysis time, enzyme concentration and hydrolysis temperature and compared
the peptone produced with commercial peptone as growth media of Escherichia
coli and Staphylococcus aureus.

A. Materials
Materials that were used were peanut meal, papaya sap, Sodium metabilsulphite,
NaCL, yeast extract, BactoTMpeptone from BD (Beckton Dickinson), BactoAgar
(BD), aquadest, Escherichia coli, Staphylococcus aureus and Nutrient Broth (BD).
B. Methods
Enzyme Preparation
Enzyme preparation was obtained by mixing papaya sap with 1.4% sodium
metabilsulphite and 0.3% NaCl in proportion of 1:1. The mixture was then
gently stirred, filtered and dried in 50–55°C, and milled. The crude enzyme was
characterized by analyzing its enzyme activity using casein as substrates. Yield of
papain enzyme obtained was 19.8% with protease activity of 5,057.47 U/g min.
Determination of Hydrolysis Time and Enzyme Concentration

Peanut meal was added with aquadest of 1:2 (b/v) and papain enzyme of 0.2%;
0.4%; and 0.6%. The mixture was mixed well and hydrolyzed for 4, 6 and 8 h.
Hydrolisis was performed at temperature of 60°C in incubator. This step was
stopped by enzyme inactivation by heating the samples at 85°C for 15 minutes
in water bath. The samples were then filtered in 225 meshes and centrifuged at
4,000 rpm for 15 minutes to obtain soluble fraction. Hydrolisis performance was
determined by analyzing ratio of Total of Soluble Nitrogen to Nitrogen Total of
material using Kjedhal method.
Determination of Hydrolisis Temperature

After hydrolysis, time and enzyme concentration were obtained and the research
was continued by determining hydrolysis temperature. Hydrolysis was carried
out in temperature of 50, 55 and 60°C. Performance of hydrolysis was measured
by determining ratio of Total of Soluble Nitrogen to Nitrogen Total of material
using Kjedhal method.
Determination of Amino Acid Content in Peptone Produced

The peptone produced was analyzed by HPLC to determine the amino acids
content.
Application of Peptone Produced as Bacterial Growth Media

The peptone produced was analyzed to be used as growth media of bacteria and
compared with commercial peptone (BactoTMpeptone, BD). The bacteria used
were Escherichia coli and Staphylococcus aureus.
Liquid medium for growing bacteria was prepared by mixing 0.5% yeast
extract, 1% peptone, and 1% NaCl. The using of peptone samples that were
produced from peanut meal hydrolysate was set up by equalizing the total of
nitrogen samples with total of nitrogen commercial peptone. Medium was
sterilized at 121°C for 15 minutes. Bacteria were inoculated to 10 ml of medium
containing peptone produced and then incubated at 37°C for 24 h and the optical
density (OD 620 nm) was measured as indicator of bacterial growth.
Bacterial growth as Total Plate Count was also measured by plating bacteria
on agar plate medium using peptone produced and compared with commercial
peptone. The composition of media was the same as liquid medium but it was
added by 1.5% Bacto Agar. After inoculation, the agar plates were incubated
at 37°C for 48 h. The number of colony was counted by using Colony Counter
Quebec.
Experimental Design
Experimental design used in this research was Randomized Factorial Design
which performed in two steps namely Randomized Factorial Design two factors to
determine hydrolysis time and enzyme concentration; and Randomized Factorial
Design one factor to determine the effect of hydrolysis temperature. The results
that showed significant different value was analyzed further using Duncan test.

Hydrolysis Time and Enzyme Concentration
The best hydrolysis condition was determined based on ratio of Total of Soluble
Nitrogen (NTT) to Nitrogen Total of Material (NTB). Higher Ratio of NTT/
NTB meant that hydrolysis process was optimally occurred [2].
Figure 1 showed the effect of hydrolysis time and enzyme concentration on
ratio of NTT/NTB. Based on statistical analysis, it was determined that hydrolysis
time and enzyme concentration significantly affected ratio of NTT/NTB (p <
0.05). The highest ratio of NTT/NTB was obtained at 4 h of hydrolysis time with
enzyme concentration of 0.4%. Duncan test result showed that 4 h hydrolysis
time was significantly different with 6 h and 8 h of hydrolysis time. Enzyme
concentration of 0.4% was significantly different with enzyme concentration of
0.2% but not significantly different with enzyme concentration of 0.6%.
Hydrolisis time is one factor that affects the enzyme stability, which tends
to decrease along with hydrolysis time [3]. In addition to that, [4] stated that
hydrolysis rate and nitrogen recovery will increase along with the increasing of
enzyme concentration. Ratio of NTT/NTB which relatively low with increasing
Figure 1. The effect of hydrolysis time and enzyme concentration on

ratio of NTT/NTB. Different superscript indicated significant differ-
ence (p < 0.05).
hydrolysis time can be affected by decreasing enzyme stability. Besides that, [5]
stated that enzymatic hydrolysis rate decrease and reaches stationary phase when
there is no more hydrolysis process occured. Soluble protein hydrolysate will be
obtained in early stage of hydrolysis, but although an amount of enzyme is added
on stationary stage of hydrolysis process, there will be no increase of hydrolysate
yield. On that condition, product inhibition will be occurred. More over, the
decrease of enzymatic reaction rate can be occurred by decreasing specific peptide
bond, enzyme inactivation and competition between substrates with peptide
obtained from hydrolysis process [6].
Hydrolysis Temperature
Figure 2 showed that hydrolysis temperature affected ratio of NTT/NTB. The
highest ratio of NTT/NTB was obtained on hydrolysis temperature of 55°C.
Statistical analyzes followed by Duncan test showed that hydrolysis temperature
of 55°C was significantly different with hydrolysis temperature of 50°C and
was not significantly different with hydrolysis temperature of 60°C. Therefore,
hydrolysis temperature of 55°C was decided as the best temperature for peanut
meal peptone production using crude papain concentration of 0.4% for 4 h.
The increase of temperature affects chemical reaction rate due to the
increase of kinetic energy between reactant. This phenomenon also occurred in
enzymatic reaction which involves substratesand enzymes. However, because
an enzyme is protein, the increase of enzymatic reaction will be performed on
certain temperature. If hydrolysis temperature is too high, protein enzyme can be
denaturated, tertiary enzyme structure can be disrupted and the enzyme catalytic
activity will decrease [7].
Figure 2. The effect of hydrolysis temperature on Ratio

of NTT/NTB. Different superscript indicated significant
difference (p < 0.05).
Amino Acid Content in Peptone Produced

The concentration of amino acids in peptone produced was different with
commercial peptone (BactoTMpeptone). Peanut peptone contained three highest
amino acids, namely 1.63% glutamic acid, 0.87% aspartic acid and 0.78%
arginine while the lowest amino acid was methionine (0.06%). Commercial
peptone contained the highest amino acid namely glycine amounted 2.03%.
The different of amino acid content is caused by the difference of raw material
used in peptone production. [8] stated that BactoTMpeptone was produced by
using of animal protein as raw material and hydrolyzed by pancreatic enzyme.
Application of Peptone Produced as Bacterial Growth Media

Escherichia coli
Testing result of peptone produced on formulation of liquid media showed
that peanut peptone produced had the higher ability for growing of E. coli than
BactoTMpeptone. This can be showed by the results of turbidity determination
of the culture (OD620 nm) which relatively higher than commercial peptone
(Figure 3a).
Statistical analysis showed that OD620 nm of culture broth using peanut
peptone produced was significantly different with commercial peptone. Different
with liquid media, application of peanut peptone produced in solid media showed
that the growth ability was not significantly different with commercial peptone
(Figure 3b).
According to [9], amino acid serine, aspartic acid, and glutamic acid are amino
acids which are very important for bacterial growth. Peanut peptone produced
contained higher glutamic acid and aspartic acid than commercial peptone, so
those could support better cells growth of bacteria.
Staphylococcus aureus
Figure 4a showed testing result of peanut peptone produced for growing of
Staphylococcus aureus. The result showed that peptone produced had higher ability
to support growth of S. auerus than commercial peptone. Statistical analysis
showed that the growth response of peanut peptone produced was significantly
different with commercial peptone.
The same with its response to E. coli, application of peanut peptone in solid
media for growing Staphylococcus aureus showed that they were not significantly
different (Figure 4b). By testing the peanut peptone produced for growing of
E. coli and S. aureus both in solid and liquid media, generally, peanut peptone
produced has similar ability with commercial peptone BactoTMpeptone from BD.
*Peptone on hydrolysis temperature: 50°C (S50); 55°C (S55); 60°C (S60); and commercial
peptone (C)
(a) (b)
Figure 3. Growth for Escherichia coli in (a) liquid media and (b) solid media. Different superscript
indicated significant difference (p < 0.05).
*Peptone on hydrolysis temperature: 50oC (S50); 55oC

(S55); 60oC (S60); and commercial peptone(C)
Figure 4a. Growth of Staphylococcus aureus in liquid

media. Different superscript indicated significant
difference (p < 0.05).
IV. CONCLUSION
Peanut peptone can be produced by hydrolysis of peanut meal using crude papain
enzyme. Production process of the peptone can be performed by hydrolysis
condition using 0.4% of crude papain for 4 hat temperature of 55 oC. The yield
peptone produced was 40.8%. Amino acids content in peanut peptone that was
obtained had high glutamic acid, aspartic acid and arginine. Generally, peanut
peptone produced has similar ability with commercial peptone BactoTMpeptone
for growing of E. coli and Staphylococcus aureus in liquid and solid media.
V. ACKNOWLEDGMENT
This research was funded by Directorate General of Higher Education, Ministry
of National Education from the scheme of Student Creativity Programme 2014.
*Peptone on hydrolysis temperature:

50 °C (S50); 55 °C (S55); 60 °C
(S60); and commercial peptone (C)
Figure 4b. Growth of Staphylococcus

aureus in solid media. Different super-
script indicated significant difference
(p < 0.05).
VI. REFERENCES
1) Badan Pusat Statistik. (2014). Tabel impor menurut komoditi. Retrieved from
http://www.bps.go.id/exim-frame.php?kat=2&id_subyek=08&notab=50
2) Wijayanti, A. T. (2009). Kajian penyaringan dan lama penyimpanan dalam
pembuatan fish peptone dari ikan selar kuning (Caranx leptolepis) (Master’s
thesis). Bogor, Indonesia: Institut Pertanian Bogor.
3) Rose, A. H. (1980). Micobial Enzyme and Bioconversion (Economics
Microbiology Vol. 5). New York, US: Academic Press.
4) Benjakul, S. and M. Morrisey. (1997). Protein hydrolysates from pasific whit-
ing solid wastes. Journal of Agricultural and Food Chemistry, 45, 3423–3430.
5) Shahidi, F., Xio-Qing, H. and J. Synowiecki. (1995). Production and
characteristics of protein hydrolysates from capelin (Mallotus villosus). Food
Chemistry, 53, 285–293.
6) Netto, F. M. and M. A. M. Galeazzi. (1998). Production and characterization
of enzymatic hydrolysates from soy protein isolate. Lebensmittel-Wissenschaft
& Technologie, 31 (8), 624–631.
7) Thenawidjaja, M. (1984). Pengantar Kinetika Enzim. Bogor, Indonesia:
Institut Pertanian Bogor.
8) Becton, Dickinson and Company. (2009). DifcoTM & BBLTM Manual of
Microbiological Culture Media 2nd ed. Sparks, Maryland: BD Diagnostics.
9) Selvarasu, S., Wei Ow, D. S., Lee, S. Y., Lee, M. M, Weng Oh, S. K., Karimi,
I. A. and D. Y. Lee. (2008). Characterizing Escherichia coli DH5α growth
and metabolism in a complex medium using genome-scale flux analysis.
Biotechnology and Bioengineering, 102, 923–934.
Potency of IAA Hormone Produced by
Endophytes Bacteria Isolated from Shorea
selanica on Supporting the Growth of
Paraserinthes falcataria
Tiwit Widowati*, Sylvia J. R. Lekatompessy, Nuriyanah and Harmastini Sukiman

Research Center for Biotechnology-LIPI
Jl. Raya Bogor KM 46, Cibinong, Bogor, Indonesia
Abstract
Indole acetic acid is a key hormone for various aspects of plant growth and development. The
objectives of this study were to screen endophytes bacteria from Shorea selanica that are able
produce IAA hormone and determine the effect of IAA hormone on the growth of Paraserianthes
falcataria. Eight isolates of endophytes were studied by growing them in Nutrient Broth medium
supplemented with Tryptophan. Isolates SSBt1 and SSBt2 had ability to produce the highest IAA
with concentration about 40 µg/ml. Both isolates were studied on producing IAA at different pH
for 120 hours and were also selected for determining their capability on supporting the growth of
P. falcataria in semi solid media. The results indicated that IAA produced by endophytes bacteria
isolated from S. selanica are able to induce elongation of primary root and stem, numerous of
lateral and sub lateral roots of P. falcataria.
Key words: IAA, Endophytes bacteria, Paraserianthes falcataria
I. INTRODUCTION
Shorea selanica is a Dipterocarp family, which has a high economic value. It is
valuable for joinery, furniture, paneling, flooring and plywood manufacture [1].
The resin, called damar, is used in traditional medicine, while the bark is used for
tanning. Dipterocarpaceae is one of forest plants which have suffered a reduction
in population mainly because of timber exploitation. The conservation efforts for
these species have been initiated in Indonesia.
Shorea is one of featured trees that recommended by Indonesian government
for rehabilitation and management of natural and industry forest. The aims of
forest management are to establish a healthy, prospective and sustainable forest
[2]. One of the components that were developed in the forest management
program is the use of microbes to support the growth of seedlings. A healthy
forest ecosystem inhabited by beneficial organisms such as microbes producing
plant growth regulators [3]. It is reported that the rhizosphere of soil ecosystems
* Corresponding author. Phone: +62-21-8754587. Email: tiwitwidowati@yahoo.com
93
will be inhabited by beneficial organisms, including microbes producing plant

growth regulators. Microbe,s mainly bacteria, can live symbiotically with plants,
free-living in the soil or around plant roots (rhizosphere) [4].
Endophytic bacteria can be defined as those bacteria that colonize the internal
tissue of the plant without harming the host plant. These bacteria play a role in
supporting the growth of plants in several ways, such as through the secretion
of plant growth regulators, phosphate solubilizing, siderophore production and
supply of nitrogen [5]. Growth hormones, such as auxin, cytokinins, gibberellins,
ethylene and absisic acid are organic compound in low concentration that affect
the growth, differentiation and development of plants [6].
Auxin is a plant hormone that regulates several physiological processes, such
as stimulates cell division and elongation, root initiation, seed germination and
seedling growth. IAA is an endogenous auxin hormone that is synthesized in stem
and roots. L-tryptophan is an amino acid as precursors for biosynthesis of auxins
in plants and microbes [7]. Biosynthesis of IAA-producing microbes in the soil
can be stimulated by the tryptophan derived from root exudates. This precursor
contains active compounds that stimulate the growth of microbes in producing
secondary metabolites.
The utilization of endophytes bacteria as phytohormone that isolated from
forest plant in the tropics is still limited. It is necessary to study the application of
IAA-producing bacteria on stimulating plant growth. In this study, the bacteria
were selected to determine their capability on producing IAA hormone and
compatibility on supporting the growth of Paraserianthes falcataria root.
II. MATERIALS AND METHODS

Isolation and screening of IAA production
Shorea selanica samples were obtained from Forest Research of Mount Dahu,
Bogor. Isolation of endophytes bacteria from the stems and leaves of Shorea
selanica was done according to [8]. Isolation and screening of endophytic bacteria
was performed in the Laboratory of Plant Symbiotic Microbes, Biotechnology
Research Center of LIPI. Bacteria isolates were selected for IAA production in vitro
by Dey et al. [9] method. These pure cultures were grown on 10 ml of Nutrient
Broth (NB) containing 0.5 mM L-tryptophan. These cultures were incubated at
28°C with shaking at 150 rpm for 24 hours and then harvested by centrifugation
at 10,000 rpm for 10 minutes. IAA production was tested by colorimetric using
a reagent Salkowsky [10]. Reagent Salkowsky contained 150 ml H2SO4, 250
ml aquades, 7,5 ml FeCl3.6H2O 0.5M. Two ml of supernatant was mixed with
2 ml of reagent Salkowsky. The suspension was incubated at room temperature
in dark for 60 minutes and then Optical Density (OD) was measured with a
spectrophotometer at wavelength of 530 nm. The appearance of a pink color

indicated IAA production. The value of IAA produced was compared with the
IAA standard.
Culture optimization for the production of IAA

The isolates, which produce the highest IAA, was studied to identify the optimal
condition for IAA production. IAA concentration was measured using different
parameters. The effect of incubation time on IAA production was studied by
cultivating the isolates in NB media containing 0,5 mM L-tryptophan. Samples
were observed every 24 hours for 5 days. The effect of pH on IAA production
was studied by cultivating the isolates in NB containing 0,5 mM L-tryptophan
at different pH levels ranging from 4.0–7.0 for 3 days.
Biological Test on the Growth of P. falcataria

Another experiment was done to see if IAA producing Shorea selanica can induce
rooting in another plant species. Isolates which produce the highest of IAA were
grown on NB medium and incubated for 24 hours. Seedlings of Paraserianthes
falcataria were soaked in 10 ml of bacterial suspension for 30 minutes. Each
seedling was inserted into tube containing 25 ml of agar 1%, then added 1
ml of bacterial suspension in each tube. On the control treatment, seedlings
of P. falcataria were added with NB. Each treatment consisted of 5 replicates.
Measurements of growth were done after 2 months planting, consisted of stem
length, root length, number of lateral and sub lateral roots.
III. RESULT AND DISCUSSION

Eight endophytic bacteria have been obtained from the stems and leaves of S.
selanica. Morphologically, there is a difference round colony size ranging 1–3
mm and color variations (white and pink) (Table 1).
Table 1. Colony color of endophyte bacteria from Shorea selanica
No Code of isolate Color of colony
1 SSBt1 White
2 SSBt2 White
3 SSKK2 White
4 SSKK4 White
5 SSKK6 White
6 SSDt1 Pink
7 SLBt1 White
8 SLBt2 Pink
Differences of shapes, sizes, color and the amount of endophytic bacteria

obtained, related to the type and age of plant, the type of infected tissue, sampling
time and environment. Environment where the plant grows also affects the
composition and structure of microbial species that colonize the roots, stems,
twigs and leaves [11].
All of the isolates were selected capability to produce the IAA hormone. There
are 3 isolates that able to produce IAA ranged from 15–43 µg/ml, whereas the
other isolates could not produce IAA.
Table 2. IAA production of endophyte bacteria from S. selanica
No Code of isolate IAA production (µg/ml)
1 SSBt1 39,89041
2 SSBt2 43,00685
3 SSKK2 -0,4863
4 SSKK4 -2,69863
5 SSKK6 5,061644
6 SSDt1 -3,59589
7 SLBt1 -3,4863
8 SLBt2 -5,31507
Isolates that were able to produce IAA indicates that it can synthesize trypto-
phan contained in the media to become IAA. The difference of IAA concentration
produced by bacteria might be caused by differences of bacteria ability to utilize
tryptophan. These differences are influenced by differences of indole pyruvate
decarboxylase enzyme activity. The concentration of IAA produced also depends
on the activity and the number of cells, nutrients and substrate availability of
tryptophan in the medium. The presence of tryptophan in the medium causing
microbes can produce IAA and other compounds in large quantities [12].
Optimization of the production of IAA from isolates SSBt1 and SSBt2 that
produce the highest IAA, started after 24 hours and reached a maximum after 72
hours and then decreased slowly (Figure 1). IAA is a secondary metabolite that is
excreted by the microorganisms nearing the end of the growth phase or during
the stationary phase. According to Lestari et al. (2007) [13], at the beginning of
growth, there are many nutrients so that IAA production is high and continues to
increase, but not significantly and consistently until the end of the growth phase.
According to Kresnawaty et al. (2008) [14], at 24-hours incubation time,
production of IAA was still minimal because it is still in the logarithmic phase
and the content of enzymes to convert tryptophan is still low. At 72-hours
incubation time, the highest IAA was produced because isolate was at the end
of logarithmic phase and the enzymes used to synthesize tryptophan into IAA is
quite high and active in line with the growth rate. The enzyme involved in the
Figure 1. Effect of incubation time on IAA production from isolates SSBt1 and SSBt2
synthesis of IAA including tryptophan monooxygenase, IAM hydrolase, indole-

pyruvate decarboxylase and IAAld dehydrogenase. Declining of IAA production
after 72-hours incubation time caused the isolates to enter the death phase. This
decline is caused by release of IAA degrading enzyme like IAA peroxidase and
IAA oxidase [15].
The maximum production of IAA was at pH 5 (Figure 2). There was significant
relationship between the growth of bacteria and IAA production where pH
influenced the function of enzyme system and dissolves the important compounds
for bacteria growth. pH is too acidic or alkaline and this is not suitable for IAA
production.
Biological test showed that the growth of P. falcataria treated with inoculants
was higher than control. The height of plant and length of roots (Figure 3) looked
significantly different.
IAA produced by SSBt1 and SSBt2 isolates showed the ability of bacteria
to stimulate the primary roots growth of P. falcataria, where the roots that were
treated by endophyte bacteria was 65% longer compared to control. Plant heights
of treatment were 45% higher than control. There was a significant relationship
between auxin produced endophyte bacteria by in vitro with the growth of P.
falcataria seeds. This indicated that auxin was produced both of isolates causing
improvement of root system to resulted greater biomass.
Seeds of P. falcataria were inoculated with SSBt1 and SSBt2 isolates stimulated
the growth of lateral roots number compared to controls (Figure 4). The ability
of bacteria to produce high IAA may be related to the high level of IAA in the
roots. Endophyte change IAA on the roots that contribute in the development
Figure 2. Effect of pH on IAA production of isolates SSBt1 and SSBt2
Figure 3. Plant height and root length of P. falcataria
of root hairs and root number in plants. Interaction between IAA-producing

bacteria and plants resulted in plant growth promoter [16].
Auxins were synthesized naturally by plants from amino acid tryptophan.
The ability to synthesize IAA spread on soil bacteria and bacteria associated with
plants [17]. IAA was secreted by plant growth promoter bacteria directly through
Figure 4. Number of lateral and sublateral root of P. falcataria
stimulation of plant cell elongation or cell division or indirectly influenced by

ACC deaminase enzyme activity. ACC deaminase in bacteria was involved in
root elongation of seed [10]. The synthesis of plant growth regulator microbe is
an important factor for soil fertilization.
IV. CONCLUSION
This study showed that bacteria SSBt1 and SSBt2 isolated from stem of Shorea
selanica had the capability to produced IAA on medium containing tryptophan.
Both isolates were also effective and compatible to stimulate elongation of P.
falcataria roots.
V. REFERENCES
1) Sakai, C., Subiakto, A., Heriansyah, I. and H. S. Nuroniah. (1999). Reha-
bilitation of degraded forest with Shorea leprosula and Shorea selanica. In S.
Kobayashi, J. W. Turnbull, T. Toma, T. Mori, and N. M. N. A. Majid (Eds.),
Rehabilitation of Degraded Tropical Forest Ecosystems. Workshop Proceedings
2-4 November (pp. 191–195). Bogor, Indonesia: CIFOR.
2) Soekotjo. (2007). Komponen silvikultur intensif dalam rangka membangun
hutan yang sehat, prospektif dan lestari. Jakarta: Dirjen Bina Produksi
Kehutanan, Departemen Kehutanan RI.
3) Hindersah, R. and T. Simarmata. (2004). Potensi rhizobakteri Azotobacter
dalam meningkatkan kesehatan tanah. Jurnal Natur Indonesia, 5, 127–133.
4) Glick, B. R. (1995). The enhancement of plant growth by free-living bacteria.

Canadian Journal of Microbiology, 41, 109–117.
5) Bandara, W. M. M. S., Seneveratne, G. and S. A. Kulasooriya. (2006).
Interactions among endophytic bacteria and fungi: effects and potentials.
Journal of Biosciences, 31, 645–650.
6) Kiba, T., Kudo, T., Kojima, M. and H. Sakakibara. (2010). Hormonal control
of nitrogen acquisition: roles of auxin, abscisic acid and cytokinin. Journal of
Experimental Botany, 62, 1399–1409.
7) Tarabily, K., Nassar, A. H. and K. Sivasithamparam. (2003). Promotion of
plant growth by an auxin producing isolate of the yeast Williopsis saturnus
endophytic in maize roots. Paper presented at the Sixth U. A. E. University
Research Conference, 2003.
8) Tomita, F. (2003). Endophytes in Southeast Asia and Japan: their taxonomic
diversity and potential applications. Fungal Diversity, 14, 187–204.
9) Dey, R., Pal, K. K., Bhat, D. M. and S. M. Chauhan. (2004). Growth promo-
tion and yield enhancement of peanut (Arachis hypogeal L.) by application
of plant growth promotion rhizobacteria. Microbiological Research, 159,
371–394.
10) Patten, C. L. and B. R. Glick. (2002). Role of Pseudomonas putida indoleacetic
acid in development of the host plant root system. Applied and Environmental
Microbiology, 68, 3795–3801.
11) Araujo, W. L., Marcon, J., Maccheroni, W., Elsas, J. D., Vuurde J. W. L. and
J. L. Azevedo. (2002). Diversity of bacterial populations and their interaction
with Xylella fastidiosa in citrus plant. Applied and Environmental Microbiology,
68, 4906–4914.
12) Lee, S., Encarnation, M. F., Zentalla, M. C., Flores, L. G., Escamilla, J. E.
and C. Kennedy. (2004). Indole-3 acetic acid biosynthesis is deficient in
Gluconacetobacter diazotrophicus strains with mutations in cytochrome c
biogenesis genes. Journal of Bacteriology, 186, 5384–5391.
13) Lestari, P., Susilowati, D. N. and R. I. Riyanti. (2007). Pengaruh hormon
asam indol asetat yang dihasilkan oleh Azospirillum sp terhadap perkembangan
akar padi. Jurnal AgroBiogen, 3, 66–71.
14) Kresnawaty, I., Andawarih, S., Suharyanto and T. Panji. (2008). Optimization
and purification of iaa produced by Rhizobium sp. in latex serum media
supplemented with tryptophan from chicken manure. Balai Penelitian
Bioteknologi Perkebunan, 76, 74–82.
15) Datta, C. and P. S. Basu. (2000). Indole acetic acid production by a Rhizobium
species from root nodules of leguminous shrub, Cajanus cajan. Microbiological
Research, 155, 123–127.
16) Spaepen, S., Fanderleyden, J. and R. Remans. (2007). Indole acetic acid in
microbial and microorganism-plant signaling. FEMS Microbiology Reviews,
31, 425–448.
17) Verma, S. C., Ladha, J. K. and A. K. Tripathi. (2001). Evaluation of plant
growth promoting and colonization ability of endophytes diazotrophs from
deep water rice. Journal of Biotechnology, 91, 127–141.
Production and characterization of
the Biosurfactant by the formation of
glycolipid isolated from Pseudozyma
hubeiensis Y10BS025
Martha Sari*, Fifi Afiati and Wien Kusharyoto

Research Center for Biotechnology-Indonesian Institute of Sciences (LIPI)
Jalan Raya Jakarta-Bogor Km. 46, Cibinong, Indonesia
Abstract
The present study demonstrates the production and properties of a biosurfactant isolated from
P. hubeiensis Y10BS025. Biosurfactants are produced by a variety microorganism to produce
renewable resources which have a unique properties. P. hubeiensis was cultivated in a medium
containing glucose and soybean oil to induce and intensify biosurfactant (bioemulsifier) synthesis.
To confirm the ability of isolate in glycolipid biosurfactant production, TLC pattern, emulsification
test and stability of the biosurfactant was investigated. The strain Y10BS025 produce bioemulsifier
and exihibit emulsification index (E24) of 47–72% with oils tested and resultant emulsion
found more stable compare to chemically made surfactant. This strain is a potential candidate
for microbial enchanced oil recovery.
Key words: P. hubeiensis, Biosurfactant, Glycolipid, TLC, Emulsifier
I. Introduction
There is a considerable interest in the development of biobased materials, such
as biosurfactants molecule from the global environmental. Microbes are well
known for their varied bioactive properties, include production of secondary
metabolites and bioactive compounds [7]. Biosurfactant are a structurally diverse
group of surface active molecules synthesized by various microbial genera like
bacteria, fungi and yeast [1]. Biosurfactants can aggregate at interfaces between
fluids having different polarities, such as water and oil, leading to the reduction
of interfacial tension, therefore become a good candidates as enhanced oil
recovery [6]. Due to these superior characteristics, biosurfactants have potential
use in pharmaceutical industries, cosmetics, food and energy saving technology
[10]. Several type of biosurfactant have been isolated and characterized, based
on glycolipids, phospholipids, lipopeptides, natural lipids, fatty acids and
lipopolysacharides properties. The principle screening of biosurfactant production
* Corresponding author. Phone: +628754587. Email: martha.biotek@gmail.com
103
is finding new structures with strong interfacial activity, high emulsion stability,
and good solubility [4].
Yeast glycolipids biosurfactant, mannosylerythritol lipids (MELs) are one of
the most promising biosurfactants known and are abundantly produced from
vegetable oils by Pseudozyma [8]. The strain showed the ability to produce
assayable biosurfactant activity [9]. Sesame oil is the best substrate for glycolipid
biosurfactant production; however, the production and recovery processes are
very complicated. Therefore, we used soybean oil as raw material for producing
glycolipid biosurfactant using P. hubeiensis instead of sesame oil. The purification
was done using different solvents, and it determined the active emulsion properties
of the biosurfactant within 30 days stability compared with chemicals surfactant,
Triton X at room temperature.
II. Method/material
A. Biosurfactant production
P. hubeiensis YB10BS025 in this study was obtained from Indonesian Institute of
Sciences (LIPI). Standard procedure for the production of glycolipid by fermenta-
tion was adopted from Sari et al., 2013. Seed culture were prepared by growing
cells in medium (40 g glucose/l, 3 g NaNO3/l, 0.3 g Mg SO4/l, 0.3 KH2PO4/l, 1 g
yeast extract/l) at 30°C on a reciprocal shaker (200 rpm) for 2 days. Seed culture
(0.1 ml) were transferred to Erlenmeyer flask containing 100 ml basal medium
using 40 g soybean oil/l as substrate. Cultivation was performed at 30°C, for 8
days using agitation at 150 rpm. After cultivation. The biosurfactant was harvested
from the supernatant, after the cells were separated by centrifugation at 6,000
rpm for 20 min and washed twice with distilled water under sterile conditions.
B. Isolation of glycolipid biosurfactant

The glycolipid biosurfactant, MELs produced were extracted from the culture
medium with an equal amount of ethyl acetate (1:1, v/v) for 1 h [5]. The organic
layer was separated and evaporated. Its volume was measured and concentrated
using vacuum evaporation. The concentrate of the glycolipid was washed with
300 ml n-hexane-methanol-water (1:6:3) to remove the remaining oil and fatty
acids (top phase) and then purified by silica gel (Wako gel C-200) column
chromatography using a gradient elution of chloroform/acetone (10:0-7:3,
v/v) mixture as solvent systems [2]. A separation of residual fatty acid was
achieved by using methanol and hexane. The extracts were analyzed by thin layer
chromatography (TLC) on a silica gel plate G-60 (Merck) with a solvent system
consisting of chloroform-MeOH-NH4OH (65:15:2, v:v:v). The compounds on
the plates were located by charring at 110°C for 5 min after spraying an anthrone
reagent as previously reported [3].
C. Assessment of bioemulsification index (E24) & Stability of emulsion

Bioemulsification index of culture sample was measured by adding 2.0 ml of
various hydrocarbon (sesame oil, canola oil, olive oil, crude oil, and soya oil)
to the same amount of culture, vortex for 30 min in individual tubes, and kept
at room temperature. Emulsification index (E24) was calculated after 24 h, by
measuring the height of emulsion layer with respect to original volume and
expressed as percentage.
For the assessment of stability of emulsion, extract purified obtained from
experiments are kept at room temperature for the period of 0–30 d. Sample
exhibiting nilphase separation were considered as high stable emulsion [7].
III. R esults and discussion

In this study, P. hubeiensis Y10BS025 was tested for ability to produce glycolipids
biosurfactant using soybean oil as substrate. We found that glycolipid biosurfactant
are abundantly produced by P. hubeiensis Y10BS025 with wide variety of ratio of
eluent in isolation systems. The mixture glycolipids were extracted from the total
culture suspension of P. hubeiensis Y10BS025, and purified by solvent diversifica-
tion system. The key components of the major glycolipid were checked into five
main fractions eluted base on the solvent mixture (Figure 1). As shown in Figure
1, the partial glycolipid contains those five fractions, named A, B, C, D, and E.
On TLC, the major glycolipid spot from the strain P. hubeiensis Y10BS025
was selected based on the anthrone reagent positive spots as those in the brown
bead of silica gel plate. The fraction of glycolipid was eluted mainly with fraction
chloroform/acetone (B). This result indicated that P. hubeiensis Y10BS025 is able
to use glucose as the carbon and soybean oil as substrate to provide glycolipid
biosurfactant. We also detected some glycolipid spot that eluted together with
residual oils in the chloroform/acetone fraction (C) when analyzed on TLC
(blue beads). In the extract from the broth culture with combination glucose and
soybean oil, by product of the residual oil were separated by using methanol (D)
with subsequent threefold extraction by n-hexane.
The varied solvent extraction is the most commonly used technique for isola-
tion and purification of biosurfactant spot on silica gel plate. The choice of method
for the purification of a particular biosurfactant depends on its ionic charge, its
solubility in water, and on whether the product is cell bound or extracellular [3].
Yeast strain belonging to genus Pseudozyma showed potential emulsification
activity. Figure 2 shows that different emulsification indexes of the crude
Figure 1. TLC pattern of the extracellular products from

soybean oil by P. hubeiensis Y10BS025 under different
fractions (A) crude ethyl acetate, (B) fraction chloroform/
acetone 10:0, (C) fraction chloroform/acetone 7:3, (D)
methanol fraction, (E) hexane fraction.
A B C D E
biosurfactant produced by P. hubeiensis strain Y10BS025 in a comparison with

Triton X using different oils. The crude biosurfactant was found to be able to form
stable emulsion between water and various oils (soybean oil, sesame oil, crude
oil, canola oil). Results showed that soybean oil has maximum activity against
all of test oils. The emulsifying index of the biosurfactant ranged from 47–72%.
In all oils tested, supernatant supplemented with soybean oil gave a better yield
was achieved 72% of emulsification index compared to synthetic supernatant,
Triton X (57%). On other hand, crude oil were found to be good substrates for
emulsification by the glycolipid biosurfactant from P. aeruginosa strain SP4 [6].
With regard to stability of emulsions, we observed emulsion (without emulsi-
fiers, with bioemulsifiers of Y10BS025, and synthetic emulsifiers). Amongst the
three bioemulsifier tested, the Y10BS025 bioemulsifier exhibits the good stability
emulsion and equivalent with synthetic surfactant without any phase separation
(Figure 3). However, Radhakrishnan et al., 2011 reported stability of emulsion
formation with crude oil up to 90 d. In general, stability of emulsion depends
on various physical and biochemical factors, include distribution of emulsion,
size of particals, external force, ion strength, structure, molecular weight of the
emulsifier, pH, metal ions, and finally the temperature.
IV. CONCLUSION
In this study, P. hubeiensis Y10BS025 was found to produce glycolipid biosurfac-
tant from a fermentation medium containing soybean oil as substrate. The major
product was identified on the fraction chloroform/acetone that appears as positive
brown bead spot in silica plate. The crude biosurfactant showed comparable
physicochemical properties in term of the emulsification index compared to
synthetic surfactants (Triton X). The emulsion found stable without any phase
separation till 30 days. This findings indicated that the present strain have great
potential for microbial enchanced oil recovery (MEOR) applications.
Figure 2. Emulsifying index of the bioemulsifiers of yeast

strain with various oils, compared with chemical emulsifier,
Triton X.
Figure 3. Comparison of emulsion stability of (a) without emulsifier,

(b) with bioemulsifiers Y10BS025, and (c) with synthetic emulsifiers,
observed before and after 30 days of incubation.
V. Acknowledgement
We would like to thank Dr. Atit Kanti (Indonesian Institute of Science) for
provision of isolate in this study.
VI. R eferences
1) Khopade, A., Ren, B., Liu, X. Y., Mahadik, K., Zhang, L. and C. Kokare.
(2012). Production and characterization of biosurfactant from marine
Streptomyces species B3. Journal of Colloid and Interface Science, 367, 311–318.
2) Konishi, M., Nagahama, T., Fukuoka, T., Morita, T., Imura, T., Kitamoto,
D. and Y. Hatada. (2011). Yeast extract stimulates production of glycolipid
biosurfactants, mannosylerythritol lipids, by Pseudozyma hubeiensis SY62.
Journal of Bioscience and Bioengineering, 111 (6), 702–705.
3) Liu, Y., Koh, C. M. J. and L. Ji. (2011). Bioconversion of crude glycerol to
glycolipids in Ustilago maydis. Bioresources Technology, 102, 3927–3933.
4) Morita, T., Konishi, M., Fukuoka, T., Imura, T., Yamamoto, S., Kitagawa,
M. and D. Kitamoto. (2009). Production of a novel glycolipid biosurfactants,
mannosylerythritol lipids, by Pseudozyma parantarctica and their interfacial
properties. Journal of Bioscience and Bioengineering, 105 (5), 493–502.
5) Morita, T., Ogura, Y., Takashima, M., Hirose, N., Fukuoka, T., Imura, T.,
Kondo, Y. and D. Kitamoto. (2011). Isolation of Pseudozyma churashimaensis
sp, a novel ustilaginomycetous yeast species as a producer of glycolipid
biosurfactant, mannosylerythritol lipids. Journal of Bioscience and Bioengineering,
112 (2), 137–144.
6) Pornsunthorntawee, O., Wongpanit, P., Chavadej, S., Abe, M. and R.
Rujiravanit. (2008). Structural and physicochemical characterization of
crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from
petroleum contaminated soil. Bioresource Technology, 99, 1589–1595.
7) Radhakrishnan, N., Kavitha, V., Madhavacharyulu, E., Gnanamani, A.
and A. B. Mandal. (2011). Isolation, production and characterization of
bioemulsifiers of marine bacteria of coastal Tamil Nadu. Indian Journal of
Geo-marine Science, 40, 76–82.
8) Sari, M., Kanti, A., Artika, M. and W. Kusharyoto. (2013). Identification
of Pseudozyma hubeiensis Y10BS025 as a potent producer of glycolipid
biosurfactant mannosylerythritol lipids. American Journal of Biochemistry and
Biotechnology, 9 (4), 430–437.
9) Sari, M., Kusharyoto, W. and M. Artika. (2014). Screening for biosurfactant

producing yeast: Confirmation of biosurfactant production. Biotechnology,
13, 106–111.
10) Takahashi, M., Morita, T., Wada, K., Hirose, N., Fukuoka, T., Imura, T. and
D. Kitamoto. (2011). Production of sophorolipid glycolipid biosurfactant
from sugarcane molasses using Starmerella bombicola NBRC 10243. Journal
of Oleo Science, 60 (5), 267–273.
APPLICATION OF MARKER ASSISTED SELECTION
AND SENSORY TEST FOR SELECTING AROMA ON
F2 PROGENY OF RICE DERIVED FROM CROSSING
BETWEEN CIHERANG X BASMATI
Santika Saria, Nono Carsonob,*, Murdaningsih Haerumanb and Farida Damayantib
Graduated Student, Master Program in Plant Breeding
a
Faculty of Agriculture, Universitas Padjadjaran, Jatinangor, Sumedang, Indonesia

Phone/Fax +62227796316
b
Laboratory of Plant Biotechnology and Breeding
Faculty of Agriculture, Universitas Padjadjaran, Jatinangor, Sumedang, Indonesia
Phone/Fax +62227796316
Abstract
Aroma is one of the characters that influences consumer acceptance in the market. Tracing
aromatic rice genotype on F2 segregated population by applying marker assisted selection
(MAS) in combination with phenotypic assessment (sensory test) is highly demanded since it
will effectively assist in finding desired genotypes. This experiment aimed to select individual
plant of F2 progeny from Ciherang (high productivity) with Basmati (aromatic rice), based
on the molecular markers and sensory test. Two hundred and thirty-three F2 rice plants were
screened. Molecular marker of Bradbury and RM223 were applied to detect fragrance gene (fgr).
The fgr gene corresponds to the BADH2 (betaine aldehyde dehydrogenase) that is responsible
for aroma metabolism in fragrant rice varieties. One percent of KOH test was used to detect
2-acetyl-1-pyrroline aromatic compound. The experiments were conducted at the Laboratory of
Plant Biotechnology and Breeding, Faculty of Agriculture, Universitas Padjadjaran. Twenty-one
genotypes were detected by molecular markers; meanwhile, fifteen genotypes were identified by
sensory test. The calculation of Spearman’s correlation resulted a value of r = 0.51 and r = 0.49
for RM223-sensory test and Bradbury’s primer-sensory test, respectively, indicated a moderate
relationship between molecular marker and sensory test. Genotype #7, 37, 49, 66, 89, 175, 206,
and 212 were confirmed by both molecular marker as well as sensory test. These genotypes are
recommended for further experiment in developing aromatic rice.
Key words: Aroma, Correlation, MAS, Rice, Sensory test
I. INTRODUCTION
The fragrance of aromatic rice is preferred by consumers all over the world. This
type of rice is highly demanded and it has a premium price in both domestic
and international markets. Rice grain aroma results from the production of many
biochemical compounds [1], but the most important compounds is 2-acetyl-1-
* Corresponding author. Phone: +6281395567514. Email: ncarsono@mail.unpad.ac.id
111
pyrroline (2AP), which is considered to be a potent aroma constituent [2]. A

single recessive gene located on chromosome 8 has been identified as the gene
responsible for the aroma trait [3,4], because it has a key role in the synthesis of
2AP [3]. The identified gene has been named differently by different groups as
BAD2 [3,5], BADH2 [6], and Os2AP [4]. The 2AP is actually detected in all parts
of the rice plant, except in the roots [7,8]. The following sensory methods have
been applied to determine the aroma in rice: chewing several seeds or cooking a
sample of seed from individual plants [9], heating leaf tissue in water or eluting
the aroma from leaf tissue with diluted potassium hydroxide (KOH) [10,11] and
heating several half-cut seeds in fresh water [12]. Chemical methods are available
which involves smelling leaf tissue or grains with solution of KOH or I2KI [10],
but these can cause damage to the nasal passages. A panel of analysts is required
to distinguish between fragrant and non-fragrant samples. However, those sensory
evaluation methods are not consistent or reliable because the aroma is subjected
to human preference.
Molecular markers, especially functional marker, which linked to economically
important traits, have been extensively exploited to assist breeders in selecting
individuals with target traits and these markers are useful with respect to the
aromatic trait in rice breeding programs. By using these markers, the difficulty in
evaluating the aroma phenotype is minimized and accuracy of selecting desired
plants is increased. Markers associated with the fgr locus could be used to assist
in distinguishing aromatic and non-aromatic rice varieties for marker assisted
selection (MAS) [13,14]. A number of marker were identified that are closely
linked to fgr [14,15] and Bradbury et al. [5] suggested that a gene encoding
putative betaine aldehyde dehydrogenase (BAD2) is most likely to be the fgr gene.
The objective of this study was to select the F2 progeny of rice plants from
crosses between Ciherang (high-yielding cultivar) with Basmati (aromatic rice),
having valuable traits: fragrance which assessed by molecular and sensory test.
II. MATERIALS AND METHODS

A. Plant materials
Two hundred and thirty three of F2 individuals derived from a cross between
Ciherang (high-yielding cultivar) and Basmati (aromatic rice) and their parents:
Ciherang and Basmati, as check, were used in this experiment. Dehulled F3 seeds
were subjected to sensory test.
B. DNA preparation and molecular analysis

The genomic DNA was isolated from young leaves according to the protocol
[16]. Concentration of DNA was estimated using a spectrophotometer (Rayleigh
UV-9200) at a wavelength of 260 nm and 280 nm. Molecular marker for detecting
aromatic trait was carried out using RM223 [17,18] and Bradbury’s marker [19].
Termocycler PCR machine (Eppendorf ) was used. Total volume of PCR mixture
was 25 µL, consisted of 9,5 µL Nuclease-free water, 12,5 µL Go Taq® Green
Master Mix (Taq DNA polymerase, 400 µM dATP, 400 µM dGTP, 400 µM
dCTP, 400 µM dTTP, and 3 mM MgCl2), 1 µL Forward Primer, 1 µL Reverse
Primer, and 1 µL Template (DNA) 20 ng. PCR profile RM223 was 95°C for
5 min, followed by 35 cycles of 94°C for 60 seconds, 55°C for 30 seconds and
72°C for 60 seconds, and finally by 5 min at 72°C for final extention. Bradbury’s
primer was performed at 95°C for 5 min (initial denaturation); then for 35 cycles
of 95°C for 1 min; 58°C for 30 sec; 72°C for 1 min followed by 72°C for 5 min.
The amplification products were separated on 1.5%–2% Agarose gel. Gels were
then run for 75 minutes at 70V in 0.5 x TBE buffer. Agarose gel was stained
with ethidium bromide solution for 30 minutes. DNA fragments were visualized
under UV light and photographed using gel doc (Syngene Inc.).
C. Sensory test
Aromatic characteristic of rice varieties was identified by testing individual grains
or cooked rice. For the sensory test of grains, 1% KOH solution was applied to
the tissue [10]. One milligram of rice flour from single grain was soaked in 1.5
ml tube with 50 µl of 1% KOH solution at room temperature. After 30 minutes,
the tube was opened and immediately smelled. The presence or absence of aroma
was scored. Each individual sampel was evaluated by two persons.
D. Data analysis
Molecular data in terms of PCR band derived from image visualization system
using Geldoc under UV transilluminator (captured by Genesnap software pro-
gram) was interpreted. The images were then analyzed using a GeneTool software
(Syngene, UK) for ensuring the differences in size of each band. Molecular data
were scored as presence (1) and absence (0) according to band pattern of parent.
Sensory test for aroma was done as aroma presence (1) or aroma absence (0) for
each marker and data was entered in binary data matrix as discrete variables.
Selection was done on individual as detected by both methods. Spearman’s
Rank correlation coefficient was applied to identify and to test the strength of a
relationship between two sets of data.

In this study, the detection and identification of specific markers for the aroma trait
in rice was aimed attempts were made to identify a specific band of DNA amplified
product which would enable to distinguish aromatic rice from non-aromatic

ones. Bradbury’s primers were designed for allele specific amplification. External
primers produced a fragment of approximately 580 bp as a positive control for each
sample. Internal and corresponding external primers generate a 355 bp fragment
from a non-aromatic allele and a 257 bp fragment from an aromatic allele. Three
possible outcomes were obtained from the application of those four primers. In
all cases, a positive control band that approximately 580 bp was produced. In the
first case, it was indicated that individual homozygous non-fragrant had 355 bp
band. In the second case, it was indicated that individual homozygous fragrant
had 257 bp band. Meanwhile, in the last case, the occurrences of both bands
(355 bp and 257 bp) indicated that the individual was heterozygous non-fragrant.
Figure 1. PCR product visualization of several individuals from F2 population

using primer Bradbury et al. (a) and RM223 (b) to detect frg gene (NF: non-
fragrance, F: fragrance).
RM223 is one simple sequence repeats (SSR) or microsatellite marker. The

fragments amplified ranged from 90 to 190 bp [19]. This polymorphic marker
is used to distinguish between the fgr gene and its allelomorph conferring non-
aromatic property. The 120 bp band corresponds to an allele fragrance and the
160 bp band corresponds to non-fragrance allele.
Those two primers were screened for DNA polymorphism among parent. The
primers were successfully amplifying the target locus of fgr gene from Ciherang,
Basmati, and were able to distinguish aromatic from non-aromatic genotypes
(Figure 1). Among 233 F2 individuals that scored based on the Bradbury’s marker,
21 plants were found to be homozygous aromatic lines, 209 plants and 1 plant
were found to be homozygous and heterozygous non-aromatic, respectively (Table
1). Based on RM223 marker, 20 plants were found to be homozygous aromatic
line, 209 plants and 7 plants were found to be homozygous and heterozygous
Table 1. Comparison on detection of aroma using molecular markers and sensory test for 233
genotypes derived from a cross between Ciherang x Basmati
Parent Genotype (F2 and F3) Number

Markers Recurent Donor Homozygote Heterozygote Homozygote of
Ciherang Basmati Non-Fragrance Non-Fragrance Fragrance plant
Primer Bradbury - + 209 1 21 231
PrimerRM223 - + 204 7 20 231
Sensory test (1% KOH) - + 216 0 15 231
Note : (-) non-fragrance, (+) fragrance, F2 used for molecular analysis, F3 used for sensory test
non-aromatic, respectively (Table 1). This suggests that aromatic is controlled

by a single recessive [7,10].
The presence or absence of aroma in the rice grain was assessed for 233 F3
grains derived from a cross between Ciherang x Basmati, including both parents
identified by the sensory test using 1% KOH solution. In Table 1, fifteen genotypes
of F3 have been identified having aroma trait. The concentration of 2AP in rice
is affected by environmental conditions [20]. It means that the aroma strength
in aromatic rice may decrease and/or disappear when rice grows in unfavorable
conditions. In this study, grains of F3 progeny were tested for aroma in order
to avoid the confusion due to segregation among grains in the same panicle.
Meanwhile, there was a considerable variation among the panelists in the etima-
tion of the strength of aroma. In the present study, the focus was only on the
presence or absence of aroma and no quantitative determination was performed.
Moreover, sensory assessment of aroma in rice is still favorable method applied
in some studies.
The two molecular data sets were significantly correlated (r = 0.72) according to
Spearman’s Rank correlation coefficient. The molecular data were also significantly
correlated to the phenotypic data (sensory test) (r = 0.51 for RM223-sensory test
and r = 0.49 for Bradbury’s primer-sensory test, p-value < 0.0000). These results
suggested that molecular markers and sensory test can be applied in selection of
aromatic rice. This study confirmed that the sensory test using single grain and
1% KOH solution is a suitable method for rapid identification of aroma in rice.
At the early development stages of rice plant, a small amount of leaf tissue could
be assessed before maturity by the molecular markers for earlier estimation of
aroma and for reducing the number and segregation of seed samples. Genotype
#7, 37, 49, 66, 89, 175, 206 and 212 were selected by both molecular marker as
well as sensory test. These selected genotypes will be recommended as a promosing
genotypes and utilized for backcrossing and pyramiding breeding programs.
In this study, we followed both sensory tests and molecular marker methods to
detect the presence or absence of aroma in F2 and F3. The F2 and F3 individuals
which were classified having the aroma alleles also showed presence of aroma in
sensory tests. Although they had aroma alleles and showed presence of aroma
in sensory tests, they varied from light aroma to strong aroma in leaf and grain.
However, some of the F3 individuals did not exhibit aroma in grain aromatic
test, although F2 had aroma alleles. In another cases, those F2 were classified as
absent of aroma alleles, surprisingly produced aroma in grain aromatic tests at
F3. Therefore, there were only less than 50% F2 that classified as aromatic or non-
aromatic rice by using molecular method was well agreed with sensory methods in
grain. In Hien et al. [21] report, they detected a coincidence among conventional
methods, which is 1.7% KOH sensory test, and molecular marker analysis in the
classification between aromatic and non-aromatic rice, but sometimes molecular
markers could not classify heterozygous and homozygous genotypes due to
molecular nature. However, in a research by Bounphanousay et al. [22], they
used the chemical analysis (detect 2AP) with molecular marker analysis. They
reported that the molecular markers are well agreed with the chemical analysis
in most of the rice varieties, except some contrasting results such as in a local
aromatic rice variety, Kai Noi Leuang. It was detected producing aroma but
was identified as homozygous non-aromatic by molecular marker analysis. They
claimed that different gene location might be responsible for the observed aroma
in rice or would be the presence of another major aromatic compound. In Myint
Yi et al. [23] investigation, they mentioned that the variation in the sensory score
may cause by minor genes or environmental factors or some rice varieties may
carry the minor QTLs which would have an influence on rice aroma. As sensory
quality has always been an important consideration in rice improvement (Paule
and Powers, 1989), [24] integration of sensory methods and molecular markers
is reliable, fast and cost-effective ways essential for rice breeders to evaluate rice
aroma in the breeding programs.
Different aroma score from light to strong which were found in F2 and F3,
are homozygous aromatic, were reported in our experiments. The aroma in rice
might also be controlled by minor genes or minor QTLs [23]. Lorieux et al. [8]
reported that aroma is a quantitative trait. A major gene on chromosome 8 and
two QTLs on chromosomes 4 and 12 regulated the formation of aroma in the
rice cultivars Azucena. However, more experiments need to be carried out to
confirm the minor genes effect and their location.
Environmental factors might be another important role in determining the
aroma in rice. In our observation, it was expected that F2 individuals detected
with aroma gene (BADH2) supposed to produce aroma in grain at F3 seed.
This might be due to high temperature during their grain filling and ripening
stage [25]. During the period of grain filling to ripening, the temperature of the
planting field is around 22–29°C at day time. High temperature may also affect the
accumulation of 2-acetyl-1-pyrroline. The concentration may be very low when
the grain exposed under high temperature for a long time. Further experiments
can be carried out by controlling the temperature during the maturity period to
confirm the accumulation of 2-acetyl-1-pyrroline in rice grains. Aroma is mainly
controlled by a major gene, but it is easily influenced by environmental conditions
such as soil type, cultural practices, and temperature during the grain filling stage,
storage conditions, as well as storage time [26,27]. During our observation in rice
field, we found some F2 plants emitted pleasant aroma from the flowering organs,
especially in the morning from 8 a.m. to 10 a.m. However, pleasant aroma which
we smelled in field during flowering was a result of a large number of compounds
present in a specific proportion. In aromatic varieties, pleasant aroma is not only
associated with cooked rice. It also means that aroma emit at different stages of
rice plant growing. Whether volatile aromatic compounds released in field at the
time of flowering differ from those released after cooking is an important question
to be answered in further investigation.
IV. CONCLUSION
Based on two methods in detecting aroma trait (molecular marker and sensory
test), genotype #7, 37, 49, 66, 89, 175, 206 and 212 were selected for fgr gene.
V. ACKNOWLEDGEMENTS
Authors wish to thank Directorate General of Higher Education, Ministry of
Education and Culture in providing a research grant (Hibah Stranas) that was
awarded to Farida Damayanti.
VI. REFERENCES
1) Petrov, M., Danzart, M., Giampaoli, P., Fayre, J. and H. Richard. (1996).
Rice aroma analysis: discrimination between a scented and non scented rice.
Sciences des Aliments, 16, 347–360.
2) Buttery, R. G., Ling, L. C. and B. O. Juliano. (1982). 2-Acetyl-1-pyrroline:
an important aroma component of cooked rice. Chemistry and Industry, 12,
958–959.
3) Bradbury, L. M. T., Fitzgerald, T. L., Henry, R. J., Jin, Q. and D. L. E.
Waters. (2005). The gene for fragrance in rice. Plant Biotechnology Journal,
3, 363–370.
4) Kumar, N. (2009). An optimal spatial sampling design for intra-urban popula-
tion exposure assessment. Atmospheric Environment, 43 (5), 1153–1155.
5) Vanavichit, A., Tragoonrung, S., Toojinda, T., Wanchana, S. and W.
Kamolsukyunyong. (2008). Transgenic rice plants with reduced expression
of Os2AP and elevated levels of 2-acetyl-1-pyrroline. US patent 7,319,181.
Washington, DC: U.S.
6) Bradbury, L. M. T., Gillies, S. A., Brushett, D. J., Waters, D. L. E. and R. J.
Henry. (2008). Inactivation of an aminoaldehyde dehydrogenase is responsible
for fragrance in rice. Plant Molecular Biology, 68, 439–449.
7) X. Niu, Tang. W , Huang. W, Ren. G, Wang. Q, Luo. D, Xiao. Y , Yang. S,
Wang. F, Lu. BR, Gao. F, Lu. T, and Liu. Y. (2008). RNAi-directed down-
regulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production
in rice (Oryza sativa L.). BMC Plant Biology, 8, 100–109.
8) Lorieux, M., Petrov, M., Huang, N., Guiderdoni, E. and A. Ghesquière.
(1996). Aroma in rice: genetic analysis of quantitative trait. Theoretical Applied
Genetics, 93, 1145–1151.
9) Yoshihashi, T. (2002). Quantitative analysis on 2-acetyl-1-pyrroline of an
aromatic rice by stable isotope dilution method and model studies on its
formation during cooking. Journal of Food Science, 67, 619–622.
10) Dhulappanavar, C. V. (1976). Inheritance of scent in rice. Euphytica, 25,
659–662.
11) Sood, B. C. and E. A. Siddiq. (1978). A rapid technique for scent determina-
tion in rice. Indian Journal of Genetics and Plant Breeding, 38, 268–271.
12) Hien, N. L., Yoshihashi, T., Sahardi, W. A. and Y. Hirata. (2006). Sensory test
for aroma and quantitative analysis of 2-acetyl-1-pyrroline in Asian aromatic
rice varieties. Plant Production Science, 9, 294-297.
13) Wanchana, S., Kamolsukyunyong, W., Ruengphayak, S., Toojinda, T.,

Tragoonrung, S. and A. Vanavichit. (2005). A rapid construction of a physical
contig across a 4.5 cM region for rice grain aroma facilities marker enrichment
for positional cloning. Science Asia, 31, 299–306.
14) Garland, S., Lewin, L., Blakeney, A., Reinke, R. and R. Henry. (2000).
PCR-based molecular markers for the fragrance gene in rice (Oryza sativa
L.). Theoretical and Applied Genetics, 101, 364–371.
15) Jin, Q. S., Waters, D., Cordeiro, G. M., Henry, R. J. and R. F. Reinke. (2003).
A single nucleotide polymorphism (SNP) marker linked to the fragrance gene
in rice (Oryza sativa L.). Plant Science, 165, 359–364.
16) Doyle, J. J. and J. L. Doyle. (1990). Isolation of plant DNA from fresh tissue.
Focus, 12, 13–14.
17) Lang, N. T. and B. C. Buu. (2008). Development of PCR based markers for
aroma (fgr) gene in rice (Oryza sativa L.). Omonrice, 16, 16–23.
18) Kirbia, K., Islam, M. M. and S. N. Begum. (2008). Screening of aromatic
rice lines by phenotypic and molecular markers. Bangladesh Journal of Botany,
37 (2), 141–147.
19) Bradbury, L. M. T., Henry, R. J., Jin, Q., Reinke, R. F. and D. L. E. Waters.
(2005). A perfect marker for fragrance genotyping in rice. Molecular Breeding,
16, 279–283.
20) Yoshihashi, T., Nguyen, T. T. H. and N. Kabaki. (2004). Area dependency
of 2-acetyl-1-pyrroline content in an aromatic rice cultivar, Khao Dawk Mall
10. Jarq-Japan Agricultural Research Quarterly, 38, 105–109.
21) Hien, N. L., Yoshihashi, T., Sarhadi, W. K., Thanh, V. C., Oikawa, Y.
and Y. Hirata. (2008). Evaluation of aroma in rice (Oryza sativa L.) using
KOH method, molecular markers and measurement of 2-acetyl-1-pyrroline
concentration. Japanese Journal of Tropical Agricultural, 50 (4), 190–198.
22) Bounphanousay, C., Jaisil, P., Sanitchon, J., Fitzgerald, M. and N. R. S.
Hamilton. (2008). Chemical and molecular characterization of fragrance in
black glutinous rice from Loa PDR. Asian Journal of Plant Sciences, 1, 1–7.
23) Myint, Y., Nwea, K. T., Vanavichit, A., Chai-arree, W. and T. Toojinda.
(2009). Marker assisted backcross breeding to improve cooking quality traits
in Myanmar. Field Crops Research, 113, 178–186.
24) Paule C. M. and J. J. Power. (1989). Sensory and chemical examination of
aromatic and nonaromatic rices. Journal of Food Science, 54, 343–346.
25) Itani, T., Tamaki, M., Hayata, Y., Fushimi, T. and K. Hashizume. (2004).
Variation of 2-acetyl-1-pyrroline concentration in aromatic rice grains
collected in the same region in Japan and factor affecting its concentration.
Plant Production Science, 7 (2), 178–183.
26) Champagne, E. T. (2008). Rice aroma and flavor: a literature review. Cereal
Chemistry, 85 (4), 445–454.
27) Goufo, P., Duan, M., Wongpornhai, S. and X. Tang. (2010). Some factors
affecting the concentration of the aroma compound 2-acetyl-1-pyrroline in
two fragrant rice cultivars grown in South China. Frontiers of Agriculture in
China, 4 (1), 1–9.
Development of Rice Lines Resistant to
Brown Planthopper with Aromatic Traits:
Selection Based on Molecular Marker
Anggia Puspa Asria, Nono Carsonob,* and Suseno Amienb
a
Student, Master Program in Agronomy, Universitas Padjadjaran
Jl. Raya Jatinangor KM 21 45363, Sumedang, Phone/Fax. ( +6222) 7796316, Indonesia
b
Associate Professor, Faculty of Agriculture, Universitas Padjadjaran
Jl. Raya Jatinangor KM 21 45363, Sumedang,Phone/Fax. ( +6222) 7796316, Indonesia
Abstract
Development of rice lines that are resistant to BPH is an alternative pest management. However,
most of resistant varieties is unfavorable, whereas aroma, flavor and texture are actually major
factors that consumer preferred. Selection for two different characters at the early generation (F2)
is time consuming; therefore, molecular assisted selection needs to be performed to speed up the
selection process. The objective was to obtain F2 progeny derived from Sintanur, aromatic rice,
as a recurrent parent and PTB-33, brown planthopper resistant rice, as a donor parent by using
brown planthopper resistant and aromatic markers. This experiment was done at the Laboratory
of Plant Analysis and Biotechnology, Faculty of Agriculture, Universitas Padjadjaran. A number of
261 rice plants of F2 progeny were analysed using RM589 and RM8213 for brown planthopper
resistance, while aromatic was detected by Bradburry’s primer. There were 125 genotypes which
were selected by brown planthopper resistance markers, and eight of them were selected by using
RM589, RM8213, and Bradburry’s primer. Eight selected genotypes i.e. SP35, SP55, SP84,
SP87, SP141, SP151, SP237, and SP261 are recommended for the next generation for brown
planthopper resistant and aromatic rice breeding program.
Key words: Aromatic, Brown planthopper, Molecular marker, Rice, Selection
I. Introduction
Brown planthopper/BPH (Nilapavarta lugens Stal) is one of the most destructive
pests, which causes significant losses of rice yield [1]. Development of rice lines
that are resistant to BPH is an alternative pest management. However, most
of resistant varieties is unfavorable, e.g. IR74 [2], whereas, consumer prefer to
choose rice which have good aromatic, flavor, and texture [3]. Because of that,
rice breeding and followed by selection of rice which are resistant to BPH with
aromatic trait are needed.
PTB-33 is a rice variety from India and has several Bph resistant genes such
as Bph3 and bph4 [4] and quantitative Bph (QBph). Meanwhile, Sintanur is
* Corresponding author. Phone: +6281395567514. Email: ncarsono@mail.unpad.ac.id
121
high yielding aromatic rice that could reach up to 7 ton.ha-1 from Indonesia
[5]. Combining two different characters by hybridization followed by selection
of desired genotypes are difficult and time consuming, so the right and fast
breeding methods are needed. Molecular marker offers an easy way to overcome
such problem since it speed up the selection process with high accuracy, and not
influenced by the environment [6] and development stage of plant.
Simple sequence repeat (SSR) markers that are associated with resistant to
BPH are RM589 and RM586 (linkage to Bph3 and bph4 genes) [7,8], RM8213
and RM5953 which are associated to Qbph4 and Bph17(t) [9]. For detecting
aromatic trait, Bradburry’s specific marker is widely applied. Its marker contains
of Internal Fragrant Antisense Primer (IFAP), External Sense Primer (ESP) for
detecting aromatic and Internal Non-fragrant Sense Primer (INSP), External
Antisense Primer (EAP) for detecting non-aromatic [10]. The objective was to
obtain F2 progeny derived from Sintanur, aromatic rice, as a recurrent parent
and PTB-33, brown planthopper resistant rice, as a donor parent by using brown
planthopper resistant and aromatic markers.
II. M aterial and Methods

A. Plant material
Two hundred sixty one plants from a cross between Sintanur (recipient) and
PTB-33 (donor) were used in this research. This experiment was done at
the Laboratory of Plant Analysis and Biotechnology, Faculty of Agriculture,
Universitas Padjadjaran.
B. Molecular Marker Analysis

Total genomic DNA was extracted according to Dellaporta’s method [11] with a
slight modification. PCR amplification was perfomed using DNAs from 261 F2
plant genotypes and each parent. Component reactions for PCR amplification
containing 1 µl 20 ng DNA template, 9.5 µl KAPA2GTM Fast ReadyMix (DNA
Taq polymerase (0.25 U), dNTPs (0.2 mM), MgCl2 (1.5 mM)), 1 µl each primer.
For detecting resistant to BPH, SSR primer RM589 and RM8213 were used
and Bradburry’s primer was used for detecting aromatic trait. The PCR program
for RM589 and RM8213 primers was performed at 94°C for 5 min (initial
denaturation); then for 36 cycles of 94°C for 1 min; 55°C for 1 min; 72°C for 1
min followed by 72°C for 7 min. Different from SSR primers, Bradburry’s primer
was performed at 95°C for 5 min (initial denaturation); then for 35 cycles of 95°C
for 1 min; 58°C for 30 second; 72°C for 1 min followed by 72°C for 5 min. PCR
products were separated on gel electrophoresis in 3.0% agarose gel at 75 volt for
90 min (SSR primers) and 1.5% agarose gel at 70 volt for 60 min (Bradburry’s
primer). Visualization was done by staining on 0.2 µl/ml Ethidium-Bromide for

30 min and used gel documentation system (G-Box from Syngene) for analyzing
DNA banding pattern.
C. Data Analysis
DNA banding pattern of examined genotypes was then compared to its DNA
banding pattern of PTB-33 (for RM589 and RM8213) and of Sintanur for
aromatic trait (Bradburry’s primer). When the DNA banding pattern matched
with their parents, marked them by plus (+) and vice versa.
D. Results and Discussion

Visualization of DNA banding pattern was prior conducted by brown planthopper
resistant markers to select F2 plant that supposed to be resistant to BPH. F2 that
had DNA banding pattern same as PTB-33 were selected. There were two F2 plants
that were selected by RM589 (a) and five plants that were selected by RM8213
(b) (Figure 1). PCR product size of RM5889 (detected Bph3 and bph4) was 186
bp [8] and 177 bp for RM8213 (detected Qbph4 and Bph17(t)) [9]. Plant that
had Bph3 and bph6 genes was resistant to BPH biotype 4 [12] and PTB-33 had
resistance to BPH biotype Indian or biotype 4 [13]. Using PTB-33 as the donor
parent is the best choice for developing rice lines that are resistant to BPH.
(a)
(b)
Figure 1. DNA banding pattern visualization by RM589 (a) and RM8213 (b); (respectively,
from left to right of Ladder 100 bp (L): Sintanur (SN), PTB-33, nine number of F2 progeny
plant, and Ladder)
Figure 2. DNA banding pattern visualization by Bradburry’s primer;

(respectively, from left to right of Ladder 1kb (L), Sintanur (SN), PTB-
33, 23 number of F2 progeny plant)
Based on DNA pattern visualization, there were 125 genotypes of F2 progeny

plant that were selected by RM589 and RM8213, but just 27 genotypes that were
selected by both primers. Then, the number of 125 genotypes were analyzed by
Bradburry’s primer. Similarity of Sintanur DNA banding pattern that appeared
on progeny were selected. PCR product size of aromatic trait was 257 bp and
355 bp for non-aromatic [10]. In Figure 2, Sintanur was 257 bp (aromatic) and
PTB-33 was 355 bp (non-aromatic). On F2 progeny plant, there were three types
of band i.e. DNA banding pattern same as Sintanur, PTB-33, and have both of
them. DNA banding pattern that had two bands were heterozygous non-aromatic
[10], so it was not selected for aromatic trait. Sintanur had aromatic trait, but it
is susceptible variety to BPH so it would be better when it combined with other
resistant varieties.
There were eight selected genotypes based on molecular markers (RM589,
RM8213, and Bradburry’s primer) i.e. SP35, SP55, SP84, SP87, SP141, SP151,
SP237, and SP261. All of the selected genotype are promising lines that could
be further examined for brown planthopper resistant and aromatic rice breeding
program and also could be parents for pyramiding.
III. Conclusion
Selection based on molecular marker method would be an alternative approach for
rice breeding program on early generation especially on segregating populations.
Developing rice lines that are resistant to BPH is one of the concern of rice breeder.
Resistant rice lines to BPH that have another special character, like aromatic, have
to be developed in order to answer consumer needs.
IV. Acknowledgement
Authors wish to thank Directorate General of Higher Education, Ministry of
Education and Culture in providing research grant (Hibah Stranas) that is awarded
to Nono Carsono, Ph.D.
V. R eferences
1) Baehaki, S. E. and I. N. Widiarta. (2009). Hama wereng dan cara pengenda-
liannya pada tanaman padi. In Padi: Inovasi Teknologi Produksi. Buku 2 (pp.
347–3833). Jakarta: LIPI Press.
2) Baehaki, S. E. (2007, August). Perkembangan wereng coklat biotipe 4. Tabloid
Sinar Tani.
3) Rohaeni, W. R., Anna, S. and M. I. Ishaq. (2012). Preferensi responden
terhadap keragaan tanaman dan kualitas produk beberapa varietas unggul
baru padi. Informatika Pertanian, 21 (2), 107–115.
4) Jairin, J., Phengrat, K., Teangdeerith, S., Vanavichit, A. and T. Toojinda.
(2007). Mapping of a broad-spectrum brown planthopper resistance gene,
Bph3, on rice chromosome 6. Molecular Breeding, 19, 35–44.
5) Suprihatno, B., Daradjat, A., Satoto, S. E., Suprihanto, Indrasari, S. D. and
W. I. Putu. (2010). Deskripsi Varietas Padi. Subang: Balai Besar Penelitian
Tanaman Padi.
6) Lakshmikumaran, M., Das, S. and P. S. Srivastava. (2003). Application of
molecular markers in Brassica coenospecies: comparative mapping and tagging
(abstract). Biotechnology in Agriculture and Forestry, 52, 37–68.
7) Jena, K. K. and D. J. Mackill. (2008). Molecular markers and their use in
marker-assisted selection in rice. Crop Science, 48, 1266–1276.
8) Jairin, J., Teangdeerith, S., Leelagud, P., Phengrat, K., Vanavhicit, A. and T.
Toojinda. (2007). Detection of brown planthopper resistance genes from
different rice mapping populations in the same genomic location. Science
Asia, 33, 347–352.
9) Sun, L., Su, C., Wang, C., Zhai, H. and J. Wan. (2005). Mapping of a major
resistance gene to the brown planthopper in the rice cultivar Rathu Heenati.
Breeding Science, 55, 391–396.
10) Bradbury, L. M. T., Henry, R. J., Jin, Q., Reinke, R. F. and D. L. E. Waters.
(2005). A perfect marker for fragrance genotyping in rice. Molecular Breeding,
16 (4), 279–283.
11) Dellaporta, S. L., Wood J. and J. B. Hicks. (1983). A plant DNA miniprepara-
tion: version II. Plant Molecular Biology, 1 (4), 19–21.
12) Baehaki, S. E. and D. Munawar. (2008). Uji biotipe wereng coklat, Nilaparvata
lugens STAL. di sentra produksi padi. Seminar Nasional Padi. 347–359.
13) Santhanalakshmi, S., Saikumar, S. and S. Rao. (2010). Mapping genetic lokus
linked to brown planthopper linked to rice Oryza sativa L. International Jounal
of Plant Breeding and Genetics, 4 (1), 13–22.
SCSER
Assessment of E-waste Recovery Facilities in
Selangor and Kuala Lumpur, Malaysia
Nurul Ain Mohd Nordin* and P. Agamuthu

Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
Abstract
The fast development of the world has increased the use of electrical and electronic equipments
(EEE). EEE is often being upgraded to more sophisticated device and users keep changing their
out of date equipment to a new product and increase EEE waste (e-waste) generation. The
implication of the increment of e-waste and its improper disposal will lead to major issues to
health and environment. Material recovery facility (MRF) of e-waste is the most critical element
in e-waste industry. This article attempts to assess e-waste recovery facilities in Selangor and Kuala
Lumpur, Malaysia. A total of 15 questionnaires were sent to MRF through email and interviews.
The results show that many challenges are faced by recovery facilities to be in e-waste industry.
E-waste recycling in Malaysia is still at infancy as there is lack of specific regulation on e-waste,
lack of MRF that carry out complete recycling process and poor e-waste disposal by public. At
the same time, this research helps us to understand various activities going on within MRF and
thus, encourage the formulation for a proper e-waste management strategy in Malaysia
Key words: E-waste, Material recovery facilities, Management, Malaysia
I. Introduction
In this era of modernization, electrical and electronic equipments (EEE) have
made our daily life more convenient. However, one of the consequences of the
rising of EEE is the generation of its waste. Waste of electric and electronic
equipments (WEEE) or e-waste is electric and electronic products that meet
their end of useful life (Sthiannopkao & Wong, 2012). According to Herat &
Agamuthu (2012), the world generates around 20–50 million tonnes of e-waste
annually and most of them are from Asian countries.
A. E-waste in Malaysia
Globally, e-waste issue has become an emerging problem that should be placed
under scrutiny (Widmer et al., 2005). This situation has affected many developing
countries, including Malaysia (Babington et al., 2010). Malaysia has became
an importer and exporter of e-waste as the geographic location of the country
is located in the middle of international e-waste trade route that makes it an
* Corresponding author. Phone: +6014-8029382. Email: nurulain.nordin90@gmail.com
129
attractive target for e-waste smugglers (Tengku-Hamzah, 2011). According to

Puckett (2005), Malaysia is listed as one of the countries that receive e-waste
from United States. The major issue of e-waste in Malaysia is its high amount
and will continue to increase exponentially (Afroz et al., 2013). Department
of Environment (DOE) Malaysia recorded 78,278.05 metric ton of e-waste in
2012. Malaysian citizens are not aware of the proper disposal of e-waste. This
lack of awareness will lead to leaking of hazardous content in e-waste and thus
harming the environment. Malaysia also suffers from an increasing volume of
illegal e-waste recycling by irresponsible party.
B. Material Recovery Facility

Material recovery facility (MRF) is a critical element within e-waste recycling
industry. At MRF, collected e-waste will be more valuable as marketable output
products including reusable systems/components (working and repairable elec-
tronics) and secondary scraps (plastics, metals and glass) (Kang & Schoenung,
2006). There are also precious metals, such as gold, silver and copper that can
be recovered from e-waste. DOE, Malaysia has encouraged the establishment of
e-waste recovery facilities and 147 recovery facilities have been established so far.
II. Methodology
The main methods for this research were survey questionnaire and interview
among managers of e-waste recovery facilities. It was followed by site visit and
observation to the recovery facilities. This study focused in Klang Valley, which
includes Malaysia capital city, Kuala Lumpur, and the most populous state in
Malaysia, which is Selangor.

In Malaysia, recovery facilities are classified into “full and partial”. Full recovery
facilities are those with capacity to recycles all part of e-waste they receive, while
partial recovery facilities are those with limited capability to recycle all parts of
e-waste (Babington et al., 2010). There are five full recovery facilities and ten
partial recovery facilities involved in this study.
A. Type of e-waste collected at recovery facilities

Figure 1 shows that Klang Valley generates various type of e-waste. The result
shows that the most generated e-waste is PC. Nowadays, most electronic users
use laptops/notebooks as another alternative to PCs. Therefore, their unused PCs
were eventually becoming wastes. Most organizations and institutions change their
old PC model to more sophisticated PC that is evolving today. From the results,
it is indicated that the majority of recovery facilities collect their e-waste from
commercial organizations like corporate companies and government agencies
(see Figure 2).
B. Source of e-waste collected at recovery facilities

Source of e-waste collected at recovery facilities are shown in Figure 2. Corporate
companies and government agencies contribute the most e-waste thrown to
recovery facilities with 30% each. Both sources provide a huge supply of e-waste
to recovery facilities as they are big scale organisations, in parallel to its high
usage of electronic devices. Electrical and electronic factories produce 20% of
e-waste, while smaller amount are from household and public. Poor collection
from household and public indicates that large amount of e-waste is stored at
household level without being collected. There might be a possibility that most
public just throw away their e-waste with other municipal waste.
Figure 1. Type of e-waste collected at re- Figure 2. Source of e-waste collected at recov-
covery facilities ery facilities
C. Residues generated by recovery facilities

There are six different types of residues generated by recovery facilities, which are
plastics, metals, glasses, PVC, fibre and dust/other waste (Figure 3). Plastics, metals
and dust/other waste are the highest types of residue generated which contribute
to 23% each. Usually plastics and metals will be sold to plastic factory and metal
scrap company, respectively. Dust and other waste will be sent to Kualiti Alam,
the only company in Malaysia that is licensed to dispose hazardous waste.
Figure 3. Residues generated by recovery facili- Figure 4. Challenges faced by recovery facilities
ties
D. Challenges faced by recovery facilities

The challenges faced by recovery facilities are shown in Figure 4. The most
prominent challenge cited are “high price of e-waste to be collected” and “low
amount of e-waste that can be collected”. Most e-waste generator demand a high
price for their e-waste when sold to recovery facilities. The recovery facilities feel
burdened by this situation as they might not obtain profitable income at the end.
Furthermore, the amount of e-waste that can be collected was quite low. This is
parallel with the other challenge, which is “lack of awareness from public”. In
reality, the generation of e-waste was higher than expected. However, the public
did not have enough knowledge to properly dispose their e-waste and eventually
the e-waste will end up in municipal landfill.
E. Economic Value of E-waste

From the survey conducted towards the e-waste contractors, the estimated best
market price of e-waste is RM 3.00/kg depending on the condition of e-waste.
Based on the best price scenario, the total value of e-waste collected by 15 recovery
facilities within Klang Valley is RM 529,890.00. If estimation has been made that
the average value of e-waste collected by one recovery facility per month is RM
35,326.00 (total value/no. of recovery facilities involved), then the estimated total
value of e-waste that is collected by whole recovery facilities within Klang Valley
(30 recovery facilities) is RM 1,059,788.57. This value of e-waste only applies to
e-waste generators that send their e-waste to recovery facilities. There are many
other generators that do not send their e-waste to recyclers. Therefore, imagine
how much money is lost when e-waste is dumped at the landfill.
IV. Conclusion
MRF is the main element in e-waste industry; their management is still at infancy
stage. The Department of Environment (DOE) Malaysia must have a proper
scrutiny on the individual contractors before issuing license. DOE must also
make a strict regulation, not only to the large organization, but also to the public
on disposing e-waste. The study suggests that e-waste collection should include
household instead of focusing on industries and organizations alone. By having
a connection between e-waste recovery facilities and municipal council, e-waste
collection around household level can be significantly improved.
V. Acknowledgement
I would like to express my greatest appreciation towards my supervisor, Prof.
Agamuthu Pariatamby, for spending his precious time, ideas and efforts in helping
me in doing this research. I am grateful to all respondents from material recovery
facilities and the officer from DOE Malaysia. I am also grateful to Universiti of
Malaya. This study was sponsored by UM grant Postgraduate Research Fund
P0005-2013B. Furthermore, I would also like to thank the Solid Waste Lab
members for giving me guidance and support. Their kindness is greatly appreci-
ated. Consideration and supports from family and friends are always remembered.
VI. R eferences
1) Sthiannopkao, S. and M. H. Wong. (2012). Handling e-waste in developed
and developing countries: initiatives, practices, and consequences. Science of
The Total Environment, 463–464, 1147–1153.
2) Herat, S. and P. Agamuthu. (2012). E-waste: a problem or an opportunity?
Review of issues, challenges and solutions in Asian countries. Waste Manage-
ment and Research, 30, 1113–1129.
3) Widmer, R., Oswald-Krapf, H., Sinha-Khetriwal, D., Schnellmann, M.
and H. Böni. (2005). Global perspectives on e-waste. Environmental Impact
Assessment Review, 25, 436–458.
4) Babington, J., Siwar, C. and M. Ahmad Fariz. (2010). Bridging the Gaps: An
E-waste management and recycling assessment of material recycling facilities
in Selangor and Penang. International Journal of Environmental Science, 1 (3),
383–389.
5) Tengku-Hamzah, T. A. A. (2011). Making Sense of Environmental Governance:
A study of E-waste in Malaysia (Doctoral’s thesis, Durham University.
6) Puckett, J. (2005). The Digital Dump: Exporting Re-use and Abuse to Africa.
Seattle: Basel Action Network.
7) Afroz, R., Masud, M. M., Akhtar, R. and J. B. Duasa. (2013). Survey and
analysis of public knowledge, awareness and willingness to pay in Kuala
Lumpur, Malaysia–a case study on household WEEE management. Journal
of Cleaner Production, 52, 185–193.
8) Department of Environment. (2012). Malaysia Environmental Quality
Report.
9) Kang, H. Y. and J. M. Schoenung. (2006). Economic analysis of electronic
waste recycling: modeling the cost and revenue of a materials recovery facility
in California. Environmental Science and Technology, 40, 1672–1680.
Public Perception on Current Waste
Management System: A Malaysian case study
S. H. Fauziah* and S. F. Ser

Institute of Biological Sciences, Faculty of Science, University of Malaya
Abstract
The fact that public should be socially and financially prepared is undeniable to ensure an effective
implementation of waste management system in a country. This paper deliberates on public
perceptions towards the efforts to improve a waste management, including pay-as-you-throw
scheme. The study was conducted through distribution of questionnaires on issues related to
current waste practices and public understanding on various concepts in waste management
hierarchy to 759 randomly selected respondents in Peninsular Malaysia. Statistical analysis was
conducted using SPSS to correlate the socio-economic background of the respondents with their
perception and level of awareness. With 96% confidence level, this study indicates that 50% of
the respondents were in agreement that individual environmental awareness dictates personal
environmental behaviour, including waste generation and participation in recycling activities. As
for the legislations related to waste management in Malaysia, majority (68%) was not aware of
the existence of such regulations and provision. This is followed by 19% that felt that the lack of
enforcement is the causal factor towards ineffective waste management system. On the statement
that people become more attentive towards the environment only when they are threatened by
the negative impacts are supported by 86% of the respondents. This indicates that in general,
public tends to be ignorance of various environmental issues if there is no direct implication to
them. The study concludes that understanding on issues related to environment in general, and
waste management in particular, is highly influenced by the education level and attentiveness
of the community.
Key words: Recycling, Public participation, Pay-as-you-throw scheme, Solid Waste Management
Act 2007
I. Introduction
Fast progress in technology has resulted with many countries in the world
benefiting the consumer-based lifestyle [1]. Consumption patterns and choices
in technology influenced the impact on environment [2]. Development resulting
from industrial revolution causes waste management to be a crucial issue globally.
An increase of 1% GDP is estimated to increase municipal solid waste generation
by 0.69% [2].
* Corresponding author. Phone: +603 7967 6739. Email: fauziahsh@um.edu.my
135
Urbanization and economic development makes the waste management a

critical concern due to the fact that the increase in the quantity of waste will lead to
health problems, portraying poor sanitation as well as downgrading aesthetic value
[3]. Therefore, it is imperative to manage waste properly to prevent detrimental
impacts to the environment.
Most of the developing nations in the world are facing huge challenges in
improving the country’s waste management system. Among the most critical
challenges are the rapid increase in waste generation and the absence of appropri-
ate and effective waste management policy. Daily waste generation which had
exceeded 3.5 million tonnes in 2010 might double up by 2050 should no drastic
actions are taken [4].
It is reported that human population worldwide can produce thrice the
amount of waste today by 2100 [5]. The amount of waste produced by the
human population is undeniably on the rise with very limited portion undergoing
transformational changes of materials [5]. The waste issue will get more serious
particularly in developing countries where the cataclysm of dumping sites might
outbalance proper waste management strategies. Mexico City and Shanghai was
reported to generate more than 10,000 tonnes of waste everyday and this will
continue to increase due to lack of initiatives on waste reduction [5]. This is evident
in many parts of Asian countries where the population is expanding rapidly and
the needs for more dumping grounds are unavoidable [6].
While developing nations struggle to cope with the countries’ waste manage-
ment, cities in many developed nations had established various strategies to tackle
waste issues in which waste management is programmed to generate income rather
than consuming the profit of waste managers. San Francisco city for example,
create “Zero Waste” program via appropriate policy implementation to eliminate
waste from landfills, while 565,000 tonnes of waste are diverted from industries
in Kawasaki, Japan [5,7]. This is possible only with the implementation of an
integrated solid waste management with the aid of suitable policy framework,
proper available technologies, ability and capacity to establish a system and
appropriate financial assistance from the authorities [8].
The 3R concept (Reduce, Reuse and Recycle) has been practiced throughout
the globe in order to achieve sustainable waste management [9,10]. However, it
can be very challenging to developing countries where environmental concern
is of less priority than that of economic development. As a result, many of these
nations resort to the cheapest waste disposal method available, landfilling [11].
Landfilling is known to be the simplest waste disposal option, but with the
least preference. With expanding human population, land scarcity made landfilling
unfavourable due to the competition of landuse. However, landfill is still necessary
since regardless of type of treatment used, there would still be unwanted residues
that need disposal [11]. Thus, the need for landfill is undeniable. However, the
matter of concern is the impacts of landfilling, as well as improper waste disposal,
towards the health of the environment. Improper managed disposal sites have been
constantly reported over the years to cause environmental pollution [12,13,14].
Landfill leachate, namely the effluent generated from waste disposal
activities, was reported to contain pollutants like monocyclic hydrocarbons and
organopesticides, and heavy metals which undoubtedly can negatively impact the
aquatic life [15]. Therefore, it is highly imperative that waste disposal sites are
properly managed in a sustainable manner to prevent any form of environmental
pollutions. Yet, it can be a real challenge if a country is not prepared to embrace
the technology. In order to move towards sustainable waste management practices,
it is necessary to highlight the appropriate driving factors.
Environmnetal drives, human drives, institutional drives and economic drives
are among the highlighted factors in many developed nations with approriate
mechanism of sustainable waste management [9]. These factors had been proven
to drive the country’s waste management system towards a more sustainable
practice [9].
Regardless the types of driving challenges existed, public plays an up most
important role in ensuring the success of an implemented strategies. The fact
that public should be socially and financially prepared is undeniable to enable a
proposed waste management strategy to be effective. Therefore, it is important
that planning of an effective waste management system should incorporate
the importance of the general public as the main stakeholders since this is the
group that initiates the waste generation process. It is necessary to understand
the behavior, the belief and the practices of the general public, so any planning
will take into consideration the possible need and expected responses after the
implementation of any waste management scheme.
In many developing nations in particular, the lack of information about the
public perspective on various environmental issues including waste management
has led to the failure of many environmental programmes, such as recycling
program [11]. Uncertainties in solid waste data, including outdated or inaccurately
estimated using regional averages, lack of a global database on waste production
and non-transparency among the stakeholders give rise to the unsuccessful
planning strategies [16].
Similar to many developing countries in the world, Malaysia too has been
struggling to improve the country’s waste management system. The introduction of
3R program in early 1980s and again in early 2000 has only increased recycling rate
to 5%, indicating the lack of public participation in the voluntary program [11].
This is mainly due to the absence of appropriate rules and regulations pertaining
to waste management in the country. As a result, a ‘not-bothered’ attitude plays a
major deciding role that public was not responsive. Yet, in 2007, contamination
of landfill leachate into the water catchment area in Klang Valley had caused
a huge public uproar that Solid Waste and Public Cleansing Management Act
2007 was passed by the Parliament [11]. The chain reaction from the event in
2007 has created a more environmental conscious public which pushed the
government to continuously improve the waste management system via a major
step of federalizing waste management sector in the country. Drastic improvement
from the management point of view has been recorded [11]. However, it is
somewhat hampered when it involves the participation from the general public.
Among the most glaring issue is the failure to improve the 5% recycling rate in
the country. In addition to that, the passed Act 2007 also includes the clause
on the implementation of Pay-As-You-Throw (PAYT) scheme. However, it is
yet to be implemented that the outcome is still uncertain. Undeniably, public
plays a crucial role in ensuring the success of any waste management program.
Therefore, it is important that their perception is taken into consideration prior
to the implementation of a program in a country. This paper aimed to investigate
the public perceptions towards efforts to improve a waste management including
PAYT scheme in particular and other waste related-environmental issues in
Malaysia.
II. R esearch Methodology

The study was conducted through dissemination of questionnaires on issues
related to current waste practices and public understanding on various concepts
in waste management hierarchy.
The questionnaires were divided into four sections with 55 questions in total.
The sections include background information, enquiries on level of knowledge
on various issues related to environment, awareness and knowledge on the past
and current programs run by the government in waste management and finally
on their level of satisfaction on the current waste management system in their
respective areas.
Reliability tests were conducted prior to actual distribution of questionnaires to
determine its reliability and coherency. The reliability test involved 30 respondents
which were selected randomly within Klang Valley. Cronbach’s Alpha was utilized
to determine the reliability of the set questionnaires.
For the actual survey, stratified random approach was adopted which identified
the number of respondent in each state of Peninsular Malaysia according to the
population distribution. Selangor, which represent the most densely populated
state (24.4%), has the largest number of respondents for this study, followed by
14.7% in Johor, 10.3% in Perak and other states accordingly.
A total of 759 respondents were randomly selected from cities in Peninsular
Malaysia to attempt the questionnaires given. Statistical analysis using SPSS were
conducted to correlate the socio-economic background of the respondents with
their perception and level of awareness.
III. R esults and Discussions

With 96% confidence level, this study indicates that 50% of the respondents
were in agreement that individual environmental awareness dictates personal
environmental behaviour including waste generation and participation in recycling
activities. Others were either unsure or disagreed. Majority of the respondents
believed that they are active participants of recycling if they practice any form
of recycling. The survey revealed that individual believed that they are recycling
regardless the volume involved. Many of the respondents indicated that they
recycled newspaper or aluminum cans only.
As for the legislations related to waste management in Malaysia, majority
(68%) was not aware of the existence of such regulations and provision. As a
result to that, many failed to understand that it is their responsibility to reduce
the generation of waste, and to reduce the total waste to be sent to landfill for
disposal. More than 50% of the respondents think that environmental awareness
plays a major role in determining the generation of waste by individuals. Figure
1 shows the likely factors which can influence the waste generation habit among
Malaysians.
Figure 1. Public opinion on the contributing factors to waste

generation among individuals in Malaysia
The survey exposed that only 32% of Malaysian public can correctly identify
their waste collection service provider. This low percentage is probably contributed
by the lack of dissemination of information by the waste collection service provid-
ers. In Peninsular Malaysia, three main consortia, namely Alam Flora Pvt Ltd.,
SWM Pvt Ltd., and E-Idaman Pvt Ltd., have been awarded with the consession
to manage the municipal solid waste in Peninsular Malaysia.
Since all residential areas are to be serviced efficiently, many sub-contractors are
hired to cater the need of the population, under the three consortia. As a result,
many small contractors run the operation under the appointment of the consortia.
This could also be the contributing factor to the public confusion on the correct
waste consortia responsible in their area. In fact, the study showed that 68%
failed to identify the waste collection service provider in their neighbourhood.
Additionally, this negligence is probably resulting from the ‘not bothered’ attitude
since there is no direct financial implication involved.
As for quality of services provided, 79% indicated that current waste
management system in the country is satisfactory. This generally is due to the
strict key performance index (KPI) imposed by the regulating bodies to ensure
that the waste managers execute their responsibility appropriately. Significant
improvements have been reported since the passing of Solid Waste and Public
Cleansing Management Act 2007 [11]. This is because the regulating body namely
the Minsitry of Urban Wellbeing, Housing and Local Government has the right
to revoke the license of any waste consortia should they fail to deliver the required
KPI. Thus, to ensure smooth renewing of waste management concession, the waste
consortia has been diligently improving their services to the public, particularly
for waste collection. For example, many of the consortia tend to upgrade their
garbage truck to generate a good image to the general public.
Since main complaints from the general public revolve around the untidiness
of garbage truck, leakage of leachate from garbage trucks and inconsistent
frequency of collection due to garbage truck break-down, replacement with
tip-top condition garbage trucks resolved the problem. However, the method of
waste treatment and disposal is given less priority since it does not involve the
public directly. As a result, pollution from disposal sites including landfill persists.
Landfilling being the main method of disposal in Malaysia is not a well known
fact by the majority of Malaysians. Only 37% of the respondents know that the
waste collected from their premises will be disposed off into landfills. Yet, the
ignorance of 73% of the respondents probably is attributed to the fact that public
is less concern about the fate of the waste they disposed. The “out of sight and
out of mind” probably is the reason that the majority is oblivious of the disposal
Figure 2. Knowledge on waste disposal method practiced in Malaysia among

respondents in Peninsular Malaysia
method practiced in Malaysia. Figure 2 depicts the public knowledge on waste

disposal methods in Malaysia.
Approximately, 75% actually believed that incineration is being practiced. In
reality, incinerators in Malaysia mainly cater for the disposal of hazardous waste
and very small volume of municipal solid waste in small islands, while the bigger
incinerators for municipal solid waste is still at its planning stage.
On issues related to factors that contribute to waste management problem in
Malaysia, 42% admitted that public attitude is the main cause. This is followed by
19% that felt that the lack of enforcement is the causal factor towards ineffective
waste management system. Figure 3 illustrates the public perception on the
contributing factors for waste management problem in Malaysia.
Indifferent attitude among the general public has always been the main reason
that lead to the failure of a program. Since public is the main generator of waste, it
is very important that they play a crucial role in any waste management program.
This includes recycling, waste reduction strategies and many more. Reduction on
the use of plastic bags (‘No-Plastic Bags’ on Saturday), for example has managed
to reduce the use of plastics bags by consumers. Rather than because consumers
are resorted to use alternative bags during shopping, the reduction was actually
due to avoidance among public to purchase the bags (RM 0.20 is charged for
each plastic bags on Saturday) or avoidance to do any shopping on Saturday. In
other words, instead of the reason to save the environment, the reduction on
plastic bags usage is heavily due to economic concern among the consumers.
Figure 3. Public perception on the contributing factors to waste management

problems in Malaysia
This is because for the majority of the public, economy is of higher priority than
environmental justification [11].
Only 12% feel that lack of education among Malaysian is the reason why
problem in waste management emerged. This probably is because ignorance
from lack of knowledge among the public can lead to lack of attentiveness and
refusal to embrace improvement. On the other hand, 9% blamed insufficient
number of facility being the factors that lead to problem in waste management.
It is also true based on the fact that waste generators are not able to participate
in waste management program if facilities provided are insufficient. This can be
supported by many cases of indiscriminate waste dumping when existing waste
bins had been filled up to the rim.
As for statements that people become more attentive towards the environment
only when they are threatened by the negative impacts are supported by 86%
of the respondents. This indicates that in general, public tend to be ignorance
of various environmental issues if there is no direct implication to them. Thus,
people in the ‘comfort zones’ are less susceptive to environmental issues in general.
This lead to the reason why the ‘Not In My Back Yard’ or NIMBY syndrome are
very strong among Malaysians.
Many developed nations had charged the public the service fees to manage
the waste generated based on the amount produced. Pay-as-you-throw (PAYT)
scheme has been reported to be very effective Japan, Korea and many parts of
EU. However, it is an unfamiliar concept in Malaysia where the cost of waste
management is embedded in the annual assessment fee paid by premise owners
to the local municipalities. As a matter of the fact, many of the general public
is oblivious of this situation. This study had ruled out that approximately 59%
Figure 4. Public acceptance on PAYT system in Malaysia
of the respondents believed that they are not charged for the waste management
services. To make matter worse, 93% of the general public is clueless of the cost
incurred to manage the waste they generate everyday. This is probably contributed
to the absence of information on the actual process of waste management and the
cost involved to deal with its collection, treatment and disposal. Thus, it is highly
recommended that the local government and non-governmental organization
(NGOs) to step in to provide the information to the general public on related
issues in waste management. Not only it will be an eye-opener to the public but
also allow them to consider carefully how to deal with their wastes.
Only 47% of the respondents thought that PAYT can be implemented in
Malaysia, while the remaining 53% were skeptical of this scheme. This is likely
due to the fact that Malaysians can be very fussy when it involved their finance.
Therefore, the need to pay for the amount of waste generated may not be appealing
since similar assessment such as sewage treatment service in the past 15 years has
been very problematic in its implementation. 43% of the respondents also think
that PAYT is not fair. Figure 4 depicts the public acceptance on PAYT system
in Malaysia.
The results obtained from this study indicated the importance of public
understanding and awareness to mould the genuine belief towards environmental
conscious society.
IV. Conclusions
The study concluded that public understanding on various issues related to
environment in general, and waste management in particular is highly influenced
by the attentiveness of the community. PAYT scheme generally is not well accepted
among the Malaysians. Nevertheless, many believed that laws and regulations can
assist in moulding the right attitude of the general public towards the improvement
in waste management in the country.
V. Acknowledgement
The authors would like express their gratitudes to respondents that participated
in this project. Appreciation also goes to the National Department of Solid Waste
(JPSPN) under the Ministry of Urban Wellbeing, Housing and Local Government
for the provision of token for the respondents in the survey. Finally, special thanks
go to Institute of Biological Sciences, Faculty of Science, University of Malaya,
for the financial assistance given to complete this study.
VI. R eferences
1) Bhada-Tata, P. and D. Hoornweg. (2012). What a waste: A global review of
solid waste management. World Bank Report.
2) Satterthwaite, D. (2009). The implications of population growth and urbaniza-
tion for climate change. Environment and Urbanization, 21 (2), 545–567.
3) Lau, V. L. (2004). Case study on the management of waste materials in
Malaysia. Forum der Geookologie, 15 (2), 7–14.
4) Hoornweg, D., Bhada-Tata, P. and C. Kennedy. (2013). Waste production
must peak this century. Nature, 502, 615–617.
5) World Bank. (2013). Global Waste on Pace to triple by 2100. Retrieved from
http://www.worldbank.org/en/news/feature/2013/10/30/global-waste-on-
the-pace-to-triple.
6) Hoornweg, D. and P. Bhada-Tata. (2012). What a Waste: A global review of
solid waste management. World Bank Report.
7) SF Environment. (2014). Zero Waste. Retrieved from http://www.sfenviron-
ment.org/zero-waste
8) UNEP. (2009). Developing Integrated Solid Waste Management Plan: Assessment
of Current Waste Management System and Gaps Therein. United Nations
Environment Programme.
9) Agamuthu, P., Khidzir, K. M. and S. H. Fauziah. (2009). Drivers of sustainable
waste management in Asia. Waste Management and Research, 27 (7), 625–633.
10) Agamuthu, P., Fauziah, S. H., Khidzir, K. M. and L. P. Chong. (2008). Issues
and challenges of 3Rs in the Asia and Pacific regions”. Proceedings of the Asian
Productivity Organization Conference 2008. Japan, 6–9 October 2008.
11) Fauziah, S. H. and P. Agamuthu. (2012). Trends in Sustainable Landfilling
in Malaysia, a developing country. Waste Management and Research, 30 (7),
656–663.
12) Palmiotto, M., Fattore, E., Paiano, V., Celeste, G., Colombo, A. and E.
Davoli. (2014). Influence of a municipal solid waste landfill in the surrounding
environment: Toxicological risk and odor nuisance effects. Environmental
International, 68, 16–24.
13) Nguyen, N. S., Satoshi, S., Ishigaki, T. and M. Ike. (2012). Microorganisms
in landfill bioreactors for accelerated stabilization of solid wastes. Journal of
Bioscience and Bioengineering, 114 (3), 243–250.
14) Magda, M., El-Salam, A., Gaber, I. and Abu-Zuid. (2014). Impact of
landfill leachate on the groundwater quality: A case study in Egypt. Journal
of Advanced Research,( In Press).
15) Fauziah, S.H., Emenike, C. U. and P. Agamuthu. (2013). Leachate risk
and identification of accumulated heavy metals in Pangasius suitchi. Waste
Management & Research, 31 (10), 75–80.
16) Tchobanoglous, G. and S. E. Vergara. (2012). Municipal solid waste and
the environment: A global perspective. Annual Review of Environment and
Resources, 37, 277.
Biomass Flow and Carbon Sequestration
in an Organic Farm
L. Hong Yeng* and P. Agamuthu

Institute of Biological Sciences, Faculty Science, University of Malaya
Abstract
Biomass is believed to improve soil fertility and carbon sequestration by enhancing soil organic
matter (SOM) content. Organic farm uses large amount of biomass input to replace chemical
fertilizer. Thus, it is considered a sustainable option for greenhouse gas (GHG) mitigation in
agriculture. There are inconsistent findings on the increase in soil carbon concentrations in
organically managed soil and this has become a hotly debated issue. In this paper, biomass carbon
flow of an organic farm was evaluated with material/substance flow analysis (MFA/SFA) for
evidence of carbon stock. Annually, there was 3,046 ton ha-1 y-1 of biomass input, which accounts
for 73% of total farm input. The only biomass output from organic farm was harvested vegetables
which was 112 ton ha-1 y-1. The substance flow shows a total of 156 ton ha-1 y-1 of carbon stock
within the system and this indicates that the farm system was playing a role as carbon sink. In
addition, carbon dioxide and carbon monoxide emission is also one of the major culprits of
carbon outflow from farm system which comprised a total of 172 ton ha-1 y-1 of carbon per year.
Moreover, the soil carbon concentration of the organic farm increased 29% during the study
period. Despite the carbon outflow, results indicate that organic farm management practices
focused large amount of biomass input had increased soil carbon concentration.
Key words: Organic, Vegetable farm, Material/substance flow analysis, STAN
I. Introduction
Biomass is believed to improve soil fertility and carbon sequestration by enhancing
soil organic matter (SOM) content and terrestrial ecosystem is an ideal reservoir for
carbon sequestration that could offset the CO2 emission due to human activities
[1]. Thus, IPCC has identified biomass application in soil as the promising tool
to capture and store carbon at terrestrial reservoir [2]. Crop residue, bio-solid,
fertilizer and manure are the common biomass applied at farm which contributes
to soil carbon sequestration [3]. Organic farm is believed to be carbon sequester
because biomass application is a common practices there.
Several reports discussed on the benefit of conversion from conventional farm
to organic farm in regards to carbon sequestration [4,5,6]. However, none draw a
* Corresponding author. Phone: +6012-6538938. Email: nicolelhy5134@gmail.com
147
definite conclusion. There are reports reveal that soil carbon sequestration through
conversion from conventional farm to organic farm is a temporary solution [7].
This article presents a case study of carbon flow in an organic farm. The farm
level analysis was chosen for three reasons. First, farm management plays a major
role in carbon sequestration. Proper farm management can create carbon sink
by encouraging carbon sequester practices [8]. Second, it provides information
about the flow and stocks of material and substances within farm system. Third,
it provides fundamental information for the development of farm management
strategies at farm level.
This analysis was conducted based on laboratory results done which present
the onsite situation for this particular study. The goal of this article are to (1)
identify key process and pathway, (2) determine the carbon stocks, (3) develop
recommendation for goal-oriented farm management in order to ensure maximum
carbon stock and minimize carbon output from the system.
II. M aterial and Methods

This study utilized the combination of field sample analysis, onsite survey and
material/substance flow analysis (MFA/SFA) method to achieve objectives.
Material/substance flow modeling was performed using modeling software STAN
2.5 (subSTance flow ANalysis) based on the field data collected [9].
A. Material/Substance Flow Analysis

The basic theory of MFA/SFA is that output is derived from input which also
knows as mass balance (Eq. 1, 2 and 3) [11,12].
Balance equation:
Σ inputs = Σ outputs + change in stock (1)
Transfer coefficient equation:
Output = transfer coefficient output x Σ inputs (2)
Stock equation:
Stock Period i+1 = stock Period i+change in stock Period i (3)
MFA/SFA is a dynamic approach that analyses material/substance flows or

any stock accumulation over a period of time based on mathematical probabilistic
distributions. MFA/SFA identifies the associations between input, output, process,
inventory and stocks. Five fundamental steps of MFA process has been followed
in this study [10,11]: (1) Determination and selection of research objective and
monitoring parameters; (2) Identification of system boundaries, scope and time
frame; (3) Identification of key pathways, processes and stocks; (4) Material flow
modelling; (5) Mass balance.
B. Farm Description
The system boundary is within an organic farm (2°56’56.59”N, 101°53’25.69”E)
which located at area with an elevation of 75 meter. The major soil groups there
are Typic Hapludult. It is managed in accordance to the standards enacted
by the regulation of Malaysia Organic Scheme based on Malaysian Standard
MS1529:2001 (the production, processing, labelling and marketing of plant
based organically produced foods).
C. Sample Analysis
Composite samples of soil, vegetable, compost, fertilizer, manure, organic waste
and water were sampled from the field through random sampling method and
analysed at the laboratory [12,13]. Solid samples were dried at 65 °C for a period
of 72 hour and sieved (1 mm mesh), and were analysed with Perkin Elmer
CHNS/O Series II 2400 for carbon content [14]. The carbon content of water
samples were analysed HACH DR/4000 (HACH procedure method 10128) and
gran alkalinity method [15].
Carbon dioxide and carbon monoxide emission was measured with 2 mm
acrylic static chamber (32cm x 22cm x 22cm) [16].
III. RESULTS AND DISCUSSION

A. Material Flow Model
Three processes have been defined: Farm Land; Vegetable Processing and
Packaging; and Husbandry. The process Farm Land is the pre-harvest stage which
involves planting activities while the process Vegetable Processing and Packaging
is the post-harvest stage. The process Husbandry does not participated in farm
production but was included in the model because organic waste was recycled as
animal feed. Based on the biomass input and output identified, a biomass balance
was established (Eq. 4).
Biomass Balance = (Bokashi Compost + Compost + Peat Moss + Vermi-
compost) – Harvested Crop (4)
Figure 1 shows the compost (2,757 ton ha-1 y-1) and Bokashi compost (276
ton ha-1 y-1) were the major biomass input in the farm system while harvested
vegetable (112 ton ha-1 y-1) was the main biomass output. The MFA model shows
that the farm recycled organic wastes generated from the post-harvest processing
as animal feed (6.15 ton ha-1 y-1).
Figure 1. Material flow model for organic farm, 2011–2013
B. Carbon Flow Analysis

The STAN model shows the biomass input has contributed to 366 ton ha−1 y−1
of total carbon into the farm system. There are reports questioning the benefit of
carbon input in carbon sequestration. This is because the carbon input increases
soil carbon but in the same time, encourages microbial decomposition activity
which leads to higher carbon emission [4]. Thus, the STAN model (Figure 2)
included carbon gaseous emission in the model to evaluate the effect of gaseous
emission in carbon sequestration. According to the STAN model, the largest
carbon flows out from the farm system was through carbon monoxide and carbon
Figure 2. Carbon flow analysis of organic farm, 2011–2013

dioxide gaseous emission which accounts for 81% of the total carbon output from
the farm system. The rest of the carbon output was vegetable sold to the consumer.
Each year, around 156 ton ha−1 y−1 (dstock) of carbon has stocked into the
farm system. This indicates the farm has the potential to be carbon sequester due
to high input and low output of biomass. This result agrees with the carbon flux
modelling by Vleeshouwers and Verhagen (2002) where the usage of organic
matter input increases carbon input and resulted to 1.50 ton ha−1 y−1 of carbon
flux [8]. To further investigate, the soil carbon concentration during the study
period was measured and the results shows the increment of 29% in terms of soil
carbon concentration. This result is higher than the result reported by Leifeld and
Fuhrer (2010) where 2.2% increase in soil carbon concentration [5].
IV. Conclusion
The carbon flow model in this study implies that the organic farm has the potential
to be carbon sink even when carbon emissions were taken into account for the
carbon flow analysis. Further research shall be conducted at other organic farm
located at different region to generate regional data.
V. Acknowledgement
The authors are grateful to University of Malaya (IPPP Project No: PV009-2012A)
for providing the financial support for executing the work. Special thanks to
Department of Agriculture Malaysia (Permanent Food Production Areas, TKPM-
Ulu Yam), SPC Organic farm, Green Oasis Sdn. Bhd., Mr. Zulklifi, Mr. David,
Mr. Thiam Kong Seng, Mr. Teo Wai Kit, and Ministry of Higher Education
Malaysia (MyPhD programme).
VI. R eferences
1) Luo, Z., Wang, E. and O. J. Sun. (2010). Soil carbon change and its responses
to agricultural practices in Australian agro-ecosystems: a review and synthesis.
Geoderma, 155 (3), 211–223.
2) Sims, R. E. H., Schock, R. N. and A. Adegbululgbe. (2007). IPCC Fourth
Assessment Report: Climate Change 2007 Working Group III: Mitigation of
Climate Change, Chapter 4: Energy Supply. IPCC.
3) Paustian, K., Parton, W. J. and J. Persson. (1992). Modeling soil organic
matter in organic-amended and nitrogen-fertilized long-term plots. Soil Science
Society of America Journal, 56 (2), 476–488.
4) Janzen, H. H. (2006). The soil carbon dilemma: Shall we hoard it or use it?
Soil Biology and Biochemistry, 38 (3), 419–424.
5) Leifeld, J. and J. Fuhrer. (2010). Organic farming and soil carbon sequestra-
tion: what do we really know about the benefits? Ambio, 39 (8), 585–599.
6) Liu, R., Xu, J. M. and C. E. Clapp. (2013). Carbon Sequestration in Organic
Farming. In Functions of Natural Organic Matter in Changing Environment
(pp. 377–379). Netherlands: Springer.
7) Smith, P. (2004). Carbon sequestration in croplands: the potential in Europe
and the global context. European Journal of Agronomy, 20 (3), 229–236.
8) Vleeshouwers, L. M. and A. Verhagen. (2002). Carbon emission and
sequestration by agricultural land use: a model study for Europe. Global
Change Biology, 8, 519–530.
9) Cencic, O. and H. Rechberger. (2008). Material flow analysis with software
STAN. Journal of Environmental Engineering and Management, 18 (1), 3–7.
10) Baccini, P. and P. H. Brunner. (2012). Metabolism of the Anthroposphere:
Analysis, Evaluation, Design, 2nd ed. (pp. 15–40). Cambridge, MA, USA: The
MIT Press.
11) Brunner, P. H. and H. Rechberger. (2004). Practical handbook of material
flow analysis. The International Journal of Life Cycle Assessment, 9 (5), 337–338.
12) Voll, M. and O. Roots. (1999). Soil water sample collector. Environmental
Monitoring and Assessment, 54 (3), 283–287.
13) Migliaccio, K. W., Li, Y. C., Trafford, H. and E. Evans. (2006). A simple
lysimeter for soil water sampling in south Florida [Online]. Retrieved from
http://trec.ifas.ufl.edu/kwm/files/pdf/publications/edis_ABE361.pdf.
14) Pereira, M. G., Espindula, A. Jr., Valladares, G. S., Cunha dos Anjos, L. H.,
de Melo Benites, V. and N. Schultz. (2007). Comparison of total nitrogen
methods applied for Histosols and soil horizons with high organic matter
content. Communications in Soil Science and Plant Analysis, 37 (7–8), 939–943.
15) Wetzel, R. G. and G. E. Likens. (2010). The Inorganic Carbon Complex:
Alkalinity, Acidity, CO2, pH, Total Inorganic Carbon, Hardness, Aluminum.
In Limnological Analyses, 3rd ed. (pp. 113–135). New York: Springer.
16) Parkin, T. B. and R. T. Venterea. (2010). USDA-ARS GRACEnet Project
Protocols Chapter 3. Chamber-Based Trace Gas Flux Measurements 4.
Sampling Protocols. USDA-ARS, Fort Collins, CO, 3-1. [Online]. Retrieved
from: http://www.usmarc.usda.gov/SP2UserFiles/person/31831/2011%20
Parkin%20and%20Venterea%20Trace%20Gas%20Protocol%20Revi-
sion%20Final.pdf
Biomass Gasification for Power Generation
Using Dual Chamber Circulating Fluidized
Bed Reactor
Haifa Wahyu*, Imam Djunaedi, M. Affendi and Sugiyatno
Research Centre for Physics, Indonesian Institute of Sciences
Jln. Cisitu-Sangkuriang 21/154D, Bandung 40135 Indonesia
Abstract
This paper presents an investigation on using dual chamber circulating fluidized bed reactor for
biomass gasification in power generation. Gasification process has been around for many years for
charcoal production from biomass or gas production from biomass or coal. The technology varies
from simple downdraft reactor or more complex system, such as circulating fluidized bed and
combined cycle. Gasification technology is an alternative solution to conventional steam power
plant to generate electricity from biomass waste. Its use can be combined with diesel generator
or gas engine where the gas products from biomass gasification system can replace up to 70%
of diesel oil while the gas can be used as 100% fuel in a gas engine. This will help reduce the
electricity cost generated from diesel engine. A major problem that still occurs in a gasification
reaction is the formation of tar. The most effective way to eliminate tar is by complete burning.
In circulating fluidized bed, tar can be captured using sand media. The tar covered sand will be
brought to the second chamber to be completely burned to remove the tar. This way, the plant
can be operated continuously without interruption. For a commercial power plant, it is important
that the plant operational is reliable. A circulating fluidized bed can be used to perform this task.
In this work, a dual chamber reactor is developed so that tar removal can be done continuously.
We have constructed a test plant with the capacity of 30 kg biomass per hour. This work includes
the gasification of several biomass types, that is wood sawdust, coffee husk, empty fruit bunch,
rice husk and vertiver root. On average, the plant is able to produce fuel gas with the composition
of H2 around 4%, CO between 10 to 12%, CO2 around 12% and CH4 around 2%. The tar
produced is less than 50 mg. Most biomass produced similar gas composition, except for the
rice husk. The rice husk must be mixed with other type of biomass to enable the production of
fuel gas satisfactorily.
Key words: Biomass gasification, Circulating fluidized bed, Dual fuel power generation
I. Introduction
Biomass gasification is an important process that can be an alternative to direct
combustion. In some cases, lignocellulosic material is hard to burn in a conven-
tional furnace, therefore it must be processed by thermal cracking to produce
* Corresponding author. Phone: +62-22-2507771. Email: haifa.wahyu@gmail.com, haifa.wahyu@lipi.

go.id
153
gas and liquid. Gaseous product consists of reactive substances mainly of carbon
monoxide and hydrogen, which is called synthesis gas (syngas). Syngas can be
used as gas fuel in diesel engine or processed further as raw material for chemical
productions. Biomass, such as rice husk, has been used as fuel in gasification
process, especially in countries where rice fields are abundant as in China [1].
Gasification of rice husk is usually done in a downdraft gasification reactor
[1,2]. Other type of reactor was also studied for gasification of rice husk, such as
fluidized bed [3], a dual distributor fluidized bed [4] and a cyclone gasifier [5].
Each type of reactor has the advantages and disadvantages. Downdraft gasifier is
more simple and easier to operate although less efficient, while fluidized bed and
cyclone gasifier are more complicated but more efficient. However, in all types
of reactors, the formation of tar is always a problem. The most effective way to
remove the tar is by complete burning. In order to remove the tar in the downdraft
gasifier, Affendi et al. [2] cleaned the equipment manually and burned the tar.
For a continuous application, downdraft gasification is a little cumbersome
since tar is cleaned when the system is shut off and the downstream components
must be disassembled. Furthermore, other type of fuel may not suitable to be used
in a downdraft gasifier. An example is wood sawdust gasification which provides
better results in a fluidized bed reactor [6]. This also applies for other material,
such as coal gasification, where an integrated gasification combined cycle has
been developed in New Zealand [7] and Austria [8] to cope with large capacity
power producing system. Circulating fluidized bed which is based on entrained
flow has the advantage of producing clean tar free gas continuously, while the
ash is produced in the form of inert slag [9].
The present work aims to investigate the use of a circulating fluidized
bed system using atmospheric air as a reacting agent to gasify several types of
biomass. We have developed a dual chamber circulating fluidized bed for biomass
gasification [10]. The system is expected to deal with the elimination of tar
and the production of gas continuously. This paper provides the results of the
gasification of sawdust, coffee husk, rice husk and empty fruit bunch. The actual
gas composition was analyzed using Orsat gas analyzer.
II. Method
The experiment was conducted in a dual chamber circulating fluidized with the
capacity of 30 kg/hour (Figure 1). The reactors are divided into two chambers
gasification chamber and combustion chamber. Gasification chamber is painted
in blue color while combustion chamber is painted in orange. Gasification
process is held in the blue chamber using sand as the heating media. The sand is
also functioning to capture the tar formed during the gasification process. The
combustion chamber is used to burn sand coated tar to completely convert the
tar into flue gas. Each reactor is equipped with dust cyclone to separate the gas
from the sand and dust. The sand is recirculated in the system, clean sand from
the combustion chamber is returned to the gasifier to perform the next cycle of
gasification process. Biomass enters the gasifier through the biomass feeder and
brought into the chamber using screw feeder.
Table 1. Design parameter of the dual chamber circulating fluidized bed
Parameter Value
Reactor diameter, mm 20
Reactor height, mm 2,250
Minimum fluidization velocity, m/s 0.5-0.8
Biomass flowrate, kg/hour 15-25
Sand flowrate, kg/hour 15
Figure 1. Dual chamber circulating fluidized bed for

biomass gasification
The following table contains the design parameter of the CFB reactor.
In this experiment, we used biomass that is common in agricultural industries
in Indonesia, such as rice husk, wood sawdust, empty fruit bunch and coffee
husk. The following table shows the calorific value of each type of the biomass.
Table 2 Calorific value of biomass

Types of biomass (ar) CV (kJ/kg)
Empty fruit bunch 18,960
Rice husk 15,719
Vertiver root 18,422
Wood sawdust 18,036
Coffee husks 18,422
Table 3 contains the properties of the sand used as the heating media.
Table 3 Sand properties

Properties Sand
Type Quartz
Average particle diameter (µm) 385
Density (kg.m-3) 2,650
Porosity 0.46
Sphericity 0.78
The results are shown using data on reaction temperature and syngas analysis.
Orsat gas analyzer was used to record the gas composition, while K-type ther-
mocouple was used to measure reaction temperature. The reaction temperature
during the experiment was recorded at several places. The reaction temperature
was taken at the inlet, in the middle of the reactor and at the syngas outlet.
The reactor was heated up until about 400 °C before the biomass entered the
gasification reactor.
III. R esults A nd Discussions

The results of the experiments show the composition of combustible gas for
each type of biomass measured using Orsat gas analyzer. The experiments were
compared with the theoretical results whose gas composition are as follows: CO
12%, H2 4.0% and CH4 3.0%.
The experimental results of the syngas composition are slightly different from
that of the theoretical values. The percentage of CO is about 2% lower during
the experiment than that of the theory. The percentage of CH4 is higher during
the experiment than that of the theory, about twice. The production of hydrogen
gas is the same for all the biomass, except in wood sawdust. The hydrogen
production is as twice as much for wood sawdust. CO2 and CnHm production
varies slightly among the biomass. Although the percentages are different, the
whole results indicate the ability of the equipment to produce CO and H2 gases
which is crucial in biomass gasification. To prove the flammability of the gas, the
syngas was burned at the outlet of the reactor. The flame was quite stable during
the experiment.
The following picture shows the burning of the syngas at the outlet.
Figure 4 shows the temperature profile in the reactor. The figure shows the
reaction temperature taken progressively from the middle of the reactor during
the experiment. The temperature shows the gradient on hourly basis. It shows
that the reaction temperatures are similar for all biomass.
Figure 3. The flaring of syngas at the outlet

Figure 2. Syngas composition compared with
theoretical values
Figure 4. The profile of the reaction temperature in the middle of the

gasifier taken after heating up until up to 4 hour reaction
IV. Conclusion
The results of the gasification experiments on several types biomass in the dual
chamber circulating fluidized bed are in agreement with the theoretical values.
Syngas can be produced successfully in this type of reactor indicated by the
composition of the gas and the burning capability of the gas products. Based on
the experimentals results, it can be concluded that a dual chamber circulating
fluidized bed can be used to produce gas fuel that can be applied in diesel
generators for electricity production.
V. Acknowledgement
The authors would like to thank the organizer of the Conference for the invita-
tion to present this paper. The authors would also like to express their thanks
to colleagues at the Research Centre of Physics LIPI for the support provided.
VI. R eferences
1) Lin, K. S., Wang, H. P., Lin, C. J. and C. I. Juch. (1998). A process develop-
ment for gasification of rice husk. Fuel Processing Technology, 55, 185–192.
2) Affendi, M., Sugiyatno, Suhartono and I. Djunaedi. (2009). Kajian tekno-
ekonomi pengoperasian PLTD-sekam 100 KW Di Haurgeulis, Indramayu.
Laporan Akhir Tahun 2009, Kegiatan Program Kompetitif LIPI. Pusat
Penelitian Fisika- LIPI.
3) Ramírez, J. J., Martínez, J. D. and S. L. Petro. (2007). Basic design of a
fluidized bed gasifier for rice husk on a pilot scale. Latin American Applied
Research, 37, 299–306.
4) Mansaray, K. G, Ghaly, A. E., Al-Taweel, A. M., Hamdullahpur, F. and V.
I. Ugursal. (1999). Air gasification of rice husk in a dual distributor type
fluidized bed gasifier. Biomass and Bioenergy, 17, 315–332.
5) Zhao, Y., Sun, S., Che, H., Guo, Y. and C. Gao. (2012). Characteristics of
cyclone gasification of rice husk. International Journal of Hydrogen Energy,
37, 6962–6966.
6) Kumaran, V. (2007). Design of a low cost fluidized bed gasifier for sawdust
gasification in rural China. Energy from Biomass and Wastes, 421–825.
7) Brown, J. (2006). Biomass Gasification: Fast Internal Circulating Fluidised
Bed Gasifier Characterisation and Comparison (ME Thesis). New Zealand:
University of Canterbury.
8) Institute of Chemical Engineering TU Wien Austria. (2011). The FICFB-
gasification system. Retrieved from http://www.ficfb.at/
9) Higman C. and M. van der Burgt. (2008). Gasification, Second Edition.
Amsterdam: Elsevier.
10) Wahyu, H., Djunaedi, I., Affendi, M. and Sugiyatno. (2013). Design,
Simulation and Experiment of Circulating Fluidized Bed Reactor for
Biomass Gasification. Proceedings of the International Seminar on Biorenewable
Resources Utilization for Energy and Chemicals 2013. Bandung, 9–11 October
(p. 243). Bandung, Indonesia.
Roof Mounted Micro-Wind Turbine for
Power Generation in Coastal Housing in
Semarang, Indonesia
Dany Perwita Sari*

Research Center for Biomaterials, Indonesian Institute of Scienes (LIPI)
Jl. Raya Bogor km.46, Cibinong, Bogor 1691, Indonesia
Abstract
Mounted micro-wind turbines have the potential power saving in coastal housing. In this paper,
the meteorological data of five year wind velocity and wind direction of Semarang, Indonesia,
were used to find out the wind energy potential. From wind direction evaluation at a height of 10
m above the ground level, it was found that the highest wind power potential is on north wind.
This research aims to present the result of Computational Fluid Dynamics (CFD) simulations
for identifying wind velocity and wind flow of two kind roof profiles: gable roof and hip roof.
Result shows that the wind flows are strongly dependent on the profile of the roof. It was also
concluded that roof mounted micro-wind turbine is suitable for electric wind application, which
can reach 20% more than wind velocity approach.
Key words: Micro-wind turbine, Wind velocity, CFD, Wind energy, Semarang, Indonesia
I. Introduction
The government of Indonesia commits to reduce fossil fuel consumption up to
26% by the year 2020 [1]. The increasing interest in renewable energy has led
to a desire to explore wind energy. Since the majority of Indonesia’s population
lives in coastal areas, implementing micro-wind turbine for housing has the
potential to make a significant contribution to government’s targets. Semarang
that has coastal housing which is famous with the traditional roof design has been
presently chosen for the case study. Semarang forecasting within 5 years period
(2008–2012) [2] shows that coastal housing where is located along northern area
of Semarang has huge potential for power generation.
With high mean wind velocity and low levels of turbulence (coastal area),
the micro-wind turbine installation will become one of the potentially low-cost
renewable sources of energy. However, wind flow and turbulence intensity at the
roof level strongly depend on the roof profile [3]. Otherwise, if a turbine is situated
in the wrong location on the roof; possibly the power output is not significant [4].
Roof shape is one of the main factors affecting the installation of roof mounted
* Corresponding author. Phone: +62-8132-5693808. Email: dany.perwitasari@gmail.com
161
wind direction wind direction

building position 0° building position 0°
wind direction
wind direction
building position 0°
Figure 1. Roof profile, from left to right: gable roof, building position 0°;
gable roof, building position 90°; hip roof, building position 0°; hip roof,
wind turbine [5]. Coastal housing in Semarang has a unique roof profile, such as
gable roof and hip roof. Based on this condition, this paper attempts to address
one of the main factors for building integrated micro-wind turbine which is
wind velocity around the roof. Computational Fluid Dynamics (CFD) is used
to analyze air flow and wind velocity over the roof profile to ascertain the most
power productive location for micro-wind turbine.
II. CFD Simulations

CFD was used to simulate wind flow above gable roof and hip roof, which
represent the basic shapes of traditional coastal housing in Semarang (Figure 1).
This modeled was assumed as residential houses 45 m2 (4 m x 9 m). Simulations
were undertaken with two different wind direction (Figure 1), with direction 0°
parallel to the roof profile and 90° cross to the roof profile.
Table 1. Percentage of wind speed and wind direction in Semarang, Indonesia (2008-2012) [6]
Wind Speed Wind Direction To-tal
(km/hr) N E SE S SW NW (%)
7-9 13,3 11,7 6,67 3,33 5 8,333 48,33
10-12 10 5 6,67 3,33 1,67 10 36,67
13-15 - - - 1,67 - 1,667 3,333
16-18 - 1,67 - - 1,67 6,667 10
19-20 - - - - - 1,667 1,667
Total (%) 23,33 18,33 13,33 8,333 8,333 28,33 100
From Table 1, wind direction evaluation at a height of 10 m above ground

level was found that the highest wind power potential is on north wind (7–9 km/
hr). The roughness classification is using ASCE (1999) [7] with smooth terrain
roughness. Wind profile for CFD analysis in Semarang can be expressed as:
(1)
Where U is wind velocity (m/s), Uref is wind velocity reference (m/s), Z is the
gradient height (m), Zref is the gradient height reference (m) and α is power law
exponent. From the equation, velocity profile could be configured (Table 2). The
wind speed profile measured in the CFD using the power law of an exponent of
0.125. The mean wind speed was 5 m/s at 10 m above the sea level.
III. Wind Power Density of Semarang City, Indonesia

Wind power density is a measure of the energy available in the wind at some
region. Wind turbine power of a wind generator [11] can be expressed as:
(2)
where:
Pturbine = the wind turbine power,
Ρ = the air density (kg/m3)
Cp = the coefficient of performance,
A = the swept area of the blades (m2),
V = free wind speed (m/s).
Two different micro-wind turbines, D400 Stealth Gen (swept area 1.1 m)
and Renewable Devices Swift Turbine (swept area 2.1 m), have been carried out
to investigate wind power density. The wind power density resulted from two
different micro-wind turbines that were given in Table 2. As shown, the majority
Table 2. Velocity profile and wind power density using D400 Stealth and Renewable Device
Swift micro-wind turbine.
Height Avarage Velocity Wind Power Density (W/m2)
(m) (m/s) D400 Stealth Gen Renewable Devices Swift Turbine
(d=1.1 m) (d=2.1 m)
1 3,749471 35,37414 67,53245
2 4,088827 45,87459 87,57876
3 4,301403 53,40801 101,9607
4 4,458898 59,49198 113,5756
5 4,58502 64,68442 123,4884
6 4,690714 69,26162 132,2267
7 4,781975 73,38338 140,0955
8 4,862462 77,15155 147,2893
9 4,934581 80,63561 153,9407
10 5 83,88531 160,1447
11 5,059925 86,93771 165,972
12 5,115259 89,82121 171,4769
13 5,166696 92,55815 176,7019
14 5,21478 95,16647 181,6814
15 5,259948 97,66077 186,4433
of wind power density in Semarang is categorized as fair wind resources [8]. This
is largely due to the inlet wind velocity. Wind power density function of the cube
(third power) of the wind speed (2). If the wind speed is doubled, power in the
wind increases by a factor of eight. This relationship means that small differences
in wind velocity lead to large differences in wind power density.
IV. R esult and Discussion

Specifying the optimum wind power density of roof mounted wind turbine
depends on the wind velocity. CFD is used for predicting wind velocity and wind
flow above the roof profiles. Figure 3a shows the average wind velocity result for
gable roof in 0° position. The simulation is expressed the wind velocity increase
in the height 1m above roof surface (7 m/s to 8 m/s) then stable in the height
10 m above the roof surface. No increase wind velocity after passing gable roof.
Hip roof in 0° position result (Figure 3b) records similar with gable roof in 0°
position result. Case study model two changes building position from 0° to 90°
(Figure 1). Figure 4a shows the average wind velocity contour of gable roof. The
wind velocities are stable and almost similar with wind velocity approach. This
contrasts with hip roof with 90° position (Figure 4b), which wind velocity increase
20% more than wind velocity approach (8 m/s up to 8.50 m/s).
Figure 3a. Average wind velocity contour for gable roof at 0° position
Figure 3b. Average wind velocity contour for hip roof at 0° position
V. Conclusion
Coastal area has the potential for wind energy. It has been found that wind velocity
depends on the roof profiles as well as the wind direction. From meteorological
data at Semarang, Indonesia, it was found that the highest wind power potential
is on north wind with average wind velocity 5 m/s in the height 10 m from the
ground surface. A comparison between two traditional roof profile, gable roof and
hip roof, suggest that hip roof is more suitable in Semarang. The wind velocity
above gable roofs has lower velocity compared to hip roof profile. Based on the
velocity above the roof, hip roof with 90° position is the most favorable shape in
Semarang, Indonesia which could increase wind velocity 20% bigger than wind
velocity approach (wind profile). Future studies of wind velocity above other roof
profiles will be covering different building shapes and places.
VI. Acknowledgement
This paper was developed and delivered with data support from Meteorological
Department (BMKG) Semarang City and Indonesian Institute of Sciences (LIPI).
VII. R eferences
1) National Standarization Agency of Indonesia (BSN). (2011). Presidential
Regulation (Peraturan Presiden) No. 6/2011: National plan to reduce
greenhouse gas emission.
2) Badan Meteorologi Klimatologi dan Geofisika (BMKG) Stasiun Klimatologi
Semarang. (2013). Wind speed, solar radiation intensity, temperathure and
humidity in Semarang, Central Java, Indonesia years 2008–2012.
3) Johansen, O. (2011). The spatial diffusion of green building technologies: the
case of Leadership in Energy and Environment Design (LEED) in the United
States. International Journal of Technology Management and Sustainable, 10
(3), 251–266.
4) Ayhan, D. and S. Saglam. (2012). A technical review of Building-mounted
wind power systems and a sample simulation model. Renewable and Sustain-
able Energy Reviews, 16, 1040–1049.
5) Abohela, I., Hamza, N. and S. Dudek. (2013). Effect of Roof Shape, Wind
Direction, Building Height and Urban Configuration on The Energy Yield
and Positioning of roof Mounted Wind Turbine. Renewable Energy, 50,
1106–1118.
6) Sari, D. P., and W. B. Kusumaningrum. (2014). A Technical Review of

Building Integrated Wind Turbine System and a Sample Simulation Model
in Central Java, Indonesia. Energy Procedia, 47, 29–36.
7) American Wind Energy Association (AWEA). Wind Classes [Online].
Retrieved from www.awea.org/fag/basicwr.html.
8) Soeripno, S. (2012). Wind energy potential and development in Indonesia.
In Proceeding of The 2nd Clean Energy Power Asia Denpasar-Bali. 14–15 May
2012.
PERFORMANCE OF A RADIAL TURBINE FOR SMALL
ORGANIC RANKINE CYCLE POWER GENERATION
SYSTEM
Maulana Arifina,*, Bambang Wahonoa and Ari Darmawan Pasekb
a
Research Center for Electric Power and Mechatronics – LIPI
Komp. LIPI Gd. 20, Jl. Cisitu No.21/154D, Bandung 4015, Jawa Barat
Phone: 022 2503055, Fax: 022 2504773
b
Faculty of Mechanical and Aerospace Engineering – Bandung Institute of Technology
Jl. Ganesha No.10 Taman Sari, Bandung 41032, Jawa Barat
Phone: 022 2504243, Fax: 022 2534099
Abstract
Coresponding to global environmental problems and energy crisis in recent years, a method to
choose the optimal working fluids which improves performance of power generation with Organic
Rankine Cycles (ORC) is required. This paper investigates the optimal working fluids for ORC
with focus on thermo-fluid. In the ORC system, radial turbine component is highly influential
in resulting high and low performance. This paper discusses comparative study of radial turbine
using R134a and R123 as working fluids. Comparative study consisted of numerical analysis to
determine the performance of radial turbine for ORC system. Numerical study was carried out in
area of fluid flow turbo-expander rotor radial with R134a and R123 as the working fluids. Analysis
was performed using two turbulence models, the k-epsilon and SST (shear stress transport). The
results shows analysis with grid of 250000 (fine grid), turbulence model SST at steady state, mass
flow rate of 0.4 kg/s, torque of 15000 rpm, inlet pressure of 5 bar, inlet temperature of 373 K,
and working fluid of R134a produces power of 6,7 kW whereas R123 produces power of 5,5 kW.
Key words: Organic Rankine Cycle, Radial turbine, Shear Stress Transport
I. Introduction
Renewable energy technologies use natural resources, such as sunlight, wind, rain,
tides and geothermal heat, all of which are naturally replenished. Climate change
concerns coupled with high oil prices are driving research and development on
renewable energies. The ORC uses organic fluid as the working fluid to provide
higher thermal cycle efficiency compared to the conventional steam Rankine cycle
at resource temperatures below 300 °C. ORC has been studied as the utilization
of waste heat recovery [1,2], solar energy [3], the combination of heat and power
(CHP) [4], geothermal [5] and heat recovery from the exhaust gases from the
engine [6]. The results of experimental studies show that the small-scale units
* Corresponding author. Phone: +6222 2503055. Email: mfilzah@gmail.com, maul004@lipi.go.id
169
ORC showed a promising performance for small-scale power generation especially

in remote areas, because the ORC has reliability, a wide output power range, the
availability of large component parts, as well as the reduced number of rotating
parts, so that ORC’s construction is more compact and smaller compared to
conventional power plants.
Most studies have focused on the study of the thermodynamic cycle of the
ORC and the selection of the working fluid, with particular attention to the ef-
ficiency of power generation. On the other hand, there are relatively few published
works on the design and optimization of turbomachinery equipment. Basically,
the relevant power range (5–5000 kW) two options are proposed: turbine axial
or radial turbines (often called turbo-expanders). The latter option is considered
more attractive, because it allows better performance at a lower scale. Therefore,
knowledge of radial turbine in ORC systems required further study.
II. M aterial and methods

The performance characteristic of the radial turbine for small organic rankine
cycle was evaluated using ANSYS by analyzing the torque and power output on
relationship between mach number and rotational speed of rotor radial turbine
with R134a and R123 as working fluid. The main components of radial turbine
are rotor, shown in Figure 1. Table 1 shows operational condition for parameter
input on analysis CFD with ANSYS.
Figure 1. Geometry radial rotor turbo-expander for small organic

rankine cycle system
Table 1. Parameter input on analysis CFD

Parameter Value
Temperature inlet (K) 353-423
Mass flow (kg/s) 0.1-1.0
Pressure outlet (atm) 1
Temperature outlet (K) 313
A. Simulation with Different Grid

In simulation with different number of grids, the objective is to know how to
influence the amount of output generated grid. Effect of grid number is the
expected outputs of the model with a certain number of grid which can be more
accurate and the selected model is used as a model for subsequent simulations.
Figure 2 shows grid model for analysis CFD and Table 2 shows grid number.
B. Simulation with two turbulence models

ANSYS uses an element-based finite volume method linked to pressure-based
coupled solver for solving the hydrodynamic equations (u,v,w,p) [7]. A second-
order discretization scheme has been applied to these equations while a first-order
upwind advection scheme has been used for turbulence equations. The turbulence
intensity has been initialised at a medium level of 5%. This value has also been
used at the inlet of the rotor.
Numerical study is carried out in area of fluid flow rotor radial turbine
with R134a and R123 as the working fluid. Analysis was performed using two
turbulence models, the k-epsilon and SST (shear stress transport).
mainstream
downstream
upstream
Figure 2. Grid model for CFD computations


A. Simulation with Different Grid
Simulation with different grid showing the output results of numerical simulation
using the finite volume method represent a power of radial turbine as a function
of rotational speed. Figure 3 shows three model grid flow field predictions for
radial turbine using R123 as working fluid with rotational speed of 20000 rpm.
In model 3 (250,000 grid), a strong vortex can be seen forming on the pressure
surface soon after the leading edge, separating flow from the hub surface and
moving it up the blade toward the tip. After calculation process are solved by
ANSYS, torque and power radial tubine will be generated from the results of
calculations and represented in Table 2.
Model 1 Model 2 Model 3

Figure 3. Three model grid flow field predictions for radial turbine using R123 as working fluid
with rotational speed of 20,000 rpm
Table 2. Power and efficiency for rotor radial turbo-expander with different grid number
Model 1 Model 2 Model 3
Number Grid 20000 100000 250000
Fluid : R123
20,000 rpm
Torque (Nm) 0,32 0,35 0,36
Power (kW) 6,56 7,08 7,21
Efficiency 0,63 0,64 0,65
Time consumed 6 minute 30 second 11 minute 56 second 28 minute 05 second
Fluida R134a
20,000 rpm
Torque (Nm) 0,44 0,47 0,48
Power (kW) 7,94 8,60 8,53
Efficiency 0,63 0,64 0,65
Time consumed 6 minute 21 second 11 minute 54 second 27 minute 30 second
Table 3. Time computation, iteration number on turbulence models for radial turbine small
ORC with R123, 20,000 rpm
Model Time consumed Iteration Power (kW)
k-epsilon (k-ɛ) 28 minute 200 6,1
SST 28 minute 200 7,2
Figure 4. Velocity contour for turbulence models (a) k-epsilon and (b) SST
0 rpm 15000 rpm
20000 rpm 30000 rpm
Figure 4. Velocity contour for steady flow radial turbine with R123 as working fluid on various rotational
speed
0 rpm 15000 rpm
20000 rpm 30000 rpm
Figure 5. Velocity contour for steady flow radial turbine with R134a as working fluid
on various rotational speed
B. Simulation with two turbulence models

Table 3 shows the computation time and the number of iterations in the k-epsilon
and SST turbulence models. In Figure 4 and Figure 5, models with SST have
good swirl flow prediction on blade passage and the significant power generated
differences occurred.
In the k-epsilon turbulence model, the power is generated at 6.1 kW while
the SST turbulence models generated power of 7.2 kW. The increase in power
for the k-epsilon model of turbulence model to SST reaches 18%. It is to be
considered for the selection of turbulence models.
IV. Conclusion
Performance of radial turbine for small Organic Rankine Cycle system has been
evaluated using 3D flow field analysis method. Grid element analysis yields 3
models, which the 250,000 grid has good results. The analysis used two turbulence
models, and SST models has good results with mass flow rate of 0.4 kg/s, 15,000
rpm, 5 bar inlet pressure, and 373 K inlet temperature. Working fluid R134a
produces power of 6,7 kW, with total efficiency-to-static (ηts) 0,71, and R123
produces power of 5,5 kW, with total efficiency-to-static (ηts) 0,66.
Figure 6. Rpm vs Power of working fluid R134a and R123 performance
V. Acknowledgement
Authors would like to thank all the members of the Research Centre for Electrical
Power and Mechatronics and members of Thermodynamics Laboratory of
Mechanical Engineering Faculty, Bandung Institute of Technology, for any
assistance that has been given.
VI. R eferences
1) Hung, T.-C. (2001). Waste heat recovery of organic Rankine cycle using dry
fluids. Energy Conversion and Management, 42 (5), 539-553.
2) Gnutek, Z. and A. Bryszewska-Mazurek. (2001). The thermodynamic analysis
of multicycle ORC engine. Energy, 26 (12), 1075–1082.
3) Manolakos, D., Papadakis, G., Kyritsis, S. and K. Bouzianas. (2007). Experi-
mental evaluation of an autonomous low-temperature solar Rankine cycle
system for reverse osmosis desalination. Desalination, 203 (1-3), 366–374.
4) Schuster, A., Karellas, S., Kakaras, E. and H. Spliethoff. (2009). Energetic
and economic investigation of Organic Rankine Cycle applications. Applied
Thermal Engineering, 29 (8-9), 1809–1817.
5) Kanoglu, M. (2002). Exergy analysis of a dual-level binary geothermal power
plant. Geothermics, 31 (6), 709–724.
6) Talbi, M. and B. Agnew. (2002). Energy recovery from diesel engine exhaust
gases for performance enhancement and air conditioning. Applied Thermal
Engineering, 22 (6), 693–702.
7) Ventura, C. A. M., Jacobs, P. A., Rowlands, A. S., Petrie-Repar, P. and E.
Sauret. (2012). Preliminary Design and Performance Estimation of Radial
Inflow Turbines: An Automated Approach. Journal of Fluids Engineering, 134,
031102.
Effect of Reaction Time and Cellulase
Loading on Dilute Alkali Pretreatment of
Sugarcane Bagasse to Produce Fermentable
Sugars for Bioethanol Production
Triyani Fajriutamia,* and Rizky Rissa Bellab

Research Center for Biomaterials, Indonesian Institute of Sciences (LIPI)
a
Jl Raya Bogor KM 46, Bogor, Indonesia

b
Bina Putera Nusantara Vocational High School
Jl Sukarindik 63A, Tasikmalaya, Indonesia
Abstract
Lignocellulosic materials, which consist mainly of cellulose, hemicellulose and lignin, are among
the most promising renewable feedstocks for the production of bioethanol. Its production typically
involves a hydrolysis-fermentation route, which has three main steps: pretreatment and hydrolysis
to get fermentable sugars, fermentation to produce bioethanol and a separation process to obtain
highly concentrated bioethanol. The pretreatment step has been recognized as a technological
bottleneck for the cost-effective development of bioprocesses from lignocellulosic materials. In
this work, we analyze the potential of dilute alkali pretreatment of sugarcane bagasse, followed by
enzymatic hydrolysis for fermentable sugars production when using 1:20 ratio between substrate
and 1% NaOH. Alkali pretreatment of sugarcane bagasse at 121 °C and reaction time of 7.5, 15,
30, 60 and 90 minutes were investigated. Furthermore, the enzymatic hydrolysis using cellulase 10
and 20 FPU/g substrate was examined. The result shows that the highest lignin loss was 74.95%
when sugarcane bagasse was pretreated for 60 minutes. The enzymatic hydrolysis of pretreated
sugarcane bagasse for 48 hours and 20 FPU/g of cellulase loading produced the highest yield of
fermentable sugars of 49.11%.
Key words: Lignocellulosic, Sugarcane bagasse, Alkali pretreatment, Cellulase hydrolysis, Ferment-
able sugars
I. Introduction
Lignocellulosic materials have been recently considered as promising sources of
bioethanol [1]. An advantage of lignocellulosic materials is the avoidance of the
competition with food production. Furthermore, there are abundant source for
lignocellulosic materials including waste materials. Lignocellulosic materials are
characterized by three components, which are cellulose, hemicelluloses and lignin.
These components are complex polymers. Lignin acts as a wall covering hemicel-
lulose and cellulose; hemicellulose do not have a defined form, and cellulose
* Corresponding author. Phone: +62-2187914509. Email: triyani@biomaterial.lipi.go.id
177
is the most difficult component to access. Among the available lignocellulosic

materials, sugarcane bagasse has been considered as a high-potential candidate
for bioethanol production in Indonesia because it is available in abundance in
sugarcane mills [2].
Three principal steps to convert lignocellulosic materials into bioethanol are
pretreatment, hydrolysis and fermentation. Pretreatment of lignocellulosic materi-
als to remove lignin and hemicellulose can significantly enhance the hydrolysis of
cellulose [3]. According to the literature [4,5,6,7], pretreatment is the bottleneck
into the development of bioprocess from lignocellulosic materials.
One of the major aspects in bioethanol production is the energy consumption,
so the pretreatment method should be as energy efficient as possible to contribute
in the success of the overall process. Alkaline pretreatment methods are receiving
much attention due to relatively cheap, less energy intensive and effectiveness
towards many feedstocks rather than other pretreatment methods [8,9]. Alkaline
pretreatments lead to delignification, disruption of structural linkages, decrystal-
lization of cellulose and depolymerization of the carbohydrates [10,11].
Enzymatic hydrolysis and fermentation are known to be environmentally
friendly processes for converting lignocellulose to ethanol [12]. Enzymatic pro-
cesses under development are supposed to have roughly equal costs today, but
the costs can be decreased further. Therefore, most studies focus on enzymatic
hydrolysis [13]. The fermentation step, on its turn, does not yet convert all sugars
with equal success. Future overall performance depends strongly on development
of cheaper and more efficient microorganisms and enzymes for fermentation.
In this study, we investigated the effect of reaction time at 121°C from dilute
sodium hydroxide pretreatment of sugarcane bagasse followed by the effect of
cellulase loading on hydrolysis process for its reducing sugar production.
II. Method
A. Raw Material
Sugarcane bagasse from sugar factory in Subang, West Java-Indonesia was milled
and sieved to get particles of 40–60 mesh, then it was stored in sealed plastic bag
at room temperature until it was ready to be used for pretreatment. The sugarcane
bagasse was measured for its ash and moisture content by gravimetric method
and extractives content by Soxhlet extraction as well as its lignin, cellulose and
hemicellulose content.
B. Pretreatment
Sodium hydroxide (NaOH) at concentration of 1% (w/v) was used to pretreat 5
g milled sugarcane bagasse samples at 1:20 ratio (substrate: NaOH solution) in
250 mL erlenmeyer flask. Treatments were performed in an autoclave at 121°C

with reaction time of 7.5, 15, 30, 60 and 90 minutes. Water is used to substitute
the alkaline solution as a control. After pretreatment reaction, the samples were
filtered to separate the solid from the liquid (pretreated hydrolysate). Pretreated
solids were washed with water until the filtrate registered a neutral pH and sealed
in plastic bags to retain moisture. Then some wet samples were refrigerated for
further Scanning Electron Microscope (SEM) analysis using Phenom ProX
Desktop SEM and subjected to enzymatic hydrolysis. The other ones were dried
at 60°C and determined for its lignin contents after pretreatment. Lignin loss
was also calculated.
C. Enzymatic Hydrolysis
Enzymatic hydrolysis of pretreated sugarcane bagasse was carried out using a
commercial cellulase (Meicellase from Meiji Seika, Japan). Vial bottles (20 ml)
containing 0.1 g (dry weight) delignified sugarcane bagasse was mixed with
cellulase solutions (10 and 20 FPU/g), 0.1 ml of sodium azide (20 mg/ml) to
inhibit microbial contamination, and weight was made to 10 g mark with 0.05 M
citrate buffer of pH 5. The vials were incubated at 50°C in orbital shaker at 150
rpm for 48 h. The vials were placed horizontal in shaking incubator to increase
surface area contact between substrate and enzyme. Reducing sugar concentration
was determined by Nelson-Somogyi method.

A. Characterization of Sugarcane Bagasse
Sugarcane bagasse is a polydisperse particulate. Most of the bagasse weight is in the
form of the so-called fiber and rind particles, which have high length-width ratios
(lengths up to a few centimeters) and correspond mostly to stalk fibrovascular
bundles [14]. The sugarcane bagasse in this work was milled and sieved before
chemical analysis because of the heterogeneous and fibrous nature of sugarcane
bagasse. Milling is an important step in the development of conversion routes
for production of cellulosic biofuels and co-products [14].
Characterization of sugarcane bagasse was carried out to determine its major
of principal components. The chemical composition of initial sugarcane bagasse
(before pretreatment) used in this study is presented in Table 1. The carbohydrate
fraction (holocellulose fraction) of sugarcane bagasse was 61.96% of the total
biomass, which consist of 34.48% alpha-cellulose and 27.48% hemicellulose.
The major component of sugarcane bagasse was alpha-cellulose, a polymer of
glucose, which is very potential as a sugar source for ethanol production. The lignin
content of sugarcane bagasse was 22.45%, comparable to the lignin content of
Table 1. Chemical contents of the raw sugarcane bagasse

Components Percentage (%)a
Ash content 2.13 ± 0.3756
Extractive in EtOH-Benzene 1.58 ± 0.1983
Klason lignin 22.45 ± 0.0095
Holocellulose:
Alpha-cellulose 34.48 ± 0.2032
Hemicellulose 27.48 ± 0.2032
composition percentages are on dry-weight basis
a
hardwoods (18–25 %) [15]. This high lignin content is the main reason of alkali
(sodium hydroxide) pretreatment which would be applied to sugarcane bagasse.
The main effect of alkali pretreatment on lignocellulosic biomass is delignification
by breaking ester bonds cross-link lignin and xylan, thus increasing the porosity
of the biomass [16].
B. Effect of Reaction Time of Pretreatment

Alkali delignification of sugarcane bagasse is to expose cellulose polymers for
hydrolysis and bioethanol production. Pretreatment refers to complete or partial
degradation of lignocellulosic biomass to expose cellulose polymers for convenient
cellulose hydrolysis into sugars [7]. Alkali treatments were used for delignification
of sugarcane bagasse before hydrolysis by cellulase.
Concentration of NaOH used in this study was 1% (w/v) for pretreatment/
delignification of sugarcane bagasse for different reaction time. Lignin contents
of treated bagasse were determined. The degradation of lignin in the pretreated
sugarcane bagasse is represented as the component loss of lignin, shown in Figure
1. Figure 1 shows that the increase of reaction time affected the loss of lignin
from 7.5 to 60 minutes. Maximum delignification (74.95%) was caused by 1%
NaOH treatment for 60 minutes (Figure 1). Varga et al. [17] reported 95%
reduction in lignin content as a result of pretreatment of corn stover with 10%
NaOH for 1 hour in the autoclave. The higher reduction level than this work
result may be attributed to a higher NaOH concentration of 10%, which in this
study was limited to 1%.
SEM data reveal difference between the raw and pretreated samples. Morpho-
logical changes of raw and alkali pretreated sugarcane bagasse were examined by
SEM to evaluate the structural modification of the surface. Figure 2 shows the
scanning electron micrographs of raw and alkali pretreated sugarcane bagasse. The
raw samples have a compact rigid structure and highly ordered fibrils, while the
pretreated samples showed a distorted structure. Moreover, the microfibrils were
separated from the initial connected structure and fully exposed, thus increasing
the external surface area and the porosity of the sugarcane bagasse. By hand-touch
Figure 1. Lignin loss after NaOH 1% pretreatment of sugarcane bagasse at 121 °C
Figure 2. SEM of sugarcane bagasse (a) before and after 1% NaOH pretreatment
at 121 °C for (b) 7.5 minutes, (c) 15 minutes, (d) 30 minutes, (e) 60 minutes, (f )
90 minutes
of the material, we also felt that pretreated sugarcane bagasse was much softer than
the untreated one. Zhang and Cai [18] treated rice straw with 2% NaOH and
reported reduction in lignin content of before and pretreated straw from 14.9%
to 9.5% respectively and also mentioned that after treatment with NaOH the
basic tissue become severely shrank.
The effectiveness of different pre-treatment methods including alkali, acid and

chlorite pretreatment of lignocellulosic feedstock’s for improving the hydrolysis of
cellulose has been evaluated by Gupta et al. [19]. Mild alkali treatment conditions
caused complete conversion of cellulose I to cellulose II, which is a more stable
form with antiparallel chain structure in NaOH solution [20]. In another recent
study, Liu et al. [21] reported that an alkaline pretreatment can improve the
cellulose hydrolysis by modifying the cellulose crystalline structure.
C. Effect of Cellulase Loading on Hydrolysis

The delignified bagasse were then treated for 48 h at 50 °C with cellulase enzyme
solution, at enzyme loadings of 10 and 20 FPU/g. Cellulase is a mixture of several
enzymes. There are at least three major groups of cellulases that involved in the
hydrolysis process: 1) endoglucanase, which attacks regions of low crystallinity in
the cellulose fiber, creating free chain-ends; 2) exoglucanase or cellobiohydrolase,
which degrades the molecule further by removing cellobiose units from the free
chain-ends; and 3) β- glucosidase, which hydrolyzes cellobiose to produce glucose
[16].
The effect of enzyme concentration on the hydrolysis of alkali-pretreated
sugarcane bagasse is shown in Table 2 and Figure 3. Reducing sugar concentration
is shown in Table 2 and reducing sugar yield is shown in Figure 3. The reducing
sugar concentration is amount of reducing sugar found on the 1 L of liquid
hydrolysate after the hydrolysis. Meanwhile, the reducing sugar yield is the total
amount of reducing sugar produced when 100 g pulp of pretreated sugarcane
bagasse was hydrolyzed.
Cellulase concentration of 10 FPU/g resulted yield of fermentable sugars,
which are 40.30%, 41.21%, 41.69%, 45.90%, 43.23% for 7.5, 15, 30, 60,
90 minutes of reaction time of pretreatment, respectively. Meanwhile, cellulase
concentration of 20 FPU/g resulted yield of fermentable sugars, which are 41.85%,
46.29%, 41.69%, 49.11%, 45.10% for 7.5, 15, 30, 60, 90 minutes of reaction
time of pretreatment, respectively. Based on these results, during 48 hours of
hydrolysis of 60 minutes pretreatment, the 20 FPU/g of cellulase loading resulted
Table 2. Reducing sugar concentration of hydrolysate after cellulase hydrolysis of pretreated
sugarcane bagasse
Reaction Time Reducing Sugar Concentration (g/L)
(minutes) 10 FPU/g 20 FPU/g
7.5 4.99 5.18
15 4.41 4.95
30 5.03 5.03
60 4.09 4.37
90 4.92 5.13
Figure 3. Reducing sugar yield after cellulase hydrolysis of pretreated sugarcane bagasse
in highest yield of fermentable sugars of 49.11%. It is revealed that longer reaction

time of pretreatment at 121°C to 90 minutes did not increase the yield of sugars.
Generally, enzymatic hydrolysis efficiency of cellulose correspondingly
increased with pretreatment temperature, reaction time and alkali concentration.
McIntosh and Vancov [6] reported that temperature had a significant impact with
121°C being more acquiescent to cellulose hydrolysis than 60°C. Comparable
glucose yields were also observed with a pretreatment time of 60 minutes.
Moreover, when alkali strengths were below 2%, no advantage was observed
when reaction times increased to 90 minutes.
IV. Conclusion
As expected, higher enzyme loading results in higher yield of sugar. In the present
study, the highest hydrolysis was obtained at enzyme concentration 20 FPU/g.
The dilute alkali pretreatment resulted in a high sugar yield of sugarcane bagasse
associated with the high reduction in lignin and the increase in cellulose content.
There may be opportunities for further process optimization, finding the right
pretreatment condition and/or enzyme combinations and dosages. Considering
its abundance and high sugar potential, sugarcane bagasse is an excellent feedstock
for ethanol production.
V. Acknowledgement
We gratefully acknowledge the financial support provided by DIPA LIPI 2013
for this work. We express our gratitude to Mr. Raden Permana Budi Laksana and
Mr. Sudarmanto for their technical assistance.
VI. R eferences
1) Conde-Mejíaa, C., Jiménez-Gutiérreza, A. and M. El-Halwagib. (2012).

A comparison of pretreatment methods for bioethanol production from
lignocellulosic materials. Process Safety and Environmental Protection, 90 (3),
189-202. http://dx.doi.org/10.1016/j.psep.2011.08.004
2) Hermiati, E., Mangunwidjaja, D., Sunarti, T. C., Suparno, O. and B.
Prasetya. (2010). Pemanfaatan biomassa lignoselulosa ampas tebu untuk
produksi bioetanol. Jurnal Litbang Pertanian, 29 (4), 121–130.
3) Goshadrou, A., Karimi, K. and M. J. Taherzadeh. (2011). Bioethanol
production from sweet sorghum bagasse by Mucor hiemalis. Industrial
Crops and Products, 34 (1), 1219–1225. http://dx.doi.org/10.1016/j.ind-
crop.2011.04.018
4) Cheng, K., Cai, B., Zhang, J., Ling, H., Zhoua, Y., Geb, J. and J. Xua.
(2008). Sugarcane bagasse hemicellulose hydrolysate for ethanol production
by acid recovery process. Biochemical Engineering Journal, 38 (1), 10–109.
http://dx.doi.org/10.1016/j.bej.2007.07.012
5) Alvarado, M., Terra, J., Gernaey, K. V., Woodley, J. M. and R. Gani. (2009).
Biorefining: computer aided tools for sustainable design and analysis of
bioethanol production. Chemical Engineering Research and Design, 87 (9),
1171–1183. http://dx.doi.org/10.1016/j.cherd.2009.07.006
6) McIntosh, S. and T. Vancov. (2010). Enhanced enzyme saccharification of
Sorghum bicolor straw using dilute alkali pretreatment. Bioresource Technology,
101 (17), 6718-6727. http://dx.doi.org/10.1016/j.biortech.2010.03.116
7) Asgher, M., Ahmad, Z. and H. M. N. Iqbal. (2013). Alkali and enzymatic
delignification of sugarcane bagasse to expose cellulose polymers for sac-
charification and bio-ethanol production. Industrial Crops and Products, 44,
488–495. http://dx.doi.org/10.1016/j.indcrop.2012.10.005
8) Xu, J., Cheng, J. J., Sharma-Shivappa, R. R. and J. C. Burns. (2010). Lime
pretreatment of switchgrass at mild temperatures for ethanol production.
Bioresource Technology, 101 (8), 2900–2903. http://dx.doi.org/10.1016/j.
biortech.2009.12.015
9) Chaudhary, G., Singh, L. K. and S. Ghosh. (2012). Alkaline pretreatment
methods followed by acid hydrolysis of Saccharum spontaneum for
bioethanol production. Bioresource Technology, 124, 111–118. http://dx.doi.
org/10.1016/j.biortech.2012.08.067
10) Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y. Y., Holtzapple, M.
and M. Ladisch. (2005). Features of promising technologies for pretreatment
of lignocellulosic biomass. Bioresource Technology, 96 (6), 673–686. http://
dx.doi.org/10.1016/j.biortech.2004.06.025
11) Sun, Y. and J. Cheng. (2002). Hydrolysis of lignocellulosic materials for
ethanol production: a review. Bioresource Technology, 83 (1), 1–11. http://
dx.doi.org/10.1016/S0960-8524(01)00212-7
12) Balat, M. (2011). Production of bioethanol from lignocellulosic materials
via the biochemical pathway: A review. Energy Conversion and Management,
52 (2), 858–875. http://dx.doi.org/10.1016/j.enconman.2010.08.013
13) Hamelinck, C. N., van Hooijdonk, G. and A. PC. Faaij. (2005). Ethanol
from lignocellulosic biomass: techno-economic performance in short-,
middle- and long-term. Biomass and Bioenergy, 28 (4), 384–410. http://
dx.doi.org/10.1016/j.biombioe.2004.09.002
14) Driemeier, C., Oliveira, M. M., Mendes, F. M. and Edgardo O. Gómez.
(2011). Characterization of sugarcane bagasse powders. Powder Technology,
214 (1), 111-116. http://dx.doi.org/10.1016/j.powtec.2011.07.043
15) Rowell, R. M., Pettersen, R. and M. A. Tshabalala. (2012). Cell wall
chemistry. In R. M. Rowell (ed.), Handbook of Wood Chemistry and Wood
Composites, 2nd ed. (pp. 33–72). Florida: CRC Press.
16) Silverstein, R. A., Chen, Y., Sharma-Shivappa, R. R., Boyette, M. D. and
J. Osborne. (2007). A comparison of chemical pretreatment methods for
improving saccharification of cotton stalks. Bioresource Technology, 98 (16),
3000–3011. http://dx.doi.org/10.1016/j.biortech.2006.10.022
17) Varga, E., Scengyel, Z. and K. Recaey. (2002). Chemical pretreatments of
corn stover for enhancing enzymatic digestibility. Applied Biochemistry and
Biotechnology, 98–100 (1-9), 73–87. http://dx.doi.org/10.1385/ABAB:98-
100:1–9:73
18) Zhang, Q. Z. and W. M. Cai. (2008). Enzymatic hydrolysis of alkali-
pretreated rice straw by Trichoderma reesei ZM4-F3. Biomass and Bioenergy,
32 (12), 1130–1135. http://dx.doi.org/10.1016/j.biombioe.2008.02.006
19) Gupta, R., Khasa, Y. P. and R. C. Kuhad. (2011). Evaluation of pretreatment
methods in improving the enzymatic saccharification of cellulosic materials.
Carbohydrate Polymers, 84 (3), 1103–1109. http://dx.doi.org/10.1016/j.
carbpol.2010.12.074
20) Xu, F., Shi, Y. C. and D. Wang. (2012). Structural features and changes of
lignocellulosic biomass during thermochemical pretreatments: a synchrotron
X-ray scattering study on photoperiod-sensitive sorghum. Carbohydrate Poly-
mers, 88 (4), 1149–1156. http://dx.doi.org/10.1016/j.carbpol.2012.01.041
21) Liu, M., Wang, H., Han, J. and Y. Niu. (2012). Enhanced hydroge-
nolysis conversion of cellulose to C2–C3 polyols via alkaline pretreatment.
Carbohydrate Polymers, 89 (2), 607–612. http://dx.doi.org/10.1016/j.
carbpol.2012.03.058
Performance of Microbes Consortium On
Single-Chamber Microbial Fuel Cell as
Electricity Generation
Diana Rahayuningwulana,*, Dani Permanaa and Herlian Eriska Putraa
Research Center for Chemistry, Indonesian Institutes of Sciences,
a
LIPI Campus Cisitu, Bandung, Indonesia
Abstract
Microbial Fuel Cell (MFC) systems use microbes to convert organic compounds, as in food
wastewater treatment, and could produce direct current. Single microbe has showed their
performance as good biocatalysts on previous researches, both on synthetic media or wastewater.
This research studied 1-liter single-chamber MFC (SCMFC) using microbes as consortium on
tofu wastewater, since it has high organic pollutant value (COD as O2). Variation of consortium
concentration consists of three single microbe, Saccaromyces cereviceae, Saccaromycopsis fibuligera,
and Escherichia coli that previously acclimated with the wastewater. Result shows that tofu
wastewater, as substrate for the consortium decreases 76% COD value compared to the blank on
variation 1. This SCMFC system also produced maximum current at 0.25 mA with consorsium
SF2SC1:EC1 during 40hours.
Key words: SCMFC, Microbes, Consortium, Tofu wastewater, Current
I. Introduction
Microbial fuel cell would be an alternative of renewable energy source, where
bacteria as biocatalyst source on oxidizing organic and/or inorganic matter and
produce electricty. Electron produced by bacteria activity from its substrate would
be transferred into anode (negative pole) to cathode (positive pole) by conductor
and resistor.
In MFC, bacteria catalyse oxidation process from reducted substrate release
electrons from respiration cell to anode, which flows by external circuit loop to
cathode chamber and produce current. Every electron produced, a proton could be
transferred via electrolyte (liquid phase) to maintain current continuity. Electron
and proton react with oxygen in cathode chamber, which catalyzed by common
catalyst, as platinum, to form water.
Based on previous research [1], electrons can be transferred to the anode by
electron mediators or shuttles, direct membrane associated electron transfer, or
* Corresponding author. Phone: +62-222503051. Email: dian009@lipi.go.id
187
so-called nanowires produced by the bacteria, or undiscovered means. Bacteria

gain the energy by electron transfer from substrate reduction on low potential
(as glucose) to electron acceptor on higher potential (as oxygen).
Basic and economic design consists of two chambers MFC of H-shape, i.e.
two bottles connected completed by separator, e.g. cation exchange membrane or
simple salt bridge. Main component consists of anode, cathode, and electrolyte.
Factors that influence MFC operation system are temperature, pH, electron
acceptor, surface area, reactor dimension and operation time. Substrates also
determine the performance of MFC, for example acetate, glucose, biomass
lignocellulose, beer wastewater, starch, cellulose and chitin [2,3]. Most of MFC
operating on neutral pH to maintain growth condition of microbes on current
production [4]. Ionic strength also influences the conductivity of suspension on
MFC that impacts to internal resistance and its performance [4].
Wastewater could be functioned as electron donor, to be placed in anode
chamber, and have large potential energy, for example domestic wastewater
with–32.80 kJ/eq [5].
Based on recent research using different types of wastewater and inoculum
source [6], current density showed below 2 mA/cm2. High current density resulted
from two-chamber MFC using chocolate wastewater and graphite as electrodes, ie.
0.302 mA/cm2 [7], which COD 1,459 mg/L and using activated sludge inoculum.
Single inoculum of microbe has been studied and showed their result on
previous research. S. cereviceae on double chamber MFC using glucose yeast
extract medium with riboflavin mediator [8], S. cereviceae on salt-brigde MFC
using rice-rinsing wastewater [9], E. coli on salt bridge MFC on glucose and
brewey wastewater [10]. S. fibuligera application on decomposed amylum and
produced amylase had been studied [11], and potential as biocatalyst on MFC.
Since mixed culture gives better organic degradation in wastewater treatment
performance [12,13], this research aim to determine the influence of consortium
microbes variation to current production and organic removal on aerobic single-
chamber MFC using tofu industry wastewater. Tofu industry as one of traditional
food industry in Indonesia produced 15–20 liter wastewater/kg soybean with
BOD dan COD concentration approximately 65 gr and 130 gr/kg soybean
[Potter, C.S., M and Gani A., 1994 in [14].

A. Reactor
This research used MFC reactor consists of single 1 liter-glass tube, which anode
chamber filled with 900 mL wastewater and 100 mL isolate microbes suspension
and using air cathode. Tofu industry wastewater was from process PT X, Bandung
Figure 1. Scheme of Single Chamber MFC
with COD concentration 7,600–10,000 mg/L (pH 4.0–5.6 and conductivity

9.6 mS).
Batch reactor operated aerobically on temperature 25+0.1°C. Electrodes
connected to multimeter SANWA and data collected every 4 hours as shown
Figure 1, with COD sample collected every 12 hours.
Carbon plate 5 mm of 4 cm x 7 cm was placed as the anode and cathode, as
their good performance and inexpensive material electrode for MFC [15].
Salt-bridge, as cation exchange, was clamped between anode and cathode
chamber. Salt bridge was prepared from 11.6 gram sodium chloride added on 5%
(b/v) commercial agar powder and diluted in 100ml aquadest, and then boiled
and stirred homogeniously. Then, agar solution was poured to the 3 cm diameter
pipe and allowed to solidify.
B. Microbes
Microbes were consortium of S. cereviceae, S. fibuligera, and E. Coli, obtained
from Biochemistry Laboratory, University of Padjadjaran, Bandung, Indonesia.
Enrichment of microbes consortium used standard methods [16]. Before operat-
ing, 100% wastewater on MFC, activation and acclimatization was conducted
for each microbes with comparison from 25, 50, until 75% of tofu wastewater
to MFC medium.
The medium and inoculum medium were used for S. cereviceae, S. fibuligera,
and E. coli cultivation was YEPD (Yeast Extract Potato Dextrose) medium.
Liquid YEPD medium placed in closed Erlenmeyer. It was sterilized at 121 °C of
temperature and 15 psi of pressure for 15 minutes with an autoclave (Hirayama
HL36 AE, Japan) [17].
Composition of YEPD (yeast extract peptone dextrose) medium for MFC
medium and inoculums medium were 0.5% (w/v) of yeast extract (Becto and
Dickinson), 0.5% (w/v) of bacteriological peptone (Becto and Dickinson),
0.3% (w/v) of ammonium sulfate (Merck), 0.3% (w/v) of potassium dihydrogen
phosphate (Merck), 2% (w/v) of glucose (Sygma-Aldrich), and 1.5% (w/v) of
agar (Becto and Dickinson) [17].
Inoculum of S. cereviceae, S. fibuligera, and E. coli was made by took a loop
of S. cereviceae, S. fibuligera, and E. coli pure culture from agar slant, and then
inoculated into sterile YEPD inoculum medium with 25 mL of volume. Then,
inoculum medium incubated for 18 hours at 30°C and shaken at 150 rpm in a
shaking incubator (Certomat B Braun). The ratio of Erlenmeyer size of to the
volume of the culture volume was maintained at 4:1 to maintain the availability
of dissolved oxygen. The entire MFCs inoculum was transferred to the medium
of MFCs. The volume of MFCs inoculum that added to MFCs medium was
10 % (v/v) of 1000 mL of MFCs medium. Concentration of microbes was 106
CFU/mL.
Since consortium concentration might influenced the electricity generation,
microbe of S. fibuligera (SF), S. cereviceae (SC), and E. coli (EC), was made on
consortium ratio between 1) SF2 : SC1 : EC1, and 2) SF1 : SC2 : EC1. Those
consortiums were tested on 100% tofu wastewater on single chamber MFC and
compared to control reactor without consortium addition.
C. Analysis
Data collection of Optical Density (OD) was set every 4 hours and 12 hours
for Chemical Oxygen Demand (COD) analysis sample. Microbes growth was
approached by detecting its optical density, where the measurement of OD (λ 560
nm) using spektrofotometer LW Scientific V325XS. COD sample was analized
using closed reflux titrimetry SNI 6989.2:2009 method.
Collected data was analized for microbes performance on acclimatization and
running MFC, COD removal, current, and voltage measurement.

A. Acclimatization
On acclimatization, microbes were adapted step by step into wastewater char-
acteristic, in order to prevent any shock loading. This 0% wastewater, increased
into 25%, 50%, and 75% wastewater was done until OD concentration showed
constant graph, where indicated their growth not significantly different between
each microbes, as shown in Figure 2. On 0% wastewater ratio, S. cereviceae was
the easiest adaptable microbe than two other microbes. It reached its maximum
growth in exponential phase on 20 hours, where the microbe growth, the cell
division and increasing cell numbers run very fast, and also cell biomass increased.
Although suddenly decreased into death phase.
At 75% wastewater ratio, those three microbes need only 12 hours for
exponential phase before entered the stationary phase, as the indicator of limited
nutrient and demand of substrate supply [18]. This phase also indicate that
microbes consortium ready for wastewater substrate.
S. cereviceae also reached maximum growth better than S. fibuligera and E.coli,
either on ratio 25% wastewater or 50%. On 75% ratio, E. coli and S. fibuligera
showed their performance approached to S. cereviceae.
(a) (b)
(c) (d)
Figure 2. Growth Curves in Acclimatization Stage of (a) 0% ; (b) 25%; (c) 50%; (d) 75%
wastewater to substrate
S. cerevisiae or Baker’s yeast was able to produce bioethanol in medium of

acclimatization and medium of MFC. As we know, ethanol is toxic for micro-
organism. During ethanol fermentations, yeast cells suffer from various stresses.
Not only yeast cells suffered various stresses, but also bacteria cells like E. coli.
S. cerevisiae have ethanol tolerance and can survive in medium that ethanol
existed. However, high ethanol concentrations cause a problem, in that the
fermentative microorganisms have limited ethanol tolerance. S. cerevisiae can
survive in maximum concentration of ethanol of 18 % (v/v) [19]. Growth curve
and cells amount of S. cerevisiae are higher than E. coli and S. fibuligera. It is
possibly caused by the ability of S. cerevisiae to growth in ethanol stress but E.
coli and S. fibuligera were not.
B. MFC Operation
At the end of acclimatization stage, the consortium microbes were tested into
single-chamber MFC with 100% tofu wastewater. Organic matter in wastewater
was oxidized by microbes, the electron released and transferred to electrode (anode)
[20]. This electron was transferred (determined as current), and open circuit
voltage (OCV) was compared with control reactor without microbes applied.
Current production on both variations was shown in Figure 3. Compared to the
control reactor, there was a significant difference and variation with SF1:SC2:EC1
gave better result in current production. Control reactor produced 0.11 mA
current at the maximum performance.
Figure 3. Current Production

In this study, compared to control reactor, consorsium addition increased

current into 0.15 mA for SF2:SC1:EC1 and into 0.25 mA for SF1:SC2:EC1 in
16 hours. The consortium addition influenced current production, almost 127%.
Mix culture as biocatalyst sinergy treated on the substrate. Utilization consortium
culture consists of S. fibuligera and E.coli in tofu wastewater as substrate, which
resulted maximum current 0.23 mA, and it had increased from 0.19 mA maximum
current when using E.coli only [21], this study gave higher result. The advantages
of using microbes consorsium are microbes could degrade the substrate sequently,
consortium can increase the rate of subsrate degradation overall, and consortium
allow microbes find the easiest thermodynamic path or process [22].
S.cereviceae is a facultative microbe that could respirated under aerob and
anaerob condition, and could use media containing high glucose and protein, as
in tofu wastewater, as source of its metabolism.
Based on Figure 4, maximum voltage 0.432 volt and 0.505 volt were resulted
by SF2:SC1:EC1 and SF1:SC2:EC1 variation respectively, in 16 hours. Compared
to double chamber using potassium fericyanate (Rohan, 2013), maximum voltage
using E. coli reached 0.534 V in 2 days.
Consortium allows microbes to find the easiest thermodynamic path or
process [22], so that consortium could oxidize the wastewater substrate faster
than single microbes.
Other fuel cell application, i.e. hydrogen fuel cell and dan Direct Ethanol Fuel
Cell (DEFC) resulted 0.95 volt and 0.6 volt, respectively [23]. Base on that fuel
cell, MFC system could be developed by optimatization of reactor configuration,
microbes performance, and electrode material.
To calculate the electricity energy produced during 40 hours, average of
current and voltage in Joule’s law multiplied by time during MFC operation P
(mW-hour) = V(volt) x I_(mA) x time (hour). In control reactor yielded 0.60
mWh, in SF2:SC1:EC1 and SF1:SC2:EC1 are 1.14 and 2.03 mWh, respectively.
C. COD Removal
As the current generated during reactor running, the tofu wastewater as substrate
tend to decrease on its organic concentration by the time, expressed as COD. This
advantage of MFC system was applied as one of wastewater biological treatment.
According to growth curve during acclimatization stage, in first 12 hours the
consortium entered growth phase rapidly so that the degradation of substrate
was maximum. After that, rate of cell decay and divide tend to similar, indicated
stationer phase had began. The substrate was utilized by microbes for their
metabolism. Since available substrate was limited, it caused the tendency of
microbes to decay, so that the COD value became fluctuated.
Figure 4. Voltage Measurement during Reaction
Overall COD removal efficiency resulted from SF2:SC1:EC1 and

SF1:SC2:EC1 variations were 76% and 58% respectively, gave better result than
51% on control reactor during 48 hours, as shown in Figure 4.
There is still challenge to increase the current and voltage production by
single chamber MFC system, either by effective proton exchange, reactor design
and reactor configuration or stacking, considering the volume of tofu wastewater
that produced every day.
IV. Conclusion
Performance of microbes consortium, S. cereviceae, S. fibuligera and E. coli, on
single-chamber microbial fuel cell as electricity generation with tofu wastewater
as substrate has been studied. Acclimatization was an important stage to adapt the
microbes to wastewater in order to increase the overall removal efficiency. Overall
COD removal efficiency with SF2:SC1:EC1 variations were 76%; it gave better
result than 51% on control reactor during 48 hours. Addition of SF2:SC1:EC1
into substrate resulted maximum current 0.25 mA in 16 hours.
V. Acknowledgement
We would like to thank to Djaenudin for his valuable outlook, as well as Oman
Rohman and Mahyar Ependi for wastewater sampling.
Figure 5. COD Removal
VI. R eferences
1) Logan, B. E., Hamelers, B., Rozendal, R., Schroder, U., Keller, J., Freguia, S.,
Aelterman, P., Verstraete, W. and K. Rabaey. (2006a). Microbial Fuel Cells:
Methodology and Technology. Environmental Science & Technology, 40 (17),
5181–5192.
2) Das, S. and N. Mangwani. (2010). Recent developments in microbial fuel
cells: a review. Journal of Scientific & Industrial Research, 69, 727–731.
3) Shen, et al. (2014).
4) Liu, H., Cheng, S., Huang, L. and B. E. Logan. (2008). Scale-up of
membrane-free single-chamber microbial fuel cells. Journal of Power Sources,
179, 274–279.
5) Sawyer, Clair N. (2003). Chemistry for Environmental Engineering and Science.
New York: McGraw Hill, Inc.
6) Pant, D., Bogaert, G. V., Diels, L. and K. Vanbroekhoven. (2010). A review
of the substrates used in microbial fuel cells (MFCs) for sustainable energy
production. Bioresource Technology, 101, 1533–1543.
7) Patil, S. E., Surakasi, V. P., Koul, S., Imjulwar, S., Vivek, A., Shouche, Y. S.
and B. P. Kapadnis. (2009). Electricity generation using chocolate industry
wastewater and its treatment in activated sludge based microbial fuel cell and
analysis of developed microbial community in the anode chamber. Bioresource
Technology, 100, 5132–5139.
8) Arbianti, R., Hermansyah, H., Utami, T. S., Zahara, N. C., Trisnawati, I.

and E. Kristin. (2012). The Usage of Saccharomyces cerevisiae in Microbial
Fuel Cell System for Electricity Energy Production. Journal of Chemistry and
Chemical Engineering, 6, 814–819.
9) Permana, D., Putra, H. E., Rahayuningwulan, D., Djaenudin and H. R.
Hariyadi. (2013a). Pengolahan Limbah Cair Cucian Beras dengan Sistem
Salt Bridge Microbial Fuel Cell (SBMFC) dengan Saccaromyces cereviceae
sebagai Sumber Katalis. In Prosiding Seminar Nasional Jurusan Biologi FMIPA
UNPAD, pp. 198–205.
10) Rohan, D’souza, Deepa, V., Rohan, G. and B. Satish. (2013). Bioelectricity
Production from Microbial Fuel using Escherichia Coli (Glucose and Brewery
Waste). International Research Journal of Biological Sciences, 2 (7), 50–54.
11) Chi, Z., Chi, Z., Liu, G., Wang, F., Ju, L. and T. Zhang. (2009). Saccharomy-
copsis fibuligera and Its Applications in Biotechnology. Biotechnology Advances,
27 (4), 423–31.
12) Juang, D. F. (2012). Organic Removal Efficiencies and Power Production
Capabilities of Microbial Fuel Cells with Pure Cultures and Mixed Culture.
APCBEE Procedia, 1, 2–7.
13) Nimje, V. N., Chen, C. Y., Chen, H. R., Chen, C.C., Huang, Y. M., Tsenga,
M. J., Cheng, K. C. and Y. C. Chang. (2012). Comparative bioelectricity
production from various wastewaters in microbial fuel cells using mixed
cultures and a pure strain of Shewanella oneidensis. Bioresource Technology,
104, 315–323.
14) Sani, E. Y. (2006). Pengolahan Air Limbah Tahu menggunakan Reaktor Anaerob
Bersekat dan Aerob (Magister’s thesis). Ilmu Lingkungan UNDIP.
15) Zhou, M., Chi, M., Luo, J., He, H. and T. Jin. (2011). An Overview of
Electrode Materials in Microbial Fuel Cells. Journal of Power Sources, 196,
4427–4435.
16) Hogg, S. (2005). Essential Microbiology. West Sussex: John Wiley & Sons.
17) Permana, D., Hariyadi, H. R., Herlian, E. P., Juniaty, W., Rachman, S. D. and
S. Ishmayana. (2013b). Evaluasi Penggunaan Metilen Biru Sebagai Mediator
Elektron pada Microbial Fuel Cell dengan Biokatalis Acetobacter aceti. Jurnal
Kimia Molekul, 8, 78–88.
18) Metcalf and Eddy. (2009). Wastewater Treatment. New York: McGraw-Hill.
19) Bai, F. W., Anderson, W. A. and M. Moo-Young. (2008). Ethanol fermentation
technologies from sugar and starch feedstocks. Biotechnology Advances, 26,
89–105.
20) Kim, In S., Chae, K. J., Choi, M. J. and W. Verstraete. (2008). Microbial
Fuel Cells: Recent Advances, Bacterial Communities and Application Beyond
Electricity Generation. Environmental Engineering Research, 13 (2), 51–65.
21) Permana, D. and Rida. (in press). Saccharomycopsis fibuligera and Escherichia
coli as Biocatalyst in Single Chamber Microbial Fuel Cell. Journal of Techno
logy.
22) Notodarmojo, S. (2005). Pencemaran Tanah dan Air Tanah (pp. 203–205).
Bandung: Penerbit ITB.
23) Dewi, E. L., et al. (2013). Implementation of Bio-ethanol for Direct Ethanol
Fuel Cell (DEFC) System. Presented at the 4th International Conference on
Fuel Cell and Hydrogen Technology. Yogyakarta, Indonesia.
Heat Release Analysis of a Two Cylinders IN
Diesel Engine Fuelled with Ethanol-Diesel
Blends
Yanuandri Putrasari*, Arifin Nur, Achmad Praptijanto and Aam Muharam

Research Centre for Electrical Power and Mechatronics, Indonesian Institute of Sciences
Komplek LIPI, Jl. Cisitu No. 154D/21 Gd. 20, Bandung 40135, Jawa Barat, Indonesia
Abstract
An experiment on the application of ethanol-diesel blends as fuel in diesel engine was carried
out at various engine loads and ethanol percentages. The experiments were performed using neat
diesel fuel of 2.5%, 5%, and 7.5% ethanol-diesel blends with 1,500 rpm of engine working speed
at three different loads namely 0, 10 and 40 Nm. Several engine parameters data, such as torque,
fuel consumption, cylinder pressure, air intake flow, radioator coolant temperature, the exhaust
gas temperature, lubricating oil temperature and exhaust emission were collected. The combustion
or heat release of the engine then was calculated and analyzed. The results show several interesting
features from heat release phenomena in every combustion process from different fuel blends.
The results indicate that the increase of ethanol fraction in the ethanol-diesel blends causes the
maximum values of cylinder pressure and heat release value to increase as well.
Key words: Heat release, Combustion, Ethanol, Diesel
I. Introduction
Diesel engine has a high thermal efficiency and produces higher power that can
save more fuel compared to gasoline engine due to its high compression ratio.
Therefore, diesel engines are commonly preferred used on large buses, trucks, heavy
duty equipment, agricultural equipment and industrial machinery. However,
diesel engines also produce, gaseous pollutants, such as carbon monoxide (CO),
carbon dioxide (CO2), sulphur oxides (SOx), nitrogen oxides (NOx), unburned
hydrocarbons (HC) and particulate matter (PM) when using the common diesel
fuel that derived from fossil or living matter of a previous geologic time. Beside
produces high emission, the existence of fossil fuel in the world is estimated
to decrease from year to year. Due to this, condition the usage of renewable
alternative fuels are needed to replace and reduce the exhaust gas pollutants and
fossil-based fuel consumption.
Nowadays, ethanol becomes popular as a potential alternative fuel for vehicles
because it is a renewable and oxygenated biofuel. Ethanol can be produced from
* Corresponding author. Phone: +62-82120136976 Email: yanu001@lipi.go.id
199
sugar cane and others renewable energy sources. The other advantage of ethanol
as engine fuel for vehicles is that ethanol has greater power density than all others
alternative fuels.
However, due to its properties that different from normal gasoline or neat
diesel fuel, it is necessary to make engine modification in the utilization of ethanol
as fuel both for spark ignition or compressed ignition engine. According to Hansen
[1], the addition of ethanol to diesel fuel affects certain key properties, such as
blend stability, viscosity and lubricity, energy content and cetane number. The
two mentioned later are suspected mainly to influence to combustion process in
the combustion chamber of the diesel engine. The simple way to utilize ethanol
as fuel for existing diesel engine without modification is by blending with main
diesel fuel.
Recently, with the advancement of technology, many researchers conducted
experiment and investigation on ethanol as fuel for diesel engine by modifying
the engine or manipulating the ethanol as ethanol-diesel blends [2-12]. Among
them there was Xing-cai et al. [12] that investigated the effect of cetane number
improver of ethanol diesel blends on heat release rate and emissions of high
speed diesel engine. Different percentages of cetane number improver (0, 0.2,
and 0.4%) were added into the blends. The results show that the ignition delay
prolonged, and the total combustion duration shortened for ethanol-diesel blends
fuels when compared to normal diesel fuel. Meanwhile, Rakopoulos et al. [2]
studied about combustion heat release analysis of ethanol or n-butanol diesel fuel
blends in heavy-duty DI diesel engine. The study used ethanol in proportions
of 5% and 10% or n-butanol in 8% and 16% (by vol.) with the engine working
at three loads and at engine speeds of 1,200 and 1,500 rpm. It can be reported
from the study that the ignition delay is increased, maximum cylinder pressure
are slightly reduced and cylinder temperatures are reduced during the first part
of combustion.
Related to this research, previous author’s study [13] reported about perfor-
mance and emission characteristic on a two cylinder DI diesel engine fuelled with
ethanol-diesel blends. The report shows that the engine performance increased
and emission of CO, HC and smoke were decreased. Based on this background,
the aim of this study is to determine the heat release analysis of the two cylinder
direct injection diesel engine fuelled with ethanol-diesel blends.
In this study, determination of the combustion process in the diesel engine can
be conducted by using heat release analysis. Combustion process stated as heat
release at every crank angle position. The heat release can be calculated during
compression stroke and combustion stroke. In this condition, both of intake and
exhaust valves are closed. The heat release analysis estimates the amount of heat
or energy that added to the combustion chamber to obtain the pressure variation
in the combustion chamber. The heat release formula according to the first law
of thermodynamics single zone models [14] is:
(1)
Where γ is heat specific ratio, cp/cv. The appropriate range of γ for the heat
release analysis of diesel engine is 1.3 to 1.35 [14]. p is combustion chamber
pressure, and V is cylinder volume (volume clearance of top death centre plus
stroke volume). Cylinder volume based on crank angle position can be obtained
using the following formula:
(2)
where r is crank throw that equals to a half of piston stroke, l is long of
connecting rod, Vc is clearance volume, B cylinder diameter and θ is crank angle
position.
II. Methodology
A. Fuel Preparation
Normal diesel fuel and three ethanol-diesel blends on volume base namely
2.5%, 5%, and 7.5% then called E2.5, E5, E7.5 respectively, were used in this
study. Ther raw material of neat diesel fuel purchased from Indonesian national
oil corporation, while 99.6% purified ethanol and “SPAN 80” sorbytan methyl
ester surfactant as blends stabilizer were obtained from local supplier. Additional
information about properties of diesel fuel and ethanol are presented in Table 1
according to Rakopoulos et al. [15].
Ethanol-diesel blends were prepared by using a blender machine in a certain
desired dose for 15 minutes at 250 rpm to obtain the homogeneity of the blends.
To maintain the stability of the blends, 1% of surfactant, measured from total
blends was added during the blending process. The fuel preparation was conducted
just before the test started to run, to make sure the blends in stable condition.
B. Engine and Instruments Installation

A two cylinder direct injection stationary diesel engine was selected for this study.
The engine specifications data are presented in Table 2. The study was carried
out by setting-up the engine on an engine test bed. Fuel balance, emission meter,
smoke meter, pressure sensor crank angle sensor and temperature sensor for
intake and exhaust manifold were also installed. To manage the rpm and engine
Table 1. Properties of diesel fuel and ethanol [15]

Properties of diesel fuel and ethanol Diesel Ethanol
Density 20 °C, kg/m3 837 788
Cetane number 50 5-8
Kinematic viscosity at 40 °C, mm2/s 2,6 1,2
Surface tension at 20 °C, N/m 0,023 0,015
Lower calorific value, MJ/kg 43 26,8
Specific heat capacity, J/kg°C 1850 2100
Boiling point 180-360 78
Oxygen, % weight 0 34,8
Latent heat of evaporation, kJ/kg 250 840
Bulk modulus of elasticity, bar 16000 13200
Stoichiometric air-fuel ratio 15,0 9,0
Molecular weight 170 46
loads the engine was coupled with eddy current dynamometer. Fuel balance
used for fuel consumption measurement, and flow of air intake was measured
using hotwire anemometer. Meanwhile, the pressure sensor and crank angle
sensor were combined using engine indicating system to measure the pressure of
the combustion chamber and crank angle position. The schematic diagram for
experimental set up is presented in Figure 1.
Figure 1. Schematic diagram of experimental se-tup

Table 2. Engine basic data

Engine parameters Basic data
Model and type Fujikawa 295D, diesel four stroke
Number of valve 4
Air charge system Naturally aspirated
Cylinder / type 2 / Vertical
Volume (cc) 1630 cc
Diameter x stroke 95 x 115 mm
Compression ratio 19:1
Maximum torque 96.9 Nm at 1500 rpm
Maximum power 13.5 kW at 1500 rpm
Fuel system Direct Injection 195 bar
C. Engine Testing Procedure and Heat Release Analysis

To obtain heat release value of diesel engine, the pressure and volume data of
combustion chamber for every crank angle position is required. The data of
combustion chamber pressure and crank angle position can be obtained by engine
testing. The engine was operated using prepared fuels at 1,500 rpm. The loads
were set on 0, 10 and 40 Nm using dynamometer panel control. The parameter,
such as fuel consumption, air intake consumption, oil engine temperature, air
intake and exhaust temperature, coolant temperature both intake and outlet of
radiator, and emission of CO, HC and smoke were measured. The pressure of
combustion chamber was measured every 1° crank angel position and recorded
during 36 cycles. The main required data specifically combustion chamber pressure
and crank angle position then collected from engine indicating system. The
amount of pressure data from indicating system is about 720 data. To calculate
the heat release, it was used the mean pressure data from 36 cycles recorded data.
Meanwhile, the volume of combustion chamber in every crank angle position
was also calculated manually using Formula 2 aided by Microsoft Excel software.
The γ value is assumed constant during the combustion process. To fulfill all
requirements data, the measurement of bore, stroke, compression ratio, length
of connecting rod, and the gap between piston and cylinder liner were also
conducted. The heat release value then calculated and analyzed.

A. Cylinder Pressure
Figure 2 shows the cylinder pressure of diesel engine using E0, E2.5, E5 and
E7.5% (a) with 0 Nm of load, (b) with 10 Nm of load and (c) with 40 Nm of
load. It can be seen from the Figure 2 (a), (b) and (c), although the engine was
run in different load, for all the peaks, value of cylinder pressure increases due to
the increase of ethanol fraction in the blends. The significant influence of ethanol
fraction to the cylinder pressure of diesel engine occurred on the E5 and E7.5%,
while E2.5% caused no significant influences.
This is happened on all of loading variation namely 0, 10 and 40 Nm. The
increasing of ethanol fraction caused the graph seen a little bit shifted to the right.
It means that ingnition delay increase and start of combustion postphoned. This
condition suspected caused by the addition of ethanol that will increase the cetane
number of the blends, then increase the ignition delay which lead the increasing
the amount of fuel burned in the premixed burning phase. In detail, when the
engine run without load as seen in Figure 2 (a), the highest pressure peak is E
(a)
(b)
(c)
Figure 2. Cylinder pressure at (a) 0, (b) 10 and (c) 40 Nm
5% followed by E 7.5%, E 2.5% and the lowest is E0. Meanwhile, in the diesel
engine when run with 10 Nm of load, the value of cylinder pressure for E 7.5%
and E 5% are almost same, higher compared with the others. Lastly, in the diesel
engine that was run in 40 Nm of load, the highest pressure cylinder value is for
E7.5 followed by E5%, then followed by E2.5 and E0.
B. Heat Release
Figure 3 gives the heat release curves of diesel engine fuelled with neat diesel and
ethanol-diesel blends at (a) 0, (b) 10 and (c) 40 Nm of loading variation. From
of all the graph it can be seen the most distinguished features that the beginning
stage of heat release process is delayed and the maximum value of heat release
increases with increase in the ethanol fraction of ethanol-diesel blends. The
increasing of ethanol fraction in the ethanol-diesel blends caused the decreases
of cetane number of the fuels. The decreasing of cetane number influnce to
the increasing of ignition delay period which will involve to the enhancement
amount of combustible mixture during the ignition delay period. This condition
will results the increasing of the fuel burned in the premixed burning phase and
produces higher maximum cylinder pressure that involves increasing of heat
release. Furthermore, the high volatility of ethanol causes the fueld more evaporate
to be, therefore this will also increase the amount of combustible mixture during
the ignition delay period. The higher the ethanol fraction of ethanol-diesel blends,
the larger the amount of combustible mixture occurred.
(a) (b)
Figure 3. Heat release at (a) 0, (b) 10

and (c) 40 Nm
(c)
IV. Conclusions
From the complete experiment and heat release analysis in this study, several
conclusions can be drawn as follows:
1) The value of cylinder pressure increases due to the increase of ethanol fraction
in the blends.
2) The increasing of ethanol fraction caused the cylinder pressure graph seen a
little bit shifted to the right. It means that ignition delay increase and start
of combustion postponed.
3) The beginning stage of heat release process is delayed and the maximum value
of heat release increases with increase in the ethanol fraction of ethanol-diesel
blends.
V. Acknowledgement
This project is financially supported by Indonesian Institute of Sciences-Research
Centre for Electrical Power and Mechatronics through the Kompetitif LIPI
2011/2012 research project. The assistance of Mr. Ahmad Dimyani, ST. and
Mr. Mulia Pratama, ST. in conducting the experiments in internal combustion
engine laboratory is also highly appreciated.
VI. R eferences
1) Hansen, A. C. et al. (2004). Ethanol diesel fuel blends a review. Bioresource
Technology, 96, 277–285.
2) Rakopoulos, D. C. et al. (2011). Combustion heat release analysis of ethanol
or n-butanol diesel fuel blends in heavy-duty DI diesel engine. Fuel, 90,
1855–1867.
3) Song, C. et al. (2010). Carbonyl compound emissions from a heavy-duty
diesel engine fueled with diesel fuel and ethanol–diesel blend. Chemosphere,
79, 1033–1039.
4) Lee, W.-J. et al. (2011). Assessment of energy performance and air pol-
lutant emissions in a diesel engine generator fueled with water-containing
ethanol–biodiesel–diesel blend of fuels. Energy, 36, 5591–5599.
5) Lei, J. et al. (2012). A novel emulsifier for ethanol–diesel blends and its effect
on performance and emissions of diesel engine. Fuel, 93, 305–311.
6) Armas, O. et al. (2011). Effect of an ethanol–biodiesel–diesel blend on a
common rail injection system. Fuel Processing Technology, 92, 2145–2153.
7) Sayin, C. and M. Canakci. (2009). Effects of injection timing on the engine

performance and exhaust emissions of a dual-fuel diesel engine. Energy
Conversion and Management, 50, 203–213.
8) Çetin, M. et al. (2009). Emission characteristics of a converted diesel engine
using ethanol as fuel. Energy for Sustainable Development, 13, 250–254.
9) Pidol, L. et al. (2012). Ethanol–biodiesel–Diesel fuel blends Performances and
emissions in conventional Diesel and advanced Low Temperature Combus-
tions. Fuel, 93, 329–338.
10) Park, S. H. et al. (2011). Influence of ethanol blends on the combustion
performance and exhaust emission characteristics of a four-cylinder diesel
engine at various engine loads and injection timings. Fuel, 90, 748–755.
11) Hulwan, D. B. and S. V. Joshi. (2011). Performance, emission and combustion
characteristic of a multicylinder DI diesel engine running on diesel–ethanol–
biodiesel blends of high ethanol content. Applied Energy, 88, 5042–5055.
12) Xing-cai, L. et al. (2004). Effect of cetane number improver on heat release
rate and emissions of high speed diesel engine fueled with ethanol–diesel
blend fuel. Fuel, 83, 2013–2020.
13) Putrasari, Y. et al., (2013). Performance and emission characteristic on a two
cylinder DI diesel engine fuelled with ethanol-diesel blends. Energy Procedia,
32, 21–30.
14) Heywood, J. B. (1988). Internal combustion engine fundamental. McGraw-Hill.
15) Rakopoulos, D. C. et al. (2008). Effects of ethanol–diesel fuel blends on the
performance and exhaust emissions of heavy duty DI diesel engine. Energy
Conversion and Management, 49, 3155–3162.
An Evaluation For Enzymatic
Saccharification of Fast-growing Tree
Species from Secondary Forest in West
Kalimantan
Lucky Risantoa,*, Danang Sudarwoko Adia, Masakasu Kanekob, Yosuke

Kurosakib, Deden Girmansyahc, Ruliyana Susantic, Euis Hermiatia and
Takashi Watanabeb
Research Center for Biomaterials, Indonesian Institute of Sciences,
a
Jl. Raya Bogor Km 46, Cibinong, Bogor 16911, Indonesia

b
Laboratory of Biomass Conversion, Research Institute for Sustainable Humanosphere
(RISH),
Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
c
Research Center for Biology, Indonesian Institute of Sciences,
Abstract
Fast growing tree species can be considered suitable raw material for the production of second
generation bioethanol due to their promising biomass sustainability. However, until now, there is
less information regarding the bioethanol utilization from these species. In this study, we examined
the enzymatic hydrolysis of potential fast growing tree species that have been previously prescreened
from fifteen species. Fast growing tree species were taken from secondary forest concession area
of PT. Sari Bumi Kusuma in West Kalimantan and Eucalytus globulus was used as a standard
wood. The experimental work was carried out using hydrothermal, diluted sodium hydroxide
(NaOH), ammonia (NH4OH) and maleic acid (MA) pretreatment with 30 minutes of heating
duration, and several conditions of temperature and concentration. Based on the results of the
saccharification after hydrotermolysis pretreatment, Ilex cissoides has the highest yield of sugars
among the other fast growing tree species, from pulp saccharification was obtained 8.16 g/100g-
biomass when pretreated at 190 °C, and sugars from the filtrate saccharification was obtained 5.30
g/100 g-biomass, thus making total yield of sugars 13.46 g/100 g-biomass. When I. cissoides was
pretreated with diluted NaOH, NH4OH and MA, the yield of sugars were increased. The highest
yield of sugars and saccharification level for I. cissoides was obtained 18.04 g/100 g-biomass and
29.50 g/100 g-pulp after pretreated with 0.5% MA at 180 °C. I. cissoides have a potential as raw
material for bioethanol production if we used pretreatment using diluted MA, due to a similar
saccharification result when compared with E. globulus as standard.
Key words: Bioetanol, Enzymatic saccharification, Fast growing species, Lignocelluloses, Pretreatment
* Corresponding author. Phone: +62-21-87914511. Email: Lrisanto@biomaterial.lipi.go.id
209
I. Introduction
The search for alternative energy sources fuel oil is currently the world’s attention
related to declining availability of energy resources of petroleum fuels [1]. One
of the most potential renewable energies that have attracted worldwide attention
is bioethanol.
Bioethanol can be produced from a variety of raw materials and through a
number of ways. Basically, bioethanol production is processed from conversion
of carbohydrates into sugars and fermentation of these sugars to ethanol. In ad-
dition, bioethanol can also be produced from plant materials containing complex
cellulose. Cellulose is the most abundant source of carbohydrates on earth, which
is the main component of the plant structure, such as wood. Consequently,
cellulose has the potential to be one of the alternative raw materials because it is
more abundant and less expensive than food crops, especially from fast growing
tree species [2].
Fast-growing tree species have been well-known for plantation forest, thus the
sustainability of these species is propitious. They can grow easily in plantation
forest, such as the forest concession area of PT. Sari Bumi Kusuma in West
Kalimantan. The benefits of these species are faster growth than the wood species
from natural forest and they also have a short period of harvesting. On the other
hand, imperfect wood quality makes them are less interest to be further explored.
Therefore, the study on these species is necessary to give more information and
it will enhance the use of the woods [3].
Woody biomass is seen as a promising raw material for future feed stock
for bioethanol. Apperently, one of the main challenges for the production of
bioethanol from woody biomass is to develop of an efficient pretreatment. The
purposes of the pretreatment are to remove lignin and hemicellulose, reduce
cellulose crystallinity, and increase the porosity of the materials [4].
There are several woody biomass investigations for the renewable fuel,
such as Eucalyptus globulus [5-7], pine [8], and Japanese cedar [9]. Romani
et. al. [5] reported that up to 94% of carbohydrates were converted mono- or
oligo-saccharides in the hydrolysis media for E. globulus after pretreated using
hydrothermal at 220°C. However, the utilization of these species has been limited
to pulp and paper making.
In previous study, Ilex cissoides, Tristaniopsis whiteana, Evodia latifolia,
Horsfieldia crassifolia, and Adinandra collina were obtained potential species from
the saccharification result among the 15 species of topical fast growing trees
from PT. PT. Sari Bumi Kusuma [10]. Considering to lower saccharification
result achieved from the screening methods, we tried another pretreatment
methods. In this study, we examined again these potential fast growing trees using
hydrothermolysis pretreatment and we compared it with E. globulus as a standard

wood. Furthermore, we treated promising wood species with microwave assisted
pretreatement using diluted base and acid.

A. Biomass Collection and Preparation
The wood samples were collected from Sari Bumi Kusuma (SBK) concession area,
Central Kalimantan, Indonesia. The minimum diameter of wood is greater than
10 cm. The wood samples were cut, chipped and air-dried at room temperature.
After that, the wood chips are grinded to past a 32-mesh screen; finally the wood
meal that holds at 40-mesh screen was collected. Eucalytus globulus was used as
a standard wood.
B. Chemical Composition
The chemical analyses of raw material were conducted using two replicates per
sample. The ash content was measured by combusting the wood meal at 525°C
base on TAPPI Standard T 211 om-02 [11]. The wood meal was extracted in the
Soxhlet apparatus with a mixture of ethanol and benzene (1:2) according to the
TAPPI Standard T 204 cm-97 [12]. The extractive-free wood meal was oven-dried
at 60 °C for overnight. Acid insoluble lignin (AIL) and acid soluble lignin (ASL)
content were determined according to the National Renewable Energy Laboratory
(NREL) Chemical Analysis and Testing Task Laboratory Analytical Procedures
#003 [13] and #004 [14], repectively. Holocellulose content was analyzed ac-
cording to Wise et al. [15] and the alpha-cellulose content by Rowell et al. [16].
C. Hydrotermolysis Pretreatment
Wood meal around 1.1 g (air-dried basis) was placed in microwave vial, and then
added with water until 20 g in total. The sample was degassed in the vacuum
desicator for 5 min. The pretreatment was performing using a 2.45 MHz
microwave reactor (Initiator ver. 2.0, Biotage Co. Ltd.) with a 400 W magnetron,
stirring rate at 900 rpm and pre-stirring for 30 s. The sample was exposed to
microwave irradiation at 180 & 190°C for 30 min of holding time, and then
cooled until room temperature. After that, the pretreated sample was filtered to
get pulp fraction and the filtrate fraction for xylose sacch. The pulp fraction was
washed with distilled water and the yield of pulp was calculated. Each pretreatment
condition was repeated three times. Two promising wood species that resulted
higher sugar yield were further pretreated with diluted base and acid.
D. Microwave Assisted Pretreatment with Diluted Sodium Hydroxide,

Ammonia Solution, and Maleic Acid
In this steps, the wood powder was pretreated using diluted base and acid to
increase the sugar yield from the woods, such as sodium hydroxide (NaOH)
(0.5 wt%), ammonia solution (NH4OH) (7 and 14 wt%), and maleic acid (MA)
(0.5; 1.0; and 1.5 wt%). The wood samples that we used were sample No. 31
and 38; also E. globulus as standard. All pretreatment conditions were the same
as hydrotermal pretreatment, except the degassing step was eliminated and the
reaction temperature were 170 °C (NH4OH) and 180°C (NaOH and MA). Each
pretreatment condition was repeated two times.
E. Pulp Saccharification
The pulp fraction was hydrolysed with CTEC2. The cellulase enzyme loading
was 5 FPU/g-substrate. Enzymatic hydrolysis was performed at a substrate
concentration of 2 wt% in 0.05 M sodium succinate buffer pH 5.0 at 50 °C on
a rotary shaker (NTS-4000C, Rikakikai, Japan) at 140 rpm for 48 h.
F. Filtrate Saccharification
Ten gram of filtrate fraction was hydrolysed using HTEC2. The endoxylanase
enzyme loading was 3 mg/g substrate, then added with 0.05 M sodium succinate
buffer 0.05 M; pH 5.0; until 20 g in total. Enzymatic hydrolysis was performed
on a waterbath shaker at 50 °C; 140 rpm; for 48 h.
Total sugar yield was measured using HPLC base on the weight of original
wood; except for MA pretreatment was determined by Somoygi-Nelson method
[17].

A. Chemical Composition of Raw Material
Table 1 presents major chemical compositions of woody biomass; it showed
that the holocellulose content of potential fast growing trees were 71.42, 71.98,
68.45, 69.12, and 71.91% for I. cissoides, T. whiteana, E. latifolia, H. crassifolia
and A. collina, respectively.
B. Hydrotermolysis Pretreatment
The hydrothermolysis pretreatment for the woody biomass are eco-friendly
pretreatment methods. Sample was contact with water at elevated temperature
and pressure. Hydrotermolysis can cause breakdown of lignocellulose structure,
this due acetic acid from hemicelluloses can improve delignification and break
the lignin-cellulose matrix. Higher temperature would increase of weight loss due
to degradation of hemicellulose and lignin. From figure 1, the highest weight

loss was obtained 25.66% from sample I. cissoides 190°C, but still lower than E.
globulus (37.68%) as standard.
Generally, all the wood samples that pretreated with hydrotermolysis at 190
°C showed increasing in saccharification level compare with wood samples that
pretreated at 180°C. The highest yield of sugars from pulp saccharification was
obtained 8.16 g/100g-biomass from sample I. cissoides using 190 °C, and sugars
from the xylose saccharification was obtained 5.30 g/100g-biomass, thus making
total sugars 13.46 g/100g-biomass. Meanwhile yield of sugars from E. globulus
as standard at the same conditions from pulp and xylose saccharification were
obtained 21.92 and 10.48 g/100 g-biomass, making total sugars 32.40 g/100
g-biomass. It seems that recalcitrant of E. globulus is more easy to reduce than
the other wood samples when using hydrotermolysis.
C. Effect Microwave asissted pretreatment with maleic acid, ammonia

solution, and sodium hydroxide
As we can see from hydrotermolysis pretreatment, I. cissoides and E. latifolia have
potential to be raw material than the other wood samples. So in this step we added
diluted NaOH, NH4OH, and MA to increase the sugar yield from the woods.
Microwave-assisted pretreatment with maleic acid (conc. 0.5%; 180 °C) of all
the wood samples have a highest weight loss (Figure 3) than ammonia solution
(conc. 14%; 170°C) and sodium hydroxide (conc. 0.5%; 180 °C). This due dilute
acid pretreatment can accelerating solubilization of the hemicellulose, and also
have an advantage for conversion of the solubilized hemicellulose into sugars.
Meanwhile, alkali pretreatment can increases the swelling of cellulose and the
solubilization of lignin [18].
Table 1. Chemical composition (wt% of oven dry) of biomass
E. globu- I. cis- T. E. latifo- H. crassi- A. collina
Composition lus [wt%] soides whiteana lia [wt%] folia [wt%]
[wt%] [wt%] [wt%]
Ash Content 0.66 0.97 1.15 0.82 0.96 0.76
Extractive in EtOH- 2.34 1.83 1.98 1.76 1.87 1.61
Benzene
Lignin:
AIL 23.72 26.45 25.74 28.34 27.34 23.93
ASL 1.34 1.56 1.45 1.87 1.94 1.27
Holocellulose: 72.72 71.42 71.98 68.45 69.12 71.91
α-cellulose 42.89 40.97 41.87 40.28 41.19 42.52
Hemicellulose 29.83 30.45 30.11 28.17 27.93 29.39
45
40
35
30
25
20
15
10
0
180°C 190°C 180°C 190°C 180°C 190°C 180°C 190°C 180°C 190°C 180°C 190°C
globulus
E. globulus I. cissoides
cissoides T. whiteana
T. whiteana E. latifolia
E. latifolia H.crassifolia
H. crassifolia A. collina
A.
Sugar Yield from Pulp Sacch. [g/100 g-biomass]

Sugar Yield from Filtrate Sacch. [g/100 g-biomass]
Total Sugar Yield from Pulp and Filtrate Sacch. [g/100 g-biomass]
Weight Loss [wt%]
Figure 2. Effect of temperature to the yield of sugars determined by HPLC (a) and to the
yield of reducing sugars determined by Somoygi-Nelson Methods (b) after enzymatic of
wood samples hydrolysis using hydrotermolysis pretreatment.
The highest yield of sugars was obtained 18.04 g/100 g-biomass and sac-
charification level was 29.50 g/100 g-pulp from I. cissoides when pretreated with
maleic acid (conc. 0.5%; 180°C) and have similar level with E. globulus as standard,
were obtained 18.54 g/100 g-biomass and 30.06 g/100 g-pulp for the yield of
sugars and saccharification level. It seems that recalcitrant of I. cissoides can be
easier to reduce with addition of reagent such as MA. I. cissoides also gave similar
level when compare with E. globulus after pretreated with NH4OH (Figure 3).
45
40
35
30
25
20
15
10
5
0
E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia
E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia E. globulus I. cissoides E. latifolia
170°C; 14% NH4OH.

NH4OH 180°C; 0.5% NaOH 180°C; 0.5% M.A.
Total sugar from Pulp Sacch. [g/100 g-pulp]

Total sugar from Pulp Sacch. [g/100 g-biomass]
Weight Loss [wt%]
Figure 3. Effect of microwave-assisted pretreatment with diluted base and acid to the yield
of sugars after enzymatic of wood samples.
30
25
20
15
10
0
170°C;7%;
170°C; 7% 170°C; 14%;
170°C; 14% 170°C;
170°C;7%;
7% 170°C; 14%;
170°C; 14% 170°C;
170°C;7%;
7% 170°C; 14%;
170°C; 14%
NH4OH
NH 4 OH NH4OH
NH4 OH NH4OH
NH 4 OH NH4OH
NH4 OH NH4OH
NH 4 OH NH4OH
NH4 OH
E. globulus I. cissoides E. latifolia
E. globulus I. cissoides E. latifolia
Total sugar from Pulp Sacch. [g/100 g-pulp]

Total sugar from Pulp Sacch. [g/100 g-biomass]
Weight Loss [wt%]
Figure 4. Effect of microwave-assisted pretreatment with different concentration of ammonia

solution (NH4OH) to the yield of sugars after enzymatic of wood samples.
50
45
40
35
30
25
20
15
10
5
0
180°C; 0.5% 180°C; 1.0% 180°C; 1.5% 180°C; 0.5% 180°C; 1.0% 180°C; 1.5% 180°C; 0.5% 180°C; 1.0% 180°C; 1.5%
M.A. M.A. M.A. M.A. M.A. M.A. M.A. M.A. M.A.
E.g.
E. globulus 31
I. cissoides 38
E. latifolia
Reducing Sugar from Pulp Sacch. [g/100g-pulp]

Reducing Sugar from Pulp Sacch. [g/100g-biomass]
Reducing Sugar from Filtrate Fraction after MW Assisted [g/100g-biomass]
Reducing Sugar from Filtrate Sacch. [g/100g-biomass]
Total Reducing Sugar from Pulp and Filtrate Sacch. [g/100 g-biomass]
weight loss [wt%]
Figure 5. Effect of microwave-assisted pretreatment with different concentration of maleic

acid (MA) to the yield of reducing sugars after enzymatic of wood samples.
D. Effect Microwave-asissted pretreatment with different concentration

maleic acid
The organic acids, such as MA can be an alternative for increasing the sugar yield
from the woods. In general, there is no significant different for the saccharification
between when using 0.5; 1.0; and 1.5% of maleic acid for each wood samples.
The highest yield of sugars and saccharification level was obtained 18.04 g/100
g-biomass and 29.50 g/100g-pulp from I. cissoides when using 0.5% MA, mean-
while the yield of sugars and saccharification level from E. globulus was obtained
19.20 g/100 g-biomass and 32.11 g/100 g-pulp, respectively. An increament of
the concentration would increase the saccharification level, but the sugar yield
did not raise (Figure 5), this is due to the increase in weight loss.
E. Effect Microwave-asissted pretreatment with different concentration

ammonia solution
The benefits to use NH4OH are swelling of the lignocellulose material, selective
to remove lignin, low interaction with carbohydrates, and easy to remove
because NH4OH was very volatile. The saccharification after microwave assisted
pretreatment with 7% NH4OH was higher than pretreated with 14% NH4OH
(Figure 4). The highest yield of sugars and saccharification level were obtained
15.42 g/100 g-biomass and 20.84 g/100g-pulp from I. cissoides, meanwhile yield
of sugars and saccharification level from E. globulus was obtained 18.48 g/100
g-biomass and 23.40 g/100 g-pulp, respectively.
IV. Conclusion
Based on the results of the saccharification after hydrotermolysis and microwave
assisted pretreatement diluted base and acid, it can be concluded that I. cissoides
have a potential as raw material for bioethanol production if we used microwave
assisted pretreatement using diluted MA, due a similar saccharification result
when compared with E. globulus as standard.
V. Acknowledgement
The guidelines for citing electronic information as offered below are a modified
illustration of the adaptation by the International Standards Organization (ISO)
documentation system and the American Psychological Association (APA) style
and finalized in Information for IEEE Transactions, Journals, and Letters Authors.
VI. R eferences
1) Liu, S. and C. Lin. (2009). Development and perspective of promising
energy plants for bioethanol production in Taiwan. Renewable Energy, 34
(8), 1902–1907.
2) Limayem, A. and S. C. Ricke. (2012). Lignocellulosic biomass for bioethanol
production: Current perspectives, potential issues and future prospects.
Progress in Energy and Combustion Science, 38 (4), 449–467.
3) Adi, D. S., Risanto, L., Damayanti, R., Rullyati, S., Dewi, L. M., Susanti,
R., Dwianto, W., Hermiati, E. and T. Watanabe. (2014). Exploration of
Unutilized Fast Growing Wood Species from Secondary Forest in Central
Kalimantan: Study on the Fiber Characteristic and Wood Density. Procedia
Environmental Sciences, 20, 321–327.
4) Sun, Y. and J. Cheng. (2002). Hydrolysis of lignocellulosic materials for
ethanol production: a review. Bioresource Technology, 83 (1), 1–11.
5) Romaní, A., Garrote, G., Alonso, J. L. and J. C. Parajó. (2010). Bioethanol
production from hydrothermally pretreated Eucalyptus globulus wood.
Bioresource Technology, 101 (22), 8706–8712.
6) Romaní, A., Garrote, G., Ballesteros, I. and M. Ballesteros. (2013). Second
generation bioethanol from steam exploded Eucalyptus globulus wood. Fuel,
111, 66–74.
7) Romaní, A., Ruiz, H. A., Pereira, F. B., Teixeira, J. A. and L. Domingues.
(2014). Integrated approach for effective bioethanol production using whole
slurry from autohydrolyzed Eucalyptus globulus wood at high-solid loadings.
Fuel, 135, 482–491.
8) Cotana, F., Cavalaglio, G., Gelosia, M., Nicolini, A., Coccia, V. and A.
Petrozzi. (2014). Production of Bioethanol in a Second Generation Prototype
from Pine Wood Chips. Energy Procedia, 45, 42–51.
9) Baba, Y., Tanabe, T., Shirai, N., Watanabe, T., Honda, Y. and T. Watanabe.
(2011). Pretreatment of Japanese cedar wood by white rot fungi and etha-
nolysis for bioethanol production. Biomass and Bioenergy, 35 (1), 320–324.
10) Hermiati, E., Risanto, L., Adi, D. S., Kaneko, M., Daidai, M., Kurosaki, Y.,
Susanti, R., Girmansyah, D., Damayanti, R., Rulliaty, S., Dewi, L. M. and T.
Watanabe. Screening of Tropical Fast Growing Wood Species for Bioethanol
Production. In Proceedings of the SABH 2012, pp. 75–78.
11) TAPPI T 211 om-02. (2002). Ash in wood, pulp, paper and paperboard:
combustion at 525 °C. Atlanta, USA: TAPPI Press.
12) TAPPI T 204 cm-97. (1997). Solvent extractives of wood and pulp. Atlanta,
USA: TAPPI Press.
13) Templeton, D. and T. Ehrman. (1995). Determination of Acid-Insoluble Lignin
in Biomass. National Renewable Energy Laboratory LAP #003.
14) Ehrman, T. (1996). Determination of Acid-Soluble Lignin in Biomass. National
Renewable Energy Laboratory LAP# 004.
15) Wise, L. E., Murphy, M. and A. A. d’Addieco. (1946). Chlorite holocellulose,
its fractionation and bearing on summative wood analysis and on studies on
the hemicelluloses. Paper Trade Journal, 122, 11–19.
16) Rowell, R. M. (2005). Handbook of Wood Chemistry and Wood. Florida, USA:
CRC Press.
17) Somogyi, M. (1952). Notes on sugar determination. Journal of Biological
Chemistry, 195, 19–23.
18) Alvira, P., Tomas-Pe, E. and M. J. Negro. (2010). Pretreatment technologies
for an efficient bioethanol production process based on enzymatic hydrolysis:
A review. Bioresource Technology, 101, 4851–4861.
a Novel Microwave-Biological Pretreatment
Effect on Cellulose and Lignin Changes of Betung
Bamboo (Dendrocalamus Asper)
Widya Fatriasaria,b,*, Wasrin Syafiic, Nyoman J. Wistarab, Khaswar Syamsud,

Bambang Prasetyae and Muhammad Adly R. Lubisa
a
,Jl. Raya Bogor KM 46, Cibinong, Bogor 16911, Indonesia
b
Departement of Forest Product Technology, Faculty of Forestry, Bogor Agricultural University
(IPB)
PO Box 168, Bogor 16001, Indonesia
cDepartement of Forest Product Technology, Faculty of Forestry, Bogor Agricultural
University (IPB) PO Box 168, Bogor 16001, Indonesia
d
Departement of Agroindustrial Technology, Faculty of Agricultural Engineering and
Technology, Bogor Agricultural University, IPB
e
Institute of National Standarization, Manggala Wanabakti Building Blok IV
Jl. Gatot Subroto Senayan, Jakarta, Indonesia
Abstract
This study was to evaluate the effect of microwave-biological pretreatment of betung bamboo
on the characteristic changes of cellulose and lignin. Chemical component analysis, FT IR
spectroscopy, X-Ray diffraction, SEM-EDX analysis was used to analyze the characteristic changes
after pretreatment. The microwave-biological pretreatment caused the weight loss, lignin and
hemicellulose removal. FT IR spectra indicated that the microwave pretreatment for 12.5 min
(330 W) with 5% inoculum loading caused loss of the absorbed O-H and conjugated C-O. This
treatment also affected C-H2 scissoring motion lost in 5 and 10 min (330 W) with 5% inoculum
loading. Aromatic skeletal of lignin (1605 cm-1) did not appear in microwave pretreatment for 5
and 10 min (330 W) and 10% inoculum loading. The lowest absorbance of lignin (1512 cm-1)
in 5% inoculum loading was founded in pretreatment for 5 min (330 W). Absorbance Syringil
propane units were lower than guiacyl moties which indicates that syringyl was more soluble
than that of guiacyl. The crystallinity of cellulose in 5% of inoculum loading tends to decrease,
while the 10% of inoculum loading is since versa. SEM images illustrates that the pretreatment
disrupted the fiber structure (more porous and soften structure). EDX analysis shows that there
was lost in minor element constituent of pretreated bamboo. Crystalline allomorph of 5, 10 and
12.5 min (330 W) with 5% inoculum loading shows Iα (triclinic) structure.
Key words: Betung bamboo, Microwave-fungal pretreatment, Crystalline allomorph, Functional
groups, Crystallinity index
* Corresponding Author. Phone: +21-87914511. Email: widya_fatriasari@yahoo.com
219
I. Introduction
To improve sugar monomer production from lignocellusic materials (LM), it
is required to break down the lignin and crystalline system as major inhibitor.
The combination of pretreatments is attractive method to enhance reducing
sugar yield. One of pretreatment kinds is microwave-biological pretreatment in
which this pretreatment included in the environmental friendly pretreatment
sources. Microwave-assisted alkali pretreatment on rice straw had been reported
previously [1]. The combination microwave with acid [2] and hydrogen peroxide
[3] pretreatment had been also conducted before.
Bamboos belonging have high potency as bioenergy source. These plants
have high biomass production and grow mainly in Asia [4]. This production
is higher than that of the other energy crops [5,6]. One of the most important
bamboo species in Indonesia is betung bamboo [7] with high fiber morphological
characteristic and good chemical component content [8].
The microwave pretreatment facilitates to increase the substrate porosity,
surface area and soften the substrate. This heating derived from microwave source
causes the vibration of polar molecules and creates hot spot with inhomogeneous
materials lead to improve the substrate structure [9,10]. Application of biological
pretreatment using white rot fungi such as Trametes versicolor to depolymerize the
lignin polymer and then solubilize it. Lignin degrading enzyme can be secreted by
fungus to degradde on the LM [13]. The positive effect of microwave-biological
pretreatment can increase the enzymatic accesibility due to more opened substrate.
In our paralel study on biological and microwave pretreatment of betung
bamboo showed that the lignin and hemicellulose degradation was reported
[15,16].These changes improved the reducing sugar yield compared to control
[17,18]. Therefore, this research was foccused to evaluate the characteristic changes
after combination of microwave-biological pretreatment. The detail characteristic
analysis of pretreatment combination can used to understand the difference effect
single and combination pretreatment.
II. M aterial /methods

A. Material Preparation
Fresh and barkless 2 year-old bamboo betung (Dendrocalamus asper) produced
from bamboo garden of Research Center for Biomaterials LIPI Cibinong, Bogor,
Indonesia was used as samples. It was cut into chip and then processed by drum
chipper, ring flaker, hammermil and disk mill to obtain bamboo powder size of
40–60 mesh.
B. Microwave Pretreatment
Oven microwave SHARP P-360J (S) set at 2,450 MHz and power output of
1,100 Watt was used in this treatment. Previously, as much as 1 g of oven dried
powder was inserted into the teflon tube, then added distilled water to obtain
a solid-to-liquid ratio (SLR) 1:30 (w/v) and then was stirred for 15 minutes.
Subsequently, the samples were transferred and irradiated for irradiation time of
5, 10 and 12.5 minutes at 330 watt and 5 minutes at 770 watt. After microwaving
finished, the pulp removed and immediately ice water cooled for 15–20 minutes
and then was filtered to separate solid residue out.
C. Biological Pretreatment
The solid fraction resulted from microwave pretreatment was subjected to
biological pretreatment following previously method described by Fatriasari et
al.[12]. The samples were incubated at 27°C up to 30 day with 5 and 10% of
inoculum loading (IL). They were then washed with distilled water for removing
the fungus. The residues of these samples were analyzed the structure characteristic
changes after microwave-biological pretreatment.
D. Cellulose, Lignin and Morphological Characteristic Changes

The characteristic changes after microwave-biological pretreatment was evaluated
following method described by Fatriasari et al. [12]. These characteristics consisted
of chemical component changes, X-ray analysis Diffraction (XRD) (crystalline
allomorph and crystallinity index), Fourier Transform Infrared Spectrometry
(FT IR) analysis (determination of functional groups), and morphological
characteristic (SEM images) and Energy Dispersive X-ray Spectroscopy (EDX)
analysis.
E. Data analysis
All the experiments were conducted in triplicate and the results were presented
as mean ± standard deviation.

A. Chemical Component Changes
The chemical component loss of pretreated bamboo was presented in Table 1.
The microwave-biological pretreatment caused the weight loss of the samples.
It might be related with heating of microwave caused by vibration of polar
molecules in solution. The lignin polymer attacking due to enzyme degrading
lignin in the biological pretreatment is also affected this loss. All treatments
decreased the lignin content, inwhich lignin loss of 10% IL was higher than
Table 1. Chemical component loss after pretreatment

Component loss ([wt%)
MV-Bio Pretreatment
Weight alpha cellulose Hemicellulose Klason lignin
7.68± (3.21)± 3.14± 13.078±
A 1.80 0.84 0.96 2.74
13.79± 8.32± 12.94± 23.46±
B 0.13 4.42 3.95 4.0
15.27± 6.89± 9.22± 24.59±
C 0.65 0.00 1.75 1.19
7.84± (5.52)± 3.23± 14.08±
D 0.06 2.44 0.21 4.81
4.37± (8.57)± 3.77± 43.39±
E 0.31 1.83 1.14 0.10
7.42± (7.08)± 1.88± 44.65±
F 1.11 1.37 0.14 2.92
6.25± (7.06)± 44.21±
G 2.23 2.05 12.72±4.24 5.46
3.85± (15.79)± 38.51±
H 1.64 4.26 5.17±1.39 1.62
MV: Microwave, Bio: Biological, A: 5 min (330W) with 5% IL, B: 10 min (330 W) with
5% IL, C: 12.5 min (330W) with 5% IL, D: 5 min (770 W) with 5% inc.loading, E: 5 min
(330W) with 10% IL, B: 10 min (330 W) with 10% IL, C: 12.5 min (330W) with 10% IL,
D: 5 min (770 W) with 10% IL, W: Weight, A: Alphacellulose, H: Hemicellulose, KL: Klason
lignin
that of 5%. It means that the combination pretreatment with higher IL is more
effective to degrade lignin. Pretreatment also caused hemicellulose removal.
It related with the deconstruction of carbohydrate-lignin complex as result of
the partial removal of lignin and hemicellulose accessing the disruption of the
hydrogen bond between cellulose [16].
In most treatment condition, the treatment caused increase in cellulose
contents. It suggested with volumetric heating allows greater volume of bamboo
contacts directly. The delignification effect of lignin was also increase more removal
of amorphous part and left cellulose rich. Furthermore, the beneficial aspect of
microwave irradiation could enhance the lignin degradation and provide the
potential of exposing cellulose and increasing cellulose contents [1].
B. Carbohydrate Changes after Microwave Irradiation of Bamboo

The infrared spectra (IR) in the 800–4,000 cm-1 region for pretreated bamboo
was shown in Figure 1A and B. A strong broad absorption at 3,441–3,286 cm-1 is
observed to H-bonded OH group derived cellulose I. The microwave-biological
pretreatment caused broadening of OH group band associating the O-H group
weaknesses [17].
Figure 1. FTIR spectra of bamboo pretreated Microwave-biological pretreatment (A) 5% and

(B) 10% inoculum loading
The microwave energy delivery to polysaccaride via molecular interactions with

electromagnetic field [18] was responsible it. Moreover, the polar molecul vibration
and ion movement generate heat and extensive collisions accelerating chemical,
biological and physical processes [19]. The saponification of intermolecular
ester bonds cross-linking xylan and other components and decreasing of O-H
band intensity caused by its consumption in this reaction was also contributed
it [23]. Lignin structure can be observed in the region of 1,589, 1,511, 1,462,
and 1,420 cm-1 [9].
There are no changes in functional groups and the treatment only shifted the
wavenumber of identified peaks. This phenomenon was also reported previously
in biological pretreatment [12]. A hydrogen bonded (O-H) stretching absorption
was identified at 3,420 cm-1 (1) and C-H stretching absorption was seen at 2,920
cm-1 (2). The finger print peaks were 1,736 cm-1 (3) for unconjugated C=O in
xylans, 1,635 cm-1(4) for absorbed O-H and conjugated C-O, aromatic skeletal
in lignin at 1,605 cm-1(5) and 1512 cm-1 (6), C-H deformation at 1,462 cm-1 (7),
1,426 cm-1 (8) for C-H2 scissoring in cellulose, 1,378 cm-1 (9) for C-H deformation
in cellulose and hemicellulose, 1,328 cm-1 (10) for C-H vibration in cellulose
and C1-O vibration in syringyl derivatives, 1,246 cm-1 (11) for guaiacyl ring and
C-O stretch in lignin and xylan, 1,164 cm-1 (12) for C-O-C vibration in cellulose
and hemicellulose, 1,110 cm-1 (13) for aromatic skeletal and C-O stretch, 1,053
cm-1 (14) for C-O stretch in cellulose and hemicellulose, the band at 897 cm-1
(15) for C-O-C stretching at the β-glycosidic linkage characteristics in cellulose
[21], and 833 cm-1 (16) for C-H vibration in lignin [22].
The microwave pretreatment 12.5 min (330 W) and 5% IL caused the
absorbed O-H and conjugated C-O lost from spectra. This treatment also affected
C-H2 scissoring lost in 5 and 10 min (330 W) with 5% IL. Aromatic skeletal
of lignin did not appear in microwave pretreatment for 5 and 10 min (330 W)
inoculated with 10% IL.
The decrease in intensity at 1,328 cm-1 (deformation combination of syringyl
and xylan) was lower than at 1,246 cm-1 (guaiacyl of lignin) after combination
pretreatment. It means that syringyl moties are more reactive than that of guaiacyl
ones. It is caused by much metoxyl group content in syringyl and syringyl was
more degradable to microorganisme compared to guiacyl. More over, syringyl
has degree of polymerization, thus it is easier to be relocated. The higher syringyl
to guiacyl (S/G) ratio means greater delignification rate which can improve
enzymatic hydrolysis [16].
C.Morphological Characteristics of Pretreated Bamboo

The morphological structural changes on the surface of pretreated bamboo were
demonstrated in Figure 2. The pretreatment destroyed the bamboo structure
demonstrated by any holes, cracks, rugged and broken faced as well. The structure
is more soften and porous, thus it will increase the possibility contact between
substrate and enzyme in hydrolysis process. The partial lignin depolimerization
and hemicellulose removal in cell wall caused the disorganized morphology, with
greater exposure of the fibers. This finding was in line with the morphological
observation after microwave pretreatment on bagasse [9] and biological pretreat-
ment on bamboo [12].
Figure 2. SEM images of microwave-biological pretreated bamboo (A) 5%

and (B) 10% inoculum loading
The changes of element constituent in weight after microwave-biological

pretreatnent based on EDX analysis was listed in Table 2A and B. As structural
component of cell wall, O was dominated compared to C. Beside that, LM also
consist of minor element constituent as inorganic constituent in ash content.
Fe, P, Ca, Na, Si was founded in pretreated bamboo. N was still founded in all
treatments using 5% IL.
Pretreatment with 10% inoculum loading gave more effect to remove minor
element.
Table 2A. Element constituent of pretreated bamboo with 5% inoculum loading
MV
A B C D
Element w/w %
BP
5% Inoculum loading
C (carbon) 16.9±0.4 13.8±0.7 13.1±0.5 12.4±1.2
O (oxygen) 73.5±0.6 71.6±0.8 79.2±0.5 72.7±1.5
Fe (ferro) - - - 6.4±3.8
Na (Natrium) 9.3±1.5 7.9±2.2 7.6±1.8 7.5±4.2
Si (Silikon) 0.4±4.8 4.6±1.2 - -
Ca (Calsium) - 1.2±3.1 - -
P (Phospor) - 0.9±4.2 - -
Total 100 100 100 100
Table 2B Element constituent of pretreated bamboo with 10% inoculum loading

MV
E F G H
Element %
BP
10% inoculum loading
C (carbon) 11.4±1.2 25.5±1.5 12.3±1.0 14.6±0.8
O (oxygen) 81.1±1.3 66.5±2.5 80.4±1.1 77.9±1.0
Fe (ferro) - - - -
Na (Natrium) 7.5±3.6 - 7.3±3.3 7.5±3.1
Total 100 100 100 100
D. Crystalline Allomorph of Pretreated Bamboo

Table 3 presented crystalline allomorph of pretreated bamboo analyzed by XRD.
To determine cellulose allomorph was used by Z-discriminant, in which
the Iβ (monoclinic) and Iα (triclinic) type were characterized as Z < 0 and Z > 0
[23]. Eventhough, microwave-biological pretreatment with 10% IL gave higher
lignin loss than that of 5% IL; however monoclinic structure was founded in this
treatment. The transformation in triclinic phase was founded in pretreatment
Table 3. Crystalline allomorph of pretreated bamboo

Crystallite allomorph
Crystal al-
MV-Bio Pretreatment D d
z lomorph
(101) nm (10-1) nm
A 0.612 0.525 13.44 Iα
B 0.639 0.550 38.27 Iα
C 0.574 0.552 -73.83 Iβ
D 0.656 0.553 62.29 Iα
E 0.581 0.512 -27.75 Iβ
F 0.589 0.546 -44.55 Iβ
G 0.589 0.522 -20.78 Iβ
H 0.598 0.516 -1,14 Iβ
A, B and D. Improvement a better effect on hydrolysis performance of Iα phase

was expected in this treatment. This phase was more degradable than Iβ [24]
meta-stable and also more reactive than Iβ [25].
E. Crystallinity Index (CI) of pretreated bamboo

The crystallinity during microwave-biological pretreatment was presented in
Table 4. The enzymatic hydrolysis is mostly affected by CI [26]. To enhance sugar
release in the enzymatic hydrolysis, the crystalline system of LM should be opened
through pretreatment process. Disruption the hydrogen bond by increasing
splitting effect on crystalline region and maximize amorphous expansion can be
conducted by microwave pretreatment. This effect can be improved by delignifica-
tion on the lignin polymer by biological process. The lignin and hemicellulose
loss (Table 1) described it. In biomass, hemicellulose and lignin are amorphous
while cellulose is crystalline. It means that pretreatment give more effect on the
amorphous region than that of crystalline ones. Microwave irradiation for up to
5 min resulted higher CI both on 5 and 10% IL. It might be related with the
severe pretreatment tend to remove more amorphous fraction. Thus it makes the
increase of the crystalline proportion.
Table 4. Crystallinity index of pretreated bamboo

CI
MV-Bio Pretreatment
Crystal (Fc) Amorphous (Fa) CI
A 1.347 1.858 42.02
B 0.774 1.172 39.77
C 0.996 1.544 39.20
D 1.096 1.572 41.07
E 0.898 1.354 39.89
F 0.638 0.898 41.55
G 1.175 1.640 41.74
H 1.255 1.444 46.50
IV. Conclusion
Microwave-biological pretreatment on bamboo caused the structural modification
demonstarted by the absorbed O-H and conjugated C-O loss from spectra in
pretreatment for 12.5 min (330 W) with 5% IL. Aromatic skeletal of lignin did
not appear in microwave pretreatment for 5 and 10 min (330 W) inoculated with
10% IL. The lowest absorbance of lignin in 5% IL was founded in pretreatment
for 5 min (330 W). The higher reactivity of syringyl moties caused syringyl was
more soluble than that of guiacyl ones. The CI of cellulose in 5% IL tended to
decrease while the 10% IL was since versa. The pretreatment disrupted the fiber
confirmed by SEM image. Moreover, it founded that 10% IL more affected to
remove minor element constituent. Monoclinic structure has transformed into
triclinic structure in the 5, 10 12.5 min (330 W) with 5% IL.
V. Acknowledgement
The research was financially supported by Ministry of Research and Technology
(RISTEK) for PhD scholarship. The author expresses the gratitude to PT. MTI
for capturing the SEM image.
VI. R eferences
1) Singh, R., Tiwari, S., Srivastava, M. and A. Shukla. (2014). Microwave–as-
sisted alkali pretreatment of rice straw for enhancing enzymatic digestibility.
Journal of Energy, 2014, 1–7.
2) Singh, R., Tiwari, S., Srivastava, M. and U. Mina. (2013a). Effect of
combination of microwave and hydrogen peroxide (H2O2) pretreatment on
enzymatic saccarification of rice straw,” International Journal of Environmental
Engineering and Management, 4 (5), 529–542.
3) Singh, R., Tiwari, S., Srivastava, M. and A. Shukla. (2013b). Performance

study of combined microwave and acid pretreatment method for enhanc-
ing enzymatic digestibility of rice straw for bioethanol production. Plant
Knowledge Journal, 2 (4), 157–162.
4) Kant, P. (2010). Should bamboos and palms be included in CDM forestry projects?
Institute of Green Economy, New Delhi, India. IGREC Working paper No.
IGREC-07:2010.
5) Sathitsuksanoh, N., Zhu, Z., Templeton, N., Rollin, J., Harvey, S. and Y. H.
P. Zhang. (2009). Saccarification of a potential bioenergy crop, Phragmites
australis (common reed), by lignocellulose fractionation followed by enzymatic
hydrolysis at decreased cellulose loadings. Industrial and Engineering Chemistry
Research, 48, 6441–6447.
6) Zhang, Y. -H. P. (2008). Reviving the carbohydrate economy via multi-
product biorefineries. Journal of Industrial Microbiology and Biotechnology,
5, 367–375.
7) Dransfield, S. and E. A. Widjaja. (1995). Plant resources of south-east Asia no.
7. Bamboos (p. 189). Leiden, Nederlands: Backhuys.
8) Fatriasari, W. and E. Hermiati. (2008). Analisis morfologi serat dan sifat
fisis-kimia pada enam jenis bambu sebagai bahan baku pulp dan kertas. Jurnal
Ilmu dan Teknologi Hasil Hutan, 1 (2), 67–72.
9) Chen, W-H., Tu, Y-J. and H-K. Sheen. (2011). Disruption of sugarcane
bagasse lignocellulosic structure by means of dilute sulfuric acid pretreatment
with microwave-assisted heating. Applied Energy, 88 (82), 2726–2734.
10) Hu, Z. H. and Z. Y. Wen. (2008). Enhanching enzymatic digesbility of
switchgrass by microwave-assisted alkali pretreatment. Biochemical Engineering
Journal, 38 (3), 369–378.
11) Messner, K. and E. Srebotnik. (1994). Biopulping: an overview of develop-
ments in an environmentally safe paper making technology. FEMS Microbiol-
ogy Reviews, 13, 351–364.
12) Fatriasari, W., Syafii, W., Wistara, N., Syamsu, K. and B. Prasetya. (2014a).
Characteristic changes of betung bambu (Dendrocalamus asper) pretreated by
fungal pretreatment. International Journal of Renewable Energy and Develop-
ment, 3 (2), 133–143.
13) Fatriasari, W., Syafii, W., Wistara, N., Syamsu, K. and B. Prasetya. (2014b).
Lignin and cellulose changes of betung bamboo (Dendrocalamus asper)
pretreated by microwave assisted heating. Under review on Advance Material
Research.
14) Fatriasari, W., Syafii, W., Wistara, N., Syamsu, K. and B. Prasetya (2014c).
Digestibility fungal pretreated betung bamboo fiber. Accepted for publication
at Makara Seri Teknologi, 18 (2).
15) Fatriasari, W., Syafii, W., Wistara, N., Syamsu, K. and B. Prasetya. (2014d).
Performance of microwave pretreatment on enzymatic and microwave
hydrolysis of betung bamboo (Dendrocalamus asper). Accepted for publication
at Jurnal Teknologi Indonesia.
16) Li, X., Ximenes, E., Kim, Y., Slininger, M., Meilan, R., Ladisch, M. and C.
Chapple. (2010). Lignin monomer composition affects Arabidopsis cell-wall
degradability after liquid hot water pretreatment. Biotechnology for Biofuels,
3, 27.
17) Goshadrou, A., Karimi, K. and M. J. Taherzadeh. (2011). Improvement
of sweet sorghum bagasse hydrolysis by alkali and acidic pretreatment. In
Proceedings pf the Wood Renewable Energy Congress, pp. 374–380.
18) Galema, S. A. (1997). Microwave chemistry. Chemical Society Reviews, 26
(3), 233–238.
19) Sridar, V. (1998). Microwave radiation as a catalyst for chemical reaction.
Current Science, 74 (5), 446–450.
20) Nomanbhay, S. M., Hussain, R. and K. Palanisamy. (2013). Microwave-
assisted alkali pretreatment and microwave assisted enzymatic saccarification
of oil palm empty fruit bunch fiber for fermentable sugar yield. Journal of
Sustainable Bioenergy System, 3, 7–17.
21) Nelson, M. L. and R. T. O’Connor. (1964). Relation of certain infrared
bands to cellulose crystallinity and crystal lattice type.Part II a new infrared
ratio for estimation of crystallinity in cellululoses I and II. Journal of Applied
Polymer Science, 8, 1325–1341.
22) Cheng, D., Jiang, S. and Q. Zhang. (2013). Effect of hydrothermal treatment
with different aqueous solutions on the mold resistance of Moso bamboo
with chemical and FTIR analysis. BioResources, 8 (1), 371–382.
23) Sassi, J-F., Tekely, P. and H. Chanzy. (2000). Relative susceptibility of the Iα
and Iβ phases of cellulose towards acetylation. Cellulose, 7, 119–132.
24) Wada, M. and T. Okano. (2001). Localization of Iα and Iβ phases in algal
cellulose revealed by acid treatments. Cellulose, 8, 183-188.
25) O’Sullivan, A. (1997). Cellulose: The structure slowly unravels. Cellulose, 4,
173–207.
26) Rezanka, T. and K. Sigler. (2008). Biologically active compounds of semi-
metals. Phytochemistry, 69 (3), 585–606.
The emergence of biogas technology for
reducing rural poverty: Empirical studies in
java island
Lutfah Ariana*
Center for Science and Technology Development Studies, Indonesian Institute of Sciences
Gedung A PDII, 4th floor, Jl. Gatot Soebroto Kav 10 Jakarta, Indonesia
Abstract
Many people in Indonesia, like in many developing countries, have lack of access to economical
and convenient energy sources. For various reasons, energy services provided by the government
or the private sector are difficult to access by those living in remote areas. When accessible, the
communities–mostly the poor–are burdened by the expensive price of the services, leading to an
even more economically vulnerable state. Although sustainable energy services will not solve the
underlying cause of poverty, its limited availability will hinder the pathway to prosperity. This
paper analyzes the arising development programs of biogas technology in some rural regions in
Java as one of the solution. These programs are mainly developed in a wide range of affordable
and appropriate technologies to address energy poverty. One of the actors in this program is the
Indonesia Domestic Biogas Programme, or commonly called BIRU. However, successful project
types and technologies have often faced barriers that prevent them from scaling up and becoming
widely disseminated. This paper briefly describes some of the internal and external obstacles faced
by biogas user in rural areas, characteristics of successful users and some empowerment programs
and strategies undertaken by various stakeholders in the development of biogas technology in
Indonesia.
Key words: Biogas, Rural, Program, Cost efficiency, Energy
I. Introduction
Indonesia is to be known as one of oil producer in the world; however, depletion
of oil reserve and decretion of environmental quality, has caused some rural people
to utilize renewable energy as alternative energy source. Biogas is one of some
renewable energy sources, that is produced during anaerobic digestion of organic
substrates, such as manure, sewage sludge, liquid manure of hens, cattle, pig,
organic waste from market and so on [5]. Biogas production enables a sustainable
agriculture with renewable and environmental friendly process system. It ranges
several benefits, such as eliminating greenhouse gas, reduction of odor, betterment
of fertilizer and production of heat and power.
* Corresponding author. Phone: +6281578723901 Email: juffrow@yahoo.com, lutf001@lipi.go.id.
231
Production and utilization of biogas has several environmental advantages

such as produces renewable energy source, reduces the release of methane to the
atmosphere, and can be used as a substitute for fossil fuels. It is also known as a
high quality digestate that can be used as a fertilizer.
In India and China, biogas technology development has been running fastly.
In 1998, India had 12 million units biogas processing instalation for generating
1,7000 MW electricity. While China had 9 million units with estimation gas
production of 6,2000–1,45000 million m3. Indonesia has potential of biogas
energy 684.83 MW with installed capacity of 0.06 MW or only 0.009% was
utilized [1,6].
Actually, biogas has long been known in Indonesia. Since 1970s, the Indone-
sian people have known biogas. Nonetheless, dissemination and demonstration
of biogas is still needs to be done in various parts of Indonesia so that people get
to know and have interest in biogas. This paper considers the development of
biogas in Indonesia is slower in the appeal of other countries in Asia or Africa.
The availability of fuel wood and fossil energy sources is abundant in the first
period to make slow progress of biogas in Indonesia.
However, in Indonesia, biogas technology development has a good prospect
because there are a lot of agribusiness regions in rural areas, such as special region
for animal husbandry, integration region of palm oil and cattle, and agropolitan
that has main commodity of animal husbandry [2]. Although the technical
viability of small-scale biogas technology has repeatedly been proven in several
Asian countries, mass dissemination of this technology has not been accomplished
in Indonesia.
Some impediments related to develop biogas are its availability, security of
supplies, price, ease of handling and ease of use. In addition, other factors like
technological development, introduction of subsidies, environmental constraints
and legislation play the role bringing its development [4].
Many studies emphasized the case of biogas because its strategic position as
alternative energy resources in current years. In addition, the possible benefits
and risks associated with the implementation of biogas require further analysis
especially related to its potential to alleviate poverty in rural regions. A study
need to be conducted to identify the current condition of biogas implementation
in different rural communities and it should be focused on its efficiency of the
digesters produced. This paper provides information about the material inputs
of some practices of biogas development which will allow an social analysis of
potential costs and benefits of using biogas digesters.
II. Method
This study is a kind of social research that emphasized the qualitative approach
in collecting the data. By interviewing several key informants in rural region,
this study identify the arising condition in initiating biogas development, and
examine the influential factors that determine the sustainable development of
such technology. In addition, this study conducted three case studies of different
regions for showing the variety of different success factors. Multiple case studies
differed in term of their historical and environmental condition in adopting
biogas technology. The other characteristics emerged among the rurals because
of the availability of ownership of cattle and financial support. However, some
impediments are found in compelling the data since the justification of the
validity are quite difficult. Therefore, triangulation is considered to be preferable in
explaining the depth of information and the breadth of data that will be analysed.
III. Biogas for rural development

The benefit of biogas is actually a lot. In terms of technology, biogas may reduce
the risk of environmental pollution by reducing the use of fossil energy. From
the economic side, the presence of biogas makes the availability of energy sources
that can reduce daily energy costs. Biogas can also be used to run lights (similar
lamps strongking), a fuel stove for cooking and others.
Biogas is built specifically for households owning cattle. Every household
should be able to provide at least 30 kg of cow dung. The cow dung as a source
is to be converted into gas. The amount of dirt as much as it should at least be
provided by 3 cows. So each household must have at least 3 cows.
By cooperating with the Indonesian Ministry of Energy and Mineral
Resources and SNV Netherlands Development Organisation through Hivos has
implemented Indonesian Domestic Biogas Program (IDBP), also known as the
BIRU programme. The first phase (2009–2012) has been funded by the Dutch
embassy in Indonesia. This programme seeks to distribute 8,000 biogas digesters
as a local sustainable energy source by developing a commercial, market-oriented
sector that also provides job and business opportunities for masons and partner
organisations in construction. The IDBP focuses on implementation through a
multi-stakeholder sector development approach, creating a market-based biogas
Table 1. Production of Biogas by the Type of The Slurry
Type of the slurry Volume of production
Cow/buffalo, m3/kg 0.023 – 0.040
Pig, m3/kg 0.040 – 0.059
Chickens/birds, m3/kg 0.065 – 0.116
Source: Sulaeman, dkk, (2009)

Table 2. Equivalence Value of Various Types of Energy Compared to Biogas

Type of Energy Equivalency Value compared to 1 m3 Biogas
LPG 0,46 kg
Kerosene 0,62 liter
Solar 0,52 liter
Gasoline 0,80 liter
Dried woods 3,50 kg
Source: Sulaeman, dkk, (2009)
sector, involving locally trained contractors and masons who are supported by
vocational training institutions, in this case is Rural Development Institute of
Technology or known as Lembaga Pengembangan Teknologi Pedesaan (LPTP)
Solo. LPTP Solo is an extension agencies and manufacture biogas reactor in Yogya,
Boyolali and Klaten. This institution received funds from Germany (Burda), the
Netherlands (HIVOS), CORDEC (Netherlands), Ministry of Environment,
Ministry of Energy and Mineral Resources and the local Government. LPTP
focuses on rural technology provider in the fields of energy, food, agriculture and
environmental management and have a basic strategy ‘to build future projects’.
This LPTP perform activities such as sustainable agriculture (biogas), waste
management, economy, organic wastewater treatment (from industry, hospitals,
households) and the engineering workshop. In addition, LPTP is a center of
sustainable energy development, which consists of biogas, firewood saving stoves,
biodiesel and coal briquettes.
In order to produce biogas, it needs a digester. One digester could reduce
methane gas emission by decomposition of organic matter from agriculture and
animal husbandry sectors. The raw material for producing biogas is cow dung
that has been fermented and then resulted methane gas.
A farmer who invests in biogas can have his investment back within around
three years if the household only uses the biogas, and even within two years if the
bioslurry is applied appropriately (leading to higher yields and lower dependency
on chemical fertiliser). A digester can serve its owner for 15 to 20 years with
minimum maintenance costs.
IV. R esult and Discussion

A. Biogas for Remote People in Cepogo, Boyolali
The village that has already implemented biogas technology is located in District
Cepogo, Boyolali, Central Java. This village consists of 18 hamlet (at the Pillars
of Citizens). One hamlet average at least have 30 cows. The average production
of milk cows in the village between 12 to 20 liters per day with medium quality.
The history of the formation of biogas user community began in 1992, by

introduction of biogas program BIRU, grant financing in the form of subsidies
from the German GTZ; Rp700,000 to build a biogas digester household with size
6 m3. The grant was distributed to farmers who have a minimum of 3 cows. In
addition, they should contribute to build biogas as much as Rp400,000 and the
rest of the cost would be supported by the Dutch Non Governmental Organization
named Borda, which partnered with LPTP Boyolali to build a biogas reactor
at the farmer’s yard. With a capital of Rp1,100,000 (1992), farmers received a
biogas reactor completed with the installation and the stove. The amount of the
reactor is equivalent to buy one adult cows at that time.
In 2011, there were about 40 families (KK) with the amount of as much as
10 pieces of biogas reactor. At the beginning of the year of manufacture of 1991,
there were 13 pieces of the reactor. There were three biogas reactors that could
not be used due to several things, including ranchers moved house, owned cattle
ranchers sold, and changed jobs into tofu and tempeh producers.
Of the 10 existing biogas reactors currently used almost all of them just for the
sake of cooking and some have been used as an additional means of illumination
with the use of patromak electricity if dead. No biogas is converted into electricity
in this village. Reactors were built by various capacity ranging from 3 m3, 6 m3,
9 m3 and 13 m3 depending on the number of animals owned. For farmers who
have 8 cows, the amount is 13 m3 reactor.
Figure 1. Biogas for Rural Energy

Figure 2. Identity for Owning Biogas for Rural Household
Since 2010, household biogas program (BIRU) with a subsidy of Rp2 million
of Hivos was introduced by LPTP (Hivos partners in Boyolali) to the village
community Cepogo. Until 2011, there were only 3 families who had build biogas
reactor. Requirements to obtain subsidies from Hivos biogas among other breeders
must have at least 2 cows.
B. Biogas in Merapi Erruption Area, Balerante, Klaten

The first biogas made of concrete built in 2003 with the assistance of PT. Sari
Husada at the home of a breeder who is on of the board of Sarana Makmur
Cooperative. After the post-eruption of Mount Merapi (2010), the biogas
digester could still operate. Until 2011, Balerante has about twelve biogas
digester that has been operating, including four digesters located in the hamlet
of Balerante, namely one comes from grants Cordaid and Hivos three with
subsidies. The other three digesters located in the hamlet of Kaligompyong,
with details of one of the grants Cordaid, one from the subsidy of Hivos and
one from the grant of Ministry of Environment. Four digesters located in the
hamlet Sidorejo, comprised three digesters with subsidies from Hivos and one
from Cordaid’s grant. The only one digester located in the hamlet Kendalsari
was supported by the grants from Cordaid. The total population of about 136
families Balerante village, and about 90% of that amount has livelihood as
Figure 3. Biogas for Energy Substitution (LPG)
farmers, with an average ownership of cattle around 2–7 cows per family.
After the eruption of Mount Merapi mid-year 2010, the village of Balerante
received several programs to improve the environment and economy of central and
local government, the program known as “Early Recovery”. Biogas development
program included as one of the activities.
Biogas is already operating in Balerante almost entirely used for the benefit of
cooking. Average savings obtained is equivalent to the use of four LPG cylinders
(3 kg) every week for owners with a large family (5–8 people) and about 4 LPG
cylinders every month for a family of very small (2–3 people). In that time(2011),
in Balerante the average price of 3 kg LPG tube is Rp18,000. Minimal savings
made by a small family in a month is Rp72,000.
C. Bio-waste Energy in Pesantren Al-Hikmah, Gunung Kidul

Biogas in rural region namely Sumber Rejo located in Pesantren Al Hikmah was
built by Kyai H. Harun Al Rashid. In 2011, this Pesantren has 400 students, and
in 2010, build a wastewater treatment plant (WWTP) with the size of biogas
digester 9 m3 and the raw materials of human sanitary waste. Development
funding mechanism came from the Ministry of Public Works project which
granted for Rp100 million, the Department of Public Works District of Rp200
million, and the boarding schools provide Rp4 million. The assistants construction
of this project is LPTP.
Biogas in this boarding school used as cooking fuel in the kitchen. The benefits
of this biogas is a portion of the cost savings to purchase firewood Rp1,500,000
per month (50% of total demand). Currently, the kitchen boarding, in addition
to using biogas, is still using firewood at a cost of Rp1,500,000.
In addition, with the biogas and wastewater treatment program, the cost to
suck human waste holding tank (“safety tank”) is reduced. Before, there was this
program every 3 months must pay about Rp900,000 to suck or dispose of waste.
But this time the cost is no more.
D. Biogas for Ranchers in Nongkojajar, Pasuruan

Nongkojajar located in Pasuruan regency of East Java. Nongkojajar divided into
12 villages and an agricultural and dairy cattle breeding. A total of 11 villages are
producing dairy cow and a village is bull breeders and vegetable farmers. First
development of biogas is started in 1987 by the Department of Agriculture of
East Java Province. Until 2011, there are about 40 biogas built on the initiative
of the citizens with the assistance of several grants for government projects, or
with their own capital in the form of loans from the cooperative. In 2009, Hivos
collaborated with KPSP Setia Kawan developed biogas in this area. Hivos trained
around 22 biogas technicians certified and successfully educate approximately
60 energy companion. Hivos program, providing subsidies worth Rp2 million
to build a biogas digester and the rest is around Rp4 million to be met by
developing breeder reactors which will measure approximately 6 m3 of biogas.
If the size of biogas digester to be built is larger, the funds required will also be
greater. Many ranchers who enjoy Hivos’ subsidy also take benefit from the loan
of cooperatives in developing biogas digester. In further, the cooperative worked
with KLH GEF Program run by Bank Syariah Mandiri provide assistance with
low interest rate (9% per year) for the farmers. Farmers pay off the debt (for
example the size of digester 8 m3) for biogas development for Rp42,000 per
10 days. These mechanisms is considered is more appropriate in facilitating the
farmers to develop biogas [3].
In this case, Hariyanto, a head of a household in this rural, has 4 oxen with
13 liters of cow milk production per day. Cow dung produced per day is 30 kg /
day / cow. With size 6 m3 of biogas, the gas produced was channeled to the two
houses. The biogas is used for cooking, lighting and water heaters.
Before using biogas, the family was using 2 tubes size 12 kg LPG and kerosene
by 15 liters for cooking. A tube 12 kg LPG used for heating water for 3 months.
After using biogas, the family was able to make savings of Rp300,000 for cooking
and Rp25,000 for energy water heater. Waste (slurry) from waste digester of 100
kg per day can be used for garden fertilizer. Estimated savings to buy fertilizer
can be pressed up to 30%. Price manure slurry if sold at around Rp300 per kg.
V. Conclusion
Utilization of alternative energy sources biogas from livestock animals and
other impurities can reduce the cost of electricity consumption in remote areas.
Utilization of alternative energy is able to create energy independence for rural
communities so that in the long run it can reduce poverty for people who are
vulnerable to energy availability.
Development program of biogas technology can improve active participation
of various stakeholders ranging from government, donors, development agencies,
technology, and society. However, the sustainability of the development of biogas
technology is still impeded by affordability level of capital for early initiation
of construction of the digester, the technical aspects of waste treatment is still
insufficient demand, and scale up technologies that require a huge cost, especially
for the needs of the industry.
The Indonesian Ministry of Energy and Mineral Resources is looking into
options for faster upscaling by providing higher subsidies or at least with a
segmented market, in which certain target groups have higher subsidies. This will
provide accelerated access to renewable energy for many farming households, but
will limit the possibilities of developing a market-based biogas sector. Intensive
discussions about the way forward are ongoing.The main conclusions of the study
may be presented in a short Conclusions section, which may stand alone or form
a subsection of a Discussion or Results and Discussion section. The conclusion
section should lead the reader to important matter of the paper. It also can be
followed by suggestion or recommendation related to further research.
VI. Acknowledgement
This study is part of a research funded by Ministry of Research and Technology
through “Program Peningkatan Peneliti dan Perekaya (PKPP)”, or namely Incentive
Program in 2011. The author are deeply appreciated to the research team (coordi-
nated by Wati Hermawati) for the support and teamwork. The data is mainly sourced
from Research Report entitled “R & D Funding Pattern and Implementation
of New Energy-Renewable in Indonesia Case Studies: Microhydro power and
Biogas”.
VII. R eferences
1) Department of Energy and Mineral Resources. (2004). The International
Workshop on Biomass and Clean Fossil Fuel Power Plant Technology. Sustainable
Energy Development and CDM. Jakarta, January 13–14, 2004
2) Ditjen Pengembangan Peternakan, Ditjen Bina Produksi Peternakan,
Departemen Pertanian. (2003a). Pengembangan Kawasan Agribisnis Berbasis
Peternakan.
3) Hermawati, W. et al. (2011). Pola Pembiayaan Litbang dan Implementasi EBT
di Indonesia, Kasus: PLTMH dan Biogas. Laporan Penelitian Program PKPP
Ristek 2011.
4) Koopmans, A. (1998). Trend in Energy Use. Expert Consultation on Wood
Energy, Climate and Health. Phuket, Thailand, 7–9 October, 1998.
5) Widodo, T. W. and A. Hendriadi. (2005). Development of Biogas for Small
Scale Cattle Farm in Indonesia. Paper presented in the International Seminar
on Biogas technology for poverty reduction and sustainable development,
Beijing, China, 18-20 October 2005.
6) Widodo, T. W. and A. Nurhasanah. (2004). Study on Biogas Technology
and its development potency in Indonesia. Proceeding of National Seminar
on Agricultural Mechanization. Bogor, 5 August 2004.
SCMG
Assessment of Erosion Potentials on Various
Cropping Patterns Using USLE: Case of Subang
Region, West Java
R. I’anatus Sholihah*, Dyah R. Panuju, Enni D. Wahjunie, Bambang H.

Trisasongko
Department of Soil Science and Land Resource, Bogor Agricultural University
Jl. Meranti, Darmaga Campus, Bogor 16680, Indonesia
Abstract
Degraded lands in Subang regency have been increasing within this decade. Land inventory
conducted by the government shows that degraded areas expanded from 7,785 ha in 2011 up
to 9,581 ha in a year. Improvements of this type of land, primarily due to erosion, are necessity
in order to support intensive agriculture. This research aims to study erosion in various cropping
pattern based on land units in four districts of Subang region, West Java, which is important for
conservation planning. The research indicates that there are 19 land units formed by combining
land capability classes and land use types. The result shows that erosion in the test site varies from
0.30 to 133.19 ton ha-1y-1. Actual erosion from USLE is found higher than tolerable soil loss,
meaning that conservation is urgent to maintain land productivity.
Key words: Erosion, Land unit, Soil and Water conservation, USLE
I. Introduction
Agricultural sector in Indonesia has laid the base for the nation’s economy.
Population increase escalates the demand for land to maintain food production.
Nonetheless, land resource is fixed and limited with competing interest to other
utilizations, such as industries and residences. Throughout the nation, land use
changes have been reported to give significant impact on soil erosion, because
it serves the interaction between natural environment and human activities [1].
Inevitable alteration urges the uses of marginal or unsuitable lands for agriculture,
or alternatively the intensification of agricultural fields. The main challenge of
the latter is uncontrolled soil erosion, which in turn ignites further problems
including pollution.
Since the last century, soil erosion has been considered a serious environmental
problem. Majority of pasture and agricultural fields in the world faces problems
of erosion [2]. Humid tropic climate in Indonesia triggers high amount of
erosion, especially in intensive agricultural fields. Despite its importance, soil
* Corresponding Author. Tel: +62-251-8422325. E-mail: rizqiianatus_99@yahoo.com
243
erosion considering local environmental settings has yet been thoroughly studied.
Estimation of the loss due to soil erosion is often difficult to quantify since it
involves many factors, including climate, land cover, soil, topography and human
activities [1].
Erosion potential is often assessed using USLE [3], which is one of the
simplest methods to date. This paper explores the model to analyze various
cropping patterns based on land units, which in turn be able to construct suitable
conservation strategies for agriculture sustainability.
II. Method/material
A. Study Area
Subang is situated in West Java, surrounded by Indramayu and Sumedang regen-
cies in the East, while Purwakarta and Karawang are in the West border. Java sea
lies in the north, and emerging industrial regency of Bandung Barat in the south.
Geographically, Subang is located between 6°11’-6°49’S and 107°31’-107°54’E.
To be able to focus on the problem, only four districts are considered, namely
Cipeundeuy, Kalijati, Pabuaran, and Patokbeusi (Figure 1). These districts are
located in wavy landform with various cropping pattern, at the altitude between
50–500 m above sea level.
Figure 1. The Study area: Subang region

B. Method
Various data are utilized for this analysis. Each data processing is taken in sequence
as follows.
1) Determining Land Unit

Land unit is formed by combining land capability and land use maps through
boolean overlay. In this research, land use types consisting of rice field (sawah),
mixed garden (kebun campuran) and dry land agriculture (tegalan) are considered.
As a result, polygons containing land use types for each land capability classes are
constructed, which will serve as the primary unit for further analysis.
2) Erosion Soil Analysis

Soil erosion potentials using USLE, firstly communicated by Wischmeier and
Smith [4], is evaluated in this paper. The model, consisting of six factors, can be
expressed by the following equation:
(1)
Where A is the amount of soil erosion in ton ha-1 y-1, R is rainfall erosivity, K
is soil erodibility, LS is topographic factor, comprises of slope steepness and slope
length, C is cropping management and P is soil conservation practice.
a) Rainfall erosivity (R)
Rainfall is one of driving forces of soil erosion. Its erosivity and temporal rainfall
pattern deliver a direct impact to soil erosion [5]. In this study, we used Bols
method by considering available data to calculate the rainfall erosivity. Bols
equation proposed for Indonesia is shown below [6]:
R = 6.119 x Rf1.21x Rn-0.47 x Rm0.53 (2)
Where R is monthly erosivity, Rf is total monthly rainfall, Rn is sum of rainy
day per month and Rm is the maximum rainfall during 24 hour in the observed
month.
The R-factor for each land unit was then spatially estimated through interpola-
tion using Inverse Distance Weighting (IDW) technique.
b) Soil erodibility (K)
Erodibility is susceptibility of soil to erosion that influenced by many factors, such
as chemical, mineralogical, biological properties of soil as well as physical terrain
attributes, hydrological and climatic factors [7]. The value of K was computed
using the following equation:
K = [( 2.1 x M 1.14 x 10-4) x (12-a)) + (3.25
(b-2) + (2.5(c-3)) x 1.293% (3)

Where M is (Svf+St)(100-Cf ), a is percentage of soil organic matter, b is
structural code of the soil, c is permeability class code of the soil, Svf, St, and Cf
are percentage very fine sand, silt, and clay fraction respectively.
Physical soil characteristics which represent K-factor value need to be
considered. In this study, we used soil texture, percentage of soil organic matter,
and permeability class code of the soil to estimate K-factor.
c) Topographic factor (LS)
Topography influences surface runoff rate, therefore it is essential to quantify LS
properly [8]. Slope steepness and its length are measured in the field for some
locations, while the rest are calculated from existing slope map, provided by the
Citarum-Ciliwung River Basin Management Agency. Slope length and steepness
were then calculated using Wischmeier-Smith equation:
LS = l1/2 (0.0139+0.0965 S+0.00139S2) (4)

Where l is slope length in m, S is the slope steepness in percent.
d) Cropping management and soil conservation practice (CP)
The C- and P-factors are derived from Abdurachman et al. [9] study according to
actual land use. In this research, C-factor was weighed by considering cropping
period in a year. C-factor from cropping pattern in dryland (tegalan) was computed
by considering monthly R-factor, and then was spatially modeled using IDW.
The equation to calculate C-factor from various cropping pattern in dryland
agriculture (tegalan) is:
(6)
Where Ci is the average of C-factor of certain cropping pattern, Ri is monthly
R-factor, Ci is C-factor [9].
3) Assessing Tolerable Soil Loss (TSL)

Tolerable soil loss was computed using Hammer equation [10]:
TSL = ED/T (7)
Where TSL is tolerable soil loss in ton mm/yr, ED is equivalent depth in mm,
T is soil usage (400 years).
Figure 2. Land use types in Subang region

A. Description of Land Cover and Land Units
Result of visual image interpretation from ALOS AVNIR-2 is summarized in
Figure 2. Delineation of land cover considered image interpretation keys such as
color, texture, association, shape, etc. Verification by ground survey indicates that
there are seven land use types, i.e. water body, forest, mixed garden, real estate,
plantation, rice field and dryland agriculture.
Figure 2 shows that rice field (sawah) dominates research site of about
19313.73 ha or 53% of total area. Irrigated rice fields are located in Patokbeusi,
while Kalijati and Cipeundeuy districts are dominated by rain-fed rice fields.
Pabuaran district has both of them equally. Mixed garden and dryland fields are
often utilized by local people for cash crops, fruits, softwood timber and others.
Rice and dryland fields as well as mixed garden are the main source of income.
Table 1. Land capability classes in research area.
Acreage
No Land capability
Ha %
1 IIe 329.92 0.92
2 IIIc 13044.11 36.27
3 IIIe 9944.18 27.65
4 IIIw 1514.40 4.21
5 IVe 1843.97 5.13
6 IVw 6414.81 17.84
7 VIe 2874.09 7.99
Table 2. Distribution of land units.

Land units code Land units Acreage (ha)
IIe-KC IIe-Mixed Garden 108.22
IIe-SW IIe-Rice Field 88.40
IIIc-KC IIIc-Mixed Garden 1709.93
IIIc-SW IIIc-Rice Field 1577.93
IIIc-TG IIIc-Dryland 78.81
IIIe-KC IIIe-Mixed Garden 173.50
IIIe-SW IIIe-Rice Field 2726.49
IIIe-TG IIIe-Dryland 67.44
IIIw-KC IIIw-Mixed Garden 235.18
IIIw-SW IIIw-Rice Field 456.90
IIIw-TG IIIw-Dryland 39.35
IVe-KC IVe-Mixed Garden 182.22
IVe-SW IVe-Rice Field 338.28
IVw-TG IVw-Dryland 7.30
IVw-KC IVw-Mixed Garden 127.61
IVw-SW IVw-Rice Field 2466.90
VIe-KC VIe-Mixed Garden 148.34
VIe-SW VIe-Rice Field 154.24
VIe-TG VIe-Dryland 7.05
Total 10694.09
Land management should conform to land capability to maintain land

productivity. In this study, land capability data were generated by combining
slope and soil type maps. Land capability in this region consists of seven classes
(Table 1).
The research area is dominated by land capability IIIc class about 13,044.11
ha in undulating terrain and the soil type is Latosol. The main characteristics
of land capability IIIc class are medium to high permeability with fairly high
climate resistance.
Land use and land capability maps designates land unit in research area. Land
units arranged at district level are presented in Table 2.
Table 2 indicates that there are 19 land units available in this test site. Land
unit IIIe-Rice Field is the highest area about 2,726.49 ha, consisting of rice field
with land capability IIIe class. This land unit is distributed on almost all districts,
mostly located in Pabuaran. This is probably due to the fact that this district is
situated between flat and undulating landform. In addition, majority of soil type
is podsolic, which is sensitive to erosion. Many farmers utilize the land for rice
field because Pabuaran relief is rather flat, while water has been sufficient.
Land unit VIe-Dryland (VIe-TG) is the smallest area covered in the test site,
i.e. about 7.05 ha. This land unit is located in steep, undulating terrain, hence it
is seldom utilized for agriculture.
Table 3. USLE individual factors
Land units R K LS C P
IIe-KC 3224.77 0.22 0.05 0.25 1.00
IIe-SW 3482.25 0.22 0.05 0.01 1.00
IIIc-KC 2513.08 0.04 0.43 0.41 0.65
IIIc-SW 2130.91 0.04 0.44 0.04 0.70
IIIc-TG 2259.17 0.04 0.47 0.50 0.70
IIIe-KC 1839.04 0.36 0.40 0.41 0.56
IIIe-SW 1784.96 0.43 0.32 0.01 0.81
IIIe-TG 1937.13 0.54 0.43 0.42 0.50
IIIw-KC 2295.83 0.26 0.26 0.28 0.55
IIIw-SW 2314.91 0.25 0.25 0.01 0.82
IIIw-TG 2230.39 0.25 0.47 0.65 0.60
IVe-KC 3463.29 0.04 1.55 0.41 0.60
IVe-SW 3422.90 0.15 1.57 0.01 0.83
IVw-KC 1732.07 0.27 0.09 0.29 0.65
IVw-SW 1468.17 0.27 0.09 0.01 0.88
IVw-TG 1601.97 0.04 1.55 0.65 0.35
VIe-KC 2704.89 0.09 2.88 0.31 0.73
VIe-SW 2877.96 0.07 2.76 0.01 0.85
VIe-TG 2632.15 0.04 2.27 0.77 0.80
B. Soil Erosion in Subang Region

Preparation of soil conservation plan requires erosion data, which is often
measured in-situ. In addition, conservation planning also needs to consider soil
tolerable loss [11]. Prior assessing TSL, soil erosion needs to be calculated. Factors
influencing soil erosion are presented in Table 3.
Values shown in Table 3 are then applicable to estimate soil erosion potentials
for each land unit. Erosion potentials and their tolerable soil loss quantifications
are presented in Table 4.
Tolerable soil loss in the research area spans about 14 up to 40.50 ton ha-1y-1.
This indicates that high annual erosion rate applies in the research area was still
Table 4. Erosion potentials and tolerable soil loss (TSL) in ton ha-1y-1 for each land unit
Land units Erosion TSL
IIe-KC 8.67 14.00

IIe-SW 0.37 14.00
IIIc-KC 10.96 39.46
IIIc-SW 0.77 40.50
IIIc-TG 13.19 39.00
IIIe-KC 57.11 30.75
IIIe-SW 1.78 29.75
IIIe-TG 91.03 28.00
IIIw-KC 34.84 21.68
IIIw-SW 1.04 21.77
IIIw-TG 103.01 19.68
IVe-KC 48.04 36.00
IVe-SW 6.83 24.40

IVw-KC 7.33 29.10
IVw-SW 0.30 29.79
IVw-TG 20.31 36.00
VIe-KC 123.21 30.60
VIe-SW 5.70 32.47
VIe-TG 133.19 36.00
tolerable. This result signifies that recent situation allows reasonable support for
plant growth [12].
Table 3 also shows the average of actual erosion in the study area, which varies
from 0.30 up to 133.19 ton ha-1y-1. Land unit type IVw-SW has the lowest soil
erosion mainly because of very low erosivity of rainfall at gentle slope region.
The highest erosion rates based on Table 3 is in land unit type VIe-TG. This
land unit is located in Jalupang village, Kalijati district, which has wavy to hilly
terrain. Land use type of this land unit is dryland agriculture (tegalan) which is
often cultivated in steep slope (26–40%), with high rainfall. Based on information
presented in Table 3, there are seven land units which have soil erosion level of
more than their tolerable soil loss. In these cases, soil conservation is urgently
needed. Soil erosion has positive correlation with slope steepness, cropping
condition and conservation practice, which is in line with previous research [13].
Land use without proper conservation practice in VIe-TG is likely to expose
high-level of erosion.
In contrary, twelve land units have actual erosion lower than tolerable soil
loss. Hence, these areas require further maintenance to sustain current condition.
Seven land units, i.e. IIIe-KC, IIIe-TG, IIIw-KC, IIIw-TG, IVe-KC, VIe-KC,
and VIe-TG have actual soil erosion prediction exceeding tolerable soil loss
(A>TSL). Soil and water conservation planning is therefore a necessity, probably
by considering alternative crop management and soil conservation techniques.
Soil and water conservation specifically planned to each land units is important
for region with actual erosion higher than soil tolerable loss. Options for soil and
water conservation include planting cover crops, constructing multilevel canopies
of different plants as well as terrace making [14].
IV. Conclusion
Assessment of erosion potentials using USLE in Subang region shows that some
land units have actual erosion exceeding tolerable soil loss value. Therefore, soil
and water conservation strategies such as crop management are critical to this
site. Various cropping patterns on cultivated field have a greater influence on
actual soil erosion or soil loss. This is due to its direct relationship with human
activities which are responsible in land use alteration. The research summarizes
that USLE is simple model which can be used to provide information on soil
erosion. Utilization of this information, in turn, leads to a better soil and water
management planning.
V. Acknowledgments
We would like to thank the Citarum Ciliwung River Basin Management Agency
and Agricultural Land Resources Agency for the data sets used in this research.
Finally, we thank to anonymous reviewers of which their comments significantly
improve the paper.
VI. R eferences
1) Alkharabsheh, M.M., Alexandrilis, T.K., Bilas, G., Misopolinos. N, and
Silleos. N. (2013). Impact of land cover change on soil erosion hazard in
northern Jordan using remote sensing and GIS. Procedia Environmental
Sciences, 912–921.
2) Pimentel, D., Harvey, C., Resosudarmo, P., Sinclair, K., Kurz, D., McNair,
M., S. Crist, L. Shpritz, L. Fitton, R. Saffouri, and R. Blair. (Feb, 1995).
Environmental and economic costs of soil erosion and conservation benefits.
Science, 267, 5201, 1117-1123.
3) Sonneveld, B.G.J.S. and M.A. Nearing. (2003). A nonparametric/parametric
analysis of the Universal Soil Loss Equation. Catena, 52, 9-21.
4) Wischmeier, W.H., and D.D. Smith. (1978). Predicting Rainfall Erosion
Losses: A Guide to Conservation Planning, Washington: United States Depart-
ment of Agriculture, 58.
5) L. Liu and X.H. Liu. (2010). Sensitivity analysis of soil erosion in the Northern
Loess Plateau. in Procedia Environmental Sciences, 134-148.
6) Aflizar, Afrizal, R., and T. Masunaga. (2013). Asessment erosion 3D hazard
with USLE and surfer tool: a case study of Sumani Watershed in West Sumatra
Indonesia. Tropical Soils Journal, 18, 81-92.
7) Perez-Rodriguez, R., Marques, M.J., and R. Bienes. (2007). Spatially vari-
ability of the soil erodibility parameters and their relation with the soil map
at subgroup level. Science of the Total Environment, 378, 166-173.
8) Beskow, S., Mello, C.R., Norton, L.D., Curi, N., Viola, M.R., and J.C.
Avanzi. (2009). Soil erosion prediction in the Grande River Basin, Brazil
using distributed modeling. Catena, 79, 49-59.
9) Abdurachman A, S. Abuyamin, and U. Kurnia. (1984). Soil and Crop
Management to Conservation. Bogor: PPT Bogor.
10) Hammer, M.J., and K.A. Mac Kichan. (1981). Hydrology and Quality of Water
Resources. New York: John Willey and Sons.
11) Dariah, A., Rachman, A., and U. Kurnia. (2004). Erosion and dry land
degradation in Indonesia. Soil Conservation Technology on Dry Land Slope,
Bogor: Research Center and Soil and Agroklimat Research, 1-8.
12) Arsyad, S. (1989). Soil and Water Conservation. Bogor: IPB Press.
13) Halim, R., Clemente, R.S., Routray, J.K., and R.P. Shresta. (2007). Integration
of biophysical and socio-economic factors to assess soil erosion hazard in the
Upper Kaligarang Watershed, Indonesia. Land Degradation and Development,
4, 453-469.
14) Dewi, I.G.A.S.U., Trigunasih, N.M., and T. Kusmawati. (Jul, 2012). Erosion
prediction and soil and water conservation planning in Saba River Basin.
E-Journal Tropica Agrotechnology, 1, 12-23.
New Stage of International Collaboration
on Climatological Observation
Manabu D. Yamanakaa,b,*
a
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan
b
Graduate School of Science, Kobe University, Kobe, Japan
Abstract
Based on collaborations with Japanese scientists (principal institution: JAMSTEC) for three
decades, Indonesian Government (principal agency: BPPT) has installed the Maritime Continent
Center of Excellence (MCCOE) which operates weather and wind-profiling radars, develops/
installs climate buoys, and informs/investigates local/global climatic variations. We have been
clarifying various scientific results on the multiple-scale climate variability which is triggered in this
Maritime Continent region and is governing the global climate. This type of truly international
high-level collaboration is necessary to watch/understand to complex climatological issues.
Key words: Climatological observation, Multiple scales, International collaboration
I. Introduction-Climate of the L and-Sea Coexsisting

Planet E arth
The climate of a planet is maintained by an energy balance between the solar
heating and the planetary infrared cooling [1][2]. The solar heating is dependent
on the distance from the sun, and the infrared cooling is dependent on the
planetary temperature. Thus, the planetary temperature is basically determined
by the solar distance, and the Earth is only one habitable planet with liquid
water. The solar heating on the Earth is with annual and diurnal cycles and
latitudinal dependence, and it is decreased by reflection by earth’s surface and
clouds (parasol effect). The Earth’s infrared cooling is partially returned to the
Earth (greenhouse effect), of which the anthropogenic increase is being watched
with critical interest by international community as so-called global climatic
change. The balance mentioned above does not hold locally, with over-heating
and -cooling in the equatorial and polar regions, respectively, and the equatorial
over-heating is also due to hydrological cycles (latent heating) associated with
a peak 2,000 mm/year of rainfall produced by clouds with spatially/temporally
small scales. These imbalances are compensated by large-scale atmospheric and
oceanic circulations transporting heat poleward, through which the climate and
* Corresponding Author E-mail: mdy@jamstec.go.jp. URL: http://aoe.scitec.kobe-u.ac.jp/~mdy/index-e.

html
253
its variability in tropics affect substantially those in extratropics and therefore the
whole Earth’s climate.
The Indonesian maritime continent (IMC) [3] is a miniature of our land-sea
coexisting planet Earth. Firstly, without an interior activity, the Earth becomes
an even-surfaced “aqua-planet” with both atmosphere and ocean flowing almost
zonally, and solar differential heating generates (global thermal tides and) Hadley’s
meridional circulations with ITCZ along the equator as observed actually over
open (Indian and Pacific) oceans in the both sides of IMC. ITCZ involves
intraseasonal variations or super cloud clusters moving eastward [4][5].
Secondly, the lands and seas over the actual Earth have been keeping the area
ratio of 3:7 (similar to that islands and inland/surrounding seas in IMC), but
their displacements have produced IMC near the equator, which turns equatorial
Pacific easterly current northward (Kuroshio) and reflects equatorial oceanic
waves inducing coupled ocean-atmosphere interannual variations, such as El
Niño-southern oscillation (ENSO) [6] and Indian-Ocean dipole mode (IOD)
[7], or displacements of Walker’s zonal circulations. These are correlated with
climate variations over IMC [8][9][10][11] (see Figure 1).
Figure 1. Correlatons with ENSO and IOD (left) and simultaneous correlation with SST (right)
for rainfall averaged for 9 stations in around Jakarta [11].
Thirdly, because IMC consists of many large/small islands with very long
coastlines, many narrow straits become a dam for the global (Pacific to Indian)
ocean circulation, and the land-sea heat capacity contrasts along the coastlines
generate the world’s largest rainfall with diurnal cycles (sea-land breeze circula-
tions) [12][13][14][15][16][17][18][19] (see Figure 2). The diurnal cycles
are dominant in the rainy season (austral summer in Java and Bali) because
rainfall-induced sprinkler-like land cooling reverses the trans-coastal temperature
gradient before sunrise, and subsequent clear sky on land until around noon
provides solar heating depending on season [8][9]. These processes lead to rapid
land/hydrosphere-atmosphere water exchange [20], local air pollutant washout,
Figure 2. Geographical distributions of power-spectral intensities for cloud-top temperature variation

components with 6 hour-2 year periods (left) and morning-evening rainfall difference (right top) [16],
and a mechanism of diurnmal cycle over IMC [19].
and transequatorial boreal winter monsoon (cold surge) [21][22][23]. In El

Niño years, the cooler sea-surface temperature suppresses the morning coastal-sea
rainfall, and often induces serious smog over IMC.
II. Observation Strategy

Based on climatological features mentioned in the previous section, high-resolution
observations/models covering both over islands and seas are necessary. During
FY2005-09, observation networks have been installed both over the land and
ocean of IMC during these two decades under collaborations by Japan Agency
for Marine-Earth Science and Technology (JAMSTEC) and Kyoto University
in the Japanese side and the Agency for the Assessment and Application of
Technology (BPPT), the Indonesian Institute for Space and Aeronautics (LAPAN)
and the Meteorological, Climatological and Geophysical Agency (BMKG) in
the Indonesian side. On land of IMC, based on several foregoing projects, a
radar-profiler network observation project called Hydrometeorological Array
for Interaseasonal Variation-Monsoon Automonitoring (HARIMAU) have been

carried out started [24]. Over the Indonesian exclusive economic zone (EEZ)
surrounding IMC, collaborations of installation and maintenance of buoys, as
well as research vessel observations, have been continued as a part of the global
network of Tropical Ocean Climate Study (TOCS) [25]. (see Fig. 3)
During FY 2009-2013, another 5 year international (Japan-Indonesia)
project entitled “Climate Variability Study and Societal Application through
Indonesian-Japan Maritime Continent COE: Radar-Buoy Network Optimization
for Rainfall Prediction (SATREPS-MCCOE project) has been carried out as
a Science and Technology Research Partnership for Sustainable Development
(SATREPS), supported financially by Japan Science and Technology Agency (JST)
and Japan International Cooperation Agency (JICA). The project is carried out
by JAMSTEC, under collaborations with Kyoto University and Kobe University
in Japanese side and with BPPT, BMKG and LAPAN in Indonesian side.
In order to keep observations of this important region permanently, it must
be promoted independently by Indonesia which becomes a G20 country now.
For this purpose the SATREPS-MCCOE project has promoted establishment
of an international research center (MCCOE) by the Indonesian Government
(Output 1). The MCCOE has three functions: observations by radars (Output 2)
and buoys (Output 3), data management (Output 4), and researches on regional
Figure 3. The Maritime Continent Center of Excellence (MCCOE) established under a

SATREPS project, which is operating the HARIMAU radar-profiler network [24] and an
InaTRITON station of the TOCS buoy network [25].
Figure 4. An example of observation networks proposed for the Year of Maritime Continent
(YMC) [26]. .
rainfall (Output 5) and global climate (Output 6). A building was constructed
at the National Research and Technology District (PUSPIPTEK) in Serpong (20
km southwest from the central Jakarta) completed by the Indonesian Government
and society under organization between BPPT, BMKG and LAPAN, as well as
researchers in universities and other agencies. The MCCOE has been opened in
November 2013 and a homepage has been constructed at BPPT: http://neonet.
bppt.go.id/satreps/
III. Conclusion-Future Scopes

As being promoted at MCCOE, the climatological observations in the next
step should be independent and more completed by each country under more
international (not bilateral) framework. A typical example is the project called
“Year of Maritime Continent”, which is now being proposed for 2017–2008 [26]
(see Figure 4). Through such activities in the next step, science and technology
specialized for equatorial/tropical climate with conditions/characteristics different
from extratropics (such as electronic parts invulnerable for very humid/hot envi-
ronment, description on non-annual periodicities, and so on) must be developed/
established by Indonesia and surrounding ASEAN countries. Completing them
and covering the whole tropical/equatorial region with a half of earth surface
will lead to a breakthrough for understanding/perdiction of the global climate.
IV. Acknowledgment
The author acknowledges many colleagues of Japan and Indonesia for collabora-
tions described here. In particular Dr. Fadli Syamsudin of BPPT has provided
comments from Indonesian side and Dr. Kunio Yoneyama of JAMSTEC has
provided information on YMC. The author’s participation at this meeting was
supported by JST.
V. R eferences
1) Hartman, D.L. (1994). Global Physical Climatology. San Diego: Academic
Press.
2) Stocker, T.F., Qin, D., Plattner, G.-K., Tignor, M., Allen, S.K., Boschung,
J., Nauels, A., Xia, Y., Bex, V., and Midgley, P.M. (Eds.). (1968). Cambridge
University Press, Cambridge, United Kingdom and New York, NY, USA,
2013. Intergovernmental Panel on Climate Change (IPCC), Climate Change:
The Physical Science Basis. Contribution of Working Group I to the Fifth
Assessment Report of the Intergovernmental Panel on Climate Change C. S.
Ramage, Role of a tropical “maritime continent. The atmospheric circulation,
Mon. Wea. Rev., 96, 365−370.
3) Madden, R.A. and Julian, P.R. (1972). Description of global-scale circulation
cells in the tropics with a 40−50 day period, J. Atmos. Sci., 29, 1.109−1.123.
4) Hayashi, Y.-Y. and Sumi, A. (1986). The 30−40 day oscillations simulated
in an “aqua planet” model. J. Meteor. Soc. Japan, 64, 451−467.
5) Philander, S.G. (1990). El Niño, La Niña, and the Southern Oscillation.
Academic Press.
6) Saji, N.H., Goswami, B.N., Vinayachandran, P. N., and Yamagata, T. (ND).
A dipole mode in the tropical Indian Ocean. Nature, 401, 360−363.
7) Hamada, J.-I, Yamanaka, M.D., Matsumoto, J., Fukao, S., Winarso, P.A., and
Sribimawati, T. (2002). Spatial and temporal variations of the rainy season
over Indonesia and their link to ENSO. J. Meteor. Soc. Japan, 80, 285−310.
8) Okamoto, N., Yamanaka, M.D., Ogino, S.-Y., Hashiguchi, H., Nishi, N.,
Sribimawati, T., and Numaguti, A. (2003). Seasonal variation of tropospheric
wind over Indonesia: Comparison between collected operational rawinsonde
data and NCEP reanalysis for 1992−99. J. Meteor. Soc. Japan, 81, 829−850.
9) Kubota, H., Shirooka, R., Hamada, J.-I., and Syamsudin, F. (ND). Interan-
nual rainfall variability over the eastern maritime continent. J. Meteor. Soc.
89A, 111−122. Japan.
10) Hamada, J.-I., Mori, S., Yamanaka, M.D., Haryoko, U., Lestari, S.,
Sulistyowati, R., and Syamsudin, F. (2012). Interannual rainfall variability
over northwestern Jawa and its relation to the Indian Ocean dipole and El
Nino southern-oscillation events. SOLA, 8, 69−72.
11) Hashiguchi, H., Fukao, S., Tsuda, T., Yamanaka, M.D., Tobing, D.L., Sribi-
mawati, T., Harijono, S.W.B., and Wiryosumarto, H. (1995). Observations
of the planetary boundary layer over equatorial Indonesia with an L-band
clear-air Doppler radar: Initial results. Radio Sci., 30, 1.043−1.054.
12) Renggono, F., Hashiguchi, H., Fukao, S., Yamanaka, M.D., Ogino, S.-Y.,
Okamoto, N., Murata, F., Harijono, S.W.B., Kudsy, M., Kartasasmita,
M., and Ibrahim, G. (2001). Precipitating clouds observed by 1.3-GHz
L-band boundary layer radars in equatorial Indonesia. Ann. Geophys., 19,
pp. 889−897.
13) Murata, F., Yamanaka, M.D., Fujiwara, M., Ogino, S.-Y., Hashiguchi, H.,
Fukao, S., Kudsy, M., Sribimawati, T., Harijono, S.W.B., and Kelana, E.
(2002). Relationship between wind and precipitation observed with a UHF
radar, GPS rawinsonde and surface meteorological instruments at Kototabang,
West Sumatera during September-October 1998. J. Meteor. Soc. Japan, 80,
347−360.
14) Wu, P.-M., Hamada, J.-I., Mori, S., Tauhid, Y.I., Yamanaka, M.D., and
Kimura, F. (2003). Diurnal variation of precipitable water over a mountaneous
area in Sumatera Island. J. Appl. Meteor., 42, pp. 1.107−1.115.
15) Mori, S., Hamada, J.-I., Tauhid, Y.I., Yamanaka, M.D., Okamoto, N.,
Murata, F., Sakurai, N., and Sribimawati, T. (2004).Diurnal rainfall peak
migrations around Sumatera Island, Indonesian maritime continent observed
by TRMM satellite and intensive rawinsonde soundings. Mon. Wea. Rev.,
132, 2.021−2.039.
16) Sakurai, N., Murata, F., Yamanaka, M.D., Hashiguchi, H., Mori, S., Hamada,
J.-I., Tauhid, Y.-I., Sribimawati, T., and Suhardi, B. (2005). Diurnal cycle of
migration of convective cloud systems over Sumatera Island. J. Meteor. Soc.
Japan, 83, 835−850.
17) Araki, R., Yamanaka, M.D., Murata, F., Hashiguchi, H., Oku, Y., Sribimawati,
T., Kudsy, M., and Renggono, F. (2006). Seasonal and interannual variations
of diurnal cycles of local circulation and cloud activity observed at Serpong,
West Jawa, Indonesia. J. Meteor. Soc. Japan, 84A, 171−194.
18) Wu, P.-M., Hara, M., Hamada, J.-I., Yamanaka, M.D., and Kimura, F.
(2009). Why heavy rainfall occurs frequently over the sea in the vicinity of
western Sumatera Island during nighttime. J. Appl. Meteor. Climatol, 48,
1.345−1.361.
19) Sulistyowati, R., Hapsari, R.I., Syamsudin, F., Mori, S., Oishi, S.T., and
Yamanaka, M.D. (2014). Rainfall-driven diurnal variations of water level in
the Ciliwung River, West Jawa, Indonesia. SOLA, 10, to appear.
20) Wu, P.-M., Hara, M., Fudeyasu, H., Yamanaka, M.D., Matsumoto, J.,
Syamsudin, F., Sulistyowati, R., and Djajadihardja Y.S. (2007). The impact
of trans-equatorial monsoon flow on the formation of repeated torrential
rains over Java Island. SOLA, 3, 93−96.
21) Hattori, M., Mori, S., and Matsumoto, J. (2011). The cross-equatorial
northerly surge over the maritime continent and its relationship to precipita-
tion patterns. J. Meteor. Soc. Japan, 89A, 27−47.
22) Wu, P.-M., Arbain, A.A., Mori, S., Hamada, J.-I., Hattori, M., Syamsudin, F.,
and Yamanaka, M.D. (2013). The effects of an active phase of the Madden-
Julian oscillation on the extreme precipitation event over western Jawa Island
in January 2013. SOLA, 9, 79−83.
23) Yamanaka, M.D., Mori, S., Wu, P.-M., Hamada, J.-I., Sakurai, N., Hashi-
guchi, H., Yamamoto, M.K., Shibagaki, Y., Kawashima, M., Fujiyoshi,
Y., Shimomai, T., Manik, T., Erlansyah, Setiawan, W., Tejasukmana, B.,
Syamsudin, F., Djajadihardia, Y.S., and Anggadiredja, J.T. (2008). HARIMAU
radar-profiler network over Indonesian maritime continent: A GEOSS early
achievement for hydrological cycle and disaster prevention. J. Disaster Res.,
3, 78−88.
24) Ando, K., Syamsudin, F., Ishihara, Y., Pandoe, W., Yamanaka, M.D., Masu-
moto, Y., and Mizuno, K. (2010). Development of new international research
laboratory for maritime-continent seas climate research and contributions to
global surface moored buoy array. Proceedings of the “OeanObs’ 09: sustained
Ocean Observations and Information for Society” Conference (Annex), Venice,
Italy, 21−25 September 2009 [J.　Hal, D. E. Harrison and D. Stammer
(eds)], ESA Publication WPP-306..
25) Yoneyama, K. (2014). Private Commnunication.
Simultaneous Correlation Analysis
of Australian Summer Monsoon Index (AUSMI)
against Rainfall in Bali Region
Subekti Mujiasiha)* and I Gede Agus Purbawab)
a
Balai Besar MKG Wilayah III Denpasar
Jl. Raya Tuban, Kuta 80362, Kabupaten Badung, Bali,Indonesia,
b
Banyuwangi Meteorogical Station
Jl.Jaksa Agung Suprapto No.152, East Java, Indonesia
Abstract
Rainfall is one of the most important parameters of weather and climate which needs to be studied
in depth because it has a large degree of variability. Its pattern and characteristics in Bali region
are influenced by the Asian and Australian Monsoon. Furthermore, monsoon is considered as
the most significant phenomena in affecting rainfall in Indonesia region. From previous studies,
Australian Monsoon Index (AUSMI), one indicator of the Asian-Australian monsoon activity,
can capture rainfall variability in Australia and the Indonesia maritime continent. In this study
we analyzed the relationship between AUSMI and rainfall in Bali region using simultaneous
correlation method. The used data was monthly averaged rainfall from 36 rainfall post station
during 30 years since 1979–2008. With expectations, we can know how strong influence of
AUSMI against rainfall, so that we can use AUSMI as one of predictors to predict rainfall. The
result shows that the AUSMI greatly affects rainfall in this region. The degree of influence varies
in range 0.5–0.8 of correlation, coefficient, spatially and temporally.
Key words : AUSMI, Simultaneous correlation, Predictor, Bali
I. Introduction
Indonesia is a maritime region. This is indicated by the composition of its territory
which consists of a vast ocean with interspersed islands large and small and also
the position is right around the equator. That type of climate is generally known
as the continental maritime climate. Continental maritime climate which is
owned by Indonesia actually has a comparative advantage than other country,
including the year-round warm climate, rainfall variability between regions
and rich in solar radiation with irradiation time range from 3–10.5 hours and
solar radiation intensity 235–535 cal/cm2/day [1]. Variability of rainfall as one
characteristic of the continental maritime climate, can also be used to determine
the time of the rainy season and dry season, apart from the wind circulation
pattern [2]. Climatology Meteorology and Geophysics Agency (BMKG) defines
* Corresponding Author.Tel: +62-85313131831. E-mail: subekti.mujiasih@bmkg.go.id.
261
the average monthly rainfall of 150 mm/month for wet season and the average
monthly rainfall less than 150 mm/ month for dry season [3]. Based on rainfall
pattern in Indonesia which has three types such as monsoon, equatorial and local
types, then Bali has a monsoon rain pattern [4]. Furthermore, by using double
correlation method, rainfall pattern in Bali is included in monsoon region [5].
According to BMKG, Bali is included in the category of regions receiving the
highest rainfall over 3,000 mm/year [6]. Based on research on average rainfall
Indonesia for 32 years (1961–1993), the rainfall pattern in Bali increased in
period of November to March and decreased in April to October [5]. Generally,
rainfall in wet season in Bali depends on atmospheric phenomena activity both
globally and regionally.
Global scale factor includes the occurrence of ENSO (La Nina or El Nino),
Dipole Mode, and the Madden Julian Oscillation (MJO). Regional scale factor
includes monsoon activity, including Asian Cold Surge, Tropical Disturbance
such as a vortex or a tropical storm, the ITCZ and so on. Whereas, local factors
include Orographic shape, land-sea breeze, mountain-valley winds, convectivity
and atmospheric stability [7].
The influence of regional scale atmospheric happens when cold surge activity
from Asia becomes the cross-equatorial flow in Indonesia region in south of the
equator, or when MJO is active in Indonesian territory or when tropical cyclones
are formed around the western and northern Australia. These phenomenons
cause rain duration tends to occur in a long and sustained period in Bali. Even
in extreme case, rain events can get all day with moderate to heavy intensity and
strong wind. However, when the global and regional phenomenon are decreasing,
rain events tend to be dominated by local factors. One of local factors causing
Bali has different climate of each region, although composed of the island and
surrounded by the sea, is orographic of each region. It means that climate pat-
terns in the highlands tend to be different from the area near the coast. In these
circumstances, it usually occurs in the intensity of light rain and sunny to cloudy
weather conditions during the day in most of the region. Rain around the coastal
areas generally occurs at night until in the morning. Whereas, in mountainous
areas or terrain, rainfall occurs in the late morning or in the afternoon.
In relation to the pattern of monsoon rainfall, rainfall variability in Australia
and the Indonesian Maritime Continent, can be detected through AUSMI.
AUSMI is dynamic monsoon index, which is based on 850 hPa zonal wind
averaged over the area (5°S–15°S, 110°E–130°E) [8]. According to AUSMI Data,
1948–2009 shows the highest monsoon index happened in peak of wet season
month (DJF), whereas the lowest index happened in peak of dry season month
(JJA)[9]. AUSMI research for the Indonesian region has already been done using
TRMM [10] especially in Tabing, West Sumatra [11]. Related to the various
climate patterns over Bali, high rainfall and AUSMI ability to recognize rainfall
variability, the authors intend to explore the relationship between AUSMI and
rainfall in Bali by using correlation method. This method is used to determine the
relationship between two or more variables and magnitude of the relationship [12]
.This method has also been used to examine the influence of ENSO on Pacific
Basin precipitation [13]. Hopefully, the results of this study can be considered in
predicting rain in Bali and surroundings by using AUSMI as one of the predictors.
Furthermore, determination of wet and dry seasons preliminary might be more
accurate by considering AUSMI as indicator of monsoon.
II. Data and Methodology

A. Rainfall Dataset
The data used is monthly rainfall observation rate of 36 rain gauge stations. Data
periods are during 30 years for 1979–2008. Then, data was preprocessed by table
and categorized by month and year.
B. Australian Summer Monsoon Indices (AUSMI) Dataset

Broad-scale Australian monsoon index (AUSMI) describing multi-time scale
variations is defined by using 850 hPa zonal wind averaged over the area (5 °S–15
°S, 110 °E–130 °E). This circulation index reflects monsoonal rainfall variability
over Northern Australia and maritime continent. The index can be used to
depict the seasonal cycle (for instance the onset) and measure the intraseasonal,
interannual and interdecadal variations of the Australian monsoon [8]. AUSMI
data set is monthly rate during 30 years for 1948–2008. AUSMI dataset can be
downloaded freely from Asia Pacific Data Research Center website [14].
AUSM Index = U850(110-130E,15S-5S) (1)[15]
Figure 1 AUSMI coverage area [8]

From two dataset above, we obtained monthly rainfall rate and monthly
AUSMI rate. Next step, these data were processed using correlation method.
Where, R (x,y) is correlation coefficient between X (AUSMI) and Y (Rainfall). x is
monthly AUSMI rate and y is monthly rainfall rate (see Equation 2). The criteria
for the correlation coefficient is as follows: if the price of r (x,y) approaches +1,
it means the relationship between the two variables is stronger and proportional
nature. If the price of r (x,y) close to -1, it means that the relationship between
the two variabel is stronger and inversely nature. If the price of r (x, y) ≥ +0.5 or
≤ -0.5, it means the relationship between the two variables is strong enough. If
the price of r (x, y) ≤ ≥ +0.5 or -0.5, it means the relationship between the two
variables is weak. Finally, the calculated correlation of data series was mapped
both series (averaged 30 years) and monthly.

In this research, authors give 3 results. They are Series Correlation Map (figure
2) Monthly Correlation Map and Linear Regression for Tejakula and Ngurah
Rai. We displayed monthly correlation map such as March (figure 3) and July
(figure 4). Author compared the two types against observation monthly rainfall
in 2012 (figure 5) and 2013 (figure 6) and Ausmi Analysis 2012–2013 (figure 7).
Figure 2. Series Correlation Map

Figure 3. March correlation map.
Figure 4. July Correlation map.

Figure 5. Observation rainfall Pos Station in Bali Region - 2012
Figure 6. Observation rainfall Pos Station in Bali Region - 2013

Figure 7. Ausmi Analysis August of 2012 - 2013
A. Series Correlation Map

Series Correlation map (figure 2) shows that all Bali regions have correlation
coefficient above 0.5. It means influence of monsoon is strong enough and linear
with rainfall. It causes rainfall increasing in December, January, February (DJF)
and decreasing in June, July and Auguts (JJA).
Generally, this pattern happened in Bali region for observation rainfall in
2012 (figure 5) and 2013 (figure 6). This is similar with pattern of AUSMI
Analysis of 2012–2013 (figure 7). The AUSMI pattern shows highest monsoon
index happened in months of peak of wet season (DJF), whereas lowest index
happened in months of peak of dry season (JJA)[9]. And also, based on research
on average rainfall Indonesia for 32 years (1961–1993), the pattern of rainfall
in Bali increased in the period in November–March and decreased in April to
October [5].
B. Linear Regression
Beside correlation, this research also has resulted series of regression equation for
each region. In this report, we displayed Ngurah Rai and Tejakula Raingauge
Pos Station. These stations have a positive linear relation with AUSMI. Linear
regression equation for Ngurah Rai is Rainfall is equal to 30.11*AUSMI+223.53
with Determination coefficient 0.57. For Tejakula, Rainfal is equal to

28.3*AUSMI+179.42, with Determination coefficient 0.62.
C. Monthly Correlation Map

Beside Series Correlation Map, we also created monthly correlation map from
series data. In this research we displayed only two maps of March and July since
they have anomaly. The first anomaly is March in 2012. First, March Monthly
correlation map (figure 3) shows Karang Asem has strong enough correlation
(0.5–0.7). It means these areas may be affected by AUSMI. It means the area
should have decreased monthly observation rainfall in March as AUSMI pattern.
But, Observation in Karang (figure 8) does not show it. It means, March correla-
tion map is not suitable for Karang Asem.
Figure 8. Karang Asem - March Anomaly 2012
Second, March monthly correlation map (figure 3) shows Badung has strong
enough correlation (0.5-0.7). It means these areas may be affected by AUSMI.
It means the area should have decreased monthly observation rainfall in March
as AUSMI pattern. However, observation in Badung (figure 9) does not show it.
It means, March correlation map is not suitable for Badung.
Third, In other hand, it shows different result for Buleleng. It has weak
correlation (0–0.3) in figure 3. It means Buleleng may not be affected by AUSMI.
It means the area should have increased monthly observation rainfall in March as
Figure 9. Badung - March Anomaly 2012
Figure 10. Buleleng - March Anomaly 2012
AUSMI pattern. Buleleng observation result (figure 10) shows March is higher
than February. It means March correlation map is suitable for Buleleng.
Fourth, as the literature, The AUSMI pattern shows highest monsoon index
happened in months of peak wet season (DJF), whereas lowest index happened
Figure 11. Badung - July Anomaly 2013
in months of peak of dry season (JJA)[9] and AUSMI 2012-2013 (figure 7).
However, for monthly observation rainfall in 2012, March is peak of wet season.
The rainfall trend of DJF and MAM period should be decreasing in March.
However, it is not decreasing in March. The March anomaly happened at 18
locations (81%) from 22 locations of observation in 2012. These are some
examples, such as Karang Asem (Selat Duda), Badung (Ngurah Rai), Buleleng
(Gerokgak, Sukasada, Tangguwisia). The areas got March monthly rainfall higher
than February. This is not similar with AUSMI pattern that March should have
lower than February.
The Other anomaly is July monthly rainfall in 2013. First, Badung has weak
correlation (0–0.3) in figure 4. It means the area may not be affected by AUSMI.
It means the area should have increased monthly observation rainfall in July.
Badung observation result (Figure 11) shows July is higher than June. It means
July correlation map is suitable for Badung.
Second, other area also gets July anomaly 2013 is Jembrana. It has weak
correlation (0–0.3) in figure 4. It means the area may not be affected by AUSMI.
It means the area should have increased monthly observation rainfall in July.
Jembrana observation result (Figure 12) shows July is higher than June. It means
July correlation map is suitable for Jembrana.
Third, other area of anomaly July monthly rainfall in 2013 is Tabanan. It has
weak correlation (0–0.3) in figure 4. It means the area may not be affected by
AUSMI. It means the area should have increased monthly observation rainfall
Figure 12. Jembrana – July Anomaly 2013
Figure 13. Tabanan – July Anomaly 2013

in July. Tabanan observation result (figure 13) shows July is higher than June. It
means July correlation map is suitable for Tabanan.
Fourth, as literature, July should have lowest monthly rainfall. But, the
observation in July shows different result. There is an increasing monthly rainfall
in July. The July anomaly happened in 32 locations (70%) from 41 locations
of observation in 2013. These are some examples such as Badung, Jembrana
(Nursasari), Bangli, and Gianyar (Kemenuh).
From the above explanation, there are some different result in some areas
between monthly correlation map, observation and AUSMI pattern. It may
be influenced not only regional factor like monsoon but also orographic factor
(local factor).
IV. Conclusion
Australian Summer Monsun (AUSMI) affecting rainfall in Bali, has various
influences in spatially and temporally with strong positive correlation (r > 0.5).
There is some different result in some areas between monthly correlation map
and observation. It may be influenced not only regional factor like monsoon but
also orographic factor (local factor). AUSMI can be used as one of predictor
for predicting rainfall and season in Bali. It is necessary long series data for each
season to get more accurate influences of Monsoon to rainfall. In the future, it is
better if research involving other factors for rainfall predicting such as sea surface
temperature in Bali.
V. R eferences
1) Syahbuddin, H. (2005). Jangan Lupa Swasembada Pangan. Majalah Inovasi
Online, 4, XII.
2) Ramage C. (1971). Monsoon Meteorology. International Geophysics Series, 15.
San Diego, CA: Academic Press.
3) BMG.(2000). Guidance of Season prediction and Analysis.
4) Tjasyono, B. (2004). Klimatologi. Penerbit ITB.
5) Aldrian, E. and Susanto, RD. (2003). Identification of three dominant
rainfall regions within Indonesia and their relationship to sea surface
temperature. International Journal of Climatology, 23, 1435–1452.
6) BMG. (2006). Peningkatan Pemahaman Informasi Iklim. BMG. Jakarta.
7) Pusdiklat BMKG. (2012). Bahan Ajar Diklat Teknis Analisa Cuaca permukaan.
8) Kajikawa, Wang, Y. B., and J. Yang. (2010). A Multi-time scale Australian
Monsoon Index. International Journal of Climatology.
9) Link http://apdrc.soest.hawaii.edu/projects/monsoon/ausmidx/index.html.
Accessed on 01 January 2014
10) Nuryanto, DE. 2012. Relationship between Indo-Australia Monsoon with
Seasonal rainfall Variability Indonesia Maritime Component Spatially based
on analysis Result of TRMM Satellite Data. Journal of Meteorology and
Geophysics, 13, 2, 91-102
11) Link https://www.academia.edu/7473108/Full_Paper. Accessed on 10
August 2014
12) Hernowo, B. 1997. Metode Korelasi Akademi Meteorologi dan Geofisika.
Jakarta
13) Link http://www.cpc.noaa.gov/pacdir/cont_chp6.html. Accessed on 01
January 2014
14) Link http://iprc.soest.hawaii.edu/users/ykaji/monsoon/seasonal-monidx.
html. Accessed on 01 January 2014
15) Link http://iprc.soest.hawaii.edu/users/ykaji/monsoon/definition.
html#ausm. Accessed on 01 January 2014
Developing Strategy for Monitoring and
Decision Support System for Smoke Haze
Trans-boundary Problem within ASEAN Region
Sheila Dewi Ayu Kusumaningtyas*

*
Agency for Meteorology, Climatology, and Geophysics (BMKG)
Jl. Angkasa I, No. 2, Kemayoran, Jakarta, Indonesia
Abstract
Land clearing for plantation and the subsequent simple approaches of biomass burning which
occur in many provinces in Indonesia, especially in Riau province increases during the past decades
and reduces the quality of air in the region. The associated trans-boundary haze pollution issue
has been a political debate and creates tensions among neighboring countries. When biomass
burns, certain aerosol pollutant is emitted to the atmosphere. The present study aims to describe a
concerted effort in monitoring the challenge of smoke haze distributions in the region to improve
early warning and minimizing the unprecedented impact on economy, health and environment.
The use of satellite remote sensing and in-situ measurement from Aerosol Robotic Network
(AERONET) Program in determining trans-boundary haze pollution in South East Asia will be
shown. Other political and operational field instruments to support the mechanism to put out
fires are applied in the region. The haze pollution that is detected from observation of aerosol
loading in Singapore during dry period in June 2013 is used as case study. Necessary data of
air quality and remote sensing are analyzed to provide comprehensive picture of the problem.
Hysplit Trajectory Model has also been applied to study the dispersion of smoke haze pollution.
According to the remote sensing satellite, smoke haze originate from Riau during fire period of
June 2013 had affected neighboring countries such as Singapore, Malaysia, and floated up to the
southwestern edge of the Philippines. This study suggests a further extension of such a project
in order to monitor further the disturbing low quality of air due to smoke fire. Ultimately, a
decision support system within the ASEAN countries is needed to have data exchange and join
analyses in detecting, monitoring and minimizing the risk of the problem.
Key words: Vegetation fires, Trans-boundary haze pollution, AERONET, Hysplit Model
I. Introduction
Over the last 20 years, fires have risen not only global attention but also Indonesia
since causing serious damage on environment and economy especially following
the 1997/1998 El Nino Southern Oscillation (ENSO) event which devastated
up to 25 million hectares of land worldwide. Fires are considered a potential
threat to sustainable development because of their direct effect to ecosystem, their
contribution to carbon emission and their impact to biodiversity [1].
* Corresponding Author.Tel: +6281381430023.E-mail: sheila_bmg@yahoo.com
275
Vegetation fires are common phenomena during dry periods in Indonesia,

especially in Riau. However, it is getting wors in line with the increasing of rapid
deforestation and land clearing. According to Anderson and Bowen [2], over the
last 5 years some 30% of the fires detected in Sumatra have been in Riau province.
Riau in all probability routinely has the greatest frequency of vegetation fires of
any province in Indonesia in terms of number of fires per square kilometers. The
province is quickly and rampantly logging its remaining forests. The dry land
forests are now nearly exhausted and attention has already turned to the swamp
forests. Like the dry areas, the land is then converted to other purposes, mainly
oil palm. Conversion is a major and planned component of Riau’s development
strategy.
The use of fire as a tool to clear land for agriculture is a long established
practice in Indonesia. When this tool is used by farmers in years of average or
above average rainfall, the practice poses little threat to the environment. This
kind of tool is considered cheap, easy, and effective and has been done not only by
small-holder farmers but also estate crop companies to clear thousands of hectares,
in the main for oil palm, is a much more worrying problem [3]. Anderson and
Bowen [2], also wrote that there is a strong correlation between fire numbers /
area burnt and the land clearing activities of oil palm companies and Sumatra
has been hard-hit when compared to Kalimantan and elsewhere in Indonesia.
Agency for Meteorology, Climatology, and Geophysics of Indonesia (BMKG)
records a considerable number of fires occur in Riau almost every year and
increasing during some dry periods. In June 2013, through observation from
Terra and Aqua MODIS Satellite, BMKG recorded up to 3,400 hotspots occurred
in Riau Pro-vince [4]. A number of hotspot as an indicator of fires has given
negative contribution to the environment, social, economy, and health as well as
trans-boundary haze pollution. Great and massive fires which occurred in several
countries like Indonesia, Brunei Darussalam, Malaysia, Philippine, Singapore,
and Thailand in 1997–1998 have destroyed more than 9 million hectares of
land in which 6,6 hectares was biomass [1]. Periods of smoke haze pollution in
Indonesia, some of it spreading to neighboring countries now regular events and
are likely to become more severe in the immediate future. In Southeast Asia, the
concern with the impacts of fires is particularly significant, as exemplified by
the signing of the Agreement on Trans-boundary Haze Pollution by the country
members of the Association of Southeast Asian Nations (ASEAN) in June 2002
in Kuala Lumpur.
According to Kunii et al., [5], the south west monsoon wind contributes
to the occurrence of cross-equatorial transport of smoke haze originate from
Indonesia to neighboring countries causing regional pollution which contain
high concentration of aerosol. When biomass burns, certain pollutant, such as
greenhouse gasses, hydrocarbon substances, and aerosol mixture contain black

carbon, organic compound, sulfate and nitrate, are emitted to the atmosphere.
According to Yokelsen et al., [6], in a large cloud of smoke caused by biomass
burning identified the presence of very high concentrations of PM10. This is
supported by research of Holben et.al., [7] which states that biomass burning in
the tropics produces high carbon aerosol pollution especially in the dry season
in August and September. A similar study by Artaxo et al., [8] which measures
the increase in the concentration of fine particulate matter (diameter <2.0 µm)
during dry season as a result of the biomass burning and allows the transport of
pollutants. The high concentration of aerosols can reduce visibility and increase
the risk of disruption of health.
Atmospheric aerosols absorb incoming solar radiation and scatter infrared
radiation emitted from the earth. The influence of atmospheric aerosols on solar
radiation is very dependent on the distribution size, shape, concentration, and
optical properties [9]. In this case, the aerosol optical thickness or commonly
referred to as aerosol optical depth (AOD) is one of the important parameters
to study the presence of aerosols in the atmosphere and its impact on air quality,
health and environment, as well as to monitor the vegetation fires.
Considering remarkable impact of vegetation fires to environment and to
overcome smoke haze pollution within ASEAN region, strategy for monitoring
and decision making system is required to build. The present study aims to describe
a concerted effort in monitoring the challenge of smoke haze distributions in
the region to improve early warning and minimizing the unprecedented impact
on economy, health and environment. The use of satellite remote sensing and
in-situ measurement from Aerosol Robotic Network (AERONET) Program in
determining trans-boundary haze pollution in South East Asia will be discussed.
The haze pollution that is detected from observation of aerosol loading in
Singapore during dry period in June 2013 is used as case study. Necessary data
of air quality and remote sensing are analyzed to provide comprehensive picture
of the problem. Moreover, this paper also discusses policy responses addressing
on trans-boundary haze pollution.

To study the smoke haze episode in Riau, analyses on the optical properties of
smoke haze with the aid of an AERONET Sun Photometer was conducted. The
AERONET program is a federation of ground-based remote sensing aerosol
networks established by NASA. To study events such as the June 2013 trans-
boundary smoke haze episode, the atmospheric radiation data used in this paper
have been taken from Singapore site. The AERONET Singapore site is located
at the National University of Singapore (1.30° N, 103.77° E, 79 m above mean

sea level) [10]. This atmospheric site was established as part of the cooperative
framework of the Seven South East Asian Studies (7-SEAS) mission created in
2007 [11]. The purpose of this frame-work is to engage the participating countries
on a regional multi-years campaign set to study the regional aerosol, cloud and
radiation environment [12]. Since this site is situated off the southern part of the
Malay Peninsula and north of the Indonesian Archipelago, it is ideally positioned
for monitoring regional pollution events, such as trans-boundary biomass burning
emissions, clouds, local anthropogenic emissions and climate variability [10].
Sun photometer instrument CIMEL Electro-nique CE-318A measures the
intensity of solar radiation at the wavelengths 340, 380, 440, 500, 675, 870, 1,020,
and 1,640 nm and then the results are used to calculate the value of AOD. To
analyse impact of regional smoke haze pollution, necessary data of air quality and
visibility parameter in Singapore are needed. Concentration of PM2,5 is taken
from National Environment Agency of Singapore, while visibility data is obtained
from Meteorological Service Singapore. Hotspot parameter as an indication of
biomass burning was derived from Terra satellite and Aqua MODIS originate
from NASA and were collected by BMKG. While Hysplit Trajectory Model has
been applied to study the dispersion of smoke haze pollution.

Period of this study is focused in June 2013 in Riau Province, since it was the
month with the highest number of hotspots throughout the year and was suspected
causing regional air pollution to neighboring countries.
3.1 Fire Activity

Active fire (i.e. hotspot) detection from remote sensing satellites is based on
the detection of the thermal infrared radiation emitted by fires. This method
is considered as the most suitable and effective way to detect spatio temporal
distribution of fire activity given the large spatial coverage of remote sensing
satellites. In this work, hotspots detection by Terra satellite and Aqua MODIS
originated from NASA and were taken from BMKG’s database. Figure 1 shows
hotspot detected during June 2013. As can be seen from that, detected hotspots
were mostly concentrated at the province of Riau. Based on data collection, there
were a total of about 3,487 hotspots observed from satellites Aqua and Terra
MODIS with most events on June 19, 2013 with the number of fires reaching
1393 locations.
The concurrent plot of hotspot together with the AOD (Figure. 2) shows that
the increase in number of fires followed by a rise in the value of AOD (showed by
Figure 1. June 19th, 2013: Hotspots distribution map over Sumatera observed
by Terra and Aqua MODIS Satellite as collected by BMKG. The color dots
represent the level of confidence.
trend line). As stated by Artaxo, et al., [8] that an increase in the concentration of
fine particulate matter (diameter <2.0 m) during the dry season as a result of the
fire biomass. However, there is time lag in which the AOD has the highest value
on 26 and 30 of June between the peaks generated by hotspot source emission
(mostly on June 19th, 21st, 23rd, and 24th). So it appears that there is an increasing
level of AOD corresponding to decreasing number of fire hotspot counts on the
end of June. This may be explained as follows: firstly, the measurement of AOD
is taken from Singapore site where it is located 300–400 km from Riau. Aerosol
takes relatively short time to be transported to Singapore. Secondly, according to
Salinas [10], unlike surface flaming combustion and peat fires which are considered
to be the main source of fuel for this event, hotspots tend to be persistent and can
smolder underground long after the surface flames have subsided. Furthermore,
smoke can still be released into the atmosphere even though no direct hotspot
can be observed or attributed to it. Thirdly, even if the amount of fire hotspot
counts has truly decreased, the observed AOD peak on day 30rd, might be the
result of the cumulative effects of stagnant (aged smoke) aerosol generated earlier
(days 19th and 21st respectively) with fresh smoke contributions from day 22nd
onwards. In other tropical biomass burning regions (e.g. Amazon, Africa) burning
takes place from various sources and is so intense that fresh and aged smoke are
often found on a combined state [13].
A qualitative way of evaluating the regional trans-boundary smoke transport
patterns is to perform trajectory of smoke haze dispersion using Hysplit Model.
One of BMKG policy that has been operationalized in terms of handling forest
fire smog is doing trajectory modeling dispersion smog. Results of running the
three-dimensional trajectory modeling with Hypslit model showed that the
distribution of fire haze in Riau in June 2013 led to the East and Northeast regions
towards Singapore which is shown on Figure 3. Besides applying Hysplit Trajectory
Model, to strengthen the occurence of regional smoke haze, observation using
satellite is ex-tremely important (Figure 4). As can be seen from the Figure 4 that
AOD observation using MODIS Satellite shows that aerosol emission as a result
of smoke haze from Riau moved to Singapore and Malaysia. Furthermore, on days
23rd June, smoke haze with high level of AOD floated up to the south western
edge of the Philippines. According to Kunii et al., [5] and Heil and Goldammer
[14], the low-level, southern monsoon wind circulation which common during the
main burning season (dry season) produces northward, cross-equatorial transport
of fire emissions from Indonesia, particularly towards Singapore and Malaysia.
3.2 Properties of Aerosol and Air Quality Condition in Singapore

AOD parameter retrieves from Sun-Photometer observation is useful to study
the properties of aerosol. Furthermore, from its derivative, the source of aerosol
could be traced.
As mentioned earlier, the presence of aerosol will impact the air quality
condition. To study impact on ambient air quality in Singapore as a result of
regional smoke haze pollution, concentration of PM2.5 and visibility were also
assessed. The reduction of visibility was widely used as an indicator for ambient
air quality during the smoke-haze episode.
1600 30 Jun 2.5

1400
26 Jun 2.0
1200
AOD (500 nm)

1000 1.5
Hotspot
800
600 1.0
400
0.5
200
0 0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Hotspot AOD (500 nm) Linear (AOD (500 nm))
Figure 2. Hotspot counts (vertical bar) in Riau Province plotted together with AOD (line) taken
from Sun-Photometer measurement at Singapore site for June 2013.
Figure 3. Trajectory smoke haze dispersion by Hysplit Model. Left side is trajectory apply on 19 June, while
the right side is trajectory during whole June 2013.
Correlation among visibility, AOD, and PM 2.5 concentration is given

on Table 1. Aerosol optical depth (AOD) on lower wavelength (340 nm) has
negative and higher correlation with visibility compared than higher wavelength.
The increasing of AOD is followed by decreasing of visibility. Higher correlation
between AOD and visibility is found on 340 nm in which small particles of aerosol
is sensitively detected on lower wavelength. According to Ward [15] and Novakov
et al., [16], vegetation fires produce particle in the fine size range (PM2.5). To
support the finding, correlation between visibility and PM 2.5 are also calculated
and showing negative and high correlation in three locations. Mean concentration
of PM2.5 during June 2013 was 64 µm/m3 with highest concentration on days of
21st. Pollutant Standard Index (PSI) on this day was recorded at 239 point which
remain very unhealthy as an impact of fires occurred in Riau.
Figure 4. Observation of AOD using Aqua MODIS Satellite from 17 June to 25 June 2013.
Colours represent level of AOD.
Tabel 1. Correlation among visibility, AOD parameter, and PM2.5 concentration during
June 2013. Visibility data were measured from three different locations in Singapore. Aerosol
Optical Depth (AOD) in many different wavelength is retrieved from Sun-Photometer located
at National University of Singapore. Average PM2,5 concentration was resulted from measure-
ment in five locations all over Singapore.
AOD (nm) PM 2.5
Visibility
1640 1020 870 675 500 440 380 340 (µm/m3)
Changi 0.06 -0.06 -0.12 -0.23 -0.37 -0.42 -0.45 -0.47 -0.78
Paya lebar 0.04 -0.07 -0.13 -0.25 -0.40 -0.45 -0.48 -0.51 -0.78
Seletar 0.03 -0.09 -0.15 -0.26 -0.41 -0.45 -0.49 -0.51 -0.74
3.3 Policy Responses on Handling and Managing Fires

In order to tackle the recent hotspots and haze occurrence, coordination of various
agencies in Indonesia is needed. This section will discuss the policy responses of
Indonesian agencies particularly BMKG and many efforts which has been taken
to minimize impacts of smoke haze both at the national level and ASEAN region.
At national level, BMKG has important roles in providing services on weather,
climate, and air quality information to support sustainable development. In the
area of smoke haze, BMKG develops and integrates systems to predict fires and
to track them once they start. Related to that, several policies implemented in
BMKG among others are (Figure 5):
1) Fire Danger Rating System (FDRS) developed by JICA, Canada, Ministry
of Forestry and the National Disaster Management Agency (BNPB). BMKG
disseminates FDRS based on real time and 7 days prediction to several agencies
i.e Ministry of Forestry, BNPB, as well as to ASEAN. This information and
then used by related agencies to take action on fire prevention.
2) Climate early warning such as drought early warning system, which is online
and automatic. It is accessible using a smartphone app.
3) Information on extreme weather such as dry spell, wet spell and tropical
cyclone.
4) Smoke haze trajectory dispersion using Hysplit Model.
5) Hotspot detection using Aqua and Terra MODIS satellite.
6) AEROSOL program collaboration with NASA to study aerosol physical
and optical properties. Three observation sites in Jambi, Pontianak, and
Palangkaraya have been established.
7) CATCOS Program (Capacity Building and Twinning for Climate Observing
Systems) for measurement of aerosols in the Global Atmospheric Monitoring
Station Kototabang Hill, West Sumatra.
8) Greenhouse Gas Monitoring Network in 15 regions.
9) Monitoring of PM 10 with Beta Attenuation Monitoring (BAM) in eight
provincial capi-tals of vulnerable forest fires (Pekanbaru, Palangkaraya
Pontianak, Jambi, Palembang, Medan, Balikpapan and Banjarmasin).
At regional level, haze pollution is one of the major and ongoing problems in
ASEAN region and becoming a political issue. All matters related to smoke haze
issue is coordinated by ASEAN through the regional and national haze action
plans and related technical assistance programs in which Indonesian government is
intensively involved. Several instruments have been developed to address the issue,
such as the ASEAN Agreement on Trans-boundary Haze Pollution (AATHP)
which emphasizes on co-operation in its approach. According to Nurhidayah [17],
arguments that emerge regarding why Indonesia has not ratified yet the agreement
because the effectiveness of a treaty or agreement required the participation and
compliance of “targeted state.” Some argue that even if Indonesia ratifies this
Agreement this would not solve haze pollution problem. This is due to complex
problem management of natural resources and environment governance in
Indonesia. In fact, to be effective the Agreement requires capacity at national,
provincial, muni-cipal and village levels to implement the Agreement.
Fire Danger Rating

System (FDRS)
Climate Early
Warning
BMKG
Policies Information on
Extreme Weather
Smoke Haze
Trajectory
Dispersion
Smoke haze &
Hotspot Detection
forest fire
AERONET
Program
CATCOS Program
Green House
gasses Monitoring
Monitoring of
PM10
Figure 5. BMKG Policies on smoke haze and forest fire
However, despite of all political issue, BMKG as a science basis institution

with its role in providing services and information on weather, climate and air
quality has obligation to monitor fires and to analyze it in order to minimize
impact. These could be done through observations, advance technology, and join
analyses in detecting, monitoring and minimizing the risk of the problem with
national and international party in particular ASEAN member countries. Such
monitoring and observation as elaborated in this paper could be an input and
support for decision maker to take further action.
IV. Conclusion
As shown in this paper that during June 2013 biomass burning that occured
in Riau Province caused the trans-boundary haze pollution over neighboring
countries. Measured parameters such as Sun-photometer AOD, in-situ PM 2.5
particulate concentration, and visibility in Singapore were consistent with the

presence of trans-boundary biomass burning smoke. Observation through satellite
also justified the ground based on observation which showed that high value of
AOD increased from the early of June to the end of month and covered Singapore,
Malaysia, even Philippines. Good correlation also was showed between visibility
and AOD measurement in 340 nm as well as with PM 2.5 concentrations. These
conclude the source of fine aerosol come from vegetation fires.
To prevent fire such as development and integration systems and to predict
and monitor fires, several policies have been taken. Further research on fire
dynamics study and air pollution related to smoke haze is needed in order to
minimize the impact.
V. Acknowledgment
Sincere thanks are expressed to those agencies and persons who supported the
author’s work in providing data and valuable inputs, in particular to Prof. Dr.
Edvin Aldrian for the guidance and remarkable support; colleagues from BMKG
named Mizani Ahmad and Gian Gardian; AERONET Program for processing and
archiving the Sun Photometer data; and National Environment Agency Singapore.
VI. R eferences
1) L. Tacconi. (2003). CIFOR Occasional Paper No. 38, Bogor, Indonesia:
Center for International Research. Fires in Indonesia: Causes, Costs and Policy
Implications.
2) I.P. Anderson and M.R. Bowen. ( 2000). Fire Zones and the Threat to the
Wetlands of Sumatra, Indonesia. Forest Fire Prevention and Control Project.
European Union and Ministry of Forestry. [Online]. Available: http://
dephut.net/Halaman/Perlindungan%20Dan%20Konservasi/FFPCP/PDF/
Firezone_and_the_threat_to_the_wetlands_of_Sumatra.PDF
3) J. Miettinen, and S.C., Liew. (2009). Burn-scar patterns and their effect
on regional burnt-area mapping in insular South-east Asia. Int. J. Wildland
Fire, 18, 837–849.
4) Link http://satelit.bmkg.go.id/satelit/image/HOTSPOT/2013/06/).
5) O. Kunii, S. Kanagawa, I. Yajima, Y, Hisamitsu, S. Yamamura, T. Amagai,
and I.T. Ismail. (2002). The 1997 Haze Disaster in Indonesia: Its Air Quality
and Health Effects. Archives of Environment Health, 57, 16–22.
6) R. J. Yokelson, T. Karl, P. Artaxo, D. R. Blake, T. J. Christian, D. W. T.

Griffith, A. Guenther, and W. M. Hao. (2007). The Tropical Forest and
Fire Emissions Experiment: Overview and Airborne Fire Emission Factor
Measurements. Atmospheric Chemistry Physics, 7, 5175–5196.
7) B. N. Holben, D. Tanr, A. Smirnov, T. F. Eck, I. Slutsker, N. Abuhassan,
W. W. Newcomb, J. S. Schafer, B. Chatenet, F. Lavenu, Y. J. Kaufman, J.
Vande Castle, A. Setzer, B. Markham, D. Clark, R. Frouin, R. Halthore,
A. Karneli, N. T. O’Neill, C. Pietras, R. T. Pinker, K. Voss, and G. Zibordi.
(June, 2001). Journal of Geophysical Research. [Online]. 106 (D11), 12067-
12097. Available: http://onlinelibrary.wiley.com/doi/10.1029/2001JD900014/
pdf
8) P. Artaxo, F. Gerab, M.A. Yamasoe, and J. V. Martins. (1994). Fine Mode
Aerosol Composition at Three Long-term Atmos-pheric Monitoring Sites
in the Amazon Basin. Journal of Geophysical Research: Atmospheres, 99,
22857-22868.
9) T. Budiwati, Sumaryanti, and I. Sofiati. (2001). Karakteristik Optik Aerosol
di Bandung. Kontribusi Fisika Indonesia,12 No. 4. Okt.
10) S.V. Salinas, B.B. Chew, J. Miettinen, J.R. Campbell, E.J. Welton, J.S. Reid,
L.E. Yu, and S.C. Liew. (2013). Physical and Optical Charac-teristics of
the October 2010 Haze Event over Singapore: A Photometric and Lidar
Analysis. Journal of Atmospheric Research, 122, 555–570 .
11) B.N. Chew, S.C. Liew, R. Balasubramanian, L.E. Yu, J.S. Reid. (2009). Seven
Southeast Asian Studies (7-SEAS): atmospheric supersite in Singapore, in
30th Asian Conference on Remote Sensing (ACRS 2009). Asian Association
of Remote Sensing. Beijing, China.
12) J.S Reid, et. al. (2013). Observing and under-standing the Southeast Asian
aerosol system by remote sensing: An initial review and analysis for the Seven
Southeast Asian Studies (7SEAS) program. Atmos. Res., 122, 403–468 (this
issue).
13) T.F. Eck, B.N. Holben, J.S. Reid, N.T. O’Neill, J.S. Schafer, O. Dubovik, A.
Smirnov, M.A. Yamasoe, and P. Artaxo. (2003). High aerosol optical depth
biomass burning events: a comparison of optical properties for different
source regions. Geophys. Res. Lett., 30 (20), 2035.
14) A. Heil and J.G. Goldammer. (2001). Smoke-haze pollution: a review of the
1997 episode in Southeast Asia. Regional Environmental Change, 2, 24–37.
15) D.E. Ward. (1990). Factors influencing the emiss-ions of gases and
particulate matter from biomass burning, In: J. G. Goldammer (ed) Fire
in the tropical biota. Ecosystem processes and global challenges. Ecological
Studies, 84, 418–436.
16) T. Novakov, H. Cachier, J.S. Clark, S. Macko, and P. Masclet. (1997).
Characterisation of par-ticle products of biomass burning combust-ion. In:
J.S. Clark, H. Cachier, J.G. Goldam-mer, B. Stocks (eds) Sediment records
of biomass burning and global change. NATO ASI Series No. 1, Global
En-vironmental Change, 51, 117–144.
17) L. Nurhidayah. (2013). Legislation, regulations, and policies in Indonesia
relevant to addressing land/forest fires and transboundary haze pollution: A
critical evaluation. Asia Pacific Journal of Environmental Law, [online]. Vol.
16, pp 15-239. Available: http://search.informit.com.au/documentSumma
ry;dn=779978732417466;res=IELHSS
The Potential Impact of Carbon Monoxide
Emission to The Community Health In The
Vicinity of Baranangsiang Toll Gates
Yudith Vega Paramitadevia,*, Arief Sabdo Yuwonob, Meiske Widyartib
Diploma Program, Bogor Agricultural University
a
Jl. Kumbang No. 14, Bogor, Indonesia

b
Department of Civil and Environmental Engineering, Bogor Agricultural University
PO BOX 220, Bogor, Indonesia
Abstract
Over the past decade, emission from carbon monoxide (CO) has risen due to the increase of
vehicles per year. Bogor as a weekend tourist city has a heavy burden in terms of the volume of
motor vehicles. Object in this study is Baranangsiang Bogor toll gateway where queue of motor
vehicles often happens and allegedly produces CO pollutants.
This study was conducted to simulate the CO concentration by the method of Finite Length
Line Source (FLLS) around Baranangsiang Bogor toll gateway and recapitulate types of diseases
associated with CO impacts on communities around the toll in line with the pattern of 6%
increase of vehicle volume per year.
Based on the results of measurements and simulations conducted in four sampling points
within 20 m and 190 m from the sources of pollution on 29 August to 1 September 2013 , the
concentration of CO is still within the range of quality standards in accordance with Regulation
No. 41 Year 1999 which is 634–9,189 μg/Nm3. Dispersion of pollutants CO dominantly heading
Eastwards with the wind speed measurements 1.5–5.2 m/s and atmospheric stability class B.
Kampung Sawah RT 02 RW 07 is higher exposed with CO. Recapitulation of medical records
showed that suspected CO intoxication disease cases are more common in Kampung Sawah than
IPB Baranangsiang settlements.
Key words: Carbon Monoxide, CO Intoxication, Dispersion CO, Toll Gates Baranangsiang
I. Introduction
Carbon monoxide emitted by various vehicles to the air is one of air pollution
causes. The dispersion of pollutants in the air affects the regional air quality
that adversely impacts the public health [1][5]. Nowadays, in most countries,
air pollution caused by the vehicular exhaust emission has increased due to the
increasing amount of vehicle per year as a result of the increasing of transportation
consumer needs [13,16,20].
* Corresponding Author.Tel: +6285-717-99-555-6. E-mail: vega_paramitadevy@yahoo.com
289
Bogor, a tourist destination city on weekend, has quite heavy burden in terms
of vehicle volume. Some places with high CO concentrations in Bogor are the toll
gates, intersections and main roads [17]. The object in this study is Baranangsiang
Bogor toll gates. At the gates, heavy traffic often occurs and allegedly produces a
lot of CO gas. CO gas increasing occurs when motor vehicle running at low speed
due to braking frequency [1,15]. Based on [6] finding at Surabaya entrance toll,
it has been stated that the highest concentration of CO was 7,845 μg/Nm3 on
weekdays while on holidays was 8,708 μg/Nm3. For concentrations higher than
11,700 μg/Nm3 during 10 hours of exposure, CO may cause negative impacts
on human health such as chest pain, chronic lung disease, flu-like symptoms
and headache [4].
Based on the problems mentioned before, a more in-depth formulation
regarding CO pollutant dispersion in the vicinity of Baranangsiang Bogor toll gates
is needed. Thus, simulation of CO pollutant dispersion is required. In this study,
Finite Length Line Source (FLLS) mathematical model was used. It is a derivative
of Line Source Gaussian model with Visual Basic programming for calculating
CO concentration. Summary of medical record disease from CO intoxication
was also carried out to determine the potential impact of CO pollution.

A. Time and Location of CO Pollutant Measurements
Data collection and measurement of CO sample was carried out in four points in
the vicinity of Baranangsiang Bogor toll gates (managed by PT. Jasa Marga) for
a week from August 26 to September 1, 2013. Two points within ±20 m apart
perpendicular to highway axis point, located at either side of the roadside. Next
two points within ±190 m perpendicular to highway, were located in KPP IPB
Baranangsiang 4 and Kampung Sawah settlement RT 02 RW 07. CO sampling
was conducted on weekdays and weekends, four (4) times a day for a week. The
duration of each measurement was one hour.
The criteria for determining the location of ambient air quality samples refer
to National Standard, namely SNI No. 19-7119.6-2005 on The Determination
of Test Sample Location for Controlling of Ambient Atmosphere Quality. Figure
1 shows the location of pollutant CO measurement. CO sample analysis was
carried out in Laboratory of Aquaculture IPB in September–October 2013.
B. Materials
Materials used in this research consist of primary and secondary data.
1). Primary data, such as:
a) CO pollutant concentration based on direct field measurement.
b) Meteorological data at the measurement time such as temperature, wind
direction and speed, relative humidity.
c) Coordinate of measurement points and toll axis point.
2) Secondary data, such as:

a) Numbers of vehicle passing through toll gates for three years (2010-2012),
obtained from PT Jasa Marga, Jagorawi Branch.
b) Meteorological data from Meteorology Station Class I Dramaga and
Meteorology Station Class III Citeko, i.e. temperature, relative humidity,
wind direction and speed, and radiation intensity from 2008-2012.
Figure 1 Location of Pollutant CO Measurement
C. Tools
Tools used in this study consist of data processing devices, in-field measurement
devices, and laboratory equipment for analysis. The devices are listed below:
1) Data processing devices, such as Windows-based CPU. Software used in this
process are:
a) Visual Basic 6.0 for simulation.
b) Global Mapper 11.0 for sampling points plotting.
c) Surfer 8.0 for visualizing of CO dispersion.
2) Measurement devices in field covered impinger, thermometer, anemometer,

spectrophotometer, tripod, manual counter, GPS, and portable CO meter.
3) Analysis equipment in laboratory including flasks, beakers, pipettes, and test
tubes. Chemical solution KI was needed to absorb CO and Iodine Standard
as primary solution.
D. Methodology
This study consist of five (5) phases, i.e. CO concentration measurements, CO
concentration analysis in laboratory, forming Line Source Gaussian dispersion
modeling using Finite Length Line Source (FLLS) method, model visualization
of CO pollutant dispersion pattern and recapitulation of medical records at
three public health centers near sampling points. The laboratory analysis of CO
concentration refered to SNI No. 19-4848-1996 on Pentoxide Method.
The calculation of emission load refered to [14] with the speed considered
varied from 0–60 km/hour and the duration of vehicles maintenance in India
and Indonesia was relatively similar between 10 and 15 years.
FLLS method determined the concentration of gaseous pollutant including
its dispersion by dividing the segments of line source into its smallest segments.
Having gained the smallest segments, then the distance between receptor and
lined source was calculated in order to determine the dispersion parameter of
each receptor [3,7,12]. FLLS equation according to [9] is listed below:
where :
(1)
Description :
Q : Pollutant source rate. At the finite point source, unit g/sec is used. At
the finite line source, unit g.m/sec is used.
ū : Wind speed at the-x (m/sec).
σz : Concentration dispersion parameter at the-z (m).
z : Position of Z in Cartesian coordinate (in this study, z=0).
H : Effective height ofemission source (in this study, H=0).
B : The rate of road length to dispersion parameter ().
The calculation was performed on model using equation (1) including emis-
sion load to obtain Q, wind speed to obtain, road length and receptor location
to obtain σy and σz as well as B1 and B2. Data visualization phase using Surfer
software in the form of coordinate data, thus pollutant dispersion patterns could
be observed based on isopleth from modeling. Recommendations were given if
the resident medical records showed a positive correlation with the isopleth map.

A. Traffic Density Condition In the Vicinity of Baranangsiang Bogor Toll
Gates
Baranangsiang Bogor toll gates are one of the Jagorawi toll gates managed by PT.
Jasa Marga. There are 9 toll booths, which 4 booths are for ticketing locations
(Entrance), other 4 booths are for payment points (Exit), and another one is a
stand-by booth which can be operated as entrance or exit booth. The total volume
of vehicle entering and exiting the toll gates in Bogor reaches 9 to 10 million units
per year [11]. The average number of vehicle passing by a toll gate is 270 units per
hour. On weekends, the average numbers of vehicle passing by Baranangsiang toll
gates can reach 70,000 units/day. On the other hand, on weekdays the average
is about 55,000 units. Traffic density that often occurs on holidays is caused by
family travel activity with Bogor as the main destination.
According to data from [11] during 2010-2012, the most dominant type
of vehicle passing by the gates is private vehicles. They were diesel-driven and
petrol-driven cars with two axles or Class I vehicle which cover about 97% of
total numbers of vehicle. The second most dominant vehicle found was Class II
vehicle such as diesel-driven small trucks and buses with two axles (3.05%). And
the remaining 0.17% was Class III vehicle while Class IV and V were 0.03%
and 0.02%, respectively.
B. Simulation of Modeling Results

In this study, software was developed using Visual Basic 6.0 program in order
to obtain accuracy and simplicity for calculating CO concentrations model.
Simulations for CO were carried out in July–September using Surfer 8.0 program
which is illustrated in Figure 2 (a-c). Meteorological outputs and traffic conditions
data used in the simulation are the average of five-year data in that specified
month, with the average wind speed is 1.31 m/sec, the average wind direction
is to the East at about 296°, atmospheric stability Class B, the average numbers
of vehicle is 2559 units/day.
The highest concentration of CO pollutant dispersion was 4,000 μg/Nm3 at 20
m at right roadside. According to the FLLS formulation, wind distribution factors
(a)
(b)
(c)
Figure 2 Simulation of CO in (a) July 2013; (b) August 2013 and (c)
September 2013
were influenced the dispersion of pollutants. The smaller the angle perpendicular
to the road, relative speed to the road was small, too. Consequently, the multiplier
factor (K) decreases and the concentration of CO model become higher than CO
observed. Pollutant was dispersed to the East direction towards Kampung Sawah.
C. Medical Record Analysis of Community in The Vicinity of Baranang-

siang Toll Gates
Based on the simulation results, the dispersion of pollutant CO dominantly moved
toward the East. The residential population point exposed to higher CO was
Kampung Sawah RT 02 RW 07. The highest concentration of CO in July 2013
was 1,200 mg/Nm3. At another point, namely IPB Baranangsiang 4 Settlement,
the highest concentration of CO in the same month was 1,150 mg/ Nm3.
The number of vehicles in the Greater Jakarta increased by nearly 6% per year
[19], indicating concentration of pollutants CO increase, too. Thus, although
the concentration of the simulation results was still below the quality standard
of 24 hours i.e 10,000 mg/Nm3, the analysis of medical records of residents was
still being done.
Urban communities have HbCO levels in the blood of about 2–6% [8,10],
symptoms of CO intoxication will be felt receptors after HbCO levels in the
blood reaches 10% [2]. CO intoxication symptoms similar to the flu and diseases
associated with respiratory infections, difficulty breathing (relationship to the
lungs, hereinafter referred to exacerbation disease of chronic obstructive lung)
and chest pain (relationship to disease in the vessels of the heart/ cardio vascular)
[4,18]. This study has used the data of upper respiratory illness (URI), exacerbation
of chronic obstructive pulmonary (COPD) illness i.e emphysema, asthma and
chronic bronchitis also vascular system diseases such as ischemic cardiac disease.
Medical records obtained from three public health centers in the city of Bogor
i.e the North Bogor Health Center near the IPB Baranangsiang 4 Settlement,
the East Bogor Health Center near Kampung Sawah Housing RT 02 RW 07
and the Pulo Armyn Health Center that is located in Pulo Armyn Padjadjaran
Street. Figure 3 shows the summary results of the medical records at the three
health centers in diseases associated with CO intoxication.
Medical record refers to the data in Figure 3 was obtained from the number
of occurrences of the disease in the population who live in the catchment area of
each health center. The number of events due to the isemic cardiac diseases was
low, especially in the area of IPB Baranangsiang 4 Settlement. A high number of
patients with isemic cardiac diseases were biggest in Kampung Sawah. The same
thing was found in URI and COPD.
Figure 3 Medical Report of CO Intoxication Disease Event from January 2012 to March 2013
Dispersion of pollutants CO moves toward to the East, but the conclusion

that the prevalence of respiratory disease, lung disease and cardiovascular in
Kampung Sawah is higher than in the Baranangsiang 4 Settlement should be
studied further. This is due to the HbCO analysis was not performed on receptors
in this region. According to the research results of [8], Indonesia rarely carried
out an examination of HbCO in patients. Consequently, patients were exposed
to pollutants CO has come in the acute situation when taken to the nearest
health center.
IV. Conclusion
The simulation results in July–September 2013, with the highest CO concentra-
tion of 4,000 mg/Nm3 in the right roadside. The simulations also indicate the
dispersion of pollutants CO eastward namely Kampung Sawah.
Summary medical record indicates a high number of patients with the
incidence of isemic cardiac disease, URI and COPD respectively 8, 858 and 424
events during the period from January to March 2013 in the village of Kampung
Sawah Baranangsiang.
The relationship between the high incidence of the disease in Kampung Sawah
and the dispersion of pollutants CO needs to be studied further.
V. Acknowledgment
Authors appreciate to Program Diploma IPB for the funding support of the
research in PT Jasa Marga (Persero) Jagorawi Branch.
VI. R eferences
1) Abbasloo A., Kaljahi, J. F., and F. Esmaeilzadeh. (2012). Prediction of three
dimensional CO concentration distribution in Zand tunnel of Shiraz using
Computational Fluid Dynamic (CFD) method. J. Appl. Environ. Biol. Sci.,
1,67–76.
2) Adir Y, Merdler, A., Haim, S.B., Front, A., Harduf, R., Bitterman, H. (2009).
Effects of exposure to low concentrations carbon monoxide on exercise
performance and myocardial perfusion in young healthy men. Occup Environ
Med, 56, 535–538.
3) Batterman S.A., Zhang, K., and R. Kanonowech. (2010)Predictions and
analysis of near road concentration using a dispersion model. Environmental
Health, 29, 1–18.
4) Clarke S., Shian, C., and C.V. Murray. (2012). Screening for carbon monoxide
exposure in selected patient groups attending rural and urban emergency
departments in England: a prospective observational study. British Medical
Journal Open, 2, 877–887.
5) Emad A. A., Sayed, M. E. S., and K. O. Kaseem. (2010). Computer
Simulation For Dispersion Of Air Pollution Released From A Line Source
According To Gaussian Model. In the Proceedings of the 4th Environmental
Physics Conference,, 63–70.
6) Endrayana P. L. E., and B. Widodo. (2010). Simulasi model dispersi polutan
karbon monoksida di pintu tol. In the Proceedings of the Seminar Nasional
Penelitian FMIPA UNY, 1–9.
7) Goyal P. and R. Khrisna. (2010). A line source model for Delhi. Transportation
Research Part D, 4, 241–249.
8) Handa P. K. and D. Y. H. Tai. Carbon monoxide poisoning: a five year review
at Tan Tock Seng Hospital Singapore. Ann Acad Med Singapore, 34, 611–614.
9) Hassan H., Singh, M.P., Gribben, R.J., Srivastava, R.M., Radojevic, M., and
A. Latif. (2006). Application of Line Source Air Quality Model to The Study
of Traffic Carbon Monoxide in Brunei Darussalam. ASEAN J. on Science and
Technology for Development, 17, 59–76.
10) Homer C. D., Engelhart, D. A., Lavins, E. S., Jenkins, and Amanda. (2005).
Carbon monoxide-related deaths in a metropolitan county in the USA: an
11-year study. J. Forensic Science International, 149, 159–165.
11) Jasa Marga. 2008-2012. Data Rekapan Volume Lalu Lintas Tol Jagorawi. PT.
Jasa Marga Cabang Jagorawi,.
12) Lin J. and Y.E. Ge. (2006). Impacts of traffic heterogeneity on roadside air
pollution concentration. Transportation Research D, 11, 166–170.
13) Mayer H.. (2009). Adaptive neuro-fuzzy modeling for prediction of ambient
CO concentration at urban intersections and roadways. Atmospheric Environ-
ment, 33,. 4029–4037.
14) Mittal M.L. and P. Sharma (2006). Anthropogenic emission from energy activi-
ties in India: Generation and source characterization Emission from vehicular
transport part II, 3-4, 15–22. USAID.
15) Mukherjee P. and S. Visvanathan. (2006). Carbon monoxide modeling from
transportation sources. Chemosphere, 45, 1071–1083.
16) Nagendra S.M.S. and M. Khare. (2012). Artificial Neural Networks in
Vehicular Pollution Modelling. Transportation Research D, 9, 163–173.
17) Pasha, A.. (2011). Simulasi Dispersi Gas Karbon Monoksida (CO) Dalam
Gardu Tol Menggunakan Computational Fluid Dynamics (CFD) Studi Kasus:
Gerbang Tol Bogor. B.Sc. thesis. Bogor Agricultural Institute, Bogor.
18) Prockop L. D. and R. I. Chichkova. (2007). Carbon monoxide intoxication:
an updated review”, Journal of the Neurological Sciences, 262, 122–130.
19) Rosdiana H.. (2009). Menggagas model proyeksi penerimaan PKB dan
BBNKB. Jurnal Ilmu Administrasi dan Organisasi, 16, 147–159.
20) Sharma P. and M. Khare. (2011). Line Source Emission Modeling. Transporta-
tion Research D, 6, 179–198.
The Mechanism of Dry Mid-Atmosphere in The
Western Maritime Continent during Rainy
Season in 2014
Supari a,*, A.M. Setiawan a, E. Makmur b, A. Sopaheluwakan a, Siswanto c,

W. Sulistya a
a
Center for Climate, Agro and Marine Climate - Agency for Meteorology Climatology and
Geophysics (BMKG)
b
Center for Research and Development - Agency for Meteorology Climatology and
Geophysics (BMKG)
c
Center for Aviation and Marine Meteorology - Agency for Meteorology Climatology and
Geophysics (BMKG)
Jl. Angkasa I no 2 Kemayoran, Jakarta, Indonesia
Abstract
In the late January to mid-March 2014, which is normally peak of rainy season, massive forest
fire cases were monitored over North-Western part of Maritime Continent resulting serious
environmental impact such as a huge pall of smoke, poor air quality and flight cancellation.
This paper is aimed to investigate the atmospheric setting of January-March 2014 based on site
observations and global re-analysis data which were associated to the fire cases. It was observed
that rainfall decreased up to 30 % than its long-term average. The evolution of vertical profile of
atmosphere based on sounding data from Medan (96035), Ranai (96147) and Pangkalpinang
(96237) show a dramatic drop of humidity in the mid-atmosphere (700–500 mb) starting from
the third week of January. The synoptic features of low-level atmosphere exhibit an intensified
northeasterly winds and a propagation of cold air over the South China Sea suggesting an intrusion
of dry sub-tropical air. The diagnosis of meridional wind component reveals that over the region,
the circulation was dominated by sinking air that suppressed the convection process, indicating
anomaly of Hadley cell. The negative phase of Madden-Julian Oscillation (MJO) is assumed to
play another role where the wide suppressed convection area was observed based on outgoing long-
wave radiation (OLR) data. These findings suggest that monitoring mid-atmosphere condition
is relevant for smoke trajectory forecast and fire danger early warning.
Key words: Forest fire, Maritime continent, Dry atmosphere.
I. Introduction
The forest fire case that occurred in the North-western Maritime Continent
during late January to mid-March 2014 was very unusual in term of timing
since it occurred during peak of rainy season. Although forest fire is almost yearly
hazard over the region, it usually occurs during dry season, mostly on peat lands
* Corresponding Author.Tel: +62-81315689645. E-mail: supari@bmkg.go.id
299
Figure 1. Top - Observed hotspot data from MODIS Terra-Aqua satellite during January – March
2014. Only data with at least 80 % confidence level are plotted. Gray area denotes Riau Province.
Bottom – temporal evolution of hotspot number over Riau Province, the most affected area due
to smoke-haze pollution during the event.
and peat-swamp forest [1]. Forest fire cases in Indonesia are generally caused by
illegal human activity, including land clearing and accidental fires. Nevertheless,
the massive forest fire case will only occur when atmosphere as contributing
factor is dry enough [2]. In fact, during January–March 2014 which should be
wet months, thousands of hotspots data were monitored over almost the entire
of Sumatera and Borneo Islands as displayed in Figure 1.
Time series of hotspots number over Riau Province shows that massive hotspot
data were monitored started from mid-February till mid-March. On 11 March,
it was detected 1,149 hotspots over the province. The impact was not surprising
then, where a huge pall of smoke leading poor air quality strikes the area. Besides
causing serious health problem, the smoke also caused flight cancellation due to
minimum visibility. The economic loss is expected around USD 1 billion.
Climate over the region is mainly controlled by monsoon systems so that strong
annual rainfall variability is a key feature with wet season during October–March
and dry season during April–September. However, the semi-annual variability is
also notable characteristic over there [3] due to local response to the southward
and northward movement of the inter-tropical convergence zone (ITCZ). Dry
season is period when forest fire risk will increase and become much more risky
during ENSO year [4].
Forest fires in Indonesia have been investigated for long time both focusing
on the impact [1], [5], [6] and also focusing on the causes of fires [7]. Different
with previous studies, the analysis of climatic condition related to fire case is the
core of the present work. The aim of this study is therefore to gain insight on the
mechanisms that triggered dry condition which is favorable then for forest fire
cases, by using remote sensing data, in-situ meteorological observations and global
re-analysis data as well. The output of recent case studies, like the one presented in
this paper, is expected to be a crucial input in term of issuing warnings. It should
be noted that forest fire is truly potential treat for sustainable development due
to direct impact on environment including carbon emission [4].
II. M aterial
Data used in this study were collected from many sources. Modis/Tera-Aqua
hotspot data [8] was produced by the University of Maryland and provided by
NASA FIRMS operated by NASA/GSFC/ESDIS with funding provided by
NASA/HQ. It is available on-line (free) https://earthdata.nasa.gov/active-fire-
data#tab-content-6.
Rainfall observation and upper air sounding data were gathered from BMKG.
Relative humidity data at pressure levels were obtained from ECMWF-ERA
Interim Re-analysis [9]. Vertical velocity parameter was obtained from Japan
Re-analysis provided by Japan Met Administration (JMA) [10]. The outgoing

long-wave radiation (OLR) data were obtained from the National Oceanic
Atmospheric Agency (NOAA) Interpolated OLR [11]. OLR data characterized
the strength of longwave radiation from the earth’s surface (under clear sky
conditions) or from the top of clouds (under cloudy conditions). Lower values
of OLR indicated more enhanced convective activity under cloudy conditions.
Picture displaying OLR data is provided by Berau of Meteorology, Australia.

A. Rainfall Observation
The spatial distributions of accumulated rainfall at 10-days scale indicate that
starting in the mid of January, the Northwestern Maritime Continent consistently
received very low rainfall. In the late of January, Riau, Jambi, Bangka Belitung
Province that are located in the eastern part of Sumatra got only less than 10 mm
per 10-days, very much below normal. This condition continued till the second
week of March resulting in very dry surface condition (Figure 2).
With dominant strong northerly monsoon wind, January and March should
be very wet over those regions by receiving rainfall at least 200 mm per month.
February indeed is the driest month among other wet months (DJFM), especially
for eastern part of Sumatra Island due to weakening of monsoon. However, the
normal value of monthly total is at least 100 mm. This February, therefore, was
extremely dry because monthly total rainfall was only 28 mm for Medan Station
(North Sumatera Province), 14 mm for Pekanbaru Station (Riau Province),
40.0 mm for Ranai (Riau Island province), 26.0 mm for Jambi Station (Jambi
Province) and 47.8 mm for Pangkalpinang Station (Bangka Belitung Province).
B. Moisture Content
The upper air sounding data (not shown) reveals notable difference condition of
relative humidity related to the event. It was observed from met station of Medan
(WMO No. 96035), Ranai (WMO No. 96147) and Pangkalpinang (WMO
No. 96237) that the humidity of atmosphere decreased dramatically. Although
there was no significant feature at the surface, we found interesting feature at
mid-atmosphere level.
At the 700 mb pressure level, relative humidity was relatively high around
80 % during first and second week of January. However, it dropped significantly
up to 30 % starting from third week of January. The similar pattern was also
found at 500 mb. In Ranai, the reduction of humidity even could be recognized
at lower level, 850 mb.
Figure 2. Observed monthly rainfall for the first 10-days of February (top), the second 10-days
(midlle) and the last 10-days (bottom).
Figure 3. Spatial distribution of relative humidity (%) at 700 mb for the first 10-days of
January (top), the second 10-days (midlle) and the last 10-days (bottom).
Figure 4. High – Latitude plot of vertical velocity averaged over 100 – 110 E, describ-
ing configuration of Hadley circulation for the first 10-days of January (top-left), the
second 10-days (top-right), the last 10-days (bottom-left) and first 10-days of February
(bottom-right). Updraft (downdraft) fraction relates to supporting (blocking) condi-
tion for convective activity.
Spatial map of relative humidity over Southeast Asia confirms that the low
humidity actually extended southward from great Asia Continent (Figure 4).
At the 700 mb, relative humidity in the first 10-days of January (1–10 January)
was generally categorized by dry air (less than 40 %) located in the 15 N and
higher latitude. In the last 10-days (21–31 January), contour of relative humidity
of 40 % propagated southward and went up to 5 N. Over South China Sea, it
even reached equator line. At the low-level atmosphere we found an intensified
northeasterly wind particularly over South China Sea and a propagation of cold
air (not shown). This anomalous appearance triggers a hypothesis of intrusion
of dry sub-tropical air mass.
Linked to the observed rainfall data, the dry condition at mid-atmosphere
describes clearly that there was not enough moisture content in the atmosphere
to support low cloud development that was expected to generate rainfall.
C. Hadley Circulation
It is interesting to track the responsible condition for minimum convective activity
as found in the previous section. Since the relative humidity at mid-atmosphere
displays southward propagation of dry air mass, we investigate then the detail
north-south atmosphere circulation i.e. Hadley cell. It is well known that tropical
area is the region in which the up draft phase of Hadley cell is dominant. This
common state gives favorable environment for convective activity to enhance
deeply. During the event, we observed surprisingly the anomalous configuration
of Hadley cell (Figure 4).
In contrast with condition of the first 10-days of January where the up draft
phase of Hadley cell dominated region between 13 S–10 N, on the second
10-days of January, the up draft phase was only observed over 10 S–0/Equator
line. The down draft phase, on the other hand dominated strangely over wide
area, from equator line to 40 N. The major circulation over the studied area was
consequently a sinking air mass that damped convective activity.
D. Role of MJO
Such background favourable conditions related to intra-seasonal phenomena like
MJO (Madden-Julian Oscillation) may also contribute for drying atmosphere.
Despite spatially large cloud system during the active phase of MJO could increase
intense precipitation in the Maritime Continent through long-lived convection
[12], [13], the negative phase of MJO, on the other hand will bring less rainfall
and drier condition [14].
OLR data observation shows that the active phase of MJO, which is character-
ized by large area of enhanced convection, developed over the Indian Ocean during
Figure 5. Reconstructed OLR field over tropical area from August 2013 – March 2014. Source
: BoM.
late November as shown in Figure 5. It propagated eastward slowly then to the

West Pacific Ocean by early January. Over Indonesia, negative anomalies of OLR
were observed during late January and mid-February. Afterward, strong positive
OLR anomalies followed as a signal of passive phase of MJO. This suppressed
convection was still monitored till late March and is expected being responsible
as another factor for drying atmosphere over the studied region. The interaction
between Hadley cell anomaly with this passive phase of MJO blocked convective
activity and therefore atmosphere with poor moisture dominate the region.
IV. Conclusion
The January–March forest fire over nort-western Maritime Continent is considered
as unusual event since it occurred during peak of rainy season. This anomalous
forest fire case associated with dry condition over the region which was character-
ized by very much below normal rainfall condition. The analysis state that this
atmospheric setting was triggered by strange configuration of Hadley cell. The
sinking air dominated over region wich disable convective activity. However the
mechanism causing this circulation anomaly is still not clear. The passive phase
of MJO that associates with suppressed convection might also play another role
in conditioning dryier atmosphere.
VI. R eferences
1) Harrison, M. E., Page, S. E., and S. H. Limin. (2009). The global impact of
Indonesian forest fires. Biologist, 56, 156–163.
2) South Asia Association of Regional Cooperation (SAARC) - Disaster Manage-
ment Centre (SDMC), Fire Disasters,” India. [Online]. Available: http://
saarc-sdmc.nic.in/pdf/fire.pdf
3) Aldrian E., and R. D. Susanto. (2003). Identification of three dominant
rainfall regions within indonesia and their relationship to sea surface
temperature. Int. J. Climatol, 23, 1435–1452.
4) Tacconi, L. (2003). Fires in Indonesia: Causes, Costs and Policy Implications.
CIFOR, Indonesia, CIFOR Ocassional Paper No. 28.
5) Glover, D., and T Jessup. (2006). Indonesia’s Fires and Haze: The Cost of
Catastrophe, Singapore. Institute of Southeast Asian Studies, 1–21.
6) Heil, A. (2000). The 1997–1998 Air Pollution Episode in Southeast Asia
Generated by Vegetation Fires in Indonesia. IFFN, No. 23, 68–71.
7) Dennis, R., Huffman, A., Apllegate, G., von Gemmingen, G., and K.
Kartawinata. (2001). Large-scale fire: creator and destroyer of secondary
forests in western Indonesia. Journal of Tropical Forest Science, 13, 786–799.
8) Kaufman, Y. J., Ichoku, C., Giglio, L., Korontzi, S., Chu, D. A., Hao, W. M.,
Li, R.-R., and C. O. Justice. (2003). Fire and smoke observed from the earth
observing system MODIS instrument - products, validation, and operational
use. Int. J. Rem. Sens., 24, 1765–1781.
9) Dee, D. P., Uppala, S. M., Simmons, A. J., Berrisford, P., Poli, P. et al.
(2011). The ERA-Interim reanalysis: configuration and performance of the
data assimilation system. Q. J. R. Meteorol. Soc., 137, 553–597
10) Ebita, A., Kobayashi, S., Ota, Y., M. Moriya, Kumabe, R., Onogi, K., Harada,
Y., Yasui, S., Miyaoka, K., Takahashi, K., Kamahori, H., Kobayashi, C., Endo,
H., Soma, M., Oikawa, Y., and T. Ishimizu. (2011). The Japanese 55-year
Reanalysis “JRA-55”: an interim report. SOLA, 7, 149–152.
11) Liebmann, B., and C. A. Smith. (1996). Description of a complete (inter-
polated) outgoing longwave radiation dataset. Bull. Am. Meteorol. Soc., 77,
1275–1277.
12) Hyun Oh, J., Kim, K.Y., G. H. Lim.(2012). Impact of MJO on the diurnal
cycle of rainfall over the western Maritime Continent in the austral summer.
Clim. Dyn., 35, 1167 – 1180.
13) Wu, P., Arbain, A. A., Mori, S., Hamada, J., Hattori, M., Syamsuddin, F., and
M. D. Yamanaka. (2013). The effect of an active phase of the Madden-Julian
Oscillation on the extreme precipitation event over western Java Island in
January 2013. SOLA, 9, 79 – 83.
14) Barlow, M., and D. Salstein. (2006). Summertime influence of Madden-Julian
Oscillation on daily rainfall over Mexico and Central America. Geophys. Res.
Lett., 33, L21708.
309
Design of Automatic Measurement
Instrument for Water Discharge On Drainage
Monitoring System
Retno Tri Wahyuni*

Electronic Departement, Polytechnic of Caltex, Riau
Jl. Umban Sari No. 1, Pekanbaru, Riau, Indonesia
retnotri@pcr.ac.id
Abstract
The drainage system is one of infrastructures that is developed to prevent local flooding in urban
area. The urban drainage management system should be implemented overall. One of drainage
management component is monitoring. The monitoring system integrates meteorological data
in form of rainfall data, and also data water level, and water discharge. The water discharge is
monitored to observe whether drainage can handle the water flow. The water discharge is also a
variable that is considered in design of drainage. Design of automatic measurement instrument
of water discharge consists of propeller, magnetic sensor, water level sensor, and micro controller.
Water discharge of drainage is obtained by multiplying the velocity of water flow (v) by wet cross
section area (A). Wet cross section area is calculated using hydraulic formula depends on the type
of drainage. The magnetic sensor changes the propeller rotation to electric signal for calculating
velocity of water flow. The water level measured by an ultrasonic sensor.
Key words: Water discharge, Automatic, Drainage
I. Introduction
Flood is one of disasters that have occurred in almost all regions in Indonesia.
Based on data from Badan Nasional Penanggulangan Bencana (BNPB) since
1815 to 2014 flood has the largest percentage compared to other types of disasters
that is equal to 38% [1]. One type of flooding is puddles flood. It is caused by
drainage that can’t dispose a lot of water coused by very high rainfall. The solution
is to build adequate drainage. In general, the management of drainage system
in many urban in Indonesia is still partial, so it doesn’t resolve the problem of
puddles flooding compeletelly. The urban drainage management system should
be implemented overall, starting from the stage of survey, investigation planning,
land acquisition, construction, operation and maintenance, institutional support,
financing, community participation, evaluation and monitoring [2]. If those
components of urban drainage management doesn’t work well, the performance
* E-mail: retnotri@pcr.ac.id
311
Figure 1. Block Diagram System
of drainage can’t work optimally. In fact, one of weakness in urban drainage

management is monitoring. The monitoring method is done manually, so it has
human error and poor data management system, therefore the automatic drainage
monitoring system should be implemented.
In drainage monitoring system, one of variables monitored is water discharge.
The water discharge is monitored to observe if drainage can handle the water
flow. The water discharge is also a variable considered in designs of drainage.
Some methods are implemented to measure water discharge of drainage, one of
them is to measure the velocity of water in drainage using current meter. In this
method, people measure velocity of water using current meter in some points
based on procedure of measuring. The procedures consist of laying the propeller at
a certain depth, cross-sectional area measurements, speed propeller measurement,
recording and calculating the data record.
In this research, process of water discharge measurement will be implemented
on integration system that consists of sensor, tranducer and controller so the
process done automatically. The automatic measurement instrument for drainage
can be implemented on automatic drainage monitoring system so it can help on
drainage management system.
II. Method
The design of automatic measurement instrument for water discharge of drainage
consists of sensor, tranducer, and controller. The sensor will sense the velocity of
water flow using propeller. The propeller rotation will be changed to electrical
signal by magnetic sensor as tranduser. The electric signal will be inputted to
microcontroller and processed based on algorithm calculation of water discharge
of drainage. Another input for calculating water dischare of drainage is level of
water and widht of drainage. The water level will sense using ultrasonic level
sensor and the widht of drainage will inisiate manually using keypad. Figure 1
describes block diagram of the system.
The flow chart of system that implemented

in micro controller described in Figure 2. First
process in the algorithm is to chose the type
of drainage. There are 3 types of drainage i.e:
rectangle, trapezoidal and circle. After choos-
ing the type, input the variables needed for
calculating the wet cross sectional area that is
different for every type of drainage, ex: width of
drainage for rectangle, gradient for trapezoidal
type, diameter for circle type etc. The next
process is water level measurement by level
sensor and velocity of water flow measurement.
The variable and result of water level sensor will
be used for calculating wet cross sectional area.
Finally, the last result of velocity of water flow
measurement and result of process calculation
for wet cross sectional area will be used for
calculating water discharge of drainage.

A. Metereological Aspect on Drainage
Monitoring System
Irrigation and drainage is one of the purposes in
water management. Based on technical report
“Climate and Meteorological Information
Requerements for Water Management”, the
comment for data requirement for irrigation
and drainage about spatial density must
represent demand and supply area variations,
measurement interval daily for design manage-
ment and operation, real time monitoring
needed in multipurpose systems.
In fact, the climate information present
is not widely used by water management [3].
In this research the metereological will be
integrated with hidrological data for monitoring
condition of drainage. For example, Figure 3
describes rainfall intensity from weather stations
Figure 2. Flowchart of system
of Pekanbaru. This data should be used for
reference to design drainage in Pekanbaru. The
Figure 3. Intencity Rainfall on Pekanbaru Weather Station

(Source: Pekanbaru Weather Station, BMKG)
data from rainfall sensor, water level sensor and water discharge of drainage will
be combined to describing the drainage condition.
The specific discuss in this paper only about automatic instrument for
measuring of water discharge on drainage. The complete discussion about drainage
monitoring system describe in [4].
B. Velocity of Water flow Measurement Instrument

The design of velocity of water flow measurement instrument use propeller for
sensing the velocity of water flow. The design of propeller based on propeller of
commercial current meter. That is a no new design of propeller in this research.
Figure 4 describe the design of propeller that was used.
Figure 4. Design of Velocity of Water flow Measurement Instrument (hori-

zontal position)
The propeller coupled with part that magnetic sensor is placed there. When the
propeller rotates caused by water flow, the magnetic sensor also rotates together.
Figure 5 describes the block diagram for velocity of water flow measurement
instrument.
The magnetic sensor has different response when detecting North pole or
South magnetic pole. The type of magnetic sensor used in this design is IC
UGN3503. The IC is cheap but reliable to detect magnetic field. Figure 6 describes
output of IC when detecting magnetic sensor with varying distance . From that
figure, we can see that IC UGN 3503 can use to detect North pole or South pole.
From that chart we can consider of the placement of magnetic sensor and magnet.
Magnetic
Sensor
Propeller Gear box

System
Figure 5. Block Diagram of Velocity of Water

flow Measurement Instrument
Figure 6. Output of IC UGN3503

The voltage will be inputed to micro controller as interrupt timer. The timer
will count based on the interrupt signal. The counting result will change to velocity
of water flow with equation 1.
60 60
ω= (1)
t
ω : angular velocity of propeller/water flow (rpm)
t : time for one rotation (second)
The angular velocity is changed to linear velocity with standar international unit
with equation 2.
v = ωr (2)
v : linier velocity (m/s)

r : radius of plate (m)
C. Water Level Sensor

The water level sensor is generated based on ultrasonic principle. The transmitter
of ultrasonic sensor transmits ultrasonic signal and the water surface reflects the
signal to the receiver that produces electric signal. The duration of ultrasonic signal
propagation from transmitter to receiver is counted by timer of microcontroller.
The result of the timer will be changed to water level with equation 3.
vt vt
s =h− (3)
2
v : velocity propagation of ultrasonic signal (m/s)
t : duration of ultrasonic signal propagation (s)
s : level of water (m)
h :distance between ultrasonic sensor to the bottom of drainage channel (m)
D. Calculating Algorithm for Water Discharge of Drainage

Water discharge is defined as volume rate of water flow in some wet area. Water
discharge is obtained by multiplying the velocity of water flow (v) by wet cross
section area (A).
Q = vA (4)
Wet cross section area is calculated using hydraulic formula. There are three
types of cross section channel drainage i.e.: trapezoidal, rectangle, and circle [5].
Figure 7 describes the type of cross section channel drainage. The height variable
(‘y’at rectangle and trapezoid type,‘d’ at circle type) obtained from measurement
result of water level sensor. The formula to calculate wet cross sectional area can
be seen in equation Table 1.
Tabel 1. Formula for Calculating Wet Cross Sectional Area
Type of Drainage Formula A (m2)
Rectangle by (4)
Trapezoidal (b+xy)y (5)
Circle 1/2(Ø–sin Ø)D2 (6)
Rectangle
Trapezoidal
a
y
x
b
Circle
B
D
d
Ø
Figure 7. Type of Cross Section Area
IV. Conclusion
1) The monitoring system integrates meteorological data in form of rainfall data,
and also water level as well as water discharge data.
2) Design of automatic measurement instrument of water discharge consist of
propeller, magnetic sensor, water level sensor and micro controller.
3) Water discharge of drainage is obtained by multiplying the velocity of water
flow (v) by wet cross section area (A).
4) Wet cross section area is calculated using hydraulic formula depends on the
type of drainage.
V. Acknowledgment
Thanks to Indonesia General Directorate of Higher Education who fund this
research through “Hibah Bersaing 2014” program.
VI. R eferences
1) Badan Nasional Penanggulangan Bencana. Frequency of Disaster. [Online].
Available: http://dibi.bnpb.go.id. Retrieved May 2014.
2) Rauf, Syafruddin. (2012) “Pemetaan Jaringan Drainase Berbasis Quantum
GIS Open Source di Kota Makassar”, Prosiding Hasil Penelitian Fakultas
Teknik Universitas Hasanuddin, Vol 6, No 1. 2012. [online]. Available:
http://journal.unhas.ac.id/index.php/prostek/article/view/766/657
3) Dent, James E. (2012). Climate and Meteorological Information Requere-
ments for Water Management. World Meteorological Organization, Geneva,
Switzerland, Tech. Rep. WMO-1094,1-9.
4) Wahyuni, R.T., Wijaya, Y.P., dan D. Nurmalasari. (2014). Design of Wireless
Sensor Network for Drainage Monitoring System. Innovative and System
Design and Engineering, 5, no.5. 6-13.
5) Nizar, C. (2013). Pengertian Hidrolika. [Online]. Available: http://www.
ilmusipil.com/pengertian-hidrolika.
ACEHSEIS, a local seismic experiment in Bener
Meriah and Central Aceh
Muzli Muzlia,*, Muksin Umarb, c, Klaus Bauerb, Masturyonoa, Jaya

Murjayaa, Rakhindro P. Mahesworoa, Sigit Pramonoa
Meteorological, Climatological and Geophysical Agency of Indonesia (BMKG)
a
Jl. Angkasa I No. 2 Kemayoran – Jakarta Pusat 10720

b
German Research Center for Geosciences (GFZ-Potsdam)
Telegrafenberg 14473 Potsdam Germany
c
University of Syiah Kuala (UNSYIAH)
Jl. Teuku Nyak Arief, Darussalam – Banda Aceh
Abstract
Following the destructive 2013 Mw 6.2 Bener Meriah earthquake in Aceh, we conduct a temporary
seismic experiment in Bener Meriah and Central Aceh. The experiment is a collaboration work
between GFZ-Potsdam, BMKG and Unsyiah. We deploy temporarily six months 30 short period
seismic sensors. The sensors are stationed with a distance of about 5 to 7 km apart. This dense
network is expected to record the local seismic, particularly from the source of Sumatran fault and
other local faults near Central Aceh. The aim of this project is to identify the seismic structure and
faults in Aceh. Understanding the local seismic structure of the region will significantly contribute
to future disaster mitigation in this region. Moreover, this study could also be used to fill the gap
of a comprehensive investigation of the large picture of the Sumatra area of the previous studies
from Lake Toba to the southern part of the Sumatra Island.
Key words: Seismic experiment, Sumatran fault, local earthquake, tomography
I. Introduction
The tectonics setting of northern part of Sumatra particularly Aceh is controlled
by subduction in the western part of Sumatra as well as great Sumatran fault. The
pressure of Indo-Australian plate to Sumatra Island which lay on the Eurasian plate
yields the local faults along Sumatra island. Sumatran fault is the longest fault in
Indonesia. Moreover, if we look more detail to the complexity of Sumatran fault,
we will find that there are many other small faults around the great Sumatran fault.
Aceh is one of the region which has relatively large number of small faults. There
are several small faults in Aceh, for instance Seulimum fault, Aceh fault, Batee
fault and Tripa fault [1]. Therefore this region is very interesting to be investigated.
Due to the complexity of tectonics setting, Sumatra, particularly Aceh becomes
a dangerous and high risk area of the earthquakes disaster. This disaster can arise
* Corresponding Author.Tel: +62-21-4246321 ext. 8200. E-mail: muzli@bmkg.go.id
319
Figure 1. Sensors location of AcehSeis project, mainshock of the Mw 6.2 Bener Meriah earthquake
and historical seismicity in Aceh during 2009-2013.
from the shock of earthquake or tsunami following the powerful earthquake that
originated in the ocean floor. The largest earthquake in Sumatra that ever occurs
is dated December 26, 2004 earthquake with a moment magnitude of 9.0. The
earthquake has become a historical record as one of the largest magnitude of
earthquake in the world. The earthquake caused the most devastating tsunami
causing casualties and properties damages very much [2].
In addition to the above effects, Sumatra earthquake has a domino effect to
the large earthquake at subduction zones along Sumatra island. The earthquake
activity in the Sumatra region increased significantly after the mainshock of
Sumatra earthquake, both the Sumatra earthquake aftershock occurrence in
subduction zones as well as activities on the Sumatra fault. Several earthquakes
follow the mainshock of Sumatra earthquake originating in the subduction zone
are for instance the Mw 8.5, 2005 Nias earthquake [3,4], the Mw 8.4, 2007
Bengkulu earthquake [5] and the Mw 7.8, 2010 Mentawai earthquake [6,7].
Besides subduction earthquake, a series of large earthquakes with the sources in
Sumatra fault occurs after the main Sumatra earthquake occurrence.
Figure 2. Aftershocks distribution of the Mw 6.2, 2013 Bener Meriah earthquake [9].
In Aceh, several strong earthquakes with the sources on the great Sumatra
fault or small local faults that occur after the sequence of Sumatra earthquake
period are for instance the Mw 6.0, 2013 Mane earthquake and the Mw 6.2, 2013
Bener Meriah earthquake. The overall seismicity rate in Aceh due to local fault
activities is represented on the figure of shallow earthquake epicenter distribution
in the period 2009–2013 [8] as shown in Figure 1.
Among the earthquakes above, one of the most interesting to be investigated
is Bener Meriah earthquake. The earthquake occurred on 2 July 2013 with a
magnitude of 6.2. The epicenter was on land at the geographic coordinates 4.7N
96.61E with the source depth of 10km. The devastating earthquake in Sumatra
mainland is generally sourced from the main Sumatra fault. Unlike in general,
Bener Meriah earthquake occurred at a location about 80 kilometers east of the
main Sumatra fault. This earthquake shows that other small faults have been
activated locally in Aceh outside of the main Sumatra fault. BMKG survey team
has conducted a survey to this earthquake aftershock in the period from 2 to 8
July 2013. The aftershock distribution is shown in Figure 2 [9].
Based on the BMKG report, the location of the main earthquake and the
aftershock series were not located along the main Sumatra fault but rather
occurred along the secondary fault system in the east of the Sumatra fault. This
phenomenon becomes interested to be investigated deeply. Understanding the
tectonic setting of the region will significantly contribute to future disaster

mitigation in the regions. Therefore, we propose a seismological study to better
understand the tectonic features of the region. This new study could also be
used to fill the gap of a comprehensive investigation of the large picture of the
Sumatra area of the previous studies from Lake Toba to the southern part of the
Sumatra island [e.g. 10].
II. AcehSeis
On collaboration work between German Research Center for Geosciences (GFZ-
Potsdam), Meteorological, Climatological and Geophysical Agency of Indonesia
(BMKG) and University of Syiah Kuala (Unsyiah), we conduct an experiment
of local seismic in Bener Meriah and Central Aceh, Aceh province. The network
consists of thirty short period seismic sensors distributed in the area of Bener
Meria and Central Aceh (see Figure 1). The sensors belong to and imported
from GFZ-Potsdam, Germany. The specification of the sensors uses “CUBE”
data logger, three components 100Hz seismometers. We use Mark products L-4
seismometers with the power supply from solar panels. The network is introduced
as AcehSeis network. This network is operated temporarily for six months from
August 2014 to January 2015.
A. Objectives
The objectives of AcehSeis project are to identify the local faults and seismic
structure in Aceh. Imaging the local travel time as well as attenuation tomography.
To complete the 1D and 3D database of velocity model. To identify the possibility
caldera of Danau Laut Tawar. To fill the gap of overall comprehensive investigation
of great Sumatra fault along Sumatra island from northern to southern part.
B. Methods
Several methods are poposed to be applied using data of AcehSeis project, include
1) Travel times of P and S wave arrivals will be used to derive the seismicity
distribution at high resolution [e.g. 11]. HypoDD method [12, 13] will be
applied to localize the better accuracy of hypocenters. The magnitudes of the
earthquakes will be determined and analyzed.
2) The polarity of first arrival waveforms are used to derive focal mechanisms
and to estimate the stress field in the region [e.g. 14].
3) Travel times of P and S waves are used to determine 1D and 3D Vp and Vp/
Vs velocity structures in the study area.
4) Waveform spectral properties of P and S waves are used to derive the attenu-
ation structure of the target region [e.g. 15].
5) Long-term registrations will allow us to recover the ambient noise generated
surface waves from the data, and to apply inversion methods for the imaging
of surface wave velocities and S velocity structure [e.g. 16].
C. Time Schedule
The project was prepared during April to June 2014. The installation was done
in July 2014. Data collecting and stations services will be done monthly from
September 2014 to January 2015. Deinstallation will be done in February 2015.
The data analysis will be started from October 2014.
III. R esults
The figures below show the process of deployment of seismometers in July 2014
(see Figure 3) and data collection in September 2014 (see Figure 4).
Figure 3. Seismometer installation in Central Aceh on July 2014.

Figure 4. Station service and data collection in September 2014.
Figure 5. Waveforms of earthquake event on August 1st, 2014 with the magnitude 4.9
and depth of 10 km. The event was located at 4.37N and 96.6E.
Several earthquakes have been recorded during the period of July to September
2014. One of relatively large magnitudes was an event on August 1st, 2014 with
the magnitude 4.9. The earthquake was located at 4.37N and 96.6E with the
depth of 10 km. Figure 5 shows the event was recorded by the whole 30 stations.
The signal to noise ratio of the records are very large, the waveforms are in a very
good quality.
IV. Conclusions and E xpected impacts

The network has been running well. Several events have been recorded during the
period of station running. At this moment we collect the data, check the quality
and localization process.
From the seismicity location and relocation as well as the focal mechanism
analysis, we could derive the detailed fault structure of the region. This is important
for the BMKG database and earthquake disaster mitigation.
The combination of the previous and the new data could be used to determine
the regional 1D and 3D model of Sumatra and the distribution of active faults.
The new regional 1D and 3D model could be assigned in the procedure of the
localization of the on shore earthquakes in Sumatra.
The seismic structure could be used to understand the volcanic activities in
the region. The seismic structure also provides the information of the sedimentary
basin as well as the possibility caldera of Danau Laut Tawar. Together with
the detailed fault structure, geological observation could be used to further
investigate the seismic hazards area and the possibility of landslides triggered by
the earthquakes. It will significantly contribute to future disaster mitigation in
the region.
V. Acknowledgement
We thanks to the local government as well as the ministry of energy and mineral
resources of Indonesia (ESDM) branch office central Aceh for supporting the
project. The project is funded by GFZ-Potsdam and supported by BMKG and
Unsyiah.
VI. R eferences
1) Wulandari, B.R. & N. Hurukawa (2013). Relocation of large earthquakes along
the Sumatran fault and their fault planes. Bulletin of the International Institute
of Seismology and Earthquake Engineering ISSN 0074-655X CODEN
IISBB2, vol. 47, pp. 25-30 [6 page(s) (article)] (1/4 p.)
2) Stein, Seth., & Emile A. Okal. (2005). Speed and size of the Sumatra earthquake.
Nature 434, 581–582.
3) Nalbant, Suleyman S., Steacy, Sandy, Sieh, Kerry, Natawidjaja, Danny &
John McCloskey. (2005). Earthquake risk on the Sunda trench, Nature 435,
756–757.
4) Walker, K. T., M. Ishii, and P. M. Shearer. (2005). Rupture details of the 28
March 2005 Sumatra Mw 8.6 earthquake imaged with teleseismic P waves,
Geophys. Res. Lett., 32, L24303, doi:10.1029/2005GL024395.
5) Borrero, Jose C., Weiss, Robert, Okal, Emile A., Hidayat, Rahman, Suranto,
Arcas, Diego, and Vasily V. Titov. (2009). The tsunami of 2007 September 12,
Bengkulu province, Sumatra, Indonesia: post-tsunami field survey and numerical
modeling, Geophys. J. Int. (2009) 178 (1): 180-194 doi:10.1111/j.1365-
246X.2008.04058.x
6) Lay, T., C. J. Ammon, H. Kanamori, Y. Yamazaki, K. F. Cheung, and A. R.
Hutko. (2011). The 25 October 2010 Mentawai tsunami earthquake (Mw 7.8)
and the tsunami hazard presented by shallow megathrust ruptures, Geophys. Res.
Lett., 38, L06302, doi:10.1029/2010GL046552
7) Muzli, M. (2013). Combined evaluation of strong motion and GPS data for
analyzing coseismic deformation caused by strong earthquakes, PhD Thesis,
(Scientific Technical Report ; 13/06), Potsdam: Deutsches GeoForschun-
gsZentrum GFZ, 142 p. DOI: http:// doi.org/ 10.2312/ GFZ.b103-13067
8) Sari, N. (2013). Hypocenter relocation of earthquakes in Aceh using
HypoDD, unpublished results. STMKG.
9) Tim Survey BMKG. (2013). Laporan gempabumi Bener Meriah 2013,
unpublished reports, BMKG.
10) Masturyono, R. McCaffrey, Wark, D. A., Roecker, S. W., Fauzi, Ibrahim,
G., and Sukhyar. (2001). Distribution of magma beneath the Toba caldera
complex, north Sumatra, Indonesia, constrained by three-dimensional P wave
velocities, seismicity, and gravity data, Geochem. Geophys. Geosyst., 2, 1014,
doi:10.1029/2000GC000096
11) Muksin, U., Bauer, K., and C. Haberland. (2013a). Seismic Vp and Vp/Vs
structure of the geothermal area around Tarutung (North Sumatra, Indonesia)
derived from local earthquake tomography. Journal of Volcanology and Geo.
Research 260, 27–42.
12) Waldhauser, F. and W.L. Ellsworth. (2000). A double-difference earthquake
location algorithm: Method and application to the northern Hayward fault, Bull.
Seism. Soc. Am., 90, 1353–1368.
13) Waldhauser, F. (2001). HypoDD: A computer program to compute double-

difference earthquake locations, USGS Open File Rep., 01–113
14) Muksin, U., Haberland, C., Bauer, K., M., Weber. 2013b. Three-dimensional
upper crustal structure of the geothermal system in Tarutung (North Sumatra,
Indonesia) revealed by seismic attenuation tomography. Geophysical Journal
International 195, 2037–2049.
15) Muksin, U., Haberland, C., Nukman, M., Bauer, K., M., Weber. 2014.
Detailed fault structure of the Tarutung pull-apart basin in Sumatra-Indonesia
derived from local earthquake data. Journal of Asian Earth Sciences (submitted).
16) Stankiewicz, J., Ryberg, T., Haberland, C., Fauzi, D. Natawidjaja. (2010).
Lake Toba volcano magma chamber imaged by ambient seismic noise tomography.
Geophys. Res. Lett. 37, L17306.
Selection of Global GMPEs models
for Seismic Hazards Assessments in Indonesia
(Case Study Sumatra-Java area)
Ariska Rudyantoa,*, Phil Cummins b,c, Hadi Ghasemic, I Nyoman Sukantaa
Badan Meteorologi Klimatologi dan Geofisika,
a
Jl. Angkasa I, No.2 Kemayoran Jakarta Pusat, 10720 Indonesia

b
Research School of Earth Sciences-The Australian National University
Bld 142 Mills Road Acton ACT, 0200 Australia
c
Geoscience Australia
Cnr Jerrabomberra Ave and Hindmarsh Drive, Symonston ACT, 2609 Australia
Abstract
This study focuses on investigation published by Ground Motion Prediction Equations (GMPE)
which is appropriate to be used in Indonesian earthquake hazard assessment, especially for study
area in Sumatra-Java region. The relevant GMPEs compared in this study are based on the
resemblance of geologic and tectonic conditions of the regions where the GMPEs were developed
to the study area. Twelve GMPEs have been considered in this study, consisting of nine GMPEs
derived for subduction-zone event types (intraslab and interface regimes) and three GMPEs
derived for the crustal regime. The analysis of GMPEs in this study was done using the graphical
analysis of residuals between the observed ground motion value and the corresponding values
predicted by each GMPE. The visual analysis of the statistical graphs presented in this study
indicates four GMPEs (Youngs (1997), Zhao (2006), Kanno (2006) and Lin-Lee (2008) that
match with the recorded data well, while the others have poor ft with the data. In this study, we
also rank the GMPEs using the quantitative method proposed by Scherbaum et.al (2004). The
Scherbaum e.al (2004) scheme shows that comparison of PGA/PSA with threshold value 0.0005
m/s2 gives a better output than using all data. In this study, we found that among all models,
only the Youngs (1997) and Zhao (2006) models provide predictions that are consistent with
the data from BMKG’s network.
Key words: Ground Motion Prediction Equations (GMPE), Seismic hazard assesment, Strong-ground
motion.
I. Introduction
Indonesia is one of the most seismically active country in the world. The
Indonesian tectonic system is highly complex, resulted from the interaction of
three major plates: the Eurasia plate in the north-northwest, the Indian-Australian
plate in the south and the Pacific plate in the east. This tectonic activity results in
rugged topography, frequent earthquakes (sometimes also followed by tsunamis)
* Corresponding Author.Tel: +062-8563322617. E-mail: ariska.rudyanto@bmkg.go.id, rudyantoariska@gmail.com
329
and volcanic eruptions that affect not only in Indonesia but also the surrounding
of Southeast Asian region as well. In addition, the tectonic setting in Indonesia
includes dozens of active faults spread throughout the country.
As a part of efforts to improve seismic hazard assesment, the Indonesian
government suported the development of the latest Indonesian seismic hazard
map in 2010 [1]. This map followed the International Building Code 2009 (IBC
2009) [2] recommendation in presenting spectral hazard maps. This work utilised
many new findings from various research fields related to the seismic activity and
the tectonic setting of Indonesia.
Some of the most important inputs to this map are appropriate attenuation
models for earthquake ground motion, known as Ground Motion Prediction
Equations (GMPEs). GMPEs are represented as functions of the earthquake
source, propagation path and local site conditions. However, due to the lack of
strong ground motion records for Indonesia, there is no current Indonesia-specific
GMPE available for seismic hazard studies and for this reason, they were forced
to rely on GMPEs developed for other tectonically active regions [3].
Since 2006, the Indonesian Agency for Meteorology, Climatology and
Geophysics (BMKG) has operated a seismographic network of approximately
158 broadband seismographs (InaTEWS, 2010) [4]. Subsequently, BMKG
supplemented this seismograph network with acceleropgraphs, in order to monitor
strong ground motion caused by earthquakes. This strong-motion network has
grown steadily since its establishment in 2007. Many earthquakes recorded by
this strong-motion network since its establishment provide a potentially useful
source of unique data that could be used to constrain GMPEs used in Indonesian
seismic hazard assessments and impact analysis [5].
II. Strong Motion Data

Strong-motion records used in this paper come from events with greater magni-
tude than or equal 5 and epicentral distance less than or equal 500 km. In this
study, we are interested in Sumatra and Java islands region inside the area of 2o
North–11o South and 90o East–116o East. In the development of a strong-motion
database, it is important to know what type of earthquake generated the ground
motion at a particular site. The classification of the earthquake plays a vital role
to understand the characteristics of ground motion attenuation because ground
motion from different types of earthquake will attenuate differently at similar
distance due to e.g., their different stress drop and path effects.
Figure 1. Cross-section of the subduction zone in Java (up) and Sumatra (below)
segment and distribution of earthquakes used in this study.
A. Earthquake Classification
Commonly, for seismology and engineering purposes, earthquakes are classified
into 3 different categories: interface, intraslab and shallow crustal earthquakes.
The scheme to classify the events is not only based on the focal depth and focal
mechanism, but it also should consider the geometry of the slab. Along the
Indo-Australia subduction zone in the Sumatra and the Java segments, events are
classified as intraslab events if they have depths more than 40 km, while crustal
events are defined as those that is located in the distance more than 200 km from
trench and with depth less than 40 km. For events less than 200 km from the
trench and with depth less than 40 km, they will inspect carefully to determine
whether they are intraslab or megathrust/interface events using focal mechanism
information. For example if the focal mechanism is thrust, they are classified as
interface; if not, they are classified as unknown [6].
Based on this approach, it was found among 249 earthquakes considered in
this study 63 earthquakes categorizes are interface/megathrust, 161 earthquakes
are intraslab, 4 earthquakes are crustal and 21 earthquakes are unknown types. The
database used in this study consists of 3,090 records from about 249 earthquakes
(Figure 1).
B. Data Distribution
According to the magnitude-depth distribution, the data are primarily dominated
by shallow earthquakes with depth less than 60 km (more than half of the
records) and only a few records were for event with depths more than 150 km.
Furthermore, the data mostly came from small earthquakes with magnitude less
than or equal to magnitude 6 and only a small amount of data were recorded for
events of magnitude greater than or equal 7. The strong motion records in this
study were dominated by the earthquake from the subduction zone (i.e. intraslab
and interface regimes). Only a few strong-motion records comprise the crustal
regime. Moreover, intraslab event records comprise almost a half of the data.
III. Data Processing

In addition to selection of data based on geographic region (i.e., Sumatra-Java)
distance to event (less than 500 km), pre-processing was applied to remove data
with non-standard errors from the database. Following selection of data and
pre-processing, the processing stages used in this study involve: mean removal,
baseline adjustment and instrumental correction, band pass filtering, integration
of the acceleration to obtain velocity and displacement and calculation of response
spectra of acceleration, velocity and displacement waveforms. The processing
approaches used in this study followed the scheme proposed by Boore et al.
(2002) [7] and Boore and Bommer (2005) [8].
An outline of this procedure was as follows (Boore, et al., 2005) [8]: (1)
Compute mean of the pre-event portion of the record (stopping a second or
so short of the estimated firrst arrival) and subtract that mean from the whole
record (the zeroth order correction), (2) Integrate to velocity, (3) Fit a quadratic
to velocity, starting at the time of the first arrival and constraine to be 0.0 at
the start time, (4) Remove the derivative of the quadratic from the zeroth order
corrected acceleration, and (5) filtering. In most cases, the long and short-period
noise in the accelerograms are removed by filtering. An acausal Butterworth
filter of fourth order was applied in this study for baseline adjustment and noise
reduction in the accelerograms.
In this study, the desired ground motion parameters were computed for each
set of acceleration, velocity and displacement records as peak ground acceleration
(PGA, in m/s2), peak ground velocity (PGV, in m/s) and peak ground displace-
ment (PGD, in m). The spectra were calculated using 5% damping and for about
100 frequencies, spanning from 0.25 hz up to 25 hz. The calculation of these
spectra followec the scheme by Nigam and Jennings (1968) [9].
IV. Goodness-of-fit of Selected GMPEs

A. Selected GMPEs
GMPEs are commonly used to describe the efects of source, propagation medium
and local site condition on ground motion. Since the dataset is dominated by
strong ground motion records from the subduction zones (intraslab or interface
earthquake type), the GMPEs considered in this study are also dominated by
those derived for subduction type earthquakes.
Nine GMPEs considered in this study are derived for subduction-zone event
types (intraslab and interface regimes) using worldwide or regional strong-motion
records. These models are Youngs et,al. (1997) [10], Atkinson and Boore (2003)
[11], Garcia et. al. (2005) [12], Zhao et. al. (2006) [13], Kanno et. al. (2006)
[14], Lin and Lee (2008) [15], Hong et. al. (2009) [16], Arroyo et al. (2010)
[17] and Gupta et al. (2010) [18].
B. Analysis of Residual and Statistical Measurement

The analysis of ground motion residuals plays an important role in the selection
of appropriate GMPEs. If the error term in a GMPE is consistent with the
observed data, then the model is considered valid. A graphical residual analysis
is a useful tool for validating the model even though there are many others that
can be used (NIST/SEMATEC, 2012) [19]. The graphical analysis of residuals
is worthwhile because it is efective in discerning patterns in the residuals, but it

is not recommended to use this technique as a quantitative analysis for selection
of GMPEs.
The normalised residuals used in this study are defined as the diferences
between the observed ground motion value (either both peak ground accelerations
(log(obs.value)- log (predict.value) (1)

R=
(standard deviation of predict.value)
or peak spectral accelerations) and the corresponding values predicted by each

GMPE, and divided by the corresponding standard deviation of the GMPE.
In this study, we focus on observing the patterns of residual variance. For that
purpose, we employ histogram plot as a graphical residual analysis. The histogram
of the normal (Gaussian) distribution can be used to assess whether the variance
of the data is normally distributed or not. The assumption that the measurement
variables follow or at least approximate log normal distributions has been tested
empirically (NIST/SEMATEC, 2012) [19].
Since the graphical residual analysis cannot provide a quantitative measure-
ment of misfit, it is necessary to apply other statistical tests for the model selection.
In this study, we employ the likelihood-based measurement known as the LH
test. This test is a recent technique developed solely to investigate and evaluate
GMPEs (Scherbaum et al.l, 2004) [20]. The LH test is a data-driven test that
calculates likelihood parameters and not only quantifes the model fit, but also
the shape of the underlying distribution of model residuals.
The LH value used in this approach is as follows:
where Erfc is the error functions, and Res is stands for the normalised residuals.
The Erfc is define by:
V. Conclusion
Since the strong ground motion dataset used in this study is dominated by the
data associated with the subduction-zone regime (intraslab and interface) and
only a small amount comes from crustal environment, it is not adequate to apply
such statistical tests to data from crustal earthquakes.
Figure 2. Distribution of normalised residuals and associated likelihood for interface (left) and
intraslab (right) regime (from upo to down respectively) of A. Youngs (1997); B. Zhao (2006);and
C. Lin-Lee (2008) at PGA for all dataset
Based on plots comparing normal (Gaussian) distributions with histogram

normalised residuals, only three GMPEs give a good matching (Figure 2). Those
three GMPEs are Youngs (1997), Zhao (2006) and Lin and Lee (2008). These
results are similar both for PGA and PSA at 1 second period, and also the same
for both intraslab and interface earthquake regimes. These analysis express the
suitability of the use of those three GMPEs in the area of study.
Table 1. Summarised mean, median and standard deviation of different GMPEs for PGA and
their ranking using Indonesian strong ground motion dataset.
The analysis of capability of selected GMPEs using all dataset based on

Scherbaum et.al (2004) scheme shows that all selected GMPEs are unacceptable
(Table 1 and Table 2). The larger variability on residuals might be because of poor
quality of the data, especially from early years, and the lack of knowledge on site
information of the network.
Explanation for poor at between recorded data and associated predictions by
GMPEs are presented by many researchers. Boomer et.al. (2007) [21] demonstrate
that small earthquakes will produce ground motion with larger variability than
moderate and large earthquakes. Douglas et.al (2006) [39] explain that the data
from different magnitude and distance ranges between observations and those
used in development of the selected GMPEs can be responsible for the low quality
of GMPE predictions.
Accordingly, we try to investigate the capability of selected GMPEs by control-
ling some aspect of the recorded strong ground motion data. Here, we restrict the
PGA or PSA value of the data, and use only the data that have the values larger
Table 2. Summarised mean, median and standard deviation of different GMPEs for PSA T= 1
s and their ranking.
than or equal to 0.0005 m/s2 in our analysis. In general as seen in Table 10, the
performance of Youngs (1997), Zhao (2006), Kanno (2006) and Lin-Lee (2008)
GMPEs on predicting PGA of the recorded strong ground motion data with this
threshold value are better than others (Table 3 and Table 4). The Zhao (2006)
GMPE gives the best performance in PGA prediction in this analysis, both for
interface and Intraslab regimes, and is classifed as C (the lowest capability class).
Conversely, Youngs (1997), Kanno (2006) and Lin-Lee (2008) are not good
enough to be classified as C class, even though their parameters are close to the
standard values of the C class. The quality of prediction of most target models
for PSA at 1 second period show the same pattern as for the PGA prediction,
with the Zhao (2006) and Youngs (1997) GMPE has better performance, while
the others have results consistent with PGA.
Table 3. Summarised mean, median and standard deviation of different

GMPEs for PGA and their ranking with PGA more than 0.0005 m/s2.
Table 4. Summarised mean, median and standard deviation of different

GMPEs for PSA T= 1 s and their ranking with PSA more than 0.0005 m/s2.
VI. R eferences
1) Tim Revisi Peta Gempa Indonesia. (2010). Ringkasan Hasil Studi Tim Revisi
Peta Gempa Indonesia 2010. Bandung 1 Juli 2010. Laporan Studi.
2) International Code Council, Inc. (2009). Internasional Building Code.
3) Sengara, I W. (2010). Analisis hazard gempa probalistik pulau jawa untuk
masukan dalam peta Zonasi gempa Indonesia. BMKG Scientific Journal Club.
Jakarta. (in Bahasa Indonesia).
4) Inatews (2010). Indonesia Country Report. ICG/IOTWS meeting. Banda Aceh.
5) Rudyanto, A. (2013). Development of Strong-motion Database for The
Sumatra-Java Region. Thesis. ANU-Canberra.
6) Boore, D. M. and J. J. Bommer. (2005). Processing of strong-motion ac-
celerograms: Needs, options and consequences, Soil Dynamics and Earthquake
Engineering 25, 93.
7) Nigam, N.C., and P. C. Jennings (1968). Digital calculation of response spectra
from strong-motion earthquake records, Report, Earthquake Engineering Research
Laboratory, California Institute of Technology. Pasadena, California.
8) Douglas, J. (2011). Ground-motion prediction equations 1964 - 2010, PEER
Research Reports,
9) Villaverde, R. (2009). Fundamental Concepts of Earthquake Engineering,
246-260. CRC Press..
10) Youngs, R. R., Silva, W. J., et.al. (1997). Strong ground motion attenuation
relationships for subduction zone earthquakes. Seismological Research Letters
1997(1), 58–73.
11) Atkinson and David M. Boore. (2003). Empirical Ground-Motion Relations
for Subduction Zone Earthquakes and Their Application to Cascadia and
Other Regions. Bulletin of the Seismological Society of America, Vol. 93, No. 4,
1703–1729.
12) Garca D, Singh SK, Herriz M, Ordaz M, and JF., Pacheco. (2005). Inslab
earthquakes of central Mexico: Peak ground-motion parameters and response
spectra. Bull Seismol Soc Am 95(6):2272–22
13) Zhao JX, Irikura K, Zhang J, Fukushima Y, Somerville PG, Saiki T, et.al.
(2004). Site classifcation for strong motion stations in Japan using H/ V
response spectral ratio. 13th World conference of earthquake engineering. Paper
no. 278 Vancouver, BC, Canada.
14) Kanno, T., Narita, A., Morikawa, N., Fujiwara, H., and Y., Fukushima.
(2006). A new attenuation relation for strong ground motion in Japan based
on recorded data. BSSA, 96(3), 879–897.
15) Lin P-S, and C-T., Lee. (2008). Ground-motion attenuation relationships
for subduction-zone earthquakes in northeastern Taiwan. Bull Seismol Soc
Am 98(1):220–240.
16) Hong, H. P., Pozos-Estrada, A., and R. Gomez. (2009). Orientation efect
on ground motion measurements for Mexican subduction earthquakes.
Earthquake Engineering and Engineering Vibration, 8(1), 1–16.
17) Arroyo, D., GarciLa, D., Ordaz, M., Mora, M. A., S. K., Singh. (2010).
Strong ground-motion relations for Mexican interplate earthquakes. Journal
of Seismology, 14(4), 769–785.
18) Gupta, I. D. (2010). Response spectral attenuation relations for in-slab
earthquakes in IndoBurmese subduction zone. Soil Dynamics and Earthquake
Engineering, 30(5), 368-377.
19) NIST/SEMATECH. (2012), e-Handbook of Statistical Methods, http://
www.itl.nist.gov/div898/handbook/.
20) Scherbaum F, Cotton F, P., Smit. (2004). On the use of response spectral-
reference data for the selection andranking of ground-motion models for
seismic-hazard analysis in regions of moderate seismicity: The case of rock
motion. Bull Seismol Soc Am 94(6), 2164–2185.
21) Bommer, J. J., P. Staford, J. E. Alarcn, and S. Akkar. (2007). The inuence of
magnitude range on empirical ground-motion prediction, Bull. Seismol. Soc.
Am. 97, 2152-2170.
SCMIT
LED-based Spectrometer for Advanced
Chemistry Laboratory Experiments
Mary Angelie Alagaoa ,*, Dwight Angelo Bruzonb, Imee Su Martinezb,
Giovanni Tapanga
National Institute of Physics
a
University of the Philippines-Diliman, Quezon City, Philippines

b
Institute of Chemistry
University of the Philippines-Diliman, Quezon City, Philippines
Abstract
An LED-based spectrometer that can scan in the visible range is demonstrated in a classroom
environment using the Arduino platform. The performance of the device was tested and evaluated
using solutions with absorbance in the visible region. The results obtained using the fabricated
instrument were compared to theoretical values obtained using a commercial available UV-VIS
Spectrometer. The development of a low-cost LED-based spectrometer, which is an initiative
of the Versatile Instrumentation System for Science Education and Research (VISSER), serves
as a tool to improve education, especially in laboratory class experiments, since commercial
spectrometers are expensive.
Keywords: Spectrometer, Light emitting diodes, Chemical sensors
I. Introduction
Spectrometers have always kept its timeless appeal in physical and analytical
chemistry for its applications in quantitative and qualitative analysis of various
chemical solutions. Its applications include, but are not limited to, kinetic studies,
wherein it is used to determine the rate of a reaction by analyzing the absorbance
over time of a reactant or product in the visible or ultraviolet region, and analytics,
wherein it is used to determine the concentration of an analyst through either
fitting from a calibration curve using a set of standards or using the standard
addition method. However, due to the optical components that make up a typical
spectrometer, commercially available ones are expensive.
The growing demand for portable sensing devices to monitor health,
environment and security has led to the use of light emitting diodes (LEDs) as
alternative to conventional light sources [1]. Compared to other light sources,
such as fluorescent lamps and incandescent bulbs, an LED offers convenience
by having a relatively longer lifespan, smaller size, lower cost and better optically
* Corresponding Author.Tel: +63 (02) 9818737. E-mail: malagao@nip.upd.edu.ph.
343
Figure 1. Typical Schematic Diagram of a Spectrometer
stability (as light source), which makes it more useful in battery-powered or

energy-saving devices.
It was in 1970 that the use of LEDs for photometers has been conceptualized
by Barnes, et al [2]. The LEDs were coupled with a phototransistor, which serves
as the detector. Different configurations have been designed to maximize the use
of LEDs in spectroscopy. Hauser, et al., used a blue LED as the light source to
test for Cr, Mn, Zn, Fe and Cl. Results were compared to those obtained from
conventional molecular absorption spectroscopy [3]. An array of LEDs coupled
with a fiber optic cable was also used to cover a wide range of wavelengths for
colorimetric methods [4].
While it is a known fact that LEDs emit light, a study by Forrest Mims in
1992 showed that an LED could also become a detector when reverse-biased.
The range of wavelength of light emitted by an LED is shifted from the range
that can detect [5]. This discovery has led to the use of LEDs in sun photometers
to measure perceptible water and atmospheric turbidity in a wavelength range
of 555 nm to 940 nm. These sun photometers were developed for the Global
Learning and Observation to Benefit for the Environment (GLOBE) program
that compare the performance of the LED-based sun photometers to conventional
filter-based instruments [6].
Multiple LED detectors of various colors were used to determine pH and
analyze heavy metals using a tungsten halogen lamp as a light source [7]. In 2013,
a paper by Shim and Eom suggested using a pair of LEDs to measure absorbance.
Two red LEDs were placed across each other to measure the absorbance of
Bromothymol blue (BTB), a common pH indicator [7].
The emitter-detector capability of light emitting diodes can be used to develop
a spectrometer for various spectrometry experiments, such as determining the
concentration of analyses in a solution. Using only LEDs, a spectrometer can be
constructed, owing to the pressing demand for alternative instrumentation that
are economical and highly convenient.
In the Philippines, Versatile Instrument System for Science Education and
Research (VISSER) develops microcontroller-based instruments and sensors for
education and research applications. Its goal is to be able to develop reliable

instruments at a low cost. Among the instruments developed is a low-cost
spectrometer using LEDs for chemical analysis.
This study investigates the use of LEDs functioning both as emitters and
detectors in spectrometry. For signal processing, an open source software, such
as Arduino is used. The instrument can scan in the visible range or wavelengths,
making it a useful tool in performing spectrometry experiments in a laboratory
class setting where a hands-on experience on using the instrument is important
and the availability of the commercial instrument to the students is limited.

A. Test solutions
Three test solutions were used: a solution of pH 4, containing 1% KHP and 0.04%
CH2O, a solution of pH 7, containing 0.68% KH2PO4 and 0.08% NaOH, and
a solution of pH 10, containing 0.38% Na2B4O7 · 10 H2O and 0.05% NaOH,
all obtained from LabChem.
To determine the response of the instrument in varying concentrations of an
analyte, five (5) solutions of copper (II) sulphate (CuSO4) were used. Required
amounts of solid copper (II) sulphate were dissolved in 50 ml distilled water to
make standard solutions with concentrations ranging from 0.02 mol/L to 0.1
mol/L.
B. Instrument
Ten (10) different colors of off-the-shelf available LEDs were used both as the
emitter and the detector in determining the spectra of solutions. LEDs were
chosen based on the color they emit as to have a representative emitter and
detector in the visible range.
The constructed spectrometer was tested on different solutions previously
prepared. The LED emitter-detector pair were placed one across the other with
a standard quartz cuvette in-between. The relative intensity detected by the LED
detector was read using an Arduino. The schematic diagram and an image of the
prototype are shown in Figure 2.
A quartz cuvette was 4/5 filled with the test solutions and was inserted in
the fabricated device. Three trials/readings were done for each color and for each
solution. Each reading taken was the average of five consecutive outputs of the
instrument.
The spectral plot obtained using the LED-based spectrometer was compared
to results obtained using a Vernier spectrometer in order to determine the
performance of the fabricated instrument.
(a)
(b)
Figure 2. LED-based Spectrometer (a) Schematic Diagram
(b) Image of the Prototype

The spectral emission and detection of the LEDs used were obtained and shown
in Figures 3a–3e, respectively.
It can be noted that the relative intensity of its emission is higher than its
detection and the range of the wavelength the LED detects is not the same as
the range it emits.
(a) Blue LED

(b) Green LED
(c) Yellow LED
(d) Orange LED

(e) Red LED

Figure 3a-3e. Spectral emission and detection of the LEDs used in the instrument
The spectrum of a solution, Q, is a superposition of the basis spectra of the

different LEDs as shown in Equation 1.
Q = ΣaiSi(λ) (1)
where ai is the voltage reading for LED detector, i, and Si(λ) is the basis spectra
or the detection spectra of the LED detectors.
In this prototype, the highest voltage that the Arduino can detect is 3 volts,
giving us a resolution of 2.9 mV per unit. Each reading obtained per LED, given
by I, for a solution is divided over its reading on a blank solution, Io in order to
compute for the transmittance as shown in Equation 2.
%T = I/Io x 100% (2)
sing Equations 1 and 2, the transmittance plots of the test solutions were
U
obtained. Figure 4 shows the superimposed spectral plots of the solutions
obtained using the commercial spectrometer and the LED-based spectrometer.
The spectra of the different buffer solutions were compared in the wavelength
range where the transmittance peaks are expected. Results obtained using the
LED-based spectrometer are comparable to the results from the commercial
spectrometer for the blue and red buffer solutions.
The LED-based spectrometer was also tested on varying concentrations of
copper sulfate solutions. Figures 5 and 6 show the transmittance spectra for
(a)
(b)
(c)
Figure 4. Spectra of the (a) blue, (b) green and (c) red buffer solutions
the copper (II) sulphate solutions using the LED-based spectrometer and the
commercial spectrometer, respectively.
On Figure 7, it can be noted that the transmittance decreases as the concentra-
tion increases from 0.02 M of Cu2+ to 0.1 M Cu2+. However, for the solutions
with 0.08 M of Cu2+ and 0.1 M of Cu2+, the LED-based spectrometer was not
able to distinguish the varying concentration.
Figure 6. Spectral plot of the copper sulphate solutions using the LED-based spectrometer
Figure 7. Spectral plot of the copper sulphate solutions using the commercial spectrometer
Figure 8. Optimal Resolution of the LED detectors
On the other hand, Figure 8 shows that the transmittance at increasing

concentrations increases in the range 400nm to 650nm while decreases beyond
the visible range.
It can also be noted that the LED-based spectrometer failed to produce the
expected shape of the transmittance spectra of the solutions. This can be due to
the resolution of the LEDs used. At 430 nm, by fitting a Gaussian function with
varying standard deviation, thereby the spectral bandwidth, it was observed that
a blue LED detector could only reconstruct a spectrum in the blue range if it has
a spectral width of at least 30 nm. Shown in Figure 8 is the optimal resolution
of the LED-based spectrometer for each wavelength. This shows the spectral
bandwidth at which the developed LED-based spectrometer can reconstruct at
a particular wavelength.
IV. Conclusion
A spectrometer was constructed using different LEDs functioning both as the
emitters and detectors to measure transmittance of various solutions. Solutions
of varying absorbance in the visible region and concentration were tested using
the fabricated spectrometer and results were compared to values obtained from
a commercial spectrometer.
The LED-based spectrometer was able to show that at increasing concentra-
tions, the transmittance decreases. However, due to the current resolution of
the LED-based spectrometer, it was not able to distinguish all concentrations
and produce the expected transmittance spectra of the solutions. The device can
further be improved by adding more LEDs at varying wavelength in order to

cover the whole spectrum.
V. Acknowledgment
This research is under the Versatile Instrumentation System for Science Education
and Research (VISSER), a project funded by the Philippines Council for Industry,
Energy and Emerging Technology Research and Development (PCIEERD) and
the Office of the Vice Chancellor for Academic Affairs– Emerging Interdisciplinary
Research Grant (OVPAA-EIDR).
VI. R eferences
1) O’Toole, M. and D. Diamond. (2008). Absorbance Based Light Emitting
Diode Optical Sensors and Sensing Devices. Sensors, 8, 2453-2479.
2) Flaschka, H., McKeithan, C., and R. Barnes. (1973). Light Emitting Diodes
and phototransistors in photometric modules. Analytical Letters, . 6, 585–594.
3) Schawarz, M.A. and P.C. Hauser. (2001). Recent developments in detection
methods for microfabriacated analytical devices. Lab on a Chip, 1. 1–6.
4) Hauser, P.C., Rupasinghe, T.W.T and N.E. Cates. (1995). Amulti-wavelength
photometerbased on light-emitting diodes. Talanta, 42. 605–612.
5) Acharya. Y.B. (2005). Spectral and emission characteristics of LED and its
application to LED-based sun-photometry. Optics and Laser Technology,. 37
547–550.
6) Mims, F. (1992). Sun photometer with light emitting diodes as spectrally
selective detectors. Optical Society of Americal.
7) Berry, R.J. Harris, J.E., and R.R. Williams. (1997). Light-Emitting Diodes
as Sensors for Colorimetric Analyses. Applied Spectroscopy,. 51, 1521–1524.
8) Shin, Dong-Yong, and In-Young Eom. (2013). A Pair of Light Emitting
Diodes for Absorbance Measurement. Bulletin Korean Chemical Society,. 34,
No. 10.
Introduction investigation: Executive
Information System for University
Spits Warnarsa*, Sasmokoa, b, Nancy Susianna b

a
Surya University
Jl. Boulevard Gading Serpong Blok O/1 Summarecon Serpong, Tangerang, Indonesia
b
STKIP Surya
Jl. Scientia Boulevard Blok U/7 Gading Serpong, Tangerang, Indonesia
Abstract
University as higher education implementer has responsibility to accelerate their national human
capital in order to compete in highly competitive economic.The higher the national human capital,
the more prepared they are to compete in highly skilled labour. University as a knowledge keeper
should be equipped with information technology in order to establish well prepared university,
particularly to support high level management. High level management university, such as rector,
vice rector, dean and head of study program should be armed with information technology tool
which can help them when making their decisions become better and accurate. EIS (Executive
Information System) as highly DSS (Decision Support System) which is designed for high
level management is the appropriate information technology tool in order to sharp high level
management’s decision making. The EIS will be built up by 24 Indonesian national standards of
higher education, where each of standards will be generated as Key Performance Indicator (KPI)
to evaluate and justify the university performance from 3 perspectives such as education, research
and community services. The average KPI score of national standard of higher education for each
study program will show the quality assurance of each study program or faculty in university.
The study program or faculty performance should be assessed based on KPI national standard of
higher education and the higher KPI score total, the better quality of the study program. When
one of standards has poor quality then it will automatically alert the high level management.
EIS will be developed with web based programming and equipped with technology such as data
warehouse and data mining. Data warehouse should be used to extract data from university
data transaction into fact constellation schema, in order to increase performance including for
multidimensional purposing. Meanwhile, data mining will equipped with mining unexpected
patterns particularly from data warehouse. At the end, the better and accurate decision making
which is produced by high level management university will make it easy for the decision maker
to create well established higher education for the society and having high accreditation score
from Indonesian government. As a reward, the university will have a good reputation among the
competitors, invite best students, lecturers, researchers and funding as well.
Key words: Executive information system, Data warehouse for higher education, Quality assurance
of Indonesian higher education, Indonesian national standard of higher education, Indonesian higher
education accreditation.
* Corresponding Author.Tel: 62181318992388. E-mail: spits.warnarsr@surya.ac.id
353
I. Introduction
Recently, the Ministry of Education of the Republic of Indonesia has launched
12 years compulsory education, where in 2020 Indonesian citizens will have
minimum high school education [1]. The big concern of Indonesian government
upon the acceleration of their human capital shows that well higher educated
human capital as the most priority to build a nation, particularly to face ASEAN
Economic Community (AEC) in 2015. Obviously, concerning Indonesian
government in accelerating national human capital to prepare the nation to be
ready in highly competitive economic region. Nonetheless, the declaration of 12
years compulsory education is not enough in order to face element free flow of
skilled labour as one of five core elements of ASEAN single market. Indonesian
human capital should be prepared to accelerate more than 12 years compulsory
education and university as higher education has huge responsibility to develop
and maintain their national human capital.
Information technology as tools should be needed by organization in order to
run their organization activity, particularly in business or university organization.
Using information technology to support daily activity organization should
give value added benefit for the organization in order to compete with their
competitor. Those organizations that accelerate their organization activity will
be one step ahead rather than their competitor. University as high education
organization should be equipped with information technology tools in order to
run their university activities and nowadays there is no university still not using
information technology tools to support their daily university activity.
High level management as the university decision maker should be supported
with information technology. High level management such as rector, vice rector,
dean and head of study program should be equipped with high level management
application software which can help them to make sharp and better decisions.
This high level management application software should be connected and inline
between high level management university positions. It means that the data or
information input or output by head of study program should be connected and
inline with the data and information which are accessed by rector, vice rector or
dean of faculty.
Executive Information System (EIS) is proposed as high level management ap-
plication software to provide high level management with information technology
support in order to make sharp and better decisions. This EIS should be supported
with datawarehouse technology where star or snowflake or fact constellation
schema as the answer to make fast response creating reporting. In the future,
data mining technique can be implemented for mining education patterns, such
as frequent pattern, similar pattern, association rules and so on. At the end, this
EIS will help high level management to provide excellent university services for
public. Indeed, EIS is a tool which provides relevant interesting information senior
manager and different with traditional information system, tailor to executive’s
information needs and access broad range of internal and external data, easy to
use, used directly without assistance and in graphical form [16].
II. R esearch Method

This research is categorized as applied research where the outcome of this research
should be implemented in the implementation of real software application. The
research should be done in 4 steps, they are
1) Literature survey.
The literature survey should be done in three sub activities, they are:
a) Information technology literature survey.
Do literature survey for current information technology application which
is used by university high level management to make their decisions.
b) Indonesian rules literature survey
Read regulation product such as law, presiden regulation, government
regulation, minister regulation which are issued for Indonesian higher
education accreditation both of internal or external quality assurance.
c) Quality assurance survey.
Do literature survey for national and international quality assurance.
2) Mapping national standard of higher education framework
Based on Indonesian rules literature survey, 24 national standards of higher
education should be implemented as Key Performance Indicator (KPI) to
evaluate and justify the university performance from 3 perspectives such as
education, research and community services.
3) Design prototyping application of EIS
Designing EIS application which is should be done in 6 steps, as follows:
a) Collect current university database Online Transcational Processing
(OLTP) and model the database design with Unified Modeling Languages
(UML) class diagram.
b) Generate reporting based on KPI from 24 national standard of higher
education, including survey result for qualitative KPI.
c) Model database design for star or snowflake or fact constellation schema
data warehouse with Unified Modeling Languages (UML) class diagram.
d) Design Extraction Transformation and Loading (ETL) algorithm as a
process to load data from university database OLTP into data warehouse.
e) Design EIS application business process with Unified Modeling Language

(UML) use case.
f ) Design user interface to entry qualitative KPI and EIS application.
4) Implementation prototyping
The EIS application would be implemented with web based programming
like Personal Home Pages (PHP) as open source server programming, whilst
database would be implemented with MySQL database as open source
database.
III. Quality assurance of Indonesian Higher Education

Universities in Indonesia both private and public should assure the quality of their
education process, in transparent and accountable ways, in order to protect the
community interest as regulated by the law [8]. The Ministry of Education and
Culture of the Republic of Indonesia has legislated the regulation [7,9] which
regulate quality assurance in higher education should consist of both of Internal
and external (article 3 clause 1) [7].
Quality assurance of Indonesian higher education both of internal and external
should be implemented by Indonesian higher education institution and has
purposes to gain and maintain the quality of learning, research and community
services [9]. The output of internal quality assurance will be used by Indonesian
National Accreditation Agency of Higher Education Institution (BAN-PT: Badan
Akreditasi Nasional Perguruan Tinggi, http://ban-pt.kemdiknas.go.id) to establish
accreditation level of study program faculty (article 3 clause 2 sub clause f ) [6],
(article 3 clause 4) [7]. Moreover, planning, executing, managing and developing
both internal and external quality assurance should refer to national standard of
higher education [7].
BAN-PT as mentioned in article 1 clause 1 [2] is an independent evaluation
board which has a duty to establish program eligibility and/or education unit on
higher eduation with reference to national standard’s education. It is nonstructural,
nonprofit and independent body which responsible to minister of education, as
mentioned in article 2 clause 2 [2].
However, regulation of ministry of national education of the Republic of
Indonesia, number 73 year 2009 [3,4] as an old regulation is not suitable with
regulation of ministry of national education of the Republic of Indonesia, number
49 and 50 year 2014 [6,7]. In old regulation, BAN-PT has seven instruments
standard accreditation (section 3, sub section A [3] or section 2) [4] such as:
1) Standard of visions, missions, goals and objectives, including strategies for
achieving.
2) Standard of governance, leadership, management and quality assurance.
3) Standard of students and graduates.

4) Standard of human resources.
5) Standard of curriculum, learning process and academic atmosphere.
6) Standard of funding, infrastructure including information system.
7) Standard of research, community service and collaboration.
As mentioned before, both internal and external quality assurance should
refer to 24 national standards of higher education [7] which consist of 8 national
standards of education, 8 national standards or research and 8 national standards
of community services [6]. The 8 National Standards of education are:
1) Standard of graduate competence.
2) Standard of learning contents.
3) Standard of learning process.
4) Standard of learning assessment.
5) Standard of lecturer and educational personnel.
6) Standard of learning tool and infrastructure.
7) Standard of learning management.
8) Standard of learning funding.
The 8 National Standards of research are:
1) Standard of research product.
2) Standard of research content.
3) Standard of research process.
4) Standard of research assessment.
5) Standard of researcher.
6) Standard of research tool and infrastructure.
7) Standard of research management.
8) Standard of research funding and budgeting.
The 8 National Standards of community services are :
1) Standard of community services product.
2) Standard of community services content.
3) Standard of community services process.
4) Standard of community services assessment.
5) Standard of community services implementor.
6) Standard of community services tool and infrastructure.
7) Standard of community services management.
8) Standard of community services funding and budgeting.
However, Indonesia with BAN-PT as Indonesian higher education quality

assurance accreditation has been recognized by Southeast Asian Ministers of
Education Organization-Regional Centre for Higher Education and Development
(SEAMEO RIHED) which can be reached at www.rihed.seameo.org. SEAMEO
RIHED is the Regional Centre for Higher Education and Development working
for the 11 Member Countries in Southeast Asia under the umbrella of the
Southeast Asian Ministers of Education Organisation (SEAMEO). SEAMEO
RIHED promotes ASEAN Quality Assurance Network (AQAN) as a common
regional framework for Southeast Asian nations for quality assurance in higher
education [5]. AQAN by ASEAN University Network, www.aunsec.org (AUN)
promotes the harmonization of the quality assurance for ASEAN higher education
where all universities in ASEAN country can have different quality assurance
system and approach.
Indonesia by BAN-PT as quality assurance body or agency has the same model
of independent body or agency with countrie such as Cambodia, Malaysia and
Thailand, whilst countries such as Brunei, Laos, and Vietnam have centralised
government body or agency within ministry responsible for higher education.
Meanwhile, Singapore and Philippines have mixed system and Myanmar alone
does not have an established quality assurance body or agency where all quality
functions are delegated to individual higher education which has responsibility
to report to Minister of Education. BAN-PT and The Commission on Higher
Education (CHED) as Indonesian and Philippines higher education quality
assurance agencies respectively, have the same common purpose of quality by
providing information on accreditation status.
IV. K ey Performance Indicator

The 24 national standards of higher education should be generated as KPI. Each
standard will have many KPI which are used to control the quality of standard.
For example, standard of graduate competence will have KPI such as :
1) Number of students who graduate on time.
2) Employer opinion regarding alumni quality.
3) Time waiting for first job.
Each of KPI will be quantified to 5 points rating scale with quantitative
number range such as 0,1,2,3 and 4 to qualitative range score such as Very Poor,
Poor, Fair, Good and Excellent respectively. Each of KPI must have quantitative or
qualitative data such as number of students or employer good opinion regarding of
alumni quality, respectively. The score of KPI quality range refer to old regulations
of BAN-PT which can be read at copy of attachment 7 of Regulation of Minister
of National education of the Republic of Indonesia, Number 73 Year 2009 [10].
Table 1. KPI of standard of graduate competence for HCI study program

KPI of standard of graduate competence Score
HCI Study Program
1. Number of students who graduate on time 3
2. Employer Opinion regarding of alumni quality 2
3. Tine Waiting time for first job 2
KPI Score Total 2.33
Score: Very Poor(0), Poor(1), Fair(2), Good(3), Excellent(4)
Table 1 shows the example of KPI quantification of graduate competence

standard which are categorized as fair KPI when it has KPI score total 2.33. KPI
score total is KPI average from all KPI score lists (3+2+2=7/3=2.33) and KPI
score total will be rated as well to Very Poor, Poor, Fair, Good and Excellent.
Finally, based on the 24 national standards of higher education, there will
be 24 tables as shown in Table 1 that each of table will show the quality for each
national standard of higher education. Moreover, the average of all 24 tables KPI
score total will show the quality of study program based on national standard of
higher education. The higher KPI score total, the better study program quality.
Controlling of each study program quality will be based on the KPI national
standard of higher education and when one standard has poor quality then it will
automatically alert high level management. High level management, such as rector,
vice rector, dean, vice dean, head of study program and etc will be convenient
to use the university executive information system and easy to control the study
program or faculty. Meanwhile, other university stakeholder such as government,
society, community, student’s parent, should have benefit from this university
executive information system where the quality of university can be accessed in
transparent and accountable way in order to protect the community interests as
regulated in law [8].
V. EIS Database architecture

Figure 1 shows EIS database architecture with 3 data marts and 1 data warehouse.
Data mart and data warehouse are recognized as Online Analytical Processing
(OLAP) database [11]. Figure 1 shows that data warehouse technology is designed
as centralized data warehouse architecture. The data warehouse in figure 1 is
representation of national standard of higher education, whilst the 3 of data
marts are representation of national standards such as education, research and
community services.
The data marts or data warehouse will have star or snowflake or fact constel-
lation scheme, represent unnormalized database which will be used to increase
the performance to read the database records with SQL select statement. Indeed,
data warehouse technology is used in order to increase the performance of SQL
select statement since there is decreasing of number managed tables, records and
data byte [11].
The data warehouse will be loaded with the data from the 3 of data marts
with ETL (Extraction Transformation and Loading) process. Meanwhile, the 3
data marts will be loaded from OLTP/TPS (Online Transactional Processing/
Transactional Processing System) database with ETL process as well. The ETL
process should be done on extraction scheduling mode with constructive merge
loading which differ old and new records [12]. The ETL algorithm will be run
automatically at the end of office hour, parallel with OLTP daily backup database
[12].
The data mart of national standard of education will be loaded with data
from OLTP database, such as student administration, academic administration,
human resources, general affair and finance. Meanwhile, the data mart of national
standard of research should be loaded with data from OLTP database such as
human resources, general affair, finance and research. Finally, the data mart of
national standard of community services should be loaded with data from OLTP
database such as human resources, general affair, financial and community services.
Transformation of chosen OLTP database into each data mart should be
done based on standard content in each of data mart which represent between
Figure 1. EIS database architecture

national standard of education, research or community services. For example,

data marts national standard of research and community services have the same
chosen OLTP databases such as human resources, general affair and finance. Both
of standards of research and community services have standard of researcher and
community service implementer respectively.
It is possible to design without data warehouse and using only 3 data marts
to input the data warehouse before the 3 data marts. The number of data mart or
data warehouse, the chosen between centralized or uncentralized data warehouse
architecture should consider based on university needs, cost and performance.
Hardware architecture implementation for EIS database architecture, such as
the number of server should be designed as user’s requirement needs, cost and
performance. The OLTP database in figure 1 is an example where it is possible
to have different database architecture which depends on each university.
The 3 data marts and data warehouse can be accessed by novice or expert user,
where the application will provide reporting, graphic reporting, multidimensional
as the power of data warehouse. Moreover the intelligence of data warehouse could
be increased by adding data mining technique which can find pattern such as
classification, discriminant, association, frequent, similar and so on with chosen
of data mining algorithm such as Attribute Oriented Induction (AOI) [15],
Emerging pattern (EP), Attribute Oriented Induction High Emerging Pattern
(AOI-HEP) [13,14], Neural Network, Bayesian Network, Decision tree and so on.
VI. Conclusion
24 national standards of higher education as a KPI framework to build EIS
application for university will fulfill the Indonesian government regulation in
order to assure the higher education quality.
Study program quality assurance can be controlled based on this KPI national
standard of higher education framework and when one of the standard has poor
quality assurance then it will automatically alert the high level management and
surely increase the quality by fulfilling the KPI requirement. Moreover, university
high level management will be convenient and easy to use the EIS in order to
assure the quality of their study program or faculty.
University stakeholder such as government, society, community, student’s
parent should have benefit from the university EIS where the quality of higher
education can be accessed in transparent and accountable ways in order to protect
the community interest.
This research should need more exploration where KPI framework from 24
national standard of higher education should be generated more accurately where
each national standard will have more than 1 KPI. Furthermore, EIS application
infrastructure should be defined in order to show the EIS business process.
VI. R eferences
1) Damarjati., D. (2012). Mendikbud: 2013, Wajib belajar 12 Tahun &
Kurikulum baru diterapkan. [Online] accessed on 9 July 2014, from : news.
detik.com/read/2012/12/04/150638/2109092/10/mendikbud-2013-wajib-
belajar-12-tahun--kurikulum-baru-diterapkan .
2) Sudibyo., B. (2005). Agency of National Accreditation of Higher Education
institution. Regulation of Minister of National education of the Republic of
Indonesia, Number 28.
3) Sudibyo., B. (2009). Accreditation instruments of Bachelor study program.
Copy of attachment 1 of Regulation of Minister of National education of the
Republic of Indonesia, Number 73.
4) Sudibyo. B. (2009). Accreditation instruments of Bachelor study program.
Republic of Indonesia, Number 73. SEAMEO RIHED. (2012). A study on
Quality Assurance Models in Southeast Asian Countries: Towards a Southeast
Asian Quality Assurance Framework [Online] accessed on 9 July 2014, from:
www.rihed.seameo.org/wp-content/uploads/2013/FrequentlyRequested/
SEAMEO_RIHED_QA_in_SEA_report_2012.pdf .
5) Nuh., M. (2014). National Standard of Higher Education. Regulation of
Minister of education and culture of the Republic of Indonesia, Number 49.
6) Nuh., M. (2014). Quality Assurance of Higher education. Regulation of
Minister of education and culture of the Republic of Indonesia, Number 50.
7) Yudhoyono., S.B. (2012). Higher Education. Law of the Republic of Indonesia,
Number 12.
8) Yudhoyono. ,S.B. (2014). Organization of higher education and management
of higher education institution. Regulation of Government of the Republic of
Indonesia, Number 4
9) Sudibyo., B. (2009). Accreditation instruments of Bachelor study program.
Republic of Indonesia, Number 73
10) Warnars., S. (2014). Perbandingan penggunaan Database OLTP (Online
Transcational Processing) and Data Warehouse. CCIT (Creative Communica-
tion and Innovative Technology) journal, 7(4).
11) Warnars., S. (2009). Desain ETL dengan contoh kasus perguruan tinggi.
Jurnal Informatika, 10(2), 86-93.
12) Warnars., S. (2012). Attribute Oriented Induction of High-level Emerging
Patterns. IEEE International Symposium on Foundations and Frontiers of
Data Mining in conjunction with IEEE International Conference on Granular
Computing (IEEE GrC2012). Hangzhou, China.
13) Warnars., S. (2014). Mining Frequent Pattern with Attribute Oriented
Induction High level Emerging Pattern (AOI-HEP). IEEE the 2nd International
Conference on Information and Communication Technology (IEEE ICoICT
2014), Bandung, Indonesia, 144-149.
14) Warnars., S. (2010). Star schema design for concept hierarchy in Attribute
Oriented Induction. Internetworking Indonesia Journal, 2(2), 33-39.
15) Kelly., F. (2002). Implementing an Executive Information System (EIS).
DSSResources.com, 11/07/2002. [Online] accessed on 10 August 2014,
from: dssresources.com/papers/features/kelly11072002.html
Design of Implementation Delay Tolerant At
Wireless Mesh Networks using IBR-DTN and
Batman-adv
Herman Yuliandokoa,b, Sritrusta Sukaridhotob, M. Udin Harun Al Rasyidb
a
State Polytechnic of Banyuwangi
Jl. Raya Jember Km.13 Labanasem-Kabat, Banyuwangi, Indonesia
b
Postgraduate of Information Engineering and Computer, Electronics Engineering,
Polytechnic Institute of Surabaya
Jl. Raya ITS Sukolilo 60111, Surabaya, Indonesia
Abstract
Wireless technology is one of the fast growing technology. Research on wireless technology has
been widely applied, and one of them is a mesh network technology research. Mesh networking
technology is considered as one solution to the problem of network with complex equipment.
By applying a mesh network, each node has the same level with other nodes without the need
of an access point. Delay-tolerant networking (DTN) is an approach to computer network
architecture that seeks to address the technical issues in heterogeneous networks that may lack
continuous network connectivity. In our research, we use propose a design of implementation
of Batman-adv software for mesh network in addition, we also will combine and implementing
delay tolerant technology IBR-DTN to support inter-node communication. It is appropriate to
mention that previous research IBR-DTN is an efficient technology to be applied compared to
other technologies. In the end, we also include the analysis and design implementation of mesh
technology will be carried out the Batman-adv and IBR-DTN running together.
Key words: Wireless mesh, Batman-adv, IBR-DTN, Combine.
I. Introduction
Kevin Fall in “A Delay Tolerant Network Architecture for Challenged Internet”
mentioned that Delay tolerance network is a technology that is very useful for
network communication has a great challenge (obstacle) [1]. DTN can solve
intermittent connectivity problems and long or variable delay because DTN is
a technology that allows to send data packets in a network that has a difficult
environment or not continuously available connection. In this technology each
node has a network storage device so that when there is network connection
problem, the data will still be kept in storage and will be resent again when the
network has been formed again. Although DTN has any advantages, DTN also
needs long time and more energy consumption. Currently, researchers study
focuses on the architecture, algorithms, routing and network design on Delay
Tolerant [1][2][3]. While research on the Delay Tolerant Network manet is still
rare.
365
Wireless mesh is currently used in many application. It is due to dynamic

mobility user, disaster area and low cost network manufacture. Research study
in rooting protocol that performed evaluation of wireless mesh have been carried
out [4]. However, research DTN for mesh network application or ad-hoc mode
is still rarely done, and majority research DTN on mesh network is still on
simulation. For an example, Laurent and Simin research on Batman simulation
with Store-and-Forward mechanism. In this research they use NS3 simulator [5].
DTN has grown so rapidly in line with DTN applications in several fields.
In the fields of transportation, DTN is used in data transmission on vehicular
network in intelligent transport system [7]. Due to the environmental conditions
of transport that has variables, they cause unstable communications networks
as well as user data to mobile users. DTN applications in the field of health
support are also very important to support communication in long distance and
long delay. Research on Rural Telemedicine Networks Using Store-and-Forward
Voice-over- IP [6]. DTN is used to support VoIP for medical field by connecting
doctor and patients in the remote areas.
In this paper, we will make research design of implementation of DTN by
using wireless mesh network. Overview on BATMAN and DTN will explain on
the first, then we review related work on performance evaluation of BATMAN
and IBR-DTN. We present setup and results of our experiment. Finally, we
summarize our contribution and describe further work.

Wireless mesh networks (WMN) are self-organized wireless networks in which
component parts (nodes) can all connect to each other via multiple hops. In
WMN, nodes are comprised of mesh routers and mesh clients. Each node operates
not only as a host but also as a router, forwarding packets on behalf of other
nodes that may not be within direct wireless transmission range of their destina-
tions[8]. Other than the routing capability for gateway/repeater functions as in
a conventional wireless router, a wireless mesh router contains additional routing
functions to support mesh networking. To further improve the flexibility of mesh
networking, a mesh router is usually equipped with multiple wireless interfaces
built on either the same or different wireless access technologies. Compared with a
conventional wireless router, a wireless mesh router can achieve the same coverage
with much lower transmission power through multi-hop communications. In
detail, WMN should have the following properties [9]:
1) Multi-hop wireless network
2) Dynamically self-organized, self configured and self-healing
3) Resilience to device failures
4) Highly scalable
A. Routing Protocols
Wireless mesh network is an unstructured network and have a high mobility. It
means the WMN (Wireless Mesh Network) have to make sure the communication
can be done. Routing protocols are responsible for discovering, establishing
and maintaining such routes. Routing protocols for WMN are mostly based on
protocols designed for mobile ad-hoc networks. These can be classified in the
following categories [10].
1) Proactive protocols construct the routing table periodically.
2) Reactive protocols construct the routing table on-demand.
3) Hybrid protocols are a mixed design of the two approaches mentioned above.
These protocols typically use a proactive approach to keep routes to nodes
in the vicinity of the source, but for nodes beyond that area, the protocol
behaves like a reactive one.
B. BATMAN-ADV
BATMAN is a proactive routing protocol for WMN. It uses a distance-vector
approach and a routing metric which incorporates the reliability of the radio links.
Despite being developed and publicly available since 2006, BATMAN, especially
its newer batman-adv variant, has received sparse attention in the scientific
community. Optimized Link State Routing (OLSR) and BATMAN are two
popular protocol. Better Approach to Mobile Ad-Hoc Networking (BATMAN)
[11] is a new routing protocol for multi-hop ad-hoc mesh networks. BATMAN
uses a simple and robust algorithm for establishing multi-hop routes in mobile
ad-hoc networks. This protocol for WMN, based on distance vector routing
and a proactive protocol in that each node maintains a routing table containing
potential next hops to all other nodes forming the WMN.
Batman algorithm protocol can be described, that each node transmits
broadcast messages (originator messages or OGM) to inform the neighboring
nodes about its existence. These neighbors re-broadcast the OGM according
to specific rules to inform their neighbors about the existence of the original
initiator of this message and so on and so forth. Thus, the network is flooded
with originator messages [2]. An OGM is used in BATMAN to distribute node
specific information in the mesh network. This OGM is encapsulated in a raw
Ethernet frame and broadcasted in a fixed interval (1 second) on all used hardware
interfaces. The frame includes all information a receiving BATMAN node needs
to build up its routing table.
In the beginning, BATMAN is like other routing protocol which running in

layer 3 (batman). In 2007, Layer 2 routing (BATMAN Advanced or batman-adv)
was introduced, moving the daemon into the Linux kernel for better performance.
The development of batman was mostly stopped later on, focusing the efforts on
batman-adv. The BATMAN wiki [11] lists some characteristics of this design:
1) network-layer agnostic - you can run whatever you wish on top of batman-adv:
IPv4, IPv6, DHCP, IPX .
2) nodes can participate in a mesh without having an IP .
3) easy integration of non-mesh (mobile) clients.
4) roaming of non-mesh clients optimizing the data flow through the mesh (e.g.
interface alternating, multicast, forward error correction, etc.).
5) running protocols relying on broadcast/multicast over the mesh and non-mesh
clients (Windows neighborhood, mDNS, streaming, etc.).
1) Batman-adv Setup
In this research, we used Ubuntu 12.04 for operation system in each node and
we used wireless too connect each node on mesh network designed. Before setup
Batman-adv in our tools, it was important to update our tools repository for
implemented Batman in Ubuntu.
Batman-adv also has some tool and C program that why it was needed to
make sure that there was C compiler program in our nodes. This compiler will
be used to compile our program when running Batman-adv.
The software of Batman-adv could be downloaded from www.open-mesh.org.
There were some release series of Batman-adv and make sure that our Ubuntu
kernel was compatible with Batman-adv version. After downloaded, we extracted
and compiled them.
To make sure our Batman-adv could run smoothly, we had to prepare some
tools, for examples qt4-dev-tools, xconfig, libncurses5-dev, menuconfig and
oldconfig.
After we installed and prepared Batman-adv tools, then we needed to set our
interface. In this research, we used wlan0 (wireless) interface to connect nodes
each other. It was important to make sure that nodes firewall is disable.
Wireless mesh was one of ad-hoc mode wireless network. It was also needed
to create essid and channel. The essid and channel should be same for all nodes.
iwconfig wlan0 mode ad-hoc essid [essid] channel [channel]
C. Activated Batman-adv and batctl

Although we already setup Batman-adv in our nodes, it was also needed to activate
our Batman-adv to operate routing protocol. Batman-adv had one advantages
compared to the another routing protocol, Nodes which use Batman-adv for
their routing protocol could connect each other by using layer 2 or layer 3. It
means nodes in Batman-adv could ping another by using IP address or MAC
address. That was why in the Batman-adv also has batctl software to support
communication process.
Wireless mesh by using Batman-adv routing protocol was ready to be used
if nodes interface status was active. To know that our interface status was active
by accessing iface_status file on Batman-adv folder.
D. DTN Store-and-Forward
DTN overcame the problems associated with intermittent connectivity, long or
variable delay, asymmetric data rates, and high error rates by using store-and- for-
ward message switching. This was an old method, used by pony-express and postal
systems since ancient times. Whole messages (entire blocks of application-program
user data) or pieces (fragments) of such messages were moved (forwarded) from
a storage place on one node (switch intersection) to a storage place on another
node, along a path that eventually reached the destination.
There were several tools or software for DTN implementation. Delay
Tolerant Network Research Group (DTNRG) to produce DTN2 to implement
basic functional of DTN. Another Linux implementation was ION. Symbian
smart-phone usually use DASM for DTN implementation and DTNLite was
designed for sensor networks running tiny OS, but did not allow for using
common applications and programming languages. IBR-DTN was to develop
a powerful and efficient implementation that runs on embedded devices as
well as on standard Linux systems. Michael Doering in IBR-DTN: An Efficient
Implementation for Embedded Systems explains that the implementation of IBR-
DTN is more efficient. Not only the slim application itself, but also the lower
Figure 1.: Store and Forward

consumption of memory makes IBR-DTN suitable to run on embedded devices

with limited resources [12].
1) IBR-DTN Setup
There were many kinds of IBR-DTN Setup and in this paper we used repository
to setup IBR-DTN tools. We need to download Release key and make sure the
distribution as Ubuntu version which used. It can be downloaded on http://
download.opensuse.org/ after that download and install IBR-DTN tools.
E. Related Works
An extensive amount of work has been done in the field of wireless mesh network
routing protocol and delay tolerant networks. In the following, we present related
work with a focus on Batman routing protocol and IBR-DTN.
Some research on wireless mesh focus on routing protocol performance only.
Laurent et al. [5] used NS3 to make analysis and demonstrate Batman with
additional mechanism store-and-forward. They have implemented SF-Batman
(Store-and-Forward Batman) in a packet level simulation and demonstrated
its performance in a scenario that consists two region of connectivity: a well
connected mesh network and a set of sparser sub networks.
A Practical Evaluation of BATMAN Advance of the Routing Performance of
Wireless Mesh Networks in the realistic office environment [4]. It was done to
evaluate parameters which influence Batman routing performance. It was also to
demonstrate failure modes of the studied protocols.
DTN2 and IBR-DTN are two famous delay tolerant tools and Michael Doer-
ing [12], research on efficiency IBR-DTN compare to DTN2 when implemented
on embedded system. Their investigation shows that IBR-DTN is an efficient
system for embedded implementation.
D. Combine Batman-adv and IBR-DTN

In this research, we wanted to make the combination of IBR-DTN and
Batman-adv. This combination to make Batman-adv could run together with
IBR-DTN. Before that, we had to make sure that batman interface (bat0) could
detect another node.
This combination would be very useful when our node operate on challenge
network area. By using IBR-DTN the data would be kept in storage if node cannot
connect to another node and would forward the data when the node could get
a network connection to another node.
F. Implementation
1) Evaluation
The goal of our evaluation was to assess the ability of Batman-adv on wireless
mesh network in real condition and to know the IBR-DTN performance on
Batman-adv protocol. Routing protocol performance could be seen from the
indication of packet loss, delay, throughput and another aspect. These conditions
were influenced by multiple parameters, and we would discuss and consider the
location of communication. The time at which communication was taken also
can influence the performance of routing protocol.
2) Experiment Design
To get valid result in our research we made design of experiment as actual condi-
tion. We built a test bed in the storey building which put node on different room.
Table 1. Experiment Design

Hardware Nodes Asus core i3, Lenovo corei5, Compact corei3
Ubuntu 12.04 i386
Operating System
Bat0 IP and Mac
- node 1 192.168.10.20 / 6c:71:d9:7a:37:99
- node 2 192.168.10.30 / 48:d2:24:ca:d9:62
- node 3 192.168.10.50 / 00:08:ca:b1:58:b4
- node 4 192.168.10.40 / d0:df:9a:23:b4:31
Scenario 1 One node in the 3rd floor, one node in the 2nd floor, 2 nodes
in the 1st floor, and all node in fix position
in the 1st floor and both of node mobile
in the outside building (outside range), and all moving to the
building/wifi range
160
140
120
500 byte
100 750 byte
1000 byte
80 2000 byte
5000 byte
60
10000 byte
40 50000 byte
20
0
Max Delay (ms) Min Delay (ms) Rountrip Avg delay (ms)
Figure 2. Nodes Positioning

Figure 3. Delay Transmit Data From Node 1 to Node 4

We make 3 scenarios to check performance of Batman-adv and IBR-DTN on
wireless mesh communication.
Scenario 1, in this trial all nodes in fix position and separate in 3 locations.
After activated Batman-adv then try to send data packet from node 4 to node 1.
Based on standard TIPHON version [13], the performance of delay can be
classified as below table.
Refer to Table 2 condition showing that performance Batman is very good.
Scenario 2, in this scenario we want to know routing characteristic of
Batman-adv, and make test route from node 4 to node 1. Before checking route
we check potential next hope information from another node by using “batctl o”.
Nodes send OGM to inform potential next hop to neighbor node. After that
we check route transfer data as below route.
Table 2. Delay Category
Category Delay (ms) Indexs
Very good <150 4
Good 150 ~ 300 3
Fair 300 ~ 450 2
Poor >450 1
Figure 4.: Potential Next Hope Position
Figure 5.: Trace Route
Scenario 3, in this experiment node 3 and 4 are mobile until go outside of

wireless coverage area node 2 and 1, this condition node 4 can only connect to
the node 3.
On “btctl o” neighbor checking we will see only node 3 which detected by
node 4. After that node 4 try to send data to node 1 although node 4 cannot detect
node 1, and node 4, 3 and 2 already setup IBR-DTN to handle connection less
Figure 6. Nodes 3 & 4 moving more than 50 meter from building
Figure 7. Transfer data process
condition. When sending data process is occuring we are also walking toward to
the wireless mesh coverage are of node 1 and 2. This condition also influences
the data sending process from node 4 to node 1.
If we see the graphic, it shows the process of transfer data from node 4 to the
node 1. Refer to above graphic that we sent three time data from node 4 to node
1 (500 byte, 1,000 byte and 1,500 byte). In the first experiment with sending
500 byte data, the data cannot be received in the node 1 because there is no
connection between node 3 and node 2, but the data is kept on storage node 3
by using IBR-DTN mechanism. The data forward by node 3 to node 2 after there
is connection again, and it is happened on the 38th sequence. This IBR-DTN
mechanism is also happen second data sending process (1,000 byte) and third
data sending process (1,500 byte). On second data sending process, IBR-DTN
forward mechanism is happened on the 21th sequence and the 39th for third data.
IV. Conclusion
In the following we present the result of our measurement. We expect that
batman-adv is showing very good performance in relation to delay result. It can
see from maximum delay measurement only 41 ms. Refer to standard TIPHON,
that delay with 41ms measurement result is in the very good range. However, we
see that deviation of delay ratio need to be a concern. Beside that a the quantity
data transfer also influence to the delay ratio and sending data stability. It is needed
to improve research with also consider to environment condition.
The number of nodes needs to be added to get more valid data in trace route
testing. Although Batman-adv has tools and library to inform user easily about
next hop priority.
Applications of Batman-adv and combining with IBR-DTN have been work-
ing. It can see from data transfer result. But in the installations process we also
have take attention to the compatibility Batman-adv version and Ubuntu kernel.
Beside that some of Ubuntu version cannot install IBR-DTN. We also want to
continue this research with implementation this result in the embedded system.
Because it will take more advantages for communication in disaster condition.
V. Acknowledgment
This work supported by IR2C Lab project and DIKTI Funding Scholarship
(BPPDN). It will be the basis for further research application of delay-tolerant
and batman-adv.
VI. R eferences
1) Fall, Kevin. (2003). A Delay Tolerant Network Architecture for Challenged
Internets. SIGCOMM ’03, New York, NY, USA: ACM 2003, 27-34. Available
at http://doi.acm.org/10.1145/863955.863960
2) Bulut, Eyuphan. (2011). Opportunistic Routing Algorithm in Delay Tolerant
NetworksPPORTUNISTIC ROUTING ALGORITHMS IN DELAY TOLER-
ANT NETWORKS. Rensselaer Polytechnic Institute Troy, New York.
3) Zhao, Wenrui. (2006). Routing and Network Design in Delay Tolerant Networks.
Georgia Institute of Technology.
4) Seither, Daniel, Konig, Andre, and Matthias Hollick. (2011). Routing
Performance of Wireless Mesh Networks: A Practical Evaluation of BATMAN
Advanced. TechnischeUniversit,Darmstadt, Bonn.
5) Laurent, Delosieres. (2012). Simin Nadjm-Tehrani, BATMAN Store-and-
Forward: the Best of Two Worlds. Linkoping University SE-581 83, Linkoping,
Sweden.
6) Warthman, Forest. (2003). Delay-Tolerant Networks (DTNs): A Tutorial v1.1.
[Online]. Available http://www.dtnrg.org/ docs/tutorials/warthman-1.1.pdf.
7) André, Rein. (2012). TrustMANET Development and Evaluation of a Trusted
Mobile Ad-hoc Network. FachbereichMathematik, Naturwissenschaften und
Informatik der TechnischenHochschuleMittelhessen
8) Babu, Karisma, Cortes-Pena, Luis Miguel, Shah, Prateek, and Shivaranjani
Sankara Krishnan. (2007). Wireless Mesh Network Implementation. School of
Electrical Engineering Georgia Institute of Technology Atlanta, GA.
9) Ricardo, Pinto. WMM - Wireless Mesh Monitoring. [Online]. Available: http://
www.gsd.inesc-id.pt/~ler/reports/ricardopinto-midterm.pdf. Retrieved June
2014.
10) Abolhasan, M., Wysocki, T., and E. Dutkiewicz. (2014). A review of routing
protocols for mobile ad hoc networks. Ad Hoc Networks 2(1),1–22
11) Lindner, M., Eckelmann, S., Wunderlich, S., et al.The B.A.T.M.A.N. Project.
[Online]. Available: http://www.open-mesh.org/. Retrieved May 2014.
12) Doering, Michael Lahde, Sven, Morgenroth, Johannes and Lars Wolf. (2008).
IBR- DTN: An Efficient Implementation for Embedded Systems. Institute of
Operating Systems and Computer Networks, TechnischeUniversitätBraun-
schweig
13) Tiphon. (1999). Telecommunications and Internet Protocol Harmonization
Over Networks (TIPHON) General aspects of Quality of Service (QoS). DTR/
TIPHON-05006 (cb0010cs.PDF).
Development of a programmable
multipurpose forced convection type dryer
Emeline C. Guevarra*
Cavite State University
Indang, Cavite, Philippines
Abstract
Drying is considered as one of the effective methods of food preservation to prolong the storage
life of foods. This study is focused on the development of a programmable multipurpose forced
convection type dryer which is designed to dry agricultural crops. The hardware of the system is
composed of a drying chamber, heater, blower, buzzer, PIC16F877 microcontroller, liquid crystal
display, temperature sensor, and keypad. PBASIC is the language used in the development of
the software which interfaces the microcontroller with the dryer. Testing of the system was done
with banana, cassava, and malunggay leaves which are loaded to the dryer by batch. Results
showed that the microcontroller is able to control and monitor the temperature inside the drying
chamber. The heater automatically stopped once the desired temperature is reached while the
blower continuously blows the air inside the chamber. Blower and heater automatically shut off
when drying time is reached.
Key words: Artificial drying, Drying, Food dryer, Microcontroller, Temperature sensor.
I. Introduction
Drying is considered as the oldest method of preserving foods and also maintains
the best quality of the product by lowering the moisture content so that molds
and spoilage organisms cannot grow. The method can be traced back since ancient
times where Egyptian tribes preserved fish, meat, vegetables and fruits by drying
and salting. The common practice is to use a safe place to spread the food where
dry air can pass over while others drape foods on branches or on wide shallow
baskets on the roof.
Nowadays, there are several methods of drying, such as convective or direct
drying, indirect drying, dielectric drying, super critical drying, freeze drying and
natural drying. Among these methods, the most common is natural drying where
unheated forced air is used. This method of drying is slow and weather dependent
and may last from days to several weeks. This leads to the development of dryers
which can reduce the moisture content level of foods in a short period of time.
* Tel: (0915)949-1578. E-mail: emi_guevarra@yahoo.com
377
Through the development of a programmable multipurpose forced convection

type dryer, the temperature and relative humidity inside the drying chamber
is controlled. The dryer monitors and maintains the needed temperature of
agricultural products inside the drying chamber which leads to the shortened time
of drying. Small scale farmers or backyard farmers of the agricultural sector might
benefit in using the dryer since excessive production losses during harvest may
be prevented. Harvested agricultural products can be dried anytime regardless of
varying weather conditions producing better quality of agricultural products since
the growth of molds and microorganisms which cause food spoilage are reduced.
II. method/material
This study employed prototyping technique. Prototyping is a technique that proves
the effectiveness of the design, the computations, and the concepts applied, by
making an actual machine. It attempts to develop the existing machine into more
substantial purposes. Prototype model allows the identification of materials used
in the development of the system. The model also allows the simulation of the
design in terms of its functionality.
A functional or working prototype was constructed to simulate the functional-
ity of the system. Software is developed to interface the microcontroller with the
drying chamber.
Data gathering is considered to identify the different requirements needed
in the development of the system. This involves gathering data from reference
materials, such as books, researches both published and unpublished, and
literatures from the internet. Observation of existing dryers is also considered.
Figure 1 shows the system block diagram, which enables easy identification
of the different components to be used in the development of the system. This
identifies the specific component which will transmit and receive signals from
different components of the system. The hardware components used in the devel-
opment of a programmable multipurpose forced convection type dryer include
the drying chamber, fan or blower, heater, microcontroller circuit, temperature
sensor, Liquid Crystal Display (LCD) and keypad.
Heated air needed for the drying process is supplied by the heating element
within the dryer. The heat from the heating element is transferred through the
forced convection process with the help of the blower. Internal temperature is
regulated and monitored by the microcontroller through the use of a temperature
sensor.
The main function of the program is to monitor the temperature readings
of sensors and to control the proper operation of the blower and heater. Based
on the readings from the sensors, the proper operation of heater and blower is
Figure 1. System Block Diagram
considered to maintain the necessary temperature requirements for the selected

crop subjected for drying. The program is also responsible in stopping the drying
process automatically.
The machine is tested if it functioned as planned. The microcontroller is
also tested if it controls the overall operation of the dryer. Proper monitoring of
temperature inside the chamber is considered to achieve the desired output. The
system automatically shut off once the drying process has ended.
Banana, cassava and malunggay leaves are used as samples in the drying
process. Crops are cut into smaller pieces and scattered on the drying trays by
batch system, only one type of crop is dried at a certain period of time. Malunggay
leaves are removed from the stalk before it is scattered on the drying trays. Three
trials of test are conducted to get the initial moisture content of each sample and
the proper time setting for the drying process. The initial moisture content of
each crop is determined using the gravimetric method. Weights are monitored
and the initial moisture content is computed using equation (1).
MC = [(Wi - Wf ) / Wi][100%] (1)
Drying time is defined as the residence time of the sample inside the drying
chamber during the drying process. Time is determined through monitoring the
initial and final weights of the sample followed by the computation of the initial
and final moisture contents after every hour. Constant final weight record for
three consecutive readings determines the safe moisture content level at which
the crop is safe for storage.
The prototype is evaluated by experts in the field of crop processing and
machine designers. The evaluation is focused on the microprocessor’s capability
to control the temperature inside the drying chamber. For checking, preset
temperature values and read out values during the drying process is considered.
Readings are displayed on the LCD.
III. R esults and Discussions

The programmable multipurpose forced convection type dryer shown in figure 2
was made up of different components which make it capable of drying different
crops based on the samples temperature requirements at specific time.
A. Construction of the Machine

The project was composed of a drying chamber, microcontroller, relays, buzzer,
blower, heater, liquid crystal display (LCD), temperature sensor, keypad and
power supply. The input and output ports of the microcontroller were configured
to control the operation of the whole system using the software while the
Figure 2. The Programmable Multipurpose Forced Convection Type Dryer

temperature, time of drying and selection of crops were set using the keypad.
The temperature inside the drying chamber was monitored through the use of a
temperature sensor and relays were used to control the operation of the blower
and heater. Finned tube provided the needed heat while the blower blew the hot
air inside the drying chamber. Likewise, the buzzer was used to indicate that the
time for drying had ended while the LCD displayed the menu for the operation
of the dryer. The power supply was responsible for providing the necessary voltage
needed by the system.
The microcontroller switched on the blower and heater as soon as the crop to
be dried and its corresponding weight had been selected or the drying temperature
and drying time had been defined. The dryer automatically shut off once the set
time had elapsed.
The drying chamber was constructed using stainless steel, fiber glass, heat
resistant gasket, hinges, angular bars, screen and metal footings. The main
components of the chamber were the blower and the heater. The blower was a
4-pole motor which operates at 208V, 60 watts and 60 Hz. The main component
of the unit includes PIC16F877, where the software for the system was stored.
The microcontroller had five ports: Port A, B, C, D and E. Port A had 6 terminals,
Port B, C and D had 8 terminals each while Port E had 3 terminals. Ports A and B
were used as input terminals while Ports C and D were used as output terminals.
Pin 2 of Port A was used as input line for the signal coming from the
temperature sensor while pins 33, 34, 35, 36, 37, 38 and 39 of Port B were used
as input lines for the signal coming from keypad. Additionally, pins 15, 16, 17,
18, 23 and 24 of Port C were used as output lines for the signal coming from
the microcontroller to the LCD. Moreover, pins 19, 20 and 21 of Port D were
connected to the relay and pins 13 and 14 were both connected to 4-MHz crystal
oscillator, which was connected to two 27-pF capacitors for the oscillation of clock
in and out timing. The crystal oscillator was used for attuning the speed of the
timer in the microcontroller. Furthermore, pins 12 and 31 of the microcontroller
were connected to the ground and pins 32 and 11 were connected to the power
source.
The transformer, bridge rectifier diode, capacitors and the 7805 integrated
chip were the main components that comprised the power supply. The stepped-
down transformer was used to lower the voltage needed by the system from 220
VAC to 9VAC while the bridge diode converted the AC voltage to DC voltage.
Through the use of a capacitor, the unregulated output voltage had been able to
reach 12 volts which powered the buzzer and relays. Passing through the 7805
IC, the voltage was regulated to 5V and was used by the microcontroller, LCD,
and moisture sensor.
The development of the system software involved several tasks. The first
part of the program declared the 4MHz crystal oscillator, the port and the pin
assignments of the LCD. Input and output ports were initialized together with
the different variables used in the program. Subroutines for the crop selection,
entry of weight, starting of dryer, starting and stopping the blower and heater and
turning on the timer were also included. After the compilation of the program, it
was then written to the microcontroller through the use of a development board.
B. Operation of the Machine

Figure 3 shows the words “Programmable Crop Dryer” that served as a welcome
message for the user of the system which was then followed by the main menu
shown in figure 4. The main menu offered two choices that includes the selection
of predefined crops, option 1. The suggested temperature for predefined crops
was included in the program. The user just need to select the crop to be dried
and then provide the weight of the crop to be dried. For user define in the main
menu, the user had to enter the needed drying temperature of the crop together
with its drying time. Set time continue to decrement until it reached zero (0)
which indicated that the set drying time had ended. Figures 6, 7 and 8 show the
menu for temperature, hour and minutes setting.
Figure 3. The welcome message

Figure 4. The main menu
Figure 5. Menu for Temperature Setting
Figure 6. Menu for Hour Setting

Figure 7. Menu for Minutes Setting
The machine was tested if it functioned as planned. The microcontroller

was tested if it controls the overall operation of the dryer. Proper monitoring of
temperature inside the chamber was considered to achieve the desired output.
The system automatically shut off once the drying process had ended.
Testing and Evaluation

Testing procedures include several preparations, such as the preparation of samples
of selected crops for drying, the determination of actual moisture content of each
crop, the actual drying process and the determination of final moisture content of
each crop. Testing whether the microcontroller was able to control the operation
of sensor and regulates the temperature to achieve the desired output was also
considered. The evaluation of the system focused on the control of temperature
sensor, blower and heater. The microcontroller automatically controlled the
internal temperature during the drying operation.
Banana, cassava and malunggay leaves were used as samples in the drying
process. Banana and cassava were cut into small pieces while malunggay leaves
were removed from the stalk. The chopped banana and cassava as well as the leaves
of malunggay were scattered on the drying trays by batch system, where only one
type of crop was dried at a certain period of time. Figures 8, 9 and 10 show the
banana chips, cassava chips, and malunggay leaves for drying.
The initial moisture content of each crop was acquired through the use of
gravimetric method and three trials for each crop were conducted. Table 1 shows
the average initial moisture content analysis of banana, cassava and malunggay
leaves. Banana contained an average initial moisture content of 66.37%, cassava
had an average initial moisture content of 58.74% while malunggay leaves had
an average initial moisture content of 69.02%. The differences of values of
Figure 8. Banana chips for drying
Figure 9. Cassava chips for drying
Figure 10. Malunggay leaves for drying

Table 1. Average Initial Moisture Content of Banana, Cassava and Malunggay Leaves
Initial Wt. Final Wt.. Moisture
Crop Temp. (ºC) Time (mins)
(grams) (grams) Content (%)
Banana 70 1000 336.30 70 66.37
Cassava 70 1000 412.60 30 58.74
Malunggay 60 200 61.97 30 69.02
Table 2. Average Drying Time of Banana, Cassava and Malunggay Leaves

Crop Time (mins) Initial Moisture (%) Final Moisture (%)
Banana 70 66.37 12.70
Cassava 70 58.74 13.02
Malunggay 30 69.02 12.37
initial moisture content were accounted on the reason that samples had different
moisture content depending on the time when the samples were harvested. Freshly
picked crops contained higher moisture than crops that were not immediately
consumed after their harvest. Table 2 shows the drying time of banana, cassava,
and malunggay leaves. Banana chips were dried using 70°C within 70 minutes,
cassava chips were dried using 70°C within 30 minutes while malunggay leaves
were dried using 60°C within 30 minutes. Also, table 2 reflects the average final
moisture content of banana, cassava and malunggay leaves. Banana contained
an average final moisture content of 12.70%, cassava contained an average final
moisture content of 13.02 while malunggay leaves contained an average final
moisture content of 12.37%. The differences in values were due to unbalanced
distribution of moisture content throughout the sample. The final moisture
content of banana, cassava and malunggay leaves were considered safe for storage
since the moisture contents were within the range of 12% to 14%. As discussed on
the book The Storage of Tropical Products 4th edition by Jelle Hayma, crops dried
to 12% to 14% moisture were free from fungal growth. Also, it was mentioned
that when a product’s moisture content was equal to or below the safe moisture
content, the danger of attack by bacteria and fungi is negligible.
Pre-heating time of dryer was also taken into consideration during drying
operations. Warm-up time of dryer varies depending on the temperature of the
room where it was located. As observed, the pre-heating time of dryer in the
morning and at night was slower compared to pre-heating time at noon. This
was due to the fact that if the air in an area was hot, the humidity is low. The air
will have more capacity to hold water vapor thus the rate of evaporation will be
faster. On the other hand, cold air has less capacity to hold water vapor than hot
air, and the rate of evaporation of water is slower.
The cost of construction of the whole system including the labor cost in the
fabrication of the machine amounted to P 18, 786.00.
IV. Conclusion
Based on the results of the study, the microcontroller controlled the whole system
through the use of a program developed using PBASIC and the temperature inside
the drying chamber is monitored using a temperature sensor. Also, the system
regulated the temperature to achieve and maintain the desired temperature and
the time of heating for each crop which is included in the program. Moreover,
the controlled temperature is enough to dry the chosen crop since dried crops
contained 12%–14% moisture–the moisture content level for safe storage of crops
for six (6) months in tropical countries. Furthermore, the temperature of an area
where the dryer is located affects the pre-heating time of dryer.
A. Recommendations
For future development and enhancement of the programmable multipurpose
forced convection dryer, the following are recommended.
1. To use a more direct moisture content sensor that will sense the moisture
content of the crop during the process maybe used.
2. To use a device which can monitor the weight of the crops during the drying
process since weight establishes a relation between moisture content. Based on
the assumption, the user will set the desired moisture content level; a software
can then be developed to perform/compute for the desired moisture content
level before starting the drying process based on the monitored weight.
3. To add a silicon rubber gasket to minimize heat losses thus increasing efficiency.
4. To add an additional tray to accomodate additional crops/load.
V. Acknowledgement
The author wishes to extend her sincerest gratitude and appreciation to the
following individuals who contributed much to the completion of this study:
Dr. Ronaldo A. Juanatas, Dr. Gisela V. Rolluqui, and Dr. Melito Baccay, faculty
members of Technological University of the Philippines, for their invaluable
guidance and suggestions;
Dr. Divinia C. Chavez, President of Cavite State University, for her encourage-
ment and support;
To those who were not mentioned but has contributed much to the success
of this study;
Most of all, to Almighty God for His countless blessings and never ending
love that led to the fulfillment of this study.
VI. R eferences
1) Ararao, R.P. (1994). Design and Evaluation of a Multipurpose Dryer Using
Agro-waste as Fuel. Unpublished Thesis. CvSU, Indang, Cavite. p. 6
2) Hall, C.W. 1980. Drying and Storage of Agricultural Crops. The AVI Publishing,
Inc., Westport Connecticut.
3) Lopez, N.L. (1992). Developement of Heat Exchangerfor Use in Multipurpose
Dryer. Unpublished Thesis. CvSU, Indang, Cavite. pp. 13–15.
4) Nolan, P.J. (1993). Fundamentals of College Physica, 433-444 Wm. C. Brown
Communications, Inc.
5) Odigboh, E.U. (1976). Mechanization of Nigerian Cassava Production and
Processing. pp. 1–10.
6) Woodroof, J.G. (1979). Coconuts: Production, Processing and Products, 2nd
Edition. The AVI Publishing Company, Inc., Westport, Connecticut. pp.
102–107.
7) Solar Dryers. Retrieved on January 15, 2010 from http://en.howtopedia.org/
wiki/How_to_Preserve_Food_with_a_Solar_Dryer
8) Microchip PIC16F87X DataSheet. Retrieved on January 15, 2010 from http://
www.ajpic.zack.pl/processing/pdf/pic16f87x_ds.pdf
A Study of Network Speech Recognition
using TCP
Asril Jarina, *, Kalamullah Ramlia, Suryadib

a
Department of Electrical Engineering, Faculty of Engineering, University of Indonesia
Depok 16424, Indonesia
b
Department of Mathematics, Faculty of Mathematics and Natural Sciences, University of
Indonesia
Abstract
Network Speech Recognition (NSR) puts the whole recognition system in the network. The client
sends the speech data to the server where recognition is carried out. The NSR over IP network
or Internet will suffer degradation caused by packet loss and delay. TCP as a reliable transport
protocol can be used by NSR to solve the packet loss but with timeliness failure.
In this paper, we study the streaming model using TCP in order to get the satisfactory performance
of NSR in terms of timeliness factor. The timeliness factor refers to an acceptable delay where
the whole packets should be ready available for recognition on the server.
Key words: Network Speech Recognition, Performance modeling, TCP streaming, Acceptable delay.
I. Introduction
According to Peinado et al. [1], application of speech recognition implemented
on network is called as remote speech recognition (RSR). In RSR, a local device,
such as telephone, mobile phone, smart phone and personal computer, can send
speech signal or parameters over network to a remote-server that has speech
recognition engine. Thus, RSR will have several advantages, namely a simplicity of
client devices, language portability and simplicity for updating and maintenance.
As shown in Figure 1, there are two approaches to implement RSR on network:
network speech recognition (NSR) and distributed speech recognition (DSR). The
first approach resides the whole recognition system in the network from the
client’s point of view. In this approach, the client sends the speech signal to the
server where recognition is carried out. The client can optionally employ a speech
codec to compress the speech in order to make a low bitrate transmission. In the
second approach, the client has a local front-end that extracts features from the
speech signal used by server to perform recognition [1].
* Asril Jarin.Tel: +62-82114942530. E-mail: asril21@ui.ac.id
389
Figure 1. Two implementation schemes of RSR
In fact, DSR has advantages in robustness of system, since it does not

implement speech codec and it needs small bandwidth due to small feature size.
However, NSR might be more suitable for applications that need original speech
data, such as remote meeting transcription. The original data will be used by
user for correcting the sentences resulted by recognition engine. The presence of
original data can be as well as proof of the correction. Although the speech can
be reconstructed from the features by using a speech synthesizer, the original
speech data received from network is still better than the reconstructed speech.
In this paper, we are interested to study the NSR application that can receive
the whole original speech data over IP network. Implementation of NSR on IP
network is not easy since IP network is an unrealiable network. Speech recognition
on server will suffer degradation caused by packet loss or packet delay. Moreover,
most of NSR applications using speech codec experience significant degradation
of recognition due to the error propagation after packet loss occurred [2]. The
speech codec generates interframes that have dependencies each other. If a packet
loss is occurred, then it will cause information loss in decoding.
On the other hand, NSR application that does not provide an interactive
service prefers to choose packet delay than packet loss. The availability of the whole
data will lead a better speech recognition on the server. However, in perspective
of user, the delay should be reasonable, in another word, the application should
have an acceptable delay. We assume, if all packets of NSR are arrived at the time
of the acceptable delay at the server, then we could get the satisfactory performance
of the application.
TCP as a reliable transport protocol that can ensure the data transmission
without loss, but no delay can be used by NSR application to achieve its satisfac-
tory performance. Additionally, TCP is already used by popular multimedia
streaming applications such as Skype and Windows Media [18].
In order to study the performance NSR using TCP, we develop an approximate

streaming model of NSR using a specific model of TCP. We set a simplified
problem of NSR application that recognize the speech signal in utterance-by-
utterance. An utterance is defined as a speech segment that is bounded by speaker
pause. We study further the correlation between media streaming for NSR and
multimedia streaming application using TCP. Based on the study, we explore a
mathematical model that describes the behavior of the streaming of NSR using
TCP, such as the fraction of the late packets and the length of delay compared
with the acceptable delay.
The rest of this paper is organized as follows. In Section II, we describe the
NSR performance over IP network, streaming using TCP and their related work.
In Section III, we propose an approximate streaming model of NSR using TCP.
Section IV concludes our study.
II. NSR Performance and TCP Streaming

A. NSR Performance
Network Speech Recognition (NSR) is one of the implementation approaches of
remote speech recognition (RSR). The client sends the speech data to the server.
The server employs an automatic speech recognition engine (ASR) to decode the
speech data into sentences.
The performance of speech recognition of NSR is generally measured based
on its accuracy and its response time. To measure the accuracy, there are several
known measures, namely error rate (ER), percent correct (PC), word error rate
(WER), and word accuracy (WAcc). ER is usually used to measure the accuracy
of the isolated word recognition system (IWR), while the other measures are used
to measure the accuracy of the continuous speech recognition systems (CSR).
While the response time is measured by the length of delay from the time the
speech is spoken up to the time the speech is recognized into sentences.
The accuracy of NSR would have significant degradation caused by three
sources from network: speech coding, packet loss and jitter [5]. However, jitter
would not affect NSR if it doesn’t need timeliness requirement.
Focusing on degradation caused by packet loss, Perkins et.al. [6] classifies the
solution in two set of techniques to overcome packet loss on streaming audio over
IP network: sender driven techniques and receiver based techniques.
In order to improve the performance due to packet loss and speech codec,
Carmona et al. [2] implements two combination techniques: the first technique
develops a robust speech coding scheme against packet loss by combining Coded
Excited Linear Prediction (CELP) and Internet Low Bitrate Codec (iLBC), and
the second one improves the speech recognition accuracy by using feature extractor
to replace the features affected by packet losses.
Additionally, Carmona et al.[3] also introduces a packet loss concealment
(PLC) algorithm based on a cepstral compensation technique and linear
interpolation.
B. TCP streaming
TCP, the most popular transport protocol in the Internet, is conventionally
regarded as not suitable for media streaming. Multimedia streaming application is
typically sensitive to delay, but it can tolerate packet loss. For most of multimedia
application using RTP/UDP protocols, packet loss can be restored or concealed
by a number of techniques as classified by Perkins et al. [6].
Multimedia streaming application using TCP will have drawbacks due to
TCP mechanisms. As more described in literature [14], TCP is known as a
window-based protocol using mechanisms to control its sending rate in response to
network congestion. The mechanisms, such as timeout and congestion avoidance,
will vary the throughput and eventually will impact the media quality and media
playback [8].
Based on Wang et al. [7], multimedia streaming applications may get good
streaming using TCP when the achievable throughput is roughly twice the media
bitrate, with only a few seconds of startup delay. They determine the performance
of TCP streaming by knowledge of the fraction of late packets. Additionally, Wang
et al. use TCP models [10] and [11] as baseline to develop Markov models for
live and stored media streaming using TCP. Two reasons for using Markov models
are as following. First, they capture the detailed congestion control and avoidance
mechanisms in TCP including the mechanisms of the triple duplicate packets
and timeout. Secondly, they can be used in order to perform transient analysis
for stored media streaming.
Yan et al. [8] develop an analytical framework that can assess the Quality
of Experience (QoE) of multimedia streaming using TCP in terms of playback
continuity and timeliness. This analytical framework could help to estimate the
frequency of buffer overflow or underflow events if buffer sizes and the initial
buffering delays are known parameters, or conversely, to dimension the buffer
and delay appropriately.
Brosh et al. [17] develop an analytical performance model for delay of TCP.
They focus on real-time application characterized by timely and continuos data
delivery for VoIP and live video streaming.
Our work deals with NSR application that sends media data in utterance
by utterance using TCP. Thus, our work has correlation with the work of Wang
et al. [7] in terms of stored media streaming. In this paper, we use further the
methodology of Wang et al. [7] to develop our model.
III. NSR Model using TCP

A. Problem Setting
Different from multimedia streaming application, which is characterized by
presence of a playback at the client, NSR application has a speech recognition
engine on the server, which may serve various users on the back-end, such as a
human user, translator machine or software applications.
In Figure 2 we set our problem focusing on NSR without speech codec. We
avoid distortions caused by speech codec as well as disregard its latency in order
to find a simple experiment model of NSR using TCP. The client records speech
signal of a human speaker via microfon. The signal is then digitized using analog/
digital converter (ADC) and cut by a segmentor into segment data based on the
number of of zero-cross, whose amplitude is identified under threshold. A segment
data is equal to an utterance and eventually, a speech consists of utterances or
segment data.
After segmentation, the client sends sequentially the segment data or utterances
to the server over TCP channel for recognition. A speech recognition engine (ASR)
begins to process the recognition once the whole packets of each utterance are
arrived in buffer of server. We assume that the server provides a large buffer that
is sufficient for every utterance arriving successively.
Figure 3 illustrates the NSR delay per utterance. It is defined as the time
length, which is spent by the system from the time the utterance is finished
spoken until the time the utterance is finished recognized into the sentence on
the server. We assume that NSR delay covers a transmission delay, a receiving
delay and a recognition delay of packets and we don’t consider all other delays
because they are relatively too small. As shown in Figure 3, an acceptable delay
is being an indicator to obtain the satisfactory performance of the application.
Furthermore, in order to model the streaming of NSR using TCP, let us first
understand the model of TCP.
Figure 2. Problem setting: NSR using TCP that sends utterance-per-utterance to the server
Figure 3. Delay of NSR in perspective of an utterance
B. Model of TCP
TCP is a window-based transport protocol that provides reliable end-to-end data
communication. It performs two main mechanisms to regulate its sending rate
in response to network congestion: timeout and additive-increase-multiplicative-
decrease (AIMD). Both mechanisms may give significant effect on the throughput
and also delay on the data delivery. More detail about these mechanisms can be
found in literature [14]. For every packet sent by the source, TCP starts a retrans-
mission timer and waits for an acknowledgment (ACK) from the receiver. The
duration of the retransmission timer (RTO) is based on an estimate of the running
average and variance of the round trip time (RTT). The retransmission will expire
(timeout) when the sender doesn’t receive an ACK for the corresponding packet
and there are no three duplicate ACKs (TD). When a timeout occurs, the packet
is retransmitted and the window size is set to one. The retransmission timer value
(RTO) for retransmitted packet is further set to twice the value of previous timer
value. TCP doubles the timer value for each subsequent to reduce the sending
rate and adapts to network congestion. This exponential-backoff continues until
the retransmitted packet is successfully acknowledged. In congestion avoidance,
the window size increases by one packet when all packets in the current window
acknowledged.
The above TCP behavior is described by a discrete-time-Markov model by
Padhye et al. [11] and Figueredo et al. [12]. The time unit is determined from
the length of a ‘round’. A round represents the back-to-back transmission of
the current congestion window. Once all packets in the congestion window are
sent, no more packets are sent until ACKs for some or all of these W packets
are received. The reception of the ACK marks the end of the current window
and the beginning of the next round. The length of a round is assumed to be a
round-trip-time (RTT). It is also assumed, that the round-trip-time is larger than
the time to send all packets in the window. Moreover, packet losses in different
rounds are assumed to be independent and packet losses in the same round are
dependent. If a packet is lost in a round, then all remaining packets in the window
are also lost. In addition, the impact of ACKs is assumed as negligible.
C. Streaming Model for NSR using TCP

As modeled in Figure 2, the client employs the segmentor to cut the speech signal
into segment data, which is regarded as an utterance. The client stores them
temporarily in a local storage and sends them one by one sequentially to the server.
This behavior indicates that the model denotes a stored media streaming model.
Based on stored media streaming model, we propose an approximate streaming
model for NSR per utterance as shown in Figure 4. S(t) represents the number
of packets sent with a constant rate by segmentor at time t. At the server side
(ASR-server), A(t) denotes the number of packets arriving at the ASR-server at
time t. A1(t) denotes that all packets of utterance-(1) arrive in an acceptable delay,
tA, while A2(t) denotes that the server receives all packets later than the acceptable
delay. The late time is assumed in amount of D seconds.
As shown in Figure 5, we determine the unit of a speech in ‘utterances’, and
the unit of utterance in ‘rounds’ that is assumed to be a RTT of length R seconds.
We consider a speech whose length is Ls utterances and an utterance whose length
is Lu rounds.
S(t) A1(t) A2(t)
N U
N
D
mA u =2
(1)
(2)
mu
N
A
u =2
N u =2
tA D Time
t
Figure 4. Approximate streaming model of NSR over TCP per utterance
Table 1. Key Notations

Notation Definition
mA Arriving rate of utterance in average and in acceptable delay ( packets
per second).
mu Arriving rate of utterance in average ( packets per second).
R Round trip time (seconds)
Ls Length of aspeech (utterances)
Lu Length of an utterance (rounds)
tA Acceptable delay in each utterance (seconds)
t Delay of u-th utterance (seconds)
D Time difference between tu and tA (seconds)
Nu Number of packets in an utterance
NuA Number of packets in an utterance that arrive in acceptable delay
NuD Number of late packets in an utterance
NrA Number of late packets in an utterance
fu Fraction of late packets in an utterance
Figure 5. representation of length of speech and - utterance
D. Fraction of Late Packets

Based on method of Wang et al. [7], we develop a fraction of late packets in an
utterance as one of measure to denote the delay of NSR per utterance,
N uD
f u := (1)
Nu
where the numerator and denominator correspond, respectively, to the number

of late packets in an utterance and the number of whole packets in an utterance.
Since a speech consists of Ls sentences, the fraction of late packets in a speech is:
∑ ∑
Ls Ls
N uD N uD
fS := u =1
= u =1
(2)
Ns ∑
Ls
u =1
Nu
where the numerator is the number of all late packets in a speech and the
denominator is the number of whole packets in a speech.
By knowing the number of packet in an utterance, we obtain:
N uD := N u − N uA (3)
If tu and tA are measured in ‘rounds’ and become Lu dan LA, where the number
of packets per round is mu.R, and if we assume also that the packets arrive at time
0, then we obtain:
N u = Lu ⋅ m u ⋅ R
N uA = LA ⋅ m u ⋅ R
(4)
N uD = ( Lu − LA ) ⋅m u ⋅R
N uD = LD ⋅m u ⋅R
where LD is the number of additional rounds after acceptable delay.
In order to model the number of late packets based on TCP model, we define
the average number of packets per round in acceptable delay.
N rA := m A ⋅ R (5)
Next we define Ni as the accumulation of impulse reward up to i-th round
[19], where impulse reward is defined as the number of more packets per round
to the average number of packets per round in acceptable delay, NrA.
We define Nil as the number of less packets. Nil Î {0,1, … , NrA}. Nil = 0
indicates that no packets is less in that round.
Based on method of Wang et al. [7], we obtain the fraction of late packet in
an utterance as following:
∑
Lu
E[ N il ] (6)
fu := i =1
Nu
We obtain the expected number of late packets:
N rA
E[ N il ] = ∑ k ⋅ P( N il = k ) (7)
k =1
P(Nil = k) is the probability of having k less packets in the i-th round. In

order to obtain the probability P(Nil = k), we define the number of packets that
must be followed in the i-th round, Nib. According to Wang et al. [7], we obtain:
0, Ni ≥ 0
N ib :=  (8)
− N i , Ni < 0
Finally, we obtain P(Nil = k) as following:

 P ( N ib = k ), 0 ≤ k < N rA
P ( N il = k ) :=  (9)
 P ( N i ≥ N r ),
b A
k = N rA
Thus, we conclude that the number of less packets in i-th round is maximal
Nr and the number of followed packets in i-th round can be larger than NrA.
A
Therefore, P(Nil=NrA)=P(Nib³NrA).
Furthermore, in order to obtain the behavior of Ni, Wang et al. [7] provides a
table of impulse reward for four situations of TCP and Wang et al. [7] recommends
as well to use the TANGRAM-II modeling tool.
IV. Conclusion
This paper is about a study of network speech recognition (NSR) application using
TCP in terms of timeliness factor. We consider that packets arriving later than
an acceptable delay represent as timeliness factor that degrades the satisfactory
performance of NSR application. Based on performance model of multimedia
streaming application developed by Wang et.al [7], we develop an approximate
streaming model for an NSR application that recognizes the speech sequentially
utterance by utterance.
Based on our model, we conclude that the number of late packets exceeding
the acceptable delay of an utterance are caused by the smaller throughput than
the arriving rate of the utterance in an acceptable delay. This might be happened,
because there are frequent negative accumulations of the impulse reward in rounds
before the time of the acceptable delay.
For future work, we will evaluate our model in a simulation and real work
experimentation.
V. R eferences
1) Peinado, A. M. and J. C. Segura. (2006). Speech recognition over digital
channels. Robustness and standards, Willey.
2) Carmona, J. L., Peinado, A. M. and J. L. Pérez-Córdoba. (2010). Coded-
speech recognition over IP networks.Presented at FALA2010, Vigo, Spain.
3) Carmona, J.L. Peinado, A. M., Pérez-Córdoba, J. L., Gómez, A.M., and

J.A. González. (2010). Robust encoded speech recognition over IP networks.
Presented at SIMPE2010, Lisbon, Portugal.
4) Mayorga, P. Besacier, L. Lamy, R. and J. F. Serignat. (2003). Audio Packet
Loss Over IP and Speech Recognition. IEEE Automatic Speech Recognition
and Understanding Workshop, 607–612.
5) Sciver, J. Ma, J. Z., Vanpoucke, F., and V. Hamme. (2002). Investigation of
Speech Recognition over IP Channels.in Proc. ICASSP, IV-3812–IV-3815.
6) Perkins, C., Hodson, O. and V. Hardman. (1998). A survey of packet loss
recovery techniques for streaming audio. IEEE Network, 40–48.
7) Wang, B. and J. F. Kurose, P. Shenoy, and D. Towsley. (2008). Multimedia
Streaming via TCP: An analytic Performance Study. ACM Trans. Multimedia,
4, No.2, Article 16.
8) Yan, J. Muehlbauer, W., and B. Plattner. (2012). Analytical Framework for
Improving the Quality of Streaming over TCP. IEEE Trans. Multimedia, 14,
No.6. pp. 1579–1590.
9) Yan, J., Katrinis, K., May, M. and B. Plattner. (2006). Media- and TCP-
Friendly Congestion Control for Scalable Video Streams. IEEE Trans.
Multimedia, 8, No. 2. pp. 196–206.
10) Padhye, J., Firoiu, V., and D. Towsley. (1999). A stochastic model of TCP
Reno congestion avoidance and control. Tech. Rep. 99–02, Dep. Computer
Science, Univ. Massachusetts, Amherst.
11) Padhye, J., Firoiu, V., Towsley, D. F., and J. F. Kurose. (2000). Modeling TCP
Reno performance: a simple model and its empirical validation. IEEE/ACM
Trans. Netw, 8, 133–145.
12) Figueredo, D. R., Liu., B. Misra., V. and D. Towsley. (2002). On the
autocorrelation structure of TCP traffic. Comput. Netw. J. (Special Issue on
Advances in Modeling and Engineering of Long-Range Dependent Traffic).
13) Deller, J. Proakis, J., and J. Hansen. (1999). Discrete-Time Processing of Speech
Signal, Wiley-IEEE Press.
14) Stevens. W. R. (1994). TCP/IP Illustrated, 1: The Protocols. Addison-Wesley
Professional.
15) Kurose, J. F. and K. W. Ross. (2012). Computer Networking: A Top-Down
Approach 6th edition. Addison Wesley.
16) Jiang, W., and H. Schulzrinne. (2000). Modeling of Packet Loss and Delay and
their Effect on Real-Time Multimedia Service Quality. Proc. NOSSDAV2000.
Chape Hill, USA.
17) Brosh, E., Baset, S. A., Misra, V., Rubenstein, D., and H. Schulzrinne. The
Delay-Friendliness of TCP for Real-Time Traffic. IEEE/ACM Trans. Network-
ing,18, No.5. pp. 1478–1491
18) Baset, S. A. and H. Schulzrinne. (2006). An analysis of the Skype peer-to-peer
Internet telephony protocol. Proc. IEEE INFOCOM, Barcelona, Spain.
19) De Souza e Silva, E. and H. R. Gail. (1998). An algorithm to calculate
transient distribution of cumulative rate and impulse based reward, Stochastic
Models 14.3, 509–536.
Aural and Photonic Spectrum Based Digital
Pest Controller for Oryza Sativa (Rice)
Ivane Ann P. Banlawea*

a
Mapùa Institute of Technology
Muralla St., Intramuros, Manila, Philippines
Abstract
A research study was conducted in the rice fields of Western Philippines University, Aborlan,
Palawan from November 2013 to January 2014 to test a prototype of an alternative digital pest
controller for rice which eliminates the use of chemicals. Two modules were developed the day
module operation covered the determination of frequency (60Hz, 80Hz, 100Hz, 120Hz) that
attracts pests and to what sound intensity will they respond (20dB, 50dB, 80dB); the night
module operation determines which light color (white, yellow, blue, UV) and the light intensity
(10lux, 40lux, 80lux) that will attract the most number of insects. The results for the day module
operation showed that 80Hz attracted the most number of insects and that most of the insects
responded to 50dB. Rice bugs were more attracted to 80Hz while black bugs were more attracted
to 120Hz. For the night module, the most number of insects were attracted to UV, at 80 lux
intensity. The white light attracted rice bugs while white leafhoppers flocked mostly to UV, and
stem borers were inclined to yellow and blue. These results indicated that insects are diversely
attracted on different sound frequencies but are more inclined with modulated sound level and
are attracted to lights with short wavelengths on higher light intensities.
Key words: Insect pest control, sound frequency response, light color attraction, rice pests, alternative
digital pest controller.
I. Introduction
Rice is the staple food for most of the Filipinos. Throughout the years, farmers
have been using primitive devices and inorganic chemicals to reduce the pests that
attack and destroy the rice. Pests in rice are present from its germination stage up
to its final stage. Rice is more attractive to pest when it is at the panicle initiation
stage. However, there are some pests which are present in the whole life cycle of
rice plant. Rice pests contribute to 40% damage in rice crops.
Pest control practices that do not use inorganic chemicals can be a better
option to prevent pollution and degradation of the environment.
Light trapping has already been practiced by farmers; however, little studies
has been done on the color and intensity of light that would be more appealing
to insect pests. Also, most common studies regarding different sound frequency
* Corresponding Author.Tel: +63-9174552259. E-mail: ivaneannbanlawe@gmail.com
401
and sound levels focused on repelling the pests, there are yet few studies on the
attraction of these pests using the said factors, hence this study.
The main objective of this research is to make a prototype of an alternative
digital pest controller which eliminates use of chemicals. Two modules were
developed: the first module for day operation, and the second module for night
operation. The module for day operation covered the determination of frequencies
that attracted pests. The study determined the species of insects that are attracted
to the given frequency (60 Hz, 80Hz, 100Hz, or 120Hz). The module for night
operation included the determination of light colors (blue, ultra violet, white,
yellow) that attract pests, as well as the insect species that are more attracted to
a specific light color.
This study only covered pests which were available during the November
to January season of planting rice. It specifically applied to flying insects and
crawling pests that climbed the equipment. The illumination and the sound
frequency only covered a specific area and was limited up to only 80lux intensity
and 80dB sound level.

A. Machine Design
The daytime operation of this machine is shown in figure 1. The machine utilized
a Light Dependent Resistor (LDR) as the sensor, a PIC16F877A microcontroller
unit (MCU) for its switching operations and frequency generation for the day
module, a 15 watts 12V amplifier kit to modulate the sound level, an 8-ohm,
70 watts ceiling speaker as the attractant. The night time operation, shown in
figure 2, used the same LDR and MCU along with a 5W, 12V Cathode Compact
Fluorescent Lamp as the attractant and a 12V 4-inch diameter fan to push the
insect toward the collecting chamber.
The machine was made up of durable materials to ensure sustainability even
in adverse weather conditions. It was composed of a solar panel, fan, light bulb,
light activated sensors, and the circuit board which contained the microcontroller
unit (see figure 3. Diagram of connections).
The height of the machine was 9.67 ft, shown on figure 4. The machine was
designed to have adjustable height. Final adjustments were made during the
installation and pre-test. The machine was placed at the center of a plot so that
the area to be covered was larger than if it was installed at the side. The stand was
submerged into the soil and the support helped the machine to stand straight.
Figure 1. Block Diagram of Daytime Operation
Figure 2. Block Diagram of Night time Operation

1.0
SOLAR CONTROLLER 100K SPKR

SOLAR BATT OUT
1
2 DC IN
AMPLIFIER
1
LDR INPUT 2
(15 WATTS)
2
1
2
1
2
1
2
SPEAKER
1
1
J1
2
1
1 J4 VOLUME TREBLE BASS

2
1
2 1 J9
3 2 J5
DC IN
3
SOLAR
FREQ OUT
PIC16F877A
ADJUST 4 J6
5
DAY SW ITCH
J2 PIC prog
1
2
1
+
2
1
2
1
LDR
D3 LED-GREEN
CCFL FAN
BATTERY
7805
1
12V
kRPM
+88.8
J7 J8
VI
1 J3 D2 LED-YELLOW NIGHT SW ITCH

-
2 2
GND
1
CIRCUIT BOARD
DC IN
D1 LED-RED
VO
3
Fig.3. Diagram of Connections
Figure 4. Machine Body Diagram

B. Data Collection and Analysis

An area of one hectare was prepared by plowing and harrowing several times and
was planted by using the “sabog-tanim” method. The area was divided into four
plots corresponding to the number of machines used in the study.
A completely randomized design was employed in the study. There were four
sound frequencies and three sound levels for the day module, while there were
four light colors with light intensities used during the night module.
Data collection started on the Panicle Initiation Stage of the rice last November
17 and lasted until January 15 before harvesting. Insects that were accumulated
in the trapping chamber and those that are within 1 meter radius of the machine
were counted by pieces.
Data gathering was done daily with a cycle of each varying treatment (sound
intensity/light intensity) being implemented weekly until the end of the data
collection period. Sound intensity level was measured using a TES 1350A sound
level meter, and the light intensity was measured using an Alexan LX104 digital
light meter.
Descriptive analysis and Analysis of Variance (ANOVA) were used as the
statistical methods. There were two evaluations of data using descriptive analysis,
one for the day module, and the other for the night module but both had the
same analysis. The damage assessment data was also in descriptive form, while
the overall assessment for day and night operation was done using ANOVA.

A. Day Module
The actual numerical count of the insect and their corresponding percentages per
frequency, regardless of the sound level, as collected within the 9 weeks research
study is presented in Table 1.
Table 2 shows the population of pests per species under their corresponding
frequencies per treatment. Results convey that insects are more inclined to 50dB
sound intensity, with 80Hz frequency being flocked by the rice bug with an actual
count of 72 pieces, which included 6 observed cases of rice bugs mating, making
up 40% of the insect population in 50dB and 29.39% in the total count, and
with 120Hz frequency again by the black bug which accounts for 27.78% in
50dB and 20.41% in the overall population.
The table also implies that sound frequencies attract pests more than other
classification of insects. The population of pests attracted amounts to 95.51%
of the total population, while the predators make up 2.86% and the beneficial
insects are only 1.63%.
Table 1. Total Population of Insects (Day Operation)
Total Col- Highest Species Amount of Highest Specie

Percentage
Frequency lected Collected Collected
(pcs) % (SN) (pcs)
60Hz 31 12.653 Rice Bug 19
80Hz 115 46.939 Rice Bug 96
100Hz 15 6.122 Rice Bug 9
120Hz 84 34.286 Black Bug 60
Table 3. Population of Insects (in pieces) based on Sound Level of Each Frequency
Collected at
Frequency
20dB 50dB 80dB
60Hz 1 23 7
80Hz 0 76 39
100Hz 0 12 3
120Hz 1 69 14
The preference of insects regarding frequency and the sound level is shown in
Table 3. The least attraction occurred at the 80Hz and 100Hz frequencies at the
20dB sound level. These results infer that insects are attracted to varying sound
frequencies but are inclined to frequencies with modulated intensities.
Comparison of frequency by sound level was also done using the analysis of
variance and results showed that at 80dB sound intensity, 80Hz would be most
effective to attract insects, at 50dB sound intensity, 80Hz and 120Hz can both
be expected to yield significant outcome, and insects are not being attracted to
any frequency at 20dB sound intensity.
B. Night Module
Table 4 shows the population of pests per species under their corresponding
treatments of light color. It can be observed that during the 9 weeks of collection,
out of the 68 identified insect species, the lights attracted 38 different species of
insect pests.But with regards to population, the predators have the highest count
which make up 70.34%, the scavengers follow with 23.09%, and the pests only
amount to 5.39% of the total population, while the beneficial insects are only
0.13%.
Table 2. Population per Species (in pieces)

Intensity 80 dB 50 dB 20 dB
Classifica-
Name Frequency
tion 120 100 80 60 120 100 80 60 120 100 80 60
(Hz)
Beneficial soldier beetle 0 0 0 1 0 0 0 3 0 0 0 0
Pest saw fly 0 0 0 0 5 0 0 4 0 0 0 0
Pest black bug 10 0 15 2 50 2 3 1 0 0 0 0
Pest rice bug 3 1 24 3 14 8 72 15 1 0 0 1
Predator field cricket 0 1 0 1 0 0 1 0 0 0 0 0
Predator spider 1 1 0 0 0 2 0 0 0 0 0 0
Table 5 summarizes the total population of insects and their corresponding

percentage in different light colors regardless of the intensity, collected during
the 9 weeks of study.
Table No. 5 Total Population of Insects (Night Operation)
Highest Species Amnt of Highest

Total Collected Percentage
Color Collected Specie Collected
(pcs) % (SN) (pcs)
White 15007 4.815 Back swimmer 5411
Water scavenger
Yellow 26280 8.431 10462
beetle
Blue 13328 4.276 Back swimmer 6578
Ultra Violet 257084 82.478 Back swimmer 99561
Table 6 records the total counted pests in a specific light color under the
particular light intensity. These results infer that insects are more attracted to
lights with shorter wavelengths at higher light intensities.
Comparison of colors by light intensity was also done using analysis of vari-
ance, result denotes that among all the light colors, insects are attracted to ultra
violet light when it is glowing at 80lux, and remains to be the preferred color at
40lux and the attraction does not significantly vary in each color at 10lux light
intensity. Results of the comparison between the light colors at each light intensity
affirms the descriptive analysis that most insects, regardless of the intensity, are
significantly attracted to ultra violet which has a shorter wavelength as compared
to the three other light colors.
Table 4. Population per Species (in pieces)

Table No. 6 Population of Pest (in pieces) based on Light Intensity of Each Light Color
Collected at
Light Color
10lux 40lux 80lux
White 1671 4831 8504
Yellow 1198 2577 22505
Blue 1900 3002 8426
Ultra Violet 34873 99546 124940
C. Damage Assessment
Damage was assessed using the percent damage per panicle, and the yield per
square meter in grams. Random sampling was done on the four plots, and the
gathered data was presented in the following graphs.
Figure 5. Percent Damage per Panicle
Figure 5 describes the percent damage in a sample panicle. Five samples were
randomly selected from each plot and were assessed by counting the number of
damaged grains in a panicle, and these were converted into percentage values.
As the figure shows, the yellow bar which represents yellow light and 80Hz
combined has the highest damaged panicle in the four samples, with a mean of
5.58%, while the white bar representing white light and 120Hz combination
has the least damaged panicles, 3.25% on the average. Overall, the damage as
seen in the panicle was not as serious as the highest percentage was only 10%.
Figure 6 shows the yield per square meter in the gram unit. Three samples
were randomly collected for each plot and were assessed by dry weight sample
Figure 6. Yield per Square Meter in Grams
of grains. As presented in the figure, the yield samples were highest in the white
bar representing the white light and 120Hz combination with an average yield
of 297.67g, and the lowest yield on the yellow bar for yellow light and 80Hz,
averaging only 179.67g. This result coincides with the previous assessment that the
yellow bar has the highest damaged panicles and white with the lowest damage.
IV. Conclusion
As all the results imply, insects are diversely attracted to different sound frequencies
but are more inclined with modulated sound intensities and are attracted to lights
with short wavelengths on higher light intensities.
It is observed that attracted insects do not go inside the collecting funnel but
are below the funnel outside the collecting container. Thus, a collecting funnel is
not effective during day operation and there is a need to devise a more effective
catching system and other forms of attracting insect pests during the day.
The damage analysis in this study treated the gathered data as the effect of
the combination of the day and night module, thus the effect cannot be singled
out because only of the night or day module.
Since this study used only four frequencies (60Hz, 80Hz, 100Hz and
120Hz) and four light colors (UV, white, yellow and blue), another study could
be done using other available light colors and other sound frequencies to test
insect attraction and find out if other species which are not collected in the four
frequencies and light colors can be attracted to them.
Another further study could be about birds being repelled by sound frequencies
during the day. It was noted that birds do not attack the field while the study was
being conducted but it was evident that they were in nearby areas.
V. Acknowledgement
The author gratefully recognize Engineering Research and Development Tech-
nologies (ERDT) under DOST for funding the research, Western Philippines
University, Palawan for the lending the rice field, Engr. Pacifico Sariego III and
Engr. Ramon G. Garcia, the advisers, Prof. Alejandra Magay, the entomologist,
Prof. Sharon Rose Anunciado, the statistician, Dr. Jonathan W.L. Salvacion, the
dean, together with the whole faculty and staff of the School of Graduate Studies,
Mapùa Institute of Technology.
VI. R eferences
1) Way, M.J., Heong, K.L. (1994). The role of biodiversity in the dynamics
and management of insect pests of tropical irrigated rice a review. Bulletin of
Entomological Research, 84, 567-587
2) Norton, G. W., K.L Heong, J. David, and S. Savary. (2010). Rice pest
management: issues and opportunities. Rice in the Global Economy: Strategic
Research and Policy Issues for Food Security. International Rice Research Institute
(IRRI), Los Baños (Philippines).
3) Major Rice insect pests, their natural enemies and economic threshold levels.
Low-external Input Rice Production (LIRP): Technology Information Kit (IIRR,
292)
4) Graf, B, Lamb, R, Heong, K.L. and L.T. Fabellar. (1994). A simulation model
for the population dynamics of the rice leaffolders (Lepidoptera: Pyralidae)
and their interactions with rice. J.Appl. Ecol. 29, pp. 558-570.
5) Dasgupta, S, Meisner, C, and D. Wheeler. (2006). Is environmentally
friendly agriculture less profitable for farmers? Evidence on integrated pest
management in Bangladesh. Rev. Agric. Econ. 29(1):103-118.
6) Pollack, G. S., and I. Kazuo. (1999). Neural analysis of sound frequency in
insects, BioEssays, 21 (4), pp. 296 – 303
7) Lauresta, I.B. (2012). Evaluation of Electronic Rice Stink Bugs Attractant.
College of Engineering and Technology, Western Philippines University.
8) Subramanyam, B. (2001). Ultrasound and Arthropod Pest Control: Hearing is
Believing, Department of Grain Science and Industry. Kansas State University.
9) Ragudo, D.S. (2012). Design and Fabrication of Electronic Rice Pests Controller.
College of Engineering and Technology, Western Philippines University.
10) Baker, R.B. and Y. Sadovy. (1978). The distance and nature of the light trap
response of months. 1978. Nature 276, 818-821.
11) Ashfaq, M., Khan, R. A., Ahsan Khan, M., Rasheed, F. and S. Hafeez. (2005).
Insect Orientation to Various Color Lights in the Agricultural Biomes of
Faisalabad, Pakistan Entomology, 27 (1), 49-52
12) Jessica, P. and A. Curtis. (2001). Insect Response to different wavelengths of light
in New River State Park, Ashe County, North Carolina, USA.
13) Ferreira, M. T., and R. H. Scheffrahn. (2011). Light Attraction and Sub-
sequent Colonization Behaviors of Alates and Dealates of the West Indian
Drywood Termite. Florida Entomologist, 94 (2), 131-136
Development of Programmable Logic
Controller (PLC)-based Coffee Pulper for
Wet Process
Marlon R. Perenaa,*
a
Abstract
The study was a portable machine equipped with programmable logic controller capable of
pulping freshly picked coffee berries. A Programmable Logic Controller-Based Coffee Pulper,
developed for pulping a freshly picked coffee beans. It measures 42 inches in height, a length of
55 inches and 17 inches in width. The machine is limited to a fixed variety of coffee berries for
pulping which is Robusta variety.
The purpose of this study was to develop a programmable logic controller-based coffee pulper
for pulping locally produced coffee berries using the wet process. It was intended to support the
machines being used at the Coffee Processing Center of the Cavite State University and other
coffee farmers who are involved in coffee pulping activity. The evaluation process was done with
the help of different coffee farmers within Cavite and the IT experts respectively. The performance
of the machine was rated very good in terms of functionality, aesthetics, workability, durability,
economy, safety and saleability.
However, there were still some recommendations given in order to help the machine, such as
provision for sorting process of coffee beans from the unpulped and manufacturing a bigger size
of coffee pulper to accommodate more load and to improve the pulping activity.
Key words: Coffee, Coffee Pulper, Wet process, Programmable logic controller, Automation
I. Introduction
Coffee belongs to genus Coffea locally known as “kape”. It is considered one of
the most important beverages among Filipinos. Maybe this is the reason why it
became as one of the research trust of this institution. Coffee industry is also one
of the major industries that bring big income to the Philippines. The processing
of its fruit is a great industry by itself. Its potential for export is high and has
gained worldwide acceptance.
Incidentally, coffee production involves a very labor-intensive process. Coffee
travel to many places and through many sets of hands before it even gets to the
roaster. Grown on trees in mountainous, tropical equatorial regions, and harvested
* Marlon R. Perena.Tel: +639275817259. E-mail: dit0055mrperena@gmail.com
413
by hand, coffee starts out as a “cherry”. Processing is required to remove the pulp
to get the bean inside.
In the thesis entitled Testing and Evaluation of Coffee Pulper in Southern
Tagalog (2001), it was cited that there are technical aspect and economic aspect
of the pulping machine, it was emphasized that notable differences in the size
and shape of coffee berries are influenced by botanical variety and environmental
growth circumstances. Preferably, coffee berries should be graded according to size
before pulping. It wa also mentioned that the adjustment of the blades depends
on the variety of coffee since it has specific physical properties like size and shape.
However, if grading is not possible, the blade of the pulper should be adjusted to
pulp the bigger berries first. Then the coffee is passed through a sieve to separate
the pulped beans from the unpulped. When the blades were already adjusted
the unpulped coffee passed through the pulper again. Two low moisture content
(below 12% wet basis) makes the berries brittle resulting in broken beans. Too
high moisture content (above 16% wet basis) results in pressed broken beans,
overloading, and slowing down of the power source.
Any improvement in coffee processing is definitely a welcome technology to
the coffee producing farmers in the Philippines. To improve the quality of the
coffee beans and to minimize delay in processing, construction and developing
of the Microcontroller-based coffee pulper for wet process is an advantage. The
development of the machine, therefore, benefits small scale coffee producer since it
performs pulping and at the same time separating the pulp from the coffee beans.

This setion presents the project design, the conceptual model, project develop-
ment, operation and testing procedure, and evaluation procedure.
In project design, the programmable logic controller was used for the
automatic operation of the coffee pulping machine. It automatically pulps the
freshly picked coffee berries which is normally a long and tedious activity to the
coffee farmers. The project uses electrical and electronics materials. The procedure
undertaken during the fabrication and construction of the machine was based on
the design considerations shown in the isometric drawing below.
This project is a portable machine equipped with programmable logic
controller capable of pulping freshly picked coffee beans, refer to Figure 2 below.
It has made the coffee pulping activities a lot easier for the coffee farmers. The
prototype has four aluminum profile posts which serve as the foundation of the
mechanism, with necessary mounting fixtures and programmable logic controls.
Figure 1. Coffee Pulper and its Parts
Figure 2. The developed coffee pulper
The system flow operation model shown in Figure 3 was considered as a guide
of the researcher in carrying out the project study. The enumerated input and
processes greatly deliver the desired output of the researcher.
The idea and concepts was formulated because of the appropriate knowledge
on how the prototype will be constructed. The input served as strong foundation
and basis for corrective layout of the system developed. Knowledge on program-
mable logic controller, PLC language was strictly implied. Skills on how to
program the PLC to control its operation was met.
CONCEPTUAL MODEL OF THE STUDY
Input Process Output
Knowledge Design
Requirements
1. Coffee PLC- Based
Development Coffee Pulper
2. Automation
3. PLC for Wet
4.Types of Pulping Process
machine Implementation
Software
Requirements
1. PLC Languages
Hardware
Requirements
Evaluation
1. Motor,PLC
Lenze
Figure 3. Conceptual Model of the Study
For the project development, the coffee pulper for wet process is divided into
two parts, the hardware and the software components. The hardware components
consist of the PLC connections, the design, the fabrication and the construction
of the machine itself, while the software component will include the system
development and the use of Instructional List.
For the hardware components, the coffee pulper consists of different parts
such as the Main Frame, Pulper Drum, Oscillating tray/agitator, Hopper, Hopper
Shutter, Pulper Housing, Mounting of pillow block, Mounting of oscillating
tray (included: bushing, shafting, link rod eccentrics with bearing), Motor base,
Mounting of pulley.
After the fabrication of the machine, the Programmable Language Controller
(PLC) used in developing the software was Instructional List. It followed a
flowchart that indicates the step-by-step procedure/operation of the system. The
flow of how the machine works, started with the setting of the frequency to 60
and then pressing the button Run for running the machine. The freshly picked
coffee beans were put to the hopper and the machine will start to pulp. The output
will be the pulped coffee beans.
A. Operation and Testing Procedure

1) Operation Procedure
a. Turn on the motor manually.
b. Input coffee berries.
c. The pulper of the machine will then start to operate.
d. Pulp the coffee berries.
2) Testing Operation
a. Prepare a coffee samples.
b. Put the coffee berries in to the machine.
c. Determine the machine pulping capacity
d. Determine the machine performance
B. Program
The PLC language used in developing the software was Instructional List. The
following were the algorithm on how a certain operation could be done.
Step 1
1) Send 1st Data byte 0001H to register 0000H, stand for Forward Run.
2) Send 2nd Data byte 5DC0H to register 0001H, for running in 80% speed
(Max. Frequency output) is 100% (7,530H).
3) Send 8th Data byte 0001H to register 0007H, stand for control of multi-
function by RS-485 and for enable output terminals Ra-Rc and disable
MO+_MO-.
4) Then the controller is initiated; multi-function output terminals Ra-Rc
activated and acceleration is carried out to 80% of max. Output Frequency
according to Acceleration Time.
Step 2
1) Send 1st Data byte 0001H to register 0000H, stand for Forward Run.
2) Send 2nd Data byte 3A98H to register 0001H, for running in 50% speed.
3) Send 8th Data byte 0001H to register 0007H, stand for control of multifunc-
tion by RS – 485 and for enable output terminals Ra-Rc.
4) Then the multi-function output terminals MO+-MO- are disabled and Ra-Rc
activated, and deceleration is carried out to 50% of max. Output Frequency
according to Deceleration Time.
Step 3
1) Send 1st Data byte 0003H to register 0000H, stand for Reverse Run
2) Send 2nd Data byte 3A98H to register 0001H, for running in 50% speed.
3) Send 3rd Data byte 0001H to register 0007H, stand for control of multi-
function by RS-485 and for enable output terminals Ra-Rc.
4) Then the controller shall decelerate to Stop from 50% speed and reverse and
accelerate to 50% of the full reverse speed; multi-function output terminals
Ra-Rc continue Output actions.
Step 4
1) Send 1st Data byte 0000H to register 0000H, stand for stopping the operation.
2) Then decelerates from 50% Reverse to ZERO speed and stop operation. Since
the content of 0007H register has not been changed, the multi-function
output terminals Ra-Rc continue their Output actions.
C. Project Structure
Table 1. PLC Keypad consists of the following keys

Operation Keys Title Function
DATA/ENTER Data Enter Key The data will be written/
stored by pressing this but-
ton.
DSPL Display Key Display information, such as
frequency command, output
frequency
Up Key Increase setting values
Down Key Decrease setting values
RUN Run Key Start to run
STOP/RESET Stop/Reset Key Stop Running
The automated coffee pulper consists of eleven (11) parts, namely: keypad, main
frame, pulper drum, oscillating tray, hopper, hopper shutter, pulper housing,
pulley, the fan belt and the mounting. The different keys of the keypad will be
shown in Table 1.
The keypad consists different buttons such as RUN, DSPL, STOP/RESET,
DATA/ENTER, UP, DOWN and the Knob

The machine has a maximum capacity of five (5) kilograms of freshly picked
coffee beans ready for pulping. It measures 42 inches in height, a length of 55
inches and 17 inches in width. It weighs 41 kilos.
The acceptability is evaluated in terms of the Functionality, Aesthetics,
Workability, Durability, Economy, Safety and Saleability. There are fifteen (15)
coffee farmers and IT experts respectively who filled up the questionnaire on
the acceptability of the project. The coffee farmers are those who are engaged
in the field of coffee farming while IT experts are those who are knowledgeable
in the field of IT. The project prototype was tested and evaluated based on the
criteria given. The mean of each criteria and overall mean were derived from the
answers of the respondent indicated in the survey questionnaire. The formula
for solving mean is
(1)
Where :
X = mean
n = total number of participants
∑ Xi = summation of the participants
Table 2 shows that safety and functionality with a rating of 4.67 and 4.60
respectively got the highest rate. They were both rated excellent. The reason for
this is obvious that the coffee farmer evaluators have witnessed that in terms
of functionality, the machine can easily operate and user friendly. In terms of
safety, they found out that the machine structure was made up of good grade
branded resources and no hazardous or toxic materials. Economy with a rate
of 4.16, Aesthetics with 4.13 and Durability with 4.11 are next in rank with a
rating of very good while Saleability and Workability with a rate of 4.00 got the
lowest rating that could be attributed to the reason that the brand of materials
used is not easy to produce in the local market. The overall mean is 4.23 with a
corresponding rating of very good.
Table 2. Ratings of the Project Prototype (Coffee Farmers)

Design Criteria Rating
Mean Interpre
tation
Functionality 1. Ease of operation
2. Provision for comfort and 4.60 Excellent
convenience
3. User friendliness
Aesthetics 1. Color Appeal
2. Attractiveness of design 4.13 Very Good
3. Appropriateness of size
Workability 1. Availability of materials
2. Availability of technical 4.00 Very Good
expertise
3. Availability of tools and
machines
Durability 1. Quality of materials
2. Quality of workmanship 4.11 Very Good
3. Quality of design
Economy 1. Economy in terms of
materials needed
2. Economy in terms of 4.16 Very Good
time/labor spent
3. Economy in terms of
machine/s required
Safety 1. Absence of toxic/hazardous
materials
2. Absence of sharp edges 4.67 Excellent
3. Provision for protection
devices
Saleability 1. Presence of market demand
2. Accessibility for finished 4.00 Very Good
product
3. Competitiveness to price
Overall Mean 4.23 Very Good
Numerical Rating of 5 for Excellent, 4 for Very Good, 3 for Good, 2 for Fair and 1 for Poor
a
Table 3. Ratings of the Project Prototype(IT Experts)

Design Criteria Rating
Mean Interpre
tation
Functionality 1. Ease of operation
2. Provision for comfort and convenience 4.49 Very
3. User friendliness Good
Aesthetics 1. Color Appeal
2. Attractiveness of design 4.42 Very
3. Appropriateness of size Good
Workability 1. Availability of materials
2. Availability of technical expertise 4.29 Very
3. Availability of tools and machines Good
Durability 1. Quality of materials
2. Quality of workmanship 4.47 Very
3. Quality of design Good
Economy 1. Economy in terms of
materials needed
2. Economy in terms of 4.20 Very
time/labor spent Good
3. Economy in terms of
machine/s required
Safety 1. Absence of toxic/hazardous materials
2. Absence of sharp edges
3. Provision for protection devices 4.78 Excellent
Saleability 1. Presence of market demand
2. Accessibility for finished product 4.40 Very
3. Competitiveness to price Good
Overall Mean 4.43 Very
Good
Numerical Rating of 5 for Excellent, 4 for Very Good, 3 for Good, 2 for Fair and 1 for Poor
a
Based on the result presented in Table 3, it could be seen that one out of seven
criteria garnered a numerical rating of 4.78 within the range of excellent mark.
Safety was evaluated within the excellent descriptive rating bracket. The panel of
evaluators found that the machine has no toxic or hazardous materials and even no
sharp edges on it. Followed was functionality with a rate of 4.49, Durability with
4.47, Saleability with 4.40, Aesthetics with 4.42 while workability and economy
got the two lowest rate. Maybe because they found that the materials used were
not easy to produce in the local market and in terms of economy, IT experts gave
a lowest rate for this since they are not inclined in the field of coffee farming.
Table 4. Comparison of the Computed Mean by the IT Experts and Coffee Farmers
Design Criteria Mean
IT Experts Coffee Farmers
A Functionality 4.49 4.60
B Aesthetics 4.42 4.13
C Workability 4.29 4.00
D Durability 4.47 4.11
E Economy 4.20 4.16
F Safety 4.78 4.67
G Saleability 4.40 4.00
Overall Mean 4.43 4.23
Based on the 15 IT Experts and Coffee Farmers
a
Table 4 shows the comparison of the computed mean between the IT Experts
and the Coffee Farmers. Based on the result, it clearly stated that the computed
mean for the IT experts are much higher compare with the computed mean for
the coffee farmers except only for one criteria which is the functionality. The
overall mean for the IT experts is 4.43 while for the coffee farmers is 4.23 but
still they are in the range of very good.
Table 5. Evaluation by Ranking of Criteria
Ranking Criteria Rating Descriptive Rating
1 Safety 4.72 Excellent
2 Functionality 4.54 Excellent
3 Durability 4.29 Very Good
4 Aesthetics 4.28 Very Good
5 Saleability 4.20 Very Good
6 Economy 4.18 Very Good
7 Workability 4.14 Very Good
a
Evaluation instrument used was based on the Technological University of the Philippines (TUP) format
Table 5 showed the varied ranks of the criteria from the evaluators composed
of the IT experts and coffee farmers. In the table presented above, safety ranked
number one followed by functionality, durability, aesthetics, saleability, economy
and the least is workability.
An overall mean rating of 4.33 with a descriptive rating of very good signifies
a successful study that aids the problem cited to address the design.
IV. Conclusion
In consideration of the objectives of the study and the results of the project
evaluation, the following conclusions were derived.
The project was designed and found to be capable of pulping freshly picked
coffee beans and removing the pulp automatically. Thus, this study indicates
that the machine is convenient to use and user friendly as well especially for the
coffee farmers.
The constructed prototype was made and ready for commercialization since
it is made up of a high quality materials. This means that the design cost and the
fabrication cost was much lower compared to commercially coffee pulper machine
for freshly picked coffee beans. Moreover, the color appeal, structure and its size
were appealing, attractive and appropriate respectively.
This study was also tested and evaluated with a descriptive rating of very
good. This is an indication that the machine was acceptable by the clients based
on the given criteria.
It is therefore conclude that pulping coffee beans for wet process was more
advisable than for the dry process to produce a better quality of coffee beans.
A. Recommendations
For the improvement of the project, the following were recommended:
1) That the project design should be improved in terms of workability.
2) That the project be manufactured in bigger size to accommodate more load
for increase production.
3) That the sorting process of coffee beans from the unpulped be considered for
the improvement of coffee.
4) That the coffee feeding process be improved to eliminate clogging of fresh
coffee berries during pulping.
5) That the fabricated metal sieve should be extended for effective segregation
of coffee pulp from the coffee beans.
6) That the beans should be soaked in water for 24 hours for easy pulping .
7) That further research be conducted for more efficient production of coffee
pulps.
V. R eferences
1) Deal III, Walter F., XBOT. (2001). Gets A New Microchip. The Technol-
ogy Teacher The Voice of Technology Education, Journal of the International
Technology Education Association, 61, no.1. pp. 67–70.
2) Del Rosario, Amiel G. (2004). Development of a Microprocessor-Based
Automated Beverage Mixer. Technological University of the Philippines.
3) Ernacio, K. M. and Montojo, E. A. (2005). Improvement of a Motor-

Operated Coffee Pulper for Wet Processing. Unpublished BS Thesis. Cavite
State University.
4) Mott, Robert L. Machine Elements in Mechanical Design. p. 95.
5) Novero, C. B. (1995). Development and Construction & Evaluation of
Manually-Operated pulper with pulp-separator. Unpublished BS Thesis,
Cavite State University, Indang, Cavite..
6) Nuestro, Rhodora V. (2006). Development of Lid Fitting Machine Using
Programmable Logic Controller. Technological University of the Philippines.
7) NW and RAW. (1981). Automation. Funk and Wagnalls, New Encyclopedia,
pp. 35–38.
8) PCARRD. (1982). The Philippines Recommendation for Coffee, pp.15-16.
Goodwill Bookstore. Manila.
9) Rodillo, R. V. (2001). Testing and Evaluation of Coffee Pulper in Southern
Luzon. Unpublished BS Thesis.Cavite State University.
10) Sivetz, M. (1983). Coffee Processing Technology, 1. Westport Connecticut
Ave Publishing Co. Inc.pp. 21–30.
SCMST
EFFECT OF SINTERING TEMPERATURE RATE ON
PHYSICAL PROPERTIES OF POROUS TRICALCIUM
PHOSPHATE (TCP) CERAMICS
Ahmad Fadli*, Abdul Rasyid, Ricky Firmansyah

Department of Chemical Engineering, Faculty of Engineering, Riau University
Jln. HR. Subrantas Km. 12,5, Pekanbaru, Riau, 28293, Indonesia
Abstract
Porous Tricalcium Phosphate (TCP) were designed for the use in bone implant via starch-
consolidation method and the effect of sintering temperature rate was investigated. TCP suspension
were mixed with wheat particles then stirred for 1 hour. The slurries were cast into cylindrical
shaped molds and then dried for consolidation process at 100°C for 30 minutes, 80°C for 24
hours and 120°C for 8 hours. Afterward, the dried bodies were burned at 350°C for 1 hour and
continued at 600°C for 1 hour, then followed by sintering at 1100°C with termperature rate of
2,5, 8°C/minute for 1 hour. The sintered TCP bodies with shrinkage in the range 56 - 59% and
porosity in range 61–82% were obtained. Increasing sintering temperature rate from 2 to 5°C/
minute will reduce compressive strength from 0.73 to 2.89 MPa.
i. Introduction
The bones are defined as connective tissue and their function as a structural
component of the human body are well known, serves to support, protect delicate
parts and organs and provides a connection between the muscles, allowing
movement [1]. Bone defect will disturb their functions and it must be repaired.
The biomaterials were used to restore the function of traumatized or degener-
ated connective tissues and thus improve the quality of life of a patient [2].
Synthetically produced calcium phosphate ceramics and implants have an
important position among other biomaterials because they are considered to
be almost fully biocompatible with living body when replacing the hard bone
tissues [3]. Implantation of bone by using bone grafts is a known strategy for a
treatment of large bone defects which all lead to limited degree of structural and
functional recovery. However, because of the limited supply, donor site morbidity
and risk of transmission of pathological organisms impose major limits to their
widespread use [4].
Tricalcium Phosphate (TCP) are bone substitute materials that are marked
out by their high biocompatibility, favourable resorption properties and
osteoconductivity [3]. Calcium hydroxyapatite (Ca3 (PO4)6(OH)2 (HA)) and
* Corresponding Author. E-mail: abdulrasyid8793@gmail.com
427
tricalcium phosphate (Ca3 (PO4)2 (TCP)) are currently recognized as ceramics

materials that significantly simulate the mineralogical structure of bone [5]. Porous
calcium phosphate ceramics have found enormous use in biomedical applications
including bone tissue regeneration, cell proliferation, and drug delivery [6].
ii. E xperimental
The materials in this research were tricalcium phosphate powder, wheat particles,
quadest, HNO3 and astor oil. Tricalcium phosphate (Merck, German) was used
as the bioactive material. Wheat particles (PT. Indofood Sukses Makmur Tbk,
Indonesia) was used as porous agent. Aquadest (Merck, Germany) were used as
solvent. HNO3 (Merck, Germany) were used for set slurry pH at 3.5. Castor oil
was used as the lubricant for facilitating demolding.
Figure 1. Scheme of research problem

The slurries were prepared by mixing TCP and aquadest in a beaker glass.
Then, wheat particles and HNO3 were added to the slurries. The slurries were
mechanically stirred at 200 rpm for 1 hour. Subsequently, the slurries were cast
in cylindrical open stainless steel mould with 10.75 mm diameter and 15.10 mm
height and dried in an oven at 100°C for 30 minutes then continued at 80°C
for 24 hours and 120°C for 8 hours. Afterward, the dried bodies were burned at
350°C for 1 hour and continued at 600°C for 1hour, then followed by sintering
at 1,100°C with termperature rate of 2, 5, 8°C/minute for 1 hour. Scheme of
research procedure and mechanism of temperature gain were shown in Figure
1 and Figure 2. Composition of slurries and sintering temperatures rate studied
are as listed in Table 1.
iii. R esults and Discussion

To investigate the ffect of sintering temperature on the physical properties of
porous TCP, samples S1, S2 and S3 were evaluated. Measurement of shrinkage,
density, porosity and compressive strength of all samples are presented in Table 2.
Samples were sintered at 1,100°C with sintering temperature rate 2, 5, 8°C/
minute. When the sintering temperature rate increased from 2 to 8°C/minute, the
shrinkage of porous TCP decreased from 51.28 to 35.87%. Shrinkage decreases as
sintering temperature rate increases. Lower sintering temperature rate enhanced
samples densification rate, and at the same time the grain growth increases.
T (°C)
1 hour
1100
2, 5, 8°C /minute
1 hour
600
2°C/minute
1 hour
350
2°C/minute
30
Time
Figure 2. Mechanism of emperature ain in urning and intering.
Table . Slurry composition and sintering temperature Rate studied

Sintering Temp.
Slurry TCP (g) Wheat (g) Water (ml)
Rate (°C/minute)
S1 18 12 35 2
S2 18 12 35 5
S3 18 12 35 8
Table . Effect of sintering temperature rate on physical properties

Slurry Shrinkage (% vol) Density (g/cm3) Porosity (%) Compressive Strength
(°C/minute)
S1 51.28 1.23 60.90 2.89
S2 43.86 1.09 65.21 1.41
S3 35.87 0.80 74.67 0.89
Table 2 shows the samples porosity at range 60–75%. Decreases on sintering

temperature rate caused the porosity decreased, at the same time it explained
the structure of samples were more compact and the density of samples were
increased. When the sintering temperature rate increased from 2 to 8°C/minute,
the density of TCP were decreased from 1.23 to 0.8 g/cm3, this increases caused
by the particles become compact and the densification was occurred.
Table 2 also reveals that the compressive strength of porous TCP was decreased
while the sintering temperature rate increased from 2 to 8°C/minute. The
compressive strength of porous TCP was in range 0.8–2.9 MPa. The compressive
strength of porous ceramics increased as the porosity deacreased.
Figure 3. Microstructure of Bodies after Sintered with Tem-

perature Rate a) 2°C/minute b) 5°C/minute c) 8°C/minute
The microstructure of samples changed obviously with the increasing sintering

temperature rate. Figure 3 shows the sintering temperature rate increases caused
the increases on porous size and fused into larger grains. Figure 3 also explain the
changed of particles distance. The particle get spaced while the temperature rate
increase. Figure 3c show that the porous size of sample S3 was bigger than sample
S1 and S2. That indicated TCP had opened porous with good interconnectivity.
Figure 4 shows the changes on sintering temperature rate should not have
any effect on chemical structure of samples. That because samples were sintered
at same temperature (1,100°C), meanwhile the sintering temperature rate (2,
5, 8°C/minute) was used as variable in this research. The chemical structure of
samples were effected by sintering temperature [7].
Figure 4. XRD pattern of porous TCP bodies after sitered with temperaturerate
a) 2 °C/minute b) 5 °C/minute c) 8°C/minute
iv. Conclusions
Porous TCP were successfully manufactured via starch-cosolidation technique
using wheat particles as porous agent. The porosity of sintered porous TCP
obtained by this method was 60.90–74.67% and the compressive strength was
0.89-2.89 MPa. When the sintering temperature rate increased from 2 to 8°C/
minute, the shrinkage of porous TCP decreased from 51.28 to 35.87%. The
change on sintering temperature rate should not have any effect on chemical
structure of porous TCP.
v. Acknowledgement
The authors are thankful to the Ministry of National Education, Republic of
Indonesia (DIKTI) for funding this research.
vi. R eferences
1) Rivera-Muñoz, E. M., (2011)). Hydroxyapatite-Based Materials: Synthesis
and Characterization. Biomedical Engineering - Frontiers and Challenges. R.
Fazel, Ed. [Online]. Available: http://www.intechopen.com/books/biomed-
ical-engineering-frontiers-and-challenges/hydroxyapatite-based-materials-
synthesis-and-characterization. Accessed on August, 1, 2011
2) Park, S. H., Llinás, A., Goel, V. K., and J. C. Keller. (2000). Hard tissue
replacement.. The Biomedical Engineering Handbook, 2nd ed. J. D. Bronzino,
Ed. Boca Raton: CRC Press LLC.
3) Ain, R. N., Sopyan, I., and S. Ramesh. (2008). Preparation of Biphasic
Calcium Phosphate Ceramics Powders and Conversion to Porous Bodies.
ICCBT Proceedings, 245–256.
4) Saki, M., Narbat, M. K., Samadikuchaksaraei, A., Ghafouri, H. B., and F.
Gorjipour. (2009). Biocompability Study of A Hydroxyapatite-Alumina and
Silicon Carbide Composite Scaffold for Bone Tissue Engineering. Yakhteh
Med. J., 11 (1), 55–60.
5) Kivrak, N., and A. T. Cuneyt. (1998). Synthetis of Calcium Hidroxyapatite-
Tricalcium Phosphate (HA-TCP) Composite BioceramicsPowders and Their
Sintering Behavior. J. Am. Ceram. Soc., 81 (9)2245–52.
6) Abdurrahim, T, and I. Sopyan. (2008). Recent Progress on the Development
of Porous Bioactive Calcium Phosphate for Biomedical Applications. Biomed.
Eng., 1, 213–229.
7) Kang, S.-J. L. (2005). Sintering: Densification, Grain Growth and Microstruc-
ture, 280 pages. Amsterdam: John Wiley & Sons.
STUDY OF KINETICS AND THERMODYNAMICS AS
WELL AS THE EFFECT OF THE PRESENCE OF CO-IONS
IN INFLUENCING ADSORPTION CU2+ ION BY COAL FLY
ASH ADSORBENT
Ahmad Zakariaa,*, Eti Rohaetib, Irmanida Batubarab, Sutisnac, Wittri

Djasmasaria
a
Bogor Analytical Chemistry Academy
Jl. Pangeran Sogiri No.283, Bogor, Indonesia
b
Bogor Agricultural University
Jl. Raya Dramaga, Bogor, Indonesia
c
National Nuclear Energy Agency
Jl. Puspitek Raya, Serpong Tangerang, Indonesia
Abstract
The aim of this research is to define the order of kinetics model and thermodynamic parameters
such as free energy, entropy and enthalpy of adsorption process of metal ion Cu 2+ by coal
fly ash adsorbent and the effect of the presence of coexisting ion against of the efficiency of
Cu2+adsorption., Coal fly ash was obtained from steam power plants Suralaya. Experiment was
carried out at pH adsorbat, contact time and adsorbent concentration optimum that were obtained
in the previous study. Kinetics experiment was performed at various contact time 5, 15, 30, 45,
60, 75 and 90 minutes while the thermodinamyc parameters studies was done at temperature
27, 32, 37 and 42 oC. The influence of coexisting ion Mn2+ and Pb2+ to the adsorption process
was examined here. The kinetics data were evaluated using a pseudo first-order and a pseudo
second-order Lagergren equation. The results revealed that the kinetic data correlated well with
the pseudo second-order kinetics model. Thermodynamic studies indicated that the adsorption
process was spontaneous and accompanied by an increase in entropy and decrease in Gibbs energy.
The coexisting ions Pb(II) or Mn(II) decreased the adsorption capacity of coal fly ash in the Cu2+
adsorption, but increased the total adsorption capacity.
Key words: Kinetics model, Coal fly ash, Coexisting ionns, Gibbs energy, Adsorption capacity.
i. Introduction
Metals such as copper are produced by industrial metal plating, alloy, steel, dyes,
electrical wiring, insecticides, pipelines, and paint [1]. Therefore, the Government
of Indonesia through Kep-51 / MENLH / 10/1995 establishes effluent standards
for copper metal content of less than 2 mgL-1 for industrial class 1 and under
0.6 mgL-1 for plating industry [2]. The presence of Cu ions in industrial waste is
* Corresponding Author.Tel: +62-812-1333843. E-mail: alifrahmatulalam@yahoo.com
433
usually accompanied by other heavy metal ions. In the plating industry wastes,
heavy metal ions Cu is the fifth largest concentration after metals Fe, Cr, Sn, and
Zn, followed by the metal ions with smaller concentrations, namely Ni, Mn, Pb,
Cd, and Ag [3].
Some treatment methods for treating heavy metal ions in industrial effluents
have been reported in the literature [1]. Among thee methods are neutralization,
precipitation, ion exchange and osmotic technique. Those methods have several
disadvantages including high cost.
Adsorption process which employed low cost and highly available material
is one of the most promising techniques. Coal fly ash is a solid waste generated
power plants that use coal as a fuel and produced in large amount thus yields
another problem if it is not properly handled. In the year 2000, the amount of
coal ash wastes are 500 million tons per year around the world and by 2006 it
had reached 2 billion tons per year and is predicted to be increasing rapidly [4,5].
Therefore, it can increase economic value if the coal ash wastes are employed as an
active material in adsorbent process. Based on those reasons, adsorption process
using coal fly ash was a potential technique as an alternative for heavy removal
metal ions. However, less is known about temperature dependence of adsorption
kinetics and thermodynamics by fly ash.
In this research, adsorption kinetics and some thermodynamics parameters
were evaluated to describe the performance and properties of coal fly ash as
adsorbent in the Cu(II) adsorption in aqueous system. The effect of the presence
of coexisting ion (Mn2+ and Pb2+) on the efficiency of Cu2+ adsorption was also
investigated.
ii. M aterials and Methods

A. Materials and Equipment
Coal fly ash was obtained from power plants in Suralaya, and used as adsorbents.
H2SO4, CuSO4, MnCl2, Pb(NO3)2, were obtained from Merck. Cu(II) was used
as adsorbate. NaOH and HCl were used for adjusting pH. All of chemicals were
of analytical grade.
Cu(II) concentration was determined by Atomic Absorption Spectrophotom-
eter (GBC), air–acetylene as fuel and oxidant.
B. Determination of Reaction Kinetics

This experiment was performed at optimum condition found from previous
research [6]. Briefly, 50 mL of adsorbate solution with a concentration of 8 and
12 mgL-1 at pH optimum (5.5) were each added to the coal fly ash (at optimum
weight) in 100 mL erlenmeyer glass. The mixture was then agitated with a shaker
at 150 rpm for a 5, 15, 30, 45, 60, 75 and 90 minutes. For each variation time,
the sample was filtered and the filtrate was measured using the AAS to determine
the concentration of Cu (II) in solution.
Using pseudo first order and second order models, the concentration of Cu(II)
was plotted as a function of time. The correlation coefficient of each curve was
calculated and by comparing these values, the order of adsorption reaction could
be found.
C. Determination of Thermodynamic Parameters

Thermodynamic parameters are determined by carrying out the experiment at
various temperatures (27, 32, 37 and 42 0C ) while the other variables were kept
constant. This experiment was done through the same steps as describes in the
previous section (B) using optimum shaking time.
D. Adsorption Experiment for Binary Metal Systems

The adsorption experiment was carried out through the same step as described
in the previous section (B) using binary metal (Cu2+/Mn2+ or Cu2+/Pb2+ mixed
solution) as adsorb ate. The Cu(II) adsorption capacity of coal fly ash with and
without the presence of Mn2+ and Pb2+ then calculated and by comparing these
values, the effect of these coexisting ions can be evaluated. In this experiment
the adsorb ate contains 8 mgL-1 Cu(II) and 4 mgL-1 Mn(II) (or 8 mgL-1 Cu(II)
and 4 mgL-1 Pb(II) ).
E. Calculation
The amount of heavy metal adsorbed by coal fly ash was calculated using the
following equation:
(1)
Co is the initial concentration (mgL-1), Ce is the final concentration in the

solution phase equilibrium (mgL-1), Qe is adsorption capacity or the concentra-
tion of adsorbate at equilibrium (mgg-1), m is adsorbent mass and V is volume
of adsorbate.

A. Effect of Contact Time
Adsorption kinetics describes the solute retrieval rate by the adsorbent during
the adsorption reaction time. It is a crucial parameter since it determines the
efficiency of the adsorption process. Effect of contact time on the adsorption
capacity can be seen in Figure 1.
Figure 1 revealed that increasing adsorption capacity of the adsorbent

was proportional to the contact time until equilibrium has occurred at 75
minutes. The equilibrium time greatly depends on the adsorbate and the
adsorbent and can be the interaction of both. Gupta and Bhattacharyya [7]
have reported the equilibrium time of 180 minutes for adsorbates of Pb (II)
and Ni (II) with kaolin and montmorilonite adsorbents. Increase of adsorption
rate occurs at the beginning of the contact time, but after almost all sides
actively interact with metal ions, the adsorption rate decreases. Thus, there
is no significant increase in the adsorption capacity as the active adsorbent
was saturated. In this condition the adsorption rate is now only dependent on
t (minutes)
Figure 1. Effect of contact time on the adsorption capacity of Cu2+.
t (minutes)
Figure 2. Relationship adsorption capacity versus contact time for

1st order kinetics.
t (minutes)
Figure 3. Relationship adsorption capacity versus contact time for
2nd order kinetics.
the migration of metal ions in the liquid phase to the surface of the complex
adsorbent-adsorbate [8].
Based on equations 2 and 3, the first order kinetics equation models and
second-order false done by plotting t against log (qe-qt) versus t and t/qt as
Lagergren equation (Figure 2 and 3). From the linear curves, the value of the
adsorption rate constant (k), the optimum adsorption capacity prediction (qe)
and coefficient terminated can be known. In this experiment was used adsorbate
concentration Cu2+ 8 mg.L-1.
(2)
(3)
Correlation coefficient for the pseudo first-order analysis is small-

er than the second-order false. Predictive value of adsorption capac-
ity compared to that of the experimental value have error of -63%
(Table 1). It indicates that the pseudo first-order kinetics equation is less
suitable to be applied as a model for the adsorption kinetics of coal fly ash
adsorbent. The correlation coefficient (r) of the linear regression equation was >
0.99 for pseudo second order reaction (Table 1), while the range of validity test
error for adsorption capacity of coal fly ash is -1.60% to + 0.27%. It suggests
that the kinetic parameters of adsorbent satisfy pseudo second- order equation
since it has a high degree of accuracy in predicting the optimum experimental
adsorption capacity.
B. Effect of Temperature
The amount of Cu(II) adsorbed by coal fly ash increases with increasing tempera-
ture in the range of 300–315 K (Figure 4). This phenomena can be explained by
two reasons. First, the higher the temperature, the higher the kinetic energy of
metal ion. Second, the higher the temperature, the larger the amount of smaller
Table 1. Comparison of First-Order Rate Constant and the Pseudo Second-Order and Predic-
tions and Experimental Qe Values
Pseudo first-order kinetics param- Pseudo second-order kinetics
qe eters parameters
Adsor- Co
(mg/g) K2
bent (mgL )
-1
K1 qe % qe %
experimen r g/mg r
(min-1) calculations error calculations error
min
8 2.172 0.018 0.783 -63.9 0.9246 0.0890 2.178 0.27 0.9960
Fly ash
12 2.968 0.018 1.09 -63.2 0.8087 0.0660 2.921 -1.60 0.9923
Table 2. Thermodynamic Parameters of Adsorption of Cu2+ by Coal Fly Ash

Thermodynamics Parameters
Adsorbate Cu2+ Temperature
Adsorbent ΔG0 ΔH0 ΔS0
(mgL-1) (°C)
(kJmol-1) (kJmol-1) (Jmol-1)
27 -0.360
32 -1.005
Fly ash 12 38.34 129
37 -1.650
42 -2.295
Table 3. Effect of Co-Ions on the Adsorption Efficiency of Cu2+ by Coal Fly Ash Adsorbent
Initial Concentration
Adsor- Adsorption capacity (mgg-1) Adsorption Efficiency (%)
(mgL-1)
bent
Cu Pb Mn Cu Pb Mn Total Cu Pb Mn
8 0 0 2.502 - - 2.502 94.02 - -
8 4 0 2.386 1.329 - 3.715 89.78 100 -
Fly ash
8 0 4 2.312 - 1.232 3.544 86.83 - 92.53
12 0 0 3.082
Figure 4. Effect of temperature on the adsorp- Figure 5. Plot Van’t Hoff adsorption of Cu2
tion capacity of Cu2 + by coal fly ash at pH 5.5 +
(at initial concentration 12 mg L-1) by coal
fly ash
metal ions due to the reduction of hydration effects, thus the easier the metal ion
to penetrate the deeper layers of pores [9].
Enthalpy ( H0) of coal fly ash adsorption at initial adsorbate concentration
of 12 mgL-1 was 38.34 kJmol-1 (Table 2), thus the process are endothermic. Some
studies have been reported the same trend [9] for the adsorbent Penicillium
simplicissium and adsorbate Cd (II), Zn (II) and Pb (II).
Values of thermodynamic parameters of adsorption of Cu2+ by coal fly ash
adsorbent were obtained from calculation of the slope and intercept of linear
curve based on van’t Hoff equations (Figure 5).
In Table 2, the entropy change of adsorption is positive, indicating an increase
in the degree of irregularity in the adsorbent-adsorbate system, thus the metal
ions adsorbed on the adsorbent are more disordered [10]. This phenomenon in
the adsorption system is very beneficial because it can increase the stability of the
adsorbent-adsorbate complex.
The Gibbs free energy of adsorption systems is negative in all experimental
temperature conditions (see Table 2), indicating spontaneous adsorption system.
The higher the temperature, the more negative the Gibbs free energy. The increase
in temperature causes the adsorption process easier due to increasing metal ion
kinetic energy making it easier for the metal ion adsorbed on the deeper layers
of pores.
C. Effect of Co-ions
Heavy metal ions, such as Mn(II) and Pb(II) are often found in industrial effluents
with Cu(II). Therefore, the effect of these co-ions on the adsorption capacity
of Cu (II) must be considered. The experiment revealed that the efficiency and
adsorption capacity of Cu(II) ions was influenced by Mn(II) and Pb(II). The
presence of these ions in a solution of copper adsorbate decrease the efficiency
and capacity of the copper adsorption (Table 3) due to the competition between
the metal ions of Cu, Mn and Pb to form adsorbent-adsorbate complexes.
In the other hand, the presence of Pb(II) or Mn(II) ions in Cu(II) solution
improves overall adsorption capacity, so it gives an advantages to the heavy metal
ion adsorption process. This phenomenon is due to shift in the equilibrium
towards the formation of adsorbent-adsorbate complex with increasing concentra-
tion of adsorbate [7].
The mechanism of adsorption of metal ions was also due to the precipitation
of metal hydroxides on the surface of the adsorbent [3]. As supporting data, the
value of Ksp Mn(OH)2, Pb(OH)2 and Cu(OH)2 is 4 x 10-14, 3 x 10-16, and 2 x
10-19, respectively. Thus, Pb(II) settles faster over Mn ions (II), resulting in the
greater amount of Pb(II) adsorbed on the adsorbent surface, leading to increasing

in the efficiency of adsorption
IV. Conclusion
Adsorption kinetics is determined as the pseudo second-order. Adsorption
reactions tend to be spontaneous and endothermic. The presence of Mn or Pb
ions decreases the efficiency of adsorption of Cu2 + but increase the total capacity
of adsorption.
V. Acknowledgement
The authors would like to thank the Leadership Bogor Analytical Chemistry
Academy and to the supervisor in completing Master Degree Chemistry from
Bogor Agricultural University.
VI. R eferences
1) Sarkar, B., Xi, Y., Megharaj, M., Krishnamurti, G. S. R., Rajarathnam, D.,
and R. Naidu. (2010). Remediation of Hexavalent Chromium Through
Adsorption by Bentonite Based Arquad 2HT-75 Organoclays. J. Hazard.
Mater., 183, 87-97.
2) Menteri Negara dan Lingkungan Hidup. Baku Mutu Limbah Cair Kegiatan
Industri. Keputusan Menteri Negara dan Lingkungan Hidup No. Kep. 51/
Menlh/10/1995, Oct 23, 1995.
3) Ventkatiswaran, P., Vellaichanny, S., and K. Palanivelu. (2007). Speciation
of Heavy Metals in Electroplating Industry Sludge and Wastewater Residue
Using Inductively Coupled Plasma. Intl. J. Environ. Sci. Tech., 4(4), 497-504.
4) Hui, K. S., Chao, C. Y. H., and S. C. Kot. (2005). Removal of Mixed Heavy
Metal Ions in Wastewater by Zeolite 4A and Residual Products from Recycled
Coal Fly Ash. J. Hazard. Mater. B, 127, 89-101.
5) Mazari Magazine. (2009). Abu terbang batubara sebagai adsorben. [Online].
Available: http://mazarimagazine.com/2009/06/abu-terbang-batubara-
sebagai-adsorben.
6)] Zakaria, A., Rohaeti, E., Bara, I. B., Sutisna, and Y. Puurwamargapratala.
(2012)Adsorpsi Cu(II) Menggunakan Zeolit Sintetis dari Abu Terbang Batu
Bara. Prosiding Pertemuan Ilmiah IPTEK BAHAN 2012, 190-194.
7) Gupta, S. S., and G. K. Bhattacharayya. (2008). Immobilization of Pb(II),

Cd(II), Ni(II) Ions on Kaolinite and Montmorillonite Surfaces from Aqueos
Medium. J. Environ. Manage, 87, 46–58.
8) Yu, B., Zhang, Y., Shukla, A., Shukla, S. S., and K. L. Dorris. (2000). The
Removal of Heavy Metal from Aqueous Solution by Sawdust Adsorption-
Removal of Copper. J. Hazard. Mater. B, 80, 33–42.
9) Fan, T., Liu, Y., Feng, B., Zeng, G., Yang, C., Zhou, M., Zhou, H., Tan, Z.,
and X. Wang. (2008). Biosorption of Cadmium(II), Zinc(II), and Lead(II)
by Penicilliumsimplicissium: Isoteherm, Kinetics and Thermodynamics. J.
Hazard. Mater., 160, 655–661.
10) Kubilay, R. S., Gurkan, A., Savran, T., and Sahan. (2007). Removal of Cu(II),
Zn(II) and Co(II) Ions from Aqueous Solution by Adsorption onto Natural
Bentonite. Adsorption, 13 (1), 41–51.
ORGANOPHOSPHORUS (OPS) IN THE ENVIRONMENT:
EFFECTS OF REPEATED APPLICATION OF
CHLORPYRIFOSON AGRICULTURAL SOIL
C. Carol* and S. H. Fauziah
Institute of Biological Sciences, Faculty of Science
University of Malaya, 50603 Kuala Lumpur, Malaysia
Abstract
The most pervasive applied pesticide of chlorpyrifos prone to bound on suspended particles
and sediment than dissolved in water. Chlorpyrifos persistency in soil is attributed to various
environmental factors such as pH, temperature, and frequency of treatment. Therefore, it is
essential to perform studies on the interaction of chlorpyrifos in different microenvironment
in order to assess the impact caused by its application onto agricultural soil. This study aimed
to determine the behaviour of chlorpyrifos in selected agricultural soil from Klang, Selangor.
Agricultural soils from vegetable farms were sampled five times within 15 days of crop cycles in
February 2014. Results show that chlorpyrifos residues persisted in each soil sample at an average
amount of 0.06±0.02 mg kg-1. The agricultural soil contained residues of chlorpyrifos even before
the seeding of crop. This study found that pesticide applied throughout the crop growing period
and even after it has been harvested significantly increases the persistent level of chlorpyrifos in
agricultural soil. Without proper practice of pesticide application, chlorpyrifos can be persistent
and last longer in the agricultural soil.
Key words: Chlorpyrifos, Agricultural Soil.
I. Introduction
Economic benefits in utilizing pesticide to increase food production are
undeniable [1]. However, it also elevates adverse effects of pesticide residues in
the environment (water, soil and crops) to man, domestic animals, and wildlife
[2]. Moreover, significant proves of inappropriate uses of pesticides have led to
public concerns on food safety, human health, environmental contamination,
insect resistance and resurgence [1].
Organophosphorus insecticides are widely used throughout the world and
have many cases replaced organoclorine pesticide [3]. Chlorpyrifos insecticides
(O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothionate) is one of
organophosphorus broad-spectrum insecticide that is most favoured in crops
treatment by farmers in agriculture nowadays [1]. Stability and effectiveness had
made chlorpyrifos one of the most popular pesticides worldwide, but on the
* Corresponding Author.Tel: +60-16-8808908. E-mail: chiacarolchia@gmail.com
443
other hand its persistence had raised environmental concerns [4]. Chlorpyrifos
is a degradable compound and the translocation action of chlorpyrifos through
the environmental systems (soil, water, plant and animal) is highly dependent on
the environmental forces [5]. Residues of Chlorpyrifos in soils can be transported
onto surface or into groundwater, and some residues can be uptaken into crops
that grows on the site [5].
Very few literature reviews were found on the study of Organophosphorus
pesticide residue in vegetable farms in Malaysia. Chlorpyrifos residues that repeti-
tively found in vegetable according to the reports by Department of Agriculture,
Malaysia since 1991 [6] implied critical concerns on the fate of the residues in
the environment. The potential environmental pollution by chlorpyrifos should
be investigated particularly on its residue in the environment. This will allow
existing factor to influence its migration or movement actions of chlopyrifos.
Moreover, the application rate of pesticide on crops should be dependent on
farmers’ practices.
Therefore, this study is aimed to provide primary data for existing level
of chlorpyrifos residue in agricultural soil. The objectives of the study are to
determine the level of Chlorpyrifos residue in agricultural soil.
II. Methodology
This study was carried out in selected vegetable farms, namely, an organic farm
and two conventional farms (referred as conventional farm 1 and conventional
farm 2) located in East Klang, Kuala Langat District, Selangor. The farms plant
lowland vegetables to be supplied to Selangor with annual production of ap-
proximately 799 metric tons (equal to RM 1,342,500.00 output value) [7]. The
commercial formulation of chlropyrifos (trade name Dursban 75) which contains
21.2% active ingredient was used in all selected farms in this study. Soil samples
were collected five times within 15 days of crop cycle in February 2014. The soil
samples included soil collected a day before the seeding of crops (Day 0), during
the seeding of the crop (Day 1), Day 5, Day 11, and Day 15 (the day the crops
were harvested). Composite surface soils (0-20 cm) were collected within a grid
sampling plot measuring 3 ft x 3 ft. Amber bottles (62 mm x 63 mm) were used
to keep the soil which were kept in coldbox to be transported to laboratory.
Soil samples were air dried, away from light exposure for 48 hours. Dried soil
samples were then sieved (1 mm) to remove stones, plants, and root residues.
The soil phase Chlorpyrifos were extracted in ethyl acetate at 1:4.5 (w/w) soil to
solvent ratio followed by centrifugation at 5000 rpm for 9 min. Soil to solvent
ratio for each case was optimized such that there was no additional gain in the
recovery with increasing solvent [8]. The extracts were then left to settle and
filtered through a filter paper (Whatman, grade 41) containing sodium sulphate
into a round bottomed flask. The filtrate was evaporated by a rotary evaporator
to almost dryness and made up to 2 mL with acetone. Final extract was placed
into amber GC vial for chromatographic analysis.
III. R esults & discussions

This study found that all soil samples collected were positive of chlorpyrifos
residual even for those soil from organic farm. The residue detected in agricultural
soil samples collected from organic farm on Day 0 (before seeding of crops), Day
1 (seeding of crops), Day 5, Day 11 and Day 15 were 0.05 mg kg-1, 0.08 mg kg-1,
0.09 mg kg-1, 0.10 mg kg-1 and 0.08 mg kg-1 respectively. Meanwhile, samples of
soil from conventional farm 1 were 0.07 mg kg-1 on Day 0, 0.04 mg kg-1 on Day
1, 0.05 mg kg-1 on Day 5, 0.05 mg kg-1 on Day 11 and 0.05 mg kg-1 for Day 15.
On the other land, detected chlorpyrifos residues in soil from conventional farm
2 were 0.07 mg kg-1 on Day 0, 0.05 mg kg-1 on Day 1, and Day 5, 11 and 15
have the same residual level of 0.04 mg kg-1 each. Overall, the mean and standard
deviation value for the chlorpyrifos residues on soil samples collected during the
whole crop cycle from organic farm was 0.08 ± 0.02 mg kg-1, while conventional
farm 1, and convention farm 2 were 0.05 ± 0.01 mg kg-1 each.
This study observed that soil from the study site have decreasing chlorpyrifos
residue on Day 1. However, due to chlorpyrifos pesticide re-application during
pre-harvesting from Day 1 to Day 11 increased the chlorpyrifos residue level.
During harvesting of crop on Day 15, another decrease of chlorpyrifos residues in
soil was recorded. This probably resulting from the fact that chlorpyrifos residues
in agricultural soil can be affected by physical interactions. Majority of applied
pesticides, even if sprayed on crop, will eventually reach the soil [9]. Farmers
applied pesticides to vegetable crops throughout their growing season, and even
applied to soils after the vegetable crop have been harvested [10]. Therefore, this
resulted in persistence of chlorpyrifos residue. It is as well agreed by Racke [11]
that stated chlorpyrifos behaviour in soil depended on the rate of application.
Chai et al. [10] observed that the higher CPF application rate of 25 microgram
per gram (µg g-1) was resulted in decrease of CPF degradation rates. Sirisha et al.
[12] found that repeated application of chlorpyrifos to the same soil significantly
enhanced the degradation rates, which may be reasoned due to the acclimatisation
of the native microflora. The microbial adaptation to the compound enhances
the catabolism, resulting in accelerated rate of degradation, due to the utilization
of chlorpyrifos as carbon or nutrient sources. Fungal and bacterial components
(antifungal and antibacteria agent) in soil microflora affect chlorpyrifos rate of
degradation via complete mineralization [13]. Chlorpyrifos at 10–300 mg kg-1
in loamy soil would significantly reduced the numbers of aerobic di-nitrogen
fixing bacteria, total number of bacteria without affecting the fungal population
and denitrifying bacteria [14]. The result of soil samples detected at Day 15 is
agreeable with the chlorpyrifos levels reported by many authors [15,16]. In fact,
CPF residues were still detected even after 15 days of application [15,16]. The
persistency of chlorpyrifos residues in soil may cause indirect affect to the soil
quality and fertility, and eventually the crops quality [17]. Physical, chemical, and
biological properties of soils health will eventually be degraded in a long-term
[17] since chlorpyrifos binds strongly to soil, is relatively immobile, and has low
water solubility [1,2].
IV. Conclusion
In order to prevent possible damages to the environment caused by the application
of chlorpyrifos in agricultural soil, it is necessary to know the mechanisms that
rule the transport of this pesticide. Thus, from the study, the level of chlorpyrifos
in soil ranges from 0.04 mg kg-1 to 0.10 mg kg-1. More stringent practice should
be employed to prevent the active ingredients from becoming persistent in the
environment.
V. Acknowledgement
We are indebted to Department of Agricultural Malaysia (Kuala Lumpur) and
Department of Agricultural Tuaran Research Centre, Sabah, for their guidance
and support in providing sufficient data and training on laboratory analysis skill.
Our gratitude also goes to the University of Malaya, Research Grant Management
Unit for the financial help of Postgraduate Research Grant (PPP) (Grant Number:
P001–2013B) that has enabled us to complete this study.
VI. R eferences
1) Lu, M.X., Jiang, W. W., Wang, J. L., Liu, X. J., and X. Y. Yu. (2014).
Persistence and Dissipation of Chlorpyrifos in Brassica Chinensis, Lettuce,
Celery, Asparagus Lettuce, Eggplant, and Pepper in a Greenhouse PLoS ONE,
9 (6), e100556.
2) M. S. Diaz-Cruz and D. Barcelό. (2006). Highly Selective Sample Preparation
and Gas Chromatographic – Mass Spectrometric of Chlorpyrifos, Diazinon
and Their Major Metabolites in Sludge and Sludge-Fertilized Agricultural
Soils. J. Chromatogr. A, 1132 (1-2), 21-27.
3) Liu, B., McConnell, L. L., and A. Torrents. (2001). Hydrolisis of Chlorpyrifos
in the Natural Water of the Chesapeake Bay. Chemosphere, 44 (6), 1315-1323.
4) Kamrin, M. A. (1997). Pesticide Profiles: Toxicity, Environmental Impact, and
Fate, 704 pages. Boca Raton, FL: Lewis Publisher.

5) Yucel, U., Ylim, M., Gozek, K., Helling, C. S., and Y. Sarykaya. (1999).
Chlorpyrifos Degradation in Turkish Soil. J. Environ. Sci. Health B, 34 (1),
75-95.
6) Rahman, A. A., and A. Hamering. (2009). Monitoring of Pesticide Residues in
Vegetables in Sabah. Presented at Seminar on Chemicals and Pesticide Usage
in Food Productions, Kota Kinabalu, Malaysia.
7) Department of Agriculture Klang. (2014). Usahawan Pejabat Pertanian Daerah
Klang. Department of Agriculture Klang (DOA Klang) Malaysia. [Online].
Available: http://doaklang.blogspot.com/p/pegawai-pertanian-daerah-klang-
en.html.
8) Tiwari, M. K., and S. Guha. (2014). Kinetics of Biotransformation of
Chlorpyrifos in Aqueous and Soil Slurry Environments. Water Res., 51, 73-85.
9) Chu, X., Fang, H., Pan, X., Wang, X., Shan, M., Feng, B., and Y. Yu. (2008).
Degradation of Chlorpyrifos Alone and in Combination with Chlorothalonil
and Their Effects on Soil Microbial Populations. J. Environ. Sci., 20 (4),
464-469.
10) Chai, L.-K., Mohd-Tahir, N., and H. C. B. Hansen. (2008). Determination
of Chlorpyrifos and Acephate in Tropical Soils and Application in Dissipation
Studies. Intl. J. Environ. Anal. Chem., 88 (8), 549-560.
11) Racke, K. D. (1993). Environmental Fate of Chlorpyrifos. Rev. Environ.
Contam. Toxicol., 131, 1-150.
12) Sirisha, K., Mohan, S. V., and S. J. Reddy. (2006). Effect of Repeated Ap-
plications of Chlorpyrifos on Its Degradation in Surface and Subsurface Soil.
Toxicol. & Environ. Chem., 88 (3), 373-384.
13) Singh, B. K., Walker, A., Morgen, J. A. Q., and D. J. Wright. (2003). Effect
of Soil pH on the Biodegradation of Chlorpyrifos and Isolationof A Chlor-
pyrifos – Degrading Bacterium. App. Environ. Microbial., 69, 5198-5206.
14) Martínez, V. J. L., Plaza-Bolanos, P., Romero-Gonzalez, R., and F. A. Garrido.
(2009). Determination of Pesticide Transformation Products: A Review of
Extraction and Detection Methods. J. Chromatogr. A, 1216 (40), 6767-6788.
15) Konda, L. N., and Z. Pastor. (2001). Environmental Distribution of Ace-
tochlor, Atrazine, Chlorpyrifos and Propisochlor under Field Conditions. J.
Agric. Food Chem, 49 (8), 3859-3863.
16) Redondo, M. J., Ruiz, M. J., Font, G., and R. Boluda. (1997). Dissipitation
and Distribution of Atrazine, Simazine, Chlorpyrifos, and Tetradifon Residues
in Citrus Orchard Soil. Arch. Environ. Contam. Toxicol., 32 (4), 346–352.
17) Spark, K. M., and R. S. Swift. (2002). Effect of Soil Composition and Dis-
solved Organic Matter on Pesticide Sorption. Sci. Total Environ., 298 (1-3),
147–161
BONDED PrFeB MAGNET: FABRICATION AND
CHARACTERIZATION
Didik Aryanto*, Ferensa Oemry, Toto Sudiro, Ayu Yuswita Sari

Research Center for Physics, Indonesian Institute of Sciences
Kawasan Puspiptek Serpong, Tangerang, Banten, 15314, Indonesia
Abstract
We studied the effect of particle size modification on the structural and magnetic properties
of isotropic bonded PrFeB magnet. The commercial PrFeB magnetic powder was used for this
study. Particle size of the powder was modified and analyzed using high energy milling (HEM)
and particle size analysis (PSA), respectively. The milled powder was compressed and heat
cured at 230oC for 1 hour. Structural and magnetic properties of bonded PrFeB magnet were
characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and vibrating
sample magnetometer (VSM).
Key words: PrFeB, Bonded magnet, Particle size, Structure, Magnetic properties.
I. Introduction
Permanent magnets are an essential component in many fields of technology
because of their profound and wide-ranging applications and devices. In the last
few decades, rapid development of hard magnetic materials has been preceded with
the advent of rare-earth (RE) permanent magnets. The energy product ((BH)max) of
these magnets is about 60 times of that BaFe12O19 (Kichiei Sumitomo Steel) [1].
NdFeB and PrFeB are some examples of these RE permanent magnets where both
Praseodymium (Pr) and Neodymium (Nd) elements have similar stoichiometric
ratio in RE2Fe14B compounds. In addition, both Pr2Fe14B and Nd2Fe14B, which
belong to space group P42mnm, possess tetragonal structure with similar a- and
c-spacing of 0.88 nm and 1.22 nm, respectively [2]. The saturation magnetization
of Pr2Fe14B (1.56 T) is also comparable to Nd2Fe14B (1.60 T). However, PrFeB
has higher anisotropy field and higher intrinsic coercive field as compared to
NdFeB and it exhibits no spin reorientation phenomenon at low temperature
(4.2 K) where NdFeB does at temperature of 140 K [3]. Hence, it suggests that
PrFeB can be implemented for wide-range temperature applications.
It is known that coercive field, mechanical properties, and surface degradation
greatly depend on the average grain size of the magnets [4,5]. For sinter rare-earth
magnet, the mean grain size decreases as the mean particle size decreases hence,
* Corresponding Author.Tel: +62-21-7560570. E-mail: didik_phys@yahoo.com
449
its overall distribution becomes more homogeneous [6]. Following mean grain
size decreases, coercive field of the magnet would also increase. Previous studies
have reported successful attempts to manipulate the particle size of sinter NdFeB
magnet in order to improve its magnetic properties [4,7,8] Nonetheless only
few of early studies which have conducted such work on bonded PrFeB magnet.
Therefore, in this study, we carried out modification of particle size of bonded
PrFeB magnets by performing mechanical milling for a short period of time using
high energy milling (HEM).
II. E xperiment
Commercial PrFeB magnetic powder (MQP-16-7A Magnequench International,
Inc) was used for this study. The powder is a multimodal mixture of plate-like
particles (Figure 1). The original PrFeB powder (size ≈ 10–400 μm) was milled
using homemade HEM by oscillating hardened steel balls (20 mm in diameter
horizontally under ambient atmosphere for 60 and 300 seconds to investigate
Figure 1. Multimodal mixture of commercial PrFeB powders

Table 1. Mean Particle Diameter of PrFeB Powder Varies with Milling Time
Mean particle diameter (mm)
Milling time
1% 12% 25% 50% 90%
0s 3.26 58.18 89.53 128.43 222.64
60 s 2.41 12.58 23.00 46.04 98.60
300 s 0.70 7.75 11.21 17.91 34.61
the effects of different milling time. The weight-to-weight ratio of steel balls and
PrFeB powder in the mixture was around 10:1. No additional compound was
added into the mixture. The powder later was fitted into small disk mold (10 mm
in diameter and 5 mm in thickness) and compressed vertically with pressure of

4 MPa at 30°C. Afterwards, the samples underwent ageing process at 230°C for
30 minutes under argon gas environment.
III. R esult and discussion

Particle size distribution of the original and milled powders is shown in Figure 2.
It is obvious that the average particle size for milled powder decreases (see Table
(a)
(b)
(c)
Figure 2. Particle size distribution of PrFeB powder milled

using HEM for (a) 0 s, (b) 60 s, and (c) 300 s.
1) which indicates that fracture in powder is dominant process in the milling

[9]. There is also a strong relationship between average particle size and milling
time of bonded PrFeB powder. Our previous study [10] confirms that notion by
demonstrating that longer milling time will trigger agglomeration process that
may increase the particle size of the powder. This is because longer milling time
will gradually increase milling temperature of the powder and induce smaller
particles to undergo coalescence and form larger particles. For shorter milling
time,heat that produced by friction between the powder, steel balls and steel wall
cell was relatively small and insufficient to cause agglomeration process on PrFeB
powder to take place.
Figure 3 shows SEM images of bonded PrFeB magnet (a) without and with
milling treatment for (b) 60 seconds and (c) 300 seconds. The distribution, shape
and size of the powders vary with increasing milling time. The images qualitatively
shows that original bonded PrFeB magnet has the highest porosity since the
initial state of the powder was not perfectly mixed with the binder and left some
particles of the powder free from the binder because of their larger size. However,
we were unable to determine the optimum binder content in PrFeB powder to
minimize porosity level because of the complexity of compacting process that is
(a)
(b)
Figure 3. SEM images of bonded PrFeB magnet (a) without and with
milling treatment for (b) 60 s and (c) 300 s.
400
350
Sampel #C
Intensity (a.u.) 300
250
Sampel #B
200
Sampel #A
150
100
50
0
20 25 30 35 40 45 50 55 60 65 70 75 80
2θ (de gre e )
Figure 4. XRD patterns of bonded PrFeB magnet produced using milled powder with dif-
ferent milling time
also affected by oxidation during the milling process, compacting pressure and
aging temperature.
XRD diffraction pattern for PrFeB powders without and with milling treat-
ment for 60 seconds and 300 seconds are depicted in Figure 3(a), 3(b), and 3(c),
respectively. The XRD patterns demonstrate that all samples have single peak that
closely located at 44.59°; 44.58°; and 44.48°. These diffraction peaks correspond
to the (006) tetragonal orientation (JCPDS 04-006-2854). The (006) plane is
perpendicular to the [001] easy-axis direction of tetragonal crystal structure of the
matrix phase. The obtained result is similar to the result that reported in sinter
Pr16Fe76B8 powder that annealed at 1,000oC [2]. Furthermore, there is almost
no significant change in full width at half maximum (FWHM) width (see Table
2) which suggest that micro residual stress remains exist [8]. In summary, milling
process that performed in a short period of time has been proven to preserve
crystal properties of the magnet and produce bonded PrFeB magnet with higher
density as supported by SEM analysis. Despite of this fact, it should be clarified
further whether magnetic properties of the magnet have changed or not following
post-milling process on the powder.
To confirm magnetic properties change, PrFeB powder was characterized
using VSM measurement. The coercive field and remanence ratio (Mr/Ms) of
bonded PrFeB magnet without and with milling for 60 seconds and 300 seconds
are (333.4 kA/m and 0.755), (91.1 kA/m and 0.319), and (268.7 kA/m and
Table 2. XRD analysis of bonded PrFeB magnet

d-spacing FWHM 2θ
Milling time (s) Crystalline size (Å) (°)
(A) (°)
0 2.03 0.78 116 44.59
60 2.03 0.88 102 44.58
300 2.04 0.79 114 44.48
0.699), respectively. The reduction of intrinsic coercive field suggests that magnetic
properties is sensitive to milling process and depends on the average particle size
of the powder [11]. The reduction could also be attributed to oxidation during
the milling process [7]. Decreasing trend of remanence of the powder is also
observed and it follows the same tendency that exhibited by intrinsic coercive
field. The powder that underwent 60-second milling process is found to have
the lowest intrinsic coercive field and remanence ratio. The reason is still unclear
and further investigation is still needed to elucidate this peculiar phenomenon.
IV. Conclusion
Short time milling process (60 and 300 seconds) on PrFeB powder has been
proven to effectively alter the average particle size of the powder while keep
maintaining its crystal properties based on the analysis provided by PSA and XRD.
Surface images of bonded PrFeB magnet from SEM characterization provide
distinctive transition process from rough surface with higher porosity to surface
with less porosity and homogeneous particle size. The result implies that milling
process can be performed efficiently within short period of time hence, avoiding
agglomeration process becomes possible. However, the intrinsic coercive field and
remanence ratio of bonded PrFeB magnet will decrease as the result of milling
process. This observation further instills the idea that magnetic properties of the
magnet depend on the average particle size of the powder.
V. Acknowledgement
This work was supported by the DIPA-LIPI 2014 with No. Vot. 0131/
IPT.1.03/A/2014
VI. R eferences
1) Sugimoto, S. (2011). Current Status and Recent Topics of Rare-Earth
Permanent Magnets. J. Phys. D: Appl. Phys., 44, 064001(1–11).
2) Corfield, M. R., Williams, A. J., and I. R. Harris. (2000). The Effects of
Long Term Annealing at 10008C for 24 H on the Microstructure and
Magnetic Properties Of Pr–Fe–B/Nd–Fe–B Magnets Based on Nd16Fe76B8
and Nd16Fe76B8. J. Alloy Compd., 296, 138–147.
3) Mingxiang, P., Pengyue, Z., Hongliang, G., Hangfu, Y., and W. Qiong.
(2012). Synthesis, Structures and Magnetic Properties of Pr-Lean Pr2Fe14B/
Fe3B Nanocomposite Alloys. J. Magn. Magn. Mater., 324, 2953–2957.
4) Rodewald, W., Katter, M., and K. Uestuener. (2004). Coercivity and
Mechanical Properties of Nd-Fe-B Magnets in Dependence on the Average

Grain Size. Proc. 18th Int. Workshop on High Performance Magnets and Their
Applications, 486–492.
5) Nakamura, H., Hirota, K., Shimao, M., Minowa, T., and M. Honshima.
(2005). Magnetic Properties of Extremly Small Nd-Fe-B Sintered Magnets.
IEEE Trans. Magn., 41(10), 3844–3846.
6) Uestuener, K., Katter, M., and W. Rodewald. (2006). Dependence of the
Mean Grain Size and Coercivity of Sintered Nd-Fe-B Magnets on The Initial
Powder Particle Size. IEEE Trans. Magn., 42(10), 2897–2899.
7) Faria, R. N., Castro, A. R. M., and N. B. Lima. (2002). Relation Between
Grain Alignment and Magnetic Properties of Pr–Fe–B Sintered Magnets. J.
Magn. and Magn. Mater., 238, 38–46.
8) Pèrigo, E. A., Lima, N. B., Takiishi, H., Motta, C. C., and R. N. Faria.
(2008). The Effect of Key Process Parameters on Crystallographic Texture
and Magnetic Properties off PrFeB HD Sintered Magnets Produced Using
High-Energy Milling. J. Magn. Magn. Mater., 320, e36–e39.
9) Khodsiani, Z., Mansuri, H., and T. Mirian. (2013). The Effect of Cryomilling
on the Morphology and Particle Size Distribution of the Nicocralysi Powders
With and Without Nano-Sized Alumina. Powder Technol., 245, 7–12.
10) Aryanto, D., Kurniawan, C., and T. Sudiro. (2014). Studi Pengaruh Mill-
ing pada Serbuk Bonded PrFeB. Proc. Seminar Nasional Fisika 2014, to be
published.
11) Su,K. P., Liu, Z. W., Zeng, D. C., Huo, D. X., Li, L. W., and G. Q. Zhang.
(2013). Structure and size-dependent properties of NdFeB nanoparticles and
textured nano-flakes prepared from nanocrystalline ribbons. J. Phys. D: Appl.
Phys., 46, 245003 (1–5).
EFFECT OF SINTERING TEMPERATURE ON
DIELECTRIC CONSTANT OF SILICA PREPARED FROM
RICE HUSK ASH
Qudratun*, Y. Iriani, Kusumandari, S. Khoirum

Departement of Physics, Sebelas Maret University
Ir. Sutami 36A Street, Surakarta, Indonesia
Abstract
Silicon dioxide (silica) is one key material in micro- and nano-electronic industries. Silica from
rice husk ash was synthesized by sintering at 800°C and 900°C. X-Ray Fluorescence (XRF)
technique was used to determine silica content of rice husk ash. The dielectric constant obtained
through LCR meter testing. Silica content of rice husk ash at 800°C and 900°C are 90,38 (wt%)
and 90,56 (wt%) respectively. Only 87,3% porosity at 900°C but at 800°C increased to 93,5
%. Dielectric constant (K) values for samples sintered at 800°C and 900°C are 5,85 and 6,02
respectively. The value increased with increasing sintering temperature.
Key words: Dielectric constant, Rice husk Ash, Sintering.
I. Introduction
Rice husk is major waste product of the rice industry obtained during milling
of rice. It is reported that approximately 20% of rice produced rice husk [1][2].
World rice production is approximately 645 million tons. South and South East
Asia account for over 90% of world’s rice production [3]. In certain countries,
farmer burned the rice husk. This procecess would cause environmental pollu-
tion. However, this problem can be solved by recycling the waste to produce
eco-material having a high end value [1].
Rice husk ash has been used in various industries, such as steel, ceramic and
refractory, cement and contruction industries and silica source [4]. Silica called
Silicon dioxide (SiO2) is the major chemical composition of rice husk ash. Silica
is a chemical compound that is a dioxide of silicon. The melting point of silica
is 1,600°C [5].
There are many methods to get silica, such as titrimetric, precipitation and
heating. Heating processed in the material fabrication at high temperature called
sintering [6]. Okuyama defined sintering as an irreversible thermodynamic
phenomenon to convert unstable packed powder having excess free energy to
* Corresponding Author.Tel: +62-857-28462978. E-mail: qudratun@nano.or.id
457
stable agglomerates [7]. During sintering, the atom in the powder particles diffuse
across the boundaries of the particles, fusing the particles together and creating
one solid piece. Sintering phenomenon involves fusion of particles, volume
reduction, and increase in grainsize as well as decrease in porosity. Porosity is the
amount of pore in sample.
Sintering process on rice husk as at higher temperatures can remove the
unburned carbon from the ashes and crystalline silica is found, but at lower
temperature amorphous silica is found [4,8].
Silica is one key in micro- and nano-electronic industries. Silica as dielectric
metal oxide semiconductor gate has dielectric constant about 3,9. At different
temperature heating for synthesis, silica gives different silica content and impurity.
Dielectric property of the material is one of fundamental characters. Dielectric
constant is one of dielectric properties. Therefore, it is very important to investigate
dielectric constant of the impure silica [9].
In this study, silica content in rice husk ash sintered at temperature 800°C
and 900°C were determined by XRF. Dielectric constant of silica in rice husk
ash was examined by LCR meter. The porosity detected by Archimedes’ principle
with water as the medium.

Raw material (rice husk) was supplied from Karanganyar, Indonesia. Rice husk was
washed by aquades in order to remove clay and rock impurities and subsequently
drying naturally by the sun. The dried rice husk then milled by dry mill to be
powder. The powder filtered into 150 meshs. The powder was molded into a pellet.
The pellet taken in crucible and placed in nabertherm furnace at 300°C to get
black ash and heated again at 800°C and 900°C to get rice husk ash. Analysis of
silica content from rice husk ash was determined by X-Ray Flourescence (XRF).
Crystal and amorph phases of the sintered RHA pellet were identified by XRD
analysis. X-Ray Diffractometer (Bruker D8 Advance) λCu about 1,5406 Å. Porosity
calculated by equation (1). The initial weight of pellet sample was measured
by the digital electronic scale. Then, the measured pellet sample was soaked in
water in a beaker glass for 24 hours at room temperature. As the samples were
immersed in water, bubbles were observed as the pores in the samples were filled
with water. After the lapsed time, the samples were removed from the beaker glas
and allowed to dry by removing the excess water on the surfaces dry napkin prior
to weighing as wet weight.
(1)
Dielectric constant examined by LCR meter (LCR-800 Series Gwinstek).

Dielectric constant measurement performed the capacitance value. The dielectric
constant can determine by equation (1). K is dielectric constant, C is capacitance ,
A is surface area of sample, d is thickness of sample, dan ε0 is dielectric permittivity
(8,85 x 10-12 Farad.m-1).
(2)

A. XRF Analysis
XRF analysis determined silica content of rice husk ash. Table 1 shows the
percentage of silica at different temperature. Silica content of rice husk ash that
sintering at 900°C is 90,56% and 90,38% at 800°C. More silica content got at
higher temperature. It is believed that temperature give effect for percentage of
silica from rice husk ash.
Tabel 1. XRF Analysis of Silica Content at Different Temperature

Temperature (°C) Percentage of Silica (%)
800 90,38
900 90,56
Figure 1. XRD pattern of silica produced from rice husk ash at differ-
ent temperature
Tabel 2. Dielectric Constant and Porosity of the Silica at Different Temperature

Temperature (°C) Dielectric Constant Porosity (%)
800 5,85 93,50
900 6,02 87,30
B. XRD Analysis
The XRD spectra of silica produce of rice husk ash is shown in Figure 1. A broad
peak centered at 2Ө angle of 22° at intensity 3,632 confirmed the amorphous
silica at 800°C. Whereas a sharp peak centered at 2Ө angle of 22° at intensity
1,1398 confirmed the crystalline silica at 900°C. It is believed that higher sintering
temperature give effect to form crystalline silica.
C. Porosity
Table 2 shows the value of dielectric constant’s silica from rice husk ash at 800°C
is 5,85 and at 900°C is 6,02. Increament of sintering temperature increased the
dielectric constant. Porosity at 800°C is 93.50% and decrease to 87,30% at
900°C. As sintering progresses the pores become smaller, it shows at table 2 the
porosity decreasing by increases sintering temperature. Reduction of the pores
make sample become more compact. It is observed that the increment of sintering
temperature increased the dielectric constant but decreased the porosity.
IV. Conclusion
The effect of sintering temperature on dielectric constant was investigated. Increase
in sintering temperature at 900°C form crystalline silica, increasing percentage
of silica content become 90,56% and dielectric constant increase to 6,02 and
consequently the porosity decreases from 93,50% to 87,30%.
V. Acknowledgement
The authors gratefully acknowledge to The Ministry of Higher Education (DIKTI)
Student Creativity Program for Scientific Grant 2013.
VI. R eferences
1) Prasad, R., and M. Pandey. (2012). Rice Husk Ash as a Renewable Source
for the Production of Value Added Silica Gel and Its Application. BCREC,
7 (1), 1-25.
2) Kalapathy, U., Proctor, A., and J. Shultz. (2000). A Simple Method for
Production of Pure Silica from Rice Hull. Biores. Tech., 73 (3), 257-262.
3) Balakrishnan.S.S. (2006). Rice Husk Ash as a Support Material for Iron

and Ruthenium Based Heterogeneous Catalyst. Thesis, 85 pages. Penang:
Universiti Sains Malaysia.
4) Haslinawati, M. M., Matori, K. A., Wahab, Z. A., Sidek, H. A. A., and A.
T. Zainal. (2009). Effect of Temperature on Ceramic from Rice Husk Ash.
IJBAS-IJENS, 9 (9), 22-26.
5) Mbaakan, C., Onojah, A., Gbaakpen, M., and D. Tragema. (2013). Variation
of Some Physical Properties of Rice Husk Ash Refracory with Temperature.
IJSR, 2 (9), 32–26.
6) Njoku, R. E., and A. R. Kennedy. (2013). Effect of Sintering Temperature
on The Density and Porosity of Sodium Chloride Preforms for open Celled
Aluminium Foam Manufacturing. NIJOTECH, 32 (1), 117-122.
7) Okuyama, K. (2006). Fundamental Properties of Particles: Sintering. Powder
Technology: Fundamentals of Particles, Particle Beds, and Particle Generation,
213-218. H. Matsuda et al., Ed. USA: CRC Press.
8) Thuduaij, N., and A. Nuntiya. (2007). Preparation of Nanosilica Powder
from Rice Husk Ash by Precipitation Method. Chiang Mai J. Sci., 3, 206-211.
9) Cheng, Y., and L. Jiang. (2009). Study on the Dielectric Property of the
impure Silicon Dioxide Based on FEM.Proceedings of the 9th International
Conference on Properties and Application of Dielectric Material, 374-377.
SYNTHESIS AND CHARACTERIZATION OF AL-DOPED
LITHIUM TITANATE Li4Ti5O12 AS ANODE MATERIAL
FOR LI-ION BATTERY
Slamet Priyonoa, *, Achmad Subhana, Joko Triwibowoa, Bambang Prihandokoa
Research Center for Physics – LIPI
a
Kawasan PUSPIPTEK Gedung 442 Serpong, Tangerang Selatan 15314, Banten

Telp. 021- 7560570, Fax 021- 7560554
Abstract
Synthesis of Li4Ti5O12 powder doped by Al atom for lithium ion battery anodes had been done.
Al doped on Li4Ti5O12 was aimed to increase the ionic conductivity and strengthen the structure
of Li4Ti5O12. Al doped on Li4Ti5O12 had been performed by following equation Li(4 – x/3)AlxTi(5 –
O (x = 0, 0.025, 0.05, 0.075) where the Al atoms substitute Ti and Li atoms. Synthesis was
2x/3) 12
done through a solid state reaction by using Li2CO3, TiO2-anatase, and Al2O3 as raw material.
In this study, the effects of substitution of Al in Li4Ti5O12 on the structure, morphology, particle
size, surface area and electrochemical performance was studied. The XRD patterns showed that
the Al doped on the Li4Ti5O12 did not change crystal structure of Li4Ti5O12. Morphology of the
obtained powder was observed by SEM, it showed that the increasing Al doped make the powder
porous. Further observation showed that the particle size decreased to 20.32 µm, surface area
increased with highest value as 8.25m2/gr and the conductivity is increased by increasing of Al
doped. The best conductivity was 8.5 x 10-5 S/cm, the working voltage was about 1.55 V and the
best cycle stability was obtained on doping Al 0.05. The maximum specific capacity was 70mAh/g.
Key words: Li-ion batteries, Anode, Lithium titanate, Aluminium doping
I. Introduction
The lithium titanate Li4Ti5O12 will substitute graphite as anode material in lithium
ion battery system, especially in HEV and EV applications [1]. Graphite is widely
used as anode material due to its advantages properties as high specific capacity,
high conductivity, environmentally friendly and abundant on earth. Unfortu-
nately, graphite has a lower operating voltage (0 to 0.4 V) than the decomposition
voltage of the electrolyte. It will stimulate a formation of a passive layer, called
Solid Electrolyte Interface [2–3]. This layer will hinder intercalation of Li ions
into the host anode. Lithium titanate Li4Ti5O12 has a higher operating voltage,
hence there will be no SEI-layer formed on the anode surface. Lithium titanate
Li4Ti5O12 has also high rate capability [4]. Spinel Li4Ti5O12 has a theoretical
specific capacity of 175 mAh/g and exhibits excellent reversibility due to the zero
volume change during cycles or charge/discharge process. Li4Ti5O12 also shows
* Corresponding Author. Tel: +62-21-7560570. E-mail:slamet.priyono@lipi.go.id
463
excellent thermal stability and cyclic performance, making it a potential anode

material for high-power applications [4]. However, Li4Ti5O12 has a low intrinsic
electronic conductivity and low diffusion coefficient of lithium-ion. Extensive
research until now has been done to address this problem. Material modifications
has to be made to enhance the performance of this material, including the carbon
layer [5], doping metal ions and non-metals [6], hybridization with carbon and
metal powders, particle size reduction of Li4Ti5O12 [7-8].
In this research, the effect of the Al doping on Li4Ti5O12 was observed.
Synthesis was carried out through solid state process. The obtained phases of this
anode material were observed by XRD analysis. SEM analysis was carried out to
observe the morphology of the particle. Particle size and the surface of the obtained
powder were analized by particle size analyzer (PSA) and Baumer-Emmer-Teller
(BET) equipment, respectively. Electrochemical impedance spectroscopy (EIS)
was performed to observe the conductivity and the battery cylcler was used to
analyze the battery performance.
II. M aterial and methods

The raw materials are in the powder form. Lithium carbonate Li2CO3 was used
as lithium source, titanium oxide as titanium source and aluminum Oxide Al2O3
as aluminum source. After weighing, all the raw materials were mixed until
homogeny powder mixture obtained. The target compound, Al-doped Li4Ti5O12,
was prepared by conventional two-step solid-state process. The preparation of
the compound depicted in Figure 1.
Figure 1. Synthesis method and characterization of sample Al-doped Li4Ti5O12

Homogen solid mixture was heated at 700°C for 2 hours. This step was
purposed to decompose the organic content. The precursor Al-doped Li4Ti5O12
was further grounded and brought to the second heating process. This heating
process was carried out at 800°C for 4 hours and purposed to synthesize the target
compound. The obtained phase, morphology, microstructure and the surface area
of the target powder were analyzed by means of XRD, SEM, PSA and BET. To
observe the conductivity and the performance of the anode material, the obtained
powder was coated on Al-foil and assembled into the half cell wherein metallic
lithium was used as counter electrode and 1M LiPF6 in solution EC:DEC (1:1
V/V) as electrolyte. AC impedance measurement was conducted using a HIOKI
3532-50 LCR-meter. A 10mV amplitude sinusoidal wave was applied in the
tested frequency range of 10 Hz–100 kHz. The galvanostatic charge–discharge
tests were carried out at constant cut-off voltage of 1–2.4 V or 0–2.4 V using a
Wonatech WBCS 3000 battery cycler.

A. Diffraction Pattern Analysis
The analysis of XRD pattern was performed using RIGAKU equipment and
built-in PDXL software for Rietveld refinement. KCu-α was used as x-ray source
and the scanning speed of 2°/min was applied for the range 0–80°. Diffraction
pattern of samples are given in Figure 2. It shows there are two phases, i.e.
Li4Ti5O12 and TiO2 –rutile obtained.
Figure 2. Diffraction pattern of Li(4-x/3)AlxTi(5-2x/3)O12 with a) x = 0, b) x = 0.025, c) x = 0.05, d) x

= 0.075
The result of data processing using PDXL software shows that the phase
Li4Ti5O12 was formed about 90% and TiO2 rutile about 10%. The peaks cor-
responding to the Li4Ti5O12 indicates that the Al atom occupied Li and Ti places
on the Li4Ti5O12 crystal structure without changing the crystal structure. The
diffraction patterns of all samples have a diamond cubic structure with space group
Fd3m Li4Ti5O12 (JDPDS, No. 49-0207). Diffraction peak of Li4Ti5O12 occurs
at 18°, 37°, 43°, 47°, 58°, 63°, 66°, 75°, 76° and 79°. These peaks correspond to
the (111), (311), (400), (331), (511), (440), (531), (533), (622) and (444) plane.
Although the main phase has been obtained, the presence of TiO2-rutile peak
causes the powder obtained is not a single phase but a composite of Li4Ti5O12
and TiO2-rutile. The composition of the composite is given in Table 1.
Table 1. Composition of Al-doped Li4Ti5O12 obtained by analyzing
diffraction pattern of samples with PDXL-software
B. Morphology Analysis
The morphology of Li4Ti5O12 and Al-doped Li4Ti5O12 powder were analyzed
by using SEM with 15,000 times of magnification and depicted in Figure 3.
Li4Ti5O12 powder has various forms from sphere to oval. Li4Ti5O12 powder
has a tendency to get a rectangular form by increasing Al content in Li4Ti5O12.
(a) (b)
(c) (d)
Figure 3. SEM photography of Li(4-x/3)AlxTi(5-2x/3)O12 with a) x = 0,
b) x = 0.025, c) x = 0.05, d) x = 0.075
Particle size of the obtained powders was observed with CILAS 1190 equip-
ment. Filtered water was used as dispersant. Result of the particle observation is
given in Table 2. This table shows the higher Al content in LTO the lower the
particle size of the obtained powder. This phenomenon occurred due to the Al
element in Li4Ti5O12 prevent the grain growth during the heating process.
Therefore, a lattice constant of the obtained material becomes smaller and also
the particle size.
Surface area of the powder is important to facilitate electrochemical reaction
occure intensively. Specific surface area measurement was performed by BET
method. The result is given in Table 3. Compared to surface area of the sample
Li4Ti5O12, Al dopant seem to have no effect on the surface area. It is appropriate
with the SEM analysis where the surfaces of the samples are similar.
Table 2. Particle size of Li(4-x/3)AlxTi(5-2x/3)O12 with varied Al-content
Table 3. Specific Surface Area of Li(4-x/3)AlxTi(5-2x/3)O12 with varied Al-

content measured through BET method
C. Conductivity and Capacity Analysis

Conductivity and battery performance of samples are performed in the form of
half cell. Metallic lithium was used as counter electrode, LTO as working electrode
and 1 M LiPF6 in solution of EC:DEC as electrolyte. The conductivity was
measured when the half cell condition was 20% charged. Data of the conductivity
test using EIS equipment was given in a Nyquist plot as depicted in Figure 4. The
conductivity of samples tends to increase by increasing Al-dopant.
Figure 4. EIS graph of Li(4-x/3)AlxTi(5-2x/3)O12 with a) x = 0, b) x = 0.025, c) x

= 0.05, d) x = 0.075 with the frequency range 10Hz – 100kHz.
By bringing the thickness and the surface area of the working electrode into
account, the conductivity of the sample can be determined. The conductivity of
the samples is given in Table 4.
Capacity analysis had been done by applying constant current and constant
voltage to the sample. In order to get the maximum capacity of the battery,
battery was charged with 0.1C until battery potential of 2.4 Volt was reached and
further charged at constant voltage of 2.4 V for 1 hour. Discharge process was
also carried out with the same rate. This cycle procedure was 4 times repeated.
Charge–Discharge graphs of the samples are given in Figure 5a to 5d. Figure 5
shows that Sample A has an operational potential of 1.55 Volt where the potential
is constant during the discharge process. The maximum specific capacity of this
Table 4. Conductivity of Li(4-x/3)AlxTi(5-2x/3)O12 with a) x = 0, b) x =

0.025, c) x = 0.05, d) x = 0.075 where the thickness and the area of
samples are considered
sample is around 110mAh/g, exhibited at the first cycle. The discharged specific
capacity is reduced until 40% at the fourth cycle. It implies that the active material
is not really stable. Figure 5b shows that sample B reaches the maximum specific
capacity of 70mAh/g. This value is lower than sample A. However, sample B
shows more stable crystal structure. The discharge specific capacity of sample B
still remains 81% after the fourth cycle.
The presence of Al doping in the sample B was causing a decrease in capacity,
but it showed better structure stability. The bond energy of Al-O bond which
is greater than the Ti-O bond led to a better structural stability when subjected
to charging/discharging. Figure 5c shows that sample C has a capacity of only
6 mAh/g and tends to be unstable. The specific capacity was reduced until 30%
after fourth cycle. Meanwhile, Figure 5d shows that sample 5d has a capacity of
130 mAh/g and is also unstable. The specific capacity is reduced to 25% after the
fourth cycle. Capacity reduction in sample C and D are due to the large number
of Al-doped atoms; therefore, they behave as impurities which make the structure
not strong and tend to be unstable.
IV. Conclusion
The doping process of Al on Li4Ti5O12 powders is quite effective by means of
powder metallurgy method. Al tends to substitute Ti atom at low doping and
substituting Li at high doping. Al doping on Li4Ti5O12 powder did not change
the crystal structure (confirmed from the XRD results). The increase of Al doping
caused particle morphology to be agglomerated and porous. The surface area
became stable, the particle size was reduced, the conductivity was increased,
but the capacity was reduced. The stability of battery performance was obtained
from sample B (with Al doping of 0.025), with the capacity of 70 mAh/g and
the working voltage of 1.55 V.
(a)
(b)
(c)
(d)
Figure 5. charge-discharge curves of Li(4-x/3)AlxTi(5-2x/3)O12
with a) x = 0, b) x = 0.025, c) x = 0.05, d) x = 0.075
V. Acknowledgement
Authors would like to thanks to all the members of the Research Centre for Phyisc.
VI. R eferences
1) Nelson, R.F. (2000). Power Requirements for Batteries in Hybrid Electric
Vehicles. J Power Sources 91, 2-26.
2) Channu, V.S., Bobba, R., R. Holze. (2013). Graphite and graphene oxide
electrodes for lithium ion batteries, Colloids and surface A : Physicochem.
Eng. Aspects 436. 245-251.
3) Wu, K., Yang, J., Qiu, X.Y., Xu, J.M., Zhang, Q.Q., and J.Jin. (2013).
Study of spinel Li4Ti5O12 electrode reaction mechanism by electrochemical
impedance spectroscopy. Electrochemica Acta, 108, 841-851.
4) Ferg, E., Gummow, R.J., Kock, A. d., and M.M. Thackeray. (1994). Spinel
Anodes for Lithium-Ion Batteries. Electrochem. Soc. 141, (1994) L147-L150.
5) Zhang, H., Chen, Y., Li, J., He, C., and Y. Chen. (2014). Li4Ti5O12/
CNT composite anode material for large capacity and high-rate lithium
ion batteries. International Journal of Hydrogen Energy 39, 16096-16102.
6) Park, J.S., Back, S.H., Jeong, Y-II., Noh, B.Y., and J.H. Kim. (2013). Effects
of a dopant on the electrochemical properties of Li4Ti5O12 as a Lithium
Ion battery anode material. Journal Power Sources xxx, 1-5.
7) Borghols, W.J.H., Wagemaker, M., Lafont, U., Kelder, E.M., and F.M.
Mulder. (2010). Lithium Storage in Amorphous TiO2 nanoparticles. Journal
of The Electrochemical Society, 157 (5), A582-A588.
8) Cai, R., Jiang, S., Yu, X., Zhao, B., Wang, H., and Z. Shao. (2012). A Novel
method to enhance rate performance of an Al-doped Li4Ti5O12 electrode
by post-synthesis treatment in liquid formaldehyde at room temperature.
J. mater. Chem., 22, 8013-8021.
Cellulose Fibers From Oil Palm Fronds
Reinforced Polylactic Acidb Composite
Firda Aulya Syamani*, Yudhi Dwi Kurniawan, Lisman Suryanegara

Research Center for Biomaterials, Indonesian Institute of Sciences
Jl. Raya Bogor Km. 46, Cibinong, West Java, Indonesia
Abstract
The aim of this study is to evaluate the effect of cellulose fibers from oil palm fronds on the
mechanical properties of polylactic acid (PLA) composites. PLA has a great potential to replace
petroleum-based plastic, due to its high stiffness and strength are comparable to polystyrene.
Nevertheless, the low heat resistance, brittleness and slow crystallization characteristics of PLA
are its drawback. To overcome PLA limitation, one of the best solution is natural fiber addition
into PLA to produce green composite. Natural fiber from oil palm frond (OPF) is renewable and
available excessively in oil palm plantation during cultivation and harvesting time. Three major
components of natural fiber are cellulose, lignin and hemicellulose. After pulping and bleaching,
lignin and hemicellulose of oil palm frond fiber will be removed and set aside cellulose fiber which
performance as reinforcing agent in PLA composite. The green composites were prepared by
mixing OPF cellulose fibers and PLA in an organic solvent with various fibers content (5 wt%,
10 wt%, 15 wt%) and various plasticizer (5 wt%, 10wt%). Mechanical properties data show that
addition of pulp fibers improved the Young modulus of PLA composite.
Key words: Oil palm frond pulp cellulose fibers, Polylactic acid composite, Mechanical properties,
Composite morphology.
I. Introduction
Natural fibers derived from renewable natural resources, such as timber and
non-timber which are available in large quantities. This natural fiber has
advantages, such as it is able to be decomposed naturally, so it does not pollute
the environment at the end of its use. The main components of natural fibers are
cellulose, hemicellulose and lignin so that are often referred to as the lignocellulosic
materials. Cellulose is a long chain polymer, polysaccharide carbohydrate of
beta-glucose. The most common of cellulose utilization is used as paper. Currently,
cellulose was developed for use in the polymer matrix composite products, both
natural polymers (eg. starch or poly lactic acid) and synthetic polymers (such as
polypropylene or polyethylene). Current research is directed to develop a green
composite that promotes the use of natural materials as the matrix and the
reinforcing component in composite products.
* Corresponding Author.Tel: +62-21-87914509. E-mail: firda.syamani@biomaterial.lipi.go.id
473
Poly-lactic acid (PLA) is one kind of bio-based thermoplastic polyester of

lactic acid, which can be produced from fermented natural-polysaccharides
such as corn or potato starch [1][2]. Compared to other bio-based plastic, PLA
provides good clarity, high strength, moderate barrier properties and easy to
process in most equipments [3], and is thus used in wide areas of practice, such
as textile applications, food packaging, film applications and interior automotive
parts [4]. Nevertheless, the low heat resistance, brittleness and slow crystallization
characteristics of PLA are its drawback.
Many kinds of natural fibers have been applied as reinforcement in PLA
composite, such as rice straw, hemp, kenaf and oil palm empty fruit bunch fibers
[5,6,7,8]. Natural fibers in PLA matrix were applied as long fibers [9], short fibers
[7,10] and micro fibers [5,8,11,12]. Further processing of natural fibers to obtain
cellulose in form of microcrystalline cellulose, microfibrillated cellulose or nano
fibers were conducted to overcome cellulose fibers limitation when blended with
PLA [1,13,14, 15].
In this study, we used cellulose fibers from bleached soda pulp of oil palm
fronds (OPF) as reinforcement in PLA matrix. Soda pulping used sodium
hydroxide as the only chemical agent in pulp production. However, high amount
of residual lignin that is still attached to the fibers must be removed in the
subsequent bleaching operation. Concerning environmental safety, hydrogen
peroxide was used rather than chlorine in oil palm fronds pulp bleaching.
Brittleness is one of PLA drawback. Therefore, to improve composite
mechanical properties, plasticizer, such as glycerol triacetate ester was applied in
PLA matrix along with cellulose fibers, in order to increase PLA elongation. The
potential of oil palm fronds cellulose fibers and glycerol triacetate to improved
PLA characteristics are therefore explored in this study.

A. Materials
PLA trade named Ingeo™ Biopolymer 4060D was supplied by NatureWork®.
The PLA density was 1.24g.cm-3 and melting point was 140°C. Oil palm fronds
(OPF) collected from oil palm plantation in West Java, Indonesia, were used in
this study. The sodium hydroxide at technical grade was used in pulping process.
Bleaching process used technical grade of hydrogen peroxide. The ethanol and
acetone at technical grade was distilled prior to use. Glycerol triacetate (GTA)
and potassium hydroxide were supplied by Merck.
B. Preparation of Cellulose Fibers

The leaves were cut off from oil palm fronds, then the fronds were processed with
ring flaker and screened to produce strands with 1–2 cm length. The moisture
content of strands was 13%. To produce soda pulp, oil palm fronds fibers were
subjected to digester with ratios of liquor-to-materials of 8:1. Soda pulping was
carried out at 45% alkali charge. Thus, NaOH solution and water with total of
3,500 ml were added to digester after 500g OPF strands (dry basis). Pulping was
conducted in 2 h and 46 min, at 176oC. After cooling the digester, the pulp was
collected and washed with water several times until neutralized.
For the bleaching process, as much of 20 gram OPF pulp (dry basis) and
300ml distilled water were added in 500 ml erlenmeyer, then put in waterbath
at 80oC. The technical grade solution of 50% hydrogen peroxide was used as
bleaching agent. Every 60 min, 8ml of 50% hydrogen hydroxide was added into
erlenmeyer, for 3 times. After 3 h bleaching process, bleached pulp was collected
and washed with running water.
Tabel 1. Composition of the Sample

PLA GTA
Samples OPF fibers (g)
(g) (g)
Neat PLA 60 0 0
PLA/fibers 5wt% 57 3 0
PLA/fibers 10wt%/
51 6 3
GTA 5wt%
PLA/fibers 10wt%/
48 6 6
GTA 10wt%
In order to eliminate hemicellulose content, bleached pulp were reacted with

5% (w/v) potassium hydroxide in waterbath at 80oC for 2 h, then washed with
running water. The final stage to produce cellulose fibers was further bleaching
process with 8 ml of 50% hydrogen peroxide for an hour at 80oC.
C. Composite Preparation
For composite preparation, it was necessary to remove water from cellulose fibers
before mixing with PLA. Water removal were conducted in magnetic plate stirrer.
An adequate OPF cellulose fibers (refer to Table 1) was stirred in 300 ml technical
grade of 96% ethanol for 30 minutes, followed by vacuum filtration to remove the
liquid phase (water and ethanol). The process was repeated three times in order
to remove water by ethanol. Next, 300 ml acetone using the same procedure and
repeating three times was used to completely remove the water. Meanwhile, an
amount of PLA (refer to Table 1) was dissolved in 200 ml acetone. The precipitate
of cellulose fibers was suspended gradually in dissolved PLA and stirred in a beaker
until well mixed for 30 minutes. The OPF cellulose fibers-PLA-acetone mixture
was spread in trays and the solvent was evaporated at room temperature for 12h
followed by oven drying at 60°C for 24 h.
The mixture of PLA/OPF cellulose fibers and PLA/OPF cellulose fibers/GTA
were kneaded by a twin rotary mixer (rheomix, HAAKE polydrive) at 140°C,
40 rpm for 8 min. The compound was crushed into small pieces and hot pressed
into sheets at 140°C in two steps: pre- heating for 5 min and 0.5MPa for 3 min.
Afterwards, sample was cooled at room temperature for another 15 min.
D. Tensile Testing
The tensile properties of samples were measured using a Shimadzu universal testing
machine (UTM) with loadcell of 1kN. The specimen gage length of about 20 mm
was measured with a caliper for each sample upon gripping and the crosshead
speed was set at 1mm/min. The specimens were 40 mm long, 6 mm wide and
0.5 mm thick. All results presented are the average of three measurements.

The mechanical properties of the composites are evaluated by 3 parameters,
namely the tensile strength (MPa), modulus of elasticity (GPa) and elongation
(%). Furthermore, sampel cross section after tensile testing was observed by
microscope to showed how was cellulose dispersion in PLA matrix.
A. Effect of Cellulose Fibers on Mechanical Properties of PLA Composite

Tensile strength and modulus of elasticity (MOE) of PLA reinforced by oil palm
frond cellulose fibers are depicted in Figure 1. Addition of cellulose fibers into
PLA matrix could improve MOE of composite. The higher the cellulose fibers
load, the higher the composite MOE. PLA composite with 15 wt% cellulose fibers
exhibited the higher MOE than that of PLA which was 2.03 GPa, compared to
1.18 GPa.
Higher MOE value attributed to the improvement of composite stiffness
on the boundary of composite elastic properties. Cellulose fibers consist of
amourphous part and crystalline part. The crystalline cellulose has crystal stucture
that has MOE of 130–240 GPa [16], and induced fiber stiffness. The presence of
stiff cellulose fibers in PLA matrix lead to higher composite MOE value.
On the other hand, in this study the addition of cellulose fiber of OPF could
not improve PLA tensile strength. The tensile strength of PLA composite with
15 wt% OPF cellulose fiber was 42.0 N/mm2, while tensile strength of neat
2500 60
50
2000
40
1500
30
1000
20
500
10
0 0
neat PLA PLA_C5% PLA_C10% PLA_C15%
MOE (N/mm2) Tensile Strength (N/mm2)
Figure1. Modulus of elasticity and tensile strength of PLA composites with various
cellulose fibers loading
PLA was 47.9 N/mm2. Composite tensile strength was influenced by dispersion
of cellulose fibers in PLA matrix and compatibility between cellulose fibers and
PLA. In fact, PLA is a hydrophobic materials and cellulose fibers were hygrocopic
materials. Although there was an effort to remove water from cellulose fibers, it
seem that cellulose fibers still not well dispersed in PLA matrix. This condition
caused some “weak points” in composite and influenced the tensile strength. The
dispersion of cellulose fibers in PLA matrix was described in Figure 2.
Elongation of PLA composite with various cellulose fibers loading was showed
in Figure 3. The improvement of composite stiffness attributed by cellulose fibers
was also demonstrated by composite elongation reduction.
Figure 2. Cross section of PLA-15 wt% OPF cellulose

fibers composites at 200x magnification
2500 6.8
6.7
2000
6.6
1500 6.5
6.4
1000 6.3
6.2
500
6.1
0 6.0
neat PLA PLA_C5% PLA_C10% PLA_C15%
MOE (N/mm2) Elongation (%)
Figure 3. Modulus of elasticity and elongation of PLA composites with various cel-
lulose fibers loading
The decrease in elongation and tensile strength of composite compared to
pure polymers can be associated with inadequate wetting of the fibers with the
matrix [17], uneven aligning of the cellulose fibers [18] and most probable poor
adhesion between the filler and matrix [19]. The poor adhesion between matrix
and fibers initiates numerous voids at the fiber matrix interface, and the stress
transfer to the fibres which are the load bearing entities becomes inefficient leading
to lower strength and elongation values.
B. Effect of Plasticizer on Mechanical Properties of PLA Composite

One of PLA drawback was brittleness. To overcome that weakness, some plasticizer
such as glycerol triacetate was added into PLA composite. In this study, fibers
content in composite was held constant, 10 wt% and glycerol triacetate content
were varied 5 wt% and 10wt%. The addition of plasticizer could improve
composite elongation as showed in Figure 4.
As observed in Figure 4, the elongation of PLA composite was dramatically
improve in composite with 10 wt% glycerol triacetate. As a plasticizer, glycerol
triacetate molecules can penetrate into the composites and disrupt the binding
force between PLA and cellulose fibers macromolecules that makes it easy for
molecular chains to slide and move, consequently increasing the elongation.
In this study, plasticizer could improve composite elongation but decrease
composite tensile strength and MOE. As observed at Figure 5, the composite of
PLA reinforced with 10 wt% cellulose fibers without glycerol triacetate showed
higher MOE and tensile strength than PLA reinforced with 10 wt% cellulose
fibers and 5 wt% or 10% gylcerol triacetate.
400
351.6
300
Elongation (%)
200
100
6.8 6.3 6.5

0
neat PLA PLA_C10% PLA_C10%_P5% PLA_C10%_P10%
Figure 4. Elongation of PLA composites with various content of plasticizer
2500 60
50
2000
40
1500
30
1000
20
500
10
0 0
neat PLA PLA_C10% PLA_C10%_P5% PLA_C10%_P10%
MOE (N/mm2) Tensile Strength (N/mm2)
Figure 5. Modulus of elasticity and tensile strength of PLA composites with various content
of plasticizer
MOE of composite PLA-10 wt% cellulose fibers without plasticizer was

1908 MPa, whereas composite PLA-10 wt% cellulose fibers with 5wt% and 10
wt% plasticizer were 1775 MPa and 805 MPa. Tensile strength of composite
PLA-10 wt% cellulose fibers without plasticizer was 38.5 MPa, whereas composite
PLA-10 wt% cellulose fibers with 5wt% and 10 wt% plasticizer were 31.8 MPa
and 12.0 MPa. The tensile strength and MOE decline had occured possibly
due to the accumulation of glycerol triacetate at the interface between PLA and
cellulose fibers.
IV. Conclusion
The addition of cellulose fibers in PLA matrix caused higher composite MOE
value. PLA-OPF cellulose fibers composite’s tensile strength was influenced by
dispersion of cellulose fibers in PLA matrix. Plasticizer such as glycerol triacetate
could improved elongation of PLA-OPF cellulose fibers composite.
V. Acknowledgement
This research was financially supported by LIPI through Competitive Program
on Advanced Materials, fiscal year of 2014.
VI. R eferences
1) Halász, K., and L. Csóka. (2013). Plasticized Biodegradable Poly(lactic acid)
Based Composites Containing Celluloce in Micro- and Nanosize. J. Eng.,
1-9.
2) Lee, S. Y. (2010). Life Cycle Analysis: Comparing PLA Plastic Food Use Products
on the Basis of Energy Consumption. The University of British Columbia.
Vancouver, Canada. [Online]. Available: https://circle.ubc.ca/bitstream/
id/69950/SinYinLee_SEEDS_Student_Report.pdf.
3) Li, H., and M. A. Huneault. (2007). Effect of Nucleation and Plasticization
on The Crystallization of Poly (Lactic Acid). Polymer, 48 (23), 6855-6866.
4) Harris, A. M., and E. C. Lee. (2006). Injection Molded Polylactide (PLA)
Composites for Automotives Applications. Presented at 6th Annual SPE Automo-
tive Composites Conference, Troy, MI, USA.
5) Qin, L., Qiu, J., Liu, M., Ding, S., Shao, L., Lü, S., Zhang, G., Zhao, Y.,
and X. Fu. (2011). Mechanical and Thermal Properties of Poly(Lactic Acid)
Composites with Rice Straw Fiber Modified by Poly(Butyl Acrylate). Chem.
Eng. J., 166 (2), 772–778.
6) Song, Y. S., Lee, J. T., Ji, D. S., Kim, M. W., Lee, S. H., and J. R. Youn.
(2012). Viscoelastic and Thermal Behavior of Woven Hemp Fiber Reinforced
Poly(Lactic Acid) Composites. Compos. Part B-Eng, 43 (3), 856–860.
7) Graupner, N., and J. Mussig. (2011). A Comparison of the Mechanical
Characteristics of Kenaf and Lyocell Fibers Reinforced Poly(Lactic Acid) (PLA)
and Poly(3-Hydroxybutyrate) (PHB) Composites. Compos. Part A-Appl. S.,
42 (12), 2010–2019.
8) Senawi, R., Alauddin, S. M., Saleh, R. M., and M. I. Shueb. (2013). Polylactic
Acid/Empty Fruit Bunch FiberBiocomposite: Influence of Alkaline and Silane
Treatment on the Mechanical Properties. IJBBB, 3 (1), 59–61.
9) Oksman, K., Skrifvars, M., and J. F. Selin. (2003). Natural Fibers as Reinforce-
ment in Polylactic Acid (PLA) Composites. Compos. Sci. Technol., 63 (9),
1317-1324.
10) Tao, Y., Yan, L., and R. Jie. (2009). Preparation and Properties of Short
Natural Fiber Reinforced Poly(Lactic Acid) Composites. Trans. Nonferrous
Met. Soc. China, 19 (Supp. 3), 651-655.
11) Mamun, A. A., and A. K. Bledzki. (2013). Micro Fibers Reinforced PLA and
PP Composites: Enzyme Modification, Mechanical and Thermal Properties.
Compos. Sci. Technol., 78, 10-17.
12) Wong, S., Shanks, R. A., and A. Hodzic. (2007). Effect of Additives on the
Interfacial Strength of Poly(L-Lactic-Acid) and Poly(3-Hydroxy Butyric
Acid)-Flax Fibers Composites. Compos. Sci. Technol., 67 (11-12), 2478-2484.
13) Suryanegara, L., Nakagaito, A.N., and H. Yano. (2009). The Effest of
Crystallization of PLA on the Thermal and Mechanical Properties Of
Microfibrillated Cellulose-Reinforced PLA Composites.Compos. Sci. Technol.,
69 (7-8), 1187-1192.
14) Haafiz, M. K., Hassan, A., Zakaria, Z., Inuwa, I.M., Islam, M.S., and M.
Jawaid. (2013). Properties of Polylactic Acid Composites Reinforced with Oil
Palm Biomass Microcrystalline Cellulose. Carbohyd. Polym., 98 (1), 139-145.
15) Iwatake, A., Nogi, M., and H. Yano. (2008). Cellulose Nanofiber-reinforced
Polylactic Acid”, Compos. Sci. Technol., 68 (9), 2013-2106.
16) Michell, A. J., (1989). Wood Cellulose-Organic Polymer Composites.
Composite Asia Pacific 89, 19-21.
17) Khalil, H. P. S. A., Alwani, M. S., and A. K. M. Omar. (2006). Chemical
Composition, Anatomy, Lignin Distribution and Cell Wall Structure of
Malaysian Plant Waste Fibers. BioRes., 1(2), 220-232.
18) English, B. W., and R. H. Falk. (1996). Factors that Affect the Application
of Wood Fiber-Plastic Composites. Proc. Forest Product Society, 189-194.
19) Huda, M. S., Drzal, L. T., Misra, M., and A. K. Mohanty. (2006). Wood-fiber-
reinforced Poly(lactic acid) Composites: Evaluation of the Physicochemical
and Morphological Properties. J. Appl. Polym. Sci., 102 (5), 4856-4869.
ISOLATION AND CHARACTERIZATION OF LIGNIN
FROM ALKALINE PRETREATMENT BLACK LIQUOR OF
OIL PALM EMPTY FRUIT BUnCH AND SUGARCANE
BAGASSE
M. Adly Rahandi Lubisa,*, Aniva Rizkia Dewi b, Lucky Risantoa, Lukmanul

Hakim Zainia, Euis Hermiatia
a
Research Center for Biomaterial, Indonesian Institute of Science
Jl. Raya Bogor, Km.46, Cibinong, Bogor, 16911, Indonesia
b
Abstract
Lignin is one of potential biopolymers that can be obtained from pretreatment of lignocellulosic
materials. In this study, lignin was isolated from alkaline pretreatment black liquor of oil palm
empty fruit bunch (OPEFB) and sugarcane bagasse (SB) by precipitation with hydrochloric acid.
The isolated lignins were analyzed for their chemical properties, such chemical compositions,
UV absorbances, and FT-IR spectra. The results showed that the ash content of lignin from
alkaline pretreatment of SB (16.91%) was lower than from alkaline pretreatment of OPEFB
(19.19%), while the acid insoluble lignin (AIL) and acid soluble lignin (ASL) contents were
vice versa, 46.23 and 4.00% for the former, and 40.14 and 2.82% for the latter. The empirical
formula of the lignin isolates derived from the result of ultimate analysis were C9H13.8764O5.9318
and C9H14.1112O6.4321 for lignin from of OPEFB and SB, respectively. The UV spectra showed
that lignin from OPEFB had similar bands with lignin from SB. Both lignin from of OPEFB
and SB also had similar FT-IR spectra.
Key words: Lignin, Oil palm empty fruit bunch, Sugarcane bagasse, Alkaline pretreatment.
I. Introduction
One of the most challenging topics in material science is to convert biomass
waste and feedstock to highly added value-materials. Oil palm empty fruit bunch
(OPEFB) and sugarcane bagasse (SB) was the most agro-industrial residues. In
the process of extraction of palm oil from oil palm fruit, a lignocellulosic material
OPEFB is generated as a waste product. Approximately 15 million tons of this
agriculture waste is generated by oil palm milling operation annually and part of
it is burned in incinerators [1]. In Indonesia, the national sugar cane production
is 33 million tons/year and there are 58 sugar mills with a total milling capacity
* Corresponding Author. Tel: +62-21-87914511. E-mail: adlylubis89@gmail.com
483
Figure 1. Basic units of lignin.
of 195,622 tons of cane per day [2]. The amount of bagasse produced in sugar
cane processing is quite large, which is about 35–40% of the weight of cane with
a water content of 48–52%, sugar 2.5–6%, and fiber 44–48%. High fiber content
and availability of bagasse waste makes great use of bagasse as an alternative to
chemical material like lignin is strategic and promising [3].
Together with cellulose, lignin belongs to the most abundantly occurring
renewable resources. Lignin (latin lignum = wood) is the second most common
organic natural material and the real lignification substance. High material
strength develops by integration of lignin in plant cell walls and association
with cellulose fibrils by polyoses, whereby especially trees can stand upright and
absorb static and dynamic forces. Lignin components are phenylpropane units
with differing amounts of methoxy groups. The percentage of the basic units
of coumaryl (H-), coniferyl (G-) and sinapyl (S-) alcohols varies very much
depending on the botanical origin (Figure 1) [4].
Lignins are renewable and natural polymers. Five millions of metric tons of
lignins are produced in the world mostly as a non commercialized waste product
per year [5]. In pulp and paper industries, lignins are the main components of
residual liquors, notably black liquor from the Kraft process. Their principal
application is as fuel to produce energy [6]. The pulp and paper industry estimated
that 50 million tons of lignin were extracted in 2010, but only 2% has been
commercialized for the formulation of dispersants, adhesives, and surfactants
or as antioxidants in plastics and rubbers. In this way, the challenge is then to
explore the potential of this renewable resource, producing valuable functional
molecules for chemistry [7].
The objective of this research were to characterize lignin obtained from black
liquor of alkaline pretreatment of oil palm empty fruit bunch (OPEFB) and
sugarcane bagasse (SB).
II. E xperimental Methods

A. Material Preparation
OPEFB and bagasse feedstock used in the experiment was obtained from
Sukabumi and Subang. Prior to processing, raw material was dried in the open
(exposed to sunlight) for one week. The raw material then made into powder
using hammer mill. The resulting powder was then filtered with a filter of 40–60
mesh. Powder 40 mesh sieve and retained on the 60 mesh is further used as raw
material delignification.
B. Isolation of Lignin
Two hundred and fifty grams dry matter of sample was treated with 1% NaOH
(solid to liquid ratio 1:15), at 170°C for 1 h. The treatments were carried out in
a digester. At the end of the reaction, the digester was cooled for 2 h (temperature
below 70°C). Furthermore, the delignified material was filtered to obtain the
black liquor without any fibrous materials. After filtration, the solids are stored
in plastic while the black liquor is inserted into the container (such as a bucket)
as the insulating material lignin.
Hydrochloric acid ( 1 M HCl) was added in to the black liquor until reaching
pH 2 in order to precipitate the acidified lignin. The precipitate lignin were
then inserted into the freezer until there were frozen (± 24 hours). Deposition
of lignin that has been frozen were removed from the freezer, then air dried at
room temperature. The precipitate that had melted filtered using filter paper in
a vacuum filter and washed with 250 mL of distilled water. The wet lignin was
then inserted to the oven at 45°C for 24 h. Lignin oven dried was then weighed
and analyzed the chemical properties such as, lignin content (ASL and AIL),
chemical composition, UV absorption and FT-IR.
C. Lignin Characterization
Ash content of lignin was determined gravimetrically. 0.5 gram of lignin put in
porcelain cup. The sample was inserted in a muffle furnace at 525 ± 25°C for 4
h. After that, put in a desiccator for 1 hour and weighed until constant weight.
Determination of lignin content is done by treating lignin and 72% sulfuric
acid, and then stirred with a magnetic stirer for 4 hours. It was intended that a
solution of acid and lignin becomes homogeneous. Due to the color that was
too dark blackish brown, then the solution was diluted to 4% by adding distilled
water to 84 mL. Furthermore, the solution sterilized by autoclave at 121°C for 1
h. Solution samples were sterile filtered to separate the precipitate (acid insoluble
lignin (AIL)) and the filtrate (acid soluble lignin (ASL)). The precipitate was
filtered and then dried in an oven at a temperature of 105°C in order to analysis
% AIL according to NREL LAP-003 [8]. While the resulting filtrate was measured
by UV spectrophotometer with a wavelength of 205 nm, which would result in
% ASL according to NREL LAP-004 [9].
Centrifuge bottles prepared, each weighed about 5 mg of lignin and
inserted into the centrifuge bottle. After that, the mixture was added 5 mL of
dioxane:water (9:1), diluted up to 50 times and absorbance was measured by
UV-Vis spectrophotometry at wavelength range of 200–400 nm. The same
treatment as much as 5 mg of lignin was added NaOH pH 12 by 5 mL, diluted
up to 50 times and absorbance was measured with UV-Vis spectrophotometry
at wavelength range of 200–400 nm.
Functional group analysis using FT-IR were performed in an Shimadzu IR
Prestige 21 instrument by direct transmittance using potassium bromide (KBr)
pellet technique. 5 mg of lignin was ground and impregnated in 200 mg dried
KBr. Each spectrum was recorded in the range from 4000 to 400 cm-1.
Total of 6 grams of isolated lignin was prepared to run an elemental analysis.
The content of carbon, hydrogen, nitrogen and sulfur were analyzed using a
Thermo Finnigan model Eager 300 analyzer. The percentage of oxygen was
calculated by subtracting 100% with the total number of C, H, N and S. The
protein content was calculated as N(%) x 6.25. The average of double bond
equivalent (DBE) was calculated based on the elemental composition, CaHbOcSd
using formula as in Eq. 1 [10].
(1)

Ash content of lignins isolated from alkaline pretreatment black liquor of OPEFB
and SB are shown in table 1. The table shows that ash content in OPEFB lignin
(19.19) is higher than SB lignin (16.91). High ash content of both lignin caused
by dirty conditions of raw materials as well as methods of delignification and
lignin isolation that produces high levels of impurities. The main reason for this
is that the alkali lignins, isolated by traditional one step precipitation, that can
present high silicate and nitrogen contents [7].
Elemental analysis shows the composition of the carbon, hydrogen, oxygen,
nitrogen and sulfur in lignin based on a percentage of each atom. Both lignins had
similar C, H and O content (Table 1). The similarity of the elemental composition
may be due to same kind of resources as annual plant and same methods of
pretreatment and lignin isolation. Nitrogen content has been detected for both
lignin (<0.30%). The presence of nitrogen content may be due to the formation
of protein-lignin complexes during deligification process [11].
Table 1. OPEFB and SB Lignin Characterization

Analysis Parameters OPEFB Lignin SB Lignin
Ash Content (%) 19.19 16.91
Ultimate Analysis (%):
Carbon 38.65 41.25
Hydrogen 5.05 5.30
Oxygen 36.83 36.25
Nitrogen 0.28 0.24
Sulfur 0.00 0.05
Protein 1.75 1.50
DBE 2.97 3.08
Lignin (%):
AIL 40.14 46.23
ASL 2.82 4.00
The protein residues attached to both lignin suggest a strong chemical bond
between precipitated lignin and protein in non-woody plant, such as OPEFB
and SB. The protein are difficult to remove by acid precipitation and reveals that
proteins linked to lignin in the starting material are still attached with lignin
fragments during delignification [11].
Alkaline pretreatment is one of sulfur-free delignification process with organo-
solv process [7]. Sulfur content in the two lignin isolates were very low (≤0.05%),
which might come from contamination during delignification and lignin isolation.
Lignin has 9 carbon atoms, so the elemental analysis of this can be seen from
the empirical formula of the isolated lignin. The percentage of carbon, hydrogen
and oxygen in the two lignin led to empirical formulas of C9H13.8764O5.9318 and
C9H14.1112O6.4321 for lignin from of OPEFB and SB, respectively.
The number of double bond equivalent (DBE) in both lignins implies the
degree to which the lignins condenses and the presence of aromatic ring structure.
It can be seen that the number of DBEs in the two lignin were almost same, 2.97
and 3.08 for OPEFB and SB lignin, respectively. Alkali lignin have fewer double
bonds than the kraft lignin. This is due to the reaction between hydroxide and
hydrosulfide with lignin, thus generating more double bonds in kraft lignin [12].
The FT-IR spectra of the two lignins are illustrated in Figure 2. FT-IR
spectra reflects the chemical structure as well as the purity of lignins [14]. The
corresponding assignments and bands for the two lignins are presented in table
2. Both lignins show bands at 1,605–1,600 and 1,515–1,505 cm-1 which is
corresponding to aromatic rig vibrations of phenyl-propane (C9) skeleton. The
presence of band at 1,470–1,460 cm-1 , assigned to C-H deformation (asymetric)
in methyl, methylene, and methoxyl groups, confirm that both lignin aromatic
structures did not change dramatically during delignification and isolation [15].
Table 2. FT-IR Absorption Bands and Assignments for OPEFB and SB Lignins
OPEFB
Band (cm-1) SB Lignin
Assignments [10,11] Lignin
[12,13]
Band Location (cm-1)
3450 – 3400 O–H stretching (phenolic OH and aliphatic OH) 3369 3253
2940 – 2820 C–H stretching (CH3 and CH2) 2926 2928
C=O stretching (unconjugated ketone, carbonyl,
1715 – 1710 1707 1713
and ester groups)
1605 – 1600 C–C stretching (aromatic ring) 1603 1599
1515 – 1505 C–C stretching (aromatic ring 1506 1506
C–H deformation (asymmetric in –CH3 and –
1470 – 1460 1454 1450
CH2)
1330 – 1325 C–O stretching (Syringil) 1325 1323
C–O(H) + C–O(Ar) (phenolic OH and ether in
1220 1217 1223
Syringil and Guaiacyl)
1115 Ar–CH in plane deformation (Syringil) 1113 1120
C–O(H) + C–O(C) (first order aliphatic OH and
1085 – 1030 1034 1039
ether)
Figure 2. FT-IR spectra of OPEFB and SB Lignins.
In the area from 3,450 to 2,820 cm-1, similar bands appeared for the two lignin.
A wide absorption band that appeared at 3,369 cm-1 for OPEFB and 3,253 cm-1
for SB lignin can be attributed to phenolic OH and aliphatic OH groups. FT-IR
spectra also showed characteristic vibrations of typical lignocellulosic materials
Figure 3. UV Spectra of OPEFB and SB lignin.
groups C-H stretching (2,928–2,926 cm-1), C=O stretching unconjugated

(1,713–1,707 cm-1), and C-C stretching of aromatic ring (1,600 and 1,506 cm-1).
Bands at 1,330–1,325 cm-1 were attributed to syringil with C-O stretching.
Bands at 1,217 cm-1 for OPEFB and 1,223 cm-1 for SB lignin can be attribute to
phenolic OH and ether in syringil and guaiacyl. The bands observed at 1,030–620
cm-1 were attributed to hemicelluloses and silicates contribution [16].
The most significant functional group in lignin structure is the free phenolic
group because in most chemical reactions involving lignin, phenolic phenyl-
propane units are preferentially attacked [12]. UV Absorbance measurements
on lignin based on the absorption of lignin conjugation. Lignin is a good UV
absorber so that energy is transferred to be in the range of 200–240 nm to initiate
the degradation process.
Both of lignin has similar UV absorbance in dioxane-water solution, with
maximum absorption at 240 nm and weak absorption at 280nm (Figure 3).
However, in alkaline medium (pH 12 NaOH) SB lignin showed higher absorbance
than OPEFB lignin. In alkaline solution, the maximum absorption appears at
210 nm and weak absorption at 280 nm (Figure 3). Absorption occurs in the
elements of the network structure of chromophore in the lignin molecules.
Chromophore elements in lignin will appear at different wavelengths, for example

at the phenolic hydroxyl group at λ 220–240 nm, conjugated double bond at λ
200–240 nm, α carbonyl group at λ 350–353 nm, and biphenyl at λ 489–493
nm. UV absorbance of lignin correlates with phenolic hydroxyl group in lignin.
The more phenolic hydroxyl group, the higher the UV absorbance intensity [17].
IV. Conclusion
The lignin from alkaline pretreatment of OPEFB and SB has similar properties.
Both contain high ash content, and low lignin content due to the alkaline
pretreatment and one step precipitation. The percentage of carbon, hydrogen,
and oxygen in the two lignin isolates led to empirical formula of C9H13.8764O5.9318
and C9H14.1112O6.4321 for lignin from of OPEFB and SB, respectively. UV analysis
showed that both lignin has a strong absorbance at 240 nm and weak absorbance
at 280 nm in neutral conditions (dioxane water), and a strong absorbance at 210
nm and weak absorbance at 280 nm in alkaline conditions (NaOH pH 12). The
more phenolic hydroxyl group, the higher the UV absorbance intensity. The
FT-IR spectra of the two lignin isolates showed that the intensity of the bands
in the spectra is similar. Further studies on lignin isolation method is needed to
determine how to produce lignin with high grade and purity.
V. R eferences
1) Rahman, S. H. A., Choudhury, J. P., Ahmad, A. L., and A. H. Kamaruddin.
(2007). Optimization Studies on Acid Hydrolysis of Oil Palm Empty Fruit
Bunch Fiber for Production of Xylose. Journal of Bioresour. Technol., 98 (3),
554-559.
2) Hermiati, E., Mangunwidjaja, D., Sunarti, T. C., Suparno, O., and B. Prasetya.
(2010) Pemanfaatan Biomassa Lignoselulosa Ampas Tebu untuk Produksi
Bioetanol. Jurnal Litbang Pertanian, 29 (4), 121–130. (In Indonesian).
3) Fajriutami, T., Fatriasari, W., Laksana, R. P. B., and E. Hermiati. (2013). Pre-
treatment NaOH dan Hidrolisis Enzimatis pada Ampas Tebu. Pusat Penelitian
Biomaterial-LIPI, Cibinong, Indonesia, Laporan Teknik. (In Indonesian).
4) Windeisen, E., and G. Wegener. (2012). Lignin as Building Units for
Polymers. Polymer Science: A Comprehensive Reference, 1st ed., M. Moeller and
K. Matyjaszewski, Ed. Elsevier Science. 255–263.
5) Vishtal, A., and A. Kraslawski. (2011). Challenge in Industrial Application
of Technical Lignins. Journal of BioRes., 6 (3), 3547–3568.
6) Schorr, D., Diouf, P. N., and T. Stevanovic. (2014). Evaluation of Industrial
Lignins for Biocomposites Production. Ind. Crop. Prod., 52, 65–73.
7) Laurichesse, S., and L. Averous. (2014). Chemical Modification of lignin:

Toward Biobased Polymers. Prog. Polym. Sci., 39 (7), 1266–1290.
8) Templeton, D., and T. Ehrman. (1995). National Renewable Energy Labora-
tory. LAP 003, 2.
9) Ehrman, T. (1996). National Renewable Energy Laboratory. LAP 004, 2.
10) Robert, D. R., Michel, B., Gellerstedt, G., and L. Lindfors. (1984). Strutural
Changes in Lignin During Kraft Cooking. Part 3. On The Structure of
Dissolved Lignins. J. Wood Chem. Technol., 4 (3), 239–263.
11) Zhao, B. X., Dai, L., and D. Liu. (2009). Characterization and Comparison
of Acetosolv and Milox Lignin Isolated from Crofton Weed Stem. J. Appl.
Polym. Sci., 114(2), 1295–1302.
12) Ibrahim, M N. M., Zakaria, N., Sipaut, C. S., Sulaiman, O., and R. Hashim.
(2011). Chemical and Thermal Properties of Lignins From Oil Palm Biomass
as a Substitute for Phenol in a Phenol Formaldehyde Resin Production.
Carbohyd. Polym, 86, 112–119.
13) Fengel, D., and G. Wegener. (1995). Kayu: Kimia, Ultrastruktur, Reaksi-Reaksi.
Yogyakarta: Gajah Mada University Press, 729 pages. (In Indonesian).
14) Moubarik, A., Grimi, N., Boussetta, N., and A. Pizzi. (2013). Isolation and
Characterization of Lignin from Morocoan Sugar Cane Bagasse: Production of
Lignin-Phenol-Formaldehyde Wood Adhesive. Ind. Crop. Prod., 45, 296–302.
15) Nadji, H., Diouf, P. N., Benaboura, A., Bedard, Y., Riedl, B., and T.
Stevanovic. (2009). Comparative Study of Lignins Isolated from Alfa Grass
(Stipa tenacissima L.). Bioresource Technol., 100 (14), 3585–3592.
16) Garcia, A., Toledano, A., Serrano, L., Egues, I., Gonzalez, M., Marin, F., and J.
Labidi. (2009). Characterization of Lignin Obatain by Selective Precipitation.
Separ. Purif. Technol., 68 (2), 193–198.
17) Risanto, L., Hermiati, E., and Y. Sudiyani. Lignin Properties from Alkaline
Steam Explosion Pretreatment of Oil Palm Empty Fruit Bunch Fiber and
Its Application as Base Polymer of Aqueous Polymer Isocyanate for Plywood
Adhesive. Makara, to be published.
CHANGES IN THE CELLULOSE CRYSTALLINITY
DURING DIFFERENT PHASES OF DISTILLED VETIVER
ROOT CELLULOSE FIBERS DEVELOPMENT
Firda Aulya Syamania,*, Subyaktoa, Sukardib, Ani Suryanib

a
Jl. Raya Bogor Km. 46, West Java, 16911, Indonesia
b
Department of Agroindustrial Technology, Bogor Agricultural University
Jl. Raya Darmaga, Kampus IPB Darmaga Bogor, West Java, 16680, Indonesia
Abstract
Distilled vetiver roots (dVR) are agricultural by products and economically lignocellulosic
resources. To extract cellulose from lignocellulosic materials, lignin and hemicellulose have to be
separated for instance by pulping and bleaching process. Having high tensile strength, cellulose
fibers are potential to be utilized as reinforcing agent in composite materials. The main goal of
this study was to monitor changes in the cellulose crystallinity occuring during different phases
of cellulose fibers development from distilled vetiver root. Soda pulp of distilled vetiver roots were
bleached using hydrogen peroxide. The bleached pulp then was reacted by potassium hydroxide
to eliminate hemicellulose. Then the second phase of hydrogen peroxide bleaching was conducted
to produce cellulose fibers. Surface morphological study using SEM revelead that there was a
reduction in fiber diameter during cellulose fibers developments. Cellulose crystallinity was
higher at final phase of cellulose fibers development as demonstrated by FTIR and XRD analyses.
Key words: Distilled vetiver root soda pulp, Cellulose fibers, Cellulose crystallinity.
I. Introduction
Cellulose fibers are available by nature in abundant quantities, either from wood
or non wood resources. Within wood and natural fibers, cellulose fibers are
attached in lignin matrix and coherenced with hemicellulose, so that are often
referred as the lignocellulosic materials. To extract cellulose from lignocellulosic
materials, lignin and hemicellulose have to be separated for instance by pulping
and bleaching process. Having high tensile strength, cellulose fibers are potential
to be utilized as reinforcing agent in composite materials.
Cellulose fibers have high tensile strength and function as reinforcing agent in
wood (lignocellulosic materials). In nature, cellulose fibers consist of crystalline
region and amorphous region. Crystalline region is interrupted every 60 nm
with non-crystalline amorphous regions which are folded in cellulose polymer
* Corresponding Author.Tel: +62-21-87914509. E-mail: firda.syamani@biomaterial.lipi.go.id
493
chain, called “defects” [1]. The strength of cellulose fibers are due to its crystalline
structure. Cellulose crystal stucture has modulus of elasticity (MoE) of 130–240
GPa [2] and induced fiber stiffness, while the bulk modulus of cellulose is
measured as 20 GPa [3]. Cellulose modulus of elasticity depends on its crystallinity
and the interaction of amorphous and crystalline region [3].
The crystallinity index (CI) of celluloses have been examined using several
different techniques including X ray diffractometer [4,5,6], fourier transform
infra red spectroscopy [4,6], nuclear magnetic resonance [4,5]. There have also
been several methods used for calculating CI from the raw spectrographic data,
particularly for XRD.
The main goal of this study is to monitor changes in the cellulose crystallinity
occuring during different phases of cellulose fibers developments from distilled
vetiver root.
II. M aterials and Method

A. Materials
Distilled vetiver roots were obtained from essential oil industry in Garut regency,
West Java, Indonesia. The sodium hydroxide at technical grade was used in pulping
process. Bleaching process used technical grade of hydrogen peroxide. Potassium
hydroxide were supplied by Merck.
B. Cellulose Fibers Developments: Pulping, Bleaching and Purifying

Processes
The distilled vetiver roots (dVR) were washed several times, sun-dried and cut
into ± 2 cm length. The moisture content of strands was 9%. To produce soda
pulp, dVR strands were subjected to digester with ratios of liquor-to-materials
of 8:1. Soda pulping was carried out at 36% alkali charge. NaOH solution with
concentration of 495 g/L and water were added to digester by 332 mL and 3,281
mL, respectively to cook 500g dVR strands (dry basis). Pulping was conducted in
2 h and 46 min, at 176°C. After cooling of the digester, the pulp was collected
and washed with water several times until neutralized.
For the bleaching process, as much as 20 gram pulp (dry basis) and 300 ml
distilled water were added in 500 ml erlenmeyer, then put in waterbath at 80°C.
The technical grade solution of 50% hydrogen peroxide was used as bleaching
agent. Every 60 min, 8 ml of 50% hydrogen hydroxide was added into erlenmeyer,
for 3 times. After 4 h bleaching process, bleached pulp was collected and washed
with running water.
In order to eliminate hemicellulose content, bleached pulp were reacted with

5% (w/v) potassium hydroxide in waterbath at 80°C for 2 h, then washed with
running water. The final stage to produce cellulose fibers was further bleaching
process with 8 ml of 50% hydrogen peroxide for an hour at 80°C, then washed
with running water to obtain cellulose fibers.
C. Characterization of Cellulose Fibers by Scanning Electron Microscopy

The cross section and element of distilled vetiver root were analyzed by scanning
electron microscope/energy dispersive spectroscope (SEM/EDS) ZEISS EVO 50,
operated at 10 kV. Samples were coated with gold using a vacuum sputter-coater
to improve conductivity of the samples and thus the quality of the SEM images.
D. Characterization of Cellulose Fibers by X-Ray Diffractometer

XRD measurements were performed on a Shimadzu XRD7000 MAXima X-ray
diffractometer to analyze pulp crystallinity. The diffracted intensity of Cu Ka
radiation (λ= 1.54 Å; 40.0 kV and 30.0 mA) was measured in a 2θ range between
10° and 40° with scan speed of 2.0 deg/min.
To determine cellulose crystallinity index, we used a software to separate
amorphous and crystalline contribution to the diffraction spectrum using a
curve-fitting process of Lorentzian function. Crystallinity index is calculated from
the ratio of the area of crystalline peaks of 101, 021 and 002 to the total area.
E. Characterization of Cellulose Fibers by Fourier Transform Infra Red

Microscopy
The ABB FT IR MB3000 was used to analyze chemical structure of vetiver
pulp component. The analysis was run using the KBr pellet technique. The KBr
pellets of samples were prepared by mixing 2 ± 0.05 mg of pulp sample with
200 mg KBr (spectroscopy grade) in a vibratory ball mixer for 20 s. The KBr
pellets were prepared under vacuum in a standard device under a pressure of 80
kN.cm-2 for 3 min to form pellet with diameter and thickness of 13 mm and 0.5
cm, respectively. The spectral resolution was 4 cm–1 and the scanning range was
from 400 to 4,000 cm–1.

Cellulose fibers were developed from distilled vetiver fibers in 4 stages; pulping,
bleaching, purifying and second bleaching. We studied cellulose crystallinity in
every stages.
Figure 1. Scanning electron micrographs of distilled vetiver root fiber cross section.
Figure 2. SEM images of cellulose fibers development from distilled vetiver roots (a) dVR soda
pulp, (b) dVR bleached pulp, (c) dvR purified pulp, and (d) dVR cellulose fibers.
A. Morphology of Cellulose Fibers

Scanning electrone microscope (SEM) images of distilled vetiver root fiber, soda
pulp, bleached soda pulp, purified soda pulp and cellulose of distilled vetiver root
are demonstrated in Figure 1 and Figure 2. After undergoing the distillation at
temperature of 120°C for 16 hours during vetiver, others were flatten, showing
flat fibers with diameter of 20.98 µm.
During bleaching, hydrogen peroxide removed residual lignin and reacted
with non-lignocellulosic components. These reaction produced some materials
that observed at the background of fibers (Figure 2b). There was flatten fiber
observed and its diameter was 18.09 µm.
To obtain purified pulp, we reacted bleached pulp with potassium hydroxide.
It seems that hemicellulose was separated from fibers then reacted with non-
lignocellulosic components and materials at the background of fibers become
more obvious (Fig 2c). Fibers structure begun to destroyed and fiber diameter
reduced to 4.90 µm.
At the final stage of cellulose fibers development, hydrogen peroxide caused
a significant damage to cellulose fibers. Fibers structure did no longer exist and
slurred with other materials (Figure 2d). These structure would not suit to be used
as reinforcing agent in polymer matrix to develop composite materials.
B. Cellulose Crystallinity Based on X-Ray Diffraction Measurement

X-ray diffraction analysis was conducted to determine crystallinity index (Figure
3A). Distilled-dVR purified pulp and dVR cellulose fibers indicated crystallinity
of 49.76%, 51.05%, 51.31% and 58.98%, respectively.
After bleaching and purifying of distilled vetiver root soda pulp, cellulose
crystallinity index slightly increased, then significantly increased after further
bleaching to obtain cellulose fibers. Meanwhile, the cellulose CI of distilled vetiver
root fibers was 37.37%.
Figure 3. XRD (left) and FTIR (right) anlysis of cellulose fibers development from distilled
vetiver roots (a) dVR soda pulp, (b) dVR bleached pulp, (c) dvR purified pulp, and (d) dVR
cellulose fibers.
Table 1 Bands characteristics of FTIR spectra related to development of distilled vetiver root
cellulose
Peak of
Characteristics Soda Bleached Purified Assignment
Cellulose
Pulp Pulp Pulp
3587 Strecthing(γ) OH (hydrogen bond)
3564
3526
3502
3348 3418 3441 3448 Stretching(γ) OH (hydrogen bond)
2908 2901 2901 2908 γCH
1643 1643 1643 1651 -
1435 1435 1458 Bending(δ) CH2 (sym) at C-6
1427 1427 δCH2 (sym) at C-6
1373 1373 1373 1373 δCH
Peak Shift
1366 -
1335 1335 δCOH in plane at C-2 or C-3
1319 δCH2 (wagging) at C-6
1165 1165 1165 1165 γCOC at β-glycosidic
1111 1111 1111 1119 γring in plane
1057 1057 1057 1057 γCO at C-3, γC–C
1026 1026 1026 γCO at C-6
γCOC at β-glycosidic, γCCH at C-5
903 895 895 895
and C-6
663 663 663 671 δCOH out plane
Distilled vetiver root fibers contains cellulose (30.33%) and other components
which are hemicellulose (14.65%) and lignin (39.53%) [7]. Soda pulping removes
a considerable part of lignin then cause an increasing of cellulose CI in dVR soda
pulp. Then, residual lignin and hemicellulose were removed by bleaching and
purifying process, respectively. Further bleaching at the final stage of cellulose
fibers development remove amorphous part of cellulose caused a high cellulose CI.
C. Cellulose Crystallinity Based on Infra Red Absorption Measurement

(FT-IR Method)
One of drawback of measuring cellulose CI using XRD is that the separation of
amorphous background from the diffraction pattern of cellulose crystallites can
be affected by significat errors related to their small size (usually between 2.5 and
3.5 nm) [8]. FT-IR spectroscopy has been employed to provide detail about the
structural characteristics of dVR cellulose fibers. The FT-IR spestroscopic method
has also been used to analyse the chemical changes taking places in softwood as
a consequence of fungal attack [9] and of environmental exposure [10].
After pulping, delignification of dVR is observed by FT-IR spectroscopy.

Natural lignin contain the following functional groups: metoxyl, phenolic
hydroxyl, primary and secondary aliphatic hydroxyl, ketone and aldehyde groups
[11]. The infrared spectra of lignin present peaks in the range 1,200–1,300 cm-1
corresponding to the aromatic skeletal vibration. In addition, due to the presence
of functional groups such as methoxyl-O-CH3, C-O-C and aromatic C=C, peaks
in the region between 1,830 cm-1 and 1,730 cm-1 were also observed. The peak
presents at 1,730–1,740 cm-1 in the spectrum corresponding to the presence of
C=O lingkage, which is a characteristic of lignin groups [12]. Other researcher
said that lignin band were found at 1,595 cm-1 and 1,512 cm-1 for C=C streching
of the aromatic ring, at 1,463 cm-1 for CH3 bending and at 1,269 cm-1 for CO
streching in lignin (guaiacyl) and hemicellulose [13]. In this study, none of those
peaks appeared (Figure 3B). Soda pulping has succesful delignified distilled
vetiver root. Since lignin has known as amorphous components in lignocellulosic
materials, the delignification of dVR will increase crystallinity of bleached pulp,
purified pulp and finally cellulose fibers of dVR.
Typical bands assigned to cellulose were located at 1,425 cm-1 and 1,375 cm-1
for CH2 and CH bending mode, respectively. To distinguish between amorphous
and crystallised I cellullose, the bands at 1,336 cm -1 and 13,17 cm-1 for hydroxyl
in plane bending and CH2 wagging, respectively were observed. Then the band
at 1,163 cm-1 which associated with C-O-C asymetric bridge oxygen and the
band at 897 cm -1 which associated with asymetric out of phase ring streching,
were also assigned to cellulose [13]. Meanwhile, treatment with aqueous NaOH
solution of a specific concentration (~10 wt%) causes the transformation of
cellulose I into cellulose II within the crystalline domain [8]. In this study, we
observed FTIR spectra distilled-vetiver (dVR) soda pulp, dVR bleached pulp,
dVR purified pulp and dVR cellulose fibers (Table 1).
The enhanced absorption band at 1,165 cm-1 and 895 cm-1 (Figure 3B) were
observed, assigned that the b-glycosidic in bleached pulp and purified pulp were
increased in comparison with soda pulp. It means that in that sampels, more
cellulose were observed due to the elimination of lignin and hemicellulose. In
accordance to amorphous cellulose, the band at 1,335 cm-1 was observed in
bleached pulp and purified pulp. Whereas related to crystallised I cellulose, the
band at 1,319 cm-1 was observed in soda pulp. That condition explained that the
enhancement of crystallinity that pointed out by XRD analyses was not due to
the crystalline cellulose. Vetiver root contain essential oil and some metals that
might influenced the crystallinity index by XRD analyses.
In dVR cellulose sampel, the FTIR spectra of hydrogen-bonded OH stretching
vibrations at around 3,441 cm_1 were resolved into five bands for cellulose II.
The bands of 3448 cm-1, 3,502 cm-1, 3,526 cm-1, 3,564 cm-1 and 3,587 cm-1
were related to the sum of valence vibration of an H-bonded OH group, an

intramolecular hydrogen bond or intermolecular hydrogen bond.
IV. Conclusion
Soda pulping increased crystallinity index of cellulose. And after bleaching and
purifying of distilled vetiver root soda pulp, cellulose crystallinity index slightly
increased, then significantly increased after further bleaching to obtain cellulose
fibers. Those statements were confirmed by XRD and FTIR analyses.
V. Acknowledgement
This research was part of disertation and financially supported by Ministry of
Research and Technology of Indonesia.
VI. R eferences
1) de Souza, I .J., Bouchard, J., Methot, M., Berry, R., and D. S. Argyropoulos.
(2002). Carbohydrates in Oxygen Delignification, Part I: Changes in Cellulose
Crystallinity.J. Pulp Paper Sci., 28 (5), 167–170.
2) Michell, A. J. (1989). Wood Cellulose-Organic Polymer Composites.
Composite Asia Pacific 89, 19–21.
3) Cabrera, R. Q., Meersman, F., McMillan, P. F., and V. Dimitriev. (2011).
Nanomechanical and Structural Properties of Native Cellulose under
Compressive Stress. Biomacromolecules, 12 (6), 2178–2183.
4) Evans, R., Newman, R. H., Roick, U. C., Suckling, I. D., and A. F. A. Wallis.
(1995). Changes in Cellulose Crystallinity during Kraft Pulping, Comparison
of Infrared, X-ray Diffraction and Solid State NMR Results. Holzforschung,
49 (6), 498–504.
5) Zaibo, H., Kwak, J. H., Zhang, Z. C., Brown, H. M., Arey, B. W., and J. E.
Holladay. (2007). Studying Cellulose Fibers Structure by SEM, XRD, NMR
and Acid Hydrolysis. Carbohyd. Polym, 68 (2), 235–241.
6) Gumuskaya, E., Usta, M., and H. Kirci. (2003). The Effects of Various Pulping
Conditoins on Crystalline Structure of Cellulose in Cotton Linters. Polym.
Degrad. Stabil., 81 (3), 559–-564.
7) Syamani, F. A., Astari, L., Subyakto, Sukardi, and A. Suryani. (2013).
Characteristics of Strands and Pulp from Oil Palm Fronds and Vetiver Roots.
Proc. 2nd Intl Symposium for Sustainable Humanosphere, 1–7.
8) Oh, S. Y., Yoo, D. I., Shin, Y., Kim, H. C., Kim, H. Y., Chung, Y. S., Park,
W. H., and J. H. Youk. (2005). Crystalline Structure Analysis of Cellulose
Treated with Sodium Hydroxide and Carbon Dioxide by Means of X-Ray
Diffraction and FT-IR Spectroscopy. Carbohyd. Res., 340 (15), 2376–2391.
9) Popescu, C. M., Popescu, M. C., and C. Vasile. (2010). Structural Changes
in Biodegraded Lime Wood. Carbohyd. Polym, 79 (2), 362–372.
10) Popescu, C. M., Vesile, C., Popescu, M. C., and G. H. Singurel. (2006).
Degradation of Lime Wood Painting Support II. Spectral Characterisation
Cell. Chem. Technol., 40 (8), 649–658.
11) Bykov, I. (2008). Characterization of Natural and Technical Lignins using
FTIR Spectroscopy. Luleå University of Technology, Luleå, Sweden. Master’s
Thesis 2008:020.
12) Owen, N. L., and D. W. Thomas. (1989). Infrared Studies of “Hard” and
“Soft” Woods. Appl. Spectrosc., 43 (3), 451–455..
13) Lionetto, F., Del Sole, R., Cannoletta, D., Vasapollo, G., and A. Maffezzoli.
(2012). Monitoring Wood Degradation during Weathering by Cellulose
Crystallinity. Materials, 5 (10), 1910-1922.
BIOETHANOL PRODUCTION USING Saccharomyces
cerevisiae IMMOBILISED ON FRESH AND MODIFIED
SUGARCANE BAGASSE
Sita Heris Anitaa,b,*, Wibowo Mangunwardoyoc, Yopid, Maulida

Oktavianib, Raden Permana Budi Laksanab
Biology Study Program, The Graduate School, University of Indonesia
a
Kampus Baru UI Depok, Depok, 16424, Indonesia

b
Research Center for Biomaterials, LIPI, Cibinong, Bogor
Jl. Raya Bogor Km. 46, Cibinong, Bogor, 16911, Indonesia
c
Biology Department, Faculty of Mathematic and Natural Science, University of Indonesia
d
Research Center for Biotechnology, LIPI, Cibinong, Bogor
Jl. Raya Bogor Km. 46, Cibinong, Bogor 16911, Indonesia
Abstract
Fresh and modified sugarcane bagasse as a carrier of immobilized cells for ethanol production were
investigated. Modified sugarcane bagasse was obtained from serial treatments respectively namely
steam only, pressure with steam, and by combining both of steam procedure. Aside from that,
biocatalyst 1% (w/v) (fresh and modified carrier containing cells) were used as an inoculum for
ethanol fermentation. The best modified sugarcane bagasse for carrier of immobilized cells was
obtained using steam treatment for 30 minutes by improving the physical properties of carrier
that increased the ethanol yield. A maximum ethanol yield was 0.40 ± 0.01 g/g in 24-hour of
fermentation period. This maximum ethanol production showed 2 and 5 times higher result
compare to immobilized cells on fresh sugarcane bagasse and free cells system, respectively.
Key words: Bioethanol, Immobilized cells system, Sugarcane bagasse, Natural Adsorption.
I. Introduction
The ongoing effort to mass produce bioethanol from lignocellulosic biomass has
been facing technical issues. One of them is the less advance of the applicable
technology. This issue contributes to low output and hight cost in the production
of bioethanol [1]. The current technology has inefficiency issue in the pretreatment
process of lignocellulosic materials along with microorganism utilization to
metabolize pentose and hexose sugars [2] subsequently, to hydrolyze and finally
for fermenting substrates simultaneously [3]. Despite the use of bioreactors with
immobilized cell, it has been considered as a promising technology for bioethanol
production [2].
* Corresponding Author. E-mail: sita.heris@biomaterial.lipi.go.id
503
Cell immobilization is defined as an entrapment of physical cells into

specific spaces [4]. Cell immobilization technology has been recommended as an
alternative approach to increase bioethanol output and minimize its production
cost [5], [6], [7]. This technology improves the productivity of ethanol two
to three times along by increasing the cell density inside the carrier [8], [9].
According to Vucurovic and Razmovski [10], to select the most appropriate cell
immobilization application for bioethanol production, there are some points
such as the expense on carrier material and the suitability techniques that must
be considered. Materials that can be used as carriers for cell immobilization has
to be accessible, inexpensive, abundantly available in nature [6], [8], [11], [12],
less toxic to microorganisms, perform high mechanical strength, and also posses
core hollow spaces to enable diffusion process for the substrates and products
[11]. Lignocellulosic biomass that have been used for cell immobilization on the
production of bioethanol are shorgum bagasse [9], [13], orange peel [14], corn
husk [12], sugar beet pulp [10], sugarcane [11] and sugarcane bagasse [8].
The aim of pretreatment of lignosellulosic biomass for cell immobilization is to
increase the affinity of microorganism cells upon the substrates [5], to prevent the
adsorption of non-productive cells, and to allow immobilization of microorganism
viable cells [15]. Furthermore, pretreatment can decrease lignin content and
cellulose crystalinity; hence, enhancing porosity of substrate [16]. Enzymatic
pretreatment on shorgum bagasse can increase cell retention from 0.87 g/g to 1.35
g/g of dry cell weight [13]. Physical and biological pretreatment combination
on sugarcane bagasse decrease lignin content to 27.44% [17]. Pretreatment by
using pressurized steamer was reported to able to enhance porosity and retention
of the substrates [18] that was indicated by increasing of water retention and cell
adsorption [10].
The goal of this research is to study the capability of fresh and modified
sugarcane bagasse as carriers of immobilized cells for ethanol production. Modi-
fied sugarcane bagasse was obtained through steaming only process, pressurized
steaming, and as well combination of both treatment in different time period.
Bioethanol production with immobilized cell system on fresh and modified
sugarcane bagasse were then compared to yeast only (free cell system) as the
control.
II. Method
A. Microorganism and Culture Medium
The yeast Saccharomyces cerevisiae was obtained from microbiology laboratory,
Research Center for Biology, LIPI and maintained on yeast peptone dextrose
(YPED) agar slant that consist of glucose 20 g/L, yeast extract 10 g/L, peptone 20
g/L, and agar 25 g/L. Prior to use, stock culture was stored at 4°C and subcultured
every two weeks [19].
Starter culture of yeast was cultivated by inoculating one or two loop of
48-hour stock culture into 30 ml of synthetic medium consisted of (g/L): 10
glucose, 3 yeast extract, 3.5 peptone, 1 KH2PO4, 1 MgSO4.7H2O, 1 (NH4)2SO4,
pH 5 in 100 ml Erlenmeyer flask [20]. Afterwards, the culture was incubated
for 24 hours at room temperature (30 ± 2°C) on a rotary shaker at 120 rpm.
After 24 hour, the content was transferred into 270 ml of production medium
in 500 ml Erlenmeyer flask and incubated for another 24 hour. Cells were then
harvested by using centrifugation at 3,000 rpm for 10 minutes. Inoculum using
on immobilization was obtained from 100 mg of the dried cells that were dissolved
in 50 ml of sterilized 0.9% NaCl suspension.
B. Sugarcane Bagasse and Pretreatment

Fresh sugarcane bagasse was provided by Rajawali II sugar factory, Pasir Bungur,
West Java, Indonesia. Before any treatment, biomass was cut into 1–2 cm in length
and washed until clean. Fresh sugarcane bagasse was then air-dried and maintaned
at room temperature [8]. For pretreatment, 20 g of sugarcane bagasse was added
by distilled water in comparison 1:3 (w/v). Subsequently, the sugarcanes were
treated as follows: steamed only at 100°C for 30 minutes, steamed with pressure
at 121°C for 15, 30 and 60 minutes, and by combining both steam procedure.
Physical characteristic of fresh and modified sugarcane bagasse i.e. lignin content,
water content (W), water retention (H) and water absorption index (WAI) were
recorded and analyzed.
Both of fresh and modified sugarcane bagasse were hydrated by adding 93.5
ml of distilled water into 2.5 g of both materials before they are used in the
experiment. These mixtures were then incubated for 24 hour at room temperature
[10]. Afterwards, the liquid was removed and the solid products were autoclaved
at 121° C for 15 minutes. These solid were ready to be used as carriers.
C. Anatomical Characterization
Fiber dimension was measured by macerating the samples and then observed
through microscope. In this observation, fiber length, the lumen, fiber diameter,
and cell wall thickness were recorded. The measurement for fiber length was
performed 30 times, while the diameter and cell wall thickness were 15 times
each to calculate the average value. The samples were macerated according to the
method described by Schulze [21].
D. Yeast Immobilization
Yeast suspension as much as 50 ml was added to 2.5 g of sterilized fresh and modi-
fied sugarcane bagasse in 500 ml of Erlenmeyer flask. Flask then was incubated
for 24 hour at room temperature (30 ± 2°C) on a rotary shaker at 120 rpm [10].
This process is called immobilization. Afterwards, biocatalyst (carrier or sugarcane
bagasse inoculated with yeast cells) was decanted and washed by adding into 100
ml of sterilized distilled water. The liquid was removed, and subsequently, the
cell retention was measured according to Singh et. al., [8]. Biocatalyst was then
used as inoculum for ethanol fermentation.
E. Fermentation
The composition of medium for ethanol fermentation was (g/L): 100 glucose,
3 yeast extract, 3.5 peptone, 2 KH2PO4, 2 MgSO4.7H2O, 1 (NH4)2SO4, 0.3
ZnSO4.7H2O, pH 5 [20]. Three gram of biocatalyst (1% (w/v)) and yeast only
were each inoculated into 300 ml of fermentation medium in 500 ml Erlenmeyer
flask and covered by bubble traps. Batch fermentation was conducted at room
temperature (30 ± 2°C), for 24 hour incubation. During the fermentation,
samples were taken for analysis sugar consumption and ethanol concentration.
F. Analytical Method
Cell biomass was determined according to NREL LAP 008 [22] while glucose
concentration was measured by using Somogyi-Nelson method [23]. The concen-
tration of ethanol was obtained by using Shimadzu GC 14B gas chromatography
with carbowax 20M column and a flame ionization detector.
The biocatalysts (after they were washed two times with 10 mL distilled
water and allow to dry for 24 hours) were scanned by using scanning electron
microscopy (SEM) FE-SEM FEI INSPECT F50 to generate their micrograph.
G. Calculation Formulae
Lignin content was calculated by using the method of Templeton and Ehrman
[24]. Water retention (H) was noted as mass of water retained per grams of
dry mass carrier. Water absorption index (WAI) was determined according to
Mussatto et. al. [25].
Cell retention’s carrier (R) was calculated as a ratio of the cell dry mass
immobilized in the carrier to the carrier’s dry mass. The yield of ethanol (Yp/s)
was determined as per grams mass of ethanol per sugar reduction. Efficiency of
sugar conversion (Ep/s) was estimated based on a ratio of the observed ethanol
yield to the theoretical value (0.51 g/g) [8].
H. Statistical Analysis
All experiments were conducted with 3 replications and subjected to a one-way
analysis of variance (ANOVA). Statistical significance was determined in the 5%
of probability level (p=0.05).

Cell immobilization technology has been widely used in the fermentation process.
Based on the physic mechanisms of cell and the materials as a carrier that were
used, there are four category of immobilization technology i.e. flocculation,
encapsulation, entrapping mechanisms in the porous matrix, and cell adhesion
on the surface of a solid substrate [4], [26]. In this study, natural adsorption of
the cells onto sugarcane bagasse (carriers) was observed.
Table 1. Physical Properties of Fresh and Modified Sugarcane Bagassa as a Carrier

Modified Carrier
Steam Steam
Fresh Pressurized Pressurized Pressurized (30 min), (30 min),
Parameters Steam
Carrier Steam Steam Steam Pressurized Pressurized
(30 min)
(15 min) (30 min) (60 min) Steam (15 Steam (15
min) min)
Water content 7.77 ± 70.68 ± 71.65 ± 73.12 ± 69.39 ± 70.89 ± 73.39 ±
(%) 0.35 3.13* 2.4* 1.50* 3.19* 6.44* 2.57*
Water content
84.27 ± 88.32 ± 87.90 ± 86.19 ± 85.11 88.03 ±
of hydrated car- 85.93 ± 0.92
1.38 1.52* 1.86* 2.93 ±1.58 0.68*
rier (%)
Water retention 4.80 ± 7.66 ± 7.39 ±
6.48 ± 1.64 5.76 ± 0.71 6.13 ± 0.45 7.37 ± 0.47*
(g/g) 0.44 1.05* 1.19*
Water absorp- 8.58 ± 8.73 ± 21.96 ±
9.28 ± 1.10 9.26 ± 0.55 9.18 ± 0.49 8.77 ± 0.12
tion index (g/g) 0.21 1.20 0.18*
Lignin content 24.4 ± 20.84 ± 19.82 ± 19.82 ± 21.81 ±
22.38 ± 0.75 21.96 ± 0.18
(%) 1.52 0.88* 3.12* 3.12* 0.49
*significantly different with fresh carrier at 5% probability level
Natural adsorption cell into substrate occurred due to electrostatic, ionic (acid-
base) and hydrophobic (van der Waals) interactions between the cell membrane
and the substrate surface [4], [6], [26], as well as water retention properties, and
the surface characteristics of the substrate [26].
The absence of barrier between the cells and solution (fermentation medium)
enable the release and relocation of cells from susbtrate (carriers). However, the
cells movement can be minimized through the selection of the proper yeast
strains and substrates. It is suggested that selected yeasts must have the ability to
flocculate while substrates must have rough and porous surfaces with high water
Table 2. Fiber Dimension Value of Fresh and Modified Sugarcane Bagasse

Fiber Length Fiber Diameter Lumen Cell Wall Thick-
Treatment
(mm) (mm) Diameter (mm) ness (mm)
Fresh
2.8100±0.4500 0.0270±0.0060 0.0190±0.0060 0.0039±0.0114
carrier
Steam (30 min) 2.6300±0.5600 0.0260±0.0060 0.0200±0.0050 0.0034±0.0005
Pressurized (15 min) 2.6100±0.5500 0.0270±0.0050 0.0210±0.0040 0.0034±0.0008
Pressurized (30 min) 2.6200±0.6500 0.0310±0.0060 0.0230±0.0050 0.0037±0.0013
Modified Pressurized (60 min) 2.8000±0.6300 0.0280±0.0070 0.0220±0.0070 0.0034±0.0080
carrier Steam (30 min), Pres-
2.8600±0.4500 0.0290±0.0050 0.0210±0.0050 0.0035±0.0010
surized (15 min)
Steam (30 min), Pres-
2.5300±0.7500 0.0310±0.0080 0.0250±0.0080 0.0030±0.0008
surized (30 min)
Figure 1. Scanning electron micrograph 4000x of fresh (left) and modified (right) sugarcane
bagasse.
Table 3. Cell Retention on Fresh and Modified Carrier

Weight of Car- Weight of Immobized
Carrier Cell Retention (mg/g)
rier (g) Cells (mg)
Fresh carrier 2.5 13.69 ± 2.55 5.41 ± 1.06
Modified carrier
2.5 25.21 ± 0.44 10.05 ± 0.17
(steam 30 min)
retention properties [26]. From Table 1, it is shown that fresh sugarcane bagasse
has good physical properties. Water content of solid substrate were varies and
depend on their rate of water absorption. Water content affects the growth of
microorganisms, while the low water content decreases the nutrien solubility;
hence, extending the lag phase of microorganism. Whereas the overhigh of water
content reduces the porosity of substrate, thus limiting the oxygen transfer into
substrate [27]. In this study, after hydration, water content of the fresh sugarcane
bagasse increased significantly.
Water retention indicates hydrophilic properties of materials while WAI is the
quantity of water absorbed by the materials [6]. Lignin content of lignosellulosic
biomass is also important in the process of selecting an appropriate substrate
as a carrier. The excessively high of lignin content provides a full-block to the

cell walls so that its texture becomes smoother; therefore, the cells’ binding is
minimum [13]. Conversely, the low lignin content affects mechanical strength
of the carrier for cell immobilization [28].
Pretreatment of the subsbtrate as a carrier is known to enhance the affinity of
cell microorganism to substrate [5], reduce the lignin content [15], and increase
the porosity of substrate [16]. The effect of physical treatment such as steaming,
pressurized steaming, and combination of both procedure on various time interval
are shown on Table 1. The addition of distilled water on pretreatment process
increases initial water content of modified sugarcane bagasse. Its increase ranges
from 61 to 65% and significantly contrast to water content of fresh sugarcane
bagasse. After hydration process, pretreatment process by pressurized steaming
for 15 min, steaming only and combination of the steaming procedure for 30
min succeeded to give significant effect on water content and water retention of
carrier. However, pretreatments used in this study had no effect on the increase
of water absorption index. In the lignin content, there was a significant decrease
after steaming only and pressurized steaming treatment for 30 minutes i.e 20.84
± 0.88 and 19.82 ± 3.12%, respectively. Type and duration of treatment were
failed to significantly influence among carrier’s physical characteristic (P>0.05).
From the result, it can be seen that steaming treatment for 30 minutes gives
significant effect on the modified carrier’s physical properties compared to the
fresh ones. The modified carrier that were obtained after steaming treatment for
30 minutes, was then used as a carrier for cell immobilization.
Water in pretreatment process is absorbed into the carrier and then filling and
swelling its pore. As the time of steaming process, the water evaporates rapidly and
causes several cell changes such as the pores volume and fiber fragmentation [29].
Anatomical characteristic of modified sugarcane bagasse are presented on
Table 2. There are some changes on the anatomical properties of sugarcane bagasse
after treatment. The fiber length of sugarcane bagasse was shortened due to the
fragmentation process. Additionally, its fiber and lumen diameter were wider than
fresh one, indicating changes on the pores volume of modified carrier. Thickness
of the cell wall also became thinner compared to fresh one which justified the
decrease of the lignin content after treatment.
Analysis through scanning electron micrograph (SEM) was used to observe
the damage occuring in the cell wall due to the treatment. SEM observation was
shown on Figure 1. The existence of lignin promoted carrier’s cell walls were
smoother with few pores. Yet, it became rougher and more porous following the
steaming treatment.
The rough surface of carrier as materials provided wider surface area for cell
attachment. Thereby, it enabled more cells to adhere on to the carrier. Cells
retention of the fresh and modified carrier can be seen on Table 3. Cells that
adhere to modified carrier were two times higher than fresh carrier.
In this study 1% (w/v) of biocatalyst (carrier containing cells) with 100 g/L of
initial sugar was used. However, when biocatalyst were transferred to production
medium, sugar concentration dropped until 63 g/L. It was assumed due to the
carrier capability to absorb the sugar. Natural carrier are able to absorb some sugars
dissolved in the medium in order to be converted into ethanol, hence this product
Table 3. Performance of Ethanol Fermentation
Time Efficiency of Sugar Conver-
Sugar Conversion (%) Ethanol Yield (g/g)
Parameter Incubation sion (%)
(hr) 1a 2b 1a 2b 1a 2b
0 0 0 0 0 0 0
3 61.00 ± 1.99 39.39 ± 3.17 0.02 ± 0.01 0.05 ± 0.01 4.18 ± 0.18 10.22 ± 0.44
6 61.84 ± 4.99 41.93 ± 8.90 0.02 ± 0.01 0.04 ± 0.01 3.37 ± 0.18 8.09 ± 0.42
Immo- 9 67.61 ± 1.02 49.24 ± 3.92 0.06 ± 0.01 0.14 ± 0.01 12.36 ± 0.32 26.44 ± 0.69
vilized cell
12 81.15 ± 3.56 70.30 ± 6.84 0.09 ± 0.01 0.16 ± 0.01 17.64 ± 0.40 31.71 ± 0.73
on fresh
carrier 15 88.04 ± 3.03 81.13 ± 5.48 0.16 ± 0.01 0.27 ± 0.01 31.19 ± 0.13 52.78 ± 0.21
18 89.57 ±1.39 83.63 ± 2.68 0.25 ± 0.01 0.42 ± 0.01 48.54 ± 0.25 81.17 ± 0.41
21 92.59 ± 0.19 88.39 ± 0.93 0.26 ± 0.01 0.42 ± 0.01 50..71 ± 0.59 82.99 ± 0.97
24 92.98 ± 1.93 88.98 ± 3.16 0.25 ± 0.01 0.40 ± 0.01 47.50 ± 0.82 77.53 ± 1.34
0 0 0 0 0 0 0
3 41.56 ± 4.69 7.78 ± 3.32 0.06 ± 0.01 0.49 ± 0.13 11.16 ± 3.16 94.76 ± 6.73
6 42.44 ± 2.88 9.08 ± 0.88 0.14 ± 0.04 0.82 ± 0.06 26.80 ± 1.50 160.73 ± 1.17
Immobi- 9 49.18 ± 3.80 19.50 ± 8.66 0.33 ± 0.01 0.94 ± 0.06 64.18 ± 0.45 184.32 ± 1.09
lized cell
12 67.82 ± 2.05 49.15 ± 2.86 0.29 ± 0.06 0.64 ± 0.12 57.26 ± 1.29 124.66 ± 4.58
on modi-
fied carrier 15 80.89 ± 4.09 69.94 ± 5.22 0.34 ± 0.04 0.63 ± 0.08 66.66 ± 3.52 121.92 ± 5.58
18 88.94 ± 1.13 82.43 ± 2.68 0.35 ± 0.02 0.59 ± 0.04 68.28 ± 0.54 116.18 ± 7.74
21 93.48 ± 0.37 89.70 ± 0.25 0.36 ± 0.02 0.59 ± 0.04 69.76 ± 0.80 114.78 ± 7.89
24 93.70 ± 0.07 90.03 ± 0.07 0.40 ± 0.01 0.65 ± 0.01 77.51 ± 1.52 127.36 ± 2.48
0 0 0 0 0 0 0
3 18.37 ± 0.13 10.12 ± 3.51 0.08 ± 0.01 0.13 ± 0.01 15.14 ± 1.20 25.31 ± 1.70
6 28.28 ± 1.80 27.79 ± 1.19 0.09 ± 0.01 0.14 ± 0.01 17.75 ± 1.16 26.28 ± 1.24
9 37.48 ± 0.64 32.68 ± 1.36 0.08 ± 0.01 0.14 ± 0.01 14.01 ± 1.17 25.66 ± 1.58
Free cell 12 40.45 ± 1.09 34.11 ± 2.12 0.07 ± 0.01 0.14 ± 0.01 14.32 ± 0.28 26.82 ± 0.51
15 48.91 ± 0.16 45.01 ± 5.00 0.08 ± 0.01 0.13 ± 0.02 14.21 ± 1.78 24.42 ± 3.08
18 6.40 ± 0.57 64.91 ± 1.70 0.08 ± 0.01 0.10 ± 0.01 15.34 ± 1.17 20.12 ± 2.93
21 68.15 ± 0.55 68.66 ± 1.22 0.06 ± 0.01 0.09 ± 0.01 12.02 ± 0.32 16.17 ± 1.76
24 72.70 ± 0.66 77.04 ± 0.81 0.08 ± 0.01 0.11 ± 0.01 15.40 ± 0.45 21.39 ± 2.91
based on initial sugar before the addition of biocatalyst or 100 g/L

a
based on initial sugar after the addition of biocatalyst or 63 g/L

b
Figure 2. Cell concentration of free cell system and immobilized cell system in the last few hours
of ethanol fermentation (left), and initial cell concentration of modified and fresh carrier (right).
decreasing the rate of fermentation [28]. In comparison with immobilized cells

system, ethanol fermentation was conducted using free cells system with 100 and
63 g/L of initial sugar concentration. Table 4 shows the result of sugar conversion,
ethanol yield, and efficiency of conversion, ethanol yield, and sugar conversion’s
efficiency in bioethanol production by using immobilized cell approach were
significantly higher than free cell system.
Ethanol yield and sugar conversion efficiency by using immobilized cells
were about 5 times higher than using free cells and for sugar conversion was 1.3
times higher. Immobilized cells on modified carrier showed a better value than
the fresh ones. It is confirmed that ethanol yield of modified carrier was about 2
times higher than the fresh one.
The ethanol fermentation by using immobilized cell was performed better
than the free cell system. It was due to the number of cell concentration using
immobilized method was higher in the last few hours of fermentation period
Figure 2a. An immobilization method protects cells from inhibitor by forming a
biofilm between them [30]. Cell retention (Table 3) and initial cell concentration
(Figure 2b) of the modified carriers were 2 times higher than the fresh one. The
surface of rougher modified carrier and porous provides more space for cell to
adhere
IV. Conclusion
Sugarcane bagasse is potential to be used as a carrier for immobilization in the
production of bioethanol. Bioethanol yield increased by using immobilized cell.
Steaming the substrates changes anatomical structures and improves the physical
properties of the cell carrier. The use of immobilized cell on modified carrier for
several fermentation cycle requires further investigation.
V. R eferences
1) Banerjee, S., Mudliar, S., Sen, R., Giri, B., Satpute, D., Chakrabarti, T., and
R. A Pandey. (2010). Commercializing Lignocellulosic Bioethanol: Technol-
ogy Bottlenecks and Possibles Remedies. Biofuels Bioprod. and Biorefin., 4 (1),
77–93.
2) Mussatto, S., Dragone, G., Guimaraes, P. M. R., Silva, J. P. A., Carneiro, L.
M., Roberto, I. C., Vicente, A., Domingues, L., and J. A. Teixeira. (2010).
Technological Trends, Global Market, and Challenges of Bio-Ethanol
Production. Biotechnol. Adv., 28 (6), 817–830.
3) Hasunuma, T., and A. Kondo. (2012). Consolidated Bioprocessing and
Simultaneous Saccharification and Fermentation of Lignocellulose to Ethanol
with Thermotolerant Yeast Strains. Process Biochem., 47 (9), 1287–1294.
4) Kourkoutas, Y., Bekatorov, A., Bonat, I. M., Marchant, R., and A. A. Koutinas.
(2004). Immobilization Technologies and Support Materials Suitable in
Alcohol Bevarages Production: A Review. Food Microbiol., 21 (4), 377–397.
5) Santos, D. T., Sarrouh, B. F., Rivaldi, J. D., Converti, A., and S. S. Silva.
(2008). Use of Sugarcane Bagasse as Biomaterial for Cell Immobilization for
Xylitol Production. J. Food Eng., 86 (4), 542–548.
6) Razmovski, R., and V. Vucurovic. (2012). Bioethanol Production from Sugar
Beet Molasses, and Thick Juice Using Saccharomyces cerevisiae Immobilized
on Maize Stem Ground Tissue. Fuel, 92 (1), 1–8.
7) Kridponpattara, S., and M. Phisalaphong. (2013). Bacterial Cellulose-Alginate
Composite Sponge as a Yeast Cell Carrier for Ethanol Production. Biochem.
Eng. J., 77, 103–109.
8) Singh, A., Sharma, P., Saran, A. K., Singh, N., and N. R. Bishnoi. (2013).
Comparative Study on Ethanol Production from Pretreated Sugarcane Bagasse
Using Immobilized Saccharomyces cerevisiae on Various Matrices. Renew. Ener.,
50, 488–493.
9) Yu, J., Zhang, X., and T. Tan. (2007). A Novel Immobilization Method
of Saccharomyces cerevisiae to Sorghum Bagasse for Ethanol Production. J.
Biotechnol., 129 (3), 415–420.
10) Vucurovic, V., and R. N. Razmovski. (2012). Sugar Beet Pulp as Support for
Saccharomyces cerevisiae Immobilization in Bioethanol Production. Ind. Crop.
Prod., 39, 128–134.
11) Chandel, A. K., Narasu, M. L., Chandrasekhar, G., Manikyam, A., and L. V.
Rao. (2009). Use of Saccharum spontaneum (Wild Sugarcane) as Biomaterial
for Cell Immobilization and Modulated Ethanol Production by Thermotoler-
ant Saccharomyces cerevisiae VS3. Bioresour. Tech., 100 (8), 2404–2410.
12) Genisheva, Z., Mussatto, S. I., Oliveira, J. M., and J. A. Teixeira. (2011).
Evaluating the Potential of Wine-Making Residues and Corn Cobs as Support
Materials for Cell Immobilization for Ethanol Production. Ind. Crop. Prod.,
34 (1), 979–985.
13) Yu, J., Yue, G., Zhang, J., Zhang, X., and T. Tan. (2010). Immobilization of
Saccharomyces cerevisiae to Modified Bagasse for Ethanol Production. Renew.
Energ., 35 (6), 1130–1134.
14) Plessas, S., Bekatorou, A., Kountinas, A. A., Soupioni, M., Banat, I. M., and
R. Marchant. (2007). Use of Saccharomyces Cerevisiae cells Immobilized on
Orange Peel as Biocatalyst for Alcoholic Fermentation. Bioresour. Technol.,
98 (4), 860–865.
15) Kilonzo, P., Margaritis, A., and M. Bergougnou. (2011). Effect of Surface
Treatment and Process Parameters on Immobilization of Recombinant Yeast
Cells by Adsorption to Fibrous Matrices. Bioresour. Technology, 102 (4),
3662–3672.
16) Cardona, C. A., Quintero, J. A., and I.C. Paz. (2010). Production of Bio-
ethanol from Sugarcane Bagasse: Status and Perspectives. Bioresour. Technol.,
101 (13), 4754–4766.
17) Fitria, R. Ermawar, A., Fajriutami, T., and E. Hermiati. (2011). Pretreatment
of Sugar-cane Bagasse as Bioethanol Feedstock. Proc. 1st Intl Symposium for
Sustainable Humanosphere, 128–132.
18) Zheng, Y., Pan, Z., and R. Zhang. (2009).Overview of Biomass Pretreatment
for Cellulosic Ethanol Production. Int. J. Agric. & Biol. Eng., 2 (3), 51–68.
19) Yadav, K. S., Naseeruddin, S., Prasanthi, G. S., Sateesh, L., and L.V. Rao.
(2011). Bioethanol Fermentation of Concentrated Rice Straw Hydrolysate
using Co-culture of Saccharomyces cerevisiae and Pichia stipitis”, Bioresour.
Technol., 102 (11), 6473–6478.
20) Nikolic, S., Mojovic, L., Rakin, M., and D. Pejin. (2009). Bioethanol Product
from Corn Meal by Simultaneous Enzymatic Saccharification and Fermenta-
tion with Immobilized Cells of Saccharomyces cerevisiae var Ellipsoideus. Fuel,
88,1602–1607.
21) Sass, J. E., (1961). Botanical Microtechnique. 2nd ed. Iowa: The Iowa State
University Press, 228 pages.
22) Dowe, N., and J. McMillan. (2001). SSF Experimental Protocols - Lignocel-
lulosic Biomass Hydrolysis and Fermentation. National Renewable Energy
Laboratory, Tech. Rep. NREL/TP-510-42630.
23) Somogyi, M., (1952). Notes on Sugar Determination. J. Biol. Chem., 195,
19–23.
24) Templeton, D., and T. Ehrman. (1995). Laboratory Analytical Prosedure:
Determination of Acid In-Soluble Lignin in Biomass. Midwest Research
Institute for the Departement of Energy, Tech. Rep. LAP003.
25) Mussatto, S. I., Aguilar, C. N., Rodrogues, L. R., and J. A. Texeira. (2009).
Fructooligosaccharides and β-Fructofuranosidase Production by Aspergillus
japonicus Immobilezed on Lignosellulosic Materials. J. Mol. Catal. B-Enzym.,
59 (1), 76–81.
26) Verbelen, P. J., De Schutter, D. P., Delvaux, F., Verstrepen, K. J., and F. R.
Delvaux. (2006). Immobilized Yeast Cell Systems for Continuous Fermenta-
tion Applications. Biotechnol. Lett., 28, 1515–1525.
27) Prior, B. A., Du Preez, J. C., and P. W. Rein. (1992). Environmental Param-
eters. Solid Substrate Cultivation, H.W. Doelle, D.A. Mitchell, and C.E. Rolz
(Eds.), London: Elsevier Applied Science, 65–85.
28) Escobar, L. M. A., Alvarez, U. S., and M. Penuela. (2012). Yeast Immobiliza-
tion Wastes for Ethanol Production in Packed Bed Bioreactor. Rev. Fac. Ing.
Univ. Antioquia, 62, 66–76.
29) Lavoie, J.-M., Beauchet, R., Berberi, V., and M. Chornet. Biorefining
Lignocellulosic Biomass via the Feedstock Impregnation Rapid and Sequential
Steam Treatment. Biofuel’s Engineering Process Technology, M. A. dos Santos
(Ed.), InTech 685–714.
30) Verstrepen, K. J. and F. M. Klis. (2006). Flocculation, Adhesion and Biofilm
Formation in Yeasts. Mol. Microbiol., 60 (1), 5–15.
EFFECT OF PH ON EXTRACTION EFFICIENCY AND
DISTRIBUTION IN NICKEL ION SEPARATION USING
SOLVENT EXTRACTION
Askal Maimulyanti*, Anton Restu Prihadi

Akademi Kimia Analisis
Jl. Pangeran Sogiri No. 283, Bogor, West Java, Indonesia
Abstract
Nickel ion separation with dimethylglyoxime as chelating agent has been developed in liquid-liquid
extraction. Extraction is done with variation of pH in acidic, neutral and alkaline condition, from
pH 5 to pH 8. Extraction performed using a total of 12 mL chloroform solvent with one extraction
process. Nickel concentration was varied from 5 ppm to 30 ppm. The optimum amount of nickel
extracted at pH 5 and 20 ppm concentration, at pH 7 and 20 ppm, at pH 8 and 25 ppm. Using
linearity curve is more visible on the extraction of nickel at pH 8 at a concentrastion range of 5
ppm to 25 ppm. 86.30 % of nickel extracted with comparative figures value distribution at 6.29.
Key words: Solvent extraction, Nickel, Dimethylglyoxime, pH, Distribution coefficient.
I. Introduction
Extraction is a way of separating solutes through two solvents (usually liquid)
that can dissolve the substances of solutes but both these solvents can not dissolve
each other (immiscible). The sample was dissolved in ‘rafinat’ which is in contact
with ‘extractant’ so that the displacement of solute molecules due to differences
in solubility in both types of solvent [1].
Solvent extraction is commonly used to separate the desired number of
substance and the substance may interfere in the analysis. Sometimes, these
interfering cluster can be selectively extracted [2]. Separations were developed
with this method. One of which is the separation of metal ions with the formation
of chelate compounds.
Many metal ions can be separated by extraction of such chelate, nickel chelate
formation using dimetilglioksime (DMG). Reaction chelate with metal ions to
form molecules are not soluble in water, but soluble in organic solvents such as
chloroform and carbon tetrachloride [3].
Nickel ions that have been separated by solvent extraction, continued by
quantitative determination of the nickel. UV-Vis spectrophotometry analysis
* Corresponding Author.Tel: +62-251-8650351. E-mail: askal_m@yahoo.com
515
methods can be developed on the determination of nickel-dimetilglioksime.

Dimethylglyoxime complexes can also be determined by gravimetry analysis.
Determination of nickel as the dimethylglyoxime complex can be performed
with the colorimetric solid phase extraction [4]. The use of dimethylglyoxime
can also be modified on the electrode in the determination of nickel [5]. Nickel
can also form a complex with methyl 4 pentadione dioxime, and complexes with
3,5-dimetoxy-4-hydroxy benzaldehyde isonicotynoil hidrazone on spectropho-
tometric analysis [6][7].
Extraction of nickel should really be perfectly extracted for determination of
nickel content using a variety of methods of analysis. Extraction result is strongly
influenced by pH and matrix effects [8]. Nickel can be extracted well at alkaline
pH condition. Effect of pH on the extraction process can be determined by
calculating the value of comparative figures and extraction efficiency distribution.
The amount of the distribution is a ratio of the amount of substance that
extracted into the organic phase and the amount of substance that remains in
the water phase. The amount of the distribution changed because of the nature
of the distribution of the solvent, the nature of the solute and temperature.
Extraction efficiency seen from the number of substances extracted fraction
is usually expressed as a percent extraction.This research studied the effect of
pH changes on the value of comparative figures and extraction efficiency of
distribution including the amount of a substance that is extracted at pH variations.
Varied pH values ranging from 5 to 8. The pH changes will show that
the optimum pH which yields the greatest percent extracted. Value of D is a
distribution which is a comparative figure of substance amount that is extracted
comparison to the amount of a substance that remains in its original phase. And
this also means that the distribution ratio can be changed by changing the acidity
or pH. Extraction efficiency can be seen from the number of substances extracted
fraction which is usually expressed as a percent extraction.
This research studied the effect of pH changes on the value of comparative
figures and extraction efficiency of distribution which includes the substance
amount that is extracted at pH variations.
Separation of nickel with extraction methods have been developed, such
as separation with preconcentration by extraction system in water samples,
preconcentration and separation of nickel using a solid phase and extraction of
nickel using silica gel [9][10][11]. Extracted fraction (percent extracted) is the
amount that states the level of success of an extraction process. Extracted fraction is
defined as the part that is picked up from the place of origin (usually the aqueous
phase) compared to the amount of the original substance. This figure also reflects
the suitability of solvent systems that are extracted from the initial sample.
This study aims to look at the effect of pH on the distribution and number
value appeal extraction efficiency.

A. Materials and Equipment
The materials used including: nickel sulphate, nitric acid (pa), ammonia 4 N,
chloroform, distilled water, dimethylglyoxime and citric acid.
The equipment used including: a UV-VIS spectrophotometer, separator
funnel, 100 mL volumetric flask, 1 L volumetric flask, beaker glass, volume
pipette, burette, washing bottle, analytical balance and pH universal.
B. Preparation of Samples and Standards

Standard series of nickel 5, 10, 15, 20, 25, and 30 ppm were made in a 100 mL
flask with dilution of the standard solution of 100 ppm nickel. Samples were
prepared by making a solution of 10 ppm nickel.
C. Nickel Extraction at pH 5
Prepared sample solutions with concentrations of nickel, respectively 5, 10, 15,
20, 25, and 30 ppm. Pipetted 10 mL of sample into a glass cup containing 90
mL of distilled water, add 5 g of citric acid (pa).
Aqueous ammonia was added to the solution until pH 5. The solution cooled
and transferred to a separator funnel. 20 mL of dimethylglyoxime was added to
a separator funnel, let it stand 1–2 minutes. 12 mL of chloroform was added to
the solution, shaked for 1 minute, then let it stand until it separated each other.
After the stable phases, the red chloroform layer separated and the absorbance
measured at 465 nm wavelength.
D. Nickel Extraction at pH 6, 7, and 8

Treated the same test with extraction at pH 5. Aqueous ammonia was added to
the solution until pH 5, 6, 7, and 8. In various of pH , the series standar solution
must be in the same condition. The absorbance measured at 465 nm wavelength.

A. Extraction at pH Variation
Have been carried out the extraction of nickel ions by complexing dimethylgly-
oxime in various different pH. Nickel extraction at pH 5 can be seen in Figure 1.
Figure 1 shows the extraction under acidic conditions at pH 5, the increase in
absorbance is proportional to the increase in the concentration of nickel is extracted.
Figure 1. Nickel extraction at pH 5. Figure 2. Nickel extraction at pH 6.
The concentration of nickel is extracted with a variation of 5 to 30 ppm. Maximum

amount of nickel extracted at a concentration of 20 ppm. After the concentration
of 20 ppm, the absorbance is decreased. Here happen a stripping or are-release
of the bound nickel. Nickel extraction at pH 6 can be seen in Figure 2.
Figure 2 shows nickel extraction under acidic conditions at pH 6. Increase
in absorbance is proportional to the increase in the concentration of nickel
extracted. The concentration of nickel is extracted with a variation of 5 to 30 ppm.
Maximum nickel extracted at a concentration of 25 ppm, after which absorbance
decreased. The acidity of the solution slightly reduced at pH 6 are thus able to
extract optimal nickel at a concentration of 25 ppm for a one-time extraction.
Nickel extraction at pH 7 can be seen in Figure 3.
Based on Figure 3 extraction at pH 7 shows the increase in absorbance is
proportional to the increase in the concentration of nickel is extracted. The
concentration of nickel is extracted with a variation of 5 to 30 ppm. Optimum
nickel extracted at a concentration of 20 ppm. Nickel extraction at pH 8 can be
seen in Figure 4.
Based on Figure 4 nickel extracted maximum at a concentration of 25 ppm.
The absorbance has decreased after the concentration of 25 ppm. In this condition,
the extraction is not optimal anymore for the separation of nickel metal.
B. Linearity Curve Extraction

Under the influence of pH, the variation of the concentration is made on the linear
curve 5 ppm to 25 ppm. The results of the experiment can be seen in Figure 5.
The concentration giving absorbance curves were linear in the concentration
range of 0 to 25 ppm. Absorbance nickel samples and determined concentration
measured were plotting the linearity curve. Sample measurement data can be
seen in Table 1.
Table 1. Nickel Sample Measurement Results

pH Aa REb Cc Ed (%) De
5 0.110 y = 0.010x + 0.018 8.36 83.60 5.10
6 0.114 y = 0.009x + 0.006 12.00 120 -6
7 0.126 y = 0.011x + 0.002 11.27 112.7 -8.87
8 0.090 y = 0.011x + 0.005 8.63 86.30 6.29
a
absorbance, bregretion equation, cconcentration (ppm), dpercentage extraction, dvalue distribu-
tion
Figure 3. Nickel extraction at pH 7. Figure 4. Nickel extraction at pH 8.
(a) (b)
(c) (d)
Figure 5. Nickel linearity curve extraction: (a) pH 5, (b) pH 6, (c) pH 7, (d) pH 8
Based on experiments conducted, it can be seen that large percentage of

extraction on extraction with pH of 8 is 86.30% with a numeric value appeals
distribution at 6.29. The greater the value D, the better extraction because
this value shows a comparison of the amount of a substance extracted into
the organic phase to a substance that remains in the water phase. Percentage
value of more than 100 percent or negative D values indicate that the extrac-
tion process is not optimal due to the presence of ions or other compounds
that participate extracted into the organic phase. To further complete the
research, it needs to be carried further by the addition of masking agents
such as citric acid or ethylene diamine tetra acetate (EDTA) to bind metal
interferer. The amount of masking agent that is added must be taken into account
to optimize the extraction process.
III. Conclusion
Based on the research that has been done, it can be concluded that the extraction
of nickel with dimethylglyoxime performed at various pH acidic, neutral and
alkaline. The optimum nickel extraction at pH 8 with a linear curve at concentra-
tions of 5 to 25 ppm nickel. Effective extraction performed at pH 8 with the
percentage of nickel extracted by 86.30% with a numeric value distribution at
6.29.
Further studies should be carried out to determine other interferer ions that
can also extractein nickel analysis.
IV. Acknowledgement
The authors acknowledge the kindness and co-operation of the informants and
local administrators in the study area, and the support of the Akademi Kimia
Analisis Bogor.
V. R eferences
1) Ubukata, M., Mitsuhashi, S. M., Ueki, A., Sano, Y., Iwasa, N., Fujita, S.,
and M. Arai. (2010). Quality Determination of Nickel-loaded Silika Prepared
from Poaceous Biomass. J. Agr. Food Chem., 58 (10), 6312–6317.
2) Skoog, D. A., West, D. M., and F. J. Holler. (1995). Fundamental of Analytical
Chemistry. 7th ed., 870 Saunders College Pub.
3) Day, R.A., and A. L. Underwood. (1991) Quantitative Analysis, 6th ed., 685
pages. New Jersey: Practice Hall International.
4) Gazda, D. B., Fritz, J. S, and M. D. Porter. (2004). Determination of Nickel

(II) as the Nickel Dimethylglyoxime Complex using Colorimetric Solid Phase
Extraction. Anal. Chim. Acta, 508 (1), 53–59.
5) Tartarotti, F. O., Oliveira, M. F., Roberto, V., and N. R. Stradiotto. (2006).
Determination of Nickel in Fuel Ethanol using a Carbon Paste Modified
Electrode Containing Dimethylglyoksime. Microchim. Acta, 155 (3–4),
397–401.
6) Dipak, J., Gaaroe, and A. D. Sawant. (2005). Extractive Spectrophotometric
Determination of Nickel (II) using 4–Metil Pentadione Dioxime (H2MP-
DDO). J. Sci. and Ind. Res. India, 64, 581–584.
7) Bai, K. A., Vallinath, G. V. S., Chamdrasekhar, K. B., and N. Devanna.
(2010). Derivate Spectrophotometric Determination of Nickel(II) using
3,5Dimetoxy-4-hydroxy Benzaldehyde Isonicotinoyl Hydrazone (DMH-
BIH). J. Chem., 3 (3), 465–472.
8) Martopo, A., (2006). Efek Kenaikan pH pada Mekanisme Ekstraksi Cair-cair
terhadap Asam-asam Oksalat. Gradien, 2 (1), 130–133.
9) Safavi, A., Abdollahi, H., Hormozi Nezhad, M. R., and R. Kamali. (2004).
Cloud Point Extraction, Preconcentration and Simultaneous Spectrophoto-
metric Determination of Nickel and Cobalt in Water Samples. Spectrochim.
Acta A: Mol. Biomol. Spectrosc., 60 (12), 2897–2901.
10) Ghaedi, M., Ahmadi, F., and M. Soylak. (2007). Preconcentration and
Separation of Nickel, Copper and Cobalt using Solid Phase Extraction and
Their Determination in Some Real Samples. J. Hazard. Mater., 147 (1–2),
226–231.
11) Jiang, N., Chang, X., Zheng, H., He, Q., and Z. Hu. (2006). Selective
Solid-phase Extraction of Nickel (II) using a Surface-imprinted Silica Gel
Sorbent. Anal. Chim. Acta, 577 (2), 225–231.
ETHOXYLATED GLYCEROL MONOOLEATE: PALM OIL
BASED NONIONIC SURFACTANT FOR OIL-IN-WATER
EMULSION SYSTEMS
Indri Badria Adilina*, Egi Agustian, Yenny Meliana, Anny Sulaswatty

Research Center for Chemistry, Indonesian Institute of Sciences
Puspiptek Area Serpong, Tangerang, 15314, Indonesia
Abstract
The present study focuses on the application of ethoxylated glycerol monooleate as a palm
oil based nonionic surfactant for oil-in-water emulsion systems. Preparation of the nonionic
surfactant was performed in the prescence of an alkaline catalyst by reacting glycerol monooleate
with ethylene oxide at 80ºC followed by purification using acetic acid and black carbon. Results
show that the stability of emulsion formed by addition of white oil to an aqueous ethoxylated
glycerol monooleate was highly affected by the physical and chemical properties of the ethoxylated
material. Formulated emulsions using the nonionic surfactant did not demonstrate separation of
oil and water phase after centrifugation at 3,000 rpm for 10 minutes and therefore potentially
used as bio-additive materials in natural cosmetics.
Key words: Nonionic surfactant, Ethoxylated glycerol monooleate, Palm oil, Oil-in-water emulsion.
Introduction
There are many types of chemical reactions used in the manufacture of surfactants,
such as sulfonation, esterification, ethoxylation, amidation, etc. Ethoxylation in
particular is applied to obtain a surfactant which has higher solubity in water
properties or a stronger hydrophilic tail [1]. The level of solubity depends on
the amount of ethylene oxide (EO) polymerized and inserted in the structure.
Ethoxylated materials as one of the nonionic surfactants are produced in industry
by reacting EO with an organic molecule containing mobile hydrogen such as
fatty alcohols, alkyl phenols or fatty acids.
Ethoxylation of fatty esters of rapeseed oil has been reported to be carried
out over a homogenous calcium based catalyst [2]. The reaction temperature was
found to have substant influence on the obtained product distribution. With the
enhancing development of oleochemistry, palm oil and other readily renewable
materials are dominating the market with its oleochemicals. We have previously
performed the direct ethoxylation of glyceril fatty esters of palm oil which in
* Corresponding Author. Tel: +62-21-7560929. E-mail: indribadria@yahoo.com, indr002@lipi.go.id
523
dominant were glycerol mono-oleate (GMO), at 80–180ºC in the presence of

an alkaline catalyst. A degree of polymerization of n = 2, 3 was yielded in the
ethoxylated GMO material (EGMO).
Oil-in-water (o/w) emulsion systems stabilized by nonionic surfactants are
widely used in agrochemical, cosmetic, food and pharmaceutical industries [3].
The stability of these emulsion systems, however, still remains a challenging issue
to be investigated. Emulsion instability may result from physical processes, i.e.
flocculation, coalescence, Ostwald ripening and gravitation separation. The rate
of these changes can be measured by determining the size and distribution of
oil droplet in emulsion. Stokes’s law state that the velocity at which a droplet
moves is proportional to the square of its radius. The stability of an emulsion
to the gravitation separation therefore can be enhanced by reducing the size of
droplets [4].
The primary objective of this research is to investigate the critical micelle
concentration (CMC) of various EGMO materials with different chemical and
physical properties as a nonionic surfactant in an o/w emulsion system and analyze
the emulsions formed using centrifugation procedures and optical microscope
analysis.
II. E xperimental
A. Materials
Methanol, chloroform and white oil were purchased from Sigma Aldrich and used
in the emulsification process. EGMO was synthesized as previously reported. An
amount of GMO and 1% KOH were introduced into a reactor and for 2 hours
without heating. EO was then inserted and the mixture was heated at 80º C for
another 2 hours. At the end of the process, the reaction was stirred for 3 hours
in room temperature. Purification of EGMO was performed using acetic acid
and adsorption with black carbon.
B. Method
1) Preparation of O/W Emulsion
An amount of 1 g of selected EGMO was diluted into 100 ml of demineralized
water. After stirring, the mixture gradually turned into white and opaque appear-
ance and 0.5 g of white oil was introduced into 10 ml of the mixture and stirred
at 2,500 rpm in room temperature for 15–30 minutes. The o/w emulsion system
formed was then kept in room temperature for stability observation.
2) Surface Tension and Critical Micelle Concentration (CMC)

The CMC values of the surfactants were obtained by measurements of surface ten-
sion as a function of surfactant concentration at 25ºC on a Sigma 70 tensiometer
(Torsion Balance Supplies) equipped with a Pt–Ir du Noüy ring. Measurements
were performed in demineralized water. The surface tension values were taken at
the breakpoint of the surface tension vs log concentration curve.
3) Stability of O/W Emulsion

Stability of the o/w emulsion was tested using centrifuge type Kokosan H-103N
series at 3,000 rpm for 10 minutes. The emulsion stability was indicated by
homogenous phase without separation of the oil and water phase.
4) Optical Microscope Measurement

The emulsion formed was observed with an optical microscope (Zeiss with Meteor
Matrox II software). Magnification was 400x and each emulsion was measured
in a 24 hours and 14 days period. Droplet size was recorded and the stability of
emulsion was analyzed.

A. Solubility Test of Various EGMO in Water
EGMO materials were obtained as reported in previous work [5] [6]. Four
products was selected for solubility and labeled as A, B, C and D. Products A, B
and C were all produced in the synthesis at temperatures of 80ºC with different
reagent molar ratios whereas D was obtained at a higher temperature at 180ºC.
The EGMO products were dissolved in 100 ml of water with two concentration
types of 0.5% (w/v) labeled as A1, B1, C1 and 1% (w/v) labeled as A2, B2, C2.
Table 1. Appearance of Aqueous EGMO at 27°C
Entry EGMO Product Appearance
1 A1 □
2 B1 □
3 C1 □
4 D ■
5 A2 ■
6 B2 ■
7 C2 ■
□ clear, dissolve, homogenous (transparent)
■ cloudy, homogenous (white, opaque)
Table 2. Properties of EGMO Selected for Emulsification

Properties
EGMO
GMO:EO AV EV MW ρ η
Product HLB
(mol) (mg KOH/g) (mg KOH/g) (g/mol) (g/cm3) (Cp)
A 1:2 6.07 101.27 460 1.98 0.9855 112.81
B 1:6 4.29 82017 878 10.02 1.0244 135.07
C 1:10 4.14 68.26 1198 12.49 1.0368 67.04
D 1:1 12.35 105.95 - - 0.9684 235.64
Figure 1. Surface tension vs concentration graph of EGMO products.
Table 3. CMC Value of EGMO

Entry EGMO Product Log CMC (%wt/v) CMC (%wt/v)
1 A -1.9 12
2 B -1.2 56
3 C -1.3 50
4 D -1.8 16
Product D was used at a single concentration of 1% (w/v). The appearance of

various aqueous EGMO at 27ºC is shown in Table 1.
A clear dissolved mixture was produced in almost all aqueous EGMO of 0.5%
(w/v) (entries 1–3) except for product D (entry 4), meanwhile mixtures became
opaque at a higher concentration of 1% (wt/v) (entry 5–6). The differences
of these appearances are speculated to be caused by the different properties of
EGMO product, such as acid value (AV), ester value (EV), molecular weight
(MW), hydrophilic-lipophilic balance value (HLB), density, and viscosity as
summarized in Table 2.
B. Measurement of CMC Value for Various EGMO Products

The CMC value of EGMO in the current work was determined by a tensiometer
and a surface tension vs concentration graph as shown in Figure 1 was obtained
for calculation. From the collected data, it can be seen that the surface tension
decreases with the addition of surfactant concentration. The CMC was determined
by an interpolation line of surface tension and log concentration of surfactant.
The value of CMC is shown in Table 3.
C. Optical Analysis of O/W Emulsion Formed Using Various EGMO

Products
Emulsions were prepared by adding 0.5 g white oil into 10 ml of aqueous
ethoxylation product. The mixture was stirred for 15–30 minutes at 2,500 rpm.
The mixture slowly turned into a white opaque solution due to the formation
of an emulsion. The stability of these o/w emulsion systems were then tested
by centrifugation procedures and optical microscope analysis. Centrifugation
was performed at 3,000 rpm for 10 minutes, directly after the emulsions were
prepared. From the results obtained, it is evident that almost all emulsions did not
demonstrate any separation of oil and water phase, except for B1 and C1 which
became a two layer mixture after the centrifugation procedure. This indicated
that the o/w emulsions formed using various EGMO products generally gave
a good stability where the mixture remained homogeneous, white and opaque.
The o/w emulsion was further kept at room temperature and analyzed by
optical microscope analysis at two different periods of time. The first analysis was
performed after 24 hours (1 day) of storage, and the second was after 14 days (2
weeks). In the case of emulsion kept for 24 hours, no droplets were observed in
the solution when analyzed using an optical microscope. This result indicated
that emulsion A1 and B2 were only stable for a few hours after the preparation.
On the other hand, droplets with various sizes were seen for emulsion A2, C2
and D as illustrated in Figure 2 and Figure 3.
Figure 2. Optical microscope analysis of; (i) A2–24 hours, (ii) A2–14 days, (iii) C2–24 hours,
and (iv) C2–14 days.
Figure 3. Optical microscope analysis of D–24 hours (left) and D–14 days (right).
Although the droplets observed were not all homogenous with the same
size, it can be seen that a double emulsion was formed for emulsion A2. After
kept in room temperature for 14 days, however, this was no longer observed
due to coalescence. The stability of an emulsion depends gratefully on the size
of droplets distributed in the emulsification system. The increase of droplet size
results in an unstable emulsion due to coalescence of the droplets in a period of
time. Emulsion with different droplet size was also seen for emulsion C2. Smaller
droplets are seen in 24 h compared to the 14 days period indicating a higher
stability in shorter period of time. In the case of emulsion D, however, relatively
homogeneous droplets were formed in the first 24 h and no significant change
in their size was seen after keeping for 14 days demonstrating a higher emulsion
stability compared to other o/w emulsion systems.
IV. Conclusion
Ethoxylated glycerol mono oleate (EGMO) from palm oil is active as surfactants
of oil-in-water emulsion systems. The nonionic surfactant was synthesized by
direct ethoxylation of GMO using ethylene oxide at a temperature of 80⁰C in
the presence of 1% KOH as catalyst. Purification of the EGMO products by
addition of acetic acid and black carbon yielded products with greater acid value,
ester value, density and viscosity. The surface tension decreased using all of the
EGMO products and a white opaque emulsion was formed by addition of 0.5
g of white oil in 10ml aqueous EGMO. Each product gave different emulsion
system which depended on their chemical and physical properties. The emulsions
did not observe separation of oil and water phase after centrifugation at 3,000
rpm for 10 minutes and therefore are potentially used in the formulation of
natural cosmetics.
V. R eferences
1) Di Serio, S., Martino, D., and E. Santecesaria. (1994). Kinetics of Fatty Acids
Polyethoxylation. Ind. Eng. Chem. Res, 33 (3), 509–514.
2) Hreczuch, W., (2002). Temperature-related Reaction Kinetics and Product
Composition of Ethoxylated Fatty Acid Methyl Esters. J. Chem. Technol.
Biotechnol., 77 (5), 511–516.
3) Dale, P. J., Kijlstra, J., and B. Vincent. (2006). The Temperature Stability of
Single and Mixed Emulsions Stabilized by Nonionic Surfactants. Coll. Surf.
A: Physicochem. Eng. Aspects, 291 (1-3), 85–92.
4) Critical Micelle Concentration: Automated Measurement. Helsinki, Finland,
2005.
5) Laksmono, J. A., Meliana, Y., Adilina, I. B., and E. Agustian. (2008). Direct
Ethoxylation of Fatty Acid Glyceril Esters from Palm Oil as Non-ionic
Surfactants. J. Reaktor, 12, 102–106.
6) Adilina, I. B., Meliana, Y., Laksmono, J. A., and E. Agustian. (2007).
Preparation and Properties of Ethoxylated Glycerolmonooleate as Non-ionic
Surfactants. Presented at 4th International Conference on Materials for Advanced
Technologies. Singapore.
7) Holmberg, K., (2001). Natural Surfactants. Curr. Opin. Colloid In., 6 (2),
148–159.
8) Johansson, I., and M. Svensson. (2001). Surfactants Based on Fatty Acid
and Other Natural Hydrophobes. Curr. Opin. Colloid In., 6 (2), 178–188.
9) Hama, I., Okamoto, T., Hidai, E., and K. Yamada. (1997). Direct Ethoxyl-
ation of Fatty Methyl Ester over Al-Mg Composite Oxide Catalyst. J. Am.
Oil Chem. Soc., 74 (1), 19–24.
10) Santacesaria, E., Diserio, M., and R. Tesser. (1995). Role of Ethylene Oxide
Solubility in the Ethoxylation Processes Catal. Today, 24 (1–6), 23–28.
CONVERSION OF CITRONELLA OIL AND ITS
DERIVATIVES TO MENTHOL OVER BIFUNCTIONAL
NICKEL ZEOLITE CATALYSTS
Indria Badila Adilina*, Ralentri Pertiwi, Anny Sulaswatty

Abstract
Citronella oil and its derivatives were converted to menthol in a one-pot reaction system using
bifunctional zeolite based nickel catalysts. The catalysts were prepared by immobilization of nickel
on natural zeolite (NZ) and synthetic zeolite (ZSM-5) by a simple cation exchange method.
Catalytic reactions were carried out at 70ºC by first stirring the mixture in air for cyclization
of citronellal to isopulegol followed by hydrogenation of isopulegol to menthol at 2 MPa of
H2 pressure. The Ni/NZ catalyst was able to convert 24% of a citronella oil derivative yielding
9% menthol (37% selectivity), whereas Ni/ZSM5 catalyst showed lower activity of with 65%
conversion and 4% yield of menthol (6% selectivity). These zeolite based catalysts are therefore
potential materials for the conversion of biomass feedstock to value-added fine chemicals.
Key words: Bifunctional Catalysts, Zeolite, Nickel, Citronella Oil, Menthol
I. Introduction
Citronella oil is considered one of the 20 most important essential oils that are
traded worldwide [1] and widely used in the manufacture of perfume, soap and
many other cosmetics. This essential oil is categorized into two types in the global
market; Ceylon citronella oil which is provided from the Cymbopogon nardus grass
plant and Java type citronella oil obtained from that of Cymbopogon winterianus
[2]. Indonesia is the third largest world producer of citronella oil (after China
and Vietnam) with estimated production of 350 tons annually [3]. The major
components in citronella oil include citronellal, citronellol and geraniol.
Menthol is a flavor of chemical which is used extensively in food, cosmetic,
and pharmaceutical industries [4]. Natural menthol can be supplied through
extraction of Mentha arvensis plant followed by a separation process. Meanwhile,
synthetic (-)menthol is industrially produced by Takasago International Corp.
and Symrise using myricene or m-cresol as the protocol in a catalytic process
involving consecutive steps. Synthesis of menthol from citronellal, on the other
* Corresponding Author. Tel: +62-21-7560929. E-mail: indribadria@yahoo.com, indr002@lipi.go.id
531
hand, may be performed by more efective two step process, i.e. one-pot cyclisation
of citronellal followed by hidrogenation of isopulegol to menthol [5].
Acid catalysts, such as SiO2, Al-MCM-41, ZnBr2, have been reported to serve
as an efficient cyclization catalyst of citronellal. The Brønsted acid site was able
to increase the rate of isomerization of citronellal although the formation of (-)
isopulegol was still low. Zinc bromide, which gives 94% selectivity to isopulegol,
is used industrially.
The hydrogenation reaction of isopulegol to menthol requires metal catalysts
which can accelerate the reaction rate. Ni metal exhibits high activity in the
processes of cracking, isomerization, hydrogenation, as well as ring opening of
hydrocarbons and a Ni/Al-MCM-41 catalyst was reported to transform citronellal
to menthol in good selectivity [4]. The weak acid sites of Al-MCM-41 facilitated
the cyclisation of citronellal to isopulegol, and upon hydrogenation 70–75%
racemic (±)-menthol was formed. Various studies have reported the use of Ni
metal as an active metal on catalyst for the hydrogenation reaction of isopulegol
to menthol, including Ni/ ZnBr2/β-zeolite catalyst [6], NiZrS catalyst [7], and
Ni/γ-Al2O3 catalyst [8].
In this paper, we present the preparation of bifunctional nickel zeolite catalysts
from natural zeolite (NZ) and synthetic zeolite (ZSM-5) and their application in
the one-pot synthesis of menthol from citronella oil and its derivatives. NZ based
catalysts are considered more economical than other syntesized silica-alumina
compounds since these materials are abundant and readily available, whereas
ZSM-5 is commercially available and was used for comparison. Ni metal was
chosen due to its low cost compared to precious metals such as Pt, Pd, and Ru.
II. E xperimental
A. Preparation and Characterization of Bifunctional Zeolite Based Cata-
lysts
Nickel supported on zeolite (NZ, ZSM5) was prepared via an ion exchange
procedure by adding 5 g zeolite to an amount of distilled water containing 10 wt%
Ni from Ni(NO3)2.6H2O (Merck). The solution was stirred at room temperature
for 24 h. The catalyst was then dried at 100oC for 2 h and then calcined at
350oC for 2 h. Before the catalytic testing, some catalysts were reduced under
H2 flow for 2 h at 300oC and 500oC for NZ and ZSM5 catalysts, respectively.
Characterization of the catalysts was conducted by a surface area analyser, X-ray
fluorescence, and X-ray diffraction.
B. Catalytic Test and Product Analysis

The catalytic test was carried out in a 20 mL autoclave at 70oC. Citronella oil
and its derivatives (5 mmol) was added into the autoclave and 0,1 g bifunctional
catalyst was added with 5 mL cyclohexane (Merck). After 3 h, H2 was flowed with
a constant pressure of 2 MPa for another 3 h. Reaction products were analyzed
by gas chromatography using biphenyl as an internal standard (Shimadzu 14A,
column DB5-MS 30 m x 0.25 m x 0.25 μm, FID detector).

A. Characterization of Bifunctional Zeolite Based Catalysts
1) Surface Area Analyzer Measurement
The surface area, pore size, volume and pore size of the various bifunctional zeolite
based catalyst were measured by a surface area analyzer according to the BET
method. As observed in Table 1, the surface area of Ni/NZ (entry 2) and Ni/
NZ-HT (entry 3) increased slightly after immobilization and subsequent hydrogen
treatment, respectively. Meanwhile, natural zeolite as the catalyst precursor tends
to show a smaller surface area and pore volume (entry 1). This is presumably due
to a number of impurities in their framework which may cover the active surface.
In the case of Ni/ZSM5 and Ni/ZSM5-HT, the surface area was detected lower
than that of the parent ZSM-5, although the change of their pore volume and
pore size was neglectable. It is predicted that a partial blockage of porous structure
in the ZSM-5 framework occurred after the Ni loading.
Table 1. Surface Area, Pore Volume, and Pore Size of Ni/NZ and NiZSM5 Catalysts
Surface Area Pore Volume Pore Size (nm)
Entry Catalyst
(m2g-1) (cm3g-1)
1 NZ 16 0.004 2.11
2 Ni/NZ 26 0.01 2.10
3 Ni/NZ-HTa 35 0.01 2.10
4 ZSM-5 [9] 400 0.03 2.03
5 Ni/ZSM5 361 0.03 2.03
6 Ni/ZSM5-HTa 342 0.03 2.04
HT: Hydrogen Transfer
a
Table 2. Elemental Analysis of NZ Based Catalysts

Entry Catalyst SiO2 (%) Al2O3 (%) NiO (%)
1 NZ 72 15 -
2 Ni/NZ 72 14 1
3 Ni/NZ-HT 71 15 1
2) X-ray Fluorescence Analysis

Table 2 summarizes the elemental composition of SiO2 and Al2O3 in the Ni/NZ
and Ni/NZ-HT catalysts. Results show that both catalysts have a SiO2 content
higher than that of AlO3. The Si/Al ratio is the ratio of the number Si atoms
to the number of Al atoms in the zeolite framework and a higher Si/Al ratio is
known to give a higher acid behavior [10]. The Ni content was measured as 1%
for both Ni/NZ and Ni/NZ-HT catalysts.
(a) (b)
(c)
Figure 1. XRD patterns of Ni/NZ catalysts: (a) NZ, (b) Ni/NZ, and (c) Ni/NZ-HT
Figure 2. Plausible products from citronellal.

3) X-ray Diffraction Analysis

The crystalline structure of a number of Ni/NZ catalysts were determined by
XRD analysis. XRD patterns of the parent NZ and that after immobilization
of Ni are shown in Fig. 2. According to the results, clinoptilolite (2θ = 13.68°,
26.78°, 30.12°) and mordenite (2θ = 19.74°, 22.42°, 25.82°, 27.92°) phases were
detected in the NZ structure. A high peak intensity at 2θ = 26.78° indicates that
the parent NZ compose of moderite as its main constituent mineral with some
existence of clinoptilolite
Meanwhile, when compared with the Ni/NZ catalyst, no change in the
value of 2θ from was observed however, the peaks intensity increased after the
immobilization. This was suggested due to the calcination process which might
remove impurities in the NZ framework. In the case of Ni/NZ-HT catalyst, a
new peak at 2θ = 41.74° was formed, indicating that the presence of Ni metal in
the structure as a result of hydrogenation treatment.
B. Conversion of Citronellal and Its Derivatives to Menthol

Conversion of citronellal and its derivatives to menthol over various bifunctional
Ni zeolite based catalysts was carried out in an autoclave at 70⁰C, such as sum-
marized in Table 4. The catalytic activity was first tested for Ni/ZN catalyst for 24
h using cyclohexane as a solvent and 100% conversion of citronellal was obtained.
The catalyst was able to yield 7% of isopulegol. From the intermediate of menthol,
however, no formation of menthol was observed (entry 1). Other byproducts
are predicted to be citronellol, 3,7-dimethyloctanal, and 3,7-dimethyloctenal.
When toluene was used as the solvent, no significant increase in the isopulegol
and menthol yield was seen (entry 2). It has been reported that catalysts with
weak Brønsted acid and high Lewis acid sites will give a high selectivity in the
cyclization reaction of citronellal to isopulegol [11].
To determine whether the Ni metal active site in the catalyst was effective for
the hydrogenation reaction, catalytic reactions using citronellal and isopulegol as
the starting material were conducted using Ni/NZ-HT catalyst. Results reveal
than no yield of menthol was obtained when citronellal was used as the reactant
(entry 3). On the other hand, 8% yield of menthol could be obtained from
isopulegol with 24% conversion giving 37% selectivity (entry 4). From these
results, it can be observed that the Ni/NZ catalyst was indeed able to act as a
bifunctional catalyst for the conversion, although the Ni metal active site cannot
play its role more efficiently in the hydrogenation reaction since the number
of isopulegol formed from citronellal is still low. In the cases of Ni/ZSM5 and
Ni/ZSM%-HT catalysts, lower conversions citronellal were seen and only 4%
menthol was formed. Nevertheless, higher yields of 21% isopulegol were obtained
Table 3. Conversion of Citronellal and Its Derivatives to Menthol Using Various Catalysts
Conversion Yield Men- Yield Selectivity
Entry Catalyst Yield Isopulegol (%)
(%) thol (%) Others (%) Menthol (%)
1a Ni/NZ 100 7 0 93 0
2b Ni/NZ 90 4 0 86 0
3 Ni/NZ-HT 97 12 0 85 0
4c Ni/NZ-HT 24 - 8 16 37
5a Ni/ZSM5 64 21 4 43 6
6 Ni/ZSM5-HT 48 8 0 40 0
7d Ni/ZSM5-HT 77 14 2 61 3
8 Blank 0 0 0 0 0
Reaction conditions: citronellal (5 mmol), catalyst (0.1 g), cyclohexane (5 ml), 70°C, 6 h, 2
MPa H2.
a
reaction time: 24 h, btoluene as solvent, cisopulegol as reactant, dcatalyst 0.2 g
(entry 5–7). Finally, control reaction without the use of any catalysts did not give
any conversion of citronellal (entry 8) which shows that the presence of catalysts is
highly essential for the conversion of citronella oil and its derivatives to menthol.
IV. Conclusion
Bifunctional Ni zeolite catalysts were successfully prepared from natural zeolite
(NZ) and synthetic zeolite ZSM-5 (ZSM-5) by immobilization of Ni using a
cation-exchange procedure. Their catalytic activity were tested in the one-pot
synthesis of menthol from citronella oil and its derivatives. Ni/NZ-HT showed
the highest catalytic of 24% conversion yielding 8% of menthol and 6% selectivity
whereas a lower catalytic activity of 64% conversion and 4% yield of menthol was
seen for the Ni/ZSM5 catalyst. Therefore, both Ni zeolite catalysts are potential
materials for the synthesis of flavor chemicals from citronella oil.
V. R eferences
1) Lawrence, B. M. (1993). A Planning Scheme to Evaluate New Aromatic
Plants for the Flavor and Fragrance Industries. New Crops, J. Janick and J.
E. Simon (Eds.), New York: Wiley, 620–627.
2) Bakri, R., Utami, T., and I. P. Sari. (2008). Kaolin sebagai Sumber SiO2 untuk
Pembuatan Katalis Ni/SiO2: Karakterisasi dan Uji Katalis pada Hidrogenasi
Benzena Menjadi Sikloheksana. Makara, 12 (1), 37–43.
3) National Agency for Export Development (NAFED). Export News Indonesia
Vol.1/No.II/Feb 2008.
4) Nie, Y., Niah, W., Jaenicke, S., and G. K. Chuah. (2007). A tandem cycliza-
tion and Hydrogenation of (±)-Citronellal to Menthol over Bifunctional Ni/
Zr-Beta and Mixed Zr-Beta and Ni/MCM-41. J. Catal., 248 (1).
5) Mertens, P., Verpoort, F., Parvulescu, A. N., and D. D. Vos. (2006). Pt/H-
beta Zeolites as Productive Bifunctional Catalysts Citronellal to Menthol
Conversion. J. Catal., 243 (1), 7–13.
6) Nisyak, K., Iftitah, E. D., and R. T. Tjahjanto. (2013). One-Pot Transforma-
tion of (R)-(+)-Citronellal over a Bifunctional Ni/ZnBr2/β-Zeolite Catalyst.
Makara, 17 (3).
7) Cortes, C. B., Galvan, V. T., Pedro, S. S., and T. V. Garcia. (2011). One
Pot Synthesis of Menthol from (±)-Citronellal on Nickel Sulfated Zirconia
Catalysts. Catal. Today, 172 (1), 21–26.
8) Iftitah, E. D., Muchalal, M., Trisunaryanti, Armunanto, R., Psaro, R.,
Ravasio, N., Santoro, F., and L. Sordelli. (2011). A Tandem Cyclization and
Hydrogenation of (±)-Citonellal to Menthol over Mixed ZnBr2/γ-Al2O3
and Ni/γ-Al2O3 Catalysts. J. Appl. Sci. Res., 7 (5), 680.
9) Jang, A., Ma, Q., and K. Wang. (1994). Plasma Gasification of Organic Waste
and the Synthesis Gas Purification Procedures. SciTech. Online). Available: www.
aec.gov.tw.
10) Lawrence, B. M. (1993). A Planning Scheme to Evaluate New Aromatic
Plants for the Flavor and Fragrance Industries. Ne Crops, J. Janick and J. E.
Simon (Eds.), New York: Wiley, 620–627.
11) Chuah, G. K., Liu, S. H., Jaenicke, S., and L. J. Harrison. (2001). Cyclisation
of Citronellal to Isopulegol Catalysed by Hydrous Zirconia and Other Solid
Acids. J. Catal., 200 (2), 352–359.
12) Milone, C., Gangemi, C., Ingoglia, R., Neri, G., and S. Galvano. (1999). Role
of the Support in the Hydrogenation of Citronellal on Ruthenium Catalysts.
J. Appl. Cat. A., 184 (1), 89–94.
13) Trasarti, A. F., Marchi, A. J., and C. R. Apesteguia. (2013). Synthesis of
Menthols from Citral on Ni/SiO2–Al2O3 Catalysts. Cat. Comm., 32 (5),
62–66.
14) Arvela, P. M., Kumar, N., Nasir, A., Heikkila, T., Lehto, V. P., Salmi, T.,
and D. Y. Murzin. (2005). One-pot Citral Transformation to Menthol over
Bifunctional Micro- and Mesoporous Metal Modified Catalysts: Effect of
Catalyst Support and Metal. Stud. Surf. Sci. Catal., 240 (1–2), 72–81.
15) Vandichel, M., Vermoortele, F., Cottenie, S., De Vos, D. E., Waroquier, M.,
and V. Van Speybroeck. (2013). Insight in the Activity and Diastereoselectivity
of Various Lewis Acid Catalysts for the Citronellal Cyclization. J. Catal., 305,
118-129.
APPENDIX
Editorial Board
Editor in Chief
Editors/Reviewers
Prof. Dr. M. Hanafi Dr. T. Mozef Dyg H. Siti Raihani b. H. Abd
Hamid (Brunei)
B. Wahono, M.Eng. N. Huda, M.Eng. Sc. Prof. Dr. Apichit Therdyothin
(Thailand)
Dr. S. Pribadi Prof. Dr. E. Aldrian Dr. Hrin Nei Thiam (Myanmar)
Dr. Desriani Dr. Z. Mustopa Dr. Sreng Vang (Cambodia)
Dr. K.A.Z. Thosin Dr. J. Triwibowo Prof. Nguyen Quang Liem
(Viet Nam)
Dr. E. Prakasa Dr. D. Rosiyadi Dr. Ong Sim Heng (Singapore)
Dr. Dirhamsyah Prof. Dr. Dwi Listyo Mr. Shahruddin Yusof (Malaysia)
Guest R eviewers
Prof. Dr. Bambang Riyanto Dr. Ahmad Agus Setiawan
Dr. Riza Muhida Prof. Dr. Tagawa Yasutaka (Japan)
Eny Ida Riyanti, Ph.D. Dr. Fatimah, M.Si
Dr. Kustiariyah Tarman Dr. Sukma Nuswantara, M.Phil.
Prof. Dr. Khanom Simarani (Malaysia) Dr. M. Ali Sulaiman (Malaysia)
Dr. Edi Kurniawan Dr. Hilman F. Pardede (Italy)
Andrian Arizal, M.Eng. (Germany) Yunita Sari, M.Sc. (United
Kingdom)
Ahmad Mukhalson, M.Sc. (United Kingdom) Nazre Batool, M.Sc. (USA)
Dr. Bakhit A. Adoum (Chad) Dr. Muhammad Agni Catur
Bhakti
Authors Index
A. D. Pasek
Faculty of Mechanical and Aerospace Engineering – Bandung Institute of
Technology
Jl. Ganesha No.10 Taman Sari, Bandung 41032, Jawa Barat
A. Fadli
A. Jarin
Department of Electrical Engineering, Faculty of Engineering, University of
Indonesia
A. Maimulyanti
A. Mahsunah
A. Muharam
Research Centre for Electrical Power and Mechatronics, Indonesian Institute
of Sciences
Komplek LIPI, Jl. Cisitu No. 154D/21 Gd. 20, Bandung 40135, Jawa Barat,
Indonesia
A. M. Setiawan
Center for Climate, Agro and Marine Climate - Agency for Meteorology Cli-
matology and Geophysics (BMKG)
A. Nur
of Sciences
Indonesia
A. Praptijanto
of Sciences
Indonesia
A. Purwito
Departement of Agronomy and Horticulture-Faculty of Agriculture, Bogor
Agriculture University, Darmaga, Bogor
A. P. Asri
Student, Master Program in Agronomy, Universitas Padjadjaran
Jl. Raya Jatinangor KM 21 45363, Sumedang
A. Rasyid
A. Rudyanto
A. R. Dewi
A. R. Prihadi
A. Sopaheluwakan
A. Subhan
Research Center for Physics–LIPI
Kawasan PUSPIPTEK Gedung 442 Serpong, Tangerang Selatan 15314,
Banten
A. Sulaswatty
A. Suryani
A. S. Putri
Jl. Raya Darmaga Kampus IPB Darmaga, Bogor 16680
A. S. Yuwono
Department of Civil and Environmental Engineering, Bogor Agricultural
University
A. Y. Sari
A. Zakaria
A. Z. Mustopa
Jl. Raya Jakarta-Bogor Km 46, Cibinong, Bogor 16911
B. H. Trisasongko
B. Prasetya
Institute of National Standarization, Manggala Wanabakti Building Blok IV
Jl. Gatot Subroto Senayan, Jakarta, Indonesia
B. Prihandoko
Banten
B. Wahono
C. Carol
Institute of Biological Sciences, Faculty of Science
University of Malaya, 50603 Kuala Lumpur, Malaysia
Desriani
D. Aryanto
D. A. Bruzon
Institute of Chemistry University of the Philippines-Diliman
Quezon City, Philippines
D. Dewi
Biotech Center, Agency for the Assessment and Application of Technology,
Puspiptek Area, Serpong 15314, Indonesia
D. Girmansyah
D. Hardianto
D. Permana
D. Priadi
D. P. Sari
Research Center for Biomaterials, Indonesian Institute of Scienes (LIPI)
Jl. Raya Bogor km.46, Cibinong, Bogor 1691, Indonesia
D. Rahayuningwulan
D. R. Panuju
D. S. Adi
D. Waluyo
E. Agustian
E. C. Guevarra
E. D. Wahjunie
Erwahyuni E.
E. Hermiati
E. Makmur
Center for Research and Development - Agency for Meteorology Climatology
and Geophysics (BMKG)
E. Rohaeti
E. Siska
F. Afiati
F. A. Syamani
F. Damayanti
Faculty of Agriculture, Universitas Padjadjaran, Jatinangor, Sumedang, Indo-
nesia
F. Oemry
G. Tapang
National Institute of Physics University of the Philippines-Diliman
H. Danuri
Jl. Raya Darmaga Kampus IPB Darmaga, Bogor 16680
H. E. Putra
H. Ghasemi
Cnr Jerrabomberra Ave and Hindmarsh Drive, Symonston ACT, 2609 Austra-
lia
H. Sukiman
H. Wahyu
H. Yuliandoko
State Polytechnic of Banyuwangi
Jl. Raya Jember Km.13 Labanasem-Kabat, Banyuwangi, Indonesia
and
Postgraduate of Information Engineering and Computer, Electronics Engi-
neering, Polytechnic Institute of Surabaya
I. A. P. Banlawe
Mapùa Institute of Technology
Muralla St., Intramuros, Manila, Philippines
I. Batubara
I. B. Adilina
I. Djunaedi
I G. A. Purbawa
Banyuwangi Meteorogical Station
Jl.Jaksa Agung Suprapto No.152, East Java, Indonesia
I N. Sukanta
I. S. Martinez
Institute of Chemistry University of the Philippines-Diliman
J. I. Royani
J. Murjaya
J. M. Yusoff
J. Triwibowo
Banten
Kusmiati
Kusumandari
K. Bauer
K. Ramli
Department of Electrical Engineering, Faculty of Engineering, University of
Indonesia
K. Simarani
K. Syamsu
Departement of Agroindustrial Technology, Faculty of Agricultural Engineer-
ing and Technology, Bogor Agricultural University, IPB
L. Ariana
Center for Science and Technology Development Studies, Indonesian Institute
of Sciences
Gedung A PDII, 4th floor, Jl. Gatot Soebroto Kav 10 Jakarta, Indonesia
L. H. Yeng
Institute of Biological Sciences, Faculty Science, University of Malaya
L. H. Zaini
Research Center for Biomaterial, Indonesian Institute of Science
Jl. Raya Bogor, Km.46, Cibinong, Bogor, 16911, Indonesia
L. Risanto
L. Suryanegara
L. Triratna
L. Windriawati
Masturyono
M. Affendi
M. Arifin
M. A. Alagao
National Institute of Physics University of the Philippines-Diliman,
M. A. R. Lubis
Jl. Raya Bogor KM 46, Cibinong, Bogor 16911, Indonesia
M. D. Yamanaka
Japan Agency for Marine-Earth Science and Technology (JAMSTEC)
Yokosuka, Japan
and
Graduate School of Science, Kobe University, Kobe, Japan
M. Haeruman
nesia
M. Kaneko
Laboratory of Biomass Conversion, Research Institute for Sustainable Hu-
manosphere (RISH)
M. Muzli
M. Oktaviani
M. Rahayuningsih
Departement of Agroindustrial Technology, Faculty of Agricultural Technol-
ogy, Bogor Agricultural University
M. R. Perena
M. Sari
M. S. M. Annuar
M. Umar
and
University of Syiah Kuala (UNSYIAH)
Jl. Teuku Nyak Arief, Darussalam – Banda Aceh
M. U. H. A. Rasyid
M. Widyarti
Department of Civil and Environmental Engineering, Bogor Agricultural
University
Nuriyanah
N. A. M. Nordin
Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur,
Malaysia
N. Carsono
nesia
N. G. Wiranti
Departement of Agroindustrial Technology, Faculty of Agricultural Technol-
ogy, Bogor Agricultural University
N. I. S. Bokhari
N. Susianna
STKIP Surya
Prabandari
P. Agamuthu
Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur,
Malaysia
P. Cummins
Research School of Earth Sciences-The Australian National University
Bld 142 Mills Road Acton ACT, 0200 Australia
and
Cnr Jerrabomberra Ave and Hindmarsh Drive, Symonston ACT, 2609 Austra-
lia
P. C. Mawarda
Research Center for Chemistry, Indonesian Institute of Sciences, Jalan Cisitu
Sangkuriang, Bandung, Indonesia
P. G. Adinurani
Qudratun
R. Firmansyah
R. I. Sholihah
R. N. Umami
Jl. Raya Jakarta-Bogor Km 46, Cibinong, Bogor 16911
R. Pertiwi
R. P. Mahesworo
R. P. B. Laksana
R. R. Bella
Bina Putera Nusantara Vocational High School
Jl Sukarindik 63A, Tasikmalaya, Indonesia
R. Susanti
R. T. Wahyuni
Electronic Departement, Polytechnic of Caltex, Riau
Jl. Umban Sari No. 1, Pekanbaru, Riau, Indonesia
Sasmoko
Surya University
Jl. Boulevard Gading Serpong Blok O/1 Summarecon Serpong, Tangerang,
Indonesia
and
STKIP Surya
Siswanto
Center for Aviation and Marine Meteorology - Agency for Meteorology Cli-
Jl. Angkasa I no 2 Kemayoran, Jakarta, Indonesia
Subyakto
Jl. Raya Bogor Km. 46, West Java, 16911, Indonesia
Sugiyatno
Sukardi
Supari
Suryadi
Department of Mathematics, Faculty of Mathematics and Natural Sciences,
University of Indonesia
Sutisna
National Nuclear Energy Agency
Jl. Puspitek Raya, Serpong Tangerang, Indonesia
Suyanto
S. Amien
Associate Professor, Faculty of Agriculture, Universitas Padjadjaran
Jl. Raya Jatinangor KM 21 45363, Sumedang
S. D. A. Kusumaningtyas
Agency for Meteorology, Climatology, and Geophysics (BMKG)
Jl. Angkasa I, No. 2, Kemayoran, Jakarta, Indonesia
S. F. Ser
S. H. Anita
Biology Study Program, The Graduate School, University of Indonesia
and
S. H. Fauziah
S. J. R. Lekatompessy
S. Khoirum
S. Mujiasih
Balai Besar MKG Wilayah III Denpasar
Jl. Raya Tuban, Kuta 80362, Kabupaten Badung, Bali, Indonesia
S. Pramono
S. Priyono
Banten
S. Sari
Graduated Student, Master Program in Plant Breeding
nesia
S. Sukaridhoto
S. Warnars
Surya University
Jl. Boulevard Gading Serpong Blok O/1 Summarecon Serpong, Tangerang,
Indonesia
T. Ernawati
Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan
Puspitek Serpong, Tangerang, Indonesia
T. Fajriutami
Jl Raya Bogor KM 46, Bogor, Indonesia
T. Sudiro
T. Watanabe
manosphere (RISH)
T. Widowati
U. Julia
W. Djasmasari
W. Fatriasari
Jl. Raya Bogor KM 46, Cibinong, Bogor 16911, Indonesia
and
Departement of Forest Product Technology, Faculty of Forestry, Bogor Agri-
cultural University (IPB)
W. Kusharyoto
W. Mangunwardoyo
Biology Department, Faculty of Mathematic and Natural Science, University
of Indonesia
W. R. Andayanie
W. Sulistya
W. Sumaryono
W. Syafii
Departement of Forest Product Technology, Faculty of Forestry, Bogor Agri-
culturalUniversity (IPB)
Yopi
Research Center for Biotechnology, LIPI, Cibinong, Bogor
Jl. Raya Bogor Km. 46, Cibinong, Bogor 16911, Indonesia
Y. D. Kurniawan
Y. Iriani
Y. Kurosaki
manosphere (RISH)
Y. Meliana
Y. Putrasari
of Sciences
Indonesia
Y. Srikandace
Research Center for Chemistry, Indonesian Institute of Sciences, Jalan Cisitu
Sangkuriang, Bandung, Indonesia
Y. V. Paramitadevi
Diploma Program, Bogor Agricultural University
Z. Alias

Naskah Cosat Revisi FINAL 22-12-2014

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Naskah Cosat Revisi FINAL 22-12-2014

Uploaded by

Copyright:

Available Formats

Organized by Sponsored by Supported by

Electronic Storage or Usage

Innovation for better ASEAN Community:

Cataloging in Publication Data

Copyeditor : Sri Suratmini Nugroho

First Edition : December 2014

Jakarta, 20 November 2014

Prof. Dr. Estiko Rijanto

Jakarta, 20 November 2014

Dr. Agus R. Hoetman

Jakarta, 20 November 2014

Prof. Dr. M. Nasir

630 Bld PUSPIPTEK Area, Serpong, Tangerang

* Corresponding author. Email: idhajr@yahoo.com

effect of mutagens on plants. In medicinal plants, the change of mutant characters

ii. MATERIAL AND METHODS

A. Irradiation treatments, plantation and subculture of mutants

B. Determination of molecular profiles of mutants

SBLT19 {(GATA)2(GACA)2}. ISSR amplification was carried out with 10

C. Determination of pytochemical profiles

dissolved in 1 mL methanol solution and prepared to inject into the HPLC

III. RESULTS AND DISCUSSIONS

Molecular analysis showed that genomic DNA of A. paniculata mutants

chromosomal rearrangements induced by gamma radiation. While the addi-

coefficient was R2 = 0.993, which indicated a good linear regression for

Lithospermum erythrorhizon mutant (Chung et al., 2006), shikonin production

In DG275 mutant, profiles of chromatogram were identified to also have 3

Of the five chromatograms that formed by gamma rays, the effects of

(Jha et al., 2011). Increased of shikonin compounds by gamma-rays on the

4) Subramanian, R., Asmawi, M. Z. and A. Sadikun. (2012). A bitter plant

Panji Cahya Mawardaa,*, Teni Ernawatib and Yoice Srikandacea

* Corresponding author. Phone: +6285711749663. Email: panji.cahya@gmail.com

ent kind of endophytics that are capable at producing bioactive compound or

II. MATERIAL AND METHOD

III. R esult and Discussion

Figure 1. (-)-epicatechin molecular structure

Table 1 1H and 13C NMR spectral data of fraction R1

Figure 2. Antioxidant activity of (-)-epictechin

20) Zuhud, E. A. M. (2009). Potensi Hutan Tropika Indonesia sebagai Penyangga

* Corresponding author. Phone: +62-21-8754587. Email: kusmiati02@yahoo.com

vitamin C, β-carotene, glutathione, ceruloplasmin, albumin, uric acid and

II. Method/M aterial

Figure 1. Hibiscus flower

Phytochemical screening of the powdered flowers of hibiscus was conducted

Table 1. Group of treatments

Figure 2. Outline of the method of study of hibiscus flower as antioxidant

III. R esult and Discussion

Note: + = available; - = not available

Figure 3. MDA content in red blood

C. SOD Enzyme Activity

Figure 4. SOD enzyme activity in

D. Catalase Enzyme Activity

Figure 5. Catalase enzyme activity

respectively, obtained by ethanol extract of hibiscus flower (40 ppm). Ethanol is

Key words: Actinomycetes, Peptide, Serine Proteases

* Corresponding author. Phone: +603 79675843. Email: hanom_ss@um.edu.my

II. M aterials and Methods

B. Protein Separation and Concentration

C. Enzymatic Activity Assay

III. R esults and Discussion

1) after protein separation by acetone precipitation method. As the samples was

Figure 1. SDS-PAGE of pure homogenous

Table 1. Enzymatic activity assays

Table 2. Antibacterial property of