AKSW2010 Proceedings

ISBN: 78-979-97789-9-4
Research Center for Biotechnology - LIPI, Indonesian Biotechnology Consortium (KBI),

Korean Society for Biotechnology and Bioengineering (KSBB), ASEAN COST-SCB Indonesia,
University of Indonesia
2010
ii
ASEAN-Korea Symposium and Workshop
on Biorefinery Technology
for sustainable production of biofuel and industrial biochemicals

(an International Forum)
PROCEEDINGS
“Converging biorefinery to response climate change”

Jakarta, 18 – 20 February 2010
Research Center for Biotechnology-LIPI, Indonesian Biotechnology Consortium (KBI),

Korean Society for Biotechnology and Bioengineering (KSBB), ASEAN COST-SCB Indonesia,
University of Indonesia
2010
iii
© 2010 Indonesian Institute of Sciences (LIPI)
Research Center for Biotechnology*
Cataloging-in-Publication Data
Proceedings ASEAN-Korea Symposium and Workshop on Biorefinery Technology,

for sustainable production of biofuel and industrial biochemicals:
Converging biorefinery to response climate change/Yopi et al. – Jakarta : LIPI Press, 2010.
vi + 29 pp.; 21 x 29,7 cm
ISBN 78-979-97789-9-4
1. Biotechnology
660.6
Layouter : Research Center fo Biotechnology, LIPI

Publisher : LIPI Press, member of Ikapi
 Research Center for Biotechnology

Indonesian Institute of Sciences
Jl. Raya Bogor km. 46, Cibinong, Bogor, 16911
Telp. +62-21-8754587, Fax. +62-21-8754588
iv
ASEAN-Korea Symposium and Workshop
on Biorefinery Technology
for sustainable production of biofuel and industrial biochemicals

(an International Forum)
“Converging biorefinery to response climate change”
EDITORS:
CHIEF:
Yopi
MEMBERS:
Anondho Wijanarko
Asrul Muhammad Fuad
Bambang Prasetya
Dwi Susilaningsih
Heri Hermansyah
Puspita Lisdiyanti
Wien Kusharyoto
TEHCNICAL EDITORS:
Eko Wahyu Putro
Hariyatun
v
vi
TABLE OF CONTENTS
Page
Foreword from Chairman of Organizing Committee......................................................................... xiii

Encouragement Address ..................................................................................................................... xv
Welcoming Address and Opening by the Minister of Research and Technology ............................ xvii
Invited Speakers : ............................................................................................................................... 1
1. Engineering Enzymes for Bioenergy and Biorefinery (Abstract)

(Prof. Young Je Yoo) ........................................................................................................................ 3
2. New Technologies for 2nd Generation Biofuels and Biorefineries: Disintegration of Plant Cell
Walls and Characterization of Surface Carbohydrates by Fluorescent-Labeled Carbohydrate-
Binding Modules (CBMs) (Abstract)
(Prof. Takashi Watanabe) ................................................................................................................ 5
3. Biorefineries-Integrated Bio- and Chemical Processes for the Conversion of Renewables

(Prof. Klaus-D. Vorlop) ................................................................................................................... 7
4. Microalgae - Key Resources for Biofuels – (Abstract)

(Prof. Kazuhisa Miyamoto) ............................................................................................................ 23
5. Separation of Woody Biomass Components and Utilization of Separated Lignin

(Prof. Yasumitsu Uraki) ................................................................................................................. 25
6. Nanobiocatalysis and its Potential Applications (Abstract)

(Prof. Jungbae Kim)....................................................................................................................... 33
7. The Korean Biofuel Policy (Abstract)

(Prof. Seung Koo Song) ................................................................................................................. 35
8. The Status and Prospects of Bioenergy in Asia (Abstract)

(Prof. Don Hee Park) ..................................................................................................................... 37
9. AFOB (Introduction of Asian Federation of Biotechnology) (Abstract)

(Prof. Jung Keug Park) .................................................................................................................. 39
10. ABD (Introduction of Asian Biotechnology Directory) (Abstract)

(Prof. Tai Hyun Park) .................................................................................................................... 41
11. Biohydrogen as an Alternative Energy for Palm Oil Mill (Abstract)

(Assoc. Prof. Dr. Poonsuk Prasertsan) .......................................................................................... 43
12. Conversion and Refining Process of Vegetable Oil to Fuel Based Downstream Products
(Prof. Mohammad Nasikin)............................................................................................................ 45
13. Current Status and Development Perspective of Cellulose-Ethanol in Indonesia (Abstract)

(Prof. Dr. Bambang Prasetya) ....................................................................................................... 51
14. TISTR Perspective on Biofuel from Microalgae (Abstract)

(Dr. Aparat Mahakant) .................................................................................................................. 53
vii
15. Enzymatically Process for Production of Biodiesel (Abstract)
(Dr. Siswa Setyahadi) .................................................................................................................... 55
16. Conversion and Refining Process of Vegetable Oil to Fuel Based Downstream Products
(Mr. Sok Chea) ............................................................................................................................... 57
Concurrent Session and Poster Presentations :.............................................................................. 59
A. Bioenergy .................................................................................................................................... 61
1. The Challenges of Biofuel Implementation in Indonesia: Environmental Prospect (O03)

(Muhammad Kismurtono & B. Paul Naiola) ................................................................................ 63
2. Sustainable Tropical Aquaculture: Microalgal Base Energies from Indonesia (O04)

(Dwi Susilaningsih, Gunawan, Theresia Umi Harwati, Khairul Anam,
Muhammad Sidiq Habibi, Hilda Farida, Ambar Susilorukmi, Toeti S., Saenab Husain,
Chakra Roy, Zaenal Mustofa, Delicia Yunita Rahman & Bambang Prasetya) ........................... 69
3. Evaluation of the Antimicrobial Efficacy and Treatment of Tinea Pedis for SiO2-Ionized
Loess (O05)
(Moon Young Yoon, Seung Ug Hong & Jung-Keug Park) ........................................................... 75
4. In Searching for the Possibilities to Produce Bioethanol (Gasohol) as New Renewable Energy
from Lontar Palm (Borassus sundaicus L.) in East Nusa Tenggara, Indonesia (O08)
(B. Paul Naiola, N. Nurhidayat, Teuku Beuna Bardant, Tri Murningsih, S. Tursiloadi,
Joko Sulistyo & Muhammad Kismurtono) .................................................................................... 81
5. Conversion of Oil Palm Empty Fruit Bunch to Sugars by Dilute-Acid Hydrolysis for
Ethanol Production (O09)
(Ria Millati, Rachma Wikandari, Elisabeth Titik Trihandayani, Muhammad Nur Cahyanto,
Mohammad J. Taherzadeh & Claes Niklasson) ........................................................................... 89
6. Two Stage Processes of Oil Palm Empty Fruit Bunch Fiber Kraft Pulp for Bioethanol
(O10)
(Yanni Sudiyani & Teuku Beuna Bardant) ................................................................................... 95
7. Bactericidal Activity and Mechanism of Supercritical N2O (O11)

(Sungmin Mun, Youn-Woo Lee & Jeyong Yoon) .......................................................................... 99
8. Phycocyanin Production of Spirulina fusiformis Culture in an Open Space Tubular Photo-

bioreactor (O12)
(Tjandra Chrismadha, Yayah Mardiati & Mey R. Widoretno) .................................................. 103
9. Using Compost as Biofilter to Reduce N2O Emission (O13)

(Tania Surya Utami, Lila Adriaty, Heri Hermansyah & Mohammad Nasikin) ......................... 109
10. Influence of 1,4-Dioxane in Enzymatic Process for Biodiesel Production Using Crude
Palm Oil (CPO) as Substrate (O14)
(Krishna Purnawan Candra, Sukartin, Fitriani & Yuliani) ....................................................... 117
11. Interesterification of Fried Palm Oil with Methyl Acetate using Porcine pancreatic
Lipase to Produce Biodiesel (O15)
(Rita Arbianti, Heri Hermansyah, Tania Surya Utami & Ryan Indra Mukti) ............................ 119
viii
12. The Influence of Carbon Sources on Laccase Production by White Rot Fungus Marasmius
sp. in Solid State Fermentation (O19)
(Hendro Risdianto, Elis Sofianti, Suraya, Sri Harjati Suhardi & Tjandra Setiadi) ................... 125
13. Provision of Superior Genotypes of Jatropha curcas for Biodiesel Production: Integrating
Morphology and Yield Variation with DNA-Based Marker (O25)
(Enny Sudarmonowati, Y. Cahyani, N. Sri Hartati & Wahyuni) ................................................ 131
B. Biorefinery ................................................................................................................................ 139
14. Study of Carbon Dioxide Emissions through Embodied Bioenergy of Renewable Biomass
(P12)
(Young Gyu Park, HyungSuk Kim & Jung-in Kim) .................................................................... 141
15. The Production of Anaerobic Hydrogen in Chalymodomonas reinhardtii was Induced by the
Decrase of Sulphur (P13)
(Maria Omega, Matthew Timmins, Ben Hankamer & Peer Schenk) ......................................... 147
16. Implementation Fuel of Methane from Biogas for Supply Energy in Small Industry
(UKM-Tahu) (P14)
(Muhammad Kismurtono, Khoirun Nisa, Satriyo K.W. & Roni Maryana) ................................ 151
17. Study on Renewable Bioenergy Source Based on Algal Biomass Extracted Oil (P16)
(Joko Sulistyo) ............................................................................................................................ 155
18. Utilization of Lignin-Rich Biomass Waste from the Production of Bagasse Bioethanol for
Additive in Pilot Scale Production of Fiber Based Composite (P17)
(Bambang Prasetya, Eko Wahyu Putro, Hariyatun & Asep Muhamad Ridwanulah) ................ 159
19. Potency of Indonesian Marine Microalgae (Chlorophyta) as Renewable Liquid Biofuel

(Biodiesel) Producers (P18)
(Khairul Anam, Hilda Farida, Muhammad Sidiq Habibi, Dian Noverita Widyaningrum,
Dicky Gustiawanto & Dwi Susilaningsih) .................................................................................. 165
20. Environmental Factors Optimization in Photofermentation to Produce Biohydrogen by

Sanur consortia (P19)
(Muhammad Sidiq Habibi, Khairul Anam & Dwi Susilaningsih) .............................................. 169
21. In situ Acid Transesterification: an Overview Simple Method for Fatty Acids Methyl
Esters (FAME) Preparation from Marine Microalgae as a Biodiesel Feedstocks (P20)
(Asep Bayu) ................................................................................................................................ 173
22. Feasibility of Sorghum bicolor as Bioethanol Producer by Mass and Energy Balance
Analysis (P21)
(Deliana Dahnum, Haznan Abimanyu, Tami Idiyanti & Sudiyarmanto) ................................... 179
23. Uniresponse Kinetics Based on Ping Pong Bi Bi Mechanism in Biodiesel Synthesis via
non Alcohol Route in Packed Bed Reactor (P22)
(Heri Hermansyah, Rita Arbianti, Arya Yudistira & Anatta Wahyu Budiman) ......................... 185
24. Updating Bioreactor Methods for Hydrogen Production Process Through Photo Fermen-
tation (P23)
(Khairul Anam, Muhammad Sidiq Habibi & Dwi Susilaningsih) .............................................. 191
ix
25. Isolation and Screening of Lignocellulolytic Actinomycetes from Rice Straw Waste (P24)
(Heri Satria) ............................................................................................................................... 195
26. Characteristics of Electricity Production by Metallic and Carbon Anodes Immersed in an

Estuarine Sediment (P26)
(Daechul Cho, In-Hyoung Rhee, Byung-Gi Park, Ki-Seob Ahn, Jong-Soo Kim,
Nam-Jun Cho & Hyung-Jin Song) ............................................................................................. 201
27. The Translation Initiation Factor eIF1 Gene is Sensitive to Alkaline Stress in Leymus
chinensis Trin. (P44)
(Sun Yan-Lin, Hong Shn-Hae & Hong Soon-Kwan) .................................................................. 207
28. The Relationship with Translation Initiation Factor eIF1 Gene Expression and Na+ Stress
(P45)
(Sun Yan-Lin, Shin Young-Boum & Hong Soon-Kwan) ............................................................. 213
29. Transformation of Leymus chinensis Trin. by Agrobacterium tumefaciens Infection of

in vitro Cultured Callus (P46)
(Sun Yan-Lin, Shin Young-Boum & Hong Soon-Kwan) ............................................................. 217
30. Efficient Production of Taraxacum coreanum by Means of Tissue Culture for Creating a
Green Tract of Land (P47)
(Jae-Hak Kim, Young-Boum Shin & Soon-Kwan Hong) ............................................................ 223
31. Compost Maintains the Soil Properties and Supports the Sustainable Agriculture (P51)
(Rakhman Sarwono) ................................................................................................................... 227
32. Nitrous Oxide Biosorption in Biofiltration Process Using Cow-Manure Compost Based
Medium (P54)
(Cynthia Noviani, Tania Surya Utami, Heri Hermansyah & Mohammad Nasikin)................... 233
33. Preliminary Screening for Herbicidal Activity of Endophytic Bacterial Extract from Canarium
Hirsutum willd.var.hirsutum Plant Using n-Butanol Solvent (P56)
(Warda Tuharea, Martha Sari, Neti Yuliaty, Alisin Febiyanti & Ines I.C. Atmosukarto) .......... 241
34. The Use of Zeolit as a Coagulant Adding Material for Treating Waste Water of Crude
Palm Oil (P58)
(Ade Sumiardi & Rusvirman Muchtar) ...................................................................................... 247
35. Suction Rate Adjusment in Filtration Process of Media Culture Circulation for Increasing
Biomass Production of Chlorella vulgaris Buitenzorg (P62)
(Anondho Wijanarko, Heru Darmawan, Dianursanti & Mohammad Nasikin) ......................... 255
36. Determination of Hydrodynamic Parameter in Bubble Collumn Photobioreactor for Scale

up Biomass Production of Chlorella vulgaris Buitenzorg (P63)
(Anondho Wijanarko, Putu Grahita Teja Kusuma, Dianursanti & Mohammad Nasikin) ......... 259
37. Microbial Conversion of Raw Glycerol to 1,3-Propanediol by Clostridium butyricum

NRRL B-1024 (P166)
(Wien Kusharyoto, Martha Sari, Nunik Sulistinah & Bambang Sunarko) ................................. 265
38. Isolation and Rapid Characterization of Biosurfactant-Producing Mutant of Soil Microbe Isolated
from Oil Mining, Cepu (P67)
(Swastika Praharyawan, Theresia Umi Harwati & Dwi Susilaningsih) .................................... 271
x
39. Studies on Marine Biosurfactant for Useful Biological Function (P68)
(Dian Noverita Widyaningrum, Donna Fujie Rahaditha Utami & Dwi Susilaningsih)............. 275
40. Potential Marine Microbes to Produce Polysaccharide Degrading Enzyme (P69)

(Apridah Cameliawati Djohan, Ahmad Thontowi & Yopi) ........................................................ 281
41. Study of Factors Affecting the Lipase Production by Yarrowia lipolytica NRRL YB-423 in Sub-
merged Fermentation (P74)
(Asep Muhamad Ridwanuloh, Eko Wahyu Putro, Martha Sari, Hariyatun &
Wien Kusharyoto) ....................................................................................................................... 289
42. Lipase Production by Yarrowia lipolytica in Solid-State Fermentation Utilizing Agro-

industrial Residues as Substrate (P75)
(Hariyatun, Martha Sari, Eko Wahyu Putro, Asep Muhamad Ridwanuloh
& Wien Kusharyoto) ................................................................................................................... 293
43. Palm Kernel Cake Biomass Fermentation Using Stirrer Fermentor for Mannanase and Saccha-
rides Production (P76)
(Yopi, Dwi Susilaningsih, Awan Purnawan, Heri Hermansyah, Ahmad Thontowi,
Apridah Cameliawati Djohan, Swastika Praharyawan & Puspita Lisdiyanti) .......................... 297
44. Mannolytic Activities of Aspergillus sp. BL5 Grown on Different Carbon Substrates
(Ahmad Thontowi, Nanik Rahmani and Yopi)........................................................................... 303
Appendices........................................................................................................................................ 307
Appendix 1. List of participants .................................................................................................. 309
Appendix 2. The Committee ........................................................................................................ 327
Appendix 3. List of Presentations ................................................................................................ 329
Appendix 4. Index of Authors ..................................................................................................... 335
xi
xii
Foreword from the Chairman of Organizing Committee
On behalf of organizing committee we are pleased to hold the ASEAN-Korea Symposium and
Workshop on Biorefinery Technology for Sustainable Production of Biofuel and Industrial Biochemi-
cals. As we all are aware that global climate change and energy crisis have paid an international con-
cern over those issues. The increasing population pressure and demand for a better life style have led
to a greater use of energy and goods. As a consequence, the rate of consumption is day by day becom-
ing much higher than it was ever before resulting in escalating pressures on the use of available re-
sources. To secure energy demand and friendly products, the biorefinery concept is recently to be an
attractive approach to produce sustainable biofuel and industrial biochemicals.
The objective of this symposium and workshop are 1) to update the state of the art of the biore-
finery technology for production of biofuel and industrial biochemicals; 2) to share the knowledge
and experiences from outstanding experts and practical industries from ASEAN countries and Korea;
3) to streghten the capability of the ASEAN countries for the implementation of the white bio-
thecnology for green products and bioenergy; 4) to accelerate the contribution to reduce problems af-
fecting global warming; and 5) to establish South East Asia Network on Biorefinery Technology.
Organizing Committee is very proud to have around 106 papers and 16 invited papers from 38
University and Research Center from Germany, Japan, Korea, Thailand, and Indonesia. The delega-
tion of the ASEAN member state from Cambodia, PDR Lao, Malaysia, Singapore, Thailand, Vietnam
will also give the paper on the current status on biorefinery technology in ASEAN. This event is sup-
ported by ASEAN Sub-Committee on Biotechnology, The Society for Biotechnology and Bioengi-
neering (KSBB), Indonesian Biotechnology Consortium (KBI), Research Center for Biotechnology-
LIPI and Department of Chemical Engineering-University of Indonesia.
On this occasion, we would like to convey our gratitude to Minister of Research and Technology,
Republic of Indonesia and his staff for supporting this event. Also, I have the pleasure of greeting and
thanking all participants for their scientific support through their presentation of papers and posters
and discussions during symposium and workshop.
Our special thanks will be also delivered to the KSSB, University of Indonesia, Indonesian Insfi-
tute of Sciences, ASEAN Sub Committee on Biotechnology from ASEAN member state, ASEAN
Secretariat for a very wonderful cooperation.
In addition, we are delighted to thank to the sponsor PT Sinarmas Forestry, PT Barat Jaya Sen-
tosa Perkasa, PT Pandu Anugerah Analitika, PT Techcomp Indotech (Indonesia), PT Pertamina, PT
Sentra Biosains Dinamika, PT New Module Int., PT Haes Brothers, PT Trikarsa Indoinstrument. PT
Ditek Jaya, PT Abadi Nusa Semesta Usaha, PD Aneka Sarana Lab and LaRIPTEK for the financial
support. Your collaboration will be very valuable as a significant contribution to solve the global
problems.
I would like to present my personal thanks to all institutions supporting us and everybody who
have contributed and special thanks go to all Organizing Committee of the AKSW.
Finally, I would like to wish you all the best during the workshop and symposium, and may
Good blesses us all.
Jakarta, February 18th, 2010
Prof. Dr. Bambang Prasetva

Focal point SC Biotechnology ASEAN/
Chairman of Indonesian Biotechnology Consortium (KBI)
xiii
xiv
Encouragement Address
Despite the aid of accumulated techniques from biotechnology which permit the standardized quality
of human life welfare to be accelerated, we encountered a variety of many problems causing difficul-
ties in securing adequate supply of natural resources on world-wide scale and in coping with envi-
ronmental agenda such as unpredictable change in weather and climate. This situation raises demands
to supply for whole-human welfare and conservation of nature on our planet by means of elaborately
developed brand-new technologies that are capable of supporting multi-purpose solution frame. In
particular, emphasis has been placed on the environment-friendly energy that is expected to be able to
reduce the use of fossil fuels and prevent soaring of prices of natural resources such as oil and coal for
assuring envisioned future of our planet residents.
"Asean-Korea symposium and workshop on biorefinery technology for sustainable production of bio-
fuel and industrial biochemicals (AKSW 2010)" can provide the opportunities and challenges to re-
solve these issues by direct networks and communications within the Asian region. This on-going co-
operation and communications for research, education and com-mercialization in biotechnology issu-
ing biorefinery technologies can be extended to realize new paradigm of environment-energy fields.
Representing the members of the Korean Society for Biotechnology and Bioengineering, I congratu-
late this international forum, AKSW 2010, and wish that constructive and future-oriented solutions
may be presented through this forum.
Ph.D. Lim, Giobin

President-The Korean Society for Biotechnology and Bioengineering
General Director-Korea Biotech R&D Group
Professor-Department of Chemical & Biochemical Engineering,The University of Suwon, Korea
xv
xvi
Welcoming Address and Opening
by the Minister of Research and Technology
Representatives from the ASEAN Secretariate,

All invitees and participants,
Ladies and gentlemen,
First of all, let us be grateful for this precious opportunity and thank God almighty for all the blessing
we have been granted upon, for we are able to attend the ASEAN-Korea Symposium and Workshop
on Biorefinery Technology for Sustainable Production of Biofuel and Industrial Biochemicals.
I am very excited about this workshop and symposium as the mission being put forward is quite
relevant to the current needs of many countries in the world in dealing with important issues at both
regional and global levels.

It is common knowledge that global warming and climate change are results of continuous use of
fossil fuels and burning of forest and wastes that release greenhouse gases to the atmosphere. Global
warming has lead to disastrous events in many parts of the world, such as climate change, floods and
droughts. In the future, this may lead to problems in food sustainability and environmental and energy
crisis, of which in turn may result in socio-economic crisis. In light of this problem, countries around
the world are in pursuit of appropriate and feasible solutions. A significant and strategic effort
towards solving this problem is to look for alternative green energy and to replace oil-based resources
with biomass-based resources.

As a developing country, our need for energy will increase in line with progression of industrial
activities, trades and services, as well as population growth. It is encouraging to know that Indonesia
has been gifted with:
Large areas of land consisting of thousands of islands, although only a number of islands are sources
of fossil fuels,
A large number of human resources (4th largest in the world),
Massive biodiversity, both in land (2nd largest) and water (1st largest)
Currently we are in possession of abundant potential energy sources such as geothermal energy,
biomass energy, microalgae, solar energy, wind energy, hydrogen, and hydro energy.
In Indonesia, these resources have major potentials to be developed, due to advantages in terms of
geological, geographical and climate conditions.
Indonesia, along with other ASEAN member countries, is gifted with wet tropical climate that enable
larger biomass production in comparison with other regions.
It has become a necessity for us to explore, characterize, and identify potential energy sources from
microalgae, in order to develop sustainable concept in its utilization. Next in line is to cultivate the
appropriate technology according to market needs and existing environmental conditions.
It is why Clean Production technology and Zero Waste concept should become the platform in
technology advancement. In this opportunity I would like to stress the importance of publicizing and
developing this concept, giving rise to more substantial contribution of science and technology to the
production sector.
Ladies and Gentlemen,

It is common knowledge that advancement in science and technology, especially in biotechnology, in
the past two decades has given considerable contribution to the development in the fields of farming,
health, manufacturing and industries, and the environment. It has been projected that the contribution
of biotechnology in the next ten years will escalate in line with new issues arising from environmental
degradation. Innovation in the field of renewable energy is becoming an important agenda in dealing
xvii
with ever more limited sources of energy and the threat of global warming as a result of waste
emission from the use of fossil fuels.
Fast advancement in the field of biotechnology in terms of development of sustainable renewable
energy is supported by the progress of information technology. It is why we need to continually be
aware of development in global trends so anticipative action can be taken and opportunities would not
be missed. Communication between international and national experts needs to be facilitated in order
for knowledge transfer to occur resulting in advancement in the know-how of technology. Therefore,
the current process of technology adoption can be applied in production and transportation sectors,
along with other economic activities. Discussion forums on technological advancement, research
results and products are essential in hastening the progression of actual problem solving efforts. I am
also thrilled that in this opportunity, the development of Asia Network for Biorefinery Technology
will be discussed. Stronger interaction between the technology developers and the industrial sectors
will impact on faster technology implementation.
Strong network on science and technology advancement is an implementation of Act no.18/2002 on
National System on Research, Development and Application of Science and Technology. Via this
network, I hope the national innovation process is more rapid. In terms of innovation process
enhancement, Ministry of Science and Technology has been assigned by the President to supervise the
development of national innovation process. I have been informed that this workshop and symposium
has been put together with support and collaboration of many sponsors, professional organization and
researchers; an effort worth continuing in the future. I believe cultural and functional relations also
play a part in hastening realization of our vision and mission in science and technology development
for the well-being of society. Due to global competition, it is now more important than ever to work
together.

Before I finish my address, I would like to extend my gratitude towards all parties that have supported
this event: our partners from ASEAN, KOREA, our honorable guest speakers and our sponsors. I
sincerely hope there will be more similar events in the future that will give rise to new innovations
that will benefit the world and societies in it.
Finally, I hope the participants can obtain maximum benefit form the discussions that will take place
as we all aim for.
Thank you.
JAKARTA, 18 February 2010

Minister of Research and Technology, Republic of Indonesia
Suharna Surapranata
xviii
Concurrent Session and Poster Presentations
The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 59

60 Jakarta, 18-20 February 2010
A. Bioenergy

The Challenges of Biofuel Implementation in Indonesia:
Environmental Prospect
Muhammad Kismurtono1 and B. Paul Naiola2
1
Technical Implementation Unit for Development of Chemical Engineering Processes,
the Indonesian Institute of Sciences
Yogyakarta, Indonesia 55861, e-mail: m_kismurtono@yahoo.co.id
2
Research Center for Biology, the Indonesian Institute of Sciences
Cibinong Science Center (CSC)-LIPI,
Jl. Raya Jakarta-Bogor Km. 46, Cibinong 16911, West Java, Indonesia
(Correspondence to: Muhammad Kismurtono)
ABSTRACT
Biofuel production in Indonesia in 2025 could reach 15.9 billion liters and 16.5 billion liters per year of
ethanol and biodiesel, respectively. If the technology is still depended on the first generation biofuels, the land
dedicated to biofuels would be in the range of 6.6 to 11.6 million hectares. Therefore, it is no wonder that there
are widespread concerns that biofuels could end up causing more problems than they solve. Several LCA (Life
Cycle Assessment) studies reported that the effects of first generation biofuels as fuel can reduce green house
gases (GHG) and produce a higher total energy amount than that of fossil fuels. However, recent and more
comprehensive studies indicated that if the land use conversion were accounted for, biofuel resulted a much
higher of GHG emissions, especially if it was included the rainforest destruction, or conversion of peat lands.
For the above reasons, in the near term, the policy priority should be to find ways to promote sustainable pro-
duction methods for biofuel feedstock, especially how to avoid direct and indirect destruction of the Indonesian
primary forest. Moreover, policy finance should focus on research and development to promote sustainable pro-
duction methods, especially on second generation biofuels, and not on increased production of first generation
biofuels.
Keywords: biofuel, environment, green house gases emission, land use conversion, production of second ge-
neration biofuel.
INTRODUCTION permanent supply of national energy. The in-

crease of world society consciousness in using
One of the nowadays problems in indonesia, environmental friendly fuel, brings bioenergy as
a will remain as serious problem is energy, more strategy. For Indonesia, the development of
whether for domestic or industry and transporta- bioenergy may increase the ability to create and
tion. The most dominant and nationally used is imporeve bioenergy based on local resources
fuel originated from fossil fuel. In correspond to Indonesia nowadays, is one of the countries
this problem, one of the government policy is to in the world that start to develop bioenergy in-
withdraw or reducing the use of kerosene in do- dustry by the producton of biodiesel and bioeth-
mestic utilization, substituted with gas (LPG – anol. The role of bioenergy industry become
Liquor Petrol Gas). In the same direction, the more important, since during the year 2008, the
government also promoting to the diversify en- price of fossil fuel increased to more than US$
ergy need by rxploring the other alternative en- 100 per barrel. The increase in fuel price has
ergy. Alternative energy should provide condi- pushed the inflation up to two digits. On the oth-
tions such as technical and economic capabilities er hand however, some experts believed that bi-
and coping with environmental issue; for exam- oenergy may create negative effect on food
ple, the use of bioenergy (BBN oil from plants). availability and environmental conservation, in
Biofuel (BBN - Bahan Bakar Nabati), is be- terms of sustainability.
lieved as one of prospective alternative energy to Hence, what to be needed is to analyse both
be developed in the future. The development of positive and negative aspects when using biofu-
bioenergy is not only due to the reduction of na- el, and to determine which biofuel is suitable to
tional dependency on fossil fuel with a risk of be developed in Indonesia. This paper presents
regular growing up price, but also keeping the the environmental review against biofuel indus-
try, that maybe as reference when developing

biofuel in Indonesia. Not less important is the ogy that correspond to advanced processing of
arisen of environmental conflicts between estab- natural materials.
lishing biofuel and food security. Based on the discussion above, thus it is
quite clear that Indonesia nowadays, just moving
BIOFUEL into the first generation biofuel, as shown by its
initial growth. According to Indonesia Associa-
Biofuel is a common term for fuel that pro- tion of Biofuel Production (APROBI), total pro-
duced from biomass, such as plants and organic duction of first generation biofuel in indonesia
wastes. Nowadays, development of biofuel is during the year 2008 ranging around 725,000
divided into 2 steps, namely: tons, i.e. equal to 824 million litres (Tjakrawan,
First generation biofuel: 2007).
 First generation biofuel is generated from raw
materials of food plants, vegetable oil and an- PREDICTION OF BIOFUEL PRODUC-
imal fat, by using conventional technology. TION IN 2025
Common biofuel “bioethanol” used in com-
mercial scales, are generated by mixing petrol Presidential Regulation Number 5 – Year
gas with etahanol; ethanol is generated by 2006 concerning National Energy Policy, con-
fermentation of sugar or starch content mate- firming the primary energy mix of Indonesia in
rials. Raw materials used are molases, cassa- 2025, is displayed in Fig 1. Contribution from
va, sugar cane, corn, wheat and sugar bit. fossil fuel should be less than 20%, while contri-
 Biodiesel is generated by mixing diesel and bution from other resources should fulfil the rest
product generated from vegetable oil or ani- as plotted in cake diagram.
mal fat. Raw materilas used are originated Based on Fig 1, it is assumed that supply
from oilpalm, jatropha, coconut and soybean. demand of each energy primer group in 2005
and 2025 is as displayed in Table 1. As shown,
Second generation biofuel: in 2025, the demand of biofuel should be about
Second generation biofuel is generated from 166.9 millions SBM (EOB – equal oil barrel).
raw material outside food plants. They are such
as agricultural wastes, wood wastes (known as
cellulotic biofuel), microalgae, or other technol-
Fig 1. Predicted national primary energy mix of Indonesia in 2005 and 2025
Note: BaU - bussiness as usual; SBM - equal oil barrel

Table 1. Supply demand of national primary energy in year bution itself. Harrison & Emma (2005) pointed out
2005 and year 2025 (in million EOB) results of many research in this aspect shows that
Primary 2005 2025 Increase biofuel production will end with the declining of
energy world food security, while (Engelhaupt, 2007) re-
Fossil fuel 524.0 638.9 114..9 vealed the declining of water availability, the rise of
Natural gas 212.8 832.0 619.2
Coal Bed Methane, EOB 0.0 127.8 127.8 greenhouse gases (Searchinger et al., 2008), and
unconstructive ending effect on biological diversity
Coal 160.4 1099.4 939.0 (Pearce, 2005), something that should be watched in
Geothermal 23.7 167.5 143.8 Indonesia.
Biofuel 0.0 166.9 166.9
Liquid coal 0.0 80.5 80.5 ENVIRONMENTAL EFFECT

Hydro power 34.0 65.8 31.8 The development of biofuel industry in Indone-
Nuclear power 0.0 55.8 55.8 sia is expected to contribute to the whole life of the
Solar power, hydro pow- 1.6 17.4 15.8
target groups in society, national income per capita,
er, biomasses, etc
self determination and environmetal issues. Conse-
quence that should be avoided is environmental de-
Based on the assumption that first generation gradation due to biofuel promotion.
biofuel is still be used, while its composition is 40% At the present time, climate change issues are
ethanol, and 60% biodiesel, thus the demand for usually judged as global warming as a consequence
pure ethanol (100%) as much as 66.9 million SBM, of uncontrolled and big consumption of fossil fuel.
while for pure biodiesel is 100 million SBM. Due to Number of researches when applying LCA (Life
the energy content of ethanol and biodiesel are low- Cycle Assessment) technique resulted that theoreti-
er that fossil fuel, i.e. 67 and 86%; hence, in 2025, cally, the use of first generation biofuel, potentially
the annual demand of ethanol is predicted as 99.85 are able to reduce green house gases, and possessing
million barrels (equal to 15.87 billion litres) and the higher netto energy than fossil fuel. Table 3 shows
predicted biodiesel is 116.27 million barrels (18.48 the decline of CO2 when using biofuels (IGES,
billion litres) per year. 2008). Table 4 explaining the comparison of NET
Various literatures shows data on the yield of (Net Energy Value) of biofuels, where the biggest
ethanol production originated from sugarcane is total energy value expressed by sugarcane. Brazil is
6.000 l/ha/y, cassava contributed 2.070 l/ha/y, while the world leading country that applying biofuel gen-
biodiesel production originated from oilpalm may erated from sugarcane.
up to 4.600 l/ha/y. Thus Table 2 presented the pre-
Table 3. Comparison of various raw materials of biofuel corre-
dicted land demand for the production of biofuel in spond to CO2 sequestration
2025 should be not less than 13 million hectares.
Production CO2
Biofuel
Table 2. Predicted land demand for biofuel production in 2025 country (% sequestration)
2 (for E10) to 23 (for E85)
Biofuel de- Corn USA
Tipe of mand in Year Land demand in Year Thailand 63
Cassava
biofuel 2025 (million 2025 (million hectare)
litres SBM) Sugarcane Brazil 80
2.64 when raw material is
originated from sugarcane Oilpalm Malaysia 60
Biethanol 66.9 15.874 Jatropha India 80
7.67 when raw material is
originated from cassava Coconut Philippines 60
4.01 when raw material is Source: IGES (2008) - using various literatures
Biodiesel 100 18.48 originated from oilpalm
Table 4. Comparison of various biofuels due to their net energy
value
Table 2 shows that when the demand of biofuel
Raw Material Production NEN (MJ/L)
is entirelly expected from first generation biofuel, country
thus in 2025, the demand of land to be dedicated for Corn Amerika 5.89
sugarcane, cassava and oilpalm is at least between Cassava Cina 15.14
6.6 to 11.6 million hectares. Cassava Thailand 22.38
Thus, no wonder if there is much growing up of Sugarcane Brazil 41.34
Oilpalm Malaysia 37.45
awareness from many environmental guardians/ in- Jatropha Thailand 3.82
stitutions, such as NGOs claiming about the conse- Jatropha India 5.26
quences of biofuel development which end in great- Coconut Filipina 31.72
er social and environmental disaster than its contri- Note: NEN = Net Energy Value = NEV. Source: IGES (2008) –
using various literatures

Thus concerning the abovementioned positive Table 6. Greenhouse gases from biodiesel production compared
aspects of biofuel utilisation by Indonesia in the fu- to diesel electric engine
ture, it is suggested to apply by deep evaluation, and No. Biodiesel production Emission
careful steps should be taken into account. One of reduction (%)
the important factor that often fail to be noticed in 1. Peatland conversion -337
2. Primary forest conversion -20
some LCA study is the effect of increased biofuel 3 Post-logged forest land 65
production against land use convertion, especially 4. Utilisation of critical land 157
the conversion of tropical rain forest and peat land. 5. Utilisation of critical land followed 159
Recent LCA studies, were often underestimated the by improvement of environmental
management
negative effect of biofuel application against green-
house gases emission. Meanwhile, for some reasons
the LCA study did not take into account other envi- CONCLUSIONS
ronmental issues such as biodiversity lost.
Concerning land conversion, Börjesson (2008) Theoritically, there is a great chance to develop
emphasized a significant effect of land conversion biofuel in Indonesia, as far as great awareness
when planting biofuel plants against greenhouse should be taken into account. Biofuel may contrib-
gases calculation. Table 5 shows the comparison of utes to the reduction of greenhouse gases, improves
greenhouse gases emission in a range of ethanol local energy security and reduces poverty; however
production, energy consumption and land conver- there are some dark sides in biofuel issue, noted as
sion scenario, compared to gasoline. possible forest and ecosystem destruction as a result
of land function conversion.
Table 5. Greenhouse gases emission from wheat based ethanol, Due to limited availability of biofuel, the in-
compared to gasoline crease of world oil price and the demand to reduce
No. Various scenario of etha- Greenhouse gases the effect of greenhouse gases have weaken the
nol production emission (%) main purpose of biofuel. However, there should
1. Gasoline (referency) 100 have some consciousness as well that biofuel may
(referency) only be a part of solution in fuel demand problem.
2. Wheat – biomass energy– +350
The solution of this problem may be considered by
raw production – peatland
conversion invention and introducing some other energy
3 Wheat – coal energy – raw +40 sources such as electricity power for transportation,
production – coal produc- production of liquid fuel and gas other than coal,
tion hydrogen from renewable resources, solar power
4. Wheat – biomass energy – -25
raw production – grassland
and to draw the people awareness in energy conser-
production vation by practising a save energy lifestyle.
5. Wheat – biomass energy – -90 The limited availability of first generation bi-
production recovery – criti- ethanol and biodiesel tended to promote the devel-
cal land
opment of second and third generation biofuel.
However, within 10 to 15 years, a great number of
Table 5 shows that the conversion of peatland
research needed to achieve maximum research in
for wheat plantation for biofuel will release the
secnd and third generation biofuel.
greenhouse gases up to four times than using gaso-
High priority of energy policy in short term
line (fossil fuel). Hence, direct land alteration may
target should retain the supply of biofuel as alterna-
become the most critical factor in LCA calculation
tive energy sources without loose touch in environ-
to predict the effect of biofuel. Indirect effect of
mental balance such as Indonesian primary forest
land conversion, however should be estimated as
conservation.
well, even though seems difficult in LCA calcula-
Meanwhile, in financial policy regarding the
tion.
development of biofuel, direction should be for-
Wicke et al. (2008), made a study case in Ma-
warded to reseach and development in promoting
laysia for oil palm. For reference, as shown in Table
production methods, especially focused on the se-
6, they replaced diesel fuel with biofuel, produced
cond and third generation, not to broaden the pro-
by various land conversion scenario.
duction of first generation of biofuel.
Biofuel from plants was previously considered
as the better one. It is due to carbon emission when
burning the forest for biofuel plantation, was ba- REFERENCES
lanced by carbon absorption during plant growth
Arifin, Y. 2008. Analisis singkat untuk pengembangan industri
period. However, carbon emission is still taking biofuel dan dampaknya bagi pertanian dan lingkungan
place during distillation and transportation of biofu- hidup di Indonesia. http://yalun.wordpress.com.
el.

Börjesson, P. 2009. Good or bad bioethanol from a greenhouse Pearce, F. 2005. Forest paying the price of biofuels. New Scien-
gas perspective-What determines this?. J. Applied Energy. tist.
86: 589-594.
Searchinger et al. 2008. Use of U.S. croplands for biofuels in-
Engelhaupt, E. 2007. Biofuelling water problems. Environ. Sci. creases greenhouse gases through emissions from land use
Technol. change. Science. 319 (5867): 1238-1240.
Escobar, J.C. et al. 2008. Biofuel: Environment, technology and Sugiyono, A. 2008. Pengembangan Bahan Bakar Nabati untuk
food security. J. Renewable and Sustainable Energy Re- Mengurangi Dampak Pemanasan Global.
views. Doi:10.10106/j.rser.2008.08.014 http://www.geocities.com.
Harrison, G. and Emma. 2005. Food security worries could Tjakrawan, P. 2007. Indonesia biofuels industry. Sustainable
limit China biofuels. Reuters. Aspect of Biofuel Production Workshop.
IGES. 2008. Prospect and Challenges of Biofuels in Asia: Poli- Wicke, B. et al. 2008. Different palm oil production systems for
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egies, Hayama, Japan. www.iges.or.jp. Biomass and Bioenergy. 32: 1322-1337.

Sustainable Tropical Aquaculture:
Microalgal Base Energies from Indonesia
Dwi Susilaningsih1, Gunawan4 , T. Umi Harwati1, Khairul Anam1, M. Sidiq Habibi,1,
Hilda Farida.1, Ambar Susilorukmi 2, Toeti S. 2, Saenab Husain3, Chakra Roy 3, Zaenal
Mustofa1, Delicia Yunita Rahman and Bambang Prasetya1
1
Research Center for Biotechnology LIPI, Jl Raya Bogor Km. 46 Cibinong Bogor, Indonesia 16911
email: dwisusilaningsih@yahoo.com.sg or dwis002@lipi.go.id
2
Research Center for Physic LIPI, Jl Cisitu Lama, Bandung, Indonesia
3
Enlightening Indonesia, Jl Topaz 1 ruko zambrud blok E 20 Makassar, Sulawesi, Indonesia
4
University of Lambung Mangkurat, Kalimantan.
(Correspondence to: Dwi Susilaningsih)
ABSTRACT
Microalgae are photosynthetic microbes which mostly live in aqueous area and have ability to convert the
sun-light, CO2 and water into valuable compounds such as lipid, hydrocarbon, carbohydrate, protein and others.
The second generation of environmental friendly-biofuel is including the microalgae utilization, due to the mi-
croalgae ability for synthesized energy stock compounds and performed reduction of green-house gaseous (CO2,
O3, SOx and NOx). In this regard we have research on utilization of selected and screened microalgae origin
from tropical area (Indonesia) for starch and diesel oil, hydrogen and hydrocarbon sources. Four (5) strains of
marine microalgae of Scenedesmus, Tetraselmis, Chlorella, Nannochloropsis and BTM 1 have synthesized lipid
in their cells around 40-70% based on cells dry weight, in laboratory scale. Two (2) strains of acidic-hot-spring
cyanobacteria (Ctr-1 and Ctr-4) have positively excreted the hydrogen gas during cultivation with rate around
10-12 ml/100 ml formed gas units. 13 strains of marine microalgae (Chlorophytes and cyanobacteria) were syn-
thesized or deposit the hydrocarbon in their cells around 20-40% based on cells dry weight.
Keywords: biodiesel, bio-hydrogen, energies base microalgae, hydrocarbon, reduction green house gaseous,
tropical microalgae.
INTRODUCTION Like plants, microalgae use sunlight to pro-

duce energy (oil) but they do so more efficiently
Utilizing the petroleum sourced fossil fuels than crop plants. Microalgae could convert the
is now widely recognized as unsustainable be- sun-light into organic carbon storages of 5-7%,
cause of depleting supplies and the contribution which is much larger than higher plant processes
of these fuels to the accumulation of carbon di- of 2-3%. Photosynthesis uses light energy and
oxide in the environment. The future of econom- carbon dioxide to make sugars like glucose. A
ic bio-energy (biofuel) will feature as an explo- general equation for photosynthesis is:
ration of the reliable and sustainable biore- 6 CO2(gas) + 12 H2O(liquid) + photons →
sources of plants and microbes. Biofuel derived C6H12O6(aqueous) + 6 O2(gas) + 6 H2O(liquid)
from crops is a potential renewable and carbon Carbon dioxide + water + light energy →
neutral alternative to petroleum fuels. Unfortu- glucose + oxygen + water
nately, biofuel from crops, waste cooking oil and
animal fat cannot realistically satisfy even a Photosynthesis occurs in two stages. In the
small fraction of the existing demand for first phase light-dependent reactions or photo-
transport fuels. Among of the microbes groups synthetic reactions (also called the Light reac-
called microalgae is appear to be the only source tions) capture the energy of light and use it to
of renewable biofuel that is capable of meeting make high-energy molecules. During the second
the global demand for transport fuels. The mi- phase, the light-independent reactions (also
croalgal-fuel is supposed to be economic sus- called the Calvin-Benson Cycle, and formerly
tainability and environmental friendly since they known as the Dark Reactions) use the high-
are renewable, carbon neutral and easy for trans- energy molecules to capture carbon dioxide
portation mater. (CO2) and make the precursors of glucose or
other compounds.

Oil productivity of many microalgae greatly 0.014 mg CoSO4-7H2O, 0.0073 mg Na2MoO4-
exceeds the oil productivity of the best produc- H2O, 0.0025 mg CuSO4-5H2O, 0.0017 mg
ing oil crops. Yield of microalgal oil is three H2SeO3, 0.2 mg Thiamin-HCl, 0.0015 mg Biotin,
times than coconut oil and ten times from soy- 0.0015 mg Vitamin B12 and 0.18 mg MnCl2-
bean oil. The yield microalgal-oil reaches 700 4H2O.
gallon/acre compare to coconut oil of 285 gal-
lon/acre and soybean oil of 62 gallon/acre (Mi- Modification of substrate medium and cultiva-
nowa et al., 1995 and Sazdanoff, 2005). Several tion: The standard of F-2 medium, the modifica-
strain microalgae are suspected the hydrocarbon tion glucose and corn starch of 2% w/v are em-
depositor such as Botryococcus, Chlorella, Du- ployed. The cultures will grow in the cylindrical
naleilla, Nanochloropsis and diatom (Chisty, plastic reactor with the aeration and supply CO2.
2007). In addition there are many reports on Intensity of light of 30 W/m2 is continuously
hydrogen producing microalgae (Ikke et al., employed. The cells will harvested by centrifuge
1997, Miyake et al., 1997, Benneman, 1998 and or settling, therefore the cell paste will used for
Ikke et al., 1998). There are also many reports further process of trans-esterification.
on microalgae depositing starch/polysaccharides
and hydrocarbon compounds in their cells (Burja Diesel-oil producing microalgae: The technique
et al., 2002; Huntley and Redalje, 2007). How- of metabolic controlling through heterotrophic
ever, there is little report on tropical microalgae growth of microalgae were applied. To increase
for biofuel production, especially in Indonesia, the biomass and reduce the cost of alga, corn
although we are maritime country with 70% of powder hydrolysate instead of glucose was used
area is water, we have a little progress on aqua- as organic carbon source in heterotrophic culture
culture of microalgae for energy purposes. medium in fermenters. The biomass was har-
Therefore, we started for exploring the microal- vested and extracted with n-hexan and then
gal biodiversity from Indonesian water environ- transmuted into biodiesel by acidic transesterifi-
ments including marine and freshwater streams cation. The biodiesel was characterized by a high
in specific area such as polluted sea-shores, coral heating value, density, and viscosity. All the
reef bays, hot spring and others extreme habitats. methods are following (Liu et al., 2007).
Thus, we were screen the culture collection for
specific targeted goal, for instants for starch-, Hydrogen gas producing microalgae: Selected
lipid- and hydrocarbon-accumulator. Finally, we microalgae were incubated into high light inten-
would like to build the data base for Indonesian sity (around 50 W/m2) and CO2 gas (1-2 ml/min
microalgal cultures. in air). The reactor was use the cylindrical glass
connected with the glass trapping chamber for
MATERIALS AND METHODS monitoring the gas formation. Gas formed ob-
servation was detected by Gas Chromatography.
Isolation and Screening of microalgae: Water The strains which positively secreted the hydro-
samples were collected from Batam bays, Jakar- gen gas are selected for further research and
ta bays, Bali bays, Lombok bays, Ciater hot evaluations. Visually the gas formation was
spring, Sentul hot spring and ponds in Cibinong monitored by the decreasing the water volume in
areas. Density microalgae were collected by the gas trapping chamber.
planktonet (25 µm ≤ size ) and subjected into
selected media (IMK-freshwater or seawater Starch, Lipid and Hydrocarbon producing mi-
containing of rich-starch, rich-hydrocarbon croalgae: Candidates’ microalgae which survive
and/or rich-fat). Samples were incubated in the in the selected media were isolated by capillary
room temperature (28-33oC) with light intensity pippeting technique and therefore cultivated in
around 30 W/m2. After 2-3 weeks the survive the nitrogen-limited-IMK media (sea or fresh-
microalgae were isolated by capillary pippeting water base medium). Rapid growth and high li-
technique and subjected into rich-IMK-media pid or carbohydrates or hydrocarbon content
according to the initial screening media. Selected were use for further evaluation. Generally all
microalgae were use for further evaluation. 1 candidates culture were subjected into nile red
Liter of IMK medium containing 200 mg NaNO3, dye fixation, the cells which could absorb much
1.4 mg Na2HPO4, 5 mg K2HPO4, 2.68 mg the dye were selected for lipid production. Visu-
NH4CL, 5.2 mg Fe-EDTA, 0.332 mg Mn-EDTA, ally in the light microscope cells with much con-
37.2 mg Na2-EDTA, 0.023 mg ZnSO4-7H2O, tent oil out site the cells (vacuole like/bag which

shiny particles) were selected fro hydrocarbon Oleaginous microalgae: Diesel-oil producing
producing microalgae. And the cells with big microalgae strain as expecting target in these
vacuoles and rigid were choose for starch- studies was carried out by conventional pippet-
accumulator strains. ing isolation and enrichment media using several
considerations of trigger compounds which is
Assay for protein, carbohydrates, lipid and hy- taking influence of oil synthesize in the microal-
drocarbon: Qualitative test for proteins was gal cells such as starch, oily compounds and ni-
conducted using the Bradford method (1976). trogen starving medium. The results showed 14
Tests for lipid, carbohydrate, and sugar were strains out of 64 sucking microalgae are posi-
carried out using the Bligh-Dryer method (1959), tively have tendency to produce the starch or
the Carbohydrate-Kit (Boehringer, Germany), lipid with range around 20-45% base on dry cells
and the phenol-sulphuric acid assay, respectively. biomass (table 1). Due to the screening methods
The hydrocarbon assay was performed by modi- were utilize the nile-red conjugate dye perfor-
fication of lipid extraction (Bligh-Dryer method, mance, the lipid and starch accumulator were
1959) therefore subjected into Gas chromatog- flashy or bias in the visual observation under
raphy for measure the hydrocarbon fraction. microscopic view. The visualization of the cells
under microscopic view were possibly influence
RESULTS AND DISCUSSION the sucking process of the selected strain, how-
ever the microalgal candidate have two potencies
The energies industry has in past been heav- possibility for starch or lipid producer strains.
ily reliant on the slow or non-renewable materi- Further, we reconfirmed and reevaluated by cul-
als. Obviously, interest now is gaining to find ture broth assayed by manipulated medium with
renewable energies sources. Algae are being tar- free of starch or lipid content, and then the har-
geted as a means of obtaining chemicals given vested biomass were extracted and calculated for
the strong demand for fine chemicals and the the starch and lipid content.
pressures of modern sustainable agricultures. An The results also exhibited the starch and li-
algae cultivation technique that uses waste from pid accumulator which selected were mostly
other sectors is being developed by several insti- kind of the chlorophytes belong to the group of
tutions in many countries. Algae cultivation has unicellular motile prasinophyceae or non motile
the added bonus of being environmentally chlorophyceae. For instance, the Scenedesmus
friendly when waste heat from power plants used and (BTM 1) which suspected Tetraselmis-like
as well as its ability to fix carbon dioxide. How- were often occurring in the selected media. The
ever, selection the available strains for specific microscopic image in the binocular and scanning
purposes and large scale production of high electron microscope vessel were shown in the
quality targeted compounds still in problems. In Fig. 1. In these images are depicted the green
this regards, we have exploring the tropical mi- color of chloroplast, specific architecture of
croalgae of Indonesian waters environments for spine and shape of Scenedesmus, and flagella of
energy stock purposes. The isolation and screen- BTM 1 (Tetraselmis-like) perfectly. Occurrences
ing for specific targeted microalgae from various of the chlorophytes are maybe due to their sur-
waters areas in Indonesia are exposing in this vival ability in containing screening media with
reports as describes below. high starch substrates and high carbon dioxide
gas.
Table 1. Lipid producing microalgae from several environments in Indonesia.

No Sampel name Origin Starch-Lipid content
(% dry weight)
1. Chlorella sp. Jepara 23-44
2. Tetraselmis sp. Lombok 33-40
3. Nannochloropsis sp. Batam 23-38
4. Scenedesmus sp. Batam 30-25
5. BTM 1 Batam 20-36
8. CTR 1 Ciater 26-33
9. CTR 4 Ciater 21-30
10. Pari 1 Pari, Seribu Island 35-65

13. Ads.Bf.3 Bali 30-45
14. Ads.Bf.5 Bali 23-40
A B
C D
Fig. 1. Starch or lipid accumulator microalgae

A: Scanning electron microscope image of Scenedesmus sp; B: light mi-
croscope image of Scenedesmus sp; C: Scanning electron microscope of
BTM 1; D light microscope image of BTM 1; error bar 10 um.
Hydrocarbon synthesizers microalgae: Micro- lected microalgae resemble or match with com-
algal direct product fuel is attempting to the mercial hydrocarbon which is use as fuel-oil.
quick replacing fossil fuel which match on the There is interesting in the variant or diversity of
demand of recent residence of the world as re- hydrocarbon producing microalgae, we found
newable, easy and save transportation, sustain some selected strains are cyanobacteria and
and environmental friendly sources. However to some of them are green algae or chlorophytes,
find the suitable strains which is possibly culti- since the method of screening was use the en-
vate in large culture scale without high risk in richment media with high organic acid, nitrogen
controlling is difficult. We have isolated several and induction hydrocarbon compounds such as
microalgae from marine and hot-spring water cooking oil and fuel-oil. Specific character of
bodies which capable for synthesis hydrocarbon these group algae were characterized by gluti-
during their cell metabolism. The results showed nous and gleaming sheath surrounding the cells
selected microalgae synthesized hydrocarbon or colonies (Fig. 2). Mainly we identify the mi-
compound in range of 15-45% base on dry croalgal collection by morphologically view
weight. Further observation on the fraction of points such as color of chloroplast, architecture
hydrocarbon using chromatography detection of cells and shape of cells.
exhibited the fraction of hydrocarbon from se-
A B
Fig. 2. Hydrocarbon depositor microalgae

A: Light microscope image Bali 3; B: Light microscope image Lmb 3.

Hydrogen producing microalgae: Heterocyst enzymes. Hydrogen is emerging sustain energy
microalgae are capable for gas evolution such as in the future, although the safety and technology
hydrogen, oxygen and carbon dioxide, since the in development behind than other energies such
algal systems have nitrogenase and hydrogenase as ethanol and diesel.
Fig. 3. Heterocyst Cyanobacteria for hydrogen evolution.

A: Scanning electron microscope image Ctr 1; B: light microscope image Ctr 1,
error bar: 10 um.
In Conclusion, Studies on the evaluation of Chisty, Y. 2007. Biodiesel from microalgae. Biotechnology
marine green algal of Nannochloropsis which Advances ; 25: 294-306.
collected from Jepara beach, Central Java has Huntley, M. E. and D. G. Redalje. 2007. CO2 Mitigation
done. The results showed the formation of oil and Renewable Oil from Photosynthetic Microbes: A
New Appraisal. Mitigation and Adaptation Strategies
content in the strain was equal with palm oil for Global Change (2007) 12: 573–608 ; DOI:
properties but less than fossil fuel oil. The fur- 10.1007/s11027-006-7304-1.
ther study on the extraction and trans-
Ikke A, Toda N, MurakawaT, Hirata K and Miyamoto K.
esterification is needed for developing utilization 1998. Hydrogen production from starch in CO2-
the strain in field application. Fixing microalgal biomass by haloterant bacterial
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219.

Evaluation of The Antimicrobial Efficacy and Treatment of Tinea Pedis for
Sio2-Ionized Loess
Moon Young Yoon1, Seung Ug Hong2, and Jung-Keug Park3
1
Research Institute of Biotechnology, Dongguk University, Seoul 100-715, Korea
2
Department of Korean Medicine of International Hospital, Dongguk University, 410-773 Il-san, Korea
3
Department of Medical Biotechnology/Chemical and Biochemical Engineering, Dongguk University,
Wonheungkwan-E208, 26, Pil-dong 3-ga, Chung-gu, Seoul 100-715, Korea, Tel.: 82-2-2260-3365,
Fax. : 82-2-2260-8534, e-mail : jkpark@dongguk.edu
(Correspondence to: Jung-Keug Park)
ABSTRACT
SiO2-ionized loess was prepared from the reaction of loess and sodium hydroxide at 1400oC for 2 hr. The
antimicrobial activity of SiO2-ionized loess against Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
Pseudomonas aerusinosa and Trichophyton tonsurans causing tinea was examined by comparing against that of
untreated loess, and clinical efficacy was examined for the treatment of tinea pedis using soap containing
SiO2-ionized loess. Antimicrobial activity showed nearly 100% on medium containing greater than 10 mg/mL of
SiO2-ionized loess. Minimum inhibitory concentration (MIC) value against T. tonsurans was 2.5 mg/mL. How-
ever, medium containing untreated loess had no antimicrobial activity. Treatment efficacy test revealed that the
symptoms of tinea pedis decreased as the duration of use of soap containing SiO2-ionized loess increased.
Keywords : SiO2 ionization, loess, antimicrobial activity, treatment efficacy, tinea pedis
INTRODUCTION pedis can cause discomfort to patients by induc-

ing severe pruritus, odoring with hyperhidrosis,
Tinea pedis, which is the most common festering, cracking, and scaling. It is difficult to
disease caused by dermatophyte fungi, afflicts treat diseases caused by dermatophyte fungi due
15% of the world population. Tinea pedis is to their strong parasitism and propagation, as well
common among individuals with a decreased as the appearance of resistant fungi in response to
immunity and has been experienced by 33-50 % the repetitive use of antibiotics and x-rays. To
of the population. However, the frequency of treat cases of dermatomycosis such as tinea, oral
tinea pedis has not decreased in recent years, antifungal agents are often administered when
despite improved sanitary conditions. Tinea, topical remedies are ineffective. However, they
which is caused by an infection of the dermato- can cause extreme side effects in as many as 24%
phyte Trychophyton and Epidermophyton species, of the cases, such as an upset stomach and im-
is a disease that causes the festering, desquama- paired liver function. Therefore, the need for a
tion and uredo of the skin and occurs in high new antifungal agent has become increasingly
humidity environments (Noble et al., 1998; Barry, urgent and the development of antifungal agents
2003). Dermatomycosis is a common infectious from natural sources that do not exert toxicity to
disease that occurs in humans and is also referred the human body are needed (Pierard et al., 1996;
to as dermatophytosis, tinea, or superficial fungal Shegal & Sujay, 2002). In the present study, a
infections. It is caused by dermatophytes, partic- novel SiO2-ionized loess was prepared by melting
ularly those that are parasitic to corneous tissues a mixture of the loess and sodium hydroxide for 2
like hair, nails, and keratin of the skin (Ab- hr at 1400oC. The antibacterial and antifungal
delrahman et al., 2006). Dermatophytosis is efficacy of SiO2-ionized loess against Staphylo-
classified according to infection area; tinea ma- coccus aureus, Bacillus subtilis, Escherichia coli,
nus and tinea pedis when it occurs on the hands Pseudomonas aerusinosa and Trichophyton
and feet, tinea faciale when it occurs on the face, tonsurans causing tinea was studied, and the
tinea capitis when it occurs on the head, tinea treatment efficacy of soap containing
cruris when it occurs on the groin and tinea un- SiO2-ionized loess against tinea pedis was then
gium or onychomycosis when it occurs in nails. evaluated through a primary, secondary, and
Tinea pedis and tinea ungium have a long dura- safety treatment efficacy test using a standard
tion and the possibility for high recurrence. Tinea method based on the guidelines of Dongguk

University International Hospital. these strains were inoculated on each media sup-
plemented with SiO2-ionized loess and untreated
MATERIALS AND METHODS loess in amounts of 2.5 mg/mL, 5.0 mg/mL, 10
mg/mL, 20 mg/mL, and 40 mg/mL, respectively.
Preparation of SiO2-ionized Loess: To prepare Media without untreated loess and SiO2-ionized
the SiO2-ionized loess, loess (Gochang Hwangto, loess cultured under the same conditions were
Korea) containing 30% of sodium hydroxide was used as a control. The culture broth were incu-
heated to 1400oC for 2 hr using an electric furnace bated at each temperature for 18 hr and the
(Ajeon Heating Industrial Co., Korea). The number of colonies were counted. To determine
melted liquid was cooled to room temperature, the degree of the antibacterial effect in the pres-
after which it was crushed using hammer mill and ence of SiO2-ionized loess, the number of re-
ball mill (DAIHAN Scientific Co., Korea) and maining bacteria was examined. T. tonsurans was
sieved to 45 m or smaller using testing sieve cultured in sabouraud's dextrose agar medium
(Chung Gye Sang Gong Sa, Korea). containing glucose 20.0 g/L, tryptone 10.0 g/L,
and agar 15.0 g/L, at 26oC. The small inoculum of
Chemical Analysis: The chemical compositions the test organism using a sterilized toothpick was
of the SiO2-ionized loess and the untreated loess placed onto the surface of the medium plate
were determined by ICP OES (PerkinElmer DV containing various SiO2-ionized loess and loess
3300, USA) according to KS L 4007: 2006 of the concentrations. The plates were incubated for 10
Korean Standards Association (Korean Standards days, and the growth halo was examined.
Inquiry Commision, 2006). The levels of Pb, As,
Cd, and Hg were determined by ICP-OES fol- Preparation of Soap containing SiO2-ionized
lowing acid digestion, and the level of Cr+6 was Loess: Soap was prepared by adding 0.25%
determined using a UV/VIS spectrophotometer (w/w) of SiO2-ionized loess and coconut oil
(Shimadzu, Japan) to measure the absorbance at (Philippines) as the soap base from Hanil Mool-
540 nm after digestion for 1 hr at 90oC using an san Co. (Korea).
alkaline solution containing Na2CO3, NaOH,
MgCl2, and phosphate buffer, according to the US Treatment Efficacy Test: The treatment efficacy
EPA (Environment Protection Agency) 3060A of tinea pedis was evaluated through primary
method (United States Environmental Protection evaluation, secondary evaluation, and a safety
Agency, 1984). evaluation using a standard method based on the
guidelines of Dongguk University International
Antimicrobial Activity: Staphylococcus aureus Hospital. Briefly, 24 patients that were greater
KCCM 11764, Bacillus subtilis KACC 10111, than 19 years of age were screened between June
Pseudomonas aerusinosa KACC 10232, Esche- 2008 and August 2008. We excluded 2 patients
richia coli KCCM 12119 and T. tonsurans who exhibited severe tinea pedis symptoms and
KCCM 11866 was obtained from the KCCM directed them to use topical steroid agents. Soap
(Korean Culture Center of Microorganisms, Ko- was used once a day for 8 weeks continuously,
rea) and KACC (Korean Agricultural Culture with a midterm and final evaluation being con-
Collection, Korea), and was used as a reference ducted at 4 weeks and 8 weeks. For the primary
strain during the antimicrobial testing. S. aureus, efficacy evaluation, the severity of the symptoms
P. aerusinosa and E. coli were cultured on nu- of tinea pedis, such as erythema, scaling, vesicles,
trient agar containing 3.0 g/L beef extract and 5.0 pustule, exudate, crust, and pruritus, were ranked
g/L peptone at 37oC. B. subtilis was cultured on following treatment with the soap, with 0 indi-
the same medium at 30oC. The antibacterial ac- cating no symptoms, 1 indicating mild symptoms,
tivity of SiO2-ionized loess was measured by 2 indicating moderate symptoms, and 3 indicating
spreading 0.1 mL of appropriate diluted strain severe symptoms. Of these symptoms, pruritus
suspension on agar plate. The antimicrobial ef- was evaluated by volunteers due to the subjective
fects of SiO2-ionized loess were evaluated by the nature of the characterization, while the other
determination of minimum inhibitory concentra- symptoms were evaluated by a researcher. The
tions(MICs). The MIC was determined as the secondary efficacy evaluation was conducted by
lowest dilution of SiO2-ionized loess that pro- dividing the evaluation into patient evaluation,
duced more than 99% of quantitative antibacterial which was based on the patients, and investigator
evaluation value. Approximately 105 CFU/mL of evaluation, which was based on the investigator.
An evaluation score of 1 was given when the

symptoms were completely cured, while a score due to a decrease in response to ignition. The
of 2 was given in cases of marked improvement, increased level of Na2O was attributed to the
which was defined as a decrease in the overall addition of sodium hydroxide to the reaction. The
symptoms of more than 50%. A score of 3 points amounts of useful mineral components of loess,
was given in the case of moderate improvement, including Al2O3, Fe2O3, CaO, MgO, K2O, and
which was defined as less than 50% for the tinea Na2O were maintained after the ionization reac-
pedis symptoms being attenuated, while a score tion.
of 4 was assigned when the treatment had no
effect on the tinea pedis symptoms. Finally, a Table 1. Composition of untreated loess and SiO2-ionized
score of 5 was given when the symptoms wors- loess.
ened. All scores were determined by comparing
Result (%)
the symptoms at 4 weeks and 8 weeks to the
Component
symptoms at the beginning of the study. The Untreated lo- SiO2-ionized
equation for quantitative treatment evaluation is ess loess
given by MV = (S x P)/T, where MV is the mean SiO2 59.90 49.82
value, S is symptom score, P is the patient num- Al2O3 21.70 21.43
Fe2O3 5.85 1.41
ber for each symptom score, and T is the total CaO 0.08 1.61
patient number. Safety evaluation and other MgO 0.63 0.34
evaluations were conducted based on the obser- K2O 0.19 3.66
vation of abnormal symptoms such as subjective Na2O 0.58 10.16
TiO2 0.14 0.19
and objective symptoms, skin allergy, and fever. Ig. lossa 10.11 10.72
Pb n.d.b n.d.
RESULTS AND DISCUSSION As n.d. n.d.
Cd n.d. n.d.
Cr+6 n.d. n.d.
Chemical Composition: The mixture of loess and Hg n.d. n.d.
sodium hydroxide was melted at 1400oC using an a
Ignite loss
electrical furnace, which resulted in the SiO2 b
not detectable
component of loess being ionized to SiO3 form
according to the following reaction equation: Antimicrobial Efficacy of SiO2-ionized Loess:
The antimicrobial efficacy of SiO2-ionized loess
SiO2 + 2NaOH → 2Na(SiO3)2- + H2O ↑ and untreated loess is shown in Table 2. The MIC
value of the materials against S. aureus, B. sub-
Table 1 shows the heavy metals and mineral tilis, P.aerusinosa, E. coli and T. tonsurans was
components of SiO2-ionized loess and untreated 10 mg/mL, 2.5 mg/mL, 10.0 mg/mL, 5.0 mg/mL,
loess. Heavy metal components of loess such as and 2.5 mg/mL, repectively. The antimicrobial
Pb, As, Cd, Cr+6, and Hg were not detected in efficacy was increased with an increased addition
untreated loess and SiO2-ionized loess, with the of the test agent. However, media containing
SiO2 contents being 59.90% and 49.82%, re- untreated loess did not show antimicrobial effi-
spectively. The SiO2 content may have increased cacy.
Table 2. Antimicrobial activity of SiO2-ionized loess.

Untreated loess (mg/mL) SiO2-ionized loess(mg/mL)
Bacteria
0 2.5 5 10 20 40 0 2.5 5 10 20 40
S. aureus + + + + + + + + + - - -
B. subtilis + + + + + + + - - - - -
P. aeruginosa + + + + + + + + + - - -
E. coli + + + + + + + + - - - -
T. tonsurans + + + + + + + - - - - -
+, Growth ; -, No growth
Fig. 1 shows the antifungal activity of SiO2-ionized loess and untreated loess against T. tonsurans.
The antifungal activity showed nearly 100% on medium containing 2.5 mg/mL SiO2-ionized loess.
However, the antifungal activity did not show on the medium containing untreated loess.

Fig. 1. Photographs of the antifungal test results on T. tonsurans KCCM 11866 in the systems of (A) Control, (B) Untreated
loess (10 mg/mL), (C) SiO2-ionized loess (1.25 mg/mL), (D) SiO2-ionized loess (2.5 mg/mL).
The growth of B. subtilis and T. tonsurans containing the SiO2-ionized loess for 4 weeks,
was strongly inhibited by the SiO2-ionized loess. and decreased to 42.0% even further by 2.3182 at
However, SiO2-ionized loess showed week anti- 8 weeks and 4.0000 at start after 8 weeks of
bacterial activity against S. aureus and treatment. Table 4 shows the results of patient
P.aerusinosa. These results indicate that evaluation for secondary efficacy evaluation.
SiO2-ionized loess inhibit the growth of both Fifteen out of 22 individuals showed signs of
fungi and bacteria without selectivity. improvement. Specifically, no individuals were
completely cured, while 6 subjects showed
Treatment Efficacy: The treatment efficacy of marked improvement, and 9 subjects showed
tinia pedis using soap containing SiO2-ionized moderate improvement at 4 weeks, compared to
loess was evaluated using a primary treatment start. Sixteen out of 22 patients showed signs of
efficacy and secondary treatment efficacy test improvement at 8 weeks, with 2 individuals being
composed of patient evaluation and investigator completely cured, 10 individuals showing signs
evaluation. Table 3 shows the evaluation results of marked improvement, and 4 individuals
for the primary treatment efficacy, which inves- showing signs of moderate improvement. The
tigated the significant difference in the scores of mean value for patient evaluation was decreased
the tinea pedis symptoms at start, 4 weeks and 8 to 14.5% by 3.1364 at 4 weeks and 2.6818 at 8
weeks. weeks. These results demonstrate that the treat-
ment efficacy of soap containing SiO2-ionized
Table 3. Average tinea pedis symptoms score at each time loess increased as the duration of treatment in-
Tinea pedis Patient Number creased.
symptoms
scorea Table 4. Patient evaluation.
Start 4 weeks 8 weeks
Patient Number
0 0 0 2
Tinea pedis symptoms score
1 0 4 5
2 5 6 6 4 weeks 8 weeks
3 6 5 7 1 (cured) 0 2
4 2 5 0
2 (marked improvement) 6 10
5 5 1 1
6 2 1 0 3 (moderate improvement) 9 4
7 1 0 0 4 (unchanged) 5 5
8 1 0 1 5 (deterioration) 2 1
9 to 22 0 0 0 Mean valuea 3.1364 2.6818
a
Mean valueb 4.0000 2.8182 2.3182 (S x P)/T, where S is symptom score, P is the patient
a number for symptom score, and T is the total patient number
0 point, absent; 1 points, mild; 2 points, moderate; 3
points, severe; clinical symptons for score are erythema,
scaling, vesicle. pustule, exudate, crust, and pruritus,
Table 5 shows the results of investigator
respectively.
b
(S x P)/T, where S is symptom score, P is the patient evaluation for secondary efficacy evaluation at 4
number for symptom score, and T is the total patient weeks and 8 weeks. Ten out of 22 patients
number showed signs of improvement at week 4, with 0
individuals being cured, 3 individuals showing
The mean value of tinea pedis symptoms signs of marked improvement, and 7 subjects
score decreased to 29.5% by 2.8182 at 4 weeks showing moderate improvement. At week 8,
and 4.0000 at start after treatment with the soap Eleven out of 22 patients showed signs of im-

provement, with 2 individuals being cured, 4 decrease in scaling. Additionally, 4 persons ex-
individuals showing signs of marked improve- pressed skin drying and 1 individual indicated an
ment, and 5 individuals showing moderate im- increase in symptoms, including festering and
provement, compared to start. The mean value for cracking. Of 22 subjects that completed the
investigator evaluation was decreased to 12.3% normal test, the pruritus symptom score de-
by 3.5455 at 4 weeks and 3.2273 at 8 weeks. creased as the duration of treatment increased.
These results also demonstrate that the treatment Additionally, 7 individuals were found to be
efficacy of soap containing SiO2-ionized loess completely cured. Thus, an increase in the dura-
increased as the duration of treatment increased. tion of treatment led to a decrease in pruritus
symptoms.
Table 5. Investigator evaluation.
ACKNOWLEDGEMENTS
Tinea pedis symptoms score 4 weeks 8 weeks
1 (cured) 0 2 This work was supported by the Dongguk
2 (marked improvement) 3 4 University Research Fund, Small& Medium
3 (moderate improvement) 7 5 Business Administration, and Seoul Metropolitan
4 (unchanged) 9 9
5 (deterioration) 3 2 Government, Korea
Mean valuea 3.5455 3.2273
a
(S x P)/T, where S is symptom score, P is the patient REFERENCES
number for symptom score, and T is the total patient
number Abdelrahman, T., V. L. Bru, J. Waller, G. Noacco & E.
Candolfi. 2006. J Mycol Med. 16: 87.
Overall, these findings indicate that an in-
Barry, L. H. 2003. Am Fam Physician. 67: 101.
crease in the duration of treatment using the soap
led to a decrease in the tinea pedis symptoms Korean Standards Inquiry Commision. 2006. KSL 4007
(Korean Standards Association, Seoul).
score, which resulted in an increase in the treat-
ment efficacy. This may have been due to the Noble, S. L., D. Pharm & C. F. Robert. 1998. Am Fam Phy-
continuous action of SiO2-ionized loess as an sician. 58: 163.
inorganic antimicrobial material, and its re- Pierard, G. E, J. E. Arrese & C. Pierard-Franchimont. 1996.
sistance to decomposition despite being exposed Drugs. 52: 209.
to a variety of environmental factors such as Shegal, V. N. & K. Sujay. 2002. Clin. Dermatol. 20: 481.
temperature, UV irradiation, chemical and bio- United States Environmental Protection Agency. 1984.
logical resistance. The safety of the treatment was Method 3060A (Office of Solid Waste and Emergency
also investigated based on the patients individual Response, Washington DC).
opinions. Specifically, 13 individuals indicated Weckesser, S., K. Engel, B. Simon-Haarhaus, A. Wittmer, K.
that they experienced a decrease in pruritus, while Pelz & C. M. Schempp. 2007. Phytomedicine. 14: 508.
9 individuals indicated that they experienced a

In Searching for The Possibility to Produce Bioethanol (Gasohol)
as New Renewable Energy from Lontar Palm (Borassus sundaicus L.)
in East Nusa Tenggara, Indonesia
B.P. Naiola1, N. Nurhidayat1, Teuku B. Bardant2, Tri Murningsih1, S. Tursiloadi2,
J. Sulistyo1 and M. Kismurtono3
1
Research Center for Biology-LIPI, Cibinong, Indonesia, e-mail: bpnaiola@yahoo.com
2
Research Center for Chemistry-LIPI, Serpong, Indonesia
3
Technical Implementation Unit for Development of Chemical Engineering Processes-LIPI, Yogya-
karta, Indonesia
(Correspondence to: B. P. Naiola)
ABSTRACT
Our field study showed that the Rotinese and Sabunese tribes in East Nusa Tenggara (ENT) islands of Indo-
nesia posses a local wisdom, which utilized the sap tapped from the inflorescence of a wild or semi-wild growing
lontar palm or palmyra palm (Borassus sundaicus L.), to produce local traditional alcoholic drink; the so called
“laru”, with alcohol content of up to 15% ethanol, and “sopi” (up to 30 to 40% ethanol). The yeast Saccharomy-
ces cerevisiae and Candida sp. were isolated as the main agents responsible in the “traditional” fermentation. To
produce these two drinks, the processes are described as PATHWAY I and PATHWAY II. To provide sopi, the
villagers first boil the nira (sap) to produce gula aer (a viscous liquid brown sugar). The gula aer was then dilut-
ed, fermented, and distilled to produce sopi. “Sopi” has a potential to be developed to local (bio)energy. Our fur-
ther upgrade of “sopi” at laboratory level, bioethanol with a purity of up to 93% was able to be produced to create
gasohol E85, which was able to operate small static engine potentially aimed for small rural family energy con-
sumption. However, much energy cost is applied by the villagers when using firewood (gathered from the poor
savanna vegetation) in producing “sopi”. We are looking for suggestions to our innovation in constructing a so
called energy less system – i.e. using solar (cell) power in generating energy to upgrade sopi as new bioenergy,
running from the early step (i.e. boiling nira to produce gula aer) to the final step i.e. distillation. Molecular sieve
dehydration method will be employed in purifying nearly pure (99%) bioethanol originated from Borassus palm
to create a new E85 mixture.
Keywords: Bioethanol, lontar palm (Borassus sundaicus L.), sopi, solar cell, molecular sieve dehydration.
INTRODUCTION due to two factors: (1) The productivity of na-

tional oil field (both terrestrial and off-shore) has
The energy consumption in Indonesia as a decreased, in terms of the amount of oil being
developing country is increased from year to mined, (2) The capacity of new oil field sources
year. The recent data show that the annual aver- discovered are not so significant compared to the
age consumption of oil for energy in Indonesia is need of the country.
not less than 60 million kilolitres, mainly for At the same time, the consumption of oil by
transportation and electricity power (Hutasoit, the country is increased for three reasons. Firstly,
2005 in Daryanto, 2005). Most of the energy in the increase of population with annual growth of
Indonesia are originated from fossil energy. The 1.2%; secondly, the increase of industries in vari-
contribution by fossil fuel (54.4%), petrol gas ous sectors, and thirdly, the enlargement of elec-
(26.5%), coal (14.1%), micro-hydro (3.4%), geo- tricity frame, including introduction to rural areas
thermal (1.4%) and (0.2%) from other sources to nearly all over the country.
including bioenergy (Blueprint Pengelolaan En-
ergi Nasional, in Krisnamurthi, 2006). New energy resources: more problems may to
As a non-renewable energy, fossil based en- come in the future, since the estimated deposit of
ergy in Indonesia shows somewhat a decrease fossil fuel is only 15 years forward. Last year,
Indonesia already resigned as member of OPEC

countries. Thus, possible turning from oil produc- pected to substitute 5% of its energy consump-
ing to oil importing country is a fact that one tion by bio-energy.
should be aware of. As a tropical “mega biodiversity” country,
To overcome the problem of increasing oil second after Brazil, Indonesian biodiversity
consumption, steps should be taken by Indonesia, should be considered as sources of biofuel. Not
in several ways to find out new energy resources, less than 28 plant species in Indonesia are catego-
namely (1) Campaign and action for energy sav- rized as fat oil sources with potential for develop-
ings and conservation. Step in energy campaign ing new biofuel (Soerawidjaja, 2004, in Budi-
become important, due to the habit of the society, man, 2004). Table 1 summarized a number of
i.e. having very low attitude and less awareness plant species in Indonesia that may be developed
in steping into energy saving lifestyle (2) Search- into biofuel. Notes on their status is underlined,
ing for new energy (alternative energy) re- since the government is reluctant to have biofuels
sources. from food plants. For the country, there should
Apart from fossil based energy, there are not be any contradiction between food and ener-
some possibilities in increasing energy capacity gy demand in Indonesia.
from available resources such as improving coal
and geothermal, while finding new energy re- Local wisdom: as a society rich in old and varia-
sources potentially available in Indonesia, such bility of cultural background, Indonesian people,
as micro-hydro power, solar power, waves/tidal driven by local wisdom possessed by tribes, have
power and bioenergy (biofuel). utilized the richness of their wild plants growing
in their surroundings for daily needs including
Why bioenergy: one of the main reasons to pro- foods, drinks, medicines, shelters, fences, cages,
pose the development of bio-energy is mainly kitchen sets, strings, weavings, colourings, cos-
due to its effect to both rural economic and envi- metics, sweets, animal feeds, pesticides, herbi-
ronment. Since bio-energy is based on plants, due cides, fungicides, bactericides, energy sources,
to its chemical properties, it may be less polluting weapons, bails, toxins, traps, transportation kits
than fossil energy. Meanwhile, the development etc. The local wisdoms may be found in local
of bio-energy, may drive to some instant benefit appropriate technology that adapted to the local
to the rural family economy, since the villagers conditions or transfer of knowledge by daily
will take some roles during the process due to the practical uses (Naiola et al., 2007).
habit of appropriate technology used. Meanwhile, When searching for new bioenergy, one of
in searching for bioenergy as an alternative new the steps is to explore and learning from the
energy sources, may bring some benefit to Indo- “wisdom” of local tribes in Indonesia when using
nesia due to it characteristic as an archipelago biodiversity in their daily life that maybe upgrad-
country; thus possible to develop energy based ed for further bioenergy demand. After several
remote islands. Thus, it is necessary to develop visits and references exploration (Ormeling,
biofuels based on regional potential (kabupat- 1955; Naiola et al., 1992; Naiola, 2002; 2005;
en/regency, kecamatan/county and desa/village). 2006) and a long and accumulated thought, we
The development of bioenergy as an alterna- found this wisdom in one species of palm plant
tive energy resource, has strongly been recom- practiced by Rotinese and Sabunese people. A
mended by the Indonesian Government, as ex- wild species that seems fulfils the explanation
pressed by the Instruksi Presiden Nomor 1, Ta- above, is lontar palm or palmyra palm (Borassus
hun 2006 (Presidential Instruction Number 1, sundaicus L.). It seems that lontar palm is one of
2006), to 13 Ministries and all Governors and all the biodiversity richness in Indonesian dryland,
Major/Head of Regents (Walikota and Bupati), to distributes wildly in dry areas, especially in the
promote and facilitate all possibilities for biofuel savannah of East Nusa Tenggara. It has potential
development. as natural source in the development of gasohol
The “National Energy Policy” as a manual as new bioenergy.
in handling the country’s energy issues in the
future, has a target that in 2025, Indonesia is ex-
MATERIALS AND METHODS

The microbial starter used by the local people
The Rotinese and Sabunese practicing some when fermenting alcohol was analysed to identify
ways in gathering their traditional alcoholic (en- the microbes responsible in fermentation.
ergy) drink from their environment, i.e. the Bo- Further steps necessary were to run a small
rassus palm. We study this aspect in lontar palm experiment using formulated bioethanol E85
species growing wildly or semi-wildly and abun- from the lontar product in running a small elec-
dantly in savannah of East Nusa Tenggara with tric engine (generator) to generate electricity.
possibilities to improve this local wisdom by in-
serting some scientific principles to have some RESULTS AND DISCUSSION
added values, especially a new renewable energy
source. Lontar, is a mocotyledonous plant spe-
The approach was by visiting locations cies, single trunk, usually grows in group of sev-
where the traditonal Rotinese community (in eral to hundreds forming forest vegetation (Fig.
Rote Island) did their daily activities in gathering 1), in around villages or savanna areas; height up
lontar palm product for making the traditional to 20 m, bear several fan-like rosette leaves on
alcoholic drinks. We also accumulated support- top end. At about 15-20 years old, a mature lon-
ing data/information by interview. The samples tar palm produces inflorescences (bunch of flow-
of the traditional alcoholic drinks were collected, ers), and since then, it appears annually.
brought to laboratory in Bogor/Cibinong. The
samples then analysed for their alcohol content.
Fig. 1. Dense forest lontar palm vegetation in East Nusa Tenggara (left); „nira liquid“ collected
from each lontar palm tree (right).
Lontar palm distributes abundantly in East housing, food and drink. However, bioetanol
Nusa Tenggara (NTT-Nusa Tenggara Timur). production has a close relationship with four
They grow densely in two smaller islands namely products of lontar palm namely nira, gula aer,
Rote and Sabu. It is estimated that there are not laru and sopi. Nira is a white-pale sugar content
less than 10 million trees of lontar palm grow all sap (alcohol content up to 20%), obtained by tap-
over East Nusa Tenggara islands. Report of Rote- ping the mature inflorescences.
Ndao Regency Government provides information Based on the plant sap (nira), the native
on standing Fig. of lontar palm present in their people make some product; but the most known
region, i.e. about 20 thousands hectares (Badan are: (1) “Gula aer” is a kind of viscous liquid
Pusat Statistik Kabupaten Kupang, 2004). brown sugar, obtained by boiling the nira at a
Although in its wild status, since a long time quite high temperature, using firewood gathered
ago the native people of East Nusa Tenggara al- from savanna area; (2) “Laru”, is a traditional
ready make used of lontar palm for their daily drink with lower alcohol content (up to 15%,
need for various purposes. It is probably one of mainly ethanol). It is provided by direct fermen-
the only several wild plant species in the world tation of nira. During fermentation process, some
that has been extremely exploited, ranging from ingredients are soaked into nira and the fermen-

tation takes place overnight or longer. Ingredients mainly ethanol). To provide it, they diluted gula
(some kind of woods) are included for taste, but aer about 5 times, then mixed with mur. The so-
another function is believed as to eliminate un- called mur, obtained from laru fermentation, is
wanted microbes during the fermentation process actually microbial masses precipitated at the bot-
except the yeast Saccharomyces cerevisiae and tom of “fermentor”. By further distillation, sopi
Candida sp. Masses of microbial suspension usu- is produced. During distillation (boiling process),
ally precipitated at the bottom of container, and they need a high number of firewood that was
will be used again as starter in the next fermenta- grabbed from savanna areas. The whole process
tion process. During the process, no firewood is described in Fig. 3 as PATHWAY II. The
was used at all. The whole process is described in yeast (Saccharomyces cerevisiae) and Candida
Fig. 2, as PATHWAY I; (3) “Sopi”, is also an sp. were isolated as the main agents responsible
alcoholic traditional drink (up to 30 to 40%, in the “traditional” fermentation of sopi.
Pathway I
Summary of making LARU
Up to 15% ethanol
1. Climbing and tapping
Lontar (or Gewang) palms
2. Collected the Nira sap
4. “Laru”, traditional
beverages (fermented),
3. “Laru’s”processing. During
15% ethanol content.
fermentation process, some
Masses of microbial
ingredients are soaked into nira
suspension precipitated
and let the fermentation taking
at the bottom of
place overnight or longer.
container
Fig. 2. Pathway I, laru making.

1. Climbing and tapping Pathway II
Lontar (and Gewang)
palms
Summary of making SOPI,
30-40 % ethanol
SOPI,
30-40%
2. Nira’s sap ethanol
collected
Traditional
distilLation of
3. Boiled the fermented liquid
Nira sap brown sugar
4. Viscous liquid
brown sugar, up to
80% sugar
Fig. 3. Pathway II, sopi making.
Formulation of E85 based on sopi: further step The laboratory purified sopi (bioethanol)
on the estimation of bioethanol content showed was then mixed with 15% gasoline to formulate
that laru had 14-15% ethanol content , while sopi gasohol E85. During small engine (1000 W) trial,
had 30-40% ethanol content. Fig. 4 summarized the engine was able to operate stabily, and able to
further distillation of traditional sopi at laboratory supply electricity for a small family domestic
level, at temperature of 65–67°C and 300 milibar demand such as kitchen appliances, house light-
pressure, which obtained bioethanol with concen- ing and possibly small carpenter operation (Fig.
tration ranged between 87 to 93% (Bardant et al., 4).
2007).
FURTHER STUDY
OF E85
7. HOME LIGHTING
BIOETHANOL/
BIOPREMIUM
FROM
LONTAR (AND
GEWANG) PALMS
6. E85 (GASOHOL/BIOETHANOL/ 8. HOME APPLIANCES AS NEW
7. BIOPREMIUM) LONTAR POWERED ALTERNATIVE
BIOENERGY
9. SMALL HOME INDUSTRY
5. Gasohol E-85
+
4.GAS/ PREMIUM 3. RE-DISTILLED 1. SOPI, 30-40%
2. REDISTILLATING bioethanol
SOPI (ethanol 93%)
SOPI
Fig. 4. Steps in formulating E85, based on sopi of Borassus.

TENTATIVE Solar (cell) power in producing Bioethanol
from Lontarpalm
Solar cell Tank for boiling “nira” to “gula
aer” by solar energy/power
Inverter
Distillator energized by solar power
Fig. 5. Future model of boiling gula aer and bioethanol distillation from Borassus
palm, using solar power.
In conclusion, The presence of lontar palm Some benefits may be gained by using lon-
growing wildly in East Nusa Tenggara region to tar palm as new bioenergy source, as described
be utilized in the development of bioethanol for below: (1) Discovering a new alternative (bio-
gasohol, is really promising one. Compared to )energy source with lower effect to the environ-
Brazil, USA and other countries, when producing ment (in terms of air pollution compared to fossil
gasohol, they should firstly planting plants as raw fuel) as refer to global climate change – green-
materials such as sugarcane, corn, cassava and house effect (Kyoto Protocol); (2) Generation of
beet. While for the lontar palm in East Nusa income for local villagers by producing a non-
Tenggara, the Mother Nature has provided the timber forest product; (3) Promoting local gov-
sap that directly tapped from the plants in a tradi- ernment to produce their own energy based on
tionally easy manner, done by local people with their biodiversity richness; (4) This is a rural
appropriate technology. based project, thus enhancing rural economy and
The results of this study illuminated some rural technology in East Nusa Tenggara; hence it
guidance for further study such as calculating the may reduce poverty among people with the low-
ratio between nira vs gula aer, gula aer vs sopi, est income (provincial) in Indonesia, thus reduc-
to scaling up of E85 for small family house, ing poverty as sounded in MDGs purposes; (5)
small industry demands, Eco-distribution of Bo- Added-value to traditional alcoholic drinks (sopi
rassus palm to estimate their standing potential. and laru) that has been produced from generation
Since during the production of gula aer and sopi to generation by the rural families in East Nusa
a high amount of firewood had to be grabbed Tenggara; (6) Creation of more field works, es-
from savanna area, it may draw another environ- pecially to native people, and to encourage young
mental problem. Someone may not produce new people to return to village. So far, the economic
energy, while burning another energy source. value of lontar palm, is found only in gula aer;
Thus solar power in producing sopi is tentatively (7) Supporting the local gender concept, as there
under development (Fig. 5). At the same time is a strong indication of differentiation of job in
molecular sieve dehydration method will be em- nira processing. Usually men are doing jobs like
ployed in purifying nearly pure (99%) bioethanol climbing to tap and making sopi and laru. While
originated from Borassus palm to create a E85 women are responsible for making gula aer; (8)
mixture. There is a growing interest in developing energy

based bio-resources in Indonesia, following the Daryanto, A. 2005. Analisis kebijakanPemerintah di bidang
declaration of Presidential Instruction (Number energi: Penanaman jarak pagar sebaagi solusi
alternatif pengadaan sumberdaya energi terbarukan.
1-Year 2006), regarding the support of Govern- Paper disampakan dalam Seminar Nasional
ment to bioenergy development. Thus the result Pengembangan Jarak Pagar (Jatropha curcas L.)
of this Research Study Project may promote any untuk Biodiesel dan Minyak Bakar, Bogor, 22
private company to take part in the business of Desember 2005. Institut Pertanian Bogor.
bioenergy based on lontar palm in East Nusa Krisnamurthi, B. 2006. Pengembangan bahan bakar nabati/
Tenggara; (9) Reducing the dependency of local BBN (Biofuel) dan kebijakan diversifikasi energi.
government on the supply of oil by Central Gov- Paper disampaikan dalam Lokakarya Status Teknologi
Budidaya Jarak Pagar, Bogor, 11 April 2006.
ernment in Jakarta. In many cases, the electricity
power and local transportation were interfered by Naiola, B. P. 2002. (Editor). Kerjasama LIPI – Pemerintah
Daerah NTT untuk Pengembangan dan Pemanfaatan
the intermittent supply (from Java) due to weath-
Sumberdaya Lahan Savana. Laporan Akhir 2002.
er and other technical reasons; (10) If the project LIPI.
is successful in developing the PATHWAY II,
Naiola, B. P. 2005. Kajian Domestikasi Gewang (Corypha
thus it will reduce the environmental cost by cast utan Lamk.) di Savana NTT untuk Keberlanjutan
away the firewood exploitation in the savanna Pemanfaatan dan Upaya Meningkatkan Penggunaan
areas. Hence, it allow the trees to grow to their Potensinya. Laporan Akhir Tahun I. Program Riset
optimal conditions, they may take back their Kompetitif-LIPI, Tahun Anggaran 2005. Lembaga
roles as environmental agent stabilization (in soil Ilmu Pengetahuan Indonesia (LIPI), Jakarta.
erosion, water conservation and clean air). Naiola, B. P. 2006. Kajian Domestikasi Gewang (Corypha
utan Lamk.) di Savana NTT Untuk Keberlanjutan
Pemanfaatan dan Upaya Meningkatkan Penggunaan
REFERENCES Potensinya. Laporan Akhir Tahun II. Program Riset
Kompetitif-LIPI, Tahun Anggaran 2006. Lembaga
Badan Pusat Statistik Kabupaten Kupang. 2004. Rote-Ndao Ilmu Pengetahuan Indonesia (LIPI), Jakarta.
Dalam Angka 2003. Kerjasama BPS Kabupaten
Kupang dan Bappeda Kabupaten Rote-Ndao. Naiola, B.P., J. P. Mogea & Subyakto. 2007. (Editor).
Gewang: biologi, manfaat, permasalahan dan peluang
Bardant, T. B., B. P. Naiola, T. Murningsih S. Tursiloadi & domestikasi. LIPI Press, Jakarta.
K. C. Sembiring. 2007. Resume Hasil Kerjasama PP
Kimia - LIPI & PP Biologi – LIPI Dalam Naiola, B.P., R. Harahap, M. H. Siagian & M. Rahayu.
engembangan Bioetanol dari Tumbuhan Lontar 1992. Etnobotani Palm Timor: Tuak dan Gewang,
(Borassus sundaicus L.) dan Gewang Gewang Penghuni Savana Yang Setiap Mendukung Kehidupan
(Corypha utan Lamk.) Untuk Bahan Baku Manusianya. Prosiding Seminar dan Lokakarya
Biogasoline. Puslit Biologi dan Puslit Kimia LIPI. Nasional Etnobotani. 306-311. Depdikbud RI, Deptan
Laporan Studi Preliminer. Puslit Biologi-LIPI dan RI, LIPI dan Perpustakaan Nasional RI.
Puslit Kimia-LIPI. Tidak dipublikasi. Ormeling, J. F. 1955. The Timor Problem: A Geographical
Budiman, B. T. 2004. Penggunaan biodiesel sebagai bahan Interpretation of An Under-developed Island.
bakar alternatif. Dalam: Biodiesel – Energi Alternatif J.B.Wolters, Batavia and Groningen.
yang Atraktif. Prosiding Seminar Prospek Biodiesel di
Indonesia. Serpong, 12 Agustus 2004.

Conversion of Oil Palm Empty Fruit Bunch to Sugars
by Dilute-Acid Hydrolysis for Ethanol Production
Ria Millati1, Rachma Wikandari1, Elisabeth Titik Trihandayani1,
Muhammad Nur Cahyanto1, Mohammad J. Taherzadeh2 and Claes Niklasson3
1
Food and Agricultural Product Technology Department, Gadjah Mada University
Jl. Sosio Yustisia, Bulaksumur, Yogyakarta, Indonesia, e-mail: ria_millati@ugm.ac.id
2
School of Engineering, University of Borås
Borås, Sweden
3
Chemical Reaction Engineering, Chalmers University of Technology
Göteborg, Sweden
(Correspondence to: Ria Millati)
ABSTRACT
Oil palm empty fruit bunch (OPEFB) is a lignocellulosic waste from palm oil industry, which is available
in large quantity. The lignocellulosic waste can be converted to simple sugars and subsequently to ethanol as a
high value product. Ethanol is of interest because it can be used as an alternative fuel or oxygenate additive to
the current fossil fuels. In the production of ethanol from OPEFB, the material is first hydrolyzed for example
by dilute-sulfuric acid. The hydrolysis of OPEFB by 0.8% sulfuric acid at 190oC for 5 min resulted in the max-
imum xylose yield with low formation of by products (HMF and furfural). Meanwhile, the hydrolysis of OPEFB
by 0.8% sulfuric acid at 210oC for 5 min gave the maximum yield of glucose with relatively low formation of
by-products. The yields of glucose and xylose at the two conditions of hydrolysis corresponded to 13.2 and
49.8% of the theoretical yields, respectively. Based on the results from one-stage hydrolysis, two-stage hydroly-
sis was performed in order to produce high glucose concentration. The residue from the first stage was separated
and it was then further hydrolyzed by 0.8% sulfuric acid at 210oC for 5 min. Hydrolyzate produced from the
second stage was used to verify the ethanol production in the fermentation process. The result shows that etha-
nol was produced by Saccharomyces cereviceae with ethanol yields of 0.46 g/g under anaerobic condition.
Keywords: oil palm empty fruit bunch, acid hydrolysis, ethanol, Saccharomyces cereviceae.
INTRODUCTION the ash is utilized as a potassium fertilizer. How-

ever, burning empty fruit palm oil bunch to solve
With oil palm plantation and crude palm oil the disposal problem causes another environ-
(CPO) production reached 6.8 million ha and mental problem, i.e. pollution as a result of in-
19.3 million metric tons in 2008, respectively, complete combustion and very fine size of ash
Indonesia is now the biggest palm oil producer particles. Thus, utilization or conversion of these
in the world (Directorate of Plantation of the by-products to value-added products is of great
Ministry of Agriculture, 2008). At the same importance. In this work, we focused on the
time, in addition to CPO, Indonesia accumulates empty fruit bunch as the raw material to be stud-
a large amount of lignocellulosic wastes in the ied.
palm oil industry. The lignocellulose wastes in- One of growing interest in bioethanol pro-
clude the empty fruit bunch (OPEFB) (tandan duction is producing ethanol from lignocellulo-
kosong kelapa sawit, TKKS), the fronds sic wastes, which are abundant from various
(pelepah), and the trunks (batang). Having neg- sources of biomass. Bioethanol attracts interest
ligible commercial value, these wastes become because it can be used not only as chemical for
problematic. Not only they cause serious dispos- industry but also as an alternative fuel for vehi-
al problem, but they occupy a large area for stor- cles. Lignocellulosic wastes from oil palm are
age. If one ton of fresh fruit oil palm bunch pro- potential raw materials for bio-ethanol produc-
duce nearly 234 kg of CPO and 217 kg of tion, especially due to the constant availability.
OPEFB (Lacrose, 2004), about 17.9 million met- The oil palm empty fruit bunch (OPEFB) could
ric tons of OPEFB was accumulated in 2008. contain cellulose (44.2%), hemicellulose
Traditionally, the empty bunch is burned for (33.5%), and lignin (20.4%) (Azis et al., 2002).
steam and power generation for the factory and The cellulose and hemicellulose content of

OPEFB can be hydrolyzed for example by chem- In addition to sugars (glucose and xylose), the
ical hydrolysis. Hydrolysis involves cleaving the hydrolyzate was also characterized with respect
polymers of cellulose and hemicelluloses into to furfural, HMF, and acetic acid. The results
their monomers. Complete hydrolysis of cellu- from two different hydrolysis temperatures were
lose results in glucose, whereas the hemicellu- used as a basis to determine the conditions to
lose results in several pentoses and hexoses. perform two-stage hydrolysis. The hydrolyzate
Among the chemical hydrolysis methods, dilute- produced was then verified for ethanol produc-
acid hydrolysis is probably the most commonly tion using Saccharomyces cerevisiae as the fer-
applied. This method has been successfully de- menting microorganism.
veloped to degrade lignocellulosic materials with
considerably success (Taherzadeh et al. 1997). MATERIALS AND METHODS
Due to the different structure of cellulose and
hemicelluloses, dilute-acid hydrolyisis is sug- Oil Palm Empty Fruit Bunch (OPEFB): Oil
gested to be carried out in two stages. The hemi- palm empty fruit bunch used in the current work
cellulose fraction is depolymerized at lower was obtained from palm oil company, Pagar
temperature or lower retention time than the cel- Merbau, Medan, North Sumatera, Indonesia.
lulose fraction. If hemicellulose is further hydro- Fresh OPEFB was shredded and sun dried until
lyzed in one-stage hydrolysis at higher tempera- the water content was around 10%. The size of
ture or higher retention time, there is a risk that dried OPEFB was reduced up to 2 mm length to
the formed monosaccharides will be degraded obtain small fraction of OPEFB.
into by-product compounds such as acetic acid,
phenolic compounds, furfural, and 5- Hydrolysis:
hydroxymethyl furfural (HMF) according to One-Stage Hydrolysis: Oil-bath reactor system
chemical reaction illustrated in Fig 1. was used for all the hydrolysis experiments. It
consisted of steel-tube reactors, an oil-bath tank,
and a thermostat. As the heating medium, special
oil that has a melting point more less 150oC was
used. The oil-bath reactor system was equipped
with a stirrer to homogenize the temperature
within the oil-bath tank.
Fig 1. Chemical reactions in dilute-acid hydrolysis of
Prior to hydrolysis experiments, 5 g of
lignocellulose OPEFB was soaked in the tube reactors in 50 ml
of 0.2 and 0.8% sulfuric acid solution. The tube
Therefore, based on the hydrolysis reaction reactors were then immersed in the oil-bath tank
above, degradation of monosaccharides should for 5 and 15 min and at various temperatures
be avoided not only to maintain high yield of (170, 190, 210 and 230oC). After hydrolysis re-
sugars but also to avoid the inhibition effect by- action was completed, tube reactors were cooled
product compounds in the fermentation of the until room temperature. The liquor (the hydroly-
sugars to ethanol. Previously, hydrolysis of zate) was drained and samples were taken to be
OPEFB has been studied under relatively low analyzed glucose, xylose, furfural, HMF, and
temperatures (120oC) and long retention time (up acetic acid.
to 90 min) (Rahman, et al., 2006). The hydroly-
sis was carried out using relatively concentrated Two-Stage Hydrolysis: Two-stage acid hydroly-
acid of up to 6% H2SO4 with the aim to degrade sis of OPEFB was performed based on the se-
hemicelluose in connection to xylose production. lected conditions of one-stage acid hydrolysis of
The optimum H2SO4 concentration of 6% and OPEFB. After the first-stage hydrolysis was
reaction time of 15 min was found to yield xy- completed and the liquor was drained, the solid
lose about 90%. residues remained in the tube reactor. After that
The current work studied the effect of some sulfuric acid was added and the solid residue was
hydrolysis parameters, which included concen- hydrolyzed again. The hydrolyzate both from
tration of acid used, temperature, and reaction first and second stage were collected and stored
time. The temperature was studied at two levels, at 4oC before being used in the fermentation pro-
i.e. low temperature (<200oC) and high tempera- cess. Some hydrolyzate was taken as a sample to
ture (>200oC) using dilute H2SO4 (less than 1%). be analyzed for xylose, glucose, acetic acid, fur-

fural, and HMF. The samples were stored at - 60°C with 0.6 ml/min eluent of 5 mM sulfuric
20oC before analysis. acid. Glucose and xylose were analyzed using
The Yeast: The yeast S. cerevisiae ATCC 96581 HPX-87P column (Bio-Rad, USA) at 85°C with
provided by University College of Borås was ultra-pure water as eluent at 0.5 ml/min. Concen-
used in this work. The strain was kept on agar trations of glucose, xylose, ethanol, and acetic
plates made of yeast extract 10 g/l, soy peptone acid were determined by RI chromatograms,
20 g/l, agar 20 g/l, and D-glucose 20 g/l. In the while furfural and HMF were determined by UV
inoculum preparation, a loopfull of yeast was chromatograms at 210 nm.
suspended in the growth medium.
RESULTS AND DISCUSSION
Fermentation:
Inoculum Preparation: One loopfull of culture One-Stage Hydrolysis: Oil palm empty fruit
was grown in 50 ml cotton-plugged Erlenmeyer bunch was hydrolyzed in one-stage hydrolysis
flask on a shaker of 80 rpm at 30oC. The liquid using 0.2 and 0.8% sulfuric acid at 170, 190, 210
volume was 10 ml. The growth medium was a and 230oC for 5 and 15 min. The results are pre-
defined medium including 50 g/l glucose as presented in Table 1. Table 1 shows that higher
viously reported (Taherzadeh et al., 1996). After yields of glucose and xylose were obtained at
incubation for 24 h, inoculum was then inoculat- 0.8% acid compared to the yields at 0.2% acid.
ed in 300 ml cotton-plugged Erlenmeyer flask on It can be seen in Table 1 that at acid con-
a shaker of 80 rpm at 30oC. The liquid volume centration of 0.8%, temperature had a significant
was 100 ml. The growth medium was a defined effect on the hydrolysis of OPEFB. The maxi-
medium including 50 g/l glucose as previously mum yield of glucose and the by-products of
reported (Taherzadeh et al., 1996). After incuba- furfural and acetic acid occurred at temperature
tion for 24 h, 110 ml of inoculum was obtained. of 210oC. Whereas the maximum yield of xylose
and HMF occurred at temperature of 190 and
Cultivation: The second stage hydrolyzate was 230oC, respectively. If the content of glucan and
first adjusted to 5.5 by addition of 10% of xylan follow the composition according to Rah-
NaOH. It was then sterilized at 121oC for 15 man et al. (2006) (Table 2), the maximum yields
min. The cultivation was carried out in 300 ml in glucose and xylose were 13.2 and 49.8% from
a loop trap Erlenmeyer flask. The loop trap con- the theoretical values, respectively. Theoretical
tains glycerol to avoid air to diffuse into the flask glucose and xylose were calculated from the to-
but the gas outlet can still pass. Pure nitrogen tal content of xylan and glucan in the OPEFB.
was sparged through a sterile filter in order to Increasing retention time of hydrolysis from
maintain the anaerobic conditions. The flask was 5 to 15 min resulted in decrease the yields of
incubated for 72 hours at 30oC and shaked at 80 glucose at 210oC and xylose at 190oC. On the
rpm. The pH was controlled at 5.5 by addition of other hand, the yields of furfural and HMF were
10 M of NaOH. The total liquid volume was 155 significantly increased. The reason for this could
ml including 135 ml hydrolyzate, 5 ml inoculum, be that longer retention time makes the sugars
and 15 ml of a solution containing mineral solu- degrade to form furfural and HMF. This also
tion. Liquid samples were withdrawn at 0, 2, 4, probably means that 5 min hydrolysis is actually
8, 12, 24, 36, 48, 60, and 72 h, which would be enough to depolymerize cellulose at their corre-
analyzed by HPLC. sponding temperature. On the other hand, since
the concentration of acetic acid was higher at
Analysis: The samples were analyzed by HPLC retention time of 15 min than that of 5 min this
(Waters, Milford, USA), which was equipped indicates that the hemicelluloses part has been
with a RI detector (Waters 410) and a UV ab- degraded within 15 min. Thus, the optimum re-
sorbance detector (Waters 486). Ethanol, HMF, tention time should be sometime between 5 and
furfural, and acetic acid were analyzed using 15 min.
Aminex HPX-87H column (Bio-Rad, USA) at

Table 1. The yields of glucose, xylose, furfural, HMF, and acetic acid at different hydrolysis temperature, retention time
using acid concentration of 0.2 and 0.8%
T (oC) Retention time (min) Yglucose Yxylose YHMF Yfurfural Yacetic acid
0.2% acid 170 5 0.00 0.00 1.21 0.00 11.07

170 15 0.00 1.99 0.00 0.65 9.31
190 5 0.00 0.00 0.00 0.00 8.24
190 15 1.70 2.58 0.31 2.99 13.00
210 5 0.00 7.18 0.76 1.66 9.63
210 15 15.71 8.57 0.14 17.32 28.69
230 5 0.76 0.00 0.82 23.11 34.65
230 15 0.38 0.00 3.90 17.22 34.97
0.8% acid 170 5 7.76 53.20 0.00 1.02 12.08

170 15 7.81 89.76 0.58 6.65 17.54
190 5 12.12 135.94 0.86 10.47 23.67
190 15 25.32 105.65 4.97 53.49 34.18
210 5 62.70 94.42 13.69 27.44 8.98
210 15 54.67 0.00 25.95 84.21 36.48
230 5 52.54 0.00 28.16 64.12 29.51
230 15 4.50 0.00 5.99 40.10 24.72
Yield = (g/kg OPEFB)
Table 2. Main component of oil palm empty fruit bunch Fermentation by S. cerevisiae: Fermentation
fiber was carried out under anaerobic conditions at
Main fraction Composition (%)
Glucan 42.85
30oC. The experiment was conducted to verify
Xylan 24.01 ethanol production from hydrolyzate originally
Lignin 11.70 produced from OPEFB. The parameters meas-
Ash 0.52 ured during fermentation were glucose consump-
Others 20.92 tion, ethanol production, and HMF, furfural con-
Adapted from Rahman et al. (2006)
sumption (Fig 2).
Fig 2 shows that S. cerevisiae completely
Two-Stage Hydrolysis: Based on the results
consumed glucose within 24 h. Within that time,
from one-stage hydrolysis and with a goal to
ethanol was produced and the concentration was
produce as high as possible glucose concentra-
slightly increased up to 48 h. Ethanol concentra-
tion due to the inability of S. cerevisiae to assim-
tion remained constant within the last 24 h (up to
ilate xylose, two-stage hydrolysis was carried out
72 h). It can be concluded that 1 day is enough
using 0.8% acid concentration in the following
for ethanol production from the hydrolyzate by
conditions: first stage hydrolysis was performed
S. cerevisiae under anaerobic conditions. The
at 170oC for 15 min and the second stage hy-
fermentation of the hydrolyzate resulted in etha-
drolysis was performed at 210oC for 5 min. At
nol yield of 0.46 g/g. Furfural and HMF, which
the end of the first stage, the hydrolyzate was
are known as inhibitors for S. cerevisiae (Lars-
drained and the solid residue remained in the
son, 1999) could be consumed by the yeast (Fig
tube reactor. The acid was added and the solid
2). One possible explanation for this is that S.
residue was hydrolyzed again according to the
cerevisiae converted furfural into furfuryl alco-
conditions at the second stage of hydrolysis. The
hol and furoic acid by enzyme alcohol dehydro-
hydrolyzate from the second stage was collected
genase and by enzyme aldehyde dehydrogenase,
and it would be fermented by S. cerevisiae in the
respectively (Taherzadeh et al., 1999). Like fur-
subsequent process. The composition of the hy-
fural, HMF was converted by S. cerevisiae into
drolyzate from this second stage was (g/l): glu-
its corresponding alcohol, HMFAL (5-
cose 5.3; furfural 0.32; and HMF 0.32.
hydroxymethyl-furfural alcohol) (Taherzadeh et
al., 2000). The high ethanol yield and the ability
of the yeast to consume furfural and HMF sug-

gest that the concentration of those inhibitors ACKNOWLEDGEMENTS
were relatively low and in tolerable level for the
yeast used in this work. The authors are grateful to The International
Program Office of Sweden for the financial sup-
6 port of this work.
5
REFERENCES
Concentration (g/l)
4 glucose
ethanol
3
HMF Azis, A.A., Husin, M. and Mokhtar, A. 2002. Preparation of
2
Furfural cellulose from oil palm empty fruit bunches via etha-
1 nol digestion: effect of acid and alkali catalysts. J. Oil
0
Palm Res. 14 (1): 9-14.
0 20 40 60 80
Directorate General of Plantation of the Ministry of Agri-
Time (h)
culture. 2008. Palm oil statistic. Directorate General
of Plantation of the Ministry of Agriculture. Depar-
Fig 2. The profile of glucose, ethanol, HMF, and furfural tement of Agriculture of Republic of Indonesia.
during fermentation of the hydrolyzate from the
second stage using S. cerevisiae. Lacrosse, L. 2004. Business Prospects in Southeast Asia for
European Cogeneration Equipment, ASEAN COGEN
3 Seminar, EC-ASIANCOGEN Programme Phase III.
CONCLUSIONS
Larsson, S., Palmqvist, E., Hahn-Hägerdal, B., Tengborg,
C., Stenberg, K., Zacchi, G. and Nilvebrant, N. 1999.
Dilute-acid hydrolysis can be applied to The generation of fermentation inhibitors during di-
produce simple sugars from OPEFB. If the glu- lute acid hydrolysis of softwood. Enzyme Microb.
cose is the main sugar to be produced, tempera- Technol. 24: 151-159.
ture of 210oC for 5 min using 0.8% acid are the Rahman, S.H.A., Choudhury, J.P. and Ahmad, A.L. 2006.
conditions of hydrolysis could be applied based Production of xylose from oil palm empty fruit bunch
on the results in this work. On the other hand, if fiber using sulfuric acid. Biochem. Eng. J. 30: 97-103.
xylose is the main sugar to be produced, temper- Taherzadeh, M.J., Eklund, R., Gustafsson, L., Niklasson, C.
ature of 190oC for 5 min using 0.8% acid are the and Lidén, G. 1997. Characterization and fermenta-
conditions recommended to be applied. In order tion of dilute-acid hydrolyzates from wood. Ind. Eng.
Chem. Res. 36: 4659-4665.
to optimize the production of the sugars, two-
stage hydrolysis process is suggested. The first Taherzadeh, M.J., Gustafsson, L., Niklasson, C. and Lidén,
stage is intended to produce xylose from the G. 1999. Conversion of furfural in aerobic and anaer-
obic batch fermentation of glucose by Saccharomy-
hemicellulose fraction. The second stage is in- ces cerevisiae. J. Biosci. Bioeng. 87: 169-174.
tended to produce glucose from the cellulose
Taherzadeh, M.J., Gustafsson, L., Niklasson, C. and Lidén,
fraction. The hydrolyzate produced from the se- G. 2000. Physiological effects of 5-hydroxymethyl-
cond stage was successfully fermented into etha- furfural on Saccharomyces cerevisiae. Appl. Microbi-
nol by S. cerevisiae with ethanol yield of 0.46 ol. Biotechnol. 53: 701-708.
g/g under anaerobic conditions. Taherzadeh, M.J., Lidén, G., Gustafsson, L. and Niklasson,
C. 1996. The effect of pantothenate deficiency and
acetate addition on anaerobic batch fermentation of
glucose by Saccharomyces cerevisiae. Appl. Microbi-
ol. Biotechnol. 46: 176-182.

Two Stage Processes of Oil Palm Empty Fruit Bunch Fiber Kraft Pulp
for Bioethanol
Yanni Sudiyani1 and Teuku Beuna Bardant1
1
Research Center for Chemistry- Indonesian Institute of Sciences
Kawasan Puspiptek Serpong, 15314, Tangerang, Indonesia
Phone: +62-21-7560090 and Fax.: +62-21-7560549, e-mail: ysudiyani@ yahoo.com
(Correspondence to: Yanni Sudiyani)
ABSTRACT
The evaluation related to the suitable production of ethanol from oil palm empty fruit bunch (EFB) kraft pulp
were studied. Two stage processes consisting of enzymatic saccharification and fermentation using Saccharo-
myces cerevisiae MK1 has been used in this study. Substrate concentration of EFB was varied from 5 to 10%,
cellulase was added at 4 g/L substrate, while the yeast inoculum was 10% (w/v). To obtain the fermentable sug-
ar of EFB, enzymatic saccharification was done at 40oC, pH 4.5 and 5.0, 100 rpm. After saccharification the
medium were filtered by RO and then the hydrolyzate was then subjected to fermentation for 72 hours. Samples
were withdrawn at regular time intervals for analysis. Ethanol production were obtained after 24 h, however,
ethanol concentration was decreased after 60h of fermentation. The highest ethanol concentration was obtained
from samples with substrate concentration of 10% (w/v).
Keywords: oil palm EFB, kraft pulp pretreatment, saccharification, fermentation, ethanol
INTRODUCTION to ethanol (Lezinou, 1994). Application of this

method need very careful environmental assess-
Energy consumption has increased steadily ment since the liquid acid catalyst was used. En-
as the world population has grown and more zymatic hydrolysis is another option. However,
countries have become industrialized. The fossil there still so many technical problem, such us
fuels have been the major resources to meet the low of the resulted fermented sugar concentra-
increased energy demand. However, they are tion and long process duration, that make the
gradually being depleted extinction because they application is hardly feasible from the economic
are not renewable (Zhu, 2006). Therefore, there point of view.
is a great interest in exploring alternative energy This paper deals with experiment on two
sources to maintain the sustainable growth of stage processes of EFB kraft pulp for bio-ethanol
society (Rajagopalan, 2005). consisting of separate enzymatic saccharification
Indonesia is currently the greatest world and fermentation (SHF) using Saccharomyces
producer of palm oil, with 51% of the world out- cerevisiae). Separation using filtration to in-
put; therefore, palm oil constitutes a great asset crease the fermented sugar concentration was
for this country. The palm oil industry in Indo- applied in between saccharification and fermen-
nesia generates approximately 15.2 x 106 t of tation. This steps was applied in order to in-
solid wastes consisting of oil palm empty fruit crease the ethanol concentrations produced so it
bunches fiber and fruit shell every year. Empty can be feasible for the purification.
fruit bunch (EFB), one of the major solid wastes
from palm oil industry, is a complex lingo- MATERIALS AND METHODS
cellulosic material consists of 41.3 – 46.5 % cel-
lulose, 25.3 – 33.8 % hemicelluloses and 27.6 – Raw material: Oil palm EFB was collected from
32.5 % lignin. It has a great potency as a source an Oil Palm Plantation belongs to PT Perke-
of cellulose and could be utilized as raw materi- bunan Nusantara VIII, in Pandeglang, Banten,
als for the fermentative production of bio- Indonesia. After collection, the EFB was
ethanol (Sudiyani, 2008). chopped to fiber and dried then it was stored in
Conventional methods applied to biocon- sealed plastic bags at room temperature until it
version of cellulose to ethanol involve acid hy- was used for pretreatment.
drolysis of the biopolymer followed by the fer-
mentation of resulting soluble oligosaccharides

Pulping and pulp analyses: The EFB fiber was the next 1.5 hours. The soften coconut fiber was
cut into 3-5 cm length and then it was digested separated from the cooking liquor and it was
using kraft process (NaOH and Na2S 22% as washed thoroughly until free from sodium hy-
active alkali, sulfidity 30%, fiber: cooking liquor droxide. The soften coconut fiber was then fibril-
= 1:5). Digestion was conducted in two steps. lated in a disc refiner 2 times. Yield and Kappa
First, 1.5 hours to reach temperature of 165oC, Number of pulp were determined based on the
and then the temperature was kept at 165oC for standard test methods TAPPI T 236 cm-85.
EFB CHIPPING KRAFT PULPNG WASHING
EFB PULP YIELD PULP SCREENING

BEATING
KAPPA NUMBER
Fig. 1. Diagram of the present investigation.
Analyses of raw material: The EFB before and Fermentation:

after pulping process were characterized as fol- Stock culture of yeast
lows: moisture content (gravimetric), The EFB The yeast Saccharomyces cerevisiae MK 1
was determined measured for its moisture con- available at our laboratory was used in fermenta-
tent by gravimetric method and oil content tion process. Active culture of yeast was pre-
(Soxhlet extraction) by Soxhlet extraction as cultured in Potatoes Dextrose Agar (PDA) 3%,
well as its lignin, cellulose and hemicellulose agar (0.25 g), H2O (50 mL) and was incubated
contents (Goering & Van Soest, 1970). for 2 days at 37oC. This pre-cultured yeast was
used for yeast inoculums in fermentation.
Enzymatic Saccharification: Enzymatic hydrol- Fermentation reaction contained the EFB
ysis of EFB kraft pulp was carried by Meicellase hydrolizate of EFB substrate concentration (5,
enzyme commercial from Meiji Seika, Japan. 7,5, 10% w/v), 0.05M acetate buffer pH 4,5 and
Concentration of the substrate (5, 7.5, 10% w/v), 10% (v/v) yeast inoculums. The sample, nutri-
cellulase 4 g/L, and 0.05M acetate buffer pH 4,5. ent and buffer were sterilized (121oC, 20 min).
The samples were incubated at 40oC, 100 rpm The nutrient medium was composed of 10 g/L
for 48 hrs. (NH4)2PO4; 0,05 g/L; MgSO4. 7H2O, and 2 g/L
yeast extract. Fermentation experiments were
Sugar separation: Fermented sugar was separat- carried out in 250 ml Erlenmeyer flask, cultivat-
ed from water to increase the sugar concentra- ed on an orbital shaker at 100 rpm for 72 hours
tions. Separation was conducted in Reverse os- at 37oC. Samples were taken at regular intervals
mosis filter with 1400 cm2 surface and pressure for analysis reducing sugar and ethanol content.
10.000 psi at room temperature.
Ethanol analysis: Ethanol was estimated by gas
Sugar analysis: At different time intervals, 1.0 chromatography GC-9A Shimadzu, Japan, with
mL of each samples were withdrawn from hy- column PEG, SE 30 Chromosorb W 80-100
drolysates (taking care that the slurry density mesh. The column oven was operated isother-
was not disturbed) and were centrifuged for 1-2 mally at 120oC and the detector and injection
min at 3000 rpm. Reducing sugars were deter- port were kept at 150oC. Nitrogen was used as
mined by Nelson-Somogyi method using spec- the carrier gas at 30-60 mL/ min flow rate, and
trophotometer U-2000 Hitachi Japan. the combustion gas was a mixture of hydrogen

and air. All experiments were conducted in du- Lignocellulosic biomass requires pretreat-
plicate and average values reported. ment, mainly because the lignin in plant cell
walls forms a barrier against enzyme attack. An
RESULTS AND DISCUSSION ideal pretreatment reduces the lignin content and
crystallinity of the cellulose and increase surface
Chemical compositions of EFB before and af- area (Takagi et al., 1977). In order to degrade
ter: Before pretreatment total carbohydrate in the and reduce the lignin and non cellulose compo-
oil palm EFB fiber was 56.49% of the total bio- nent, EFB was made into pulp using kraft pro-
mass. It consisted of 33.25% -cellulose and cess, which involved treatment with alkali solu-
23.24% hemicelluloss. The lignin content of tion and heat at certain times and followed by
EFBs was 25.83%, which was comparable to the fibrillation using disc refiner. After this treat-
lignin content of hardwoods (Mandel et al. ment lignin and hemicellulose content was de-
1976). Thus, major component of EFB was - creased significantly to 9.96% and 10.25%, re-
cellulose (33.25%), a polymer of glucose which spectively, while the holocellulose and alfa cel-
is very potential as sugar source for ethanol pro- lulose increased to 94.37% and 84.12%, respec-
duction. tively, as shown in Table 1.
Table 1. The chemical compositions of EFBs before and after pretreatment with alkaline in kraft pulping process.
EFB Water Extractives Lignin Holocellulose Alfa cellulose Hemicellulose
(% wb) (% db) (% db) (% db) (% db) (% db)
Initial EFB 8.56 4.19 25.83 56.49 33.25 23.24
EFB Kraft Pulp 73.79 0.63 9.96 94.37 84.12 10.25
Sacharification and fermentation of EFB by The best result was obtained at the highest
SHF system: The resulted fermented sugar from substrate input as expected. Then the highest
sacharification process for 5, 7.5 and 10 % are result was compared to the SSF process. SSF is
1.11, 0.94 and 0.57 %.vol respectively. All of Simultaneous Sacharification Fermentation,
this hydrolyzate was then concentrated from which means the sacharification and fermenta-
1500 ml to 300 ml using reverse osmosis filtration conducted together. The result was shown in
tion. The resulted concentrated hydrolyzate for Fig. 3.
5, 7.5 and 10 % are 3.37, 2.84 and 1.73 % vol.
the separation process gave very high sugar loss Ethanol Concentration using SHF and SSF methods
since the filter area was used are very large. 2,50
Then the concentrated hydrolyzates were SSF
SHF
fermented using Saccharomyces cerevisiae MK 2,00
EtOH conc. (%)
1. Samples were taken every 24 hours and the 1,50

ethanol concentrations were measured. The
measurement results were shown in Fig. 2. 1,00
Ethanol
0,50
concentration (%)
2,00
1,80 0,00
1,60 0 20 40 60 80
1,40 Time (hours)
1,20
1,00
0,80 Fig. 3. Data comparison between SSF and SHF.
0,60
0,40 It was shown that the SSF process gave
0,20
0,00
slightly higher concentration than SHF. Biggest
0 20 40 Time (hr) 60 80 problem of using SHF process is the sugar loss
5% 7,50% 10% in separation step. However, SHF system did not
required additional macronutrient as much as
Fig. 2. Resulted ethanol concentrations from SHF system. SSF. Further study will be required for feasibil-
ity from economic point of view.
CONCLUSIONS

Lezinou, V., P. Christakopoulos, D. Kekos, J. B. Macris.
1994. Simultaneous saccharification and fermentation
The SHF of the conventional pretreated of of sweet sorghum carbohydrates to ethanol in fed-
EFB kraft pulp to ethanol were investigated us- batch process. Biotechnolology letters Vol. 16 No.9:
ing cellulose enzyme and S.cerevisiae. The op- 983-988.
tima concentration of EFBs was 10% (w/v) sub- Mandels, M., R. E. Andreotti & C. Roche. 1976. Measure-
strate. Under the optimum substrate concentra- ment of saccharifying cellulase. Biotechnol. Bioeng
tion the ethanol concentration reaches 1.7 % vol. Symp. 6: 21-23.
Further study is still required to find out whether Rajagopalan S., E. Ponnamplam, D. Mc calla & M. Stow-
the sugar loss will be significant if the working ers. 2005. Enhanching profitability of dry mill ethanol
volume was folded several times. SSF applica- plants. Appl Biochem Bioethnol. 120 (1): 37-50.
tion is more suitable for process using low sub- Sudiyani, Y., A. Syarifah, J. Waluyo & E. Hermiati. 2008.
strate load. Dilute acid pretreatment and enzymatic saccharifica-
tion of oil palm empty fruit bunch fiber for ethano
production. Procc. of the 4th Int. Biotechnology con-
ACKNOWLEDGEMENT ference, Bogor 5-7 August 2008. 257-264.
The authors thank to the student Dede Rop- Takagi, M., S. Abe, S. Suzuki, G. H. Emert & N. Yara.
1977. A method for production of alcohol directly
iah, who have given assistance to this experi- from cellulose using cellulase and yeast. Proceedings
mental work. This work was funded by Competi- of Bioconversion of Cellulosic Substances into Ener-
tive Project of Indonesian Institute of Sciences gy, Chemicals and Microbial Protein, ed. T. K.
(LIPI) of fiscal year 2008-2009. Ghose, I. I. T., Delhi. 551-571.
Zhu, S., Y. Wu, Z. Yu, X. Zhang, C. Wang, F. Yu & S. Jin.
REFERENCES 2006. Production of ethanol from microwave-assisted
alkali pretreated wheat straw. Process Biochem. 41:
869-873.
Goering, H. K. & P. J. Van Soest. 1970. Forage fiber analy-
sis (apparatus, reagents, procedures, and some appli-
cations). Agricultural Handbook No. 379. Agricultur-
al Research Service – United States Department of
Agriculture: Washington. DC.

Bactericidal Activity and Mechanism of Supercritical N2O
Sungmin Mun1, Youn-Woo Lee2, Jeyong Yoon1
1
Biofilm Engineering Lab., School of Chemical and Biological Engineering, Seoul National Universi-
ty, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea, TEL : +82-2-880-8927, FAX: +82-2-876-
8911 , e-mail : jeyong@snu.ac.kr
2
Supercritical Fluid Process Lab., School of Chemical and Biological Engineering, Seoul National
University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea
(Correspondence to: Jeyong Yoon)
ABSTRACT
Recently, there is growing interest with non-thermal processing due to the increased consumer demand for
nutritious and fresh-like food products. These products are required non-thermal sterilization in order to ensure
the microbiological safety without the deterioration of the product quality. Hence, supercritical CO 2 (SC-CO2)
has been widely investigated as a promising alternative non-thermal sterilization technique for foodstuffs and
pharmaceutic products, but the decrease of pH in aqueous medium after SC-CO2 treatment is a major drawback
which may cause to reduce the enzyme activities in foodstuffs and to restrict the application for pH sensitive
materials. To overcome the drawback, SC-N2O has been considered as an alternative supercritical fluid because
of no pH decrease in aqueous after treatment. However, the bactericidal effect of SC-N2O was not clearly under-
stood still despite the previous studies. In order to elucidate the exact inactivation mechanism of microbe by SC-
N2O, we examined the inactivation behavior of bacteria by SC-N2O compared to SC-CO2. Viability, the release
of cellular components, cellular proteins, enzyme activities, morphology of bacteria were investigated and com-
pared after exposure to both supercritical fluids. The inactivation of P. aeruginosa by SC-N2O was similar to
that by SC-CO2, showing more than 8 log10 CFU/ml inactivation of P. aeruginosa within 5 ~ 6 min at 100 bar
and 37oC. As additional advantages, SC-N2O extracted approximately 3 - 5 times more the cellular components
such as nucleic acids and proteins than SC-CO2 without serious decrease of enzyme activities and modification
of cell morphology compared to SC-CO2.
Keywords: Supercritical N2O, supercritical CO2, non-thermal sterilization, inactivation mechanism
INTRODUCTION croorganism without significant deteriorating the

physical and chemical quality of the juice.
Supercritical CO2 (SC-CO2) has been wide- However, little work concerning the SC-
ly investigated as a promising alternative non- N2O bactericidal application has been carried
thermal sterilization technique for foodstuffs and out, and the inactivation mechanism for the SC-
pharmaceutic products, but the decrease of pH in N2O application remains largely unclear. In order
aqueous medium after SC-CO2 treatment is a to elucidate the exact inactivation mechanism of
major drawback which may cause to reduce the microbe by SC- N2O compared to SC-CO2.
enzyme activities in foodstuffs and to restrict the
application for pH sensitive materials (Zhang et MATERIALS AND METHODS
al., 2006; Spilimbergo et al., 2007). To over-
come the drawback, SC-N2O has been consid- Pseudomonas aeruginosa PA01 was used in
ered as an alternative supercritical fluid because this study as an indicator microorganism. The
of no pH decrease in aqueous after treatment. initial population of P. aeruginosa for inactiva-
Recently, Spilimbergo et al. (2007) demonstrated tion experiment ranged from 1.2 x 108 to 3.5 x
the comparable microbicidal effect by SC-N2O 108 CFU/ml. Supercritical fluids sterilization
in comparison with SC-CO2, showing that about experiments were conducted in the high pressure
5 log inactivation of S. cerevisiae was achieved experimental system which was used in our pre-
by SC-N2O for 30 min (100 bar and 36oC). More vious study (Mun et al., 2009). Pressure, tem-
recently, Gasperi et al. (2009) performed a com- perature, mixing intensity, and working volume
parative study to investigate the effects of SC- considered as a potential important factor affect-
N2O and SC-CO2 pasteurization on the quality of ing the biocidal activity were investigated to fig-
fresh apple juice. This study demonstrated that ure out their effect on bactericidal activities. The
both SC fluids successfully inactivated the mi- viability of cells was examined by plate counting

method. To analyze the released cellular compo- treatment, the effect of increasing pressure on
nents, the treated cell suspension was centri- inactivation of P. aeruginosa was less than SC-
fuged to separate supernatant from cell pellet. CO2 treatment. Spilimbergo et al. (2007) report-
The release of cellular components such as nu- ed that the inactivation of total microbial in an
cleic acid and proteins in the supernatant was apple juice by SC-N2O treatment decreased, de-
measured by a UV-vis spectrometer at 260 and spite of small extent, as pressure increased from
280 nm, respectively. To confirm of the exist- 100 to 200 bar. They explained that a pressure
ence of released protein, the supernatant was increase over 100 bar leads to a decrease of N2O
analyzed by SDS-PAGE. diffusion inside the liquid phase and resulting
low biocidal efficiency of SC-N2O. However,
RESULTS AND DISCUSSION this phenomenon was not detected during the
inactivation of P. aeruginosa by SC-N2O as
Fig. 1 shows the P. aeruginosa inactivation pressure increased from 100 to 200 bar.
using SC-N2O as a function of pressure and two Effect of temperature by SC-N2O treatment
temperatures (37oC and 42oC) compared to SC- on inactivation of P. aeruginosa appeared to be
CO2. As shown in Fig. 1A and B, SC-N2O exhib- modest and again comparable to compared to
ited an effective disinfection power for P. aeru- SC-CO2 as shown in Fig. 1 as the temperature
ginosa that was comparable to SC-CO2. The SC- increased from 37oC to 42oC. One explanation
N2O treatment at standard condition achieved for the temperature effect was reported that in-
about 8 log CFU/ml reduction after about 6 min. creasing temperature on SC-CO2 application can
The bactericidal efficiency by SC-N2O treatment stimulate not only the diffusivity of CO2, but
was slightly increased with increasing pressure also the fluidity of the cell membrane to make
from 100 bar to 200 bar, which is comparable to penetration easier (Garcia-Gonzalez et al.,
that of SC-CO2 treatment. In case of SC-N2O 2007).
0
A B
N2O 100 bar 37oC CO2 100 bar 37oC
N2O 150 bar 37oC CO2 150 bar 37oC
o
-2 N2O 200 bar 37 C CO2 200 bar 37oC
o
N2O 100 bar 42 C CO2 100 bar 42oC
Log(N/No)
-4
-6
-8
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Time (min) Time (min)
Fig. 1. Comparison of the bactericidal effect of SC-N2O (A) and SC-CO2 (B) as a function of pressure and temperature on P.
aeruginosa. Experiments were carried out with a working volume ratio of 10% and an agitation speed of 600 rpm.
Fig. 2A and B reveal the UV absorbance of teins because of cell or cell membrane damage
the supernatants obtained from the SC-N2O caused by the SC fluid treatment. Fig. 2 shows
treatment at 260 nm and 280 nm, which repre- that the UV absorbance of the SC-N2O treated
sented nucleic acids and proteins, respectively supernatants was significantly higher (approxi-
(Hong et al., 2001). The high absorbance indi- mately 3 ~ 4 times) than the SC-CO2 treatment.
cated the larger release of nucleic acids or pro- Therefore, the greater cell or cell membrane

damage during SC-N2O treatment brought about treatments such SC-CO2, because the SC-N2O
the efficient inactivation of P. aeruginosa, even treatment had a greater extraction capacity.
without the acidic pH effect caused by SC fluid
0.12
A B SC-N2O
SC-CO2
0.09
Absorbance (OD260, 280)
0.06
0.03
0.00
0 5 10 15 0 5 10 15
Time (min) Time (min)
Fig. 2. The absorbance of the supernatants of the P. aeruginosa cell suspensions at 260 nm (A) and 280 nm (B) after both
SC-N2O and SC-CO2 treatment. Experiments were carried out with an agitation speed of 600 rpm and a working ratio of
10% at 37oC and 100 bar.
Fig. 3 shows the existence of released pro- efficiency of intracellular components than SC-
teins after both supercritical fluids treatment. CO2. The similar sterilization effect of SC-N2O
High and various protein bands were observed without pH decrease compared to SC-CO2 may
with SC-N2O treatment. Fig. 2 and 3 results ex- be caused with this high extraction potential of
hibit that SC-N2O has more higher extraction SC-N2O.
Fig. 3. SDS-PAGE analysis of the released proteins in the supernatant of the P. aeruginosa cells treated with SC-N2O (lanes
6, 7, and 8) and SC-CO2 (lanes 3, 4, and 5). Experiments were carried out with an agitation speed of 600 rpm and a working
ratio of 10% at 37oC and 100 bar (Lane 1, standard marker proteins; lane 2, untreated supernatant of the P. aeruginosa cells
as the control).
In conclusion, this study examined the in- CFU/ml inactivation. Among the reaction pa-
fluence of the SC-N2O treatment on the inactiva- rameters studied in this work, the bactericidal
tion behavior of P. aeruginosa in comparison to efficiency of SC-N2O was the most influenced
SC-CO2. The SC-N2O treatment was as effective by mixing intensity, and slightly increased with
as the SC-CO2 treatment for inactivating P. ae- increasing pressure, temperature, and with de-
ruginosa, and required about 6 min for the 8 log creasing working volume. Furthermore, a higher

extraction power for intracellular components Gasperi, F., E. Aprea, F. Biasioli, S. Carlin, I. Endrizzi, G.
such as proteins and nucleic acids was observed Pirretti & S. Spilimbergo. 2009. Effect of supercritical
CO2 and N2O pasteurization on the quality of fresh
in the SC-N2O treatment compared to SC-CO2. apple juice. Food Chem. 115:129-136.
This study clearly suggested that the SC-
Hong, S. & Y. Pyun. 2001. Membrane damage and enzyme
N2O application was a viable option over SC- inactivation of Lactobacillus plantarum by high pres-
CO2 for application, when changes in the ensure CO2 treatment. Int. J. Food Microbiol. 63:19-28.
zyme activities and pH or modification of the Mun, S., S. Jeong, J. Kim, Y. Lee & J. Yoon. 2009. Inactiva-
cell morphology were not desirable. tion of Pseudomonas aeruginosa biofilm by dense
phase carbon dioxide. Biofouling. 25:473-479.
REFERENCES Spilimbergo, S., D. Mantoan & A. Dalser. 2007. Supercriti-
cal gases pasteurization of apple juice. J. Supercrit.
Garcia-Gonzalez, L., A. H. Geeraerd, S. Spilimbergo, K. Fluids. 40:485-489.
Elst, L.V. Ginneken, J. Debevere, J. F. V. Impe & F. Zhang, J., S. Burrows, G. Gleason, M. A. Matthews, M.
Devlieghere. 2007. High pressure carbon dioxide in- LaBerge & Y. H. H. An. 2006. Sterilization using
activation of microorganisms in foods: The past, the high-pressure carbon dioxide. J. Microbiol. Meth.
present and the future. Int. J. Food Microbiol. 117:1- 66:479-485.
28.

Phycocyanin Production of Spirulina Fusiformis Culture
in an Open Space Tubular Photobioreactor
Tjandra Chrismadha, Yayah Mardiati and Mey R. Widoretno
Research Centre for Limnology LIPI

Kompleks LIPI Cibinong, Jl. Raya Bogor Km 46 Cibinong 16911, e-mail: tjandra5660@yahoo.co.id
(Correspondence to: Tjandra Chrismadha)
ABSTRACT
An experiment of Spirulina fusiformis culture for phycocyanin production in a simple open space tubular
fotobioreactor has been carried out by application of relatively cheap common available cylindrical PE plastic
column for the tube. The tube diameter of 15 cm was winded 6x10 m to channel culture aliquot between two
collector tanks which was provided by circulatory centrifugal pumps. It was placed in an open space with solar
radiation as the light source, in which the radiation was reduced about 70% by means of para net. The result
shows that S. fusiformis was successfully grow in the plastic column fotobioreactor resirculated culture in a
batch mode placed in the open space. The average growth rate was 0.040 µ/day and the weekly culture biomass
concentration was 0.44–0.512 g/l, corresponds to the culture productivity of 2.28 g/m/day. The average phy-
cocyanin content was 1.99% of the dry weight, which gives the level of productivity equivalent to 163.57
kg/ha/year. This result shows the advantage of plastic column photobioreactor in an open space to produce pig-
ment phycocyanin from S. fusiformis culture.
Keywords: algal culture, Spirulina fusiformis, photobioreactor, phycocyanin.
INTRODUCTION witzka, 1994; Chrismadha et al., 2000; Janssen

et al., 2003). Unfortunately, the efforts to devel-
Spirulina is a potential blue-green alga for op these production systems in to a commercial
natural source of food and feed, particularly in scale still face various obstacles of the environ-
terms of its high content of natural pigment mental factors, as well as the economical profi-
phycocyanin, which has been reported as high as ciency (Borowitzka, 1999; Janssen et al., 2003).
20% of the total protein (Richmond, 1988). The In tropical area, which is believed to be the most
high phycocyanin content could be of interest for prospective place to produce microalgal biomass
economical scale production, since the pigment as the area receive constantly plenty daily solar
has been reported to have various advantages, radiation along the year, is in fact suffers from
including antioxydant, antiinflamatory, necrosis over-saturated as well as high amplitude of the
cancer inhibitor, and neural cell protection daily light radiation. Beside increasing microal-
(Romay et al., 1998, 2003; Reddy et al., 2000), gal cells photorespiration, high light intensity
which is considered to have a prospective market also produce great daily themperature fluctuation
in pharmaceutical industries. This high and require a fairly expensive control mecha-
phycocyanin content is also supported by the nism (Lee, 2001). In addition, tubular photobio-
ability of the alga to growth in extreemly basic reactor is economically unattractive due to high
media up to pH 11, so that it is easier to maintain investment cost (Carlsson et al., 2007).
as monoalgal culture for a long period of time. The first trial to employ tubular photobiore-
Microalgal culture development, such as for actors for biomass production of S. fosiformis in
phycocyanin production mensioned above, up to an open space shows that the blue green alga can
this time is still obstacled by low culture produc- be successfully grown in the photobioreactors
tivity (Borowitzka & Borowitzka 1989; Carlsson (Chrismadha & Widoretno, 2009), although
et al., 2007). Accordingly, efforts to develop some indications of over saturated light was re-
microalgal cultures for phycocyanin production vealed. This paper reports a further trial to opti-
can not be only directed to optimization of the mise the algal growth and productivity in the
phycocyanin content, but also to the various photobioreactor by providing culture agitation
growth and productivity factors. One among the which was expected to keep the algal filaments
most prospective microalgal culture system is in suspension as well as to homogenize the cul-
tubular photobioreactor (Chrismadha & Boro-

ture physicochemical conditions to support a tubes to maintain the gas balance inside the col-
higher biomass and phycocyanin productivity. umns.
The culture medium was modified Zarouk
MATERIAL AND METHODS medium (Borowitzka, 1988). The inoculum was
from a 400 l tank monoculture which was poured
The blue-green alga S. fusiformis used in into the collection tank with dilution rate of 30X.
this experiment was obtained from microalgal The culture was then grown in a batch mode and
collection of the Mariculture Laboratory of Re- harvested once a week until the growth was ex-
search Centre for Oceanography-LIPI Jakarta. hausted at day 51. Sampling for biomass content
Some initial experiments showed that the alga measurement was carried out twice a week,
can be grown in a semi-open place. while those for the biochemical content were
The trial was carried out at Research Centre once a week along with the harvesting time.
for Limnology-LIPI Cibinong, where the daily The algal biomass is expressed by means of
light can reach more than 100 klux about noon the dry weight, which was determined by filter-
between 11–14 o’clock, while the daily average ing 20–100 ml culture aliquot through a tared
of the maximum light intensity are 60 klux. To Whatman GF/A filter paper. The filter paper was
reduce the impact of oversatiurated solar radia- then dried in an oven at 60oC over night and
tion, the tubular photobioreactor was placed in a weighted. The algal dry weight was obtained by
shade of para net which reduce the radiation up subtracting the weight of algal biomass contain-
to 70%. ing filter paper by the initial weight.
The tubular photobioreactor was made from Ten ml culture aliquot was also filtered
PE grade cylindrical plastic material which is through Whatman GF/A filter paper for analysis
commonly available in market and relatively of chlorophyll content, total carbohydrate, total
cheap. The used plastic column was 15 cm in protein and total lipid. The chlorophyll content
diameter with total length of 60 m, winded into was determined by extraction in 90% acetone
six parallel columns by means of u-form PVC according to Jeffrey & Humprey (1975). The
tubes. Each column edges were connected into total carbohydrate was analyzed by phenol-
two revervoir tanks of 60 l made of fibre glass sulphuric acid method following Kochert (1978),
material. The photobioreactor was filled by cul- while the total protein determination employed
ture suspension to make up 40 cm culture depht folin-phenol method of Lowrey et al. (1951).
in the reservoir tanks. Two submersible pumps The total lipid content was determined gravimet-
of 3500 l/h capacity was installed to circulate the rically according to Bligh & Dyer (1959). The
culture aliquot from the one reservoir tank to the phycocyanin measurement was carried out by
other which is then gravimetically flow trough extraction in pH 7 buffer solution (10.64 g
the plastic column back to the first reservoir K2HPO4 and 5.29 g KH2PO4 in a liter of
tank. A gas outlet was provided at every PVC aquades) according to Boussiba & Richmond
(1979).
1,2
1
Solar Radiation
0,8
(100 klux)
0,6
0,4
0,2
0
0:00:00 3:00:00 6:00:00 9:00:00 12:00:00 15:00:00 18:00:00 21:00:00 0:00:00
Tim e
Fig 1. Daily solar radiation of the experimental place

coincidence with rainy seasons, whereas the pre-
vious trial was in the peak of dry season. The
lower radiation as well as the shorter light period
in the rainy season can be responsible for the
lower biomass productivity observed in this trial.
It is in consistent with the fact that no indication
of physiological stress due to oversaturated solar
radiation, such as was observed in the previous
trial in which a remarkable cell carbohydrate
accumulation was occurred (Fig 3) (Chrismadha
et al., 2009; Torzillo et al., 2003).
Fig 2. S. fusiformis culture in the tubular photobioreactor
0,8
Dry Weight (g/l)

0,6
0,4
S. fusiformis was grown successfully in the
cylindrical plastic made tubular photobioreactor 0,2
in a resirculated batch culture under the reduced 0

solar radiation open space. The average daily 50 Protein Carbohydrat Lipid
growth rate was 0.040 /day, in which the high-
40
est growth rate was observed at day 24, which
% Dry Weight
was 0.056 /day. The weekly biomass concen- 30
trations at the harvesting time were 0.44–0.51 20
g/l, whereas the total biomass harvested along 10
the culture period was 524.70 g. This amount 0

corresponds to the culture productivity of 2.28
g/m2/day, which is much lower compared to that 3,5
Chlorophyill
previous trial without resirculation system that 3 Phycocyanin
achieved 5–8 g/m2/day (Chrismadha et al., 2,5
% Dry Weight
2009). It is also lower compared to that reported 2
by Carlsson et al. (2007) where the microalgal 1,5
1
culture productivity was 2–50 ton/ha/year.
0,5
This phenomenon can be associated with
0
shear factor induced by the culture agitation, par- 10 17 24 31 38 44 51
ticularly the circulating pump including the ex- Days
tention pipe which deliver the culture to the oth- Fig 3. Biomass and the biochemical content development
er collection tank. This shear factor caused stress of S fusiformis grown in a an open space tubular
to the microalgal cells and increased the respira- photobioreactor
tion rate. As has been previously reported, even
though culture agitation is required to avoid cell Biochemical composition of the S. fusiform-
settlement as well as to maintain the culture is culture in this trial was protein 37.8–44.3%,
physico-chemical condition, but excessive agita- carbohydrate 12.1–28.7% and fat 7.8–12.1% of
tion can give effect of increasing cell respiration the dry weight. It indicates the healthy grown
and reduce the biomass productivity (Pirt et al., cells. As has been previously reported (Chrisma-
1983; Gudin & Chaumont, 1991). Chrismadha et dha et al. 2006) in a laboratory scale experiment
al. (2000) also reported the increase of algal cell under sufficient nitrogen and phosphorus condi-
respiration in a tubular photobioreactor caused tion, the protein content of S. fusiformis was 50–
by more intensive agitation due to addition of 60% of the dry weight. At the same time, the
compartement inside. Increasing respiration rate carbohydrate content was 29–40%. There was
exhausts the cell energy which is normally useful also no observable significant change in the
for biomass formation, so as to reduce the cul- biochemical composition during the trial period,
ture biomass productivity. indicating a good culture condition during 51
The lower biomass productivity in this trial days trial period.
also can be attributed to the trial time which was

It was observed that the pigment content of support from the Government of Indonesia
S. fusiformis, both chlorophyll and phycocyanin through the National Development Budget (DI-
tend to decrease along with the culture age. In PA) year 2009. A great expression is also ad-
the early stage the chlorophyll concentration was dressed to Deni Hadiansyah for helping in the
1.18% of the dry weight and lower to 0.36% in tubular photobioreactor preparation.
the late phase of the culture. The same decreas-
ing pattern was also observed in the algal phy- REFERENCES
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late phase of the culture. A trend of chlorophyll lipid extraction and purification. Canadian J. Bio-
chem. Physiol. 37: 911-917.
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has been widely reported (Chrismadha & Boro- Borowitzka, M.A. 1988. Algal growth media sources of
witzka, 1994; Chrismadha et al., 2009). This is algal cultures. In: Borowitzka, M.A. and Borowitzka,
L.J. (Eds.). Microalgal biotechnology. Cambridge
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Borowitzka, M.A. 1999. Commercial production of micro-
deficiency has been reported to inhibit both chlo-
algae: ponds, tanks, tubes, and fermenters. J. Biotech-
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Borowitzka, M.A. and Borowitzka, L.J. 1989. Industrial
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fusiformis biomass was 1.99% of the dry weight. technology. Longman Scientific and Technical. Lon-
As the culture biomass productivity was 2.28 don. 294-315.
g/m2/day, the phycocyanin productivity can be Boussiba, S. and Richmond, A. 1979. Isolation and purifi-
calculated as much as 45.44 mg/m2/day or cation of phycocyanins from the blue-green alga
equivalent to 163.57 kg/ha/year. This level of Spirulina platensis. Archives Microbiol. 120: 155–
159.
phycocyanin productivity is largely higher com-
paring to those in laboratory scale experiment Carlsson, A.S., Beilen, J.B., Moller, R. and Clayton, D.
which accounted only 2.64 mg/l/day (Chrisma- 2007. Micro- and macro-algae: Utility for industrial
applications. CPL Press. United Kingdom. 82.
dha, 2007). This can be attributed particularly to
the remarkable higher biomass productivity in Chrismadha, T. and Borowitza, M.A. 1994. Effect of cell
density and irradiance on growth, proximate composi-
the tubular photoreactor placed in the open space tion and eicosapentanoic acid production of Phaeo-
due to the higher daily light intensity. In agree dactylum tricornutum grown in a tubular photobiore-
with this, Sukenik (1991) has reported the actor. J. Appl. Phycol. 6: 67-74.
important to maintain both high biomass Chrismadha, T., Sutapa, I.D.A., Hidayat, Rosidah and
productivity and unsaturated fatty acids content Mardiati, Y. 2000. Pengaruh cahaya intermitan ter-
when algal culture was employed for unsaturated hadap fotosintesis kultur alga Chlorella vulgaris. Ma-
fatty acids production. In addition, Vonshak & kalah dipresentasikan pada Seminar Nasional Biologi
VIII. Bandung, 17 Juli 2000.
Richmond (1985) also emphasized the important
of biomass productivity in assessing the Chrismadha, T., Panggabean, L.M. and Mardiati, Y. 2006.
economic proficiency af an algal culture. As Pengaruh konsentrasi nitrogen dan fosfor terhadap
pertumbuhan, komposisi biokimia, serta produksi
shown by this experimental result, an effort to fikosianin pada kultur Spirulina fusiformis. Berita Bi-
optimize phycocyanin production based on a ologi. 8 (3): 163-170.
blue green alga S. fusiformis culture also requires
Chrismadha, T. 2007. pengaruh periode panen terhadap
conditions that support the culture to achieve produktivitas biomassa dan kandungan fikosianoin
high biomass productivity along with its high kultur Spirulina fusiformis. Prosiding Konferensi Aq-
cell phycocyanin content. This trial shows the uaculture Indonesia 2007. 164-168.
advantage of the tubular photobioreactor to Chrismadha, T., Mardiati, Y. and Widoretno, M.R. 2009.
produce phycocyanin from S. fusiformis culture Pengaruh diameter kolom fotoreaktor terhadap per-
in open space. tumbuhan Spirulina fusiformis di tempat terbuka. Pro-
siding MAI 2009 in press.
ACKNOWLEGEMENTS Gudin, C. and Chaumont, D. 1991. Cell fragillity: the key
problem of microalgal mass production in closed pho-
tobioreactors. Biores.Technol. 38: 145-151.
This experiment is part of research activities
implementing in Research Centre for Limnology Janssen, M., Tramper, J., Mur, L.R. and Wijffels, R.H.
2003. Enclosed outdoor photobioreactors: light re-
of Indonesian Institute of Sciences with financial

gime, photosynthetic efficiency, scale up and future Reddy, C.M., Bhat, V.B., Kinarmay, G., Redding, M.N.,
prospects. Biotechnol. Bioeng. 81: 193-210. Reddana, P. and Mediastla, K.M. 2000. Selective in-
hibition of cyclooxygenase-2 by c-phycocyanin, a bil-
Jeffrey, S.W. and Humprey, G.F. 1975. New spectrophoto- liprotein from Spirulina platensis. Biochem. Biophys.
metric equation for determining chlorophyll a, b, c1, Research Comm. 277: 597–603.
and c2 in higher plants, algae and natural phytoplank-
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Borowitzka, L.J. (Eds). Microalgal Biotechnology.
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Physiological and Biochemical Methods. Cambridge bau, V. 2003. c-Phycocyanin: a biliprotein with anti-
University Press. Cambridge. 95-75. oxidant, anti-inflammatory and neuroprotective ef-
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Technol. Biotechnol. 33B: 35-58. ing the biotechnology of algal biomass production.
Plant and Soil. 89: 129–135.

Using Compost as Biofilter to Reduce N2o Emission
Tania Surya Utami1, Lila Adriaty1, Heri Hermansyah1 and M. Nasikin1
1
Chemical Engineering Department, Faculty of Engineering, University of Indonesia
Kampus Baru UI Depok, Depok 16424, Phone No. 7863516
E-mail: nana@che.ui.ac.id ; lila_adriaty@yahoo.com
(Correspondence to: Lila Adriaty)
ABSTRACT
A large quantity of pollution is the important thing to solve that caused serious environmental problems.
Many pollutants are emitted from various industrial processes and transportation activities such as particulate
CO, CO2, SO2, VOCs, Pb and NOx. Nitrogen oxides (NOx) consist of about 95% nitric oxide and about 5%
nitrogen dioxide, both of which are hazardous air pollutants and cause serious environmental problems. One of
NOx compound is N2O which is fourth biggest greenhouse gas (after CO2, CH4, water vapor), can contribute to
global warming. N2O has 320 times the global warming potential of CO2 which is predicted that will be in-
crease. Biofiltration is the last technology pollution control for removal N 2O with compost as medium filter.
This technology has advantages such as low installation and operation cost, secure operation, low energy con-
sumption, good stability and able to remove pollutant with high efficiency. Effects of N2O flow rate, water con-
tent, and usage nature and synthetic nutrient supplement in compost which is adding Nitrobacter, sp. will be
investigated towards to N2O gas reduction efficiency for 9 hours in batch system. Decreasing concentration of
N2O was analyzed with Gas Chromatograph (GC) and increasing quantity of microorganism in compost as filter
material was analyzed with Total Plate Count (TPC). The result indicates that the highest N2O gas reduction
efficiency is obtained under biofilter length 50 cm and gas flow rate 72.02 cc/min and 60% water content as
conditions for removal efficiency was achieved. The result shows that N2O gas removal efficiency could be
optimized by adding synthetic nutrient supplement in compost which’s been mixed with Nitrobacter, sp., hence
75.9 % of removal efficiency. Results of TPC show increasing bacteria population before and after biofiltration.
Keywords: biofilter, compost, N2O, Nitrobacter, sp., removal efficiency.
INTRODUCTION quired high temperatures and the use of cata-

lysts, involving high installation and operation
A large quantity of pollution is the im- costs as well as generating a large quantity of
portant thing to solve that caused serious envi- secondary waste for which manufacturers had to
ronmental problems. Many pollutants are emit- pay cleanup and disposal costs (Pandey, 2004).
ted from various industrial processes and trans- Economic and technical constraints in SCR and
portation activities such as particulate CO, CO2, SNCR methods motivated researchers to develop
SO2, VOCs, Pb and NOx. Nitrogen oxides new, cost-effective processes to removal NOx
(NOx) consist of about 95% nitric oxide and from flue gas. Biological NOx treatment has
about 5% nitrogen dioxide, both of which are been deemed a promising alternative to eliminate
hazardous air pollutants and cause serious envi- industrial waste generation and enable compli-
ronmental problems (Yang et al., 2007). One of ance with emission standards (Shuler & Kargi,
NOx compound is N2O which is fourth biggest 1992).
greenhouse gas (after CO2, CH4, water vapor), Biofilter is a biological treatment of con-
can contribute to global warming. N2O has 310 taminated air which is an emerging technology
times the global warming potential of CO2 which for air pollution control. The principle is rela-
is predicted that will be increase. Global warm- tively simple; a contaminated air stream is
ing will be influenced to climate change, is hap- passed through medium filter. Like all biological
pened because just little sun gleam could be back treatment processes, air biotreatment relies on
to atmosphere. microbial reactions for degradation of waste
In the past, traditional control technologies, compound. Biofilter systems can be operated
such as Selective Catalytic Reduction (SCR) and under ambient temperature with the use of inex-
Selective Non-Catalytic Reduction (SNCR), pensive microbial inoculate. One such system,
were applied to control NOx emissions in some the biofilter, is a kind of biochemical fixed bed
industries. However, these two processes re- reactor wherein microorganisms settle on the

surface of the filter medium material and form a ciency. The steps before doing experiments are
biofilm; the airborne substances are absorbed medium preparations which make compost as
and utilized by microorganisms (Sutanto, 2002). filter material, leakage test, flow rates calibration
Biofilter treatment has been proven effective in which know the actual flow rates and N2O cali-
treating odors and volatile organic compounds bration before using Gas Chromatography.
(VOC), such as benzene, styrene, phenols, and
alkenes. In the present study, the parameter op-
eration in biofilter will be investigated is how 3
operational parameters (flow rate, moisture, and
nutrient) effect the N2O removal efficiency.
The medium filters used in this research are 2

a compost bed medium. The compost contained 4
mixture of goat manure and wood chips was ob-
tained from Sekolah Alam Indonesia as compost
maker. Compost was sieved before use to pre-
N2O 1
vent filter blocking with filter 100 mash. Husks
were added to the compost to serve as a bulking
agent that could increase free space, reduce
compaction, and enhance ventilation in the sys-
tem. Nitrous dioxide (N2O) and air were ob- 5
tained from Samator Gas (Cikarang, Bekasi-
Indonesia) which has concentration about 15.000
ppm. Fig. 1. Schematic diagram of laboratory scale biofilter.
In this study will be investigated natural and (1: N2O gas supply, 2: Flow meter, 3-5: Sampling port, 4:
synthetic nutrient for reduction contaminated air Biofilter column).
with biofilter. Nutrient supplement was provided
to the biofilter as the source of carbon (glucose), This experiment is conducted to decrease
inorganic nutrients, and moisture. The medium N2O concentration by using biofilter which is
was added nutrient 40 mL for synthetic nutrient. investigated parameter operation such as flow
The nutrient solution (pH 8.0) contained the fol- rates, water content of filter material which is
lowing components (in 1 L of H2O): K2HPO4 conducted for 9 hours. N2O concentrations of
(0.4 g), KH2PO4 (0.15 g),NH4Cl (0.3 exhaust gas from biofiltration process are meas-
g),MgSO4·7H2O, (0.4 g), sodium acetate 2.93 g, ured by Gas Chromatography (GC) (Shimadzu,
and 2 mL of trace element solution, which con- Tokyo, Japan). Experimental gases were injected
tained (in 1 L of H2O): EDTA (50.0 g), from the column top, and the flow rate was regu-
ZnSO4·7H2O (2.2 g), CaCl2·2H2O (5.5 g), lated by a set of precision flow controllers. The
MnCl2·4H2O (5.06 g), FeSO4·7H2O (5.0 g), off gas was discharged to the atmosphere via
(NH4)6Mo7O24·2H2O (1.1 g), CuSO4·5H2O (1.57 ventilation. Total Plate Count (TPC) method is
g) and CoCl2·H2O (1.61 g). These inorganic ma- conducted to measure quantity of microbes be-
terials were selected because they had previously fore and after biofiltration.
been used to grow aerobic nitrifying bacteria
(Rene et al., 2005). Natural nutrient supplements RESULTS AND DISCUSSION
is liquid waste from cow husbandry waste (Ku-
kusan Teknik, Depok-Indonesia) Effects of flow rates variation: The graph in
Fig. 1 shows the schematic diagram of the Fig. 2 shows that longer retention time between
biofilter column. Biofilter equipment is designed the compost and N2O gases will cause decreas-
as experiment need with batch system. The flow ing in concentration of N2O from the lower bio-
meter is used which has the smallest flow rates filter column. Fig. 2 also shows that the smaller
about 70 cc/minutes. In order to get longer reten- gas flow rate the higher removal efficiency of
tion time in filter material of biofilter. Longer N2O. Fig. 2 shows that at t = 1-2 hours is the ar-
retention time will get higher N2O removal effi- ea where the unsteady flow situation through the
medium of pollutant gases. In Fig. 2 also can be

shown that the removal efficiency profile for the filter media was short. This can also occur due to
flow rate 127.22; 185.74; 232.89 cc / minute had the use of dry composting so as to facilitate N2O
a fluctuation plot at t = 1 hour and t = 2 hours. gas flow, and it caused of less moisture content
Because at these flow rates the residence time on which cause channeling (Govind, 1998).
0 1 2 3 4 5 6 7 8 9
t (hour)
Q = 72 cc/min Q = 88 cc/min Q = 105 cc/min
Q = 127 cc/min Q = 186 cc/min Q = 232.89 cc/min
Fig. 2. Effects flow rate variations to N2O removal efficiency.
Flow rate (cc/min)
Fig. 3. Comparison N2O removal efficiency in N2O flow rate variation.

(h = 50 cm, dried medium, t = 9 hours)
In Fig. 3 can be shown that highest N2O tion and degradation processes more than the
removal efficiency (RE) is occurred in lowest greater N2O gas flow rate. N2O gas removal effi-
N2O gas flow rate 72 cc/min with removal efficiency is also greater at every hour contact time
ciency, 56.7%. N2O removal efficiency tends to between the compost and N2O gas. More with
increase when flow rate is smaller because of the previous explanation, and if it is associated
N2O gas residence time in the filter medium be- with the adsorption curve in general, the concen-
comes longer and the contact time between N2O tration of a adsorbat will decrease because ab-
gas and biofilter medium is also longer. As a sorbed by the adsorbent to the removal efficien-
result the intensity of N2O gas through adsorp- cy of the N2O gas as a contaminant will increase

compared to other smaller flow rates. So it can crobial growth in it. The variation of water con-
be concluded based on the bar graph in Fig. 3 tent is 30%, 40%, 50%, 60%, and 70% (w/w)
that the smaller flow rate, the greater N2O re- weight of compost. Range variation of water
moval efficiency. content 30-70% which is based on previous stud-
ies that investigated reducing the moisture con-
The effects of water content variations: This tent in the other pollutants (Yonghui et al.,
experiment aims to find out how effects of water 1998). The height of the filter medium used was
content in the compost biofilter to reduce N2O. 50 cm with the smallest flow rate (72 cc/min)
Addition of water content, aims to increase the based on the previous result.
humidity of filter medium, which affects the mi-
C N2O (g/m3)
t (hour)
30% (w/w) 40% (w/w) 50% (w/w) 60% (w/w) 70% (w/w)
Fig. 4. The effects of water content to N2O removal efficiency.
Fig. 4 is represented concentration profiles In addition, biofilms provide essential nutrients

decrease with the variation of water content in for biological activity, and maintain moisture for
which is added to the filter. Fig. above also illus- bacterial growth. Thus, the optimum humidity in
trates the trend with the three segments of the the area will improve the performance of mi-
profile up, down and up again slowly. When the crobes in biofilms degrading N2O gas that dif-
experiment was carried out and begins to biofil- fuses into the pores of the particles will be dis-
tration process (t = 0) is assumed N2O gas con- solved into the compost in layers of biofilms and
centrations in the biofilter column is close to degraded by the microbes contained therein.
zero so that the N2O gas output concentration
also considered zero. This is why N2O removal
efficiency equal to zero. However, at t = 1 hour
N2O gas has not been detected yet, whereas at t =
2 hours there is N2O gas with very small concen-
trations, which then increases until a certain time
(6-7 hours). This is because N2O gas residence
time in filter medium is longer because increas-
ing in humidity biofilms which make medium
porosity became smaller. In Fig. 5, it can be seen
30 40 50 60 70
that highest removal efficiency is in moisture
Additional water, w/w (%)
content of 60% (w/w) with efficiency of 70.13%.
In this process the influence of humidity Fig. 5. N2O removal efficiency in water content variation.
biofilms larger compared to the use of dry com- (h = 50cm, f = 72 cc/minutes, t = 9 hours).
post. Regional biofilms on particle filter medium
is aerobic area and contains water and the mi- N2O gas removal efficiency tends to in-
crobes to the degradation of pollutants flowing. crease at the optimum water content for each

particular filter medium. Based on Fig. 5 can be by nutrition which could increase microbe’s ac-
seen that the N2O removal efficiency are the tivity to reduce N2O. The microbes needed nutri-
most effective at water content 60% (w/w) of the tion to be able to survive and increase their ac-
weight of compost. When the water content is tivities.
more than 0.6 g water / g dry weight of the me- The experiment was conducted with a filter
dium, the biofilter efficiency is reduced. Remov- medium height 50 cm, flow rate 72 cc/min and
al efficiency increases than previous experiments the addition of optimum water content of 60%
because optimum moisture in compost and po- (w/w) compost. Dissolved nutrients added as
rosity decrease which make longer residence many as the best water volume in the previous
time of N2O in the compost. As a result, more experiments. The added natural nutrients in the
intensity of N2O through adsorption and degra- form of liquid waste from dairy farms, while the
dation processes, so that N2O removal efficiency synthetic nutrient given solution consists of nu-
is also increase at every hour contact time be- trient and trace element. Nutrient solution was
tween the compost and N2O gas. So, based on given as much as 40 ml supplemented with trace
Fig. 5 concluded that at 60% water content of element solution as much as 2 ml micronutrient
compost will result in higher N2O removal effi- then dissolved into the water so that the same
ciency. with the addition water content of 60%. Given
nutritional composition consisting of K2HPO4,
Comparison synthetic and natural nutrition: KH2PO4, NH4Cl, MgSO4.7H2O, COONa. While
This experiment had a purpose to know how the trace element solution consisting of EDTA,
effects of addition natural and synthetic nutri- ZnSO4.7H2O, CaCl2.2H2O, MnCl2.4H2O,
tion. The effects of using those two nutrition de- FeSO4.7H2O, (NH4)6MO7O24.2H2O,
pend on N2O removal efficiency that was pro- CuSO4.5H2O, CoCl2.H2O. These compounds
duced. In this experiment, compost as the filter have been selected because previously used to
medium was given Nitrobacter,sp. as the nitrifi- grow aerobic nitrifying bacteria.
cation bacteria. Then, this filter medium is added
t (hour)
Nitrobacter sp. Nitrobacter sp.+ Synthetic Nitrobacter sp.+ Natural
Nutrition Nutrition
Fig. 6. Nutrition addition in the compost with Nitrobacter,sp. to N2O removal efficiency.
Fig. 6 shows the profile of the addition of time until 6-7 hours. Differences only in nutri-
natural and synthetic nutrients in the compost ents addition which increase the moisture con-
that has been given Nitrobacter,sp. to N2O re- tent of biofilter performance and degrading mi-
moval efficiency. Fig. 6 show that graph con- crobe’s performance in N2O reduction. This is
tains three segments like previous result. This is differences between addition of nutrients and
because the condition of the same filter medium water content. The difference between the two
that contains water. So that N2O distribution in experiments lies on the adsorption and degrada-
the compost is not different from the unsteady tion process in biofilter performance. In compost

which added natural and synthetic nutrition, N2O addition of synthetic nutrients are better than the
removal efficiency is higher than water content natural nutrients.
addition only.
The purpose of nutrient addition is intended Microbes testing in composting: Tests for mi-
to support the growth of nitrifying bacteria based crobial growth in compost before and after the
on previous research. Therefore, to maximize biofiltration conducted by Total Plate Count
use of nutrients, the compost is added with Ni- (TPC). TPC is one of the analysis methods to
trobacter,sp. before given nutrient solution. In determine colonies number of microbes in sam-
order to, this comparison has initial parameters, ple. TPC is one of the calculation techniques
the experiments conducted in compost with add- using Nutrient Agar (NA) as a medium for bac-
ed Nitrobacter,sp. without supply of nutrients. teria to be counted. The results of the calculation
From these parameters can be compared the ef- the TPC will be represented in terms of Colony
fect of adding nutrients both synthetic and natu- Forming Units (CFU) per gram of compost.
ral to the increased N2O removal efficiency. Based on Table 1 can be shown that after biofil-
Here is a comparison of the use of synthetic and tration has been done, the number of microbes
natural nutrition in reducing N2O. From Fig. 7 calculated by TPC method is increasing. This
can be seen the difference between three treat- can be seen from number of microbes from
ments. 5.32.109 be 1.08.1010 CFU/g.
Table 1. TPC test results before and after biofiltration.
∑ Bacteria after
Sample of TPC tests
biofiltration (CFU/g)
Compost before biofiltra- 5.32.109
tion
Compost after biofiltration 1.08. 1010
Nitrobacter sp Nitrobacter sp.+ Nitrobacter sp.+
Syntetic Natural This increase may occur as described in the
Nutrients Nutrients
previous discussion. One of them is the energy
Fig. 7. Comparison nutrients for compost to N2O removal (ATP) derived from the transformation of air
efficiency. pollutants that flow to the biofilter (Shuler &
Kargi, 1992). The result is number of bacteria
Fig. 7 shows that the highest removal effi- contained in the compost is higher than that of
ciency is in synthetic nutrients addition with N2O dry compost was also used to process biofiltra-
removal efficiencies, 76.9%. When compared tion. The existence of water content can create
with experiment without nutrient, removal effi- optimum environmental conditions for microbial
ciency 4.2% higher and when compared with the growth, thus increasing the performance of these
use of natural nutrients removal efficiency 2.2% bacteria in degrading to get more nutrients from
higher. Increased efficiency due to the reduction the results of such degradation. The phenome-
of N2O due to the addition of synthetic nutrients nons of increasing number of bacteria in the
nutrition has minerals needed in particular micompost after biofiltration strengthen analysis of
crobe microbes directly related to the degrada- degradation processes that occur in the reduction
tion of N2O. Meanwhile, natural nutrients not of pollutants in the biofilter performance. It can
have as complete mineral nutrient than synthetic. also be concluded that the compost has been
However, natural nutrient also contains many used in biofiltration process is getting better
additional microbial results from cow dung, quality. This is because increasing the number of
where the microbes are also helpful in reducing bacteria as a fertilizer plant in the compost.
biofilter performance N2O. Nutritional composi- In Conclusion, Performance of biofilter in
tion and trace element that is added contains el- this study in achieving N2O removal efficiency
ements N, S, P, Ca, K, Na, Mg, Fe, Co, and Zn. 75.9% with a medium height of 50 cm, N2O flow
According to Shuler and Kargi (1992) minerals rate 72 cc/min, 60% water content and the addi-
needed by the microbes containing S, P, Ca, K, tion of synthetic nutrients and Nitrobacter,sp. on
Na, Mg, Fe, Co, and Zn. Just as that added in compost as a filter medium. The use of both nat-
this experiment, that causes the results from the ural and synthetic nutrition can improve N2O
removal efficiency. Nitrobacter,sp. and synthetic

nutrients addition in compost can improve the Shuler, M. L. & F. Kargi F. 1992. Bioprocess engineering–
N2O removal 2.2% higher than the natural nutri- basic concepts. Prentice Hall, Englewood Cliffs.
ents. TPC test results is shown after biofiltration Sutanto, R. 2002. Penerapan Pertanian Organik. Jakarta.
done, the number of microbes that calculated by Yang, W. F., H. J. Hsing, Y. C. Yang & J. Y. Shyng. 2007.
the TPC method increasing. The Effect of Selected Parameters on the Nitric Oxide
Removal by Biofilter. J. Hazardous Materials.
148(3): 653-659.
REFERENCES
Yonghui, L., X. Quan, Y. Zhao, S. Chen & H. Zhao. 2004.
Govind, R. 1998. Biofiltration: An Innovative Technology Removal of Ternary VOCs in air streams at high
for the Future. Paper submitted to Environmental loads using a compost-based biofilter. J. Biochemical
Progress. Engineering Journal.23(1): 85-95.
Pandey, A. 2004. Concise Encyclopedia of Bioresource

Technology. The Haworth Press, Inc: New York.
Rene, E. R., D.V.S. Murthy & T. Swaminathan. 2005. Per-
formance evaluation of a compost biofilter treating
toluene vapors, Process. Biochem. 40: 2771–2779.

Influence of 1,4-Dioxane in Enzymatic Process for Biodiesel Production
Using Crude Palm Oil (CPO) as Substrate
Krishna Purnawan Candra1, Sukartin2, Fitriani2 and Yuliani1
1
Chemistry and Microbiology Laboratory, Department of Agricultural Product Technology,
Faculty of Agriculture, Mulawarman University
Kampus UNMUL Gunung Kelua, Jl.Tanah Grogot, Samarinda, Indonesia,
e-mail: kcandra_99@yahoo.com
2
Microbiology Laboratory, Center for Research and Industrial Standardization Samarinda
Jl. Biola, Samarinda, Indonesia
(Correspondence to: Krishna Purnawan Candra)
ABSTRACT
Nowadays, mass biodiesel production is usually using chemical reaction method. However, since last
decade there are many biodiesel researches using enzymatic method. In this enzymatic process, esterification
and transesterification can happen concurrently. Besides, there are many advantages using enzymatic method
compared to chemical method. As Indonesia is being on of the largest producers of Crude Palm Oil (CPO) in the
world, producing renewable energy based on plant oil is a huge potential for this country. In this report, we
show the effort to increase the yield of biodiesel production using enzymatic process with CPO as raw material
by using emulsifier. We have demonstrated a biodiesel (FAME) production by enzymatic process using
Pseudomonas cepacia lipase and Crude Palm Oil (CPO) as substrate. In this experiment, the yield of biodiesel
was still very low. Introducing of 1,4-dioxane as emulsifier in producing biodiesel using CPO as substrate did
not affected significantly the yield of biodiesel.
Keywords: CPO, Pseudomonas cepacia, enzymatic process, biodiesel.
INTRODUCTION from Sigma, kaolin as carrier for enzyme

immobilization, alkyl ester for C14:0, C16:0,
Nowadays, mass biodiesel production is C16:1, C18:0, C18:1, C18:2, and C18:3,
usually using chemical reaction method. potassium dihidrogenphosphate and potassium
However, since last decade there are many hidrogenphosphate was obtained from Merck.
biodiesel researches using enzymatic method. In
this enzymatic process, esterification and FAME production: Thirty grams (33,9 mmol)
transesterification can take place concurrently. of CPO were mixed with methanol in molar ratio
Besides, there are many advantages using of 1:9 in the presence of immobilized enzyme
enzymatic method compared to chemical using kaolin as matrix, which was already
method. Since the end of this decade, Indonesia incubated in phosphate buffer of pH 7.0. The
is being the largest producers of Crude Palm Oil mixture was incubated at 50 oC and the FAME
(CPO) in the world. This is a huge potential for was assayed at 4-10 h.
producing a renewable energy based on plant oil.
In this report, we show biodiesel production Esterase activity assay: FAME produced was
process using CPO and methanol as raw assayed by GC equipped with FID using capillay
material, which was catalyzed by lipase from column from Innowax. Sample of 1 ml was
Pseudomonas cepacia. taken up from the mixture, and centrifuged to
separate between glycerol and methylester.
MATERIALS AND METHODS Hundred l of methyl ester phase was pippeted
out and added with sodium sulphate anhydrate,
CPO as fatty acids source was obtained and then 1.0 ml of hexane was added. Following
from PTPN XIII in Paser Regency, East vortexing and centrifugation, 1 µl of the hexane
Kalimantan Province, absolute methanol as alkyl phase was then subjected to GC (inlet
source, sodium sulfate anhydrate, and hexane temperature of 220°C, detector temperature of
were obtained from Merck, Pseudomonas 275°C, and gradient program of column
cepacia lipase as biocatalystaor was obtained temperature of 150°C for 1 min, then increased

to 240°C by 15oC per min, and finally increased CPO as substrate, and other incubation method is
to 260°C by 5°C per min and holded at the still being investigated to increase the yield of
temperature for 8 min. biodiesel, including isolation of indigenous
lipase producing bacteria from waste treatment
RESULTS AND DISCUSSION of CPO manufacturing, which is still being in
progress.
Previous study showed that Pseudomonas
cepacia lipase possesed esterase activity on palm
kernel oil (Abigor et al., 2000), and we have also
demonstrated that this lipase was also active on
Crude Palm Oil (CPO) as substrate in producing
biodiesel (FAME). In this production, the
esterase activity is still stable until a molar ratio
of CPO and methanol of 1:9.
Kaolin showed a better result as matrix
compared to celite in the immobilization of
lipase from P.cepacia. The methanolysis was
optimum at 50°C, which was very suitable for
Fig 1. Enzymatic production of FAME with P. cepacia
CPO. CPO has a melting point between 31- lipase using CPO as substrate. Incubation
41°C, so that it becomes liquid at the process. temperature was at 50°C
In this experiment, the yield of biodiesel
was still very low, around 6.5% (Fig 1). To REFERENCES
overcome this problem, some emulsifier were
used in the trial to optimize the enzyme activity Abigor, R.D., Uadia, P.O., Foglia, T.A., Haas, M.J., Jones,
as described by Iso et al. (2001). However, K.C., Okpefa, E., Obibuzor, J.U. and Bafor, M.E.
although 1,4-dioxane as emulsifier could 2000. Lipase-catalysed production of biodiesel fuel
from some Nigerian lauric oils. Biochem. Soc.
increase the yield to 100%, it still could not Transaction. 28 (6): 978-981.
compete with the chemichal production method,
which could achieved a yield of 80-90%. Some Iso, M., Chen, B., Eguchi, M., Kudo, T. and Shrestha, S.
2001. Production of biodiesel fuel from triglycerides
other emulsifiers will be studied in this effort to and alcohol using immobilized lipase. J. Mol. Cat. 16:
increase the yield of FAME production using 53-58.

Interesterification of Fried Palm Oil with Methyl Acetate using Porcine
Pancreatic Lipase to Produce Biodiesel
Rita Arbianti, Heri Hermansyah, Tania Surya Utami and Ryan Indra Mukti
Department of Chemical Engineering, University of Indonesia

Depok 16424, Indonesia, e-mail: arbianti@chemeng.ui.ac.id
(Correspondence to: Rita Arbianti)
ABSTRACT
New method on biodiesel synthesis via non-alcohol route with biocatalyst was developed. This method re-
places conventional method which used alkali catalyst by replacing alcohol with alkyl acetate as alkyl acceptor
in the reaction. Interesterification of triglyceride from used cooking oil with alkyl acetate can produce biodiesel.
Alcohol substitution with alkyl acetate can enhance the biocatalyst stability during reaction process significant-
ly. The application of waste as triglyceride source is expected to enhance the economic feasibility of biodiesel
synthesis using biocatalyst. Besides, triacetylglycerol, as the by-product, has a higher economic value than glyc-
erol from alcohol route. In this research, methyl acetate was reacted with triglyceride from fried palm oil using
porcine pancreatic lipase in batch reactor. The reactants and products were analyzed by HPLC. The effect of
operating factors such as enzyme concentration, substrate ratio, operating temperature and addition of inhibitor
using free and immobilized enzyme were investigated. The results showed that porcine pancreatic lipase was
able to convert 62.78 and 53.26% of triglyceride from fried palm oil to biodiesel using free and immobilized
lipase, respectively, under optimum conditions. The optimum operating temperature that gave highest biodiesel
yield was 37°C for 50 h reaction. The highest biodiesel yield was obtained at methyl acetate to oil molar ratio of
12:1 using 4% (w/w of oil) enzyme concentration. Stability test indicated that the activity of the immobilized
biocatalyst still remained after three reaction cycles.
Keywords: Biodiesel, fried palm oil, triglyceride, porcine pancreatic lipase, interesterification, non-alcohol
route.
INTRODUCTION homogenously. In addition, biocatalyst is able to

lead to specific reaction without any undesirable
Biodiesel (fatty acid methyl esters), which side reaction such as saponification reaction, that
is conventionally produced by transesterification occurs between alkali catalyst and free fatty acid
of triglycerides with methanol has become in- (Xu et al., 2005; Hermansyaha et.al., 2008; Her-
creasingly important due to diminishing petrole- mansyahb et.al., 2008). The use of biocatalyst is
um reserves and environmental consequences of also more environmentally friendly because it
exhaust gases from petroleum fuel engines does not generate hazardous chemical waste (Xu
(Mattelbach, 1990; Noureddini & Zhu, 1997). et al., 2005; Hermansyaha et.al., 2008).
The attractive features of biodiesel fuel are: it is However, alcoholic environment causes li-
plant derived, not a fossil fuel, and as such, its pase deactivation and the stability in catalyzing
combustion does not increase current net atmos- reaction become worse (Hermansyaha et.al.,
pheric levels of carbon dioxide, a greenhouse 2008; Hermansyahb et.al., 2008). New methods
gas, biodegradable, its combustion products have had been developed to overcome enzyme deacti-
reduced levels of particulates, i.e. carbon mon- vation by replacing the alcohol with alkyl acetate
oxide, sulfur oxides, and hydrocarbons (Linko, which serves as an acceptor alkyl groups (Xu et
1998; Dossat et al., 1999; Iso et al., 2001; Kaie- al., 2005; Hermansyaha et.al., 2008; Hermans-
da et al., 2001; Shimada et al., 2002; Watanabe yahb et.al., 2008). This process was beneficial
et al., 2002). because enzyme deactivation on alcoholic envi-
Researches on biodiesel synthesis that re- ronment does not occur and the by-product, tri-
cently done lead to the utilization of biocatalyst acetylglycerol, has a higher economic value than
as the substitution of alkali catalyst which was glycerol from conventional route (Xu et al.,
used in industrial level. This condition is able to 2005; Hermansyaha et.al., 2008; Hermansyahb
decrease the disadvantages of alkali catalyst us- et.al., 2008).
age in such that product separation can be done Utilization of lipase as biocatalyst has a
easily, because product and catalyst do not mix great potential to overcome these problems.

However, lipases were easily deactivated in al- 500 rpm for 60 min. Then, 0.4 g zeolite was
cohol (Colluci et al., 2005). Recently the use of added into enzyme solution, and the mixture was
methyl acetate as an alkyl acceptor for the inter- stirrerd gently for 6 hours at 37°C. Zeolit was
esetrification of soybean oil has been reported then separated from the mixture. The
(Colluci et al., 2005; Vicente et.al., 2005; Vicen- concentration of the enzyme in the solution
te et.al., 2006). The interesterification method before and after immobilization were measured
eliminates the risk of deactivation of the enzyme by Lowry method.
by glycerol, as no glycerol is produced in the
reaction. Furthermore, products produced during Interesterification reactions: The interesterifica-
interesterification have no negative effect on the tion reactions were carried out in a 25 ml
lipase activity (Colluci et al., 2005). Erlenmeyer flask. containing a mixture of oil and
Only a few papers related to this new reac- methyl acetate and free or immobilized porcine
tion route have been reported (Dossat et al., pancreatic lipase. First, the free or immobilized
2002; Colluci et al., 2005; Vicente et.al., 2005; enzyme was dissolved in methyl acetate, then the
Vicente et.al., 2006). Methyl acetate and ethyl fried oil was added into enzyme-methyl acetate
acetate has been used as alkyl supplier in the solution. The mixture was strirred at 37°C and
synthesis of biodiesel from soybean oil, ja- 100 rpm. The effect of various operating factors
thropa and sunflower oil using Candida antarc- such as molar ratio of methyl acetate to oil,
tica lipase, C. cylindraceae lipase and porcine initial concentration of enzyme, addition of
pancreatic lipase (Dossat et al., 2002; Colluci et inhibitor, and temperature were investigated.
al., 2005; Vicente et.al., 2005; Vicente et.al., Furthermore, stability test was also conducted by
2006). using the immobilized enzyme for three reaction
In this research, the kinetics of interesterifi- cycles under the optimum condition. The
cation of- triglyceride with methyl acetate to samples were taken at certain time interval of
produce biodiesel was further studied. Fried reaction. The concetration of biodiesel was
palm oil was used as triglyceride source and por- measured by using HPLC system equipped with
cine pancreatic lipase was used as biocatalyst. C-18 column (Wakopak, particle size 5.0 μm,
The activity of the free and immobilized lipase i.d. 4.6 mm, length 0.15 m, Wakosil-GP-N6
was investigated under various conditions of Tokyo, Japan) and a UV detector (L-4000,
substrate ratio, enzyme concentration, tempera- Hitachi, Ltd., Tokyo, Japan) at 205 nm. The
ture and addition of inhibitor. The experimental temperature of the column oven was 313 K.
results showed that 62.78 and 53.26% of triglyc- Methanol, isopropanol and hexane were used as
eride from fried palm oil was converted into bio- mobile phase, and flown at 0.8 cm3 min‾1
diesel under the condition of 4% (w/w) lipase in through the column by a gradient technique.
the substrate, 1/12 mol ratio of oil/methyl acetate
after 50 h reaction using free and immobilized 2
lipase, respectively. 3
37oC 4
MATERIALS AND METHODS 1 5
6
Materials: Porcine pancreatic lipase immobi-

lized on zeolite, was puchased from ICN. This
enzyme hydrolyzes all three ester bonds of the 1. Water bath 4. Water container
triglyceride and has an activity of 7.06x108 U/kg. 2. Piping 5. Bar stirrer
3. Erlenmeyer 25ml 6. Magnetic stirrer
Refined palm oil was purchased from local
hypermarket and the fried palm oil was prepared
Fig 1. Schematic diagram of batch reactor to produce
by cooking 8 kg of chicken on 4 l palm oil. biodiesel
Methyl acetate, sodium hydroxide, palmitic acid
and other chemical were of reagent grade and
purchased from Merck Schuchardt, Hohenbrunn,
Germany.
Lipase immobilization: A specified amount of

porcine pancreatic lipase was dissolved in 4 ml
phosphate buffer (0.05 M, pH 7), and stirred at

RESULTS AND DISCUSSION 3.5
3
Ct (mol/L)
Free Lipase: In this experiment the activity of 2.5 Cd (mol/L)
Cm (mol/L)
lipase enzyme was tested in synthesizing bio-
Ci (mol/L)
2 Cb (mol/L)
diesel. Lipase was used as biocatalyst because its
1.5
ability to hydrolyze fat can accelerate the reac-
1
tion and enhance the yield in the reaction pro-
0.5
cess. The reaction took place through a non-
alcoholic route. Oil as substrate will react with 0
0 10 20 30 40 50 60
methyl acetate as donor of alkyl group in inter- t (jam)
esterification reaction. To determine the reaction Fig 3. The concentration of each component (mol/l) in the
rate of product formation, variation of time was synthesis of biodiesel by fried oil substrate using
performed in this study. The result of this exper- immobilized porcine pancreatic lipase
iment is shown in Fig 2.
The effect of temperature: Experiments were
4
performed to examine the effect of temperature
3.5
on the catalytic activity both of free and immobi-
3 Ct (mol/L)
Cd (mol/L) lized lipase in the interesterification reaction of
Ci (mol/L)
2.5 Cm (mol/L)
Cb (mol/L)
palm oil and fried palm oil with methyl acetate.
2
The reaction temperature is an important param-
1.5
eter in enzymatic catalysis. Higher temperature
1
can give a faster transformation, but too high
0.5
temperature will lead to enzyme denaturation.
0
0 10 20 30 40 50 60
The effect of reaction temperature from 25 to
t (jam) 50°C on biodiesel yield were investigated as
Fig 2. The concentration of each component (mol/l) in the
shown in Fig 4. The experimental results showed
synthesis of biodiesel by fried oil substrate using that the highest yield of biodiesel was obtained
free porcine pancreatic lipase at the temperature of 37°C using free and immo-
bilized lipase (62.78 and 53.26%, respectively).
From Fig 2 it can be seen that the concen- This temperature was considered as the optimum
tration of biodiesel reached its maximum at 50 h condition for the reaction using porcine pancre-
reaction, which resulted in 3.71 mol/l or 62.78% atic lipase.
yield under the condition of 37°C temperature,
4% (w/w) lipase based on substrate weight, 1/12
mol ratio of oil/methyl acetate.
Immobilized lipase: In this experiment, we used

lipase which was immobilized through adsorp-
tion methods in the production of biodiesel. The
result is shown in Fig 3. The biodiesel yield from
this experiment was 53.26%, lower than the re-
sult from the previous experiment. However, the
immobilized biocatalyst can be used repeatedly
Fig 4. Effect of temperature on biodiesel yield in inter-
in interesterification reactions, since it had good esterification reaction of palm oil and fried palm
stability. The operating condition in this experi- oil using methyl acetate to oil molar ratio of 12:1
ment were 50 h reaction, 4% (w/w) lipase based at 25-50°C and 100 rpm for 50 h with single step
on substrate weight, 1/12 mol ratio of oil to me- addition of methyl acetate
thyl acetate at 37°C.
The effect of substrate molar ratio: In Fig 5, the
results of the experiments showed that 62.78 and
21.49% of fried palm oil were converted to bio-
diesel at methyl acetate to oil molar ratio of 12:1
and 3:1, respectively. The highest methyl esters
yield was obtained at methyl acetate to oil molar
ratio of 12:1 using 4% (w/w of oil) enzyme con-

centration at 37°C for 50 h reaction. A large ex-
cess of methyl acetate was required in order to
shift the interesterification reaction into product
formation. Shifting the substrate molar ratio
above or below the optimum value decreased the
methyl esters yield with all of the oil substrate.
The findings of the present investigation were in
close agreement with the result of other re-
searcher (Mattelbach, 1990) who reported that
the optimum ethyl acetate to oil molar ratio of
11:1 in interesterification reaction of crude
jatropha, karanj and sunflower oil and found that Fig 6. Effect of enzyme dosage on biodiesel yield in inter-
the molar ratio greater than 3:1 led to an exces- esterification reaction of palm oil and fried palm oil
sive dilution of oil resulting in a reduced ethyl using methyl acetate to oil molar ratio of 12:1 at
37°C and 100 rpm for 50 h with single step addition
esters yield. of methyl acetate
Fig 5. Effect of methyl acetate to oil molar ratio on bio- Fig 7. Effect of inhibition by palmitic acid on biodiesel
diesel yield in interesterification reaction of palm yield in interesterification reaction of fried palm oil
oil and fried palm oil catalyzed by porcine pan- using methyl acetate to oil molar ratio of 12:1 at
creatic lipase (4% based on oil substrate weight) 37°C and 100 rpm for 50 h with single step addition
at 37°C and 100 rpm for 50 h with single step ad- of methyl acetate
dition of methyl acetate
Repeated use of immobilized enzyme: The main
The effect of initial enzyme concentration: The advantage of immobilization of an enzyme is
concentration of enzyme was varied from 1 to that an expensive enzyme can be used repeated-
4% (w/w of oil). The yield of biodiesel increased ly. Stability test of the immobilized porcine pan-
as the enzyme concentration increased as shown creatic lipase indicated that the activity of the
in Fig 6. This was because the amount of the immobilized lipase still remained after three re-
enzyme that contributed in the interesterification action cycles as shown in Fig 8.
increased. Since there was a limitation of expen-
sive enzyme, the concentration of 4% (w/w of
oil) of porcine pancreatic lipase was considered
high enough for this reaction.
The effect of inhibition: In this experiment, 1%

(w/w of oil) palmitic acid was added into the
substrate fried palm oil using free and immobi-
lized lipase. Biodiesel yield for each experiment
was shown in Fig 7. Biodiesel yield for both of
free and immobilized porcine pancreatic lipase
was 23.97 and 20.76%, respectively. These re-
sults showed that free fatty acid inhibited the Fig 8. Operational stability of lipase with methyl acetate
interesterification reaction. as the alkyl acceptor. Reaction conditions: methyl
acetate/oil molar ratio 12:1, 100 rpm, 37°C, porcine
pancreatic lipase

CONCLUSIONS Hermansyahb, H. et.al. 2008. Prosiding Seminar Nasional
Rekayasa Kimia dan Proses. ISSN: 1411–4216.
Porcine pancreatic lipase was able to con- Iso, M., Chen, B.X., Eguchi, M., Kudo, T. And Shrestha, S.
vert 62.78 and 53.26% of triglyceride from fried 2001. J. Mol. Catal. B: Enzym. 16: 53-58.
palm oil into biodiesel under the condition of 4% Kaieda, M., Samukawa, T., Kondo, A. and Fukuda, H.
(w/w) lipase in the substrate, 1/12 mol ratio of 2001. J. Biosci. Bioeng. 5-12.
oil/methyl acetate after 50 h reaction using free Linko, Y.Y. 1998. J. Biotechnol. 66: 1-50.
and immobilized lipase, respectively. Further- Mattelbach, M. 1990. J. Am. Oil Chem. Soc. 67: 168-170.
more, the stability test showed that the activity
of the immobilized lipase still remained after Noureddini, H. and Zhu, D. 1997. J. Am. Oil Chem. Soc.
74: 1457-1463.
three reaction cycles.
Shimada, Y., Watanabe, Y., Sugihara, A. and Tominaga, Y.
2002. J. Mol. Catal. B: Enzym. 133-142.
REFERENCES
Vicente, G. et.al. 2005. Ind. Eng. Chem. Res. 44: 5447-
Colluci, J.A. et al. 2005. JAOCS. 82: 525–530. 5454.
Dossat, V. et.al. 2002. Enzyme Microb. Technol. 30: 90–94. Vicente, G. et.al. 2006. Energy & Fuel. 20: 1722–1726.
Dossat, V., Combes, D. and Marty, A. 1999. Enzyme Mi- Watanabe, Y., Shimada, Y., Sugihara, A. and Tominaga, Y.
crob. Technol. 25: 194-200. 2002. J. Mol. Catal. B: Enzym. 17: 151-155.
Hermansyaha, H. et.al. 2008. Prosiding Seminar Nasional Xu, Y. et al. J. Mol. Cat. Elsevier. 32: 241-245.
Fundamental dan Aplikasi Teknik Kimia. ISSN 1410-
5667.

The Influence of Carbon Sources on Laccase Production by White Rot
Fungus Marasmius sp. in Solid State Fermentation
Hendro Risdianto1, Elis Sofianti1, Suraya2, Sri Harjati Suhardi2 and Tjandra Setiadi1
1
Department of Chemical Engineering, Faculty of Industrial Technology, Institut Teknologi Bandung
Gedung Labtek X, Jl. Ganesha 10 Bandung 40132, Indonesia, e-mail: tjandra@che.itb.ac.id
2
School of Life Sciences and Technology, Institut Teknologi Bandung
Gedung Labtek XI, Jl. Ganesha 10 Bandung 40132, Indonesia
(Correspondence to: Tjandra Setiadi)
ABSTRACT
Laccase is an enzyme that capable to degrade lignin in biomass. This enzyme has the capability to be used
as as a biological agent on lignin removal pretreatment for bioethanol production from biomass. Laccase has
been produced by white rot fungus Marasmius sp in Solid State Fermentation (SSF) using rice straw as the solid
support media. The influence of carbon sources, i.e. glucose, glycerol and molasses, on laccase production were
studied in this paper. The concentration of 0.5%, 1.0% and 2.0% were used for each carbon sources. The results
showed that the highest lacase activity was obtained within 6-10 days of cultivation. Glucose concentration of
0.5%, 1.0% and 2.0% gave the highest laccase activity were 872.0 U/L (day 6), 1516.67 U/L (9th day) and
1270.69 U/L (day 10) respectively. The highest laccase activity on using glycerol and molasses was 1422.36
U/L (at concentration of 1 % on 7th day) and 1113.19 U/L (at concentration of 2% on 8th day), respectively. This
activity was comparable to that of glucose substrate. Therefore, glycerol and molasses gave a potential chance
as carbon sources for the strategy on low cost laccase production in solid state fermentation.
Keywords: glucose, glycerol, laccase, molasses, Marasmius sp., solid state fermentation
INTRODUCTION shown to be particularly suitable for the produc-

tion of enzymes by filamentous fungi since they
Laccases (benzenediol:oxygen oxidoreduc- reproduce the natural living conditions of such
tases, EC 1.10.3.2), representing the largest sub- fungi (Couto & Sabroman, 2005). The sup-
group of blue multicopper oxidases (MCO), use port/substrate for performing SSF is essential,
the distinctive redox ability of copper ions to since the success of the process depends on it. In
catalyze the oxidation of a wide range of aro- recent years, there has been an increasing trend
matic substrates concomitantly with the reduc- towards the utilisation of organic wastes such as
tion of molecular oxygen to water (Winquista et residues from the agricultural, forestry and ali-
al., 2008; Giardina et al., 2009). Since these en- mentary industries as raw materials to produce
zymes catalyze the oxidation of a broad range of value-added products by SSF technique (Osma
phenolic and aromatic amines, laccase-mediated et al., 2007). Furthermore, most of these wastes
processes become a very promising alternative contain lignin or/and cellulose and hemicellulose,
for cellulose pulp bleaching (Risdianto et al., which act as inducers of the ligninolytic activi-
2009), effluent detoxification, phenol removal ties. Moreover, most of them are rich in sugars,
(Musatto & Roberto, 2004), discoloring textile which make the whole process much more eco-
dyes (Boehmer et al., 2006; Osma et al., 2007; nomical.
Bettin et al., 2008) and lignin removal on bio- The application of laccase enzyme in indus-
ethanol production from lignocelluloses. Laccase trial and environmental technologies, including
can be produced both of Sub-merged Fermenta- the modern concept of integrated biorefineries
tion (SmF) and Solid State Fermentation (SSF) requires significant amounts of these enzymes at
(Osma et al., 2007). low cost (Elisashvili et al., 2008). Besides using
Solid State Fermentation (SSF) or Solid the residue from agro-industrial as the support,
State Cultivation (SSC) is generally defined as a the exploration of cheap carbon sources is one of
growth of micro-organisms on solid materials in the strategies on low cost laccase production in
the absence or near absence of free water (Couto solid state fermentation. There are some low cost
& Sabroman, 2005; Winquista et al., 2008; carbon sources i.e molasses and glycerol. Sugar-
Singhania et al., 2009). SSF processes have cane molasses is a by-product of the manufac-

ture or refining of sucrose from sugarcane. In ammonium tartrate 0.4 g/L, MnCl2 0.02 g/L,
addition to sucrose, molasses contains glucose, yeast extract 0.3 g/L, CuSO4.7H2O 0.01 g/L,
fructose, raffinose and numerous non-sugar or- H2MoO4 0.007 g/L, MnSO4.4H2O 0.01 g/L,
ganic materials. While glycerol is a chemical ZnSO4.7H2O 0.006 g/L and Fe2(SO4)3 0.007 g/L.
compound also commonly called glycerin or To examine the influence of carbon sources, the
glycerine which is a sugar alcohol, and is sweet- concentration of 0.5%, 1.0% and 2.0% were
tasting and of low toxicity. It is a 10% by- used for each carbon sources. The substrate of
product of biodiesel production (via the trans- rice straw was accomplished by the medium and
esterification of vegetable oils). This process has steam sterilized at 120°C for 20 minutes prior to
led to a excess of crude glycerol in the market, inoculation. The culture was then allowed to
making the epichlorohydrin process no longer cool down. The inoculums size of 1.5 x 1.5 cm
economical (Coral, 2008). was then transferred aseptically into the flask,
Previous study showed that Marasmius sp, then cultures were incubated under the static
rice straw promoted an excellent growth for the condition for about 10-13 days fermentation time.
fungi and gave the highest laccase activity in Sample was collected everyday to determine the
SSF (Risdianto et al., 2009). Currently, there are laccase activity. Sample was extracted by adding
a few reports on utilisation of molasses and 50 mL of sodium acetate buffer (pH 4.5) and
glycerol as carbon sources in laccase production shakes at 100 rpm for 2 hours and then ground in
by SSF method. Therefore, the goal of the pre- mortar. The slurry was centrifuged at 5000 rpm
sent paper was to investigate the potential of for 10 minutes. The filtered supernatant was then
carbon source of molasses and glycerol for the used for determining of laccase activity.
production of laccase by Marasmius sp. under
SSF conditions, since the utilisation of the cheap Laccase assay: Laccase activity was determined
carbon sources would mean an important reduc- with 2,2’-azino bis (3-ethylbenzthiazoline-6-
tion in production costs. sulphonic acid) (ABTS) in 0.4 mM sodium ace-
tate buffer (pH 4.5). Oxidation of ABTS was
MATERIALS AND METHODS determined by the increase in A420 (ε420 = 36
(mM cm)-1) using spectrophotometer. One unit
Lignocellulosic substrate: Rice straw as solid of enzyme activity (U) was defined as the
substrate was obtained from farm near Bandung amount of enzyme required to oxidize 1 µmol of
city, Indonesia. The rice straw was chopped in ABTS per minute. All values reported are the
length of about 3 cm. mean of two replicates.
Microorganism: The white rot fungus Marasmi- RESULTS AND DISCUSSION

us sp was obtained from Laboratory of Microbi-
ology and Bioprocess Technology, Department Laccase activity used glucose as carbon
of Chemical Engineering, Institut Teknologi sources presented in Fig. 1. Fig 1 shows the
Bandung, Indonesia. The strain was maintained highest laccase activity was reached at concen-
on Potato Dextrose Agar (PDA) medium in the tration 1.0% (1516.67 U/L) on day 10th. A de-
Petri dish and incubated for 7 days at 28°C and crease in the laccase activity was not observed,
stored at 4°C until it was used. higher enzymes activity might be achieved if the
cultivation time was extended. For the concen-
Culture condition: Solid state fermentation was tration of 0.5%, laccase activity began to be de-
carried out at room temperature (28 ± 1°C). Five tected on 2nd day (70.97 U/L) and reached the
grams of chopped rice straw was impregnated by maximum on 7th day (1031.53 U/L) and then
20 mL of modified Kirk medium in 250 mL of decreased. The similar pattern was occurred at
flask. Composition of Kirk medium are using concentration of 2.0%. Laccase activity was
one of the carbon sources (glucose, glycerol and firstly observed on 3rd day (4.86 U/L) and then
molasses), KH2PO4 1.7 g/L, MgSO4.7H2O 0.4 achieved maximum activity of 1270.69 U/L
g/L, CaCl2 0.09 g/L, sodium acetate 2.3 g/L, di- on10th day.

1800
glucose 0.5%
1600
Laccase activity (U/L) glucose 1.0%
1400 glucose 2.0%
1200
1000
800
600
400
200
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (day)
Fig. 1. Laccase activity on using glucose as carbon source.
Laccase production using glycerol as carbon U/L on 7th day). At the concentration of 0.5%,
sources is shown in Fig. 2. Laccase activity was laccase reached its maximum value of 722.36
start detected on 4th day at concentration of 0.5% U/L on 8th day and steady enzyme activity were
(159.44 U/L) and 1.0% (113.75 U/L), while the observed afterwards. The activity of 759.31 U/L
concentration of 2.0% was firstly detected on 5th (on 7th day) was the peak value on using molas-
day (31.59 U/L). The highest laccase activity ses of 1.0%.
was reached at concentration of 2.0% (1422.36
1800
molasse 0.5%
1600
molasse 1.0%
1400 molasse 2.0%
Laccase activity (U/L)
1200
1000
800
600
400
200
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (day)
Fig. 2. Laccase activity on using molasses as carbon source.
As shown in Fig. 3, laccase production on 1.0%, the activity sharply increased up to a max-
using glycerol began on the 4th day at all concen- imum value about two-folds than that of 0.5%
tration. The peak activity was achieved at con- (1422.36 U/L) on 7th day. The maximum laccase
centration of 0.5% was 713.61 U/L on 6th day activity at concentration of 2.0% was lower than
and then decreased. For the concentration of that of 0.5% and 1.0%.

1800
glycerol 0.5%
1600
glycerol 1.0%
Laccase activity (U/L) 1400 glycerol 2.0%
1200
1000
800
600
400
200
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (day)
Fig. 3. Laccase activity on using glycerol as carbon source.
Fig. 4 shows the productivity of the three concentration of 1% glycerol has the highest
carbon sources that used in this study. The productivity values (203.19 U/L/day). Further-
productivity is the ratio of the highest value of more, the second highest productivity value is at
laccase activity to the age of cultivation. The 1% glucose (168.52 U/L/day).
250
glucose
molasse
200
glycerol
laccase activity (U/L)
150
100
50
0
0.50% 1.00% 2.00%
Concentration
Fig. 4. The comparison of maximum productivity on using glucose, molasses and glycerol.
The enzyme activity appeared after 2-5 days et al., 2006). The laccase activity about 1570
of cultivation and gradually increased achieving U/L on 20th day was obtained on using banana
a maximum values on days 6-10 and then de- skin as carbon sources by Trametes pubescens
creased probably due to specific degradation of (Mussatto & Roberto, 2004). In this study, the
laccase by extracellular protease present in the highest lacase activity was 1516.67 U/L (at glu-
culture (Mazumder et al., 2009). Elisashvili et cose concentration of 1.0% on 9th day).
al. (2008) showed that Cerena maxima was the As it was indicated, for glucose and glycer-
best laccase producer (7,620 U/L) in SSF. This ol, the increasing concentration of carbon
appreciable laccase activity was obtained in ab- sources increased the maximum activity up to
sence of specific aromatic compound (Boehmer optimum concentration and then decreased for

higher concentration probably due to substrate Couto, R. S. & M. A. Sanroman. 2005. Application of sol-
inhibition. However, increasing the concentra- id-state fermentation to ligninolytic enzyme produc-
tion. Biochemical Engineering Journal 22:211–219.
tion of molasses increased the maximum laccase
activity. The highest activity on using molasses Elisashvili, V., E. Kachlishvili & M. Penninckx. 2008. Ef-
fect of growth substrate, method of fermentation, and
and glycerol were comparable to that of glucose nitrogen source on lignocellulose-degrading enzymes
substrate. This indicated that a ligninolytic en- production by white-rot basidiomycetes. J Ind Micro-
zyme is formed as a part of secondary metabo- biol Biotechnol. 35:1531–1538.
lism. Carbohydrate starvation likewise leads to a Giardina, P., V. Faraco, P. Pezzella, A. Piscitelli, S.
rapid production of ligninolytic activity Vanhulle & G. Sannia. 2009. Laccases: a never-
(Sanchez, 2009). ending story. Cell. Mol. Life Sci. 67: 369-385.
In conclusion, the results presented in this Mazumder, S., S. K. Basu & M. Mukherjee. 2009. Laccase
paper exhibited an excellent growth of fungi on production in solid-state and submerged fermentation
rice straw and produced a high laccase activity. by Pleurotus ostreatus. Eng. Life Scie. 9 No1: 45-52.
The usage of cheap carbon sources i.e. glycerol Mussatto, S.I. & I. C. Roberto. 2004. Alternatives for de-
and molasses produced a comparable enzyme toxification of diluted-acid lignocellulosic hydrolyza-
activity to that of glucose substrate. Therefore, tes for use in fermentative processes: a review, Biore-
glycerol and molasses gave a potential chance as source Technology. 93: 1–10.
carbon sources for the strategy on low cost lac- Osma, J. F., J. L. T. Herrera & S. R. Couto. 2007. Banana
case production in solid state fermentation. skin: A novel waste for laccase production by
Trametes pubescens under solid-state conditions Ap-
plication to synthetic dye decolouration. Dyes and
ACKNOWLEDGMENTS Pigments. 75:32-37.
Risdianto, H., E. Sofianti, S. H. Suhardi & T. Setiadi. 2009.
This work was funded by Ministry of Re- “Produksi Lakase Menggunakan Fermentasi Padat
search and Technology of Republic of Indonesia (Solid State Fermentation) dari Limbah Hasil
through the scheme of Insentif Ristek 2009. Pertanian”, Prosiding Seminar Nasional Teknik Kimia
2009, Bandung.
REFERENCES Risdianto, H., K. Jonathan C., Y. Christine V.M., T. Setiadi.
2009. Evaluation of Crude Laccase Enzyme Perfor-
Bettin, F., Q. Montanari, R. R. Calloni, A. T. Gaio, M. M. mance At Pulp Bleaching Pretreatment Process. Pro-
Silveira & A. J. P. Dillon. 2008. Production of lac- ceeding of International Seminar on Sustainable Bi-
cases in submerged process by Pleurotus sajor-caju omass Production and Utilization Challenges and
PS-2001 in relation to carbon and organic nitrogen Oppurtunities (ISOMASS). Lampung University.
sources, antifoams and Tween 80. J Ind Microbial Sanchez, C. 2009. Lignocellulosic residues: Biodegradation
Biotechnol. and bioconversion by fungi. Biotechnology Advances.
Boehmer, U., S. H. Suhardi & T. Bley. 2006. Decolorizing 27:185-194.
Reactive Textile Dyes with White-Rot Fungi by Singhania, R. R., K. Patel, C. L. Soccol & A. Pandey. 2009.
Temporary Immersion Cultivation. Eng. Life Sci. 6 Recent advances in solid-state fermentation. Bio-
No. 4. chemical Engineering Journal. 44:13–18.
Coral, J. 2008. Propionic acid production by Propionibacte- Winquista, E., U. Moilanena, A. Mettäläb, M. Leisolaa & A.
rium sp. using low-cost carbon sources in submerged Hatakka. 2008. Production of lignin modifying en-
fermentation, Ph.D Thesis, Université de Provence. zymes on industrial waste material by solid-state cul-
tivation of fungi, Biochemical Engineering Journal.
42:128-132.

Provision of Superior Genotypes of Jatropha Curcas for Biodiesel
Production: Integrating Morphology and Yield Variation
with DNA-Based Marker
Enny Sudarmonowati1, Y. Cahyani1, N.S. Hartati1 and Wahyuni1
1
Research Centre for Biotechnology-LIPI
Jl. Raya Bogor Km. 46. Cibinong 16911, e-mail: s_enny@hotmail.com
(Correspondence to: Enny Sudarmonowati)
ABSRACT
Selection and grouping based on the yield parameter specifically number of fruits per bunch of Jatropha
curcas which also relates to the weight of individual fruits and the morphological performances related to shape
and colour of leaves and petioles of 60 accession numbers has been made. Four groups related fruit yield were
identified i.e. A ( ≥4), B ( ≥3), C ( ≥2), D (0-1) fruits per bunch while 5 groups consisting of 3 of petiole, 1 of
mature leaves, 2 of young leaves and 2 of shoots colours. The size of leaves could not be differentiated as most-
ly were similar. The correlation between fruit yield and RAPD marker was assessed by screening 17 random 10-
mer primers which resulted three primers i.e. OPB-07, OPB-10 and OPH-01 could be amplified. Although fur-
ther study and confirmation are needed, the results indicated that several accessions possessed different banding
patterns which might be a potential marker related to fruit yield and in the future this would also be used for
assessing that of oil content. The oil content was around 22% and 37.5% using pressing machine and chemical-
ly extracted, respectively. J. curcas clustering based on morphological character indicated that there was a cor-
relation between fruit yield and growth response.The confirmed genotype superiority would support Jatropha’s
based especially bio oil industries to increase production cost efficiency through the use of improved genotypes
and appropriate cultivation suitable for each condition area .
Keywords: Jatropha curcas, Biodiesel, genetic variation, morphological variation, RAPD markers, fruit, oil
content.
INTRODUCTION yield are not known. In intensively cultivated

areas, irrigated lands might be used for food
Jatropha is said to be a drought hardy shrub, production, recommending to direct breeding
non-demanding, tolerant to extremes, suitable to from maximum yields towards reduced input
tropical and non-tropical climate and considera- needs for those cases. For forestry projects, pos-
ble climatic changes, even up to light frost. Tree sibly as well for farm-based agro-forestry, the
borne oil seeds have always been a component shade tolerance might be the key selection crite-
of traditional agricultural systems practiced in rion to be further developed.
India. The 70 million wasteland in the country Biodiesel contains no petroleum, but it can
are available for plantation of fuel plants. Grow- be blended at any level with petroleum diesel to
ing these oil bearing plants on wastelands, as create a biodiesel blend or can be used in its pure
avenue trees and in the back yards all over the form. Just like petroleum diesel, biodiesel oper-
nation will improve the availability curcas is a ates in compression ignition engine which essen-
small tree or large shrub with smooth gray bark, tially require very little or no engine modifica-
which exudates a whitish colored watery latex, tion, because biodiesel has properties similar to
upon cut. It has large green to pale green leaves, petroleum diesel fuels. Biodiesel is considered
alternate to sub-opposite, three to five lobed with clean fuel since it has almost no sulphur, no ar-
a spirally phylotaxis (Verma & Gaur, 2009) omatics and has about 10 percent built -in- oxy-
However, the degree of domestication in gen which helps into burn fully. Among the oil
tree-borne oilseed species as a whole is at a very seeds of forest origin Jatropha curcas popularly
early stage compared to most cultivated crops. known as Ratanjayot have an immense potential
Increased domestication and increased inputs for producing oil, which finds large scale indus-
might increase pests and diseases, now assumed trial uses. J. curcas is a large soft wooded, de-
low in Jatropha influence of increased mineral ciduous, multipurpose tree of 4-7 meter height,
fertilizer and water doses on pests, oil content or

which belongs to the family Euphorbiaceae. The purpose of the study was to determine
(Pant et al., 2006). oil content and correlation analysis of pheno-
Male flowers dominate the plant; flower typic character among jatropha grouping based
visitors needed for the predominant male flowers on fruit yield.
include bees, ants, thrips and flies. To what ex-
tent bees can be produced with positive syner- MATERIALS AND METHODS
gies on Jatropha pollination and honey yields is
an open issue. Plant materials: A total of 1334 number of ac-
Plant density recommendations fluctuate cessions belongs to Research Centre for Bio-
between 1000 plants and 5000 plants per ha, technology-LIPI were collected from West Java
with no relation to genotype, cultivation method (Bogor, Cibinong, Jonggol, Bandung,
or soils. As well, there are still a number of open Sumedang), East Java and Lampung .
issues to be researched on optimal flowering and
fruiting patterns. Timing and degree of pinching, Selection based on fruit production: Selection
pruning and a close. For the micro credit based on fruit production was conducted in two
schemes, factors influencing time lag until full stages. First selection was determined based on
yield can be achieved on different lands, is of fruit number per bunch and the second selection
paramount importance however not yet known. one was determined by fruit number.
Uses as pesticide, moluscicide and for medicinal
purposes might as well require specific selection, Selection based on morphological and rapid
breeding and cultivation practices. Present ef- growth: A number of 125 plants were measured
forts in the frame of the national program leave their growth response indicated by height, diam-
the cropping system open. The focus is on un- eter and branch number at the age of 1 year
derutilized lands, but seems to expect a rapid and 4 years. Identification of morphological
increase of production through monoculture character of each group plant was identified by
planting of Jatropha on agricultural fields, and the colour variation of young and mature leaves,
mixed forestry cropping in forestry areas. shoot and petiole.
There has been little genetic improvement,
identification of elite germplasms, tissue culture Oil content and chemical composition of seed:
experiments and propagation so far nor a sys- Oil content of peeled seed was determined
tematic or coordinated capture of genetic re- through physical process by using pressing ma-
sources in seed banks for its regeneration, hybrid chine and chemical process. Seed chemical
production and sustainable cultivation. As well, composition was measured according to Indone-
preference of cuttings, seedlings (with or without sian National standard method SNI 01-2891-
polybags), or other propagation methods, the 1992, point 5,1, SNI 01-2891-1992 pointr 6,1,
level of pruning, trimming, to extend the juve- SNI 01-2891-1992 point 7,1, SNI 01-2891-1992
nile phase of the plant, and suitable spacing of point 8,2, SNI 01-2891-1992 point 11 for water
the plant has barely been optimized so far. Life- content, ash level, protein content, lipid content
time under cultivated conditions is not known and fiber.
yet. Candidate plus tree selection is the first and
most important stage in any tree improvement Genetic analysis:
programme. The selection of candidate plus DNA extraction: DNA was extracted following
plants (CPPs) is based upon various important CTAB methodsi with some modifications (Gil-
attributes associated with the species and their lies et al., 1997).
relative ranking. Relative preference between RAPD analysis: A total of 17 arbitrary decamer
various traits and scoring for each trait has been primer from OPERON TECHNOLOGIES Inc.
worked out by using the method of paired com- were used for RAPD amplification. Amplifica-
parisons for the selection of CPP in J. curcas L. tion was carried out in 25 ul reaction volume
The most important ones are seed and oil yields ( containing 10X PCR buffer (50 mM KCl, 10
Mishra, 2008). mM Tris-HCL, 1.5 mM MgCl2), 200 uM each
Random Amplified Polymorphic DNA dNTPs, 20 uM of primer, 0.5 units of Taq DNA
(RAPD) has been used ini many plant species to polymerase and 25 ng DNA template.
detect genetic variation and genetic linkage with Amplification reaction was carried out in Gen-
specific characters (Fu et al., 2003; Tarras et al., Amp PCR System 2400 (Perkin Elmer) Pro-
2007)

grammable thermal cycler under following PCR 1334 plants indicated that J. curcas collection
condition, 1 min at 95oC (pre PCR); 30 cycles of could be grouped into 4 groups namely A, B, C
30 sec at 95oC, 45 sec at 56oC and 1 min at and D which fruit number/tree were ≥4, ≥3,
72oC; 7 min at 72oC (post PCR). PCR product ≥2, 0-1, respectively (Fig. 1). Percentages of
were separated in 1.5% agarose gels using TAE plant number of each group were 19.44 %, 22.77
buffer and visualized using UV transillumina- %, 17.82 % and 39.95 % were considered as
tor. group A, B, C and D. Second fruit yield selec-
tion indicated that the highest fruit num-
Data analysis: Dendogram constructed based on ber/bunch which highest average fruit num-
morphological character data of each jatropha ber/tree (66.8) as shown in Table 1. Maximum
individual sample grouping with fruit number numbers of branches/tree of 1 year old and 4
rank was generated by using computer package years old plant were recorded in group A (Table
SPSS version 13. 1.). Plants of group A also showed highest
growth response indicated by highest plant di-
RESULTS AND DISCUSSION ameter and height range. It seems therefore,
there was a positive correlation between growth
Morphological variation, fruit yield and oil rate reflected by height and stem diameter and
content within 4 groups of J.curcas: First fruit the fruit yield.
yield selection based on fruit number/bunch of
Table 1. Fruit yield and growth response of J. curcas.

Groups Height range Total branch Primary Diameter Average Average
(cm) number branches num- range (cm) of fruit num- fruit
range of 1 ber of 4 year 4 years old ber per number
year old old plants plants plant per
plants bunch
1 year old 4 years old
A 121-202 215-290 20-34 6-8 10.83-14.33 65.88 ≥4
B 118-130 160-220 18-20 6-7 4.46-8.6 21.68 ≥3
C 123-134 175-220 16-17 5-7 5.73-8.6 9.74 ≥2
D 105-114 165-210 16-17 3-6 5.1-12.0 2.68 0-1
A B

C D
Fig 1. Variation of fruit yield.
Leaves variation of J. curcas was identified in the colour of petiole, shoot and young leaves. The colour
variations were green and green reddish (Table 1, Fig. 2). There was no significant correlation between leaves
colour variation and J. curcas fruit yield group.
Tabel 2. Variation of young and mature leaves, shoot and petiole colour.
Individual samples of Mature leaves petiole shoot Young leaves
each group
A1 Green Green Green reddish Green reddish
A2 Green Green Green reddish Green reddish
A3 Green Green Green Green
B1 Green Green Green Green
B2 Green Green Green Green
B3 Green Green Green reddish Green reddish
C1 Green Green Green reddish Green reddish
C2 Green Green reddish Green reddish Green reddish
C3 Green Green Green reddish Green reddish
D1 Green Green Green Green
D2 Green Green brownish Green Green
D3 Green Green brownish Green reddish Green reddish
A B

C D
Fig 2. Colour variation of shoot (a), petiole (b), young and mature leaves (c, d).
Fig 3. Dendogram of phenotyphic variation of four J. curcas group constructed based on height range, diameter range and
leaves variation.
A J. curcas dendogram constructed based plant canopy, collar diameter, number of primary
on height and diameter ranges and leaves varia- branches, number of secondary branches, num-
tion divided into two clusters (I and II). Cluster I ber of leaves, number of capsule, seed in gram
consisted of only group A while groups B, C and and seed oil content were grouped in the same
D were grouped in cluster II. Cluster I possessed cluster (Gohil & Pandya, 2008).
high fruit yield, primary and total branch num-
ber, height and diameter ranges. J. curcas clus- Oil content: Chemical composition analysis of
tering based on morphological character indi- oil content analysis was carried and the result
cated that there was a correlation between fruit was ranging from 25.1 % to 32.4% (Tabel 2.).
yield and growth response. Similar result was J.curcas oil content were ranging from 8.1 % to
also found on genetic diversity assessment of 45% depending on geographic distribution, cli-
Indian collection of J. curcas based on pheno- mate and soil condition (Pant et al., 2006;
typic character viz., plant height, plant canopy, Kaushik et al., 2007; Gohil & Pandya, 2008).
coral diameter, number of primary branches, Some plant individuals of group A which pos-
number of secondary branches, number of ter- sessed oil content more than 30% could be cate-
tiary branches, average leaf area, petiole length, gorized as high jatropha oil content and could be
flower bud setting and basal height for 1st selected as superior candidate for further breed-
branch initiation and oil content which is divided ing program.
into 5 clusters. The results indicated that the In- High oil content of J. curcas indicated that
dian Jatropha genotypes with most desirable this oil is suitable for non-edible vegetable oil
characters i.e. high mean value for plant height, feedstock in oleo chemical industries (biodiesel,

fatty acids, soap, fatty nitrogenous derivatives, arable soil condition and higher altitude (800-
surfactants and detergents, etc). Currently, J. 1000 m) could increase number of fruit per tree
curcas can produce 2000 liter/ha oil per annual up to 217 and oil content up to 45% (Pant et al.,
while its yield productivity vary from less than 2007). The fact that Jatropha oil can not be used
100 kg to more than 10 tonnes of seed per ha for nutritional purposes without detoxification
(Akbar et al., 2009). Because of the wild nature makes its use as energy or fuel source is very
of the plant, morphological characters, oil con- attractive as biodiesel. The oil extracts exhibited
tents and other chemical constituent vary consid- good physicochemical properties and so it could
erably among different provenance of J. curcas be useful as biodiesel feedstock and industrial
(Becker & Makkar, 2008). The estimated yield application (Akbar, 2009). Further research is
of group A with plant density 2200/ha is about needed such as analysis of physical and mechan-
413 kg. The yield productivity and oil content ical properties of seed to optimize oil recovery
of group A plant could be increased through and analysis of oil properties such as density,
improvement of various agronomic important viscosity, peroxide value, and acid value in order
factors such as planting density, nutrient and to obtain superior genotype that useful in oleo
water demand and pruning time. Combination of chemical industry of this superior clone.
Tabel 2. Chemical composition of group A of Jatropha fruit.

Individual plant Water content Ash level (%) Protein content Oil content Fiber (%)
(%) (%) (%)
A2 5.4 3.51 22.3 28.5 20
A10 3.43 3.43 24.6 31.5 31.6
A69 6.09 2.98 28.3 25.1 25.8
A5 6.1 4.03 24.1 32.4 29.7
A7 5.93 3.61 25.4 30.1 31.2
DNA finger print variation generated with Tabel 3. Number of band and polymorphism score of
RAPD marker: Seven out of 17 RAPD primers RAPD banding patterns generated in Jatropha.
used could produce amplification product and Primer Maximum scorable Score of poly-
out of seven, a total of 5 primers showed poly- band morphism
morphic bands (Tabel 3.). DNA finger printing OPB-07 8 +
OPB-10 8 +
variations based on RAPD marker were also OPE-01 7 +
detected in Jatropha germplasm collection of OPE-05 8 +
Research Centre for Fibre and Tobacco, Ka- OPE-20 7 -
rangploso, Malang, East Java (Maptuchah, OPF-10 5 +
2001). RAPD markers were able to identify PH-17 3 -
moderate to high genetic variability index among
bp
the 40 genotypes of different eco-geographic
India’s Jatropja collection (Iqbal et al., 2010).
DNA finger print variations were detected within OPB-07 OPB-10
group A and also between group A, B, C and D 12216
(Fig. 4 and 5.). In the future, it needs further 8144
5090
confirmation concerning genetic variation 4072
RAPD-based analysis in a larger number of 2036
samples of each plant individual group to identi- 1636
fy specific DNA bands or banding pattern relat-
1018
ed to yield and oil content.
506
DNA A1 A2 A1
Ladder
Fig 4. RAPD banding pattern of J.curcas generated with
OPB-07 and OPB-10 primers.

bp
bp
12216
8144
12216 5090
8144 4072
5090 3054
4072 2036
3054
2036 1636
1636
1018
1018
506
506
A3 B2 C2 DNA A5 B5 D3 C1 DNA
ladder ladder
Fig 5. RAPD banding patterns variation of group A, B, C,D of J. curcas using OPB-07 primer.
CONCLUSIONS Becker, K & H. Makkar. 2008. Jatropha curcas: A poten-

tial source for tomorrow’s oil and biodiesel. Lipid
Technoogy. 2 (5): 104-107.
On the bases of observations of morpholog-
ical character, yield, oil content and growth re- Fu, C., Y. Qiu & H. Kong. 2003. RAPD analysis for genetic
diversity in Changium smyrnioides (Apiaceae), an
sponse of J. curcas collection of Research Cen- endangered plant. Bot. Bull. Acad. Sin. 44: 13-18.
tre for Biotechnology-LIPI group A that pos-
Gilies, A. C. M, J. P. Cornelius, A. C. Newton, C. Navaro,
sessed the highest fruit number per tree (65.88),
M. Hernandez & J. Wilson. 1997. Genetic variation
and high oil content (30.1% -32.4% ) could be in Costa Rica population of tropical timber species
considered as superior tree when compared to Cedrela odorata L. assesed using RAPDs. Molec
other groups (B, C, D). Having known the genet- ecol. 6: 1113-1115.
ic pattern of J. curcas that produced high oil Gohil, R. H. & J. B. Pandya. 2008. Genetic diversity as-
yield and the growth rate, as well as the related- sessment in physic nut (Jatropha curcas L.). Interna-
ness between accessions, the superior one could tional Journal of Plant Production. 2 (4):321-326.
be potentially developed as mother plants to Kaushik, N, K. Kumar, S. Kumar, N. Kaushik & S. Roy.
provide high quality planting materials to sup- 2007. Genetic variability and divergence studies in
port biofuel industries. RAPD analysis con- seed traits and oil content of Jatropha (Jatropha cur-
firmed the correlation between morphological cas L.) accessions. Biomass and Bioenergy .31: 497-
502.
parameters (height, diameter, number of branch-
es, and yield and that superior one (group A) was Maftuchah. 2001. Analisis molekuler tanaman jarak pagar
(jatropha curcas L.) berdasarkan penanda random
in a separate cluster apart from the other. amplified polymorphic dna: optimasi kondisi reaksi
PCR-RAPD. Publikasi. Umm. ac. Id.
AKCNOWLEDGEMENTS Mishra, D.K. 2009. Selection of candidate plus phenotypes
of Jatropha curcas L. using method of paired com-
The authors would like to thank Mr. Tatang parisons. Biomass and Bioenergy. 33: 542-545.
Kuswara for assisting the chemical analysis of
Pant, K.S., V. Khosla, D. Kumar & S. Gairola. 2006. Seed
fruits, Mr. Nawawi and Ms. Nurhaidar Rahman oil content variation in Jatropha curcas Linn. In dif-
for assisting in compiling the fruit yield group- ferent altitudinal ranges and site conditions in H.P.
ing. India. Seed oil content variation in Jatropha curcas
Linn. in different altitudinal ranges and site condi-
tions in India. Lyonia. 11(2): 31-34.
REFFERENCES
Tarras, A., N. Tawatti & F. Al-Maliki. 2007. Genetic fin-
Akbar, E., Z. Yaakob, S. Kamarudin, M. Ismail & J. gerprint of some KSA date palm cultivars using
Salimon. 2009. Characteristic and Composition of modern biotechnological techniques. Bioetchnology.
Jatropha Curcas Oil Seed from Malaysia and its Po- 6(2): 263-267.
tential as Biodiesel Feedstock. European Journal of Verma, K.C. & A. K. Gaur. 2009. Jatropha curcas L.: Sub-
Scientific Research. 29 (3): 396-403. stitute for Conventional Energy. World Journal of
Agricultural Sciences. 5 (5): 552-556.

B. Biorefinery

Study of Carbon Dioxide Emissions through Embodied Energy
of Renewable Biomass
Young Gyu Park1, HyungSuk Kim2 and Jung-in Kim3
1
Department of Chemical Engineering, Daejin University 11-1 Sundandong Pocheonsi Kyungkido
487-711 Korea, Tel:8231-531-1970, Fax:8231-536-6676, e-mail: ypark@daejin.ac.kr
2
DSK Engineering, Seochodong Seochoku Seoul Korea.
3
Department of Industrial Economics, Joongang University, Ansung Kyungkido Korea.
(Correspondence to: Young Gyu Park)
ABSTRACT
In order to make the best choice between renewable energy technologies, it is important to be able to com-
pare these technologies on the basis of their sustainability, which may include a variety of environmental, and
economic indicators. This study examined the comparative sustainability of renewable technologies in terms of
their life cycle CO2 emissions and embodied energy, using life cycle analysis. The models developed were based
on case studies of biogas pilot plant in Korea. The comparative results showed that power generation of bioen-
ergy was associated with 0.96 kWh/m3biogas and the reduction of CO2 emission is 2.1 kg of CO2 /kgBiomass.
Other environmental indicators should be applied to gain a complete picture of the technologies studied. The
generation of electricity is 2.07 kWh/m3 biogas in comparison with theoretical results of 3.09 kWh/m3 (efficien-
cy of generator is 30%) based on the assumption of the removal efficiency 95% of CO 2, methane conversion
100%, efficiency of generator 30% . Final results are the production of methane: 250 m3/day, production of elec-
tricity: 770 kWh/day when used 5 m3/day of waste.
Keywords: Renewable technology, Bioenergy, Carbon dioxide, Greenhouse gas, Environmental Software, LCA.
INTRODUCTION ments will look to alternative technologies. The

objective of this study was to compare the dif-
Due to increasing concerns over greenhouse ferences in the life cycle sustainability of renew-
gas emissions and declining fossil fuel stocks, able bioenergy technologies in a Korea, using
interest in renewable energy technologies is the indicators of associated CO2 emissions. This
growing rapidly. Many countries are now look- includes the construction, maintenance, and de-
ing to invest heavily in renewable energy sources commissioning phases, and the transport associ-
in order to meet the electricity requirements of ated with each phase. It is recognized that other
their populations and reduce their contribution to factors influence the sustainability of a product,
global greenhouse gas emissions. While this but an evaluation of both life cycle CO2 and en-
trend has been progressing in Korea for some ergy will provide an initial comparison.
years now, for example with a prominent recent CO2 emissions and embodied energy can
example has been United States’ President reveal the importance of “hidden” processes and
Obama’s announcement to reduce greenhouse materials pertaining to an individual product or
gas emissions to 1990 levels by 2020, through a component. This in turn gives an indication of
cap-and-trade system. the environmental impact of each technology in
While electricity generation technologies terms of their contribution to environmental ef-
that rely on renewable resources are perceived to fects such as climate change and resource deple-
be clean, a more appropriate definition would be tion. However, because sustainability is an ex-
to say that they are “cleaner” than fossil fuel- tremely broad field, although these indicators
based technologies, by life-cycle methodology provide a salient overview of each system, rely-
which renewable generation systems still con- ing on just two indicators limits the possible
sume energy and produce emissions during the measure of sustainability. Many other environ-
construction, maintenance, and deconstruc- mental sustainability indicators (such as ecotoxi-
tion/disposal (I.e., the whole life cycle) of the city) were not quantified, and social and eco-
required infrastructure. Because the number of nomic indicators were also ignored.
sites for new large-scale biogas plant in Korea is
limited, it is likely that new electricity develop- Embodied Energy: although embodied energy

(sometimes referred to as “energy”) is not yet life cycles of the machinery carrying out these
widely used as an indicator of sustainability, it processes, as the machinery has a separate life
provides a good indication of the level of recycle. For example, transportation of a drill rig
source consumption required to create or extract and the diesel used to drill a borehole were in-
a product and to transport it to its final destina- cluded but not the components of the drill fig
tion. Embodied energy includes all primary en- itself, as it was assumed that manufacturing
ergy used by a product or process, including fuel emissions and energy would be proportional to
and electricity. These energy inputs come from the emissions and embodied energy of the mate-
various sources, such as the machinery used in rials and, in this context, the omission of manu-
the extraction of raw materials, transport of the facturing requirements would have a negligible
raw materials to the processing plant, manufac- effect on the relative performance of the com-
turing processes, and transport associated with pared technologies.
the final end use. In this study, the initial energy Fugitive emissions from geothermal fields
(intrinsic energy stored in raw materials) of the were noted, though not added to the result for
raw materials has been excluded, mainly due to geothermal fields were noted, though not added
the difficulty associated with quantifying this to the result for geothermal power generation,
energy. This, however, maintains consistency but all other “CO2 emissions” pertaining to this
with the main source of materials and process study arose from construction, maintenance, and
data used for this study (1). decommissioning of power stations, since re-
newable technologies (apart from geothermal)
Life Cycle Analysis Methodology: ISO Standard do not emit CO2 during normal operation.
14044 is the standardized method for life cycle
analysis (LCA). It consists of four steps: goal RESULTS AND DISCUSSION
and scope definition, inventory analysis, impact
assessment, and interpretation. This methodolo- Table 1 shows several scenarios to analyze
gy was followed as closely as possible for this the CO2 emissions depending on the recycle of
study. A LCA was chosen for this study, the in- renewable bioenergy. The estimated total CO2
ventories were entered into LCA software, and emissions, embodied energy, and energy genera-
the results were compared on a normalized basis tion for each of the renewable energy technolo-
(per kilowatt-hour). As suggested by SETAC, a gies were studied. The life-cycle methodology
level one approach was taken for this study, in for CO2 emissions and embodied energy were
which the third step of impact assessment was normalized to obtain the CO2 emissions and em-
not applied due to the comparative structure of bodied energy per kilowatt-hour of output.
the study. SimaPro 7 software was chosen for The electricity generation and heat genera-
this study to quantify the embodied energy and tion by bioenergy, with the lowest total life cycle
CO2 emissions for each type of generation tech- CO2 emissions and total embodied energy, had
nology. the lower global warming potential in the total
life cycle energy generation. The life cycle CO2
System Boundaries: as this study was of a com- emissions and embodied energy for electricity
parative nature, the LCA system boundaries generation were only slightly lower, compared to
were drawn around the unique aspects of each the very high values of all three calculated for
technology, and apparatuses common to all types coal-based and oil-based electricities. While
of power generation were ignored, including coal-based and oil-based electricities both
components such as paints and lubricants, as showed significantly higher life cycle CO2 emis-
well as transmission and distribution infrastruc- sions and embodied energy than bioenergy-based
ture such as switchyards. CO2 emissions and em- electricity generation, the total energy generation
bodied energy related to the manufacture of ma- for bioenergy-based electricity was lower than
terials were included but assembly or fabrication that calculated for 30% increase of biogas elec-
of whole components was not. For example, en- tricity. Coal-based and oil-based electricities’
ergy and CO2 emissions changed the final values generation benefit from their higher load factors,
by less than one percent for each technology, which allow a much greater output in the 100-
meaning that the error for each model would be year plant lifespan compared to bioenergy-based
higher than the effect of this omission. electricity generation. So, although there is a
Additionally, energy consumed and CO2 pro- much higher materials investment in coal-based
duced in processes were quantified but not the and oil-based electricities, the load factor brings

the values for both CO2 emissions and embodied (apart from maintenance, which is included in
energy into close proximity with the values for life cycle CO2). On the other hand, the biogas
bioenergy-based electricity generation. production using pig manure in the anaerobic
Overall, recycled-bioenergy electricity bioreactor was 252+20 m3/day, of which has
generation had the lowest of both CO2 emissions 68+0.9% of CH4 contents. Final technical results
and embodied energy per kilowatt-hour com- has production 1.115m3 CH4 per VS 1 kg, which
pared to the other technologies studied as shown used for the production of electricity using gen-
in Table 2. Conversely, coal-based electricity erator of consumption of 36.9 liter/day light oil.
generation had the highest values of CO2 emis- 626 kWh/day of electricity and 689 Mcal/kg VS
sions and embodied energy per unit output. The were produced. The biogas production and their
difference between biogas recycle for heat gen- properties were classified as shown in Table 3.
eration and bioenergy for heat and electricity Another study calculated a CO2 emissions
generation was difficult to determine, since factor (including fugitive emissions) of 1.96 g of
while recycled-bioenergy electricity generation CH4 /kgOM for composting. Taken altogether,
had lower CO2 emissions per kWh. Fig. 3 suggests that the greenhouse gas emis-
Fig. 2 shows the LCA result of percent con- sions for biogas generation originate primarily
tribution to total CO2 emissions and embodied from fugitive CH4 emissions. Although biogas
energy by component for bioenergy technology, facilites allow CH4 to become concentrated at the
with each component further subdivided into surface of a well, these emissions are generated
percent contribution of each life cycle phase. by natural processes rather than by the presence
Where the decommissioning phase accounts for of a power plant, and as such, they have not been
negative contribution, such as in the case of re- added to this study’s result for bioenergy power
cycling, this is shown as a negative bar on the generation. The fugitive emissions refer to op-
left-hand side of the chart. erational CH4, rather than life cycle CH4; the
As this was a comparative study and com- latter comprises the CO2 emissions resulting
ponents common to all power stations (such as from the lifespan of plant equipment and its
transmissions systems) were not considered, the maintenance. However, these additional emis-
results may be used for comparison but not as sions should be taken into account when consid-
definitive value additionally, manufacture of ma- ering the sustainability in terms of CO2 emis-
terials for each plant was quantified but fabrica- sions from geothermal fields are highly site-
tion of components from those materials was specific, and so CO2 release will vary widely
not. This means that the results calculated are between geothermal power plants. In Korea, bi-
probably slightly lower than the reality. ogas plant fugitive CH4 emissions are estimated
A previous study undertaken suggests CO2 annually as ranging around 67%, the average
emissions factors of anywhere 0.131 kg of CO2 heat being 689 g Mcal /day; Biogas production is
/0.579 kg for livestock farms and 0.109 kg of 252 Nm3/day.
CO2 /0.012 kg for fertilizer. Another study calcu- Likewise, in electric power systems, me-
lated the CO2 emissions coefficient at 0.225kg of thane released from the decomposing of flooded
CO2/1.24MJ for electricity and 0.767 kg of CO2 organic matter increases the technology’s associ-
/0.266 kg for growing pig farms (23). While the- ated greenhouse gas release. In some cases, trees
se results vary quite widely, it was felt the results are cut down before the dam is filled, reducing
obtained from this study generally conformed, the associated methane production; while in oth-
considering the comparative nature of the study. er cases, vegetation is left as is, and depending
However, total CO2 emission releases on the location of the dam, there may be more or
around 2.21kg of CO2 /0.935 kg pig-manure, less vegetation per unit area, as well as more or
during normal operation. None of the other tech- less flooded.
nologies studied release CO2 during operation
Table 1. Scenario for the reduction of CO2 emissions.
Items of Scenario Explanation of Scenario Reference

Scenario I Heat energy for the boiler and energy for the pro-
duction of electricity: Hard coal
Scenario II Heat energy for the boiler and energy for the pro-
duction of electricity: Petroleum oil

Scenario III Heat energy for the boiler: Biogas energy for the Recycle of biogas for the re-
production of electricity: Hard coal use energy of the boiler
Scenario IV Heat energy for the boiler and energy for the pro- Recycle of biogas for the re-
duction of electricity: Biogas use energy of the boiler and
electricity
Scenario V Heat energy for the boiler and energy for the pro-
duction of electricity: 30% Increase of Biogas
Table 2. LCA results of scenario.

Impact
Unit SenarioI SenarioII SenarioIII SenarioIV SenarioV
category
Greenhouse kg CO2 -0.74 -0.835 -1.17 -1.23 -1.02
Ozone layer kg CFC11 144E-8 7.84E-8 1.09E-8 1.0E-8 1E-8
Photochemical Oxida- kg C2H4 0.000411 0.000358 0.000264 0.000259 0.00308

tion
Acidification kg SO2 0.015 0.0118 0.0124 0.0123 0.0155
Eutrophication kg PO4 0.0253 0.0243 0.026 0.00266 0.00335
Table 3. Input & output data for the LCA.
Input Output Comments

3
Input materials (m /d) 5 - TS 4.9%, VS 3.6%
3
-Swine waste (m /d) 3.5 - TS 2.2%, VS 1.4%
- waste (m3/d) 1.5 - TS 12.3%, VS 10.8%
Electricity (kW/d) 84.9 626 Assuming that all biogas was converted to electricity
3
Biogas(Nm /d) - 252±20 CH4 67.9%, CO2 31.6%, H2S 0.5%
Diesel (L/d) 36.9 Used for the generator
Currently the heat produced is not used for maintaining the fa-
Heat (Mcal/d) - 689
cility
Liquid fertilizer - 5 N : 0.45%
Table 4. Estimated gas emission of 1 ton of the mixture of swine waste and food waste during the composting period of 90
days1).
Contents Estimated gas emission
Gases Unit emission2)
(/ton raw material) (/ton raw material)
NH3 127.4 g NH3-N/kg T-N 4.74 kg T-N 604 g NH3-N 733 g NH3
N2O 46.5 g N2O-N/kg T-N 4.74 kg T-N 220 g N2O-N 691 g N2O
CH4 1.96g CH4/kg OM 45.2 kg OM 85.9 g CH4
1)
The mixture ratio of swine waste:food waste was 7:3. The data were cited from Fukumoto et al23)
2)

Fig. 1. Biomass diagram to use a renewable source.
Fig. 2. LCA results for the pig manure.

100
90
Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ
80 Acidogenic Methanogenic Gas collector
CH4 (%) 70
60
50
40
30
20
10
0
0 100 200 300 400 500
Time (day)
Fig. 3. Methane production for the anaerobic fermentation of pig manure.

The Production of Anaerobic Hydrogen in Chlamydomonas reinhardtii
was Induced by the decrease of Sulphur
Maria Omega1, Matthew Timmins2, Ben Hankamer2 and Peer Schenk1
1
School of Biological Sciences, Faculty of Science, University of Queensland
St Lucia, Australia 4072, e-mail: prihtamala_omega@yahoo.com
2
Institute of Molecular Biosciences, University of Queensland
St Lucia, Australia, 4072
(Correspondence to: Maria Omega)
ABSTRACT
NMR sprectoscopy and GC-MS (gas chroamtography-mass spectrometry) and TLC were used to observe
the metabolic status of Chlamydomonas reinhardtii after sulphur depletion in order to increase the production of
H2. The promising experiments show that from 0 to 24 h of sulphur depletion, these algae consume acetate me-
dium to accumulate starch and to deposit high amount of triacylglycerides inside the cells. Between 24 and 72 h
period, the metabolism of energy fermentation indicate the plummeted pH, the production of H 2 and the increase
production of H2. During 72 to 120 h, the results show that the lowered metabolism giving rise to stabilised pH,
in spite of the remaining starch and triacylglyceride molecules. Finally, our summary suggests that energy de-
pletion does not slow down the H2 production. However, sulphur depletion or toxic fermentative products such
as formate and ethanol leads to the loss of this important function.
Keywords: NMR, GC-MS, TLC, Chlamydomonas reinhardtii, sulphur.
INTRODUCTION
There are various ability of green algae to
produce hydrogen in anaerobic environment Anaerobic conditions to produce hydrogen
(Boichenko & Hoffmann, 1994; Timmins et al., via Sulphur depletion: C. reinhardtii Stm6 cul-
2009) which is supported by the presence of tures were harvested at exponential phase by
light (Gaffron & Rubin, 1942). Some experi- centrifugation (2500xg, 3 min, 25°C) and
ments have indicated that the build-up of Hydro- washed 3x in sulphur depleted TAP medium
gen in C. reinhardtii are from the utility of two (Harris, 1989; Schonfeld et al., 2004). Cells
Oxygen which can decrease ferredoxin involving were resuspended to 15x106 cells/ml in this me-
in the proton depletion to produce Hydrogen dium (pH 7.3) and kept in 600 ml flasks. Cul-
(Happe & Naber, 1993; Forestier et al., 2003). tures were exposed to 500 µ/Em2/s consecutive
The studies of H2 yield can be achieved by mak- white light and stirred at 150 rpm. The concen-
ing anaerobic algae culture using purged inert tration of dissolved O2, H2 gas production and
gases or dark incubation followed by light expo- pH of cultures were observed for 120 h during
sure (Gaffron & Rubin, 1942; Gfeller & Gibss, sulphur depletion. Samples were taken for analy-
1984). Therefore, sulphur depletion is the best sis at 0, 24, 48, 72, 96, and 120 h after sulphur
method to induce Hydrogen yield in algae. depletion. For GC/MS, 6 samples were taken at
Moreover, the transportation of sulphur into C. each time point and for NMR, the TAP medium
reinhardtii cells are done in the form of sulfate was modified by adding high concentration of
anion and various lipids, proteins and metabo- phosphate (from 2.33 to 3.27 g/l) and 15 mM
lites are needed in this process (Pollock et al., acetate.
2005). In addition, the observation of C. rein- Concentration of dissolved O2, H2 gas pro-
hardtii genomes has concluded that a massive duction and pH of cultures were analyzed by a
amount of peptides is essential in fermentative pH electrodes (Consort, Belgium) and gas com-
pathways and hydrogen production in anaerobic position was measured by an Agilent Micro
conditions (Grossman et al., 2007). So, we did GC3000 gas chromatography.
integrative studies to understand the metabolom- NMR analysis: algal culture (1 ml) was re-
ic response of C. reinhardtii under sulphur de- moved from bioreactor and then immersed in a
pletion and anoxia in order to produce hydrogen. beaker of boiling water for 3 min, cell culture

was transferred into a tube and the debris was that the pH decline could increase hydrogenase
centrifuged at 16000xg for 3min. For NMR stud- response but affect the H2 production rates.
ies, 500 µl of supernatant was transferred to an
NMR tube and 50 µl of distilled water was add- NMR studies of the C. reinhardtii metabolic
ed. High resolution of proton NMR spectroscopy profiles over sulphur-depleted H2 production:
was measured on a 500 MHz spectrometer (Ad- For NMR analysis, a culture medium was ex-
vance 500, Bruker, Germany). tracted to harvest the extracellular and intercellu-
TLC: algal cultures (2 ml) was centrifuged lar metabolites in algal culture. Samples were
at 16000xg for 3 min. Non polar lipids was ex- taken for NMR studies at 24 h intervals during
tracted from the pellet by addition of methanol, 120 h of sulphur-depleted H2 production (Fig 2).
chloroform and water (1:1:1). Samples were
sonicated for 20 min and vortexed. Phase separa-
tion was performed by centrifugation at 16000xg
for 5 min and the choloroform layer was dried in
an evaporator. Samples were resuspended to a 20
ml chloroform and 3 µl was loaded onto a
HPTLC-HL normal phase silica gel plate (Anal-
tech) and the plate was dried and visualized un-
der UV illumination and fatty acid analysis was
performed using an Agilent 6890 GC fitted with
a 5975 MSD (Poerschmann et al., 2004).

Fig 2. Analysis of Relative abundance changes of metabo-
The production of H2, pH and O2 post sulphur lites between 0 and 120 h following sulphur depletion by
depletion: For metabolomic studies, H2 produc- NMR
tion was induced by sulphur depletion in C.
reinhardtii and the levels of H2, pH and O2 were It is interesting to observe that between 0
measured during 120 h following sulphur deple- and 120 h, the acetate metabolites is an overall
tion (Fig 1). production when H2 is still produced. Ethanol
and formate were the massive fermentative
products in the next spectral profiles as well as
succinate. Their abundance is relatively in-
creased during the time period. Glycerate and
starch increased within 24 h and then decreased
until 120 h. We summed up that a number of
metabolites could change in abundance especial-
ly polar ones. This could be due to the loss of the
other metabolites during resuspension, wash and
centrifugation steps. These could affect the
membrane integrity of the cells, so it may loss
the function.
Fig 1. H2 production, dissolved O2 concentration, and pH of
C. reinhardtii cultures after sulphur depletion Amino acids profiling of C. reinhardtii over
sulphur-depleted H2 production: The studies of
After C. reinhardtii was induced by sulphur C. reinhardtii metabolites over sulphur-depleted
depletion, there was an increase in the dissolved H2 production indicated that amino acids are es-
Oxygen (O2) and pH of the medium from 0 to 12 sential in adaptive conditions and a changing
h. After 12 h, the dissolved O2 concentration response between protein degradation, synthesis,
started to plummeted and the culture became energy needs, nitrogen and redox levels. There-
anoxic at 20 h as well as the pH began to drop. fore, detailed analysis of amino acid metabolism
The production of H2 reached a peak at 48 h co- was undertaken by growing algae in TAP medi-
incided with a drop of pH to 7.6 and then this H2 um with isotope-labelled nitrogen salts (15N)
production decreased until 120 h. We suggested during 96 h of sulphur depletion (Fig 3).

increase in amino acid abundance during anoxic
H2 yield compatible to the normal levels of its
role in anoxic conditions.
Lipid analysis of C. reinhardtii over sulphur

depletion: Soluble fraction of lipid from C. rein-
hardtii cultures was observed at 24 h intervals
after sulphur depletion by GC/MS and TLC (Ta-
ble 1).
Triacylglyceride (TAG) abundance in-
creased within 24 h during sulphur depletion and
increased steadily throughout the time period.
Fig 3. Relative abundance of 15N-labelled amino acids after While sterol esters indicated an increase during
96 h of sulphur depletion H2 production. Similarly, there was an increase
in fatty acid concentration. Taken together, we
Two amino acids (lycine and proline), per- suggested that the amount of energy accumulat-
formed an increase in 15N-labelled isotope and ed in TAG was more than in the starch over the
this trend may stem from protein degradation or 24 h of sulphur depletion although the starch
synthesis from originally amino acids. Alanine analysis was measured from cells grown in op-
and aspartate showed a small change in 15N- timized medium for NMR, not for H2 yield.
labelled isotope. Whereas glutamate and leucine
indicated a decrease in 15N-labelled isotope due
to natural turn over. So, we suggested that the
Table 1. Analysis of fatty acid composition of C. reinhardtii cells post 120 h of sulphur depletion by GC/MS and TLC
Total Fatty acid (µg/ml culture) 0h 24 h 48 h 72 h 96 h 120 h

Free fatty acids, free sterols and chlorophyl a/b 57.6
TAG, free fatty acids, free sterols and chlorophyl a/b 116.7
Sterol esters, beta carotene, TAG, free fatty acids, free ster- 106.9
ols, chlorophyl a/b
Sterol esters, beta carotene, TAG, free fatty acids, free ster- 110
ols, chlorophyl a/b
ols, chlorophyl a/b
ols, chlorophyl a/b
CONCLUSIONS Grossman, A.R., Croft, M., Gladyshev, V.N., Merchant,

S.S., Posewitz, M.C., Prochnik, S. and Spalding,
M.H. 2007. Curr. Opin. Plant Biol. 10: 190-198.
This project was used to understand the
metabolomic profiling of C. reinhardtii over H2 Happe, T. and Naber, J.D. 1993. Eur. J. Biochem. 214: 475-
481.
production. The results can identify the process
and important molecules for bioengineering to Harris, E. 1989. The Chlamydomonas sourcebook. Aca-
demic Press. San Diego, CA. 780.
increase H2 production and finally to boost the
biofuel yield. Poerschmann, J., Spijkerman, E. and Langer, U. 2004. Mi-
crob. Ecol. 48: 78-89.
Pollock, S.V., Pootakham, W., Shibagaki, N., Moseley, J.L.
REFERENCES and Grossman, A.R. 2005. Photosynth.. Res. 86: 475-
489.
Boichenko, V.A. and Hoffmann, P. 1994. Photosynthetica.
30: 527-552. Schonfeld, C., Wobbe, L., Borgstadt, R., Kienast, A., Nix-
on, P.J. and Kruse, O. 2004. J. Biol. Chem. 279:
Forestier, M., King, P., Zhang, L., Posewitz, M., 50366-50374.
Schwarzer, S., Happe, T., Ghirardi, M.L. and Seibert,
M. 2003. Eur. J. Biochem. 270: 2750-2758. Timmins, M., Thomas-Hall, S.R., Darling, A., Zhang, E.,
Hankamer, B., Marx, U.C. and Schenk, P.M. 2009. J.
Gaffron, H. and Rubin, J. 1942. J. Gen. Physiol. 219-240. Exp. Bot. 60: 1691-1702.
Gfeller, R.P. and Gibss, M. 1984. Plant Physiol. 75: 212-
218.

Implementation Fuel of Methane from Biogas for
Supply Energy in Small Industry (UKM-Tahu)
Muhammad Kismurtono1, Khoirun Nisa1, Satriyo K. W.1 and Roni Maryana1
1
Technical Implementation Unit for Development of Chemical Engineeri Processes, Indonesian Insti-
tute of Sciences, Yogyakarta 55861, Indonesia
Telp/Fax: 0274 (392570); e-mail: m-kismurtono@yahoo.co.id
(Correspondence to: Muhammad Kismurtono)
ABSTRACT
The present of CO2 in biogas does not give to contribute to the calorific or heating value and are often
washing out in purification plants in order to obtain a gas with almost 100% CH 4 and dangerous effect on envi-
ronment. The objective of this study to support procurement of energy alternative and production of methane
88.94%. DIPA LIPI of budget year 2008 / 2009 by exploiting livestock waste to produce the biogas. A first pro-
cess form biogas is liquefaction so degradation organic material, to acetate acid. The second process here in
after is gasification by bacterium of methane producer use the enzyme to break the acid become the methane.
Inpres No. 1/2006 and No. 5/2006 hitting energy newly represent the basis for base law to develop the new en-
ergy. A researching into of Iptek DIKTI-LIPI proposed 2009 year this of making of system of purification of
biogas have high grade methane, for the supply of small industry (UKM-Tahu) requirement energy. Supply in
the form of electrics use the gas generator, boiling and process frying. From various gas type which implied in
the biogas represent the source energy is methane, but composition of methane gas not optimal, a mixture com-
posed mainly of 40-70% CH4, 30-60% CO2, 0-1% H2 and 0-1% H2S (Soewarno et al., 1991). The concentration
methane which not optimal, enabling to be concentration process of “CO2 removal”, using aqueous NaOH 1M
solution, local zeolite and synthetic so that biogas have the heating value larger ones and enable for the applica-
tion of as source of raw material energy to be converted become the energy electrics by using gas generator.
Results from development can be expected a methane fuel have high grade passing process of purification
system fuel the “CO2 removal” environmental.
Keywords: Aqueous NaOH 1M, Biogas, CO2 removal, Methane, Zeolite.
INTRODUCTION with Selexol TM; (4) Chemical absorption with

amines.
The biogas is a produced by the anaerobic Alternative fuels such as alcohol, gas
decomposition of organic matter.It is primarity (LPG,CNG), and biomass derived fuel have been
composed of methane (CH4). And carbon diox- studied intensively. One of potennnnntial fuel
ide (CO2) with smaller amounts of hydrogensul- wich is abundantly available is biogas. .Biogas
phide (H2S), ammonia (NH3) and nitrogen (N2). originates from bacteria in the process of biodeg-
Usually, the mixed gas is saturated with water radation of organic material under anaerobic
vapour (Angenent, 2004). Biogas can be used for condition. It consists of a varying proportion of
all applications designed for natural gas. Not alla CH4 (Methane) ans CO2 (carbon dioxide) and
gas applications require the same standards. The traces of H2S,N2 ,CO,O2.The content of CH4 and
usage of biogas as fuel has significantly in- CO2 is a function of the matter digester and pro-
creasedinthe last years. For an effective use of cess conditions like temperature, Ph, C/ N ra-
biogas as fuel it has to be enriched in me- tio.Biogas is a clean fuel for internal combustion
thane.The is primarily achieved by carbon diox- engine, Jiang Chengqui et al. investigated the
ide removal which then enhances the energy compressed biogas and natural biogas and their
value of the gas to give longer driving distances application to diesel fuel. Biogas was com-
wich fixed gas storage volume (Angenent, pressed up to 9.8 x 106 for high pressure applica-
2004). AT present four different techniques for tion. For the low pressure operation, biogas was
upgrading of biogas are used commercially in supplied directly from the digester. The results
Sweden: (1) Absorption with water; (2) PSA showed that the diesel fuel replacement achieved
(Pressure Swing Adsorption); (3) Adsorption by connecting the high-pressure biogas directly

into the mesine is less than thatof connecting MATERIALS AND METHODS
low-pressure biogas (natural biogas). Com-
pressed biogas is becoming widely used in Swe- Research design and methods used to test
den,Switzerland and Germany. There are many removal of the CO2 from biogas stream. Under
kinds of orocess that stated in literature to re- continuous operation condition, first the biogas
moving CO2 gas which can be divided into two introduced at the bottom of the packed column,
ways; those are chemical absorption and adsorp- passing yhrough the aqueous NaOH 1M solu-
tion.Chemical absorption is an absorption which tion, flowing downwards to the solution separa-
is owed by chemical reaction where the absorbed tor. In this column the CO2 is absorbed and trans-
gas is reacted with the reactant in liquid phase, formed into aqueous NaOH 1M solu-
while adsorption is absorptionon the surface of tion.Samples of the inlet and outlet biogas were
solid particle whichis called adsorbent. The aim taken during experimental tests using gas sam-
of this study is to get operation data to get ples.The compositions of these samples were
packed column design in eliminating CO2 from determined by gas chromatography. CO2 remov-
biogas. The benefit of the research is that the al, expressed as a percentage, was calculated by
research result can be used as pattern to design dividing the difference between the inlet and
and analyses packed column for chemical ab- outlet volume compositions by the inlet.
sorption of gas CO2 from biogas, and also for
other similar system.
CONSTRUCTION OF
CONSTRUCTION OF
FIXED DOME TYPE
CO2 REMOVAL
DIGESTER
STEP I FERMENTATION
Formula of
fermentation
BIOGAS NO
YES
STEP II PURIFICATION BY
2 (TWO) COLUMN
ACTIVATION OF ZEOLITE
AND NaOH 1M
PURIFICATION PREPARATION OF
FUEL GRADE METHANE GAS BY
ABSORBTION AND ADSORPTION
TECHNIQUE
NO FUEL GRADE METHANE GAS

88.94 %(v/v)
YES
METHANE GAS FOR HEAT AND

ELECTRICITY
TESTING OF METHANE GAS

FOR MACHINE AND FURNACE OR
GRINDER

Table 1. Specification of product biogas (before treatment).
No Item Dimention Test methods Specification
1 CH4 %(v/v) GC 63.20
2 CO %(v/v) GC 11.10
3 CO2 %(v/v) GC 25.19
4 H2 %(v/v) GC 0.49
5 Impurities %(v/v) GC 0.02
Table 2. Specification of product biogas.

No Item Dimension Specification Specification Specification
(a)* (b)* (c)*
1 CH4 %(v/v) 55 - 65 55 - 75 40 - 70
2 CO %(v/v) - - -
3 CO2 %(v/v) 35 - 45 25 - 45 30 - 60
4 H2 %(v/v) 0- 1 1- 5 0- 1
5 N2 %(v/v) 0 -3 0 – 0.3 -
6 O2 %(v/v) - 0.1 – 0.5 -
* * *
Note. (a) Arifin et al., 2008; (b) www.kolumbus.fi; (c) Muryanto et al., 2006.
RESULTS AND DISCUSSION the effect of pressure, concentration of aqueous

NaOH 1 M solution in inlet absorbent and
This study we assumed steady and temperature on percentage of CO2 absorbed.
isothermal condition and the system studied in Liquid flow rate was 40 ml. S-1 , pressure was
research comprises a packed column 10 cm in varied 350 and 700 mm H2O. The gas flow rate
diameter filled with 1 cm ball or 1.6 mmpellet was held constant at 600 ml. S-1 (Table 3).
zeolite to height of 80 cm . This research studied
Table 3. Pressure of biogas from digester vs Temperature Maximum from Estimation,o C.

No Pressure of Time of Input gas to Input gas to Input gas to Temperature
biogas from burning, column column absorber burner or gas maximum from
digester, (hour) adsorber(zeolite), (NaOH,1M) generator, estimation, o C
(mm H2O) %(v/v) %(v/v)* %(v/v)*
1 350 1 54.8900* 71.8700* 88.6200* 1.750***
* * *
2 700 2 55.8994 81.8780 88.6288 1.850***
3 350 1 56.1900** 82.8700** 88.4100** 1.980***
4 700 2 60.1400** 85.8900** 88.9400** 2.000***
Note: * -
Petrolab Services; ** - Jaringan Kerjasama Kimia Indonesia Services; *** - Trial and error, formula , H = Ho 298 +
Hsensible
In conclusion, results obtained in this REFERENCES

research are inferential that the percentage of
removal CO2 was influenced by absorbent flow Angenent, L. T., et. al. 2004. “Production of Bioenergy and
rate and pressure biogas and temperature Biochemicals from Industrial and Agricultural
Wastewater”. TRENDS in Biotechnology Vol.22
maximum: 2.000o and electricity 3.000 watt. No.9 September 2004.
Anonim. “Biogas”. www.electrigaz.com
ACKNOWLEDGEMENTS
Anonim. “Biogas”. www.wikipedia.org
The authors wish to acknowledge the Anonim. 1998. “Biogas Sumber Energi Alternatif yang
donors of Insentif Riset bagi Peneliti dan Ramah Lingkungan”, Majalah Kampus Genta, Edisi
Perekayasa Tahun 2009 by DIKTI-LIPI for the 117, Thn XXXIII, halaman 35-38, Surabaya.
support of this study. Cooper, J. 2001. “Turning Carbon Directly into Electrici-
ty”, Science and Technology Review, Lawrence Liv-

ermore National Laboratory, US Department of Ener- Setyo I. And Yuli. 2005. “Reaktor Biogas Skala
gy. Kecil/Menengah (Bagian Pertama)”. ISTECS, Japan,
www.beritaiptek.com
Raven, et.al. 200. “Biogas plants in Denmark: successes
and setbacks”. Eindhoven University of Technology, United States Patent – 4042332.
The Netherlands.
United States Patent – 5013334.
United States Patent – 5174796.

Study on Renewable Bioenergy Source Based on Algal Biomass
Extracted Oil
Joko Sulistyo
Microbiology Division, Research Center for Biology - LIPI

Jl. Raya Bogor-Jakarta Km. 46 Cibinong 16911, Bogor, Indonesia, e-mail: josulisty@yahoo.com
(Correspondence to: Joko Sulistyo)
ABSTRACT
Bioenergy has built a revolutionary platform that uses photosynthetic microalgae to produce a renewable,
high-value replacement for fossil fuel petroleum. This algae biofuels requires only sunlight, CO2 (potential for
carbon credits), nutrients and non-potable water – and can be produced at massive scale on non-arable land. In
order to determine the benefits of algal oil, we first have to identify some of the problems that it could solve.
The most obvious is a shortage of petroleum reserves and supplies increasingly being attributed to peak oil. Sig-
nificant production of algae biofuels could solve a great deal of those problems. That because of carbon dioxide
is the primary input required by algae to grow, in fact, the growth of algae could displace power plant emissions.
Growing algae is also very water efficient. The best part is, algae can grow in brackish, saline and wastewater,
further reducing the amount of freshwater needed to grow it. And the nutrients in wastewater actually feed the
algae, making it possible to cultivate at any one of wastewater treatment facilities nationwide. The benefits on
cultivation of algae are no one country has a monopoly on algae production or the algae production equipment,
algae can grow in temperatures ranging from below freezing to 80ºC. It is not in direct competition with food
crops. There are a multitude of algae biofuel value-added byproducts like high-protein animal feeds, agricultural
fertilizers, biopolymers (plastic), glycerin and even bioethanol.
Keywords: renewable bioenergy, algae oil, biodiesel, carbon dioxide, sunlight.
INTRODUCTION
Microorganism and inoculum preparation:
Due to rising oil prices and growing advo- Scenedesmus dimorphus, Chlorella vulgaris, and
cacy for environmental issues, especially climate Spirulina fusiformis were maintained on agar
change, research in alternative energy sources is containing Ca(NO3)2; KH2PO4; MgSO4; Na-
on the rise. Biofuels, such as biodiesel and bio- HCO3 and trace elements or metals such as
ethanols, are being viewed as a potential alterna- FeCl3.6H2O or ZnSO4.7H2O at 4°C. The stand-
tive to conventional fossil fuels. Algae is the best ard inoculums used was in order of 5-day-old
hope in producing renewable energy in large plates. Seed culture was prepared by transferring
quantities without damaging the environment or the cell suspension into 500 ml conical flask
competing with our food supply. Algal biomass containing 200 ml of spesific medium, incubated
consists of natural oils, proteins, and carbohy- at 30°C without agitation for 48 h.
drates. Many species of algae can produce oils
comprising up to 40% or more of their dry Photobioreactor medium and condition: Seed
weight. Algal oil can be converted to biodiesel cultures medium contained the following con-
through transesterification process by using stituents (g/l): KNO3; K2HPO4; MgSO4.7H2O;
strong base catalyst and bioethanol from starch Fe solution (EDTA and FeCl3.6H2O in DW);
crops by employing several main steps including Trace Metal solution (H3BO3; NaMoO4.2H2O;
enzymatic hydrolysis and fermentation process- CuSO4.5H2O; CoCl2.6H2O; ZnCl2; MnCl2.4H2O
es. The objective of the present study is to evalu- in DW) which was sterilized and added separate-
ate the suitability of the bioenergy source based ly. To investigate the microbial lipid production,
on microbial single cell lipid by cultivating of oil 10% (v/v) of seed culture was added into aqua-
producing algal strains and converting the algal culture containing 50 liters of the same composi-
oils extracted from its biomass to biodiesel tion of the photobioreactor medium at 21 to
through transesterification process using bioeth- 25°C and the cultures were harvested after
anol derived from starch crops. 10days incubation.

Production of cell-free extract: Algal biomass remaining water will be removed from the bio-
was suspended in 20% (w/v) extraction buffer ethanol.
containing 100mM KH2PO4 /KOH (pH 7.5),
20% (v/v) glycerol and 1mM EDTA and dis- Transesterification process: The algal biomass
rupted using mortar. The suspension was centri- were harvested by filtration of 100 ml cultures
fuged at 10,000 g for 15 min at 4°C and the re- through a Whatman no. 1 filter paper and
sulting supernatant (cell free extract) was used washed with 200 ml of distilled water. The fil-
for enzyme assays. Protein concentration was tered biomass were then freeze-dried for 6 hr for
determined using BSA as standard. determination of dry weight. Dried biomass were
then ground into powder using mortar, followed
Cell dry weight determinations: For each dry by lipid extraction using hexan/isopropanol 3:2
weight determination, a 5-ml culture sample was (v/v). Lipid obtained was is then added with 5%
centrifuged in a tared tube at 3000 xg for 10 min sodium hydroxide and bioethanol to a warm so-
at room temperature and washed first with 10 ml lution of the obtained oil, and the mixture is
and then with 5 ml of 0.1 M phosphate buffer at heated (50ºC) for 4h to allow the transesterifica-
pH 7.0. The cell paste was dried at 65°C for 48 h tion to proceed. The resultant colorless or pale-
in a vacuum oven. After cooling, the tube was yellow transparent methyl esters were analyzed
weighed, and the cell dry weight (grams per li- by gas chromatography.
ter) was calculated. Reported results are the
mean values of two determinations. RESULTS AND DISCUSSION
Algal oil extraction: A 10 ml hexane- Algal biomass of C. vulgaris, S. limorphus

isopropanol solution (3:2, v/v) was added to the and S. fusiformis were harvested, filtered, freeze-
dried mycelium in a 45 ml centrifuge tube, ho- dried and followed by extraction using hex-
mogenized for 1 min, and centrifuged at 5000 an/isopropanol 3:2 (v/v). The algal lipid contain-
rpm for 10 min. This extraction procedure was ing mixtures were then centrifuged at 10.000
repeated twice for the residue and the three su- rpm for 10 min. This procedure were repeated
pernatants were combined. Ten ml of 0.47 M twice and both of obtaining supernatants were
sodium sulfate was added to the supernatant to combined and added with 10 ml of 0.47 M sodi-
break the emulsion. The upper phase containing um sulphate to obtain more optimally oil yield.
purified lipid was transferred to another tube and The obtained oils were then evaporated at 60°C
evaporated to dryness in a 45°C water bath. The to remove residual solvent and to obtain the sin-
dry weight of the residue was determined as the gle cell lipid in the flask. To determine the yield
weight of total lipids. and composition of fatty acid in the mixture, the
obtained oils were then analyzed by gas chroma-
Bioethanol fermentation steps: The major steps tography and the result is shown in Table 1.
for making bioethanol from starch crops are
comprised of milling, liquefaction, saccharifica-
tion, fermentation, distillation and dehydration.
The tubers of starch crops were first ground into
meals and then hydrolyzed with alpha-amylase
and the starchs were liquefied. The mashes from
were then cooled and a secondary enzyme (glu- Fig 1. Freeze dried algal biomass, enzymatic hydrolyzate
of tubers starch and transesterified single cell oil
co-amylase) was added to convert the liquefied (left to right)
starch to fermentable sugars. Yeast (Saccharo-
myces cerevisae) was then added to the mashes In the starch crops-to-bioethanol process,
to ferment the sugars to ethanol and carbon diox- enzymes are used to catalyze the reaction, fol-
ide for about 72 to 96 hours before the distilla- lowed by fermentation as a series of chemical
tion process was started. The fermented mashes reactions that convert sugars to ethanol. The
(containing about 10% alcohol) were then dis- basic processes for converting sugar and starch
placed to the distillation system to remove alco- crops are well-known and used commercially
hol from the solids and the water. The alcohols today. While these types of plants generally have
came from the destillation column were then a greater value as food sources than as fuel
pass through a dehydration system where the sources there are some exceptions to this.

Table 1. Yield and fatty acid composition of algal single cell oil
Algal Single Cell Lipids (%)
Fatty Acid Content
C. vulgaris S. dimorphus S. fusiformis
Caprylic 0.0 0.0 0.0
Capric 0.0 0.0 0.0
Myristic 0.0 0.0 0.0
Palmitic 2.1 1.0 0.0
Lauric 1.2 4.0 16.1
Stearic 11.0 1.2 5.0
Palmitoleic 24.2 10.0 14.2
Oleic 10.5 1.0 10.1
Linoleic 24.2 12.1 36.0
Linolenic 18.2 24.0 16.1
Arachidonic 0.5 1.2 1.0
Dehenic 1.2 0.2 0.5
EPA 2.5 18.4 6.4
DHA 6.4 20.1 2.5
The fermentation reaction is caused by CONCLUSIONS

yeast which feed on the sugars. A final dehydra-
tion step removes any remaining water from the Biodegradable biofuel extracted from mi-
ethanol. The simplified fermentation reaction crobial lipids such as algae can be used for pow-
equation for the 6-carbon sugar, glucose, is: er generation. Significant production of algae
biofuels could solve a great deal of those prob-
C6H12O6  2 CH3CH2OH + 2 CO2 lems. That is because algae has a much higher
glucose ethanol carbon dioxide
productivity potential than crop-based biofuels.
These high yields can be attributed to algae's
The transesterification process reacts the bi-
high growth rate, which is often monitored in
oethanol with the single cell lipids contained in
hours instead of days, and has inputs of only
algal oils, forming fatty acid alkyl esters (bio-
land, sunlight, water, carbon dioxide and nutri-
diesel) and glycerin. The reaction requires heat
ents those required by algae to grow. There are
and a strong base catalyst, such as sodium hy-
numerous reasons for considering the algal pro-
droxide. The simplified transesterification reac-
duction system as a source of biofuel. It does not
tion is shown below (FFAs: free fatty acids):
use depleting fossil fuel deposits, the primary
Base
source of crude oil in the world. It requires small
Algal oil + FFAs (<4%) + Bioethanol  Alkyl esters + Glycerin land and limited supplies of water for culturing,
it does not compete with food crops, has a high
The catalyst, potassium hydroxide, was dis- yield, and produces no pollution from the use of
solved into bioethanol and mixed with the pre- fertilizers or pesticides. Microalgae possess sig-
treated oil. Once the reaction is complete, the nificant quantities of lipids and oils with compo-
major co-products, biodiesel and glycerin, are sitions similar to those of vegetable oils. These
separated into two layers. One environmental large amounts of oils are made up of single cell
benefit of replacing fossil fuels with microbial lipids which are transformed into biodiesel
and starch crops-based fuels is that the energy through transesterification by employing bioeth-
does not add to global warming. All fuel com- anol fermented from starch crops. Algal oil and
bustion, including fuels produced from biomass, starch crops based bioethanol are economically
releases carbon dioxide into the atmosphere. feasible towards production of biofuel. Algae
However, because algae use CO2 from the at- have the ability to absorb carbon dioxide from
mosphere to grow by photosynthesis, the CO2 the atmosphere, thus making a significant con-
formed during combustion is balanced by that tribution to the environment.
absorbed during the annual growth of the plants
used as the biomass feedstock, and the result is REFERENCES
still substantially a reduction in net greenhouse
gas emissions. Briggs, M. 2004. Widescale biodiesel production from al-
gae. http://www.unh.edu/p2/ biodiesel/article_algae.-
html.

Cohen, Z. 1999. Chemicals from microalgae. Tylor & Pelly, M. 2000. Mike Pelly’s biodiesel method. www.jour-
Francis Ltd. neytoforever.org.
Gabrosky, M.S., McCormick, R.L., Alleman, T.L. and Her- Sheehan, J., Dunahay, T., Benemann, J. and Roessler, P.
ring, A.M. 1999. Effect of biodiesel composition on 1998. A look back at the U.S. Department of Ener-
NOx and PM emission from DDC series 60 engine. gy’s aquatic species program. Biodiesel from Algae.
Colorado Institute for Fuel and Engine Research, Colorado. USA.
Colorado.
Soedradjat, S. 1999. Dunia minyak memasuki pasca 2000.
Gabrosky, M.S. and McCormick, R.L. 1998. Combustion Lembaran Publikasi Lemigas 32 (2): 98-99.
of fats and vegetable oil derived fuel in diesel engine.
Program Energy Combination Sci. 24: 125-164. Soeroso. 2005.Kilang pngolahan BBM dioptimalkan.
Harian Pagi Jawa Pos 11 Maret.
Culshaw, F.A. 1993. The potential of biodiesel from
oilseed rape, J. Power and Energy, Proc. Instn. Mech. Solistia, W.S. 2005. Biodiesel pilot plant.
Engrs. 207: 173-17. http://www.bppt.go.id/potensial/tampilkan.php?id=7.
Graham, L.E. and Wilcox, L.W. 2000. Algae. Prentice- Zuhdi, M.F.A. 2004. Uji ketahanan motor diesel dengan
Hall. USA. bahan bakar komposisi castor methyl ester, palm
methyl ester, dan minyak solar. Prosiding Seminar
Rahman, M. 1995. Biodiesel, alternatif substitusi solar Nasional Pasca Sarjana IV. Program Pasca Sarjana
yang menjanjikan bagi Indonesia. Lembaran Pub- ITS.
likasi Lemigas. 1: 95.
Zuhdi, M.F.A. 2002. Aplikasi penggunaan waste methyl
O’connor, C.T., Forester, R.D. and Seurrell, M.S. 1992. ester pada high speed marine diesel engine. Seminar
Cetane number determination of synthetic diesel fuel. Nasional Teori Aplikasi Teknologi Kelautan. FTK
FUEL, 71. ITS.

Utilization of Lignin-Rich Biomass Waste from the Production
of Bagasse Bioethanol for Additive in a Pilot Scale Production
of Fiber Based Composite
Bambang Prasetya1, Eko Wahyu Putro1, Hariyatun1 and Asep Muhamad Ridwanuloh1
1
Research Centre for Biotechnology, LIPI
Jl. Raya Bogor Km. 46 Cibinong Science Center, Cibinong, Bogor 16911, Indonesia,
e-mail: bambang.prasetya@lipi.go.id; bambang.prasetya@gmail.com
(Correspondence to: Bambang Prasetya)
ABSTRACT
Increasing interest on utilization of lignocellulosic biomass for production of bioethanol opens posibilities
to find out an alternative to use the resulted solid waste which contains a significant amount of lignin. In labora-
tory scale we have succeeded in producing bioethanol from bagasse with ethanol content of more than 30%
from dry weight. The remaining solid waste contains 15-20%, lignin and 2-6% holocellulose of total weight
before the process. In order to develop a total utilization of biomass with zero waste concept, utilization of lig-
nin from this waste will be an interesting aspect. One of the relatively simple utilization lignin is for adhesion of
fiber composite. The objective of this research is to find an alternative process to make use of lignin in existing
commercial product. In this experiment, we used lignin bagasse from the waste of fermentation process from
hydrolyzed bagasse. 500 g waste dried at 100 oC for 2 h, and put in 100 kg reactor. Phenol solution was added
with a ratio of waste to phenol of 1:3 w/v. Before addition of phenol solution, 0.05% (v/v) of sulfuric acid was
mixed with phenol solution. The mixture was heated until 120°C for 1 h and then cooled. After cooling the mix-
ture was filtered and kept in room temperature. The obtained solution with a concentration of around 2.5% (v/v)
was used as additive in the UF and PF glue system for fiber board production in commercial factory. The prop-
erties of fiberboard showed that the additive led to increasing physical and mechanical properties of fiber board
and fulfilled SII standard.
Keywords: lignin-rich biomass waste, bioethanol production, additive, UF and PF glue system, fiberboard
composite, commercial production, bonding strength.
INTRODUCTION structure and properties which has an amorphic

three-dimensional substance is difficult to be
Lignocellulosic biomass, such as agricultur- reacted with other chemicals. Therefore, the
al residues, plantation and forestry residues are most economically implementation of lignin is
basically suitable for used as inexpensive feed- mainly not in fine chemicals. Utilization of lig-
stocks for biofuel (bioethanol, biodiesel, biooil), nin-macromolecule is in form of bulky biochem-
however the technology for this process differs icals, such as for polymer resin, adhesives, mold-
from that of the starch-to-ethanol because of the ing product. Research and development to utilize
complex chemical structures of lignocellulosic lignin for many polymeric based resin or adhe-
materials. Recent research has been conducted sives has been done since more than 3 decades.
on the pretreatment of lignocellulosic biomass Technical lignins are also produced in tremen-
before the fermentation process or growing bio- dous quantities every year as a by-product of the
mass to be used for bio-ethanol. In line with de- pulping process. Extensive work has been con-
velopment of technology process for biofuel ducted to include organosolv lignins and other
production from biomass, utilization of its by lignins with their polymeric structure of phenolic
product become a strategic way to increase eco- units in Phenol Formaldehyde (PF) resins. The
nomically feasibility. One of the most important substitution of phenol, particulary with kraft lig-
aspect is to utilize of lignin rich biomass for in- nin, in the synthesis of PF resin is the most stud-
dustrial products. ied use of lignin (Conner, 2001).
Based on the composition, lignin belongs to The hydroxymethylation of alkali lignin
the one of the most abundant, renewable natural with formaldehyde in alkaline solution was stud-
products on earth because every woody material ied by Malutan et al. (2008). The influence of
contains 18-25%. However, due to chemically reaction conditions of the hydroxymethylation of

alkali lignin was followed by modifying the (Hui et al., 2009). The effects of catalyst per-
temperature, time and the ratio of NaOH to lig- centage used in producing wood liquid on its
nin and CH2O to lignin. The reaction was fol- bond strength as phenol formaldehyde substitute
lowed by total consumption of formaldehyde. adhesive in plywood were observed by using
Phenol formaldehyde resins are the major adhe- wood liquid form wood mill of Albizia (Parase-
sives used for bonding wood panels for outdoor rianthes falcataria). The results show that bond
application. PF adhesives modified with enzy- strength testing of exterior grade plywood sam-
matic hydrolysis lignin (EHL) were synthesized ples of Meranti showed that a quite high per-
by a one-step process. The phenol component of centage of catalyst (3%) decreased bond
the PF adhesives was partially substituted by strength, while that of Keruing showed that the
EHL extracted from the residues of cornstalks higher the percentage of catalyst, the higher the
used to produce bio-ethanol. The EHL–PF adhe- bond strength. In general, adhesive composition
sives were used to prepare plywoods by hot- of 80/20, 60/40 and some 40/60 (PF/WL) can be
pressing (Jin et al., 2010). The study presents used as plywood adhesive and meet the SNI for
lignin extration from commercial black liquor plywood (Prasetya et al., 2004)
and utilize it as a partial substitute in PF resin In connection with research on development
synthesis. Shear test properties showed that phe- of bio-ethanol production from biomass, we try
nol 15% replacement with black liquor lignin to utilize of lignin from solid waste. The re-
produced comparable physical bond properties mained solid waste contains mostly lignin and
and could be used to formulate a low cost modi- holocellulose in small amount (2-6%). This re-
fied PF resin for plywood panels (Gothwal et al., search aims at developing of total utilization of
2010). biomass with zero waste concept to produce
Compared to the lignin rich-solid waste green products. One of the relatively simple uti-
from pulping waste, the lignin waste from bio- lization lignin is for adhesion of fiber composite.
ethanol process contains mostly more than 20%
of holocellulose. Therefore the utilization of it MATERIALS AND METHODS
waste needs to make all remained holocellulose
soluble. One of recommended process is to make Lignin bagasse from the waste of fermenta-
liquefaction by using solvent and catalyst. Wood tion process from hydrolyzed bagasse was used
liquefaction using phenol as the reagent solvent for this experiment. 500 g waste dried at 100oC
and an acid catalyst has long been studied as a for 2 h, and put in 100 kg reactor. Phenol solu-
novel technique to utilize biomass as an alterna- tion was added with a ratio of waste to phenol of
tive to petroleum based products. A variety of 1:3 (w/v). Before the addition of phenol solution,
general studies has been conducted on wood liq- 0.05% (v/v) of sulfuric acid was mixed with
uefaction with phenol. The effects of water, cata- phenol solution. The mixture was heated until
lyst type, catalyst concentration, liquefaction 120°C for 1 h and then cooled. After cooling, the
temperature and time, and phenol to wood ratio mixture was filtered and kept in room tempera-
have been investigated (Lee et al., 2005). The ture. The obtained solution with a concentration
molecular weight and flow properties of the liq- of around 2.5% (v/v) was used as additive in the
uefied wood have also been characterized (Alma PF glue system for fiber board production in
et al., 1998). A model compound of lignin has commercial factory. The method is shown in the
been used to demonstrate the reaction mecha- diagram below (Fig 1).
nism of lignin during wood liquefaction (Lin et The composite material was set up as fol-
al., 2001). However, a comprehensive under- lowing formulation:
standing of the mechanism of wood liquefaction Bagasse Fiber = 45%
in the presence of phenol has not yet been clear- Bamboo Fiber = 15%
ly established. Mineral perlite = 25%
The commercial application of liquefied Commercial Resin = 12.5% based solid content
wood depends on the reagent solvent used in the Additive = 2.5%
liquefaction. Wood liquefied with phenol can be Target density = 0.75 gr/cm3
directly developed into molded products or used Thickness = 9-21 mm
to produce phenolic resin or foam. Phenolic resin Pressing temperature= 140oC
is a leading adhesive for composite panels in Pressing time = 10 min
moisture-sensitive applications, such as oriented
strand board and plywood used for sheathing

Sugar cane Bagasse
Pretreatmet
(Fungal and Steam)
Bagasse
Multi Enzyme Hydrolysis
Fermentation Phenol and Catalyst
Distillation “Cake” Solid waste Reactor
Bio-ethanol Liquid
Mixing with PF/UF Resin
Fiber board Composite
Fig 1. Flow process utilization of lignin rich-waste for adhesives
RESULTS AND DISCUSSION pH increased to 6.8. The liquid obtained after

liquefaction of cake was then used for additive in
As shown in Fig 1, the cake or solid waste phenol formaldehyde and urea formaldehyde
obtained after distillation process contains high resin. Addition of liquid was to increase the po-
lignin. Conversion process of cellulose and hem- tential polymerization of resin during hot press-
icelluloses of bagasse during hydrolysis and ing of composite material. In Fig 3 the influence
fermentation resulted in a very low cellulose and of additive on mechanical strength (modulus of
hemicelluloses content, while the lignin content rupture, MOR) is shown. It is very significant
did not change. Therefore, as shown in the Fig 2 than additive increase the MOR which tested in
the lignin content of the cake was around 80% dry and in wet condition. MOR in wet condition
after fermentation. The content of the remaining indicated that bonding strength was water re-
cellulose and hemicelluloses was below 10%. sistant and still gave a relatively high MOR of
Interestingly, the composition of cake, which is composite.
rich on lignin content, was dissolved by phenol To know the whole properties of products
solution in small polyphenolic polymer. In the we compared with Australian standard (ANZ),
same time cellulose and hemicelluloses will be Japanese Standard (JIS) and Indonesian standard
hydrolyzed and dissolved in presence of catalyst. (SNI). In pilot scale production, the physical and
The obtained solution used for additives was a mechanical properties as shown at Table 1 indi-
deep brown liquid, and had a viscosity of 6.2 P cate that the resulted products fiber composite
at 25 °C; pH 3.55. After addition of glue, the has very strong in bending strength (modulus of
viscosity of the mixture declined to 4.4 poise and rupture) in wet condition. The excellent proper-

ties also shown in thickness swelling. Both prop-
erties fulfill the ANZ for flooring class, or JIS
for Class I, and SNI.
In commercial scale production, we produce
in different thickness of fiber board composite,
9, 15 and 21 cm. The testing of samples were
carried out in dry condition and also after im-
mersion in water at 70 oC (wet condition). The
results show that the fiber board with thickness 9
cm has the best properties and fulfill the SNI
standard (Table 2).
Fig 3. Comparition of modulus of rupture (Mpa) in wet

and dry condition using PF resin
One of the important of parameter in the

development of product is related to formalde-
hyde release. Many efforts have been done to
block the formaldehyde release of end products
by various method, such as thermal treatment
and by adding of chemical, which known as
catcher. Polyphenol liquid resulted in this exper-
iment can act as formaldehyde catcher. The
Fig 2. The composition of bagasse after (in black) and be- emission of formaldehyde of the fiber board
fore (in grey) fermentation show 0.1-0.2 mg/l for all types. The standard of
emission should be below 0.5 mg/l for E0, 1.5
mg/l for E1, and 5 mg/l for E2. It means the
products fulfill the zero emission of formalde-
hyde release.
Table 1. Properties of fiber composite board in pilot scale production using PF Resin
Properties Exp. Results ANZ Standard JIS Class I SNI

2
Modulus of rupture (dry) (kg/cm ) 121.44 190 180 180
2
Modulus of rupture (Wet) (kg/cm ) 90.5 42 90 90
2
Modulus of elasticity (kg/cm ) 20.06 27.5
2
Internal bond (kg/cm ) 4.47 5.5 3 3
Thickness swelling (%) 3.2 8 12 12
Table 2. Properties of fiber composite board in different thickness in commercial production using PF Resin
Properties T 9 cm T 15 cm T21 cm SNI

2
Modulus of elasticity (dry) (kg/cm ) 245 310 151 180
2
Modulus of elasticity (wet) (kg/cm ) 310 98 67 90
2
Modulus of elasticity (dry) (kg/cm ) 22,218 25,593 14,510
2
Modulus of elasticity (wet) (kg/cm ) 9,747 6,962 6,143
2
Internal bond (kg/cm ) 3.9 2.8 1.2 3
Thickness swelling (%) 8.7 7.5 7.4 12

CONCLUSIONS REFERENCES
Increasing effort to utilize the biomass for Alma, M.H., Yoshioka, M., Yao, Y. and Shiraishi, N. 1998.
bio-ethanol production will produce by-product Preparation of sulfuric acid catalyzed phenolated
wood resin. Wood Sci. Technol. 32: 297-308.
which contains mainly lignin. Lignin-rich bio-
mass from the waste of bio-ethanol production Conner, A.H. 2001. Wood adhesives. Elsevier Science Ltd.
Amsterdam, New York.
from sugarcane bagasse can be dissolved in phe-
nol and catalyst. The resulted liquid is a highly Gothwal, R.K., Mohan, M.K. and Ghosh, P. 2010. Synthe-
polyphenol and can be used as additive in the sis of low cost adhesives from pulp and paper industry
waste. J. Sci. Industrial Res. 69: 390-395.
gluing system of fiber based composite. Utiliza-
tion of additive derivate from lignin waste in- Hui, P. and Shupe, T.F. 2009. Wood liquefaction and value-
creased the physical and mechanical properties, added products.
especially wet strength and met the Australian Jin, Y., Cheng, X. and Zheng, Z. 2010. Preparation and
Standard, Japanese Standard and Indonesian characterization of phenol–formaldehyde adhesives
modified with enzymatic hydrolysis lignin. Biores.
Standard. These results can also inspire the crea- Technol. 101 (6): 2046-2048.
tion of green products and replace petrochemical
based in the future in line with increasing biofuel Malutan, T., Nicu, R. and Popa, V. I. 2008. Hydroxymeth-
ylation of alkali lignin. BioRes. 3 (1). 13-20.
production from biomass.
Lee, S.H. and Wang, S.Q. 2005. Effect of water on wood
liquefaction and the properties of phenolated wood.
ACKNOWLEGMENT Holzforschung. 59: 628-634.
Lin, L., Yao, Y., Yoshioka, M. and Shiraishi, N. 2001. Liq-
The authors thank to Mr. Zaenal Asikin and
uefaction mechanism of β-O-4 lignin model com-
Mr. Dayat for the assistance of production and pound in the presence of phenol under acid catalysis.
technical testing of products. I. Structural characterization of the reaction products.
Holzforschung. 55: 617-624.
Prasetya, B., Hermiati E. and Sudijono. 2004 The effects of
catalyst percentage used in producing wood liquid on
its bond strength as phenol formaldehyde substitute
adhesive in plywood production. J. Ilmu dan
Teknologi Kayu Tropis. 2.2.

Potency of Indonesian Marine Microalgae (Chlorophyta) as Renewable
Liquid Biofuel (Biodiesel) Producers
Khairul Anam1, Hilda Farida1, Muhammad Sidiq Habibi1, Dian Noverita Widyaningrum1,
Dicky Gustiawanto1 and Dwi Susilaningsih1
Research Center for Biotechnology, Indonesian Institute of Science (LIPI)

Jl. Raya Bogor Km.46 Cibinong 16911, Indonesia; Phone: 021 8754587, Fax: 021 8754588
e-mail: ka_anam@yahoo.com
(Correspondence to: Khairul Anam)
ABSTRACT
Indonesia has a very abundant biodiversity, included microalgae. Some previous research clarified that mi-
croalgae are able to produce the biodiesel, which is based on its biomass fatty acid. This research was using mi-
croalgae Chlorella and Tetraselmis for alternative fuel source to solve the energy crisis. Results, both microal-
gae Chlorella and Tetraselmis, show that amount of the biomass increase along with the cultivation period. On
the contrary the fatty acid was not following the biomass increasing pattern which sometimes was decreased.
Cultures was harvested on 12 weeks cultivation period, Chlorella and Tetraselmis produced dry biomass1.91
g/L and 0.35 g/L; fatty acid 13.33±5.77% and 8.00±6.92%, respectively. Two weeks cultivation period, ob-
tained dry biomass 0.30 g/L and 0.05 g/L; fatty acid 21.76±3.25% and 61.91±5.94% for Chlorella and Tetra-
selmis, respectively. Meanwhile, the treatment of cornmeal as the substituent substrate, increase the biomass but
decrease the fatty acid. Other screening activities show BTM 11 has high content of lipid and can be candidate
for biodiesel.
Keywords: microalgae, biomass, lipid, biodiesel.
INTRODUCTION concentration of EPA-DHA (Eicosapentaenoic

Acid-Docosahexanoic Acid) inside its cells (40-
Energy crisis have already called for atten- 80%) (Cohen, 1999).
tion to many countries, including Indonesia. In- Tetraselmis and Chlorella have lipid con-
creasing world’s petroleum cost, affected life tent up to 20-40% of cell weight in the normal
pattern for Indonesian people. Petroleum price cultivation condition. Manipulation of cultiva-
that increased in Indonesia directly or indirectly tion media, illumination, and shifting physiolog-
affect to increase the other needs as well, such as ic life cycle condition from autotroph to hetero-
building material, textile, health care, and trans- troph drastically to both green microalgae may
portation. The increasing of petroleum cost also lead to the increasing of lipid up to 60% of dry
indirectly affects the society lives, for example weight (Xiong & Wu, 2008). This kind of mi-
there are a lot of lay-off, jobless, and poor socie- croalgae can be found in the rich of nitrogen and
ty increased, low purchasing power, to poor nu- phosphate water, brackish and salty waters, and
trition. also can be culturized with a simple technology.
Looking back to the real affect that happens Factors that determine the success in mass cul-
in society, we try to offer one solution to de- tured marine microalgae are high nitrogen con-
crease this petroleum problem, which is using centration in media, pH 6-8, light control, well
marine microalgae native in Indonesia, to pro- mixed with stiring, and harvesting through sepa-
duce biodiesel. Photosynthesis in microalgae can ration. Marine microalgae mass production use
produce carbon source in the form of carbohy- ideal media and waste able to produced biomass
drate and fatty acids, also producing oxygen that around 40 g/m2/day and 5 g/m2/day (Westhead,
can be released into the air. Microalgae also able 2003).
to bind CO2 and nitrogen from the air, so it can Tetraselmis and Chlorella from tropic ma-
help to reduce air pollution and carbon gas emis- rine grow well and optimal in open lighting,
sion. Marine microalgae Tetraselmis and Chlo- temperature relatively hot 35-38°C, well circu-
rella, known as highly adapted photosynthetic lated CO2, and good water resource. Beach areas
green algae lived in waters have lipid reserva- have a potential to develop microalgae cultiva-
tion, protein, green pigment, vitamin, and high tion cause of good supply of water and relatively

hot temperature. High rainfall can limit the culti- fore media modification and after media modifi-
vation of microalgae, it may lead to volume cation with the addition of cornmeal. After ± 2
overflow on the pool. This problem can be weeks cultivation period, microalgae that pro-
solved with installing the transparent roof, e.g. duced in the gallon were harvested by filtering in
plastic. In addition to protect from rain, it can small purified cloth. Biomass that was filtered
also protect the culture from excessive evapora- then weighed as wet biomass weight. And then,
tion or maintain the moisture. wet biomass dried up in 50°C oven temperature
In this research, we study selected marine and weighed again to obtain dry biomass weight.
microalgae Tetraselmis sp. and Chlorella sp. in Biomass was collected for measurement of fatty
laboratory scale (10-15 litre) to produce bio- acid level. Comparison in cultivation microalgae
diesel. For addition, we do screening activities Pari C6, Pari F2, Pari A1, Btm 04, and Btm 11
and isolating to get microalgae culture to add our were held in working volume 1 litre using the
microalgae collection. same media.
MATERIALS AND METHODS Fatty acid level measurement: was done with
modification of Bligh & Dyer (1959) method.
Microalgae: were obtained from Environmental Extraction was done with adding methanol : wa-
Bioengineering Research Group, Research Cen- ter (1:1) solution into microalgae biomass (wet
ter for Biotechnology Indonesian Institute of or dry) that have been weighed. That mixture
Science (LIPI) collection, using strain Tetra- shaken for 2 hours and was added with a volume
selmis and Chlorella also some collections that of 2 parts chloroform into the methanol-water
was obtained from Indonesian water which have solution. The chloroform phase was taken and
a good growth, such as Pari C6, Pari F2, Pari A1, then centrifuged to precipitate the debris, if there
Btm 04, and Btm 11. is one. A 1 ml of centrifuged chloroform phase
was inserted to the 1.5 ml tube that has been
Cultivation: was using F2 medium which com- weighed before. Chloroform phase will be evap-
posed of stock solution (g/L water) 150.0 orated in room temperature, fatty acid then can
NaNO3, 0.0196 CuSO4.5H2O, 0.044 be weighed from the chloroform phase that
ZnSO4.7H2O, 0.022 CoCl2.6H2O, 0.360 didn’t evaporate in room temperature.
MnCl2.4H2O, 0.0126 Na2MoO4.2H2O, 22.7
Na2SiO3.5H2O, 9.0 Ferric citrate, 9.0 Citric acid, RESULTS AND DISCUSSION
11.3 NaH2PO4.2H2O, and 1 ml vitamin B12
0.01%. The stock solution then diluted 1000x Based on both strain microalgae biomass
with sea water as a solvent, this diluted solution weigh calculation result, known that cultivation
can be used as the cultivation media period affects the amount of biomass (Table 1).
(www.marine.csiro.au, 2009). Main culture was But based on the calculation of fatty acids level,
held by using Erlenmeyer flask, with 200 ml fatty acids level that obtained from 2 weeks old
working volume. Half of main culture was har- microalgae was higher than microalgae which
vested and re-grow on the gallon bottle with vol- harvested 12 weeks later, although biomass ob-
ume 10-15 litre for production, so can be obtained is bigger. This result shows that fatty acid
tained a lot of biomass. Substrate addition was production reached its highest level when in
performed to determine whether the addition of growth phase. Because of this, from the result
carbohydrates can raise fatty acids levels in cer- calculation analysis, we get information that cul-
tain microalgae. Substrate is added in the form tivation period linear with amount of biomass,
of cornmeal 10 g/l. Tetraselmis and Chlorella but opposite with amount of fatty acid concen-
cultivation in gallon was done twice, that is be- tration.

Table 1. Biomass weight and fatty acid concentration of microalgae Tetraselmis and Chlorella.
Volume of Cultivation Wet Dry Water

Strain of Substrate Fatty acid
medium (lit- period weight weight content
microalgae addition % (w/w)
tre) (week) (g/l) (g/l) % (w/w)
10 12 - 25.12 1.91 92.38 13.33±5.77

Chlorella 15 2 - 0.30 0.02 92.28 21.76±3.25
15 2 cornmeal 1.32 0.16 87.58 4.53±0.53
14 12 - - 0.35 - 8.00±6.92
Tetraselmis 15 2 - 0.56 0.05 90.84 61.91±5.94
15 2 cornmeal 2.60 0.36 86.22 5.13±0.04
From the information we need to advance omass to both microalgae strain, but decrease the
the study about cultivation period optimization fatty acid concentration.
towards amount of biomass. In this research ob- Cultivation was done to some microalgae
tained fatty acid concentration of Chlorella 18- collection too, which was collected from sea wa-
24%. It is pointed that fatty acid concentration in ters around Batam Island and Pari Island. Based
Chlorella is 14-22% (Miyamoto, 1997). on calculation of fatty acid concentration from
Research result also shows with substrate each microalgae culture in cultivation volume
addition such as cornmeal into cultivation media less than 1 litre, so we obtained data fatty acids
will increase amount of biomass in Tetraselmis level from Pari C6, Pari F2, Pari A1, Btm 04,
and Chlorella. In the other hand, from the calcu- and Btm 11 respectively are 8.16±2.07%,
lation, shows less result if compared to both mi- 26.84±5.88%, 5.76±2.59%, 32.21±4.39%,
croalgae media without substrate addition. From 40.44±3.84% (Table 2). Based on microscope
the result, we can get information that cornmeal study known that Btm 11, Pari C6, and Pari F2
addition into media will increase amount of bi- respectively belong to the species of Cyano-
phyceae, Cytonema, and Nostoc.
Tabel 2. Biomass weight and fatty acid concentration of microalgae Pari C6, Pari F2, Pari A1, Btm 04, and Btm 11.
Volume of Cultivation Dry Water

Strain of mi- Substrate Wet weight Fatty acid
medium (li- period weight content
croalgae addition (g/l) % (w/w)
tre) (week) (g/l) % (w/w)
pari C6 1 >4 - 2.24 - - 8.16±2.07

pari F2 1 >4 - 5.15 1.03 80.00 26.84±5.88
pari A1 1 >4 - 0.36 - - 5.76±2.59
btm 11 1 >4 - 1.46 0.27 81.51 32.21±4.39
btm 04 1 >4 - 0.27 0.05 82.45 40.44±3.84
Comparison of different microalgae shows verted to biodiesel from 20-60% wet weight.
that the highest fatty acids level comes from Btm Microalgae which collected from Batam island
11, results 40.44±3.84%. Based on the calcula- water have the potential as fatty acid producer up
tion of fatty acids, because of dry weight ob- to 40% dry weight.
tained was so small, so the numerator was so
small either and make the multiply factor in dis- ACKNOWLEDGMENTS
solving extract using chloroform become more
significant. So because of this, in the future, re- We would like to express our gratitude
searcher will try to uniform the extracted dry thanks to Indonesian Institute of Sciences (LIPI),
weight, so we can get the better result. Environmental Bioengineering Research Group,
In conclusion, microalgae Chlorella and RC Biotechnology LIPI. This project collaborate
Tetraselmis have a potential to become the with PT. Barat Jaya Sentosa Perkasa.
source of fatty acid producer which will be con-

REFERENCES Miyamoto, K. 1997. Renewable biological systems for
alternative sustainable energy production. FAO agri-
cultural services bulletin: 128.
Bligh, E. G. & W. J. Dyer. 1959. A rapid method of total
lipid extraction and purification. Can J Biochem Westhead, K. 2003. The introduction to the cyanobacteria.
Physiol 37:911–917. In: Whitton, B.A. and Potts, M. (eds). The ecology
of cyanobacteria. Kluwer Academic Publishers, The
Cohen, Z. 1999. (ed.) Chemicals from microalgae. Taylor
Netherlands, pp. 1–11.
& Francis Ltd., London.
Xiong, W. & Q. Wu. 2008. High-density fermentation of
Media Recipes and Preparation.
microalga Chlorella protothecoides in bioreactor for
http://www.marine.csiro.au/microalgae/methods/Medi
microbio-diesel production. Appl Microbiol Biotech-
a%20CMARC%20recipes.htm#F. 12 November
nol.78: 29-36.
2009.

Environmental Factors Optimization in Photo-fermentation
to Produce Biohydrogen by Sanur Consortia
Muhammad Sidiq Habibi1, Khairul Anam1 and Dwi Susilaningsih1
1
Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI)
Jl. Raya Bogor Km. 46 Cibinong 16911, Bogor, West Java, Indonesia 16911.
Tel. +62-21-8754587, Fax. +62-21-8754588, e-mail: ka_sidiq@yahoo.com
(Correspondence to: Muhammad Sidiq Habibi)
ABSTRACT
Green and renewable energies are the character of energy resources that required presently for substitute
fossil fuel that can reduce negative impact to environment. Hydrogen is one of prospective green energy carrier
which easily be converted to electricity without any pollution. Photosynthetic bacteria is one of microorganisms
that can produce biological hydrogen gas. With support of light and donor electrons from water, this microbe can
change organic substrate into hydrogen. Therefore, in this studies, we focused on research about effect of envi-
ronmental factors such as temperature, illumination, pH, and agitation in hydrogen production from glucose sub-
strate. Hydrogen gas was collected from photo-fermentation by using Rhodobium marinum and Sanur consortia.
Photo-fermentation using Sanur consortia exhibited the best result of 1710.3 ml H2/L from working volume 75
mL. The photo-fermentation conditions were treated on temperature 30°C, illumination 2000 lux, pH 7, and 120
rpm agitation. Currently the photo-fermentation in large scale volume conditions is studied.
Keywords: Biohydrogen, environmental factors, photo-fermentation, Sanur consortia
INTRODUCTION ies on using glucose (Miyake et al., 1984) and

organic acids (Miyake et al., 1986).
Hydrogen gas has been receiving much at- In this study, we optimize the environmental
tention as a clean and renewable energy carier factors such as temperature, illumination, pH,
that does not produce carbon dioxide in combus- and agitation in hydrogen production from glu-
tion (Li & Fang, 2008). It can be converted to cose substrate by using Rhodobium marinum
electric energy with high efficiency (Ikuta et al., NBRC 100434 and Sanur consortia. Rhodobium
1998). Using biotechnology as a means of envi- marinum NBRC 100434 used as a reference pho-
ronmentally acceptable hydrogen production has tosynthetic bacteria, whereas Sanur consortia
been a goal of researchers throughout the world (was collected from Sanur beach, Bali, Indone-
(Aoyama et al. 1998). Hydrogen production us- sia) used as test photosynthetic bacteria.
ing photosynthetic bacteria is a promising meth-
od of obtaining energy from sunlight (Wakayama MATERIALS AND METHODS
et al., 1998).
Photosynthetic bacteria that can utilize solar Rhodobium marinum NBRC 100434 and
light as an energy source and generate hydrogen Sanur consortia was grown on steril phototropic
by decomposition of water and organic matter bacteria medium, modification of Kawaguchi et
have many excellent features. Photosynthetic al. (2002) containing dissodium succinate (10
bacteria have a broad range of light intensity that g/L), yeast extract (0.3 g/L), 10 mL basal
they can use under different environmental con- medium (100x), 1.5 g NaHCO3, and 1000 mL
ditions (Turkaslan et al., 1998). These bacteria aquadest. Nitrogen gas was passed trough the
can utilize many organic wastes and contribute to medium to provide anaerobic conditions. The
waste treatment processes as they produce clean culture was incubated at 30ºC under illumination
fuel. (36 W/m2) and 120 rpm agitation. Growth was
There are several groups in the world who determined by OD (optical density) measurement
work on hydrogen production by using photosyn- at 660 nm. The Sanur consortia culture was har-
thetic microorganisms. Some of these groups vested once the OD of the culture reached OD ≥
have utilize organic wastes from the sugar indus- 1.00 by using centrifuge Sorvall RC26 plus (rotor
try (Vincenzini et al., 1982; Boliger et al., 1985; GSA, 6000 rpm, 7 ˚C, 20 minutes). The pellet
Tanisho & Ishiwata, 1994). There are also stud- was resuspended on 30 mL steril production

medium (containing glucose (10 g/L), yeast ex-
35
tract (3 g/L), 10 mL basal medium (100x), 1.5 g 30
NaHCO3, and 1000 mL distilled water. Nitrogen
Hydrogen (ml)
25
gas was passed trough the medium to provide 20
anaerobic conditions). Furthermore 10 mL of 15
Sanur consortia suspension was inoculated into 10
65 mL new steril production medium. 5

0
Optimization of temperature factor was 28 30 37 42
done with modified the temperature of reactor Tem perature (ºC)
(28, 30, 37, and 42 ºC). Optimization of pH
medium factor was done with modified the pH Fig. 2. Hydrogen production on optimization of tempera-
medium on the reactor (pH 4, 5, 7, and 9). ture factor.
Optimization of illumination factor was done
with modified the illumination (light intensity) pH medium optimization: the effect of pH medi-
that irrediating the reactor (0, 2000, and 5000 um on H2 production was also studied. The initial
lux). Whereas optimization of agition factor was pH 7 of the medium showed the highest level of
done with modified the speed of reactor rotation hydrogen production (Fig. 3). Photo-
(0, 60, and 120 rpm). Hydrogen was collected in fermentation on initial pH 7 of the medium can
a burette and the volume of the collected gas was produce 128.27 ml hydrogen. It was observed
measured. Gas chromatography and H2 Scan that pH were very critical parameters for hydro-
sensor model 2240 were used to determines the gen production (Turkaslan et al., 1998). As
composition of the collected gas. shown in Fig. 3, Sanur consortia can produce
hydrogen in the medium with pH 5-9.
160
0.4
140
0.3 120
Hydrogen (ml)
OD 660 nm
100
0.2 80
60
0.1 40
20
0
0
0 1 4 5 6 8 11 12 13 14 20 26 56 58
4 5 7 9
Incubation tim e (day/s)
pH Medium
Rhodobium marinum Sanur consortia

Fig. 3. Hydrogen production on optimization of pH medi-
Fig. 1. Growth curve of Rhodobium marinum and Sanur um factor.
consortia.
Illumination optimization: the result of illumina-
RESULTS AND DISCUSSION tion (light intensity) optimization was shown that
2000 lux induced the maximum amount hydro-
Temperature optimization: the result of tempera- gen production (Fig. 4). In this illumination,
ture optimization shown that photo-fermentation Sanur consortia can produce 17.09 ml hydrogen
on temperature 30ºC produce the highest volume during 48 hours.
of hydrogen rather than other initial (Fig. 2).
25
Total hydrogen that produced when photo-
fermentation on temperature 30ºC achieve 27.51 20
Hydrogen (ml)
mL hydrogen in 48 hours. At the end of 48 h, gas 15

production seemed to stop, probably due to a
critical nutrient depletion in the medium. 10
0
0 2000 5000
Illum ination (lux)
Fig. 4. Hydrogen production on optimization of illumina-

tion factor.

Agitation optimization: agitation process in this tute of Sciences (LIPI), on Program Renewable
photo-fermentation was done to homogenize the Clean Energy and Water Supply 2009.
medium so that Sanur consortia can consume
medium as substrate to produce hydrogen easily. REFERENCES
The result of agitation optimization was shown
that agitation with rotation speed 120 rpm can Aoyama, K., I. Uemura, J. Miyake & Y. Asada. 1998. Pho-
produce hydrogen up to 16.22 ml H2 (Fig. 4). Fig. tosynthetic bacterial hydrogen production with fer-
mentation products of cyanobacterium Spirulina
4 also showed that amount hydrogen production platensis. In: Zaborsky O, editor, Biohydrogen. Lon-
on the agitation with rotation speed 120 rpm don: Plenum Press; p.305-310.
thrice than un-agitation photo-fermentation (agi-
Boliger, R., H. Zurrer & R. Bachofec. 1985. Photoproduc-
tation with rotation 0 rpm). tion of molecular hydrogen from waste water of a
20
sugar refinery by photosynthetic bacteria. Appl Mi-
crobiol Biotechnol. 23: 147-151.
15 Ikuta, Y., T. Akano, N. Shioji & I. Maeda. 1998. Hydrogen
Hydrogen (ml)
production by photosynthetic microorganisms. In:

10 Zaborsky O, editor, Biohydrogen. London: Plenum
Press; p.319-328.
5
Kawaguchi , et al. 2002. Effect of algal extract on H2
0 production by a photosynthetic bacterium
0 60 120 Rhodobium marinum A-501: analysis of stimulating
Agitation (rpm ) effect using a kinetic model. J. Bioscience and Bioen-
ginering. 94: 62-69.
Fig. 5. Hydrogen production on optimization of agitation Li, R. Y. & H. H. P. Fang. 2008. Hydrogen production
factor. characteristics of photoheterotrophic Rubrivivax gelat-
inosus L31. Int J of Hydrogen Energy. 33:974-980.
Photo-fermentation using Sanur consortia Margaritis, A. & J. Vogrinetz. 1983. The effect of glucose
exhibited the best result of 1710.3 mL H2/L me- concentration and pH on hydrogen production by
dium or 10.18 mL H2/hL medium from working Rhodopseudomonas sphaeroides VM 81. Int J of Hy-
drogen Energy. 8(4):281-4.
volume 75 mL when treated on temperature
30°C, illumination 2000 lux, pH 7, and 120 rpm Miyake, J., X. Y. Mao & S. Kawamura. 1984. Photoproduc-
agitation. These are much higher than the report- tion of hydrogen from glucose by a co-culture of a
photosynthetic bacterium and Clostridium butyricum.
ed 4.1 mL H2/hL medium for R. gelatinosus L31, J. Ferment Technol. 62: 531-535.
using glucose (Li & Fang, 2008) and 2.2 mL
H2/hL medium for R. sphaeroides VM81, using Miyake, J., X. Y. Mao & S. Kawamura. 1986. Screening
photosynthetic bacteria for hydrogen production from
glucose (Margaritis & Vogrinetz, 1983). Current- organic acids. J. Ferment Technol. 64: 245-249.
ly the photo-fermentation in large scale volume
Tanisho, S. & Y. Ishiwata. 1994. Continuous hydrogen
conditions is studied. production from molasses by the bacterium Entero-
In conclusion, the results obtained revealed bacter aerogenes. Int J Hydrogen Energy. 19:807-
that temperature 30°C, illumination 2000 lux, pH 812.
7, and 120 rpm agitation were the optimum envi- Turkaslan, S., D. O. Yigit, K. Aslan, I. Eroglu & U.
ronmental factors to photo-fermentation in this Gunduz. 1998. Photobiological hydrogen production
study. Photo-fermentation using Sanur consortia by Rhodobacter sphaeroides O.U.001 by utilization of
exhibited the best result of 1710.3 ml H2/L medi- waste water from milk industry. In: Zaborsky O,
editor, Biohydrogen. London: Plenum Press; p.151-
um or 10.18 mL H2/hL medium from working 156.
volume 75 mL when treated on temperature
30°C, illumination 2000 lux, pH 7, and 120 rpm Wakayama, T., A. Toriyama, T. Kawasugi, T. Arai, Y.
Asada & J. Miyake. 1998. Photohydrogen production
agitation. using photosynthetic bacterium Rhodobacter
sphaeroides RV; simulation of the light cycle of
ACKNOWLEDGEMENTS natural sunlight using an artificial source. In: Za-
borsky O, editor, Biohydrogen. London: Plenum
Press; p. 375-81.
The author would like to thank Research
Group Environmental Bioengineering, RC Bio- Vincenzini, M., G. Florenzano, R. Materassi & M. R.
technology LIPI. This research was funded by Tredici. 1982. Hydrogen production by immobilized
cells; H2 photoevolution and wastewater-treatment by
Research Competitive Grant of Indonesian Insti- agar-entrapped cells of Rhodopseudomonas palustris
and Rhodosprillum molischianum. Int. J Hydrogen
Energy. 7(9): 725-728.

In Situ Acid-Transesterification: An Overview Simple Method
for Fatty Acid Methyl Esters (FAME) Preparation
from Marine Microalgae as A Biodiesel Feedstocks
Asep Bayu
Research Centre for Oceanography, Indonesian Institute of Sciences (LIPI)

Jl. Pasir Putih I Ancol Timur, North Jakarta, Phone (021) 64713850 Fax. (021) 64711948
e-mail : asepbayu@yahoo.co.id / asepbayu86@gmail.com
ABSTRACT
The objective of this research is to investigated the initial potency and effectiveness of fatty acid methyl
esters (FAME) preparation from marine microalgae Chaetoceros gracilis by using in situ acids-
transesterification method. Dried biomass C. gracilis was suspended into hexane : methanol (2:1 v/v) and
reacted with H2SO4-methanolic 1% (v/v) (80oC; 4 h). After reaction was completed, mixtures were neutralize
with KHCO3 2% (w/v) and washed with NaCl 5% (w/v). FAMEs were extracted with hexane, evaporated with
nitrogen gas and then anlyzed by Gas Chromatography-Mass Spectrometer (GC-MS). As a references,
conventional FAME preparation (ex situ transesterification) from C. gracilis was examined to by using Christie
method. GC-MS spectrum showed that in situ acid-transesterification having potency to prepared FAME from
C. gracilis since this methods given the identically peak as well as conventional method. Palmitate, palmitoleate
and miristate acid are major components in C. gracilis with 20,09 ± 4,29 %; 21,84 ± 1,46 %; 18,67 ± 1,43 %
respectively. Furthermore, in situ method was resulted low unsaturated fatty acids (32,32% vs 38,30%) and
FAME content (76,27% vs 91,14%) than ex situ method. However, in situ transesterification method was
potentiable to yield of FAME from marine microalgae since this procedure was reduced the time, waste, and
cost prodcution than ex situ transesterification method.
Keywords: Transesterification, FAME, Biodiesel, Microalgae, Chaetoceros gracilis.
INTRODUCTION simultaneously extracted and transesterified.

This procedure could significant reduction in
Generally, conventional methods for length of the total procedure, saving the time and
preparation and determination of fatty acid works. The use of reagents and solvents are
methyl esters (FAME) as a biodiesel feedstock reduced, the analysis is less expensive, easier,
from microalgae had a lengthly and cumbersome faster, and the concern about waste disposal is
procedures. These procedures required lipid avoided. Furthermore, similiarity with
extraction from biomass, transesterification, conventional transesterification, in situ
neutralization and purification (Lepage & Roy, transesterification could be catalyzed with acid
1986; Haas et al., 2004). The long times and or base (Carrapiso & Garcia, 2000; Schlechtriem
many chemical reagents that used in these et al.). Mechanism of the reaction both of this
procedures are not effective and it can increased method it same to.
the cost production a biodiesel from biomass as Currently, in situ transesterification has
microalgae. It is estimated that 70% of this cost been succesfully for fatty acids analysis from
production primarily in feedstocks production biological samples. Lepage & Roy (1986)
(Gavett & Inc., 1995).Thus, there is interest to reported this method is aplicable for fatty acids
simplification of the FAME preparation anlysis in some biological specimens (e.q.
procedures to reduce this problem. plasma, feces, bile, rat liver) that having simple
In situ transesterification or direct and complex lipid (Lepage & Roy, 1986). They
transesterification could be one of resolved this compared to the Folch procedure and showed the
problem. It can be defined as the production technique is led to increase for all of sample and
ester derivative from sample biomass without more good precission. Haas et al. (2004) showed
previous lipid extraction (Carrapiso & Garcia, in situ alkaline transesterification is an effective
2000). The separate lipid extraction from crude method for the production of fatty acid esters
sample is avoided, and fatty acids are from soybeans (Haas et al., 2004). In situ acid

transesterification was succesfully to wtih given Conventional Transesterification Metho: lipids
high FAME (90-98%) from ground soybean, rice were extracted from dried biomass using Bligh
bran and sun flowers (Ozgul & Turkay, 1993; & Dyer (1959) modified method. Dried biomass
Kildiran et al., 1996; Marinkovic & Tomasevic, was suspensed into MeOH:CHCl3:H2O
1998; Ozgul & Turkay, 2002; Ozgul & Turkay, (2,5:1,25:1) (mixtures I), shaked for 2 h and
2003). However, FAME preparation from placed for 24 h. Organic phase from mixtures I
microalgae using this method has not been was separated to other vials using Pasteur
reported. pippetes (organic I) and pellet cells were
In this research, the initial potency and extracted again with MeOH:CHCl3:H2O
effectiveness of in situ acid-transesterification (2,5:1,25:1) (mixtures II). Furthermore, organic
methode for FAME preparation from marine phase from mixtures II was separated using
microalgae will be investigated. Chaetoceros Pasteur pippetes and then mixed with organic I
gracilis was used as a sample since their have (organic II). Afterward, CHCl3-H2O (1:1) was
high lipid (oil) content that can be derivatived to added and mixtures were shaked for 2 h. After
FAME (Herman, 1991). two layers were produced, the lower phase was
separated and extracted with NaCl 5% while the
MATERIALS AND METHODS upper phase was extracted with CHCl3. The
organic phase was separated and solvent was
All glassware (Iwaki-Pyrex) were rinsed evaporated with nitrogen gas. Furthermore,
with chloroform-methanol (2:1 v/v) before used. lipids that produced were transesterified using
Sample Chaetoceros gracilis was prepared from the same procedures in previously
Mariculture Laboratory-Research Centre for transesterification method (Christie, 1993).
Oceanography LIPI. Biomass were harvested by
centrifuge method (4000 rpm, 4oC, 10 min) and GC-MS method: FAME was determined by non
dried with oven (60oC, 24 h). polar capilary coulomn (HP Ultra 2; 30 m x 0,25
In Situ Acids-Transesterification Method. mm I.D.) installed on a Agilent Technologies
Dried biomass was suspended into hexane- 6890 Gas Chromatograph with Auto Sampler
methanol (2:1 v/v) in screw-cap tube and then and 5973 Mass Selective Detector and
vortexed for 15 min. Five milliliters of H2SO4- Chemstation data system. The initial oven
methanolic 1% (v/v) was added into a mixture temperature was 65oC held for 0,05 min,
and then heated in waterbath (80oC, 4 h) with subsequently increased to 234oC at a rate of 3oC
vigorous hand-shaking for 5 min every 10 s. min-1 and then held for 2 min. Furthermore,
After reaction was completed, mixtures were temperature oven was increased again until
cooled at room temperature and neutralize with 300oC at a rate of 30oC mint-1 and held for 10
KHCO3 2%. Furthermore, mixtures were min. Temperature injector and detector were set
extracted with hexane and organic phase 250oC and 230oC respectively while helium was
separated using Pasteur pippetes to the other used as the carrier gas.
tubes. Hexane was evaporated with nitrogen gas
and FAME was placed into GC-vial containing 1 Data quantitation: FAME content was
mL hexane and it was stored at freezer until calculated based on peak area of individual fatty
injection to GC-MS. acids compared a total amount. SPSS 13.0 for
Windows was used for statistical anlysis.

DRIED BIOMASS
+ lipid extraction
(Bligh & Dyer (1959) modified method
LIPID
+ Hexane:MeOH (2:1)
+ H2SO4-metanolic 1% & Heated (800C, 4h)
MIXTURE
+ KHCO3 2% & Hexane
ORGANIC PHASE WATER PHASE

+ evaporation (N2)
FAME
Conventional (ex situ) method

GC-MS In situ method
RESULTS AND DISCUSSIONS fatty acids profile were less quantities of C-18
fatty acids as stearate and oleate. Moreover, γ-
GC-MS spectrum showed that either in situ linolenate (C18:3(n6)) or α-linolenate
acids or ex situ transesterification given the (C18:3(n3)) was not detecable for in situ
identically peak of FAME derived from method. The similiarity result was reported by
Chaetoceros gracilis (Fig. 1). Palmitate (C16:0), (Herman, 1981). This results indicated that in
palmitoleate (C16:1n7) and miristate (C14:0) situ acid-transesterification is suitable for FAME
fatty acid were detected as a three highest peaks preparation from marine microalgae as well as
in both of method. On the other hand, C. gracilis others biomass.
ex situ transesterification In situ transesterification
Fig. 1. GC-MS spectrum of fatty acid methyl esters in Chaetoceros gracilis using ex situ and in situ transesterification
method.
Table 1. Fatty acid methyl esters composition of Chaetoceros gracilis.
No ex situ transesterification in situ acids-transesterification

*
FAME %-w ± SE FAME %-w ± SE*
1 Miristate C14:0 19,61 ± 0,3 Miristate C14:0 18,67 ± 1,43
2 Pentadekanoic C15:0 0,67 ± 0,13 Miristoleate C14:1 0,28 ± 0,23
3 Palmitate C16:0 31,09 ± 2,32 pentadekanoic C15:0 0,82 ± 0,01
4 Palmitoleate C16:1(n7) 31, 74 ± 0,36 Palmitate C16:0 20,09 ± 4,29

5 Heksadekaterienoic C16:3 1,61 ± 0,27 Palmitoleate C16:1 21,84 ± 1,46
6 Stearate C18:0 1,06 ± 0,21 Heptadekanoic C17:0 1,11 ± 0,16
7 Oleate C18:1(n9) 3,13 ± 0,23 Stearate C18:0 0,62 ± 0,04
8 8,11-oktadekadienoic C18:2(n7) 0,30 ± 0,06 Elaidate C18:1n9t 2,64 ± 0,56
9 γ-linolenate C18:3(n6) 0,09 ± 0,09 Oleate C18:1n9c 0,36 ± 0,03
10 α-linolenate C18:3(n3) 0,08 ± 0,08 Behenate C22:0 0,46 ± 0,04
11 Arakhidonate C20:4(n6) 1,07 ± 0,80 Erukate C22:1n9 2,77 ± 0,49
12 Erukate C22:1(n9) 0,28 ± 0,06 Arakhidonate C20:4n6 0,40 ± 0,10
13 Behenate C22:0 0,10 ± 0,06 Lignoserate C24:0 2,19 ± 0,24
14 Lignoserate C24:0 0,30 ± 0,03 Nervonic C24:1 4,03 ± 1,48
15 Others 8,86 ± 2,19 Others 23,73 ± 2,91
*
Mean value from three replicate of sample analysis.
The FAME compositions of C. gracilis the yields of FAME. These can be derived from
were resulted by using in situ acid- sample itself or non-ester by-product of
transesterification method, mainly consisted of esterification proceses (e.q. water, gliserol) (Su-
saturated fatty acids (SFA) (SFA=43,95%) as khija & Palmquist, 1988; Herman, 1991;
well as ex situ method (SFA=52,83%) (Table 1). Christie, 1993; Kildiran et al., 1996; Suter et al.,
However, the unsaturated fatty acids 1997; Carrapiso & Garcia, 2000; Lotero et al.,
composition (UFA) derived by ex situ method 2006). In ex situ method, this problem is not to
(UFA=38,3%) was higher than in situ method be considerablle since isolated lipid was used as
(UFA=32,32%) especially of PUFA. The lowest a sample while in situ method used a biomass as
proportion of UFA were obtained for in situ a sample. Isolated lipid has a low matrix content
method showed that these method are not than a biomass since it was extracted before
suitable for FAME preparation from sample with when isolated processes (Christie, 1993).
high contained UFA. This might be due to Because of these, in situ method yielded lower
partial destruction of these fatty acids during the FAME than ex situ method.
esterification processes (Christie, 1993; Moreover, the environmental conditions of
Schlechtriem et al.). Christie (1993) noted that it reaction such as temperature, times,
must be considered since sulfuric acid as a concentrations of reagents, solvents and catalyst,
catalytic agent in this method, is a strong can affect to yields of FAME. Harsh conditions
oxidizing agent that can oxidized of polyenoic (high temperatures, heating for long time) was
fatty acids under heated temperature. Moreover, reported can cause degradation in fatty acids, but
oven drying a biomass before reaction seems can mild conditions yield low FAME recoveries
affect to because it can bring about alterations in (Sukhija & Palmquist, 1988; Herman, 1991;
unsaturated fatty acids (Lepage & Roy, 1986; Christie, 1993; Kildiran et al., 1996; Suter et al.,
Sukhija & Palmquist, 1988; Christie, 1993). 1997; Carrapiso & Garcia, 2000; Lotero et al.,
Thus, temperature of esterification processes and 2006; Schlechtriem et al.). In this preliminary
oven heated when pretreatment of biomass must studies, sample was heated at 80oC for 4 h since
be considered. acidic transesterification is carried out near the
Overall, FAME derived from C. gracilis boiling point of the methanol and usually more
using in situ acid-transesterification method was than 2 hours (Christie, 1993; Carrapiso & Gar-
lower proportion than ex situ method (76,27% cia, 2000; Lotero et al., 2006). While, solvents
and 91,14% respectively). This is indicated not plays a role in dissolving lipids in order to
all fatty acids in C. gracilis was transesterified to reacted with methanol. The effectiveness of the
be a FAME. This incomplete conversion may be solvent depends on its ability to solubilize lipids
due to the existence of matrix on the sample that (and specifically nonpolar lipids due to their
could affect esterification processes. Christie limited solubility in the methanolic medium used
(1993) was noted that FAME resulted from in for the synthesis) and to create a one-phase
situ transesterification depending on the nature system with lipids, methanol, and other reagents
of the sample especially the matrix and water (Carrapiso & Garcia, 2000). Hexane was used
content since these has an important factor on since it can solubilized a nonpolar lipid although

other nonpolar solvents such as petroleum W.W. Christie, Advances in Lipid Methodology –
benzine, dioxane and chloroform can be applied Two, The Oily Press.
(Long et al., 1988; Suter et al., 1997; Carrapiso Gavett, E., & Information Resources, I. 1995. Biodiesel: A
& Garcia, 2000; ). Moreover, methanol-sample Technology, Performance, and Regulatory Overview.
Jefferson City, Missouri: National Biodiesel Board.
molar ratios was affected to the yield as well as
solvents. Acidic condition was preferred since Haas, M. J., K. M. Scoot, W. N. Marmer & T. A. Foglia.
this is affected less by small amounts of water 2004. In situ Alkaline Transesterification: An
Effective Method for the Production of Fatty Acid
while methanol-sulffuric acid was used as a Esters from Vegetable Oils. JAOCS. 81(1): 83-89.
catalyst because it is very easy to prepare than
Herman, R. P. 1991. Lipid Production from Algae in Saline
other acid catalyst (Christie, 1993; Carrapiso &
Water Using Low Intensity Culture Technniques,
Garcia, 2000; Lotero et al., 2006). Technical Completion Report No. 258, New Mexico
However, either in situ acid- Water Research Institute, New Mexico.
transesterification or ex situ transesterification Kildiran, G., S. Ozgul Yucel & S. Turkay. 1996. In situ
yielded of FAME belows Indonesian National Alcoholysis of Soybean Oil. Ibid. 73:225–228.
Standard (SNI-04-7182-2006) for Biodiesel.
Lepage, G. & C.C. Roy. 1986. Direct transesterification of
FAME that resulted from in situ and ex situ all classes of lipids in a one-step reaction. J. of Lipid
mathod are 91.14% and 76,27% while SNI-04- Research, 27: 114-120.
7182-2006 is about 96,5%. The low yielded of Long, A. R., S. J. Massie & W. J. Tyznik. 1988. Rapid
FAME that resulted from this research showed Direct Extraction Derivatization Method for the
these elementary conditions must be optimize. Determination of Acylglycerol Lipids in Selected
Thus, further research is needed to optimize Samples Matrices. J. Food Sci. 53: 940–942, 959.
condition of reaction towards given highly yields Lotero, E., J. G. Goodwin, JR., D. A. Bruce, K.
of FAME. Suwannakarn, Y. Liu & D. E. Lopez. 2006. The
In conclusion, In situ acid- Catalysis of Biodiesel Synthesis. Catalysis. 19:41-83.
transesterification method was potentiable to Marinkovic, S. S. & A. Tomasevic. 1998.
prepared of FAME from marine microalgae Transesterification of Sunflower Oil in situ. Fuel
Chaetoceros gracilis. It will reducing the cost .77:1389–1391.
production to since it saving times, work, waste Ozgul-Yucel, S. & S. Turkay. 1993. In situ Esterification of
and chemical reagents than conventional Rice Bran Oil with Methanol and Ethanol. J. Am. Oil
method. It might be aplicable for large scale Chem. Soc. 70:145–147.
production of biodiesel from marine microalgae. Ozgul-Yucel, S. & S. Turkay. 2002. Variables Affecting the
Yields of Methyl Esters Derived from in situ
Esterification of Rice Bran Oil. Ibid. 79:611–613.
ACKNOWLEDGMENTS
Ozgul-Yucel, S. & S. Turkay. 2003. FA Monoalkylesters
from Rice Bran Oil by in situ Esterification. Ibid.
This research is a part of Researchers
80:81–84.
Candidate Programe 2009 were funded by
Research Centre for Oceanography-LIPI. I Schlechtriem, C., R. J. Henderson & D. R. Tocher. A
critical assesment of different transmethylation
would thanks to Lily M. G. Panggabean and procedures commonly employed in the fatty acid
Diah R. Noerdjito for their support in provision analysis of aquatic organisms. Research Report, Dept.
of marine microalgae Chaetoceros gracilis of Food Science, Swedish Univ. of Agricultural
culture. Sciences.
Sukhija, P. S. & D. L. Palmquist. 1988. Rapid Method for
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Analysis: Some New Extraction Techniques and in Suter, B., K. Grob & B. Pacciarelli. 1997. Determination of
situ Transesterification. Lipids. 35 (11): 1167-1177. Fat Content and Fatty Acid Composition Though 1-
min Transesterification in the Food Sample:
Christie, W. W. 1993. Preparation of ester derivates of fatty
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acids for chromatographic analysis, p.69-111. In
258.

Feasibility of Sorghum Bicolor as Bioethanol Producer by Mass
and Energy Balance Analysis
Deliana Dahnum, Haznan Abimanyu, Tami Idiyanti and Sudiyarmanto
Research Centre for Chemistry – LIPI

Kawasan PUSPIPTEK Serpong, Tangerang Selatan – Banten, e-mail: dhely_a@yahoo.com
(Correspondence to: Deliana Dahnum)
ABSTRACT
Indonesia as an agricultural country with a lot of biological wealth has a high potential as a producer of bi-
oethanol. Bioethanol is becoming hot issue to discuss in replacing fossil fuel which is currently running low. Oil
reserves that are running out forced many researchers to seek for alternative fuel derived from plants which are
renewable. Bio-ethanol, one of biofuel, is being developed extensively, and is commonly produced from food
and non-food crops. Sorghum bicolor, one of the cereal crops that has resistance to drought and high productivi-
ty, and which cultivation is relatively inexpensive, can be developed as one of the feedstocks for bioethanol pro-
duction. Juice and bagasse can be separated from stalk for further processing. Juice can be directly fermented to
produce bioethanol, while bagasse should be first hydrolized before it is fermented using Saccharomyces
cereviceae. From the results of our conducted research, it was found that the ethanol content of 9% could be
obtained from sorghum juice and that of 1% from sorghum bagasse. Through calculations and systematic analy-
sis of mass and energy, the ability or the feasibility of producing bioathanol from sorghum can be well predicted
with certainty. From mass balance calculation a 3.15% yield of ethanol could be obtained with ethanol concen-
tration of 9%, thus bioethanol based on sorghum is eligible to be developed further. The process performed here
was still not eficient, since the energy to produce bioethanol is higher than the energy content of ethanol itself.
Keywords: Sorghum bicolor, bioethanol, mass and energy balance.
INTRODUCTION magnitude of fossil fuels input (and related

emissions) relative to subsequent fossil fuel
Energy needs grow along with the increas- savings (and avoided emissions) resulting from
ing of population and industrialization. Until their use as alternatives to conventional fossil
now, energy controls an important role as almost fuels. (Malça & Freire, 2004)
all sectors are supported by the availability of One method to reduce air pollution is the
energy. The energy used comes from fossil fuel oxygenated fuel for vehicles. MTBE (Methyl
consist of petroleum, natural gas, and coal. All tert-butyl ether) is a member of a group of
of them are nonrenewable energy source that chemicals commonly known as fuel oxygenates
when used constantly will be run out. In Indone- (Fischer et al., 2005). It is a fuel additive to raise
sia, petroleum, natural gas, and coal respectively the octane number. But it is very soluble in water
contributed 51.66, 28.57 and 15.34% of energy and it is a possible human carcinogenic. it should
needs, while the rest are hydro and geothermal. be substituted for other oxygenated substances to
The continuous use of fossil fuels would increase the octane number of the fuel.
lead to increase CO2 in the air. CO2 is the result Presently, ethanol as an oxygenous biomass fuel
of combustion of fossil fuels. However, the is considered as a predominant alternative to
extent to which biofuel can displace fossil fuels MTBE for its biodegradable, low toxicity,
and net emissions of CO2 depends on the persistence and regenerative characteristic.
efficiency with which it can be produced. In fact, (Almodares & Hadi, 2009)
all processing technologies, including biofuel The experts have tried to find an alternative
options, involve (directly and/or indirectly) the fuel that can replace fossil fuels, environmental-
use of fossil fuels in their production and/or ly friendly (produce less CO2 to the environ-
operation, resulting in associated greenhouse gas ment) and renewable. Initial research was per-
emissions. Therefore, in practice, the actual formed by finding the source of natural materials
benefits of biofuels displacing their fossil fuel that can produce renewable fuels. Bioethanol is
equivalents depend, crucially, on biofuels energy one of them that can be developed and obtained
and carbon balances, which indicate the relative from the crop. Some crops that can be used as

raw material of bioethanol are corn, cassava, used a strong acid catalyst, H2SO4 96%. In this
sugarcane, sweet potatoes, sorghum, potatoes, study, will be used H2SO4 for hidrolyzed rather
and beet. (Ega & Triwiyono, 2006). Biofuels are than enzyme because the economical factor.
originated from plant oils, sugar beets, cereals,
organic waste, and the processing of biomass. Fermentation: Fermentation process of juice and
Sugar beets, cereals (wheat, corn, barley, etc) bagasse sorghum performed using urea, NPK,
and other crops can be fermented to produce al- and TSP medium with each concentration of so-
cohol (bioethanol) to be used in gasoline en- lution at 1.5% of the amount of input material
gines. (Malça & Freire, 2004) and concentrations of solid yeast is 5% of the
Indonesia has potential of plants as a very amount of input material. The result of this fer-
large source of bioethanol whether originating mentation is alcohol which was measured using
from food crops or non-food. The big potential alcoholmeter. In this research uses Saccharomy-
of non-food crop that can be used as a source of ces cereviceae for fermentation.
bioethanol is sorghum (Sorghum bicolor). Sor-
ghum is a tropical cereal plant that has 4 months Filtration: This process is used to separate a
crop duration. It is not so difficult to grow them. mixture of fermentation products. Laboratory-
All types of drained soil are able to grow this scale experiments used filter paper only, so did
plant. Sorghum can produce approximately 30 not require energy in the separation. From the
dry tons/ha per year of biomass on low quality fermentation, the filtrate will be obtained in the
soils with low inputs of fertilizer and limited form of liquid and solid which are a yeast and
water per dry ton of crop, half of that required by the fermentation medium.
sugar beet and a third of the requirement for
sugar cane or corn (Renewable Energy World, Evaporation: The purpose of this evaporation is
2000). to get more pure ethanol with less water. Anoth-
In this paper, will be analyzed the ability of er object is to separate the ethanol from its impu-
S. bicolor in producing ethanol based on mass rities.
and energy balance. The calculated energy bal-
ance is the ratio between the energy required to Distilation: The process for purify ethanol. Eth-
produce bio-ethanol with the energy produced anol was separated from water. Mixture was
from bioethanol. From this analysis it can be boiled at the ethanol boiling point temperature
seen whether S. bicolor is eligible for producing which is 78oC then cooled through a cooling
bioethanol. condenser.
MATERIALS AND METHODS Analysis of sugar and ethanol:

Sugar Analysis (Somogyi Nelson Method): Sam-
Analysis of mass and energy calculation ple solution was added into the Nelson reagents
performed after a laboratory-scale research. A and B in the ratio 25: 1. After heating for 20
Sweet Sorghum cultivation was obtained from min and cooling, the reagent arsenomolybdate
BIOTROP garden, Bogor and the seeds were and aquadest were added to the solution. Sugar
from PATIR - BATAN. level of solution was analyzed using a spectro-
photometer.
The steps of the laboratory process to produce
bioethanol: Ethanol analysis: There are many methods to
Milling: First treatment of sorghum is milling. determine the alcohol concentration in a sample
Sorghum stems were peeled and cut into 5 cm such as GC, Dicromate Oxidation Method, alco-
long and then milled and pressed using juicer. holmeter, etc. In this research, alcoholmeter was
The result of this process is juice and bagasse. used to determine the ethanol concentration. Al-
Milling is important process because have a coholmeter was chosen because it can easily
function for size reduction and opening up of the predict the existence of ethanol in the sample
the fibrous materials for futher treatment. accurately and in a short time. But, alcoholmeter
needs quite a lot of samples compared with the
Hydrolysis: Hydrolysis process of bagasse sweet other alcohol analytical methods.
sorghum was done by enzymatic and liquid acid Based on the results of experiments had
catalysts. By enzymatic process it was used cel- been done in the laboratory, calculation and
lulase enzyme and by liquid acid catalyst was analysis of mass balance and energy balance

were performed. Mass balance was calculated to produce ethanol. To support growth of the
through mass sorghum used and bioethanol re- yeast, it was also necessary to add urea, NPK,
sult obtained. Energy balance is the amount of and TSP as a nutrient.
energy required to produce bioethanol compared In contrast to the juice, a particular treat-
with the energy of bioethanol itself. From this ment is done for bagasse. For lignocellulosic
analysis, the feasibility of S. bicolor as a re- materials, a process called hydrolysis was neces-
source of bioethanol to be further developed as a sary, since lignocellulosic materials consists of
renewable fuel will be known. hemicellulose, cellulose and lignin. Hydrolysis
process has the purpose as a bond breaker, to
RESULTS AND DISCUSSION remove the lignin content and also to break
down the structure of cellulose and hemicellu-
Mass balance analysis: In general, there are two lose into simple sugar compounds. To hydrolyze
ways to obtain bioethanol, (1) By fermentation lignocellulosic materials, 1 M H2SO4 was used
of the juice of extracts the crop, and (2) by using (Zimbardi et al., -). The use of a strong acid was
hydrolyzed bagasse and fermentation. The se- selected under the consideration that the acid can
cond process is known as second generation of convert almost 90% of sugar (Badger, 2002).
bioethanol production. Bioethanol is a liquid fuel Although the use of concentrated acids required
obtained with the help of microorganisms. Mi- a relatively long time and a good washing pro-
croorganisms are used to convert glucose into cess to achieve the pH of the reaction before
alcohol which is ethanol. Usually fermentation adding the microbes in the ethanol fermentation
to produce ethanol is performed with S. process, it remains superior than dilute acid. By
cereviceae. using diluted acid, sugar degradation will occur
Fig 1 illustrates the way of producing bio- in the hydrolysis reaction and also side products
ethanol from S. bicolor. In general, the process formation that are not desired. Adjusment of pH
for ethanol production is composed of two pro- value was necessary in order to make a solution
cesses; fermentation for ethanol production and in accordance with the fermentation pH between
isolation of ethanol from fermentation broth. As 4.5 and 5.5. Adjusment of pH was performed by
seen in Fig. 1, sorghum is first separated into adding 1 M NaOH. S. bicolor showed a compo-
juice and bagasse. After separation, two different sition of 75% juice and 25% bagasse (Table 1).
treatments were performed to obtain the product.
Juice was obtained, then fermented by the yeast
Urea = 1,5 % electricity

electricity NPK = 1,5 %
TSP = 1,5 %
Yeast = 5 %
Sweet Juice
Milling Fermentation Filtration Evaporation
Sorghum
Bagasse
Urea = 1,5 %
Hydrolysis NaOH Distilation
NPK = 1,5 %
TSP = 1,5 %
H2SO4 Yeast = 5 %
Bioethanol
Fig 1. Flowchart illustrating bioethanol production from S. bicolor

Table 1. Composition of juice in S. bicolor was 9%, while from bagasse fermentation was
Component Percentage (%) 1%. Bioethanol obtained was then purified by
(v/v) distillation. In the first distillation ethanol con-
Water 75.0 tent obtained was 37%. More detail mass bal-
Glucose 7.2 ance calculation can be shown in the following
Carbohydrate 11.6
Impurities 6.2
table.
Total 100 Mass balance calculations showed that from
750 ml of juice (obtained from 1000 g stalk of
Table 2. Lignin and cellulose content in bagasse sorghum shorgum was obtained 25.49 g of ethanol or
while is converted to ethanol will get 32.55 ml of
Component Percentage (%)
(dry basis)
ethanol. Ethanol yield obtained was of 3.15%
Klason Lignin 26.5206 with a percentage of 37%.
Holo-Cellulose: Yield obtained, indeed, is slightly. Research
- α-Cellulose 26.4189 which was done is not in the optimization step to
- Hemicellulose 25.5627 obtain the maximum yield. To maximize the
Others Component 21.4978
Total 100
products of ethanol, it takes a variation of type
and number of microorganisms, pH, amount of
The next step was purification of fermenta- micronutrients, etc.
tion products. After going through filtration to While the research results from the use of
separate solid and liquid fermentation broth, the bagasse sorghum into bioethanol showed that
filtrate obtained was subjected to an evaporation ethanol concentration is very small amount, 1%
in order to separate ethanol and water compo- ethanol in 190 g bagasse. Bioethanol from ba-
nents from the remaining glucose and other car- gasse (cellulose base) has glucose levels that are
bohydrates. The final step was distillation to smaller and its process is longer compared to the
separate bioethanol from existing water. production of bioethanol from juice of sorghum.
Based on result of research, bioethanol Therefore, the influence of bagasse to produce
content obtained from fermented sorghum juice bioethanol is not too significant.
Table 3. Mass balance of bioethanol production from sorghum juice (obtained from 1000 g S. bicolor)
Component Milling (g) Fermentation (g) Filtration (g) Evaporation (g)

Water 607.50 607.50 607.50 546.75
Glucose 58.32 5.83 5.83
Carbohydrate 93.96 93.96 93.96
Impurities 50.22 50.22
Bagasse 190
NPK 12.15
Urea 12.15
Yeast 40.50
Ethanol 26.83 26.83 26.83
CO2 25.66
Total 1000 874.8 734.11 573.58
Table 4. Distillation of fermentation product of sorghum juice
Output
Input
Component Distillate Residue
x m (g) x m (g) x m (g)
Water 0.91 271.25 0.63 43.39 0.99 227.86
Ethanol 0.09 26.83 0.37 25.49 0.01 1.34
Total 1.00 298.08 1.00 68.88 1.00 229.20

Energy Balance Analysis: Energy balance cal- CONCLUSIONS
culations based on the utilization of electrical
energy is used during the process. Electrical en- From mass balance calculation obtained
ergy was used to convert the energy which is yield of ethanol was 3.15% with ethanol concen-
expected. For example, evaporator, electrical tration 9%, thus bioethanol based on sorghum is
energy is converted into heat energy. In this pro- eligible to be developed further. However, the
cess, the use of electrical energy is needed only result of calculation obtained from the energy
on the three instruments, milling, evaporator, balance of energy to produce bioethanol is great-
and distillation. er than the energy content of ethanol itself. Op-
timization of bioethanol production process is
Table 5. Energi Balance For Production of Bioetanol from needed to further improvement production effi-
Juice of Sorghum (basis 1000 g S. bicolor)
ciency.
Process P (W) t (s) Q (kJ)
Milling 350 600 210 ACKNOWLEDGEMENTS
Evaporation 60 3600 216
We express our gratitude to DIKTI project
Distillation 600 18000 10800
for its research grant on 2009 which fully
Total 1010 22200 11226 supported in this research. We thank also to
BIOTROP for providing samples of S. bicolor.
Table 5 shows the amount of heat used to
produce 25.49 g ethanol. Data obtained based on REFERENCES
experiments that have been conducted in the la-
boratory. From the mass balance calculation, Almodares, A. and Hadi, M.R. 2009. Production of
found 25.49 g of ethanol in 810 g of sorghum. bioethanol from sweet sorghum: a review. African J.
Agricultural Research. 4 (9): 772–780.
When it compared to energy balance, energy
required to produce 25.49 g of ethanol was Badger, P.C. 2002. Ethanol from cellulose: A general
11,226 kJ or 440.48 kJ/g ethanol. review. p. 17–21. In: J. Janick and A. Whipkey (eds.),
Trends in new crops and new uses. ASHS Press,
Energy content of ethanol itself is about Alexandria, VA
27.3 kJ/g ethanol. When compared with existing
processes, energy required during the process is Ega, L. and Triwiyono, B. 2006. Kajian tekno-ekonomi
produksi fuel grade ethanol dari nira aren dan kelapa
higher rather than energy content in ethanol sebagai sumber energi engine alternatif. Jakarta.
itself. This indicates that the laboratory process
Fischer, A., Oehm, C., Selle, M. and Werner, P. 2005
for ethanol is not economical. Ethanol results .Biotic and abiotic transformations of methyl tertiary
obtained from the laboratory is very small while butyl ether (MTBE). Environ. Sci.Pollut. Res. Int. 12:
the process that is done use a large electric ener- 381-386.
gy. Reith, J.H. 2002. Co-production of bio-ethanol, electricity,
When the calculation is based on a pilot and heat from biomass residues. Amsterdam,
plant or manufacture scale, the energy required Netherlands.
is obtained or ethanol would be more economi- Malça, J. and Freire, F. 2004. Life cycle energy analysis for
cal. Need for optimization of existing processes bioethanol: allocation methods and implications for
in terms of energy and raw materials will be cal- energy efficiency and renewability. Mexico.
culated to obtain competitive materials for re- Sun, Y. and Cheng, J. 2002. Hydrolysis of lignocellulosic
newable energy. materials for ethanol production: a review.
Bioresource Technol. 83: 1-11.
Zimbardi, F., Viola, E., Gallifuoco, A., De Bari, I.,
Cantarella, M., Barisano, D. and Braccio, G.
Overview of the bioethanol production. Università
degli Studi de L’Aquila. Dipartimento di Ingegneria
Chimica e dei Materiali. Monteluco di Rojo.

Uniresponse Kinetics Based on Ping Pong Bi Bi Mechanism for Biodiesel
Synthesis via Non Alcohol Route in Packed Bed Reactor
Heri Hermansyah, Rita Arbianti, Arya Yudistira and Anatta Wahyu Budiman

Depok 16424, Indonesia, e-mail: heri@chemeng.ui.ac.id
(Correspondence to: Heri Hermansyah)
ABSTRACT
Biodiesel has become an alternative of environmental-friendly fuel made of vegetable oils and animal fat
derived from renewable resources. Recently, a research on synthesis of biodiesel from waste cooking oil via non
alcohol route using an immobilized Candida rugosa lipase as biocatalyst in a packed bed reactor has been car-
ried out. In this study, a uniresponse model based on Ping Pong Bi Bi mechanism was proposed to describe the
behavior of all component involved in biodiesel synthesis reaction via non alcohol route. The two lumps reac-
tion rate constants result of this study were estimated by Runge-Kutta-Fehlberg method. The best fitting results
on experimental data yielded kinetic parameters θ1 = 0.0439 and θ2 = -16.8303 for fresh cooking oil and θ1 =
0.6485 and θ2 = 20.3453 for used cooking oil. Squared-sum of relative error of this kinetic constants were
1.1764x10-4 for fresh cooking oil and 1.1452x10-4 for used cooking oil. Sensitivity analysis showed that the re-
sulting constants have good sensitivity; with the smallest deviation was 24.64%.
Keywords: biodiesel, uniresponse, Ping Pong Bi Bi mechanism, packed bed reactor, non alcohol route.
INTRODUCTION sults. Biodiesel yield obtained reaches 90%

(Kaieda et al., 1999; Ban et al., 2001; Hama et
Biodiesel is an alternative and environmen- al., 2007). In addition, lipase from other species
tal-friendly fuel made of vegetable and animal was also used, such as Candida antarctica
oils derived from renewable resources. Biodiesel (Watanabe et al., 2000; Kose et al., 2002; Wang
is generally synthesized using palm, soybean, et al., 2007; Halim et al., 2009; Varma et al.,
sunflower, Jatropha, and other vegetable oils 2009), Pseudomonas cepacia (Noureddini &
(Knothe et al., 1996) as raw materials. Zhu, 1997; Shah & Gupta, 2007; Cheirsilp et al.,
Conventionally, biodiesel was produced 2008; Torres et al., 2008), Pseudomonas
through transesterification reaction of fluorescens (Iso et al., 2001), Thermomyces
triglyceride and alcohol which catalyzed by lanuginosa (Al-Zuhair, 2005; Yagiz et al., 2007;
alkalines (Xu et al., 2005). However, this Dizge et al., 2009), and Rhizomucor miehei
methods yield several problems such as (Dossat et al., 2002; Shieh et al., 2003; Al-
homogenously mixed between catalyst and Zuhair, 2005; De Paola et al., 2009).
product so that difficult to separate. In addition, The use of used cooking oil as raw material
alkalines give saponification reaction which is a in producing biodiesel considered due to its
side reaction in this process. Undesired side re- higher saturation level than palm oil that makes
actions are present in the synthesis. This can be the conversion process into biodiesel better
burdensome the product purification process and (Shimada et al., 1999; Nie et al., 2006; Wang et
reduce biodiesel conversion. To overcome this al., 2007; Dizge et al., 2009; Enweremadu &
problem, a catalyst which did not join in a ho- Mbarawa, 2009; Halim et al., 2009; Maceiras et
mogeneous mixture and capable of directing al., 2009). Maceiras reported the enzymatic
specific reactions to produce the desired product production of biodiesel from waste frying oil
without any side reactions is needed. with methanol using immobilized lipase
Recently, research of lipase enzymes as Novozym 435 as catalyst. The biodiesel
biocatalysts continues to be developed. The use conversion reached 89.1% at 50°C and methanol
of lipase enzymes as biocatalysts for the bio- to oil ratio used was 25:1. The reaction took 4 h
diesel synthesis is very promising since they can to complete and the catalyst concentration used
overcome several problems of alkaline catalysts. was 10% (Maceiras et al., 2009). However, the
The use of Rhizopus oryzae lipase in the trans- use of lipases as biocatalysts for the biodiesel
esterification reaction had given satisfactory re- synthesis via transesterification reaction is still a

problem. Alcohol, such as methanol, deactivated E*Q, FP, and E*G describe the enzyme-
lipase rapidly and caused bad stability in substrate complexes which its concentration is
catalyzing the reaction (Xu et al., 2005). shown on Eq. (1) to (4).
Xu et al. (2005) carried out a study on the
k1
biodiesel synthesis using methyl acetate as acyl [ EG ]  [ E ][G]
acceptor substituting methanol. Furthermore, Xu k 1 (1)
et al. (2005) used lipase as biocatalyst for bio- k k
diesel production via non alcohol route. The re- [ FP ]  P 1 [ E ][G ]
k  P k 1 (2)
sult was that, in addition to environmentally
k
friendly, lipase as a catalyst in the production of [ EQ ]  3 [ E ][Q]
biodiesel has high selectivity and efficiency in k3 (3)
the production. In addition, Du and Modi was
then investigated biodiesel synthesis via interest- While the overall enzyme mass balance shown in
erification in batch reactors (Du et al., 2004; Eq. (4).
Modi et al., 2007) while Mendes et al. (2009) [ E ]tot  [ E ]  [ EG ]  [ FP]  [ EQ]
and Ognjanovic et al. (2009) in continuous reac- (4)
tors. Combination of Eq. (1) to Eq. (4) yields Eq. (5).
Studies on the kinetics of the biodiesel syn-
thesis have been done. These kinetic studies  k k k  k 
generally focus on transesterification reaction [ E ]tot  [ E ]1   1  P 1 [G ]  3 [Q] (5)
  k 1 k P k 1  k3 
(alcoholysis) kinetics. Most studies were using
reversible second order mechanism. However, So the concentration of enzyme, [E] can be ex-
for the transesterification reaction with pressed as:
biocatalyst, some researchers suggested using a
[ E ]tot (6)
Ping Pong Bi Bi mechanism. Ping Pong Bi Bi [E] 
mechanism is the most suitable model to de- k k k  k
1   1  P 1 [G ]  3 [Q]
scribe the reaction mechanism using biocatalyst.  k 1 k  P k 1  k3
MATERIALS AND METHODS Reaction rate for intresterification:

d [G]
Assumptions taken in the reduction of rint    k 2 [ FP ] (7)
uniresponse kinetic model based on Ping Pong dt
Bi Bi is as follows: Substituting Eq. (2) into the Eq. (7) yields Eq. (8).
a. The reaction followed first-order kinetics
mechanism for each substance involved. v max
[G ]
b. Product and/or substrate inhibition were K m ,G
neglected rint  (8)
[G ] [Q]
c. Water concentration was constant during the 1 
reaction. K m ,G K i ,Q
Reaction scheme to develop a uniresponse kinet-
ics model is shown in Fig 1. Where,
k 2 k P [ E ]tot
v max  (9)
k P  k P
k  P k 1
k m ,G  (10)
k1 (k P  k  P )
k3
k i ,Q  (11)
k 3
The concentration of product [Q]:
Fig 1. Reaction scheme
[Q]  [G]0  [G] (12)

The rearrangement of Eq. (8) using Eq. (12) estimated constants of this model mechanism is
yields Eq. (13). shown in Table 1.
1 [G ]
rint  (13) 0,06
1   2 [G ] Used Cooking Oil Model
Fresh Cooking Oil Model
0,05 Used Cooking Oil Experiment
Where, Fresh Cooking Oil Experiment
vmax K i ,Q 0,04
1 
K m,G K i ,Q  [G]0 
(14)
[B] (mole/L)
0,03
0,02
K  K m ,G 
2 
i ,Q
(15)
K m,G K i ,Q  [G ]0  0,01
Kinetic parameters (reaction constants) es- 0

0 1 2 3 4 5 6
timation was determined using the linear regres- Residence Time (h)
sion method. Eq. (13) is modified to form a line-
ar equation as Eq. (16). Then, plot was made Fig 2. Curve fitting between model calculations and
between the residence times and the experimental data
concentration accessible glyceride bonds that
Table 1. Estimated value of kinetic parameters of the pro-
have not undergone reaction, [G], using the posed model (in l/mol/s).
Runge-Kutta-Fehlberg method with the parame-
ter values obtained from regression. Results Value
Parameter
from the model were then compared to the ex- Fresh Used
periments results, and the errors were calculated θ1 0.0439 0.6485
using sum square error method. θ2 -16.8303 20.3453
1 1 1  (16)
    2 To determine the sensitivity of the kinetic pa-
d [G ]  1 [G ]  1
rameters obtained, the sensitivity analysis of pa-
dt
rameter was done by changing the constant value
of the kinetics to a half or twice without changing
y = a x +b the value of other constant. Then the errors oc-
curred were observed. Parameter is said to have
RESULTS AND DISCUSSION good sensitivity if its changes result in a signifi-
cant error. Table 2 shows the results of sensitivity
The results of curve fitting between model analysis of the kinetic constants for the model of
calculations and experimental results were fresh cooking oil.
shown in Fig 2. These model calculations were
using the method of Runge-Kutta-Fehlberg with Table 2. Sensitivity analysis for kinetic constants of fresh
cooking oil
the time increment of 0.1 h for the time span of
0 to 5 h. Initial concentration of glyceride bonds Constants Value Error % deviation
accessible for interesterification was determined 0.0220 1.8412x10-6 98.43
based on the mass balance of fatty acid residue θ1 0.0439 1.1764x10-4 0
shown in Eq. (17). 0.0659 3.5787x10-4 204.20
[G]0  3 [TG]  2 [ DG]  [MG]  [ FAME ] (17) -8.4152 8.8663x10-5 24.64
θ2 -16.8303 1.1764x10-4 0
Fig 2 shows that both model calculations -25.2455 3.2114x10-3 2629.72
and experimental data showing increase of con-
centration of biodiesel as the residence time Table 2 shows that the percentage deviation
increased. This is due to the longer residence obtained if the value of each constant value raised
time of substrate in the reactor, the longer the or lowered by 50% showed a great value. The
contact between substrate and enzyme so that the smallest deviations are the parameter θ2 that have
conversion of biodiesel increased. The value of

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Updating Bioreactor Systems for Hydrogen Production Process
Through Photo Fermentation
Khairul Anam, Muhammad Sidiq Habibi and Dwi Susilaningsih
Research Center for Biotechnology, Indonesian Institute of Sciences

Jl. Raya Bogor Km. 46 Cibinong 16911, Bogor, West Java, Indonesia, e-mail: ka_anam@yahoo.com
(Correspondence to: Khairul Anam)
ABSTRACT
Clean and renewable energy systems are required to prevent environmental problems caused by the use of
fossil energy sources. Hydrogen is one of the promising green energy sources, because it is easily converted to
electricity and cleanly combustible. Photosynthetic bacteria are one among the other bacteria that can produce
hydrogen biologically. With assistance of water and light, these microbes can produce hydrogen from substrate
which comes from organic material sources such as glucose. The research on hydrogen production from glucose
was done with updating the photo-fermentation system using various bioreactors, from simple bioreactor, shaker
bioreactor, up to shaker bioreactor using gas separation to improve, stabilize and purify hydrogen production by
photo fermentation. From several experiments, simple bioreactor only produce 4 ml hydrogen gas compare bio-
reactor using shaker can produce 16 ml in 75 ml production medium consist of 1% glucose. Shaker bioreactor
using gas separation can purify the gas production into hydrogen up to 80% of total volume gases formed com-
pare without using gas separation, it’s only can purify gas production into hydrogen gas up to 40% in 500 ml
production medium consist of 1% glucose.
Keywords: hydrogen, photo fermentation, bioreactor, photosynthetic bacteria.
INTRODUCTION which comes from organic material sources,

such as glucose (Miyake, 1997).
Climate change as the consequent of the In this study, hydrogen production from
global warming caused by greenhouse gasses glucose was done with updating the photo fer-
which were produce by combustion of fossil fuel mentation system using various bioreactors,
that needed by human life. To minimize the from simple bioreactor, shaker bioreactor, up to
greenhouse gasses production, is urgently re- shaker bioreactor using gas separation. The ob-
quired to find some resources as the clean and jectives of this study are to improve, stabilize
environmental friendly energy. Hydrogen was and purify the hydrogen gas production.
one of some resources that can provide clean and
friendly energy because hydrogen did not pro- MATERIALS AND METHODS
duce CO2 in its combustion. With oxygen, hy-
drogen was converted into energy by fuel cell to Microorganism was consortia of photosyn-
produce electricity (Kawaguchi et al., 2001; thetic bacteria which were collected from Sanur
Nishio & Nakashimada, 2006). Usually, hydro- Beach, Bali. Rhodobium marinum known as the
gen was produced by fossil fuel gasification. dominant bacteria among other bacteria in this
But, now hydrogen can be produce by biological consortia. This consortia then called as Sanur
process and can be renewable (Li & Fang, consortia.
2008). This process involved microorganism that Sanur consortia was grown on steril
can produce hydrogen gas and organic material phototropic bacteria medium containing
as substrate that will be converted into hydrogen dissodium succinate (10 g/l), yeast extract (0.3
by microorganisms (Miyamoto, 1997). Hydro- g/l), 10 ml basal medium (Kawaguchi et al.,
gen can be produce by several microorganisms 2002) stock 100x, 1.5 g NaHCO3, and 1000 ml
such as photosynthetic bacteria, cyanobacter and aquadest. Nitrogen gas was passed trough the
anaerobic bacteria (Benneman, 1997). medium to provide anaerobic conditions. The
Photosynthetic bacteria are one among the culture was incubated at 30ºC under light illu-
other bacteria that can produce hydrogen biolog- mination and with or without agitation and gas
ically. With assistance of water and light, these separation. Growth was determined by OD (op-
microbes can produce hydrogen from substrate tical density) measurement at 660 nm. The Sanur

consortia culture was harvested once the OD of tle, lamp, silicone pipe, accumulator column,
the culture reached OD ≥ 1 by using centrifuge NaCl container and shaker; up to shaker
Sorvall RC26 plus (rotor GSA, 6000 rpm, 7˚C, bioreactor using gas separation which consists of
20 min). The pellet was resuspended on 30 ml reactor bottle, lamp, silicone pipe, accumulator
steril production medium that contain glucose 10 column, NaCl container, shaker and separator
g/l, yeast extract 3 g/l, 10 ml basal medium stock column (Fig 1). Hydrogen which produce in the
100x, 1.5 g NaHCO3, and 1000 ml aquadest. process was collected in a glass column and the
Nitrogen gas was also passed through the medi- hydrogen percentage of the volume from
um to provide anaerobic conditions. Further- collected gas production was measured by using
more, to get OD 1 in production medium, sever- Hydrogen detector from H2Scan-Teledyne
al volume of Sanur consortia suspension was model 2240.
inoculated and new steril production medium
was added to make 75 ml working volume in RESULTS AND DISCUSSION
bioreactor bottles.
Several runs of experiments were conduct-
ed to observe the yield of differences photo fer-
A mentation systems. Simple bioreactor that not
using shaker can only produce 4 ml of hydrogen
gas from 75 ml working volume production me-
dium which consist of 1% glucose. Bioreactor
system using shaker more efficiently produce
hydrogen and it can produce 16 ml of hydrogen
gas, four times higher than simple bioreactor.
Shaker bioreactor also consumes glucose faster
than simple bioreactor. The glucose concentra-
tion already used up in the first day of fermenta-
B
tion compare with simple bioreactor that used up
in the third day (Fig 2).
B
Fig 1. A. Simple bioreactor using no shaker with illumina-
tion from 300 W/m2 fluorescent lamps, B. Bioreac-
tor using shaker in 120 rpm of agitation with 44
W/m2 neon lamps, C. Shaker bioreactor using gas
separation column in 120 rpm of agitation and 44
W/m2 illuminations of neon lamps
Several experiment of photo fermentation

system by Sanur consortia were perform by
using various bioreactors, from simple
bioreactor consists of reactor bottle, lamp, sili- Fig 2. A. Consumption of glucose substrate, B. hydrogen
cone pipe, accumulator column and NaCl con- yield by photosynthetic bacteria using simple bio-
reactor ( ) and shaker bioreactor ( )
tainer; shaker bioreactor consists of reactor bot-

From other several runs of experiments
shaker bioreactor was updated with gas separa-
tion column. With using gas separation, the per-
centage of hydrogen in gasses formed that pro-
duce in the fermentation process can reach up to
80% compare with shaker bioreactor it was more
pure that using no gas separation column that
only can reach up to 40%. The separation was
only in the few part of fermentation, because the
purifying process involves chemical reaction.
The other gas that produce in the fermentation
was carbon dioxide react with base and concen-
trate in the column, so if more carbon dioxide
was produce more base were needed (Fig 3 and
4).
Fig 4. Hydrogen ( ) yield compare with gas production ( )

from shaker bioreactor with gas separation. Hydro-
gen production was calculated by percentage total
volume gasses formed
In these experiment, the gas production

which formed by photo fermentation in the
shaker bioreactor more higher compare with
shaker bioreactor using gas separation. But the
yields of hydrogen production comparing both
of the systems are almost the same because the
productions of hydrogen gas were the same. So,
if the gas production was collected from both
systems, shaker bioreactor using no gas separa-
tion was produce higher yield but with low per-
centage of hydrogen gas. In comparison with
shaker bioreactor which using no gas separation,
the systems that using gas separation produce
Fig 3. Hydrogen ( ) yield compare with gas production ( ) gasses which formed from fermentation lower
from shaker bioreactor without gas separation. Hy- but high percentage of hydrogen gas.
drogen production was calculated by percentage to- Compare with research that was done by Li
tal volume gasses formed
& Fang (2008) which can produce 70 ml of hy-
drogen gas from 28 mM glucose, the current
study produce almost the same in the quantity of

glucose that have been used to. In these studies, REFERENCES
the highest yield of hydrogen production can
reach up to 120 ml in 75 ml working volume Benneman, J. R. 1997. The technology of biohydrogen.
with shaker bioreactor systems. Proceedings of International Conference on Biologi-
cal Hydrogen Production. 18-30.
From these researches we can conclude that
bioreactor systems updated can improve, stabi- Kawaguchi, H., Hashimoto, K., Hirata, K. and Miyamoto,
lize and purify the hydrogen gas production. K. 2001. H2 production from algal biomass by a
mixed culture of Rhodobium marinum A-501 and
Shaker bioreactor that using gas separation gives Lactobacillus amylovorus. J. Biosci. Bioeng. 91 (3):
more yield production and pure of hydrogen 277-282.
production. Kawaguchi, H., Nagase, H., Hashimoto, K., Kimata, S.,
Doi, M., Hirata, K. and Miyamoto, K. 2002. Effect of
CONCLUSIONS algal extract on h2 production by a photosynthetic
bacterium Rhodobium marinum A-501: Analysis of
stimulating effect using a kinetic model. J. Biosci.
Shaker bioreactor was more effective to
Bioeng. 94 (1): 62-69.
convert glucose into hydrogen and produce
higher yield of hydrogen production than biore- Li, R.Y. and Fang, H.H.P. 2008. hydrogen production char-
acteristics of photoheterotrophic Rubrivivax gelatino-
actor using no shaker. Shaker bioreactor using sus L31. Intl. J. Hydrogen Energy. 33: 974-980
gas separation can purify gas production up to
80% than 40% the yield of bioreactor systems Miyake, J. 1997. The technology of biohydrogen. Proceed-
ings of International Conference on Biological Hy-
that using shaker without gas separation column. drogen Production. 7-18.
Miyamoto, K. 1997. Renewable biological systems for
ACKNOWLEDGEMENT alternative sustainable energy production. FAO -
The project is supported and funded by Re- Food and Agriculture Organization of the United Na-
search Competitive Grant of Indonesia Institute tions.
of Sciences (LIPI), 2006-2009. Nishio, N. and Nakashimada, Y. 2006. recent development
of anaerobic digestion processes for energy recovery
from wastes. J. Biosci. Bioeng. 103 (2): 105-112.

Isolation and Screening of Lignocellulolytic Actinomycetes
From Rice Straw Waste
Heri Satria1
1
Department of Chemistry, University of Lampung, Jl. Prof. Dr. Soemantri Brodjonegoro No. 1 Ban-
dar Lampung 35145 Indonesia Telp./Fax: 0721-704625
email: satria_chemistry@unila.ac.id
ABSTRACT
Bioconversion of lignocelluloses material trough microbial enzyme to produce fermentable sugar has been
given serious consideration and continuous research around the world to release renewable of source energy.
Actinomycetes from rice straw waste as source lignocellulolytic enzymes was isolated on Yeast Malt Agar
(YMA) medium supplemented with 50 µg/L nystatine and 25 µg/L streptomycine. Thirty four isolates were
purified and were screened for ligninolytic, cellulolytic, and xylanolytic enzymes production. Production of
Acid Precipitable Polymeric Lignin (APPL) in solid state fermentation used as ability to ligninolytic indicator
beside decreasing of lignin proportion in rice straw by the isolates. Screening employing 1% (w/v) CMC congo
red plate clearance assay, either to 1% (w/v) Birchwood Xylan used to identified celulolytic and xylanolytic
abilty. The results of the screening gave four isolates showed good activity. AcM-1, AcP-1, AcP-7, and AcT-4
showed APPL production (mg/gr substrate) each 27.29, 19.92, 64.50, and 48.56. For the lignin loss (%) pa-
rameter each isolate gave value 11.97, 8.74, 28.29, and 21.30 respectively. Index of cellulolytic and xylanolitic
the isolates amount 1.30 to 3.36 larger than others.
Keywords: Actinomycetes, lignocelluloses, rice straw.
INTRODUCTION solving lignin without destroying it and some of

its subunit, its exact chemical structure is diffi-
Potency of the production of lignocelluloses cult to ascertain. For this reason delignification
biomass from Indonesian agricultural activities is process that must be done before saccarifica-
rise up to 146,700 ton per year (Abdullah 2001). tion to optimum result in bioconversion lignocel-
Lampung province placed as seven higher pro- luloses to fermentable sugar (Taherzadeh &
duction ones with 4.72% amount of 71.45% total Karimi, 2008).
solid waste. Bioconversion of lignocelluloses Chemicals treatment to degrade lignin has
can potentially be increase value-added products some negative effects for environment. Biologi-
including reduction sugar that useful as source of cals treatment is the best choice that needs func-
fermentation industry to produce bio-ethanol, tional microorganism as agent to produce hydro-
improved animal feeds and human nutrients lytic enzymes. Actynomycetes is one of genus
(Howard et al. 2003). Bioethanol production that has been suggested play a role in lignocellu-
that use lignocelluloses source is second genera- lose breakdown, but studies on role of cultiva-
tion in which first generation bioethanol made tion, enzymes produced, and some mechanisms
from sugary and/or starchy resources thus pre- on breakdown still rare done. However, there is
diction has a potential to compete with food pro- a wide range of examples where Streptomyces
vision (Thomsen et al., 2007). and others Actinomycetes have been identified as
Difficulties to degradation of lignocellu- degrading lignin or lignocellulose. Some re-
loses matters are caused by strength of compact searchers had reported that Streptomyces viri-
structure made by their components. Lignocellu- dosporus T7A (Crawford et al., 1983), S. badius
lose consists of lignin, hemicellulose and cellu- (McCarthy et al., 1986), Streptomyces spp. (Ra-
lose that lignin is further linked to both hemicel- machandra et al., 1987; Yamac & Tamer, 2008)
lulose and cellulose forming a physical seal are lignocellulolytic strains.
around the latter two components that is an im- This research has objective to isolation and
penetrable barrier preventing penetration of solu- screening Actinomycetes from rice straw waste
tion and enzymes. Because of difficulty in dis that have capabilities to degrade lignocellulose.

MATERIALS AND METHODS produced APPL per gram substrate was calculat-
ed.
Rice Straw Samples: rice straw sample collected
from harvesting waste at field directly, from Screening of Cellulolytic and Xylanolytic Activ-
three difference location in Lampung province. ities: cellulolytic and xylanolytic screening were
The locations were Pringsewu, Metro, and Ta- done with plating strains on YMA containing
lang Padang. 1% CMC for cellulolytic activity, either to 1%
Birchwood Xylan for xylanolytic activity. After
Isolation and Purification Actinomycetes: acti- an appropriate incubation period at 37°C (1
nomycetes are isolated from fermented rice week), the agar medium was flooded with an
straw supplemented with extracted mineral from aqueous solution of 1% Congo red for 15 min.
soil. 500 grams of dry rice straw blended into 2- The Congo red solution was then poured off, and
3 cm (Mishra et al. 2001) and placed in plastic plates were further treated by flooding with 1M
bag. 500 ml destilled water and 5 grams of ster- NaCl for 15 min. The visualized zones of hy-
ile soil added to enrichment the medium. Fer- drolysis could be stabilized for at least 2 weeks
mentation do for 7 days at room temperature. To by flooding the agar with 1 M HCl, which
isolation Actinomycetes, 100 grams of ferment- changes the dye color to blue and inhibits further
ed rice straw diluted with 500 ml 0,85% NaCl enzyme activity. The plates were scored for a
solution contain 0,05% tween 80 further shack- clear halo surrounding the colony (Teather &
ing for 30 minutes. Dillution series is done for 1- Wood, 1982).
106 with sterile 0,85% NaCl solution. Samples
plating on Yeast Maltosa Agar (YMA) medium Analytical Methods: to determine APPL pro-
supplemented with filtered nystatine (50 µg/L) duced in submerged and solid state cultures, cul-
and streptomycine (25 µg/L). To obtain pure ture filtrate was acidified to pH 1-2 with 12M
isolate single colony were subcultured on other HCl. The resulting precipitate was collected by
YMA medium and the strain maintained as spore centrifugation (16.000 g, 30 min). After discard-
suspensions and hyphal fragments in 15% glyc- ing the supernatant, the pellet was washed twice
erol (v/v) at refrigerator until used. with acidic water, dried at 50°C for 24-48 hr
and, was weighted. From these data, produced
Screening of Ligninolytic Activity: to determine APPL per gram substrate was calculated for in-
which of the strains has higher ligninolytic activ- cubation period.
ity, amount of produced APPL was measured as The lignin content of rice straw was deter-
gravimetric way. 48 hours cultivated of two mined by the modified Klason lignin procedure.
loop strains in Yeast Maltose Broth was used to To lignin assay, 50 mg of lignocelluloses residue
inoculated flask containing 100 ml defined fer- was treated one milliliter concentrated H2SO4 for
mentation medium: 40 mesh rice straw 2.0 gr, 1 hour with occasional mixing. Then, 28 ml of
yeast extract 6.0 gr, Na2HPO4 5.3 gr, KH2PO4 distilled water was added and the suspension
1.98 gr, MgSO4.7H2O 0.2 gr, NaCl 0.2 gr, was autoclaved for 1 hour. After cooling, acid
CaCl2.2H2O 0.05 gr, soil extracted mineral 1 ml, insoluble material was collected by pre-weighed
distilled water 1000 ml, pH 7.1-7.2 (Yamac and filter paper and after several washes with dis-
Tamer 2008). Culture was incubated at room tilled water, it was dried at 70ºC. The lignin
temperature, 100 rpm for 7 days. To determinate containing filter paper was reweighed to deter-
APPL amount produced by strains, samples were mine the amount of acid-insoluble Klason lignin
taken and separated by filtration onto filter paper in residue.
(Whatman no 1) and then was washed with hot
water. Culture supernatant was added 0.1 ml RESULTS AND DISCUSSION
12M HCl per 2 ml culture medium for adjusted
pH to 1-2. The amount of APPL was measured Isolation and Purification Actinomycetes: isola-
by differences weigth of centrifuged tube. Acidi- tion Actinomycetes from rice straw were done
fied supernatant was centrifuged at 16.000 g for by plating dilution series of solid fermented rice
30 minutes use pre-weighted centrifuge tube. straw on YMA medium supplemented with 50
After discharged supernatant, the pellet was µg/L Nystatine and 25 µg/L Streptomycine. The
washed twice with acidic water, dried at 50oC for addition of Nystatine in the isolation media
24-48 hours, and was weighted. From this data, served to minimize fungal contamination, either
Streptomycine to minimize the other bacteria

bacteria except to Actynomycetes (Akaike & lates from Metro gave code Ac-Mx, 10 isolates
Hirata, 1994). The dilution factor that gave op- from Pringsewu gave code Ac-Px, and 9 isolates
timum density growth of Actinomycetes to taken from Talang Padang gave code Ac-Tx. Four
the colony for purification was among 10-3 to 10- isolates which are chosen base on their charac-
5
. For purification interest colony is chosen base teristics to degrade lignocelluloses shown in Fig.
on morphology forming, elevation, margin, and 1.
color. Thirty four isolates obtained each 15 iso-
Fig.1. Pure Actinomycetes isolate growth on YMA medium were isolated from rice straw.
Code of isolate from left to right are AcM-2, AcP-1, AcP-7 and AcT-4.
Screening of Ligninolytic Activity: lignin con- that selected Streptomyces strain their use pro-
tent of rice straw which using for this research duced APPL between 18.30 – 56.00 mg/g sub-
has determined with Klason Lignin procedures, strat and showed has correlated between APPL
the result was 22.80%. In other hand, Wyman et production and lignin degradation. It was deter-
al. (2002) reported 23,40%. mined that several Actinomycetes strains pro-
The APPL production amounts of 8 Acti- duce APPL like product from lignin and they
nomycetes strains for submerged fermentation have similar degradation mechanisms. All of the
medium during 7 days incubation period were lignin degradable strains produce APPL like
determined between 18.94 – 64.50 mg/g sub- product and APPL is not produced when ligno-
strate, the others produced bellow to 1 mg/g sub- celluloses is absent.
strate repectively. AcP-7 produced highest APPL Besides, a correlation was also determined
64.50 mg/g substrat. Beside APPL parameter, between APPL production and lignin loss. Ac-
lignin loss during fermentation determined by cording to these data, APPL production may be
Klason Lignin procedures, that compare between accepted as an evidence for lignin degradation
lignin content of rice straw before and after fer- activity of Actinomycetes. APPL production
mentation. From the data obtained, lignin loss (mg/g substrate) and lignin loss (%) performed
tendency has similar to APPL produce suggest by strains at the end of fermentation is shown in
that APPL production has correlate with lignin Fig. 2.
degradation. Yamac and Tamer (2008) reported

Fig. 2. Strains activity to degradation of lignin from rice straw determined as APPL produc-
tion (mg/g substrate) and lignin loss (%) for 7 days fermentation.
Screening of Cellulolytic and Xylanolytic Activ- between 1.03 to 3.80, and xylanolytic index were
ities: after an appropriate incubation period at 1.05 to 2.92. In Fig. 3. shows clear zone for-
37°C for 1 week, cellulolytic and xylanolytic mation by strain and Table 1. content of index of
index can be determined with Congo Red clear- cellulolytic and xylanolytic of the strain.
ance assay. The use of Congo red as an indica- Meryandini et al. (2008) have isolated cel-
tor for degradation of cellulose and xylan is the lulolytic bacteria from the soil which have vari-
basis for a rapid and sensitive screening test for ous cellulolytic index between 2.2 to 5.5, these
B-Dglucan degradation in an agar medium value have correlated with enzyme activity in
(Teather and Wood 1982). Clear zone diameter cellulose broth medium. Otherwise Zulfarina
(mm) were formed by strain thus compared with (1998), reported that Streptomyces 114I-1 which
diameter of colony (mm) to obtain index value. has higher clear zone among the others on xy-
Twenty strains that have shown ligninolytic ac- lanolityc assay, it has higher enzyme activity,
tivity tested, cellulolytic index were determined too in xylan broth medium.
AcM-2 AcP-1 AcP-7 AcT-4
AcM-2 AcP-1 AcP-7 AcT-4

Fig. 3. Formation of clear zone by four best strain were selected on YMA containing 1%CMC
(above) to selected cellulolytic activity and 1% Birchwood Xylan (below) to selected
xylnolytic activity.
Table 1. Index of cellulolytic and xylanolytic Actynomy- 7, and AcT-4 showed APPL production (mg/g
cetes strain base on comparison of diameter clear zone substrate) each 27.29, 19.92, 64.50, and 48.56.
formed (mm) and diameter of colony (mm).
For the lignin loss (%) parameter each isolate
Code of Index of Index of gave value 11.97, 8.74, 28.29, and 21.30 respec-
Isolat Cellulolytic Xylanolytic tively. Index of cellulolytic and xylanolitic the
AcM-1 3,36 1,82
AcM-2 3,80 2,00 isolates amount 1.30 to 3.36 larger than others.
AcM-3 1,57 1,86
AcM-4 2,00 1,32 ACKNOWLEDGEMENTS
AcP-1 3,60 2,64
AcP-3 1,03 1,35
AcP-4 1,48 1,55 This work was supported by University of
AcP-7 3,33 2,92 Lampung trough DIPA program under the con-
AcT-3 1,53 1,82 tract no 1896/H26/KU/2009 July 1st 2009 for
AcT-4 1,83 1,90 Heri Satria, thank to Iman and Yoanita that have
AcT-10 1,11 1,05
AcT-12 1,14 1,38
helpfully working in the laboratory.
In conclusion, isolation Actinomycetes REFERENCES

from rice straw waste obtained 34 indigenous
Abdullah, K. 2001. Biomass energy potentials and utiliza-
Actinomycetes isolates that had been successful- tion in Indonesia. Indonesian Renewable Energy So-
ly purified which were 4 isolates shown good ciety (IRES).
lignocellulolytic activities. AcM-1, AcP-1, AcP-

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Characteristics of Electricity Production by Metallic and Carbon Anodes
Immersed in an Estuarine Sediment
Daechul Cho1, In-Hyoung Rhee1, Byung-Gi Park1, Ki-Seob Ahn2, Jong-Soo Kim3, Nam-Jun Cho4
and Hyung-Jin Song1
1
Department of Energy & Environmental Engineering, Soonchunhyang University
Asan, 336-745, Korea, e- mail: daechul@sch.ac.kr
2
Department of Health & Environment, BaekSeok Culture University
Cheonan, 330-704, Korea
3
Department of Civil Engineering, Sun Moon University
Asan, 336-708, Korea
4
Department of Applied Chemical Engineering, Korea University of Technology and Education
Cheonan, 330-708, Korea
(Correspondence to: Daechul Cho)
ABSTRACT
One chambered sediment cells with a variety of anodic electrodes were tested for generation of electricity.
Metallic material for anodic electrodes was iron, brass, zinc/iron, and copper, while graphite felt was chosen as a
non-metallic electrode. Also, copper plate wound with a carbon cloth strip was used for anode. Graphite felt was
used as common cathode. The estuarine sediment served as supplier of oxidants or electron-producing microbial
flora, which evoked electrons via fast metal corrosion reactions or a complicated microbial electron transfer
mechanism, respectively. Maximum power density or maximum current density was found in the iron/zinc
electrode cell or iron cell as 6.90 W/m2 or 7.76 A/m2, respectively, while copper and brass electrodes were
followed. Interestingly, copper wrapped with carbon cloth produced better electric performance than copper
only, an increase of 60%, possibly because the cloth not only prevented the accelerating corrosion at the copper
surface by some degrees, but also helped growing electron-emitting microbes on its surface. At anodes
oxidation reduction potential(ORP) was kept to be stationary over time except at the very initial period (mostly
for sediment positioning). The current over voltage was found to be strongly correlated each other in most metal
electrodes meanwhile that was not so in the graphite felt electrode, in which the relatively weaker electricity
generation through microbial action was observed. The pH reduction found in the copper and copper/carbon
electrodes could be a sign of organic acid production due to chemical change in the sediment. The simple
estimation of interfacial, electrical resistances of corrosion oxidation or microbial action implied that a key to
the electricity generation should be in how to control corrosion rate or microbial electron transfer activity.
Keywords: estuarine sediment, metal anode, graphite felt, electricity production, microbial activity.
INTRODUCTION Sediment cell is a kind of MFCs that uses

microbial consortia spread over sediment. The
Worldwidely, energy generation research microbial consortia convert organic or inorganic
using unlimited resources such as sea waters and matter as substrate into other substances with
wastewater is going on under spotlight. some byproducts such as electrons. The
Microbial fuel cell (MFC) is uprising as a new electrons, if a closed circuit is formed, flow
trend because of its enhanced efficiency, for through to generate electricity. The following
example from addition of electron mediators and reactions are a few examples (Wilcock &
the conventional low cost. Currently, sea Kauffman, 1997; Reimers et al., 2001; Lovley,
sediment or lake sediment are being used as 2006):
another resources for MFCs because of their
intrinsically favorable oxidation-reduction C6 H12O6  6H 2O  6CO2  24H   24e (1)
potential along with their abundance. However, 6O2  24H   24e  12H 2O (2)
power obtained from MFCs remains less 10   
mW/m2, which is still weak for most of HS  S ( S )  H  2e or
(3)
applications, therefore more research for HS   4 H 2 O  SO42  9 H   8e 
improvement is still needed (Kim et al., 2002;
Du et al., 2007; Choi et al., 2009). 2Fe 2  3H 2 O  Fe2 O3  6H   2e  (4)

CH 3COOH  2H 2 O  2CO2  8H   8e  Electrodes and the apparatus: The experimental
device was shown in Fig 1. It was a one-
(5) chambered cell, in which the anode was in the
sediment, and the cathode was in saline water
On the other hand, it is well known that two
(2.8% (w/v) NaCl). The cell circuit was
electrodes with different ORP can be a nice cell
completed with 10 ohm resistor. For anodic
combination. It is true particularly for metals, for
material, iron, brass, iron-zinc alloy, copper were
instance:
used, respectively. Macroporous graphite felt
Fe  Fe 2  2e  (6) (5×2×0.5 cm3; CS Tech, ROK) and carbon cloth
1 (4×4×0.1 cm3; SCCG-5N, Nano Best, ROK)
H 2 O  2 H   O2  2e  (7) were also used for the combined electrodes.
2 Graphite felt was used as a common cathode. A
2H 2 O  2e  2OH   H 2

(8) highly conductive epoxy sealant was used in
every connections. Electric conductivity data is
In this case, iron ions can be present as Fe2+, shown in Table 1.
Fe3+, and Fe(OH)2+ in electrolytes.
This work attempts to combine the basic Table 1. Electrical conductivities of potential material for
microbial electron emission and metal corrosion anodes
reaction, resulting in a synergic improvement in
Electrode Material Electrical Conductivity (S/m)
a sediment cell. That is, graphite felt carbon
Iron 1.03 × 107
sheet was chosen for a part of anodic material 1.50 × 107
Brass
that needed good electric conductivity and 1.67 × 107
Zinc
microbial adhesion meanwhile metal pieces were Copper 5.80 × 107
cloaked in the fiber sheet for the rest of anode. Graphite felt 0.87∼2.78
Voltage and current were measured to evaluate Graphite 7.00 × 104
the performance of the cell. Aluminum 3.66 × 107
Silver 6.14 × 107
Carbon 3.7 × 10-1
MATERIALS AND METHODS Platinum 9.26 × 106
Gold 4.4 × 107
Sediment sampling: Sediment for all the Nickel 1.39 × 107
experiments was sampled at Sapgyo Bay in
Chung-nam Province. The sediment was Analysis of electricity generated: Voltage (V)
preserved in a refrigerator when it was not in and current (I) in a cell were measured with the
use. It was exposed to ambient temperature digital multimeter (Agilent, USA). Power (P)
before experiment. The averaged ORP of the and power density (ρ) were calculated using the
sediment was ranged from +145 to +160 mV. following equations:
(9)
(10)
where A is the area of the oxidative electrode.
Resistances in the cell were classified as follows:
at electrode (probably corrosive oxide film or
degree of microbial activity), in electrolyte
(including sediment), and preset resistor.
Equations (11) and (12) denote overall
resistances (Rtot) for metal electrodes and
carbonic electrodes, respectively.
(11)
(12)
Fig 1. A simple representation of the experimental setup

Fig 2. Voltage and current densities over time at different anodic electrodes: A. Fe; B. Cu/Zn; C. Fe/Zn; D. Cu; E. Graph-
ite; F. Cu/Carbon
RESULTS AND DISCUSSION A/m2 and 789 mV at its maximum, respectively.

On the other hand, those for a carbon electrode
Change of voltage or current in sediment cells: were found to be 0.2 A/m2 and 495 mV,
The electrical output was shown in Fig 2 when respectively. Table 2 summarizes maximum
metal or graphite electrodes were used. Metal values of electricity for different electrode
anode-cells exhibited 7 times higher voltage than material.
graphite anode cells, and in particular they Metal electrode cells typically showed a
showed 40 times higher than graphite ones for steeply rising high voltage and then a
current density. For example, for an iron considerably rapid fall-off after three-four days’
electrode current density and voltage were 7.76 stationary period. Metal in the sediment directly

reacted with oxygen or oxidative water to generating more electrons. The cell performance
become metal ion, producing a couple of pointed out that the corrosion tendency precisely
electrons outside. This reaction was also known represented order of ionization power of metals
to accompany formation of oxide film over the (compare Fe, Zn, and their alloys with Cu or its
metal surface which possibly could prevent alloy). The averaged power generation of
further electron production along with the graphite electrode-cells was about 0.1 W/m2,
corrosion reaction. Meanwhile, graphite felt which was lower than that reported in the
electrodes were continually generating electricity literature. Generated power is likely to
even if the microbial film development on the depending on sediment potential (such as ORP)
electrode surface was quite slow for the initial or a subtle balance between microbial consortia
short times as seen in Fig 2E. In this work it took and its substrates.
about 120 h.
1400 Fe Cu/Zn Fe/Zn Cu Cu/C Graphite felt
Table 2. Maximum power and current densities measured 1200

in this work
1000
Maximum Maximum current
Electrode
power (W/m2) density (A/m2) 800
V(mV)
Fe 6.03 7.76 600
Cu/Zn 0.19 0.72
400
Fe/Zn 6.90 5.63
Cu 1.13 1.14 200
Cu/C 2.13 1.93
0.10 0.20 0
Graphite felt
0 5 10 I(mA) 15 20 25
Well operated cells were examined via Fig 4. V-I relationship viewed by scattered data
SEM as shown in Fig 3, and adhered biofilm was
found, which meant that there might be some
microbial contribution to the electron generation
as we postulated before. To minimize the rapid
corrosion (which would short the lifetime of the
cell) and in addition to maintain the microbial
action, a combined electrode of copper, the least
in ionization power, and carbon cloth was
attempted to be in use(compare D and F).
Attachment of carbon on copper surface
evidently enhanced the cell’s lifetime as well as
the electrical performance.
Fig 5. Schematic of three possible mechanisms occurring

at different anodes. The details are found in the text
Fig 3. Bacterial cells were found on graphite electrode
surface. This SEM photo was magnified by 5000 Figs 4 and 5 show V-I relationship and
times plausible electricity generation mechanisms,
respectively. As was pointed in Fig 4 desired
Two possibilities are suggested for this: the direction for better cell performance would be
carbon cloth blocked a part of the copper from the down left end to the up right, which
surface, and retarded the overall corrosion rate; implied that a good cell should produce high
and biofilm on the cloth developed to help voltage and high current. It is true for Fe and

Fe/Zn electrodes, not for Cu and its alloys. Even Change in ORP and pH: ORPs at anodes
if sediment provides enough electrical potential represent the potential for electricity generation
there should be something more that catalyzes through an oxidation. In general, the lower is
oxidative reactions like corrosion or microbial ORP, the higher is the potential, because
metabolism related to electron emission. As anaerobic microorganism would be more active
expected, carbon cloth addition to the metal has in that environment. Fig 7 shows ORPs and pHs
shifted V-I correlation to the right more. In Fig at both of the electrodes. In overall, ORPs slowly
5, a describes a simple corrosion under oxidation dropped with time, for some case it decreased by
environment while B explains how microorga- half probably because a certain changeable
nism adheres on the electrode surface and pores ‘heterogeneity of electrons’ in a local area
inside in order to metabolize adequately for occurred due to the interface between sediment
emitting electrons out. For better effect, C, a and electrode. This phenomena need more
combined electrode, is suggested to take both research in the future. The change of pH, which
advantages: fast corrosive oxidation for possible decreases by a small amount, is explainable
high voltage and prolonged lifetime of cell via because organic matter in the sediment oxidized
microbial metabolism. to form some acids naturally (Kim, 2008).
Power density and electric resistances: Power

densities at different anodes are shown in Fig 6.
The order of power strength was found as
follows: Fe/Zn > Fe > Cu/C > Cu/Zn > Graphite.
The maximum power density was 6.9 W/m2. We
also derived Rtot and its components from each
V-I data. Each component represents sediment,
electrode interface, electrodes, electrolytes, and
an external resistor. Rbac was thousands times
greater than Rox, which meant that the
complexity of microbial action for generating
electrons extremely limited the power generation
unlikely the relatively fast oxidative, metal
corrosion reactions. When one compares
diffusion coefficient on microbially active,
graphite electrode side with that on metal
electrode side, one can simply obtain the ratio of
the coefficients as about 40 (Perry & Chilton,
2008). This implies that diffusion in electrolyte
regions might control by somewhat the main
electrical resistance in the cell.
Fig 7. Time changes of ORPs and pHs at both electrodes

during the experiments
CONCLUSIONS
Electricity generation work using some

local sediment revealed the following: 1). It was
found that 7.76 A/m2 of maximum current
density and 6.9 W/m2 of maximum power
density for Fe and Fe/Zn electrodes, respective-
ly; 2). Electric performance for metal electrodes
was well correlated to ionization power of metal;
3). The interfacial resistance occurred from
oxide film was the lowest at Fe/Zn and the
Fig 6. Power densities calculated at each anodic electride. highest at Cu/Zn. The main reason for extremely
They show maxima about in 1~4 days

high resistance found in the graphite anode-cell Du, Z., Li, H. and Gu, T. 2007. A state of the art review on
might be due to low efficiency in generating microbial fuel cells: a promising technology for
wastewater treatment and bioenergy. Biotechnol. Ad-
electrons through microbial action only; 4). vances. 25: 464-482.
Attachment of carbon cloth on the copper
Wilcock, W.S.D. and Kauffman, P.C. 1997. Development
surface enhanced the voltage and current density of a seawater battery for deep-water applications. J.
by 20 and 60%, respectively, in spite of copper’s Power Sources. 66: 71-75.
poor ionization power.
Reimers, C.E, Tender, L.M, Fertig, S.J. and Wang, W.
2001. Harvesting energy from the marine sedi-
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tric power generation and treatment efficiency of or- keep on going. Microbe. 1 (7): 323-329.
ganic matter on hydraulic retention time in microbial
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25 (1): 159-166. handbook. 5th ed. McGraw-Hill.
Kim, H.J., Park, H.S., Hyun, M.S., Chang, I.S., Kim, M.A. Kim, J.G. 2008. Removal characteristics of organic
and Kim, B.H. 2002. A mediator-less microbial fuel pollutants in microbial fuel cell. Master Thesis.
cell using a metal reducing bacterium, Shewanella pu- Keum-O Tech University.
trefaciens. Enzyme and Microbial Technol. 30: 145-
152.

The Translation Initiation Factor eIF1 Gene is Sensitive to Alkaline Stress
in Leymus chinensis Trin.
Sun Yan-Lin1, Hong Shn-Hae1, Hong Soon-Kwan1, 2
1
Department of Plant Biotechnology, Kangwon National University, Chuncheon, Korea
2
Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Korea,
e-mail: soonkwan@kangwon.ac.kr
(Correspondence to: Hong Soon-Kwan)
ABSTRACT
Adverse environmental conditions limit plant growth and drastically reduce plant productivity. To counter-
act these stresses, plants drive lots of biological actions, such as accumulating several compatible solutes, in-
crease of activities of detoxifying enzymes, and regulating the expression of some related genes. However, the
understanding of the adaptation mechanisms to stresses is far from complete and not understood well. The Hal-
ophyte Leymus chinensis Trin. is a perennial rhizome grass placed in Gramineae that is widely distributed
throughout northern China, Mongolia and Siberia. Due to its high saline tolerance, and high vegetative produc-
tivity, L. chinensis evaluates as a soil-binding plant and a major grass forage product. The initiation of protein
synthesis is a necessary process during the tolerance of stress and a conserved process in eukaryotic cells, and
the activity of the translation initiation factor eIF1 gene is known to be important in this process. Although in
vitro protein synthesis has been found sensitive to NaCl, the adaptation to stress has not been known completely.
In this study, we investigated the LceIF1 expression responses to saline and alkaline stress in this grass. We
found that LceIF1 expression occurred in nearly all parts in plants in normal conditions, and increased with the
treatment of alkaline stress, but not saline stress over 24 h stress. In addition, the wounding stress also stimulat-
ed the high expression of LceIF1. These results suggest that saline stress and alkaline stress are two kinds of
stresses, different from not only physiological characteristics, but stress signal conduction mechanisms.
Keywords: Leymus chinensis, salt stress, translation factor, eIF1 gene
INTRODUCTION that control the amount and timing of these ef-

fector molecules (Hasegawa et al., 2000; Zhu,
Salt stress, mostly caused by high NaCl 2002).
concentrations, is one of the most significant The halophyte Leymus chinensis (Trin.)
abiotic stresses for cultivated plants (Boyer, Tzvel., a perennial rhizome grass placed in tribe
1982). However, in nature, the main salt compo- Gramineae, is a plant species that has adapted to
sitions of soils are NaCl dominating in neutral thrive under natural stress conditions; it is wide-
salt soils and Na2CO3 salt dominating in basic ly distributed throughout northern China, Mon-
salt soils, and it is well-established that saline golia and Siberia (Huang et al., 2004). Because
stress and alkaline stress are significantly differ- of its intrinsic tolerance of highly alkaline-sodic
ent, not only from in their mechanism of action soil conditions (Jin et al., 2005), L. chinensis is
but also from the physiological responses of used as a soil-binding plant to protect soil from
plants (Abd El Samad & Shaddad, 1996). Salt desertification. Due to its high vegetative
stress induces water deficit, ion toxicity, nutrient productivity and protein content, this species is
imbalance and oxidative stress in plants, which used as a major forage product to meet the needs
results in growth inhibition and even plant death of grazing (Shu et al., 2005). However, climate
(Niu et al., 1995; Blumwald, 2000; Zhu, 2001). changes, overgrazing and reclamation of grass-
Some plants have inherent alkaline tolerance, but land result in severe deterioration of the grass-
little is known about their specific adaptive land ecosystem, which significantly affects hu-
mechanisms. This adaptation mechanisms in man life and causes other ecological problems.
some plants, especially some halophytic C4 Thus, a greater understanding of stress tolerance
plants, require the activity of effector molecules mechanisms would be helpful for improving the
that lead to tolerance and also regulatory pro- traits of this grass.
teins

The initiation of protein synthesis is a con- Cloning of eIF1 gene: The cDNA of LceIF1
served process in eukaryotic cells in which the was isolated by PCR using gene specific primers
ribosome is recruited to an mRNA and posi- encompassing the translation start codon and the
tioned at the initiation codon (Rausell et al., translation end codon; eIF1-F1, 5’-AAC AAC
2003). More than 12 initiation factors participate AAG TTC ATG TCT GAT-3’, and eIF1-R1, 5’-
in the assembly of the 80S ribosomal subunit AAC AAC AAG TTC ATG TCT GAT-3’. The
that is competent for protein synthesis. Eukary- PCR product was sequenced.
otic translation initiation factor 1 (eIF1) is first
found as a factor stimulating binding of Met- Sequence alignment and phylogenetic tree con-
tRNA and mRNA to the 40S ribosomal subunits struction: The deduced amino acid sequence of
(Schreier et al., 1997; Trachsel et al., 1997). The the LceIF1 gene were aligned with previously
eIF1A is found to stabilize the binding of Met- reported genes from other species. A phylogenet-
tRNA to 40S ribosomal subunits and promote ic tree was constructed using the DNAMAN soft
mRNA binding. Some studies found that transla- version 5.0 via the observed divergency method.
tion initiation as a physiological target of salt The accession numbers of the sequences used for
toxicity plays an important role on stress toler- the construction of phylogenetic tree are: AteIF1
ance in plants (Rausell et al., 2003). In this work, (BT000649) from Arabidopsis, OseIF1
we clone eIF1 gene in L. chinensis and investi- (AF094774) from rice, TaeIF1 (AF508969) from
gate the characteristics of this gene. Also, we bread wheat, ZmeIF1 (AF034944) from corn,
describe the overexpression of LceIF1 gene un- and HseIF1 (BC008710) from human.
der various salt stresses and provide data demon-
strating that overexpression of LceIF1 gene is RNA extraction and RT-PCR: Total RNA was
sensitive to alkaline stress. isolated from various treated plants using the
Trizol reagent (Invitrogen, Carlsbad, CA, USA)
MATERIALS AND METHODS according to the manufacturer’s instructions. The
NanoPhotometer (IMPLEN, UK) was used to
Plant material and growth conditions: Mature determine RNA concentration and quality. The
seeds of salt-tolerant Leymus chinensis (Trin.) quality of RNA was also assessed by agarose gel
were obtained from natural grassland in Siping, electrophoresis.
Jilin, China; these seeds have been optimized for The β-actin gene was used as control. The
environment by many years of natural evolution. LceIF1 gene was amplified using eIF1-F1, 5’-
Mature seeds were dipped with water in 4°C for AAC AAC AAG TTC ATG TCT GAT-3’, and
3 days, and those sinking to the bottom were eIF1-R1, 5’-AAC AAC AAG TTC ATG TCT
selected and grown in pots containing GAT-3’ primer set. Each PCR reaction contained
clay/vermiculite (3/1, v/v). The cultures were 1.0 μl 10 ppm of each primer and 2 μg RNA
grown one plant per pot in a 25°C greenhouse template in a total volume of 20 μl using Max-
with a 16/8 h (day/night) photoperiod and a rela- ime RT PreMix Kit (Oligo dT Primer, iNtRON
tive humidity between 45% and 70%. Seedlings BIOTECHNOLOGY, Korea), according to the
germinated in these conditions were watered dai- manufacturer’s instructions. PCR amplification
ly with Hoagland nutrient solution (Hoagland & was performed with initial denaturation at 95oC
Amon, 1950). for 5 min followed by 35 cycles of incubation at
95oC for 1 sec, 50oC for 30 sec, and 72oC for 30
Saline and alkaline stress treatments: Six- sec, with final extension at 72oC for 7 min. PCR
week-old plants cultured in the above conditions reactions were performed using the ASTEC
were subjected to saline (200 mM NaCl) or alka- PC808 PCR detection system (ASTEC, PC808,
line stress (100 mM Na2CO3, 200 mM NaHCO3, Japan). PCR products were analyzed by 1% aga-
and 200 mM NaOH) and sampled at various rose gel electrophoresis. Photos were taken and
times after the initiation of treatment (1, 3, 6, 12, analyzed by MultiDoc-It Digital imaging system
24 h) to determine the level of gene expression. (UVP, Cambridge, UK).
Six-week-old plants were watered with different
stress solutions until a final concentration of Statistical analysis: Statistically significant dif-
salts we expected was reached. Three independ- ferences between means were determined by
ent repeats were performed for each experiment. two-way analysis of variance (ANOVA) using
Three plants were analyzed in for each experi- Duncan’s multiple-range test (Duncan 1955).
ment.

A P value of less than 0.05 was considered sig- other species previously reported in NCBI Gen-
nificant. Bank database. DNA alignment was shown in
Fig. 1. We found LceIF1 gene was very similar
RESULTS AND DISCUSSION with eIF1 gene from plant species (with 79%
similarity at least), but not from human (with
Sequence analysis of the LceIF1 gene: The only 60% similarity, Fig. 2). The ORF regions of
LceIF1 cDNA sequence was isolated and com- eIF1 gene in plants have commonly 248 nucleo-
pared with the eIF1 gene among L. chinensis and tides, except in Arabidopsis 242 nucleotides.
Fig. 1. Nucleotide sequence of ORF of eIF1 gene among L. chinensis and other species, including Arabidopsis, rice, born,
bread wheat, and human.

Fig. 2. The phylogenetic tree of eIF1 gene among L. chinensis and other species, including Arabidopsis, rice, born, bread
wheat, and human.
LceIF1 gene expression in leaves treated with The expression showed a slight decrease at 3 h
various stresses: To evaluate the effect of vari- after Na2CO3 stress, but a rapid increase over 6 h
ous stresses on LceIF1 gene expression, 200 mM and reached the maximum of gene expression.
NaCl, 100 mM Na2CO3, 200 mM NaHCO3, and During 24 h stress treatment time, the gene ex-
200 mM NaOH were exogenously applied to 6- pression finally remained the level which was
week-old plants (Fig. 3). Treatment with 200 comparable with the control level. Under 200
mM NaCl did not activate the over-transcription mM NaHCO3, the gene expression had a signifi-
of the LceIF1 gene. A slight increase was found cant increase after 1 h, and decreased back to the
after 1 h of NaCl stress, stayed the expression normal level as the control level until 24 h of
until 24 h the expression began to decrease. stress. At 6 h after stress, the gene expression
Treatment with 100 mM Na2CO3 and 200 mM showed a slight increase. However, another alka-
NaHCO3 activated the LceIF1 gene. Transcrip- line stress, 200 mM NaOH did not active the
tion began 1 h after 100 mM Na2CO3 and 200 LceIF1 gene over these 24 h stress treatment
mM NaHCO3, although the gene expression time, although had a slight increase trend up to 3
caused by Na2CO3 was not significantly high. h, the change was not significant.

Fig. 3. The expression of LceIF1 gene and relative expression (%) in stressed leaves with saline stress, 200 mM NaCl and
alkaline stress, 100 mM Na2CO3, 200 mM NaHCO3, and 200 mM NaOH. The β-actin gene is shown as control. Values are
the mean of three repeated experiments. Means followed by the same letter in the same series are not significantly different
at P < 0.05 according to two-way ANOVA using Duncan’s multiple-range test.
Sensitivity of the LceIF1 gene to pH value: Un- longing to light alkalescent stress, which could
der alkaline stress, 100 mM Na2CO3 and 200 active the LceIF1 gene expression; the pH value
mM NaHCO3, the LceIF1 gene showed overex- of 200 mM NaCl used in present work was 5.51,
pression, but not under saline stress, 200 mM which did not active the gene expression, sug-
NaCl. We quantified the Na+ concentration as gesting that the LceIF1 gene expression was re-
200 mM in various stresses, indicating that the lated with pH value of stress treatment solution.
effect of saline and alkaline stress was not relat- However, it was interesting that 200 mM NaOH
ed with the Na+ influence. So the responses to alkaline stress with pH value of 12.75, did not
saline stress and alkaline stress might be related active the gene expression, which made us have
with pH value of treatment solution (Table 1). Of the idea that the LceIF1 gene was only sensitive
them, the pH value of 100 mM Na2CO3 and 200 to light alkalescent stress, but not strong al-
mM NaHCO3 were between 8.31 and 11.99, be- kalescent stress.
Table 1. The pH value of various stress treatment solution.
Values followed by the same letter in the same the gene expression had relationship with pH
volume are not significantly different at P < 0.05 value of stress treatment solution.
according to two-way ANOVA using Duncan’s
multiple-range test. ACKNOWLEDGMENTS
CONCLUSIONS This work was supported by Nutraceutical

Bio Brain Korea 21 Project Group. Following
The LceIF1 gene was isolated from leaves are results of a study on the “Human Resource
of L. chinensis and had high homology with Development Center for Economic Region
eIF1 genes from other plant species. The LceIF1 Leading Industry” Project, supported by the
gene showed high expression under alkaline Ministry of Education, Science & Technology
stress, but not saline stress. We also found that (MEST) and the National Research Foundation
of Korea (NRF).

REFERENCES Niu, X., R. A. Bressan, P. M. Hasegawa & J. M. Pardo.
1995. Ion homeostasis in NaCl stress environments.
Plant Physiol. 109: 735-742.
Abd El Samad, H. M. & M. A. K. Shaddad. 1996. Compar-
ative effect of sodium carbonate, sodium sulphate, Rausell, A., R. Kanhonou, L. Yenush, R. Serrano & R. Ros.
and sodium chloride on the growth and related meta- 2003. The translation initiation factor eIF1A is an im-
bolic activities of pea plants. J. Plant Nutr. 19: 717- portant determinant in the tolerance to NaCl stress in
728. yeast and plants. Plant J. 34: 257-267.
Blumwald E. 2000. Sodium transport and salt tolerance in Schreier, M. H., B. Erni & T. Staehelin. 1997. Initiation of
plants. Curre. Opin. Cell Biol. 12: 431-434. mammalian protein synthesis. I. Purification and
characterization of seven initiation factors. J. Mol.
Boyer, J.S. 1982. Plant productivity and environment. Sci.
Biol. 116: 727-753.
218: 443-448.
Shu, Q.Y., G. S. Liu, S. X. Xu, X. F. Li & H. J. Li. 2005.
Hasegawa, P. M., R. A. Bressan, J. K. Zhu & H. J. Bonnert.
Genetic transformation of Leymus chinensis with the
2000. Plant cellular and molecular responses to high
PAT gene through microprojectile bombardment to
salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol.
improve resistance to the herbicide Basta. Plant Cell
51: 463-399.
Rep. 24: 36-44.
Hoagland, D. R. & D. I. Arnon. 1950. The water-culture
Trachsel, H., B. Erni, M. H. Schreier & T. Staehelin. 1997.
method for growing plants without soil. Calif. Agric.
Initiation of mammalian protein synthesis. II. The as-
Exp. Stn. Circ. 347: 1-32.
sembly of the initiation complex with purified initia-
Huang Z., Zhu J., Mu X., Lin J., Pollen dispersion, pollen tion factoros. J. Mol. Biol. 116: 755-768.
viability and pistil receptivity in Leymus chinensis,
Zhu, J. K. 2001. Plant salt tolerance. Trends Plant Sci. 6:
Ann. Bot. 93 (2004) 295-301.
66-71.
Jin, H., P. Plaha, J. Y. Park, C. P. Hong, I. S. Lee, Z. H.
Zhu, J. K. 2002. Salt and drought stress signal transduction
Yang, G. B. Jiang, S. S. Kwak, S. K. Liu, J. S. Lee, Y
in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol.
A. Kim & Y.P. Lim. 2006. Comparative EST profiles
53: 247-273.
of leaf and root of Leymus chinensis, a xerophilous
grass adapted to high pH sodic soil. Plant Sci. 170:
1081-1086.

The Relationship with Translation Initiation Factor eIF1 Gene Expression
and Na+ Stress
Sun Yanlin1, Shin Young-Boum1, Hong Soon-Kwan1, 2
1
Department of Plant Biotechnology, Kangwon National University, Chuncheon, Korea
2
ABSTRACT
Adverse environmental conditions limit plant growth and drastically reduce plant productivity. To counter-
act these stresses, plants drive lots of biological actions, such as accumulating several compatible solutes, in-
crease of activities of detoxifying enzymes, and regulating the expression of some related genes. However, the
understanding of the adaptation mechanisms to stresses is far from complete and not understood well. The Hal-
ophyte Leymus chinensis Trin. is a perennial rhizome grass placed in Gramineae that is widely distributed
throughout northern China, Mongolia and Siberia. Due to its high saline tolerance, and high vegetative produc-
tivity, L. chinensis evaluates as a soil-binding plant and a major grass forage product. The initiation of protein
synthesis is a necessary process during the tolerance of stress and a conserved process in eukaryotic cells, and
the activity of the translation initiation factor eIF1 gene is known to be important in this process. Although in
vitro protein synthesis has been found sensitive to NaCl, the adaptation to stress has not been known completely.
In this study, we investigated the LceIF1 expression responses to Na+ and K+ salt treatment in this grass. We
found that LceIF1 expressed in leaves without stress, and overexpressed under Na + alkali treatment, but not Na+
saline treatment. However, under K+ treatment, LceIF1 overepression occurred either alkali or saline treatment.
These results suggest that LceIF1 gene is related with Na+ stress. This work provides good evidence implicating
LceIF1 as a physiological target of stress toxicity and a new approach to study Na + metabolic mechanisms in
plants.
Keywords: Leymus chinensis, salt stress, translation factor, eIF1 gene
INTRODUCTION lead to tolerance and also regulatory proteins

that control the amount and timing of these ef-
Salt stress, mostly caused by high NaCl fector molecules (Hasegawa et al., 2000; Zhu,
concentrations, is one of the most significant 2002).
abiotic stresses for cultivated plants (Boyer, The halophyte Leymus chinensis (Trin.)
1982). However, in nature, the main salt compo- Tzvel., a perennial rhizome grass placed in tribe
sitions of soils are NaCl dominating in neutral Gramineae, is a plant species that has adapted to
salt soils and Na2CO3 salt dominating in basic thrive under natural stress conditions; it is wide-
salt soils, and it is well-established that saline ly distributed throughout northern China, Mon-
stress and alkaline stress are significantly differ- golia and Siberia (Huang et al., 2004). Because
ent, not only from in their mechanism of action of its intrinsic tolerance of highly alkaline-sodic
but also from the physiological responses of soil conditions (Jin et al., 2005), L. chinensis is
plants (Abd El Samad & Shaddad, 1996). Salt used as a soil-binding plant to protect soil from
stress induces water deficit, ion toxicity, nutrient desertification. Due to its high vegetative
imbalance and oxidative stress in plants, which productivity and protein content, this species is
results in growth inhibition and even plant death used as a major forage product to meet the needs
(Niu et al., 1995; Blumwald, 2000; Zhu, 2001). of grazing (Shu et al., 2005). However, climate
Some plants have inherent alkaline tolerance, but changes, overgrazing and reclamation of grass-
little is known about their specific adaptive land result in severe deterioration of the grass-
mechanisms. This adaptation mechanisms in land ecosystem, which significantly affects hu-
some plants, especially some halophytic C4 man life and causes other ecological problems.
plants, require the activity of effector molecules
that

Thus, a greater understanding of stress tolerance old plants were watered with different stress so-
mechanisms would be helpful for improving the lutions until a final concentration of salts we ex-
traits of this grass. pected was reached. Three independent repeats
The initiation of protein synthesis is a con- were performed for each experiment. Three
served process in eukaryotic cells in which the plants were analyzed in for each experiment.
ribosome is recruited to an mRNA and posi-
tioned at the initiation codon (Rausell et al., RNA extraction and RT-PCR: Total RNA was
2003). More than 12 initiation factors participate isolated from various treated plants using the
in the assembly of the 80S ribosomal subunit Trizol reagent (Invitrogen, Carlsbad, CA, USA)
that is competent for protein synthesis. Eukary- according to the manufacturer’s instructions. The
otic translation initiation factor 1 (eIF1) is first NanoPhotometer (IMPLEN, UK) was used to
found as a factor stimulating binding of Met- determine RNA concentration and quality. The
tRNA and mRNA to the 40S ribosomal subunits quality of RNA was also assessed by agarose gel
(Schreier et al., 1997; Trachsel et al., 1997). The electrophoresis.
eIF1A is found to stabilize the binding of Met- Full-length cDNA of the LceIF1 gene was
tRNA to 40S ribosomal subunits and promote isolated using primer sets in previous work:
mRNA binding. Some studies found that transla- eIF1-F1, 5’-AAC AAC AAG TTC ATG TCT
tion initiation as a physiological target of salt GAT-3’, and eIF1-R1, 5’-AAC AAC AAG TTC
toxicity plays an important role on stress toler- ATG TCT GAT-3’. In present work, the LceIF1
ance in plants (Rausell et al., 2003). We cloned gene was amplified using this primer sets also.
the LceIF1 gene in L. chinensis in previous work The β-actin gene was used as control. Each PCR
and drew the conclusion that the LceIF1 gene reaction contained 1.0 μl 10 ppm of each primer
was sensitive to alkaline stress and the gene ex- and 2 μg RNA template in a total volume of 20
pression was related with pH value. In this work, μl using Maxime RT PreMix Kit (Oligo dT Pri-
investigate the characteristics of this gene further mer, iNtRON BIOTECHNOLOGY, Korea), ac-
and describe the overexpression of LceIF1 gene cording to the manufacturer’s instructions. PCR
also has relationship with Na+ stress under vari- amplification was performed with initial dena-
ous salt stresses. turation at 95oC for 5 min followed by 35 cycles
of incubation at 95oC for 1 sec, 50oC for 30 sec,
MATERIALS AND METHODS and 72oC for 30 sec, with final extension at 72oC
for 7 min. PCR reactions were performed using
Plant material and growth conditions: Mature the ASTEC PC808 PCR detection system (AS-
seeds of salt-tolerant Leymus chinensis (Trin.) TEC, PC808, Japan). PCR products were ana-
were obtained from natural grassland in Siping, lyzed by 1% agarose gel electrophoresis. Photos
Jilin, China; these seeds have been optimized for were taken and analyzed by MultiDoc-It Digital
environment by many years of natural evolution. imaging system (UVP, Cambridge, UK).
Mature seeds were dipped with water in 4°C for
3 days, and those sinking to the bottom were Statistical analysis: Statistically significant dif-
selected and grown in pots containing ferences between means were determined by
clay/vermiculite (3/1, v/v). The cultures were two-way analysis of variance (ANOVA) using
grown one plant per pot in a 25°C greenhouse Duncan’s multiple-range test (Duncan 1955). A
with a 16/8 h (day/night) photoperiod and a rela- P value of less than 0.05 was considered signifi-
tive humidity between 45% and 70%. Seedlings cant.
germinated in these conditions were watered dai-
ly with Hoagland nutrient solution (Hoagland & RESULTS AND DISCUSSION
Amon, 1950).
LceIF1 gene expression in leaves treated with
Saline and alkaline stress treatments: Six- various K+ stresses: To evaluate the effect of
week-old salt-tolerant and salt-sensitive plants various stresses on LceIF1 gene expression, 200
cultured in the above conditions were subjected mM KCl, 100 mM K2CO3, and 200 mM KOH
to saline (200 mM KCl) or alkaline stress (100 were exogenously applied to 6-week-old plants
mM K2CO3, 200 mM KOH, and 100 mM (Fig. 1). Treatment with K+ stressl did not acti-
Na2CO3) and sampled at various times after the vate the over-transcription of the LceIF1 gene.
initiation of treatment (1, 3, 6, 12, 24 h) to de- An increase was found at 6 h of KCl stress, but
termine the level of gene expression. Six-week- not significantly. Treatment with 100 mM K2CO3
214
Jakarta, 18-20 February 2010
stress and 200 mM KOH also did not induce the LceIF1 gene expression in leaves treated with
high expression of LceIF1 gene, with the contin- 100 mM Na2CO3: To examine whether the gene
uous slightly decrease over 24 h stress treatment expression was related with Na+ stress, 100 mM
time. Based on these results, the LceIF1 gene Na2CO3 was exogenously applied to plants (Fig.
could not be active by K+ stress, neither saline 2). Treatment with 100 mM Na2CO3 could active
nor alkaline stress. However, in previous study, the LceIF1 gene expression. Transcription began
we had investigated that the LceIF1 gene expres- 1 h after stress, and had a rapid decrease up to 3
sion was related with pH value, but it was h. After 6 h of stress, the gene expression
strange that it was not seen in present work. So showed a slight increase until 24 h of stress, but
the LceIF1 gene expression might be influenced not remarkably higher than the control level un-
by not only pH value but Na+ stress. der non-stress.
Fig. 2. The expression of LceIF1 gene and relative expres-

sion (%) in stressed leaves with Na+ stress, 100 mM
Na2CO3. The β-actin gene is shown as control. Values are
the mean of three repeated experiments. Means followed by
the same letter in the same series are not significantly dif-
ferent at P < 0.05 according to two-way ANOVA using
Duncan’s multiple-range test.
Relationship with LceIF1 gene expression and

Na+ stress: In previous work, the LceIF1 gene
expression showed sensitivity to alkaline stress,
with no overexpression under saline stress, but
over expression under alkaline stress after 1 h.
However, in present work, under K+ stress with
different negative ions the gene expression
showed stable levels, although the pH value var-
ied from the K+ stress treatment solution, be-
tween 6.37 and 13.84 (Table 1). The results sug-
gested that at least under K+ stress, the gene ex-
pression was not regulated by alkaline stress.
Compared with both carbonate stress, K2CO3
and Na2CO3 stress, which were differed from
Fig. 1. The expression of LceIF1 gene and relative expres- positive ions, K+ or Na+, the LceIF1 gene ex-
sion (%) in stressed leaves with K+ stress, 200 mM KCl
and alkaline stress, 100 mM K2CO3, and 200 mM KOH.
pression showed very different responses (Fig. 1
The β-actin gene is shown as control. Values are the mean and 2). Between them, the pH value of Na2CO3
of three repeated experiments. Means followed by the same (11.99) was slightly higher than that of K2CO3
letter in the same series are not significantly different at P < (10.8). However relatively higher pH value
0.05 according to two-way ANOVA using Duncan’s multi-
stress of 200 mM KOH did not active the gene
ple-range test.
expression, suggesting that the change was not
caused by pH value.

In contrast, it is suggested that the gene expres- Blumwald E. 2000. Sodium transport and salt tolerance in
sion could be regulated by Na+ stress. Combined plants. Curre. Opin. Cell Biol. 12: 431-434.
with the previous results, we drew the conclu- Boyer, J.S. 1982. Plant productivity and environment. Sci.
sion that the LceIF1 gene expression was not 218: 443-448.
only regulated by alkaline stress, but also by Na+ Hasegawa, P. M., R. A. Bressan, J. K. Zhu & H. J. Bonnert.
stress. 2000. Plant cellular and molecular responses to high
salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol.
Table 1. The pH value of various stress treatment solution. 51: 463-399.
Hoagland, D. R. & D. I. Arnon. 1950. The water-culture
method for growing plants without soil. Calif. Agric.
Exp. Stn. Circ. 347: 1-32.
Huang Z., Zhu J., Mu X., Lin J., Pollen dispersion, pollen
viability and pistil receptivity in Leymus chinensis,
Ann. Bot. 93 (2004) 295-301.
Jin, H., P. Plaha, J. Y. Park, C. P. Hong, I. S. Lee, Z. H.
Yang, G. B. Jiang, S. S. Kwak, S. K. Liu, J. S. Lee, Y
A. Kim & Y.P. Lim. 2006. Comparative EST profiles
of leaf and root of Leymus chinensis, a xerophilous
Values followed by the same letter in the same grass adapted to high pH sodic soil. Plant Sci. 170:
1081-1086.
series are not significantly different at P < 0.05
according to two-way ANOVA using Duncan’s Niu, X., R. A. Bressan, P. M. Hasegawa & J. M. Pardo.
multiple-range test. 1995. Ion homeostasis in NaCl stress environments.
Plant Physiol. 109: 735-742.
CONCLUSIONS Rausell, A., R. Kanhonou, L. Yenush, R. Serrano & R. Ros.

2003. The translation initiation factor eIF1A is an im-
portant determinant in the tolerance to NaCl stress in
The LceIF1 gene did not show high expres- yeast and plants. Plant J. 34: 257-267.
sion under various K+ stresses, but showed high
Schreier, M. H., B. Erni & T. Staehelin. 1997. Initiation of
expression under Na+ stress, suggesting that the mammalian protein synthesis. I. Purification and
gene expression was related with Na+ stress. We characterization of seven initiation factors. J. Mol.
also found that the gene expression had no rela- Biol. 116: 727-753.
tionship with pH value under K+ stress, but not Shu, Q.Y., G. S. Liu, S. X. Xu, X. F. Li & H. J. Li. 2005.
under Na+ stress, suggesting that the gene ex- Genetic transformation of Leymus chinensis with the
pression might be regulated by Na+ stress and the PAT gene through microprojectile bombardment to
pH value. improve resistance to the herbicide Basta. Plant Cell
Rep. 24: 36-44.
ACKNOWLEDGMENTS Trachsel, H., B. Erni, M. H. Schreier & T. Staehelin. 1997.

Initiation of mammalian protein synthesis. II. The as-
sembly of the initiation complex with purified initia-
This work was supported by Nutraceutical tion factoros. J. Mol. Biol. 116: 755-768.
Bio Brain Korea 21 Project Group. Zhu, J. K. 2001. Plant salt tolerance. Trends Plant Sci. 6:
66-71.
REFERENCES Zhu, J. K. 2002. Salt and drought stress signal transduction
in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol.
Abd El Samad, H. M. & M. A. K. Shaddad. 1996. Compar- 53: 247-273.
ative effect of sodium carbonate, sodium sulphate,
and sodium chloride on the growth and related meta-
bolic activities of pea plants. J. Plant Nutr. 19: 717-
728.
216
Jakarta, 18-20 February 2010
Transformation of Leymus chinensis Trin. by Agrobacterium tumefaciens
Infection of in vitro Cultured Callus
Sun Yanlin1, Shin Young-Boum1, Hong Soon-Kwan1, 2
1
Department of Plant Biotechnology, Kangwon National University, Chuncheon, Korea’
2
ABSTRACT
The Halophyte Leymus chinensis Trin. is a perennial rhizome grass placed in Gramineae that is widely dis-
tributed throughout northern China, Mongolia and Siberia. Due to its high saline tolerance, L. chinensis evalu-
ates as a soil-binding plant. Most grass crops display poor regeneration capacity of in vitro cultured callus, im-
plying that technologies such as transformation are often restricted to grass crops with high transformation fre-
quencies. In our previous study, we established a highly efficient regeneration system from somatic embryogen-
esis using mature seeds as explants. In the present study, we used this plant regeneration system for transfor-
mation. Agrobacterium tumefaciens strain EHA105, carrying the vector pCAMBIA2300 harboring a kanamycin
resistance gene as selection genes, and the 2Cys-peroxiredoxin (C2C-Prx) gene as reporter genes, was used for
transformation in wild type of L. chinensis. The results suggested that the growth status of cultured callus used
for infection was an important factor, and OD600 of the Agrobacterium solution used for infection was believed
to be the reason resulting in low frequency of transformation. In our work, we used 1 month-old callus with 7
days refresh culture to use for infection, and the infection solution was OD 600 of around 0.3-0.4 with 100 μM
acetosyringone. Successful transformation was obtained from the transformation system by GUS detection and
C2C-Prx gene insert detection. We conclude that cultured callus can be used as explants for transformation of L.
chinensis, and this technique allows for the rapid and direct generation of high quality transgenic plants.
Keywords: Leymus chinensis, 2Cys-peroxiredoxin, Agrobacterium-mediated transformation, oxidative stress,

stress tolerance.
INTRODUCTION hydrogen peroxide (Chae et al., 1994). Prxs were

originally divided into two categories, the 1-Cys
The halophyte Leymus chinensis (Trin.) and 2-Cys Prxs, based on the number of cyste-
Tzvel., a perennial rhizome grass placed in tribe inyl residues (Chae et al., 1994). Due to different
Gramineae, is a plant species that has adapted to structural and mechanistic data, 2-Cys Prxs were
thrive under natural stress conditions; it is wide- further divided into typical and atypical 2-Cys
ly distributed throughout northern China, Mon- Prxs, of which, the typical 2-Cys Prxs are the
golia and Siberia (Huang et al., 2004). Because largest class of Prxs and are identified by the
of its intrinsic tolerance of highly alkaline-sodic conservation of their two redox-actie cysteines,
soil conditions (Jin et al., 2005), L. chinensis is the peroxidatic cysteine (generally near residue
used as a soil-binding plant to protect soil from 50) and the resolving cysteine (near residue 170)
desertification. Due to its high vegetative (Hofmann et al., 2002). Typical 2-Cys Prxs
productivity and protein content, this species is which could be oxidized directly by hydrogen
used as a major forage product to meet the needs peroxide, have found as a protector against the
of grazing (Shu et al., 2005). However, climate toxic effects of hydrogen peroxide (Wen & Van
changes, overgrazing and reclamation of grass- Etten, 1997; Veal et al., 2004; Jang et al., 2004;
land result in severe deterioration of the grass- Moon et al., 2005). And other genetic studies
land ecosystem, which significantly affects hu- also have revealed that typical 2-Cys Prxs play
man life and causes other ecological problems. important roles in protecting against DNA dam-
Thus, cultivated species of this grass with image, oxidative stress, and cancer (Huang et al.,
proved traits and strong stress tolerance becomes 2003; Lee et al., 2003; Neumann et al., 2003).
urgent demands. The transgenic technique has become a
Peroxiredoxins (Prxs) are a group of ubiqui- common and convenient method for improving
tous peroxidase enzymes in which redox-active the traits of plants (Hiei et al., 1997, Akutsu et al.,
cysteine residues participate in the reduction of 2004). For transformation of L. chinensis, mi-

croprojectile bombardment transformation with netin and 2.0 g/L casamino acid. The cultures
the phosphinothricin acetyltransferase (PAT) were incubated at 28℃ under low light condi-
gene has successfully performed (Shu et al., tions (25 μE m-2 s-1) with a 16/8 h (light/dark)
2005), and later, Agrobacterium-mediated trans- photoperiod for 1 week, and then maintained
formation with wheat late embryogenesis abun- under high light conditions (70 μE m-2 s-1) with
dant (LEA) gene has also successfully performed the same photoperiod. Regenerated shoots were
(Wang et al., 2009). In our previous work, an transferred onto rooting medium for root devel-
efficient regeneration system via in vitro somatic opment until grown to 3 cm in length. Rooting
embryogenesis from mature seeds and leaf base medium contained half-strength MS basal medi-
segments of L. chinensis was established (Sun & um with 30.0 g/L sucrose and 4.0 g/L gelrite.
Hong, 2009). To improve the traits of this grass The pH was adjusted to 5.8 prior to autoclaving.
via over-expressing of 2-Cys Prx gene to im- Cultures for rooting were incubated in 28oC un-
prove the oxidative stress tolerance in L. chinen- der high light conditions (70 μE m-2 s-1) and a
sis, we transferred a sweet potato 2-Cys Prx un- 16/8 h (light/dark) photoperiod. Well-rooted
der the control of CaMV 35S promoter into L. plantlets were removed from culture medium,
chinensis via Agrobacterium-mediated transfor- rinsed with water to remove the media, and then
mation. Our results showed an efficient trans- transplanted into a mixture of sterilized soil and
formation system based on a highly-frequency vermiculite under humid conditions in a growth
tissue culture and plant regeneration system was room for 3 weeks prior to being transplanted into
established and the transgenic plants with sweet greenhouse.
potato 2-Cys Prx gene had enhanced oxidative
stress tolerance in L. chinensis. Bacterial stains and plasmids: The Agrobacte-
rium tumefaciens stain EHA105 was used in this
MATERIALS AND METHODS study. The stain EHA105 harbors plasmid
pCAMBIA2300, containing 2-Cys Prx and NptII
Plant materials: Mature dry seeds of L. chinen- gene under the control of the CaMV35S promot-
sis, used as the source of mature embryos, were er.
collected at the grassland in Siping, Jilin prov-
ince, China, with storage at 4oC. All seeds were Agrobacterium-mediated transformation: The
de-husked and surface-sterilized with 70% etha- Agrobacterium tumefaciens stain EHA105 with
nol 1 min, and then 5% sodium hypochlorite 20 the binary vector pCAMBIA2300 was grown in
min. The sterilized seeds were rinsed five times YEP liquid medium supplemented with 20 mg/L
with sterile water and then placed on callus in- rifampicin (RIF) and 50 mg/L kanamycin (KM).
duction medium; meanwhile, surface-sterilized The culture was grown at 28oC for 12 h with
seeds were germinated on Murashige and Skoog continuous shaking (225 rpm) to an OD600 of
(MS) basal medium (Murashige & Skoog, 1962) 0.4~1.0. Centrifuge and deposit the solution at
for supplying explants for callus induction from 5000 rpm for 10 min and suspend the deposition
leaves. The cultured seeds germinated within 10 with the same volume of MMA medium (MMA
days. The leaves were cut into 1 cm segments medium was composed with MS basal salt, MES
until it reached to 7 ~ 8 cm in length. 10 mol/L pH 5.6, acetosyringone (AS) 100
μmol/L, sucrose 20 g/L) for infection. Embryo-
Tissue culture conditions: The callus induction genic calli pre-cultured in callus induction medi-
medium M2 contained MS basal medium sup- um M2 for 7 d were immersed in A. tumefaciens
plemented with 5.0 g/L L-glutamic acid, 2.0 EHA105/pCAMBIA2300 suspension for 10~60
mg/L 2,4-D, 30.0 g/L sucrose and 4.0 g/L gelrite min. Excess bacteria were removed after the in-
for solidification. The pH was adjusted to 5.8 cubation and the infected calli were drained on
prior to the addition of gelrite. Mature seeds and filter paper and co-cultivated on callus induction
leaf segments were incubated for callus induc- medium M2 with 100 μmol/L AS. Co-cultivation
tion on the media at 28℃ in darkness with 60% was carried out for 3 d at 28oC in the dark. After
humidity. The calli were transferred onto fresh co-cultivation, transgenic calli were washed with
subculture medium with biweekly subculture. sterile water containing 250 mg/L cefotaxime
The embryogenic calli were transferred onto (CE) for 5 times and drained on filter paper for
regeneration medium ZN0.2 containing MS ba- removing excess water. Then transgenic calli
sal medium with 0.2 mg/L NAA, 2.0 mg/L Ki- were transferred onto M2 medium containing 20
mg/L RIF, 50 mg/L KM and 250 mg/L CE for

selection with once biweekly subculture. Plant
regeneration was carried out on ZN0.2 medium RESULTS AND DISCUSSION
with containing 20 mg/L RIF, 50 mg/L KM and
50 mg/L CE for selection. Rooting was carried Transformation and regeneration of transgenic
out on half strength MS basal medium with 50 plants: Type 1 callus, yellow, nodule-like and
mg/L KM. Transgenic shoots were grown as de- compact callus which was induced from mature
scribed above. seeds and leaf base segments of L. chinensis in
our previous study (Sun & Hong, 2009) was
DNA extraction and gene insertion assay: Ge- used in the present work. The Agrobacterium
nomic DNA was extracted from non-transgenic tumefaciens stain EHA105 with the binary vec-
and 2 transgenic plants using SDS method. PCR tor pCAMBIA2300 was using as infection solu-
analysis for the marker gene (nptII) was carried tion. As transformation efficiency was influ-
out following the method above: 1.0 μl 10 ppm enced by many factors, such as callus growth
of each primer and 10 ng DNA template in a to- status (callus age), infection solution concentra-
tal volume of 20 μl PCR action; 95oC for 5 min tion (OD600), infection time, co-culture period,
followed by 35 cycles of incubation at 95oC for 1 and the addition of AS, we used two age phase
sec, 50oC for 30 sec, and 72oC for 30 sec, with callus, two different concentration of infection
final extension at 72oC for 7 min. The nptII gene solution for improving transformation system.
was amplified using primer sets: npt2-f1, 5’- After infection with Agrobacterium solution with
GAG GCT ATT CGG CTA TGA CTG-3’, and various infection times, callus was transferred on
npt2-r1, 5’-ATC GGG AGC GGC GAT ACC co-culture medium with MS medium containing
GTA-3’. To detect the presence of the objective 2.0 mg/L 2,4-D, 5.0 mg/L glutamic acid, and 100
gene (2-Cys Prx), one gene-specific primer sets μM AS for 3 d co-culture. And then infected cal-
were designed with the following sequences: lus was transferred on callus induction medium
prx-f1: 5’-TCT AGA ATG GCG TCT GTT with selection, 50 mg/L KM, 20 mg/L RIF, and
GCT-3’ and prx-r1: 5’-GAG CTC CTA AAT 250 mg/L CE. In this experiment, we set the
AGC TGA GAA-3’, using the same PCR pro- subculture interval as 20 d and only no subcul-
gram above. PCR reactions were performed us- ture on callus induction with selection stage.
ing the ASTEC PC808 PCR detection system After 20 d selection culture on callus induc-
(ASTEC, PC808, Japan). PCR products were tion medium, callus was transferred on plant re-
analyzed by 1% agarose gel electrophoresis. generation with selection as reported above. At
Photos were taken and analyzed by MultiDoc-It the first 20 d on plant regeneration medium, in-
Digital imaging system (UVP, Cambridge, UK). fected callus showed purple spots or shoots, and
few of them green spots or shoots. After once
Southern blot hybridization analysis: Total subculture, the regenerated shoots grew up and
RNA was extracted from non-transgenic and 2 some of purple shoots began to turn green or
transgenic plants using Trizol reagent (Invitro- some green shoots began to grow. Until the
gen, Carlsbad, CA, USA) according to the manu- leaves reached 3 cm in length, we transferred on
facturer’s instructions. The NanoPhotometer rooting medium with 50 mg/L selection. Nearly
(IMPLEN, UK) was used to determine RNA 100% regenerated shoots were induced roots
concentration and quality. Twenty μg total RNA well after 10 d when transferred on rooting me-
was denatured with formaldehyde and forma- dium with selection. Well-rooted kanamycin-
mide, fractionated in a 1% agarose gel using resistant plants were transferred into pots con-
MOPS buffer and then blotted to a positively taining a mixture of sterilized soil and vermicu-
charged nylon membrane. The membranes were lite (3:1) in the greenhouse.
hybridized and detected using the DIG detection
kit (Roche Co. Germany) according to the manu- The effect of infection time with A. tumefaciens
facturer’s instructions. on transformation frequency: To test for the
effect of infection time with Agrobacterium in-
Statistical analysis: Three identical and inde- fection solution with OD600 of 0.8~1.0, older
pendent experiments were performed for each than 3-month-old callus were used for transfor-
experiment. In each replicate 85~100 mature mation under various infection time (60, 30, 25,
seeds, leaf segments or calli were used. The data 20, 15, and 10 min, Table 1). The survival prob-
presented here were the average of these experi- ability after the first selection in callus induction
ments. medium was significantly influenced by various

infection times. After 60 min infection time, cal- rate. During these transformation systems, only 2
lus used for transformation had the lowest sur- transgenic plants were obtained from the 20 min
vival probability, remarkably lower than that infection time system. And relatively high fre-
after 20 min and less than 20 min. But the quency of albino transformation was obtained
growth rates of transgenic callus through various from the 60 min infection time system, by reason
infection time transformation systems, were sim- of old age callus or long infection time or high
ilar with each other, with about three-fold growth OD600 of infection solution.
Table 1. Growth rate and transformation frequency according to various infection times (60, 30, 25, 20, 15, and 10 min).
a
Callus used for transformation in this experiment is older than 3-month-old callus. The OD600 of Agrobacterium solution
used for infection arranges between 0.8 and 1.0.
Values are the mean of three repeated experiments. Means followed by the same letter in the same series are not significantly
different at P < 0.05 according to two-way ANOVA using Duncan’s multiple-range test.
The effect of callus age on transformation fre- gene. Two transgenic plants showed clear bands
quency: To investigate whether the callus age of marker gene and objective gene, indicating
plays significant roles in transformation frequen- that this transformation system could successful-
cy, we used 1-month-old callus for transfor- ly produce transgenic plants.
mation, shorted the infection time under various In order to determine whether T-DNA inte-
infection times (20, 15, 10 min), and low OD600 gration had taken place and how many copies of
of infection solution (0.35~0.42). During 20~10 T-DNA were present in plant genome, the trans-
min infection time system, relatively high sur- genic plants were further used for Southern blot
vival probabilities after the first selection hybridization. Samples of genomic DNA were
(51.58~53.33%) were obtained from transfor- digested with HindIII and hybridized with
mation using 1-month-old callus than using 3- probes from PCR products of nptII gene. But the
month-old callus. Due to the high growth poten- T-DNA region of pCAMBIA2300 had two
tial of 1-month-old callus, not only survival HindIII cleavage site, digestion of genomic
probabilities after selection were improved, but DNAs of transformants with HindIII generated
growth rates also increased to 3.15~3.52-fold. more than relevant numbers when more than one
The highest transformation frequency was ob- copy was integrated. One or two major bands
tained from 15 min infection time system with hybridized to the probe were found in two trans-
4.93% of transformation frequency, and in this genic lines, while no hybridization signal was
transformation system, there was no albino observed in non-transformed plants, which indi-
transgenic plants, with 0.00% of albino trans- cated that one or more than one copy of gene
formation frequency. Based on these results, two, were integrated in the genome of transgenic
seventeen, two transgenic plants were success- plants. Our observations are consistent with the
fully obtained from 20 min, 15 min, 10 min in- prebious reports in which most transformants
fection time transformation system, respectively. mediated by Agrobacterium usually carried a
fairly low copy number of transgene (Dai et al.,
Molecular analysis of the transgenic plants: 2001; Zhao et al., 2002).
Leaf tissues from independent kanamycin-
resistant clones were used for PCR analyses us-
ing primers specially amplifying nptII gene and CONCLUSION
an internal 770 bp fragment of the 2-Cys Prx

A protocol was reported with successful Jin, H., P. Plaha, J. Y. Park, C. P. Hong, I. S. Lee, Z. H.
transformation and subsequent regeneration of Yang, G. B. Jiang, S.S. Kwak, S. K. Liu, J. S. Lee, Y.
A. Kim & Y. P. Lim. 2006. Comparative EST profiles
callus using kanamycin resistance gene as a se- of leaf and root of Leymus chinensis, a xerophilous
lection marker. This system involved 1-month- grass adapted to high pH sodic soil. Plant Sci. 170:
old callus and 20 min infection time with OD600 1081-1086.
=0.4~0.5. Stable integration of T-DNA into the Lee, T. H. et al. 2003. Peroxiredoxin II is essential for sus-
genome was confirmed by PCR and Southern taining life span of erythrocytes in mice. Blood. 101:
hybridization analysis with low number copy 5033-5038.
insertions. However, the development of Agro- Moon, J. C. et al. 2005. Oxidative stress-dependent struc-
bacterium-mediated transformation system will tural and functional switching of a human 2-Cys
pave the way for introduction of stress related peroxiredoxin isotype II that enhances Hela cell re-
sistance to H2O2-induced cell death. J. Biol. Chem.
genes into this grass to improve the traits in L.
280: 28775-28784.
chinensis.
Murashige, T. & F. Skoog. 1962. A revised medium for
rapid growth and bio assays with tobacco tissue cul-
ACKNOWLEDGMENTS tures. Physiol. Plant. 15: 473-497.
Neumann, C. A. et al. 2003. Essential role for the peroxire-
This work was supported by Nutraceutical doxin Prdx1 in erythrocyte antioxidant defence and
Bio Brain Korea 21 Project Group. tumour suppression, Nature. 424: 561-565.
Shu, Q. Y., G. S. Liu, S. X. Xu, X. F. Li & H. J. Li. 2005.
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PAT gene through microprojectile bombardment to
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the monocotyledonous Alstroemeria by Agrobacte- Rep. 24: 36-44.
rium tumefaciens. Plant Cell Rep. 22: 561-568.
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Chae, H. Z. et al. 1994. Cloning and sequencing of thiol- regulators and L-glutamic acid on shoot organogene-
specific antioxidant from mammalian brain: alkyl hy- sis in the halophyte Leymus chinensis (Trin.). accept-
droperoxide reductase and thiol-specific antioxidant ed. doi: 10.1007/s11240-009-9653-4.
define a large family of antioxidant enzymes. Proc.
Natl. Acad. Sci. USA. 91: 7017-7021. Veal, E. A. et al. 2004. A 2-Cys peroxiredoxin regulates
peroxide-induced oxidation and activaton of a stress-
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N. Beachy & C. Fauquet. 2001. Comparative analysis
of transgenic rice plants obtained by Agrobacterium- Wang, L. J., X. F. Li, S. Y. Chen & G. S. Liu. 2009. En-
mediated transformation and particle bombardment. hanced drought tolerance in transgenic Leymus
Mol. Breed. 7: 25-33. chinensis plants with constitutively expressed wheat
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Hiei, Y., T. Komari & T. Kubo. 1997. Transformation of rice
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Biol. 35: 205-218. a stress-induced protein with antioxidant properties, is
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Hofmann, B. et al. 2002. Peroxiredoxins. Biol. Chem. 383: hibitor of c-Abl tyrosine kinase activity. Gene Dev. 11:
347-364. 2456-2467.
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Jang, H. H. et al. 2004. Two enzymes in one; two yeast
peroxiredoxins display oxidative stress-dependent
switching from a peroxidase to a molecular chaperone
function. Cell. 117: 625-635.

Efficient Production of Taraxacum coreanum by Means of Tissue Culture
for Creating a Green Tract of Land
Jae-Hak Kim1, Young-Boum Shin1, Soon-Kwan Hong1,2
1
Department of Plant Biotechnology, Kangwon National University, Chuncheon 200-701, Korea.
2
Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701,
Korea, Tel: 033-250-6476, e-mail: soonkwan@kangwon.ac.kr
(Correspondence to: Soon-Kwan Hong)
ABSTRACT
A root cause of global warming raised global-scale problem was that the artificially produced greenhouse
gases increased. These gases have the ability to reserve a terrestrial radiation, with the consequence that Earth's
temperature will rise. The green plants absorb CO2 through photosynthesis and decomposition and in case of
exposed soil without plants, CO2 emissions will be increased by corruption of organic and carbon oxidation. In a
prompt attempt to cope the global warming, present work aims at an inquiry about creating a green tract of land
in the city surroundings and wasteland with dandelions and the tissue culture methods to supply through in a
stable manner irrespective of geographical, climatic conditions. Dandelion with a tenacious hold on life grows
naturally roadsides and ruderal sites so they adapt rapidly in any environment, aesthetic features and excellent
value as a medicinal. For this reason, calli induced from seeds collected in Kangwondo area were treated single
growth regulators making a difference of consistency. As a result, callus growth and regeneration rate was more
effective weak cytokinin in auxin types. Especially, callus weight grew in MS medium with 0.1mg/L TDZ was
22.14 g, the difference was in a whopping 10 times compared with MS medium with 0.1 mg/L IAA. Such phe-
nomena were also shown multi-shooting of a yellow dandelion. These results demonstrated TDZ was how effec-
tive whether the evidence makes.
Key words: Taraxacum coreanum, A green tract of land, TDZ
INTRODUCTION plant in the Asia, Europe and America roadsides

and ruderal sites (Lee et al., 2008). Among them,
Since the Industrial Revolution, our de- Korean dandelion (Taraxacum coreanum), a tra-
pendence on fossil fuels increases by population ditional Korean medicine, has been used in the
growth and rapid industrialization. Carbon diox- treatment of women’s disease and diuretic and
ide concentration in the atmosphere and global anti-inflammatory medicine (Cheong et al.,
average temperatures have brought a remarkable 2008). Most of the previous researches on Ta-
increase. The speed of change is too large and raxacum focused on its apomictic characteristics
socio-economic areas such as the sea level rise, and effective components, (Molinari et al., 2003)
agricultural production and shortages of water however, their strong propagation power and full
resources, human health violations, destruction vitality is fully expected to be available for the
of natural ecosystems have terrible affects, these wasteland recovery. Especially, there have been
changes within a short period requires a global a sharp rise demand for medicinal and edible use
response and interest.(Oh, 2008) Global warm- of Taraxacum coreanum recently, in Korea. In
ing caused the increase of atmospheric water other words, T. coreanum is worthy to study its
vapor and precipitation, however, the existing genetic resources for industrial application as
desertification was extended by regionalization well as its role in creating green spaces for the
of precipitation.(Joh et al., 1997) For this reason, prevention of desertification.
increasing interest for desertification prevention In a prompt attempt to cope the global
and recovery, ways to reduce greenhouse gas and warming, present work aims at an inquiry about
the technologies research on effective creating a creating a green tract of land in the city sur-
green tract of land is being proceeds.(Kim et al., roundings and wasteland with dandelions and the
2009) tissue culture methods to supply through in a
The genus Taraxacum is a member of the stable manner irrespective of geographical, cli-
family Asteraceae, subfamily Cichorioideae matic conditions.
(Lee et al., 2004) and is a common traditional

MATERIALS AND METHODS RESULTS AND DISCUSSION
Plant materials: Healthy plants of white dande- T. coreanum as positive photoblastic seeds
lion (Taraxacum coreanum) used in this study were well cultivated in dark condition. When
were collected from Gangwon province, Repu- gradually lowering the temperature, callus initia-
blic Korea in the spring of 2007 and 2009. Plants tion and cultivation in the temperature around
were transplanted to a pot and kept for breeding 25±oC were suitable and less moisture (0.4~0.5%
in the greenhouse. gelrite) slightly increased the growth efficiency.
Callus proliferation in the initial culture was
Callus induction and subculturing: Sterilized more effectively maintain at 28oC. However,
seeds were germinated in 90×20 mm Petri dishes when the temperature and humidity is higher
containing 25 ml MS medium in culture room. during cultivation, the calli are browning, while
After 3 weeks, the roots of generating seedlings this phenomenon does not exist or showed sig-
were cut in an aseptic condition and the pieces of nificantly lower at 25±oC.
plantlets were cultured on half-strength MS solid Callus weight growth rate on suspension
medium with 3% sucrose (Murashige and culture supplemented with 4 different growth
Skoog, 1962). The culture room was maintained regulators showed different results more growth
at a temperature of 28oC, a 16 h photoperiod. rate was observed withe weak cytokinin type
Embryonic callus, which formed at the surface than the strong auxin type (Fig. 1). However, as
of cutting pieces was separated from the explant a result of examine callus states under the micro-
tissue and any surrounding non-embryonic cal- scope, volume of callus significantly increased
lus, and transferred to fresh MS medium sup- in cytokinin, while number of callus increased.
plemented with 1.0mg/L 2,4-D. Under strong light conditions, regeneration
without root strongly occurred in cytokinin,
Initiation and maintenance of cell suspension TDZ. TDZ is a substitute for phenylurea com-
cultures: About 500 mg fresh weight of callus pound and has emerged as a highly efficacious
(approximately 3 month old) was transferred into bioregulant of morphogenesis in the tissue cul-
a 500 ml conical flask containing 100 ml of liq- ture of many plant species. In this study we can
uid MS medium supplemented with various clearly see that TDZ took effects on callus pro-
kinds of growth regulators. The cultures were liferation of in Taraxacum. But this phenomenon
incubated on a rotary platform shaker at 100 rpm also happened in weak auxin type and the con-
in the light condition 25oC. After 4weeks, em- trol condition was very weak. It is thought that
bryonic callus were measured fresh weight and the results were due to correlation between light
dry cell weight of callus and growth rate. and callus of T. coreanum.
Fig. 1. Growth rate of calli of T. coreanum cultured for 30 days on suspension culture media.

The plant influenced global warming, but Lee, H. H. & S. Y. Lee. 2008. Cytotoxic and Antioxidant
on the other hand it play an important role in Effects of Taraxacum coreanum Nakai. and T. offici-
nale WEB. Extracts. Korean J. Mediicinal crop Sci.
the prevention of global. Especially, Taraxacum 16(2): 79-85.
coreanum is high-value plants resources includ-
Lee, H.H., Y. S. Kim & H. Y. Park. 2007. Plant regeneration
ing aesthetic, commercial, environmentally via organogenesis from leaf explant culture of Tarax-
friendly, so in many parts of this research for acum coreanum Nakai. Korean J. Plant Medicinal
more information about this study will be per- Crop Sci. 15: 62-66.
formed. Lee, M. H., E. S. Yoon, J. H. Jeong & Y. E. Choi. 2004.
Agrobacterium rhizogenes-mediated transformation
ACKNOWLEDGEMENTS of Taraxacum platycarpum and changes of morpho-
logical characters. Plant Cell Rep. 22: 821-827.
This work was supported by Nutraceutical Lee, M. H., E. S. Yoon, S. J. Jung, K. H. Bae, J. W. Seo &
Bio Brain Korea 21 Project Group. Y. E. Choi. 2002. Plant regeneration and effect of aux-
in and cytokinin on adventitious shoot formation from
seedling explant of Taraxacum platycarpum. Korean
REFERENCES J. Plant Biotechnology. 29: 111-115.
Molinari, L., A. Busti & O. Calderini. 2003. Plant regenera-
Cheong, M. J., J. S. Yoon, Y. B. Roh, Y. B. Choi, J. S. Kim
tion from callus of apomictic and sexual lines of Pas-
& H. H. Lee. 2008. Effects of dandelion (Taraxacum
palum simplex and RFLP analysis of regenerated
coreanum) extracts on the mouse liver with acute tox-
plants. Plant Cell Rep. 21: 1040-1046.
icates by mercury chloride. Korean J. Electron Mi-
croscopy. 38(1): 01-08. Murashige, T. & F. Skoog. 1962. Revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Joh, M. S., J. W. Kim & I. U. Chung. 1998. The Numerical
Physiol Plant .15: 473-497.
simulation of the desertification induced by Global
Warming. Asia-Pacific Journal of Atmospheric Sci-
ences. 34: 65-74
Kim, H. J., Y. J. Kim, K. H. Kang, G. J. Lee & J. Y. Lee.
2009. Evaluation of soil degradation properties of
mulching mat coated by biodegradable polymer. Ap-
plied Chemistry.13: 77-80.

Compost Maintains The Soil Properties
and Supports The Sustainable Agriculture
Rakhman Sarwono1
Research Center for Chemistry – Indonesian Institute of Sciences (LIPI)

1
Komplek PUSPIPTEK, Serpong (15314), Tangerang, Phone: (021) 7560929, Fax: (021) 7560549
e-mail: rach014@lipi.go.id
ABSTRACT
In this review paper discuss the benefit of compost fertilizer in the sustainable agriculture. Compost im-
proves the soil properties, as mulch sources, and as a soil amendment to improve soil fertility. Soil improving
included the physical, chemical and biological reaction, resulted benign soil for plant growth. The main nutri-
tion element is N,P, and K as macro and trace as micro elements. Compost provides macro and micro nutrients
as plant needed. There are many advantages of organic fertilizer application compared to the chemical fertilizer
application. Organic fertilizer improve the soil structure, the sandy soil is more compact if the organic fertilizer
is applied, and also clay soil is more lighter if the organic fertilizer is applied. The nutrition content of organic
fertilizer is more complete compared to the chemical fertilizer. The long term chemical fertilizer application will
damage the soil structure and decrease the productivity. Organic fertilizer is produced from the living things is
called renewable materials such as biodegradable organic waste, municipal solid waste and agricultural waste.
Chemical fertilizer is produced from mineral, is called unrenewable sources such as natural gas, fosphate and
kalium rock. Organic fertilizer applied regularly in the organic farming to produce the healthy food and support-
ed sustainable agriculture based on the maintaining of soil mulches to ensure sustaining the soil fertilize.
Keywords: compost, nutrition, chemical, organic, fertilizer.
INTRODUCTION materials, it will finished some day. Indonesia is

a lucky country, has a lot of minerals deposite as
Fertilizer provides the crops nutrition. Ac- raw materials of chemical fertilizer. Indonesia
tually, nutrition is already available in soil and produced urea approximately 6.5 million ton a
air, but it is not ready yet used by the crops, and year, and the requirement about 5.5 million ton a
also the nutrition amount is not enough, needs year, 1 million ton surplus. There is price diver-
addition from others sources in order the crops sity between local and international market.
grow optimally. Fertilizers classified into two Consequently, there are some problems with the
groups, organic and inorganic fertilizers. Organic stock and the distribution, sometimes farmers
fertilizers are derived from living materials such have difficulty to get this fertilizer.
as animal waste, crops residue, and plantation. Nitrogen content in urea about 46%, the uti-
Inorganic fertilizers are derived from minerals lization of urea 2 Kw per hectar for a paddy rice
such as artifial fertilizer made from rock de- plantation. By increasing the fertilizer prices and
posite. the government suspend the aids to the farmer,
Plant requires approximately 16 nutrients the fertilizer price is jump and made the opera-
for optimum growth, such as carbon©, hydrogen tion cost of paddy rice cultivation increase. It
(H), oksigen (O), nitrogen (N), phosphorus (P), occurs mostly every year when the cultivation
potassium (K), calsium (Ca), Magnesium (Mg), time coming, the farmers got the difficulties.
sulfur (S) is called macro-nutrition. Micro- Those problem can be overcome by using an
nutrition such as iron (Fe), manganese (Mn), alternatif fertilizer such as compost or organic
baron (B), molybdenum (Mo), copper (Cu), dan fertilizer.
chlorine (Cl) is used in small quantity (Anon2, Application of chemical fertilizer for long
2004) term reduces the soil fertile, it may cause the
Chemical fertilizer such urea, TSP, and KCl microbial activities reduce. The microbial activi-
are made from minerals, the minerals sources are ties help the fertilizer ready used by the crops
limited. Urea is made from natural gas, TSP and (Goenadi, 2004). Microbial plays an important
KCl is made from fosphate and potassium rock rule in the nutrition used, improve the soil textu-
deposites. Mineral deposites are unrenewable re, such as N fixation, dissolved fosphate and

soil aggregate stability. If the soil nutrition is pared to chemical fertilizer that just gets basic
already enough, addition of chemical fertilizer element such as N,P, and K.
would increase the cultivation cost and environ-
mental pollution. Therefore, addition chemical Table 1. Composition of compost and vermicompost.
fertilizer should be counted exactly. Parametre Compost Vermicompost
pH 7,8 6,8
Organic and chemical fertilizers: compost is EC (mmhos/cm) 3,6 11,7
derived from biodegradable organic waste. Indo- Total kjeldahl 0,8 1,94
nitrogen (%)
nesian people is very rare to do composting for Nitrate nitrogen 156,5 902,2
chemical fertilizer substitution. Organic fertili- (ppm)
zers have many advantages compared the chemi- Phosphorus (%) 0,35 0,47
cal fertilizer. Organic fertilizer improve the soil Potasium (%) 0,48 0,7
Calsium (%) 2,27 4,4
structure but the nutrition content is lower. Nu- < 0,01 0,02
Sodium (%)
trition content in chemical fertilizer is higher but Magnesium (%) 0,57 0,46
it’s readily dissolved and leached by the run off. Iron (ppm) 11690 7563
If the soil has been chemical fertilized for a long Zinc (ppm) 128 278
time will damage their structure. Manganese (ppm) 414 475
Copper (ppm) 17 27
In order to enlarge the use of compost, Boron (ppm) 25 34
community socialization is neccessary to sustain Aluminium (ppm) 7380 7012
the soil fertility. The compost and vermicom- Source: Dickerson (2004).
post content as shown in Table 1.
Compost content is not only macro and mi- There some comparison between chemical
cro nutrient but also content of soil organic that and organic fertilizer production, as shown in
is usefull to microbial growth. Vermicompost is Table 2. Compost can be produced by the com-
also content of growth enzyme that is usefull for munity because of the simple technology and the
crops growth. Therefore, compost and ver- availability of raw materials in surrounding our
micompost have more complete nutrient com- life.
Table 2. The comparison between chemical and organic fertilizer.

Chemical fertilizer compost/organic fertilizer
1. Use high technology 1. Use simple technology
2. Unrenewable raw materials 2. Renewable material as waste Materilas.
3. Damage the soil structure in long term application. 3. Improve the soil amendment and fertility.
4. Higher external energy input 4. Lower external energy input
5. Help the environment protection
The advantages of organic fertilizer is that through the leaching of rain or irrigation. An
the excess addition is not has any risks. Compost application which is too heavy or too close to the
has slower dissolved nutrition by water and also roots of the plants may cause ―burning‖, it may
compost can be used as soil amendment. The cause economic lose and environmental pollu-
disadvantages of organic fertilizer is that the nu- tion. Application of chemical fertilizer for a long
trition is not ready used by the crops because of time reduces the soil fertility, organic soil con-
slower dissolved. The quntity of organic fertiliz- tent, and productivity.
er used to 10 to 50 times compared to the chemi- Most of soil microbes are heterotrops that
cal fertilizer used, consequently, the cost is ex- used organic as a carbon sources for it’s metabo-
pensive, the farmer is not interest to use of or- lisms, growth and reproduction. Activities of
ganic fertilizer in their plantation. But for long microbial changes corelated with the carbon in-
term organic fertilizer is more usefull than the put to the soil as the application of organic or
chemical fertilizer (Goenadi, 2004). The ad- compost. Fraser (1988) concluded that increas-
vantages of chemical fertilizer are that the nutri- ing the microbial activities corelated with the
tion is readily used by the crops and the exact increasing of organic and nitrogen in soil.
amounts of given element can be calculated When farmers apply nutrients, either in or-
(Williams, 2004). The disadvantages are that ganic or mineral form, it is to fertilize the soil,
commercial fertilizer, espescially nitrogen, is not the plant. The soil then acts as a conversion
easily washed below the level of the plant’s root system for the crops, receiving, storing, trans-

forming, tranporting and exchanging plant nutri- ready decomposed, the mulch will promote gra-
ents. It converted nutritient to readily use by the nulation, or the clinging together of soil parti-
crops. The change is happen before absorps by cles. During the decomposition of organic mate-
the root of the plant through many processes rial, soil micro-organisms secrete a sticky subs-
such as adsorption, fixation, leaching, evaporati- tance that plays an important role in soil granula-
on, reduction that processes included the chemi- tion.
cal, physical and biology processes (Anon1, Chemical effects of mulches act as soil aci-
2002; Anon2, 2004). dity. Soil pH may affected by the use of organic
Soil is a place of plant growth that provides mulches. Most organic mulches raise pH
anything what plant needs. If we have any pro- slightly, making the soil reaction more alkaline.
blems with the plant, soil condition should be Oak leaves may be acid when fresh, but as de-
look after, is that malnutrition, less water or composition progresses, the net result is an alka-
exess water or the soil structure. Plants grow in line reaction. Since organic mulches are compo-
the soil so the growing it’s deppen on the soil sed of plant materials, they add small amounts of
condition. Compost acts as soil amendment im- nutrient to the soil through decomposition. These
proves the soil structure. There are sandy or amounts have little effects on the nutrient level
heavy soil type, both condition is not good for in the soil and should not be considered a substi-
plantation. Compost will increase the aggregate tute for fertilizer.
to the sandy soil become more compact more Biological effects of mulches come from
holding-water. Heavy soil type will reduce the the mulches serve as food for many microorga-
aggregate bound makes the soil is more porous, nisms in the soil. These organisms are necessary
it’s improved the air aeration. Compost addition for maintaining and promoting soil granulation.
will improve the soil structure and suitable for A mulch also helps keep the soil temperature
plantation. constant so that the activity of the microorgan-
isms can continue at an even rate.
BENEFICIAL OF COMPOST TO THE Compost is very useful for land or soil, one
SOIL of the compost use is as mulch. Organic materi-
als as mulch retains the water from the soil sur-
Mulches building: the humus layer forms ap- face. There are many compost quality that make
proximately 4% of the topsoil. When the layer in different purposes. Compost with high fiber
falls below approximately 1%, erosion will content and larger particle can be uses as mulch.
commence and the soil is unable to retain its A compost of sewage sludge and garden refuse
moisture or nutrients. A seed that falls on soil was applied to the surfaces of raising beds. Pep-
that lacks humus has much greater difficulty in per fruit yields with compost mulch were higher
establishing itself. Mulches have some benefits than those without mulch, but lower than those
in agricultural application (William, 2003). Mul- from mulched with polyethylene (Roe et al.,
ches conserve moisture by reducing the amount 1993).
of soil water lost through evaporation. Mulches Apparently, compost with C/N ratios > 25
help maintain a uniform soil temperature. They were made primarily for such as mulches, to
act as insulator, keeping the soil warmer during control weeds and regulate soil temperature.
cool weather and cooler during the warm month. Compost with C/N ratios < 25 and N > 1%
Mulches minimize soil erosion and compaction could be uses as a sources of slow-release organ-
from heavy rain and aid in wáter penetration. ic N ( Hue, 1995).
Organic mulches are derived from plant material
that decompose to likely organic fertilizer. It ha- Compost as a soil amendment to improve soil
ve several important effects on the soil and plant fertility: soil improvement is a continual process,
growth. it often takes ten or more years to build a pro-
Physical effects of mulches alter the structu- ductive soil. Compost can be used as soil
re of the soil which usually increases root amendment, increase the organic content in the
growth. The addition of such mulches as leaves, soil, water retention and nutrient, erosion abate-
sphagnum peat moss, or shredded bark to the ment, and pH stabilization. Compost can be
soil brings an almost immediate effects. Aeration source of micro and macro nutrients (Staffela,
is improved in clay soils, and the water-holding 1997). Soil amendment should included the
capacity is increased in sandy soils. If not al- function. (1)Soil amendments improve the phys-
ical properties of soils; (2) Amendments are

mixed into the soil. Mulches are placed on the The carbon to nitrogen ratio (C/N) is also
soil surface; (3) The best soil amendments in- influents to the mineralization. Initial C/N ratio
crease water- and nutrient-holding capacity and higher than 20 may result in the immobiliza-
improve aeration and water infiltration; (4) tion of soil N during the initial decomposition
Wood products can tie up nitrogen in the soil; process. Initial C/N less than 20 may release
(5) Sphagnum peat is superior to mountain peat. mineral N during decomposition.
The best organic amendments are coarse materi- Mineralizaed N from compost, like inorgan-
als, don’t use dusty, fine peats that clog soil ic N fertilizer, will move downward in the soil
drainage. Organic amendments include sphag- profile with rainfall and irrigation, and possibly
num peat, wood chips, grass clippings, straw, move below the root zone into groundwater.
compost, manure, biosolids, sawdust and wood High application rates have been reported to re-
ash. Inorganic amendments include vermiculite, sult in leaching of NO3-N, NH4-N, and PO4-P
perlite, tire chunk, pea gravel and sand (Davis, from topsoil, with subsequent groundwater con-
2005). tamination (Poverly, 1994). The field applica-
Soil consists of a small particle binds to- tion of organic wastes such as sewage sludge and
gether that is called soil structure. If the particles manure has also been reported to cause leaching
strongly bound it’s called clay type, if the bound of NO3-N into the groundwater (Daliparthy et
is weak it’s called sandy type. Both types are not al., 1995). Untreated groundwater may cause
good for plantation. Those type needs some eurothropication in the cathment area.
treatment by addition of organic layer to change
the soil structure (Wicaksono, 1993). Organic In conclusion, compost is usefull in the
addition to sandy soil increases the binding maintaining and caring the soil fertility to sustain
force, resulted soil with good water holder. Clay the agricultural system. Compost is used to build
soil added with organic reduced the binding the mulches layer on the top of soil. The real
force, resulted soil with good porosity and aera- purpose of mulch is to protect the health of the
tion. soil. Mulch increases moisture retention, prevent
Compost increase the water holding capaci- top soil from washing away, and reduce soil
ty and the soil fertility, and also improve the soil compaction. Mulch shades out the weeds by
structure with addition of mulch layer to the covering the ground, weeds can not get enough
sandy or clay soil. In addition of helpful micro light to grow.
organisms present in the compost help plants to Compost applied as a soil amendment can
fight disease and some of the effects are quite improve the soil organic content, the water and
dramatic. nutrient retention in soil susceptible to leaching,
Compost nutrient is slow leaching com- and stabilize soil pH. Compost can be a source
pared to the artificial fertilizers. The use of com- of both macro- and micro-nutrients.
post also added the nutrient to the soil, even Application of organic/compost can main-
though in small amount compared to the artifi- tain the healthy soil and keeps the nutrient will
cial fertilizers. Compost can reduce the nutrient enhance the crop yield, maintain the soil stuc-
leaching, resulted reducing the use of artificial ture, and gave a certain quarantee of sustainable
fertilizers. agriculture. Sustainable agriculture lays on the
soil management, that the humus level can be
Compost mineralization: to provide mineral to maintained properly by application of organic
the soil compost should be mineralized to release fertilizer. Organic fertilizer plays an important
the mineral to the soil before used by the crops. rule in the maintaining of land, sustainable farm-
Compost mineralization is the biological reaction ing and agricultural system.
whereby organic nitrogen is converted into inor-
ganic forms, such as NH4-N and NO3-N. The REFERENCES
mineralization of compost generally depends on
the composition and muturity of the compost, Anon1. 2002. Why use chemical fertilizer instead of organ-
and environment conditions such as soil mois- ic fertilizers?. FITR.
http://www.fitr.state.fl.us/southo_organic_fertilizers.h
ture and temperature. After application determi- tm
nation of the rate of compost mineralization is
essential to determine proper application rate and Anon2 .2004. Proganic International Corporation.
http://www.proganic.com/products.htm
frequencies, and to predict potential N uptake by
crops and N losses.

Baskin, J. M. & C. C. B. Baskin. 1987. Environmentally Paverly,J.H. and Gates,P.B. (1994). Nitrogen mineralization
induced change in the dormancy states of buried weed studies in corn and vineyard plots treated with com-
seeds. British crop Protection conference on weeds. posts of differing C/N ration. Abstract from the Na-
2:695-706. tional Conference on solid Waste Composting Coun-
cil.
Daliparthy, J., S. J. Herbert. P. P. M. Veneman & L. J. Mof-
fitt. 1995. Nitrate leaching under alfalfa corn rotation Reganold, J. P. 1989. Comparison of soil properties as in-
from dairy manuring. Proceedings from the confer- fluenced by organic and conventional farming sys-
ence of Clean Eater-clean environment- 21 st Centu- tems. Am.J. Alternative Agriculture. 3: 144 – 145.
ry. 2(Nutrients): 39 – 42. Kansas City, M1, March 5 –
8, 1995. Roe, N. E., P. J. Stoffella & H. H. Bryan. 1993. Utilization
of MSW Composted and others Organic mulches on
Darmosarkoro,W., E. S. Sutarta & Winarna .2001. commercial vegetable crops. Compost science utiliza-
Penggunaan Kompos Tandan Kosong Sawit pada tion. 1: 73 – 84.
Tanaman semusim dan Hortikultura. Lokakarya
Pengelolaan lingkungan Pabrik kelapa Sawit. Medan, Schaller (1993) in Anon4 (2004). Apa itu Pertanian
19 – 20 Juni 2001. Organik?. http://www.plh-smk.or.id/kuri_pert.html
Davis, J. G. & C. R. Wilson. 2005. Choosing a Soil Steffela, P. J. et al.1997. Utilization of composted organic
Amendment. waste in vegetable production systems. An Interna-
http://www.ext.colostate.edu/Pubs/Garden/07235.htm tional information center for farmer in the Asia Pacif-
l ic Region.
http://www.agnet.org/library/article/tb147.html
Dickerson, G.W. 2004. Vermicomposting.
http://www.cahe.nmsu.edu/pubs/_h/h-164.html Wicaksono, R. 1993. Kompos Memperbaiki struktur tanah.
Sinar Tani, 25 September 1993, V.
Fraser, D. G. et al. 1988. Soil microbial populations and
activities under conventional and organic manage- William, D. J. 2003. Organic Mulch.
ment. J. Environ. Quality. 17: 585 – 590. http://www.hort.purdue.edu/hort/courses/hort491w/su
pplement/
Goenadi, D. H. 2004. Teknologi Konsumsi Pupuk yang
Mineral. Kompas,36, Sabtu, Mei 2004. Williams, S. 2004. Fertilizer: Application (Organic VS
Inorganic).
Hue, N. V. & H. Ikawa. 1995. Composts as a Soil http://gardenline.usask.ca/misc/fertili2.htm
Amendment.
http://www.ctahr.hawaii.edu/huen/hue_compost.htm Woese, K., D. Lange,C. Boess & K.W. Bogl. 1997. A com-
parison of Organically and Conventionally Grown
Neeson, R. 2004. Organic farming: an Introduction. Agnote Foods—Results of a Review of the Relevant Litera-
DPI-17, 2th ed. Feb.2004. ture. J.Sci.Food Agric. 74: 281 – 293.

Nitrous Oxide Biosorption in Biofiltration Process Using
Cow-Manure Compost Based Medium
Cynthia Noviani1, Tania Surya Utami1, Heri Hermansyah1 and M. Nasikin1

Kampus Baru UI Depok, Depok 16424, Phone No. 7863516
E-mail: nana@che.ui.ac.id ; cnoviani@yahoo.com
(Correspondence to: Cynthia Noviani)
ABSTRACT
Nitrous Oxide (N2O) is one of the greenhouse gases in the atmosphere after CO2, CH4 and water vapor, but
it gives the biggest contribution to global warming. This is due to its very difficult to decompose in the atmos-
phere, even relatively low concentrations. Emissions of N2O gas is known to have an impact 310 times greater
than the impact of CO2 on global warming. In order to reduce NOx emissions that are harmful to the environ-
ment, new technology which is more efficient than the conventional technology is needed. This new technology
is known as biofilter. Biofilter work by draining the contaminated air flow through a porous medium in which
contaminants in the air flow will adsorbed by biofilms and these contaminants will be oxidized to produce bio-
mass such as CO2, H2O, NO3-, and SO42-. In addition, the biofilter can support the growth of the microorganisms
present in the porous media. Biofilter also has been successfully used effectively to eliminate odors and volatile
organic compounds (VOC) such as benzene, styrene, phenol, and alkenes from various industrial processes. A
laboratory-scale biofilter was used to evaluate the effects of flow rate and depth of the filter medium on the
removal efficiency of N2O and the growth of microorganisms in the compost. Properties of the medium before
and after biofiltration and characteristics of the filter medium will also be examined. The biofilter was operated
using cow manure compost based medium with husk and coco peat as bulking agent. Research was carried out
by batch flow system for 9 hours. The result indicates that the highest N2O removal efficiency is obtained under
flow rate of 88 cm3/minutes with a depth of 50 cm by 61.35%, and elimination capacity for 14078 g/m 3h was
achieved.
Keywords: biofilter, compost, greenhouse gas, N2O, reduction efficiency
INTRODUCTION U.N. News Center, farming produces more

greenhouse gases than driving a car. Greenhouse
Most air pollution faced by many countries, gases will accumulate in the atmosphere thus
especially developing countries produced by ve- cause an increase in concentration over time.
hicle exhaust and industrial processes. Approxi- The higher the concentration of greenhouse gas-
mately 10% of air pollutants each year consist- es, the more heat radiation from Earth is trapped
ing of nitrogen oxide gas which is harmful pollu- in the atmosphere and radiated back to earth.
tants that can cause serious environmental prob- This has led to an increase in the earth's surface
lems (Yang, et.al, 2007). There are eight possi- temperature and global climate change.
ble outcomes if the reaction of nitrogen react Nitrous Oxide (N2O) is one of the green-
with oxygen, but that number is quite a lot of house gases in the atmosphere after CO2, CH4
NO, N2O, and NO2. Burning fossil fuels such as and water vapor, but it gives the biggest contri-
coal and oil release high concentration of nitro- bution to global warming. This is due to its very
gen oxides (including nitric oxide or N2O) as a difficult to decompose in the atmosphere, even
smoke. If N2O gas reaches the stratosphere, then relatively low concentrations. Emissions of N2O
the gas will destroy the ozone layer, resulting in gas is known to have an impact 310 times great-
higher UV radiation levels, the risk of skin can- er than the impact of CO2 on global warming. In
cer and also the risk of cataracts. Besides the order to reduce NOx emissions that are harmful
burning of fossil fuels, emissions of N2O are also to the environment, new technology which is
produced by livestock and agricultural indus- more efficient than the conventional technology
tries. Contribution of greenhouse gases from is needed. This new technology is known as bio-
livestock (CO2, NH4, and N2O) is known 18% filter. Biofilter work by draining the contaminat-
larger than the emissions released by all modes ed air flow through a porous medium in which
of transportation in the world (13%). According contaminants in the air flow will adsorbed by

biofilms and these contaminants will be oxidized MATERIALS AND METHODS
to produce biomass such as CO2, H2O, NO3-, and
SO42-. In addition, the biofilter can support the Biofilter equipment used in this research
growth of the microorganisms present in the po- made of acrylic material with a high dimensional
rous media (Liu, et.al, 2004). Biofilter also has column 120 cm, outside diameter 8 cm, and 7.35
been successfully used effectively to eliminate cm in diameter. This material is selected in order
odors and volatile organic compounds (VOC) to prevent leakage as effectively as possible.
such as benzene (Kardono and Allen, 1995), sty- Meanwhile, piping and junctions in the biofilter
rene (Lackey and Holt, 1996), phenol (Zilli, system is made of stainless steel which has min-
et.al, 1993), and alkenes (Morgenroth, et.al, imum connection. The use of stainless steel as
1995) from various industrial processes. the material in biofilter system aims to prevent
Biofiltration research with a batch flow sys- corrosion (Yang, et.al, 2007). This biofilter
tem for 9 hours by using a filter medium in the equipment is also equipped with a U-type water
form of cow manure-based compost and bulking manometer on the top and bottom of the biofilter
agent such as rice husks and coco peat can pro- column that serves as a pressure gauge to deter-
duce higher N2O removal efficiency. The param- mine pressure drop that occurred in the biofilter
eters that examined in this research include the column. In addition, to ensure redistribution of
effect of N2O flow rate variation and depth var- gas in the column, perforated plates are used
iations of the filter medium in the biofilter per- from acrylic material which was given the holes
formance to reduce N2O and to the growth of with a diameter of each hole is 2 mm. Perforated
microorganisms in the compost. plates are used if the filter medium depth ≥ 80
cm and placed on half of the depth of the filter
medium used.
Fig. 1. Biofilter design.
Experiment preparation begins with the biofilter system and the peak area of sample vol-
preparation of a filter medium such as filter me- ume N2O in N2O standard.
dia manufacturing process, drying, and sieving Biofiltration experiment was conducted for
the filter medium. Filter medium that will be 9 hours with the batch flow system in order to
used is cow manure-based compost with bulking evaluate the effects of N2O gas flow rate (73-233
agent coco peat and rice husk. Next is leak test cm3/minute) and depth filter medium (50, 60, 70,
which aims to ensure that N2O concentration 80, 100 cm) to N2O removal efficiency and to
decreases due to adsorption and degradation pro- the growth of microorganisms in the compost. In
cesses, not because of leaks. Next is flow meter addition, changes in the properties of the medi-
and N2O volume calibration to determine the um that occurred before and after biofiltration
actual flow rate of gas which is flow into the and characteristics of the filter medium used will
also be examined. The properties of the medium

to be studied include density, pH, water content, Large specific surface area is a favorite charac-
and porosity of compost. To measure the pH teristic for biofiltration applications. In each
value of the filter medium, 5 g of the filter medi- case, small pore size can encourage biomass
um was blended with 50 mL distilled water and growth along the surface of the medium, thus
was determined using the pH meter and pH indi- reducing the specific surface area allows the deg-
cator. Meanwhile, the water content of the filter radation of pollutants. Small pore diameter also
medium was measured by the weight loss after causes the surface of material that can be used as
the medium samples were dried at 105 ℃ for 2 substrate for microbial activity is more wide-
hours. Biofilter performance is carried out by spread (Suriawira, 2006).
using gas chromatography (GC) to analyze N2O Composition of elements in the compost-
that comes out of the filter medium. based medium was examined. The elements that
examined include the composition of nitrogen
RESULTS AND DISCUSSION (N), P2O5, K2O, sulfur (S), and organic carbon.
This test was conducted in Sucofindo laboratory,
Compost Characteristics: Characteristic of the Cibitung. Table 2 shows the composition ratio of
medium was examined by using BET and com- elements contained in cow manure-based com-
position elements contained in the compost test. post.
BET is done by using Quantachrome Autosorb
Automated Gas Sorption conducted in the La- Table 2. Composition ratio of elements contained in cow
manure-based compost.
boratory of Chemical Engineering and Nature
Products Materials (Lab. RPKA) to the filter Parameter Cow manure-based compost
medium which is used in biofiltration. Table 1 Composition (% dry
shows the results obtained from the BET of the weight)
cow manure-based compost. Nitrogen (N) 1.19
P2O5 1.7
Table 1. Characteristic of cow manure-based compost.
K2O 0.59
Characteristic of Filter Cow manure-based com- Sulfur (S) 0.18
Medium post
Multipoint BET 3.957 m2/g Organic Carbon (C) 15.39
DA Method Pore 1.8.101 Å Rasio C/N 12.93
Diameter
The appropriate adsorbate to evaluate po- Effects of Flow Rate: Research on variation of
rosity of solids in BET is nitrogen at a tempera- N2O gas flow rate carried out in order to deter-
ture of 77 K. Meanwhile, the characterization is mine the effect of gas flow rate of pollutants to
based on the size of pores, where the porosity the removal efficiency of N2O. Variations of
with radius region exceed 500Ǻ called macro N2O gas flow rate used in this study include 73,
pore, while the pores with a radius not exceed 88, 103, 128, 186, and 233 cm3/minute. The
20Ǻ defined as micro pore, and the pores of in- depth of the filter medium used in this study is
termediate size called mesopore (Autosorb Man- 50 cm. It should be noted that the depth of the
ual Book-6B). Based on the results obtained by medium that will be used is based on the mass of
BET, it is known that the pore diameter is 18Ǻ. the filter medium, 945 g for 50 cm depth. The
Thus, the pore radius of cow manure-based results of the performance of biofilter to reduce
compost is 9Ǻ. Therefore, the pores of this com- N2O can be seen in Fig. 2.
post medium can be defined as micro pore.

Q = 73 cc/minute Q = 128 cc/minute
Q = 88 cc/minute Q = 186 cc/minute

Data 15
Q= 103 cc/minute Q = 233 cc/minute
80
unsteady steady
70
60
50
% RE
40
30
20
10
0
0 2 4 6 8 10
t (hour)
Fig. 2. Flow rate variation profile to N2O removal efficiency by using cow
manure–based compost (h = 49.7 cm, m = 945 g, filter medium = dry
compost).
In this study, observation of the biofiltration If associated with adsorption breakthrough

is done every hour for 9 hours by using GC. As curves in general, the concentration of an ad-
can be seen in Fig. 2, the removal efficiency of sorbate will decrease because absorbed by the
N2O tends to increase every hour, although this adsorbent until a certain time before having
does not happen in 0 hour to 2 hours because at equilibrium adsorption. Thus, the concentration
this time the N2O flowing in the medium has not of N2O will decrease because adsorbed by biofil-
homogenous so it produced unsteady conditions. ter media at any particular time interval before
Phenomenon that occurs in Fig. 2 is the adsorp- the biofilter media came to saturation. N2O con-
tion of N2O gas. This graph also shows that the centrations will decline accompanied by im-
longer the contact time between compost and proved removal efficiency.
N2O gas, the higher the removal efficiency of In this study, the greater the flow rate will
N2O will be. This is a phenomenon that occurs at also decrease the concentration of N2O. This is
each flow rate. because the flow rate is so great that time living
N2O gas removal efficiency should increase in the filter medium is not long. Based on the
as the smaller flow rate is achieved. This is due above statement, the flow rate should be small so
to the longer residence time of N2O in the filter the decrease in N2O concentration will be great-
medium and the contact time between N2O gas er. However, this statement is not obtained in the
and biofilter medium is also longer. As a result, experiment because the smallest flow rate used
the intensity of N2O gas through adsorption and is 73 cm3/minute and it causes an unstable flow
degradation processes is more than the higher meter. Shareefdeen et al. (2005) states that the
gas flow rate. However, based on the graph in emission of pollutant gases that often fluctuates
Fig. 2, it can be seen that the removal efficiency can cause damage to the biofilter microbial pop-
of N2O produced are not in accordance with the ulation and the overall performance. Fig. 3
above statement. shows the removal efficiency of N2O obtained in
the 9 hours of biofiltration for each flow rate.

B
Data 16
80
70.1
70
61.3
55.9
60
47.1
50
42.2
% RE
40
30
24.2
20
10
0
73 88 103 128 186 233
3
Flow rate (cm /minute)
Fig. 3. Removal efficiency of N2O by flow rate variations (h = 49.7 cm,

dry medium, t = 9 jam).
In Fig. 3, it can be seen that the highest re- used in this study have a smaller porosity so that
moval efficiency obtained in the 9 hours of bio- the filter medium becomes more dense and depth
filtration is contained in the gas flow rate of 103 of the filter medium is smaller than 50 cm.
cm3/minute with N2O removal efficiency of
70.1%. However, the selection of the optimal Effects of Filter Medium Depth: Research on
flow rate to produce the highest N2O removal filter medium depth variation to N2O removal
efficiency is not only based on the results ob- carried out in order to determine the effect of the
tained by the removal efficiency at the 9 hours filter medium depth to biofilter performance in
but also through observation of the removal effi- reducing N2O. The research results from Yang et
ciency profile that can generate steady condition al. (2007) stated that the position of the higher
and high removal efficiency (Fig. 2). Therefore, column can produce a better reduction perfor-
the optimum N2O gas flow rate chosen in this mance. This is caused by the increasing number
experiment is 88 cm3/minute with removal effi- of pollutants gas come in contact with the medi-
ciency of 61.3%. um and also with the inoculated denitrification
The pressure drop that occurs at each flow microorganisms, so more gas can be reduced.
rate is not significant, except for a pressure drop This is also supported by the longer contact time
on flow rate of 186 cm3/minute. Pressure drop between the pollutant gas and the filter medium.
indicates an increase of 18 mm H2O due to the Thus it can be said that the biofilter column
gradual clogging and compost particles compac- which is higher will result in a higher reduction
tion. This has led to anomalies in the concentra- performance too.
tion of N2O removal profile, which the concen- In this study, variations in the depth of the
tration occurs to decrease immediately after the filter medium that will be tested is the depth of
gas flows into the column of the biofilter at 0 60, 70, 80, and 100 cm and done randomly, us-
hour. The depth of the filter medium expected in ing the optimum flow rate which has been ob-
this study was 50 cm. However, based on obser- tained in previous experiments by 88
vations the depth of the medium obtained is only cm3/minute. However, similar to the flow rate
49.7 cm. The difference height between expected variations, the base depth of the filter medium
and obtained is due to differences in types of used was the mass medium of 945 g for a medi-
filter media used. As mentioned earlier, a differ- um depth of 50 cm. On the use of the filter me-
ent filter medium will have a different medium dium depth ≥ 80 cm, perforated plates are used
depths based on the mass of the same filter me- in order to ensure redistribution of gas in the bio-
dium. It could be argued that the filter medium filter column so that the gas distribution in the

column will be more homogeneous. A perforated formance in reducing N2O on the depth variation
plate was placed on half of the mass media in the of the filter medium can be seen in Fig. 4.
biofilter column. The results of the biofilter per-
h = 50 cm h = 70 cm h = 100 cm
Data 18
h = 60 cm h = 80 cm
100
unsteady steady
80
unsteady steady
60
% RE
40
20
0
0 2 4 6 8 10
t (hour)
Fig. 4. Filter medium depth variation profile to removal effi-

ciency of N2O (filter medium = dry compost).
Phenomenon that occurs in Fig. 4 is the re- N2O by compost, so more concentration of N2O
duction of N2O as influenced by the depth of the can be reduced and the higher the % RE. N2O
B
filter medium. Supposedly, the greater filter me- removal efficiency of each filter medium depth
dia depth will make longer adsorption time of at the 9 hours can be seen in Fig. 5.
Data 19
70
61.3
60
53.2
50
44
37.3
36.4
40
% RE
30
20
10
0
50 60 70 80 100
filter medium depth (cm)
Fig. 5. Removal efficiency of N2O by filter medium depth varia-

tions (dry medium, t = 9 jam).

Based on Fig. 4 above and N2O removal ef- geneous until it reaches steady state is 4 hours.
ficiency results obtained in Fig. 5, it can be seen The biofiltration flow system used in this study
that there is a deviation where the greater the is a batch system that lasted for 9 hours. There-
filter media depth, the lower the removal effi- fore, in order to evaluate the removal efficiency
ciency is resulted, than the removal efficiency at of N2O produced by the filter medium depth ≥
a depth of 50 cm. This is not in accordance with 60 cm observations may take longer due to the
the statement of Yang et al. (2007) which states gas N2O takes longer to reach a stable state.
that the higher the biofilter column will result in Pressure drop that occurs at each depth of
higher reduction performance too. N2O removal the filter medium is not significant, except at a
efficiencies generated at a depth of 50 cm is depth of 70 cm where the resulting pressure drop
higher when compared with the removal effi- can be reached 8 mm H2O. This is what causes
ciency at a higher depth. This can be explained the low removal efficiency of N2O produced due
by considering Fig. 4, where the time required to the filter medium compaction so that the gas
by N2O to be homogeneous in the biofilter col- flow rate in the field will be hampered and caus-
umn until it reaches steady state at a depth of 50 es reduced adsorption happens to N2O gas by the
cm only lasted to 2 hours. Meanwhile, at a depth filter medium. Table 3 shows the readable depth
≥ 60 cm the time required by N2O to be homo- of the filter medium on biofilter column.
Table 3. The readable depth of the filter medium on biofilter column.

Medium Depth Medium depth in Column Depth Comparison
(cm)
60 cm 63.4 1.057
70 cm 69.2 0.989
80 cm (with perforated plates) 82 1.025
100 cm (with perforated plates) 99 0.99
Based on Table 3, it can be seen that at me- biofilter (Shuler and Kargi, 1992). The reason
dium depths of 100 cm and 70 cm there is medi- for the dilution is because the number of micro-
um compaction because the filter media depth is organisms in the compost is so many. Therefore,
more tightly than expected. The compaction also the higher the level of dilution, the number of
disrupt the use of perforated plates at a depth of microorganisms contained in it would be less.
100 cm because the perforated plates used lies Microorganism population will grow from the
uneven and compaction make holes in the perfo- energy (ATP) derived from the transformation of
rated plate clogged by the filter medium itself. air pollutants that flow to the biofilter. In other
This has resulted in low removal efficiency in words, the growth of microorganisms is a result
the depth of 100 cm medium. of the metabolism of pollutants. The minerals
needed by microorganisms containing N, S, P,
Development of Microorganisms in Compost: Ca, K, Na, Mg, Fe, Co, and Zn (Shuler and
The development of microorganisms contained Kargi, 1992), where the elements are generally
in the filter medium both before and after biofil- contained in the flow of air pollutants. At the
tration can be analyzed in two ways, namely pollutants that contain sulfur, nitrogen or halo-
through the method of the TPC (Total Plate gen, some of these elements would accumulate
Count). TPC (Total Plate Count) is one analyze in the system and will be reduced by microor-
method that aims to determine the number of ganisms that reduce energy autotrope from the
colonies of microbes in a sample. TPC is one of oxidation of molecules and use CO2 as a carbon
the calculation technique using nutrients (NA) as source.
a medium for microorganisms that will be Based on calculations that have been done,
counted. The results of the calculation the TPC it is known that the number of microorganisms
will be represented in terms of Colony Forming after biofiltration with flow rate variations is
Units (CFU) per gram of compost tested. 18% fewer than the number of microorganisms
Supposedly, the number of colonies after in the compost before beginning biofiltration.
biofiltration is more than before biofiltration be- This is caused by the contamination that oc-
cause of the energy (ATP) derived from the curred in diluted solution at 109 at the initial
transformation of air pollutants that flow to the compost so it increases the number of microor-

ganisms. Meanwhile, results of the number of REFERENCES
microorganism can be seen in Table 4.
Chung, Y. C., C. Huang, C. P. Tseng & J. R. Pan. 2000.
Table 4. Hasil uji TPC sebelum dan setelah biofiltrasi. Biotreatment of H2S and NH3 containing waste gases
by co-immobilized cells biofilter. Chemosphere, 41:
Sample ∑Microorganism 329–336.
(CFU/g)
Devinny, J. S., M.A. Deshusses, and T.S. Webster. 1999.
Initial Compost Before 1.35.10 11 Biofiltration for air pollution control. Lewis publish-
Biofiltration ers.
Compost after biofiltration 2.37.1010 Kardono, K & E.R. Allen. 1995. Elimination of benzene
with flow rate variation using a compost biofilter. 88th Annual AWMA
Meeting & Exhibition, 95-TP9C.01.
Compost after biofiltration 1.45.1011
with filter medium depth Lackey, L & T. Holt. 1996. Not for the birds. WEF
variation Industrial Wastewater, Vol. 4: pp 31–33.
Liu, Y., Xi. Quan, Y. Zhao, S. Chen & H. Zhao. 2004. Re-
moval of Ternary VOCs in air streams at high loads
Conclusions obtained during the experiment using a compost-based biofilter. Dalian University of
include: [1] Optimum removal efficiency Technology, China.
achieved at flow rate 88 cm3/min by 61.3%, and Morgenroth, E. et al. 1995. Nutrient limitation in a compost
depth of 50 cm is the optimum filter medium biofilter degrading hexane. 88th Annual AWMA
depth in reducing the N2O by 61.3%; [2] Mois- Meeting & Exhibition, 95-TP9C.05.
ture content (MC) or water content increased Ottengraph, S. P. P. 1977. Theoretical model for a sub-
from the initial value of 57.72% to final value of merged biological filtration. Biotechnol. Bioeng, 19:
65.1% at the end of the flow rate variations and 1411–1418.
63.65% at the end of the filter medium depth Shuler, M. L & F. Kargi. 1992. Bioprocess engineering–
variation caused by the absorption of moisture basic concepts. Prentice Hall, Englewood Cliffs.
from the gas output; [3] Biofilter performance in Suriawiria, H. U. 2006. Pupuk organik kompos dari
reducing N2O achieves optimum removal effi- sampah. Bandung: Humaniora Utama Press.
ciency at flow rate 88 cm3/min and 50 cm depth Yang, W. F., H. J. Hsing, Y. C. Yang & J. Y. Shyung.
of the medium. 2007. The Effect of Selected Parameters on the Nitric
Oxide Removal by Biofilter. National Taiwan Uni-
versity, Taiwan.
Zilli, M. et al. 1993. Phenol removal from waste gases with
a biological filter by Pseudomonas putida. Biotechnol.
Bioeng. Vol. 41: pp 693–699.

Preliminary Screening for Herbicidal Activity of Endophytic Bacterial
Extract from Canarium Hirsutum willd.var.hirsutum Plant
Using n-Butanol Solvent
Warda Tuharea1, Neti Yuliaty1, Martha Sari1, Alisin Febiyanti1, and Ines I.C.Atmosukarto1
Research Centre for Biotechnology, Indonesian Institute of Sciences

Jl. Raya Bogor Km.46 Cibinong 16911, Phone.021-8754587 Fax 021-8754588,
e-mail: wardatuharea@gmail.com
(Correspondence to: Warda Tuharea)
ABSTRACT
Herbicides are compounds that are spread on agricultural land to suppress or eradicate plants that can cause
a drop in yields and hence are useful means for controlling alien plants. Endophytic microbial metabolites
include compounds that can be utilized by the host plant to gain a selective advantage. We propose that these
can be used as a source of new natural herbicides. In vitro screening for herbicidal activity can be done using
water plants to facilitate observation. A commonly used aquatic plant in this type of study is Lemna minor. The
research presented in this paper was intended to conduct preliminary screening of numerous bacterial extracts
prepared from endophytic microbes to search for extracts with potential herbicidal activity. The results of this
study indicated that an extract prepared from the endophytic bacterium 389 B1 isolated from Canarium
hirsutum willd.var.hirsutum plant has potential as a herbicidal compound. In this case the activity appeared to be
associated with the bacterial culture broth. This was evidenced by the consistent results observed upon testing
of many extracts prepared from this endophytic bacteria which consistently yielded positive results following
exposure of the test plant to the extract for 24 hours. For further confirmation, following initial purification the
extract was characterised by TLC and column chromatography. A positive fraction was identified that had
herbicidal activity within 24 hours. Further studies will be required to identify the compound(s) responsible for
this activity.
Keywords: Herbicidal Activity, Endophytic Microbes, Bacteria, Canarium hirsutum willd.var.hirsutum
INTRODUCTION tissue. Endophytic microbes are microorganisms

that live in plant tissue without causing harm to
Agricultural land is generally planted with the host (Song, 1988). This is thought to occur
one or two types of agricultural crops. Yet other because the interaction between those
plants can often grow on the land and compete endophytic microbes and the plant host can be
for sunlight and nutrients in the soil. Herbicides mutually beneficial (mutualism). The
are compounds or material that are spread on relationship is predicted to provide a source of
agricultural land to suppress or eradicate plants plant nutrients for the microbes, and the same
that cause a decrease in yields. The existence of time the microbes are thought to produce
thus unwanted plants is not desirable. The use of bioactive compounds of benefit to the plant
herbicides can be used as a mean of controlling (Strobel et. al,1998 ).
alien plants (wikipedia.org). In vitro screening for potential herbicides
So far the utilization of biological resources, can be done using water plant known to be
especially plants, as a source of biologically sensitive to herbicide compounds to facilitate
active material has been conducted by exploring observation. The aquatic plant selected for this
the plant itself such as stems, leaves, roots and study is Lemna minor of the family Lemnaceae
fruit as a source of phytochemicals. This is because it is fast growing, where each of the
generally done by physical and chemical adult leaves will separate themselves to develop,
extraction of plant material (Hostettmann et. al, grow floating on the surface of moist soil, ponds
1991). An alternative approach to obtain and the likes.
chemical compounds without compromising the Secondary metabolites produced by
preservation of plants is to utilize the existence endophytes are likely to be useful in agriculture,
of microbes that live as endophytes within plant industry, and as pharmaceuticals. Because in the

field of biotechnology, endophytic microbes can diluted with methanol and 200 µl was added to
be used to produce metabolite compounds in the appropriate wells . The control well included
industrial scale without significant impact on the 200 µl methanol. The plate was finally dried in a
environment. This study is intended as a desiccator under vacuum. Dried well was added
preliminary study to study the potential of with 50 µl of DMSO (except in the negative
bacterial extracts as a source of herbicidal control), and the extract was resuspended by
activity. pipetting up and down. The plate was transferred
to shaker plate shaker, until the extract had
MATERIALS AND METHODS dissolved. This generally took approximately 1
hour, to each well was added by 2 ml of growth
Rejuvenation of Endophytic Bacterial Isolates: media [stock solution (MgSO4.7H2O,
Endophytic bacterial isolates were obtained from Ca(NO3).4H2O, KH2PO4, KNO3), micronutrient
Canarium hirsutum willd.var.hirsutum which is solution (H3BO3, MnCl2.4H2O, ZnSO4.7H2O,
part of the bacterial collection of PT. Indo Bio Na2MoO4.2H2O, CuSO4.5H2O), and Fe-EDTA
Pertiwi at the Research Center for Biotechnology Solution (FeCl3.6H2O, EDTA)] and the plate
LIPI. Isolates to be used were inoculated into was placed on a shaker for 30 minutes. Finally 2
Nutrient Agar (NA) in a petri dish and incubated frond Lemna were placed in each well and the
for 7 days at room temperature. plate was incubated at room temperature for 7
days. Each day the plate was observed for any
Fermentation of Endophytic Bacterial Isolates: evidence of discoloration in the plant.
Endophytic bacteria were first inoculated into 25
ml of liquid medium Nutrient Broth (NB) which Characterisation of positive extracts by Thin
has been sterilized in an appropriate-sized Layer Chromatography: Positive extracts
erlenmeyer flask, and then incubated for 5-7 identified from the first screening was further
days with shaking at 170 rpm speed at room characterised using Thin Layer Chromatography
temperature. When potential extracts have been and an apropriate eluent to use in further
identified, re-screening is conducted using fractionation of the extract by column
extract prepared from 100 ml cultures and 500 chromatography was identified. Briefly, 4µl of
ml cultures. extract was dotted on silica gel plates GF 254,
the plates were inserted in a vessel which
Extraction: Culture pH was adjusted by the contained the TLC eluent. Once the eluent had
addition of glacial acetic acid and 1N NaOH to travelled up the plate and reached the boundary
pH 3.5-4.0. Subsequently, bacterial mass was markers, the plates were removed and air dried.
harvested by centrifugation at 4000 rpm for 15 Spots were identified under UV light at 254 nm
minutes at a temperature of 4ºC to separate the wavelength.
broth from the cells. Cultures were extracted
with n-butanol (approximately half of the Fractionation by Column Chromatography:
volume of culture) with vigorous stirring using a Positive extracts were submitted for
magnetic stirrer for 1-2 hours. The mixture was fractionation by column chromatography using
subsequently transferred into a separation funnel the best eluent for separation as identified by
and left to form two layers (phases): an aqueous Thin Layer Chromatography. Columns were
layer and a butanol layer. Once separated, the cleaned and dried, and mounted perpendicular to
water phase was discarded while the butanol the stative. Glass wool was inserted into the
phase was collected and filtered. The butanol column and compressed at the base of the
phase was placed under vacum at a temperature column. Silica gel 60 that has been suspended
of 45-50ºC to obtain viscous extract 1-2 ml. The with eluent was added to the column. A total of
extract was finally transferred to an amber 2 ml of extract was added slowly. The separation
coloured bottle and transferred to a desiccator was done by gravity and eluate was collected in
under vacuum to obtain a dry extract which was 3 ml fractions. Each fraction was tested by TLC
finally weighed. The resulting extract was and TLC was stopped when the test showed no
dissolved in methanol (analytical grade) to more visible spots. Fraction yielding similar spot
obtain a concentration 20 mg/ml. patterns were combined and dried for further
testing.
Herbicide screening: Screening was conducted
using a 24-well plate. Endophytic extracts were

\RESULTS AND DISCUSSION grew at pH 5-6. Changes in pH are thought to be
crucial for successful extraction of active
This research was facilitated by access to a metabolites. From previous studies (unpublished
library of endophytic microbes isolated from data) the extraction of active ingredients using
various plants that had been collected from n-butanol was best conducted at pH 3,5-4,0. pH
several locations in Indonesia. This endophytic is a parameter that affects the growth, product
microbial library was intended as a natural formation, and stability of the fermentation
resource that can be used for various types of process. According to Fardiaz (1988)
research aiming to take advantage of Indonesia’s fermentation pH is important because the
natural resources that are unique and very rich in optimum pH should be maintained during
diversity. First, isolates were rejuvenated after fermentation.
storage and confirmed to be pure. Bacterial Substances produced by the isolates in
endophytes that originated from the walnut liquid fermentation were harvested by
tree (Canarium hirsutum willd.var.hirsutum) centrifugation that was conducted at low
were selected for this study. Rejuvenation of temperature (4ºC) in order to reduce the
bacteria was intended so that the bacterial possibility of damage to the active ingredients
metabolism is optimal for liquid culture. contained in a cell. The advantage of extracting
According to Satish (1990), the number of both the broth extract and cell extract was to
microbes increases exponentially. This determine whether the potential herbicide
exponential phase of microbial growth can last compounds were retained within the cell or
18-24 hours but can vary depending on the type secreted in the culture medium. Our results
of bacteria. Doubling occurs on a regular basis, showed that herbicide activity was found in the
as long as all existing components in the cell are broth extract. This is not surprising given that
in a state of balance. The medium used in the the bacteria will secrete organic compounds
rejuvenation was nutrient agar, as it had been aimed to influence their environment, including
found to be optimal for the growth of bacteria through their interaction with other organisms
(Wahyudi, 2001). that may compete with it. Secondary compounds
Fermentation process was carried out to may be antibacterial, antifungal, and as
obtain compounds of secondary metabolites that evidenced in this study herbicidal. Therefore,
are expected to contain herbicidal. Production of the extraction of broth was done to obtain
herbicidal compounds are expected by growing concentrated organic compounds.
the bacterium in liquid media containing all Herbicidal screening was done by using the
nutrients needed by the bacterium to obtain aquatic plants Lemna minor, a type of plants that
energy, growth, and cell formation material grows rapidly and can be used for toxic
needed for production of biosynthesis compounds. Herbicidal activity was identified by
metabolism. Production of herbicidal observing the death of Lemna plants marked
compounds was carried out for 7 days with with leaf color changes to brownish white on
shaking at room temperature. In general, the these plants (Table 1).
cultures were visibly turbide after 24 hours and
Table 1. Screening results of endophytic bacterial isolates.

Endophytic Isolates Herbicidal activity (extract volume 25 ml) * Observation result
389 B1 BE +++ Lemna died within 24 hours
389 B1 CE - Lemna lived up to 7 days
389 B2 BE + Lemna died day 5
389 B3 CE + Lemna died day 5
* +++ : very strong + : less powerful - : weak BE: broth extract CE: cell extract
Table 1 indicates that bacterial isolates from activity (Lemna plant death detected at 24
broth extract 389 B1 had the most positive hours).

Early on the Lemna appeared to be fresh In Fig. 1, the extracts are shown that have
green, but after incubation with the active the ability to influence the color of the Lemna
extract, the leaves changed color to pale yellow leaves. Appropriate controls were used to show
and gradually to a brownish white. This was that the activity was not caused by the solvent
very different when compared to other extracts used but the active ingredient itself.
which were negative in their activity where no Although crude extracts obtained using n-
change in the color of the leaves was detected as butanol extraction proved to have herbicidal
they remained green. This was seen in the activity, further research must be conducted for
control well too (Fig. 1). Because herbicidal further identify the properties of organic
activity was shown not to be caused by the compounds responsible for the activity.
solvent used in the extraction or by the organic Preliminary research was conducted
materials that exist in NB media, it was towards the purification of the active compound.
concluded that the positive activity in the extract Thin Layer Chromatography was conducted first
was due to secondary organic compounds that to identify the most optimal eluent. Results
are soluble in butanol. showed that the mixture of hexane : Aceton :
Acetic acid (50:50:2) was most optimal.
Column chromatography was performed to
separate compounds contained in the extract so
that a pure compound is obtained. From the
results of column chromatography, conducted on
extract 389 B1 BE, 5 fractions were obtained.
Fractions were dried by evaporation and then
tested again by to determine the fraction that is
active as a herbicide. From the results of testing
of 5 fractions, fractions 2, 3, and 4 were shown
to have herbicidal activity marked by color
Fig. 1. Herbicide Activity Test against Lemna Plants Using
24-well plate. changes on Lemna (Table 2).
Table 2. Results of fractions tested from broth extract of isolate 389 B1 BE.
Fraction Herbicidal activity * Observation result
1 + Lemna died 6 days
2 +++ Lemna died within 24 hours
5 - Lemna lived up to 7 days
* +++ : very strong + : less powerful - : weak
CONCLUSION ACKNOWLEDGEMENTS
Fraction 2, 3, and 4 bacterial broth extracts This research is supported by the

389 B1, which were extracted from the plant cooperation between the Indonesian Institute of
isolate Canarium hirsutum willd.var.hirsutum Sciences and PT. Indo Bio Pertiwi.
compounds were shown to have herbicidal
activity. Further research is needed to identify REFERENCES
the types of compounds that have herbicidal
activities. Fardiaz, S., 1988, Fisiologi Fermentasi, Pusat Antar
This study showed that endophytic bacteria Universitas IPB, Bogor.
sampled from plants in Indonesia have the Hostettmann. K., Hamburger M., Hostettmann M., Marton
potential to be developed as a source of natural A., 1991.” New Developments in Separation of
herbicides. This supports further research into Natural Product” , in Modern Phytochemical
Methods, Plenum Press.
endophytes as a potential source of herbicidal
compounds. Satish, G. 1990, Mikrobiologi Dasar, Ed. 3, translation of
Julius, E.S., Bina Rupa Angkasa, Jakarta, hal 81-86,
179-188.

Song, Y., 1988.” Isolation and Cultivation of Endophytic Wahyudi, P. 2001, Mikroba Endofitik : Simbiosis dalam
Fungi”, Asian Network on Microbial Researches, jaringan tanaman, NEED : Lingkungan Manajemen
Gadjah Mada University, Yogyakarta. Ilmiah, Vol.3, No.2.
Strobel, G. and Long, D. 1998,” Endophytic Microbes Herbisida. http:// id.wikipedia.org., accessed on 19
Embody Pharmaceutical Potential”, ASM News, November 2009.
Vol.63, No 5, Hal.263

The Use Of Zeolit as A Coagulant Adding Material
for Treating Waste Water of Crude Palm Oil
Ade Sumiardi1, Rusvirman Muchtar2
1
Program Study of Biology, 2Program Study of Cemistry, Faculty of Mathematics and Natural
Sciences, University of Mathla’ul Anwar, Banten.Telp/Fax. (0253) 401773,
email : fmipaunma@gmail.com
(Correspondence to: Ade Sumiardi)
ABSTRACT
CPO waste water that used in this experiment is taken from palm oil factory in Jambi. The first condition
of the waste before process is dark brown with concentration 352,6 (unit PtC 0), TSS=0,529 mg/l and pH=8. The
waste processing begin with choosing coagulant and auxilliary substance coagulant then it continued with
zeolith weight optimation as coagulant aim used jar-test methode that stiring it quickly in 5 minutes then slowly
in 10 minutes and continued with floe sedimentation in 30 minutes. The experiment result indicated optimum
zeolit weight in coagulant (Al2(SO4)3) is 700 mg with concentration 36,6 (PtC0), where as in PAC coagulant
optimum zeolit weight is 800 mg with concentration 34,6 (unit PtC 0). The best result from jar-test used in floe
fast test sedimentation that the result are in coagulant (Al2(SO4)3) without zeolit=0,0863 cm/s and PAC with
zeolit=0,0214 cm/s. The final result of the processed waste is light brown (almost clear) with concentration 36,6
(unit PtC0), TSS=0,216 mg/l and pH=7.
Keywords: Zeolit, Coagulant, Crude Palm Oil
INTRODUCTION process. CPO liquid waste cause pollution

because the content of Biological Oxygen
Zeolite is one of the minerals that have been Demand (BOD) which is very high ie about
widely used commercially in a variety of ion 25,000 mg/l and has a dark brown color so that
exchange process, catalyst, absorbent and when the waste is disposed of immediately going
molecular filters. Unique properties such as down the river stream water quality.
porous zeolites, cations and able to change a The dark color of waste water can also
large empty volume that can be utilized in damage the environment include the river of the
various fields such as chemistry, agriculture, ecosystem can change it. For this reason the use
industry and others. (Husaini and Trisna, of zeolite research for wastewater treatment of
1997). Zeolite is a hydrated silicate minerals and CPO can be used in waste water treatment
alumina have cavities connected to each other is installation palm oil industry because of the
an empty channel to direction variety, filled with potential and huge reserves and spread in some
water and ions are easily confused as sodium, parts of Indonesia.
potassium, magnesium and calcium (Arifin and
Komarudin, 1999).Zeolites have a beneficial MATERIALS AND METHODS
nature and extent of its use as an absorbent, ion
exchanger and the catalyst (Soebagjo, 1997). These research is begun by Introduction
This is because zeolites have a hydrophobic study to choose coagulant and coagulant material
group structure that can adjust the size of the adding that will be used continous research. The
entry of amolecules into the pore zeolite step of research namely selection of coagulant
framework (Arifin and Harsodo, 1999). material, selection coagulant material adding,
In this study zeolite used as raw enthusiasm optimization of zeolite doses and speed test of
coagulant (coagulant aim) in the coagulation- deposition in setling rate. Parameter of chemistry
flocculation process to reduce effluent color analysis that will be observed consist of color
CPO (Crude Palm Oil). The use of zeolites absorbance, pH, DO, BOD, and TSS. Here is the
together with the coagulant will accelerate the flow chart of research:
sedimentation flock of coagulation flocculation

Fig 1. The flow chart of research.
Coagulant Material Selection: CPO 100 ml of to precipitate out of solution for 30 minutes, take
waste added to the coagulant (alum, PAC, the clear at the end of the solution to be
FeSO4) each of 4 ml (the concentration of alum analyzed. Experiments such as these also
= 0.073, FeSO4 = 0.164 mol /L, stirring rapidly performed to PAC as a coagulant species
for 5 minutes, stirring slowly for 10 minutes and comparison.
then filtered to be analyzed of filtrat. 100 ml of
waste is put into a glass trophy plus each Sedimentation Speed Test (Setling-Rate): CPO
(zeolite, bentonite) weighing 100 mg, stirring for 1000 ml of waste that has added 40 ml of alum is
30 minutes, filtered and taken for analysis of put into the goblet, add 700 mg of zeolite,
filtrat. Added 100 ml of effluent was added 4 ml stirring rapidly for 5 minutes, stirring frequently,
of alum into the glass cup, inserted zeolite each for 10 minutes slowly, observed rate of
with size (-30 +45, -45 +60, -60 +100, -100 sedimentation rate 5-minute intervals for 1 hour,
+150, -150 mesh) weighing 100 grams, rapid analyzed the top of the solution to see the best
stirring for 5 minutes, stirring slowly for 10 color absorbance. Such experiments are also
minutes and then taken for analysis of filtrat. made to PAC as a coagulant type of comparison.
Added 100 ml of effluent was added 4 ml of
alum and 100 mg of zeolite into the glass cup, Chemical Analysis of Liquid Waste CPO:
stirring rapidly for 5 minutes, stirring slowly for Chemical analysis carried out before treatment
10 minutes, taken filtratnya with a varied and after the jar test for heavy optimization of
deposition time (10, 20, 30, 40 minutes), the best zeolite. Parameters used for chemical analysis is
results will be used for further experiments. the concentration of color, pH, DO (analysis
before determining CPO), BOD and TSS.
Weight optimization of Waste Zeolit: CPO 1000
ml was added 40 ml of alum is put into a glass RESULTS AND DISCUSSION
trophy, with the added weight of zeolite
respectively (100, 200, 300, 400, 500, 600, 700, Coagulant Selection: Coagulant selected from
800, 900, 1000 mg), stirred rapidly for 5 three types of material ie alum coagulant, FeSO4
minutes, stirring slowly for 10 minutes, allowed and PAC. The results show alum gives color

youngest of waste processed (attached). From concentration PtCo color. Zeolite weight
the analysis of waste processed color optimization is performed to determine the
concentration with alum coagulant PtCo of 39.6 appropriate weight zeolite as a coagulant aid.
units. Alum dose on the election, the results at a Adding too much or too little causes the color
dose of 4 ml and 5 ml (concentration of 0.073 concentration rises again. The best results from
mol / L) colors are not processed waste is not too Jar-Test is used to test the speed of
much different from that for the efficiency of sedimentation.
alum dose used four ml.
Objectives and coagulant dose optimization Sedimentation Speed Test (Setling Rate):
is to obtain a color condition of good arable Sedimentation rate test conducted to compare the
waste with not too large doses. Coagulation sedimentation rate between the use of coagulant
process starts when inserted in the waste alum Floc without zeolite and coagulant with zeolite.
that will happen instability colloid particles. In setling test rate, alum without zeolite, speed of
Colloidal particles will form mikrofloc and sedimentation = 0.0216 cm/sec, alum with
makrofloc but its still fragile, this caused by the zeolite = 0.0167 cm/sec. This means that 0.77
presence of Al ions that react with the alkaline times faster with sedimentation rate without the
elements of the flocculation process. zeolite while the PAC without zeolite
sedimentation velocity of 0.0863 cm/sec, plus
Selection of Coagulant Adding Material PAC for zeolite sedimentation velocity 0.0124
(Coagulant Aim): Adding coagulant material cm/sec. At the time of determining the speed of
selected from the two types of zeolite and sedimentation at the alum coagulant above the
bentonite. From the experimental results show sludge still a very fine floc moving down so that
that the zeolite provides color results with arable at the moment to be droped after 30 minutes, the
youngest PtCo concentration of 42.6 units. floc was carried. In settling-rate alum plus
Zeolites can not directly absorb the colors zeolite fine floc fewer moving so the
contained in the crude palm oil waste that must concentration of the resulting color is alum
first be added coagulant. In coagulation- without zeolite = 93,3 units PtCo, alum plus =
flocculation process, pengabungan mikrofloc be 52.3 units of zeolite PtCo. In PAC coagulant
makrofloc still fragile, to help the formation of type no visible floc smooth, concentrated color
the added material macrofloc coagulant aids the to the PAC obtained without zeolite = 39.6 units
zeolite. Zeolites act as a bridge between PtCo and PAC with zeolite PtCo = 34.3 units.
microfloc become heavier makrofloc so floc
faster precipitates (Rybacki, 1974). In the Chemical Analysis of Liquid Waste CPO: Initial
election the size zeolite, the concentration of analysis of TSS = 0.529 mg/l, final analysis TSS
color occurs in the size of the smallest -60 +100 = 0.216 mg/l. After the processing of TSS
zeolite. Zeolite grain size determines the amount decreased by 0.313 mg/l. Concentration of 352
of overall surface area, which affects the units of color PtCo after treatment for 34.6 units
effectiveness of absorption. Precipitation timing PtCo, pH before processing = 8, after the
is performed to determine the precipitation floc processing pH = 7. BOD and COD are relatively
time, the best result is deposition of wastes undetectable.
processed for 30 minutes, after 30 minutes floc
has settled all and at the dipipet floc solution so Table 1. Data analysis with the adding of palm oil waste
that there is no concentration of small color alum, FeSO4 and PAC.
obtained for 42.6 units of PtCo. No Type of Color C (unit PtCo)
Coagulant Absorbance
Optimization Zeolites with Jar-Test: In the Jar- 1 Al2(SO4)3 0,092 39,6
Test, optimization done on the type of zeolite 2 PAC 0,107 44,6
coagulant alum and PAC. Results showed the
optimum conditions on the type of zeolite weight 3 FeSO4 0,503 176,6
coagulant alum is 700 mg of zeolite for a color Description:
concentration of 36.6 units PtCo. At the PAC, 1. Coagulant dose each 4 ml in 100 ml of waste
under optimum conditions on the weight of 2. pH before treatment 8, after treatment 7 for all types of
zeolites as much as 800 mg with 34.6 units of coagulant

Table 2. Data analysis with the adding of palm oil waste alum coagulant and coagulant aid materials (zeolite, bentonite).
Material Coagulant Aids + Al2(SO4)3 Material Coagulant Aids Without Alum
No
Type of Color C Type of Color C
Coagulant Absorbance (Unit PtCo) Coagulant Absorbance (Unit PtCo)
1 Zeolit 0,101 42,6 Zeolit 0,801 276
2 Bento 1,112 46,3 Bento 0,803 276,6
Table 3. Data Results Zeolites Size Variation.

No Size Variation of Zeolit with Al2(SO4)3 Size Variation of Zeolit without Al2(SO4)3
Size of Zeolite Color C Size of Zeolite Color C

(Mesh) Absorbance (Unit PtCo) (Mesh) Absorbance (Unit PtCo)
1 -30+45 0,119 48,6 -30+45 0,861 296

2 -45+60 0,138 55 -45+60 0,670 232,3
3 -60+100 0,103 43 -60+100 0,655 227,3
4 -100+150 0,524 183,6 -100+150 0,810 279
5 -150+ 0,504 177 -150+ 0,820 282,3
Table 4. Jar Test Results.

No Coagulant Volume Volume of Doses of Zeolite Color C
(ml) Al2 (SO4)3 (mg) Absorbance (Unit PtCo)
1 1000 40 100 0,127 51,3
2 1000 40 200 0,140 55,6
3 1000 40 300 0,181 69,3
4 1000 40 400 0,195 74
5 1000 40 500 0,182 69,6
6 1000 40 600 0,111 46
7 1000 40 700 0,083 36,6
8 1000 40 800 0,109 45,3
9 1000 40 900 0,105 44
10 1000 40 1000 0,104 43,6
Description: Optimum dose of zeolite obtained at doses of 700 mg and the pH after treatment was 7.
80
70
Concentration (Unit PtCo)
60
50
40
30
20
10
0
0 200 400 600 800 1000 1200
Doses of Zeolite (mg)
Fig 1. Graph showing the relationship between the dose of zeolite with the concentration of color.

Table 5. Data Analysis of Jar-Test with PAC Coagulant.
No Waste Volume Doses of Doses of Zeolite Absorbance C
Coagulant (Unit PtCo)
1 1000 40 600 0,090 39

2 1000 40 700 0,087 38
3 1000 40 800 0,077 34,6
4 1000 40 900 0,082 36,3
5 1000 40 1000 0,083 36,6
39,5
39
Concentration (Unit PtCo)
38,5
38
37,5
37
36,5
36
35,5
35
34,5
34
0 200 400 600 800 1000 1200
Doses of Zeolite (mg)
Fig 2. Graph showing the relationship between the doses of zeolite with color absorbance in
the jar-test with a PAC coagulant.
Table 6. Data analysis speed test of deposition (settling-rate) with alum coagulant.
No Coagulant Al2(SO4)3 without Zeolite Coagulant Al2(SO4)3 with Zeolite
Time (minutes) High Deposition Time (minutes) High Deposition
1 5 27,5 5 27
2 10 27 10 25,5
3 15 25,5 15 16,5
4 20 22,5 20 10,5
5 25 19,5 25 7,5
6 30 14,5 30 5,5
7 35 10,5 35 5,5
8 40 8,5 40 5,5
9 45 7,5 45 5,5
10 50 6,5 50 5,5
11 55 5,5 55 5,5
12 60 4,5 60 5,5
Description: Height 28.5 cm solution in 500 ml measuring cup with a diameter of 5 cm.
1. Absorbance Al2(SO4)3 without zeolite at minute of 30 = 0.253, concentrations = 0.93 units PtCo
2. Absorbance Al2(SO4)3 with zeolite at minute of 30 = 0.130, concentrations = 52.3 units PtCo

30
25
High Depotition (cm)
20
15
without zeolit
10 add zeolit
0
0 10 20 30 40 50 60 70
Time of Depotition (minutes)
Fig 3. Graph showing the relationship between the alum coagulant adding of zeolites and with
adding of zeolite on the settling rate test.
Table 7. Data analysis speed tests of floc deposition on the type of PAC coagulant.
No PAC Coagulant without Zeolite PAC Coagulant with Zeolite
Time (minutes) High Deposition Time (minutes) High Deposition

1 5 26,5 5 25,8
2 10 25,5 10 21,5
3 15 23 15 18
4 20 20,5 20 14,5
5 25 17,7 25 13,5
6 30 16,6 30 12,5
7 35 16 35 11,5
8 40 15,8 40 10,5
9 45 15,7 45 10,3
10 50 15,6 50 9,7
11 55 15,5 55 8,9
12 60 15,4 60 7,9
Description:
1. PAC Absorban without zeolite at minute of 30 = 0.092, concentrations = 39.6 (PtCo units)
2. PAC Absorban with zeolite at minute of 30 = 0.076, concentrations = 34.3 (PtCo units)

30
25
High depotition (cm)

20
15
10
0
0 10 20 30 40 50 60 70
Time (minutes)
without zeolit add zeolit
Fig 4. Graph showing the relationship between PAC coagulant without the adding of zeolite
and with the adding of zeolite in settling rate test.
ACKNOWLEDGMENTS Hrenovi, J. et al., 2003. Use of Natural Zeolite to Upgrade

Activated Sludge Proccess. Food Technol.
Biotechnol. 41(2): 157 – 165.
This research is funded by the Project on
Higher Education Research Directorate General Husaini & Trisn., 1997. The Utilization of the Cikalong
Natural Zeolites for a Textile Industrial Waste Water
of Higher Education Ministry. Treatment. Technology Research Development
Center, Bandung.
REFERENCES Mumpton A. F. 1999. Uses of Natural Zeolites in
Agriculture and Industry: Proc. Natl. Acad. Sci. USA.
Arifin, M & Komarudin. 1999. Zeolites. Research and Rahman A. R. & A. H. Mahvi. 2006. Use of Ion Exchange
Technology Development Center for Mineral, for Removel of Amonium: a Biological Regeneration
Bandung. of Zeolite. Global Nest Journal. Vol. 8. No.2.
Arifin, M. & Harsodo. 1999. Natural Zeolite: Potential, Rybacki, R. L. 1974. Characteristic of Waste Water, Water
Technology, Usability and in Indonesia. Economic Treatment Section. Petrolite Corporation.
Report of Mining Material No. 57, PPTM Bandung.
Rybacki, R. L. 1974. Coagulation Theory, Water Treatment
Gruatt, K. 2003. The Art of Zeolite Application. Applying Section. Petrolite Corporation.
Crystal Right – Water Right. Inc.
Soebagjo. 1997. Structure and Properties of Zeolites.
Thermochemistry Conversion Laboratory Department
of Chemical Engineering ITB Bandung.

Suction Rate Adjustment in Filtration Process of Media Culture
Circulation for Increasing Biomass Production
of Chlorella vulgaris Buitenzorg
Dianursanti, Anondho Wijanarko, Heru Darmawan, and Mohammad Nasikin
Bioprocess Technology Study Program, Faculty of Engineering, University of Indonesia

Kampus Baru UI-Depok, Depok 16424, Indonesia, e-mail: mail: anondho@che.ui.ac.id
(Correspondence to: Anondho Wijanarko)
ABSTRACT
Nutritional content of Chlorella vulgaris Buitenzorg biomass is suitable for holistic food supplement be-
side fact of its capability to reduce CO2 a global warming pollutant by photosynthesis. Cause of it is impossible
to use lightening alteration for solving self shading problem during cellular growth in daily solar lightening; a
solution using cellular filtration treatment was done in this experiment. Cellular cultivation is operated in Bub-
ble Column Photo bioreactor at temperature of 29°C; Pressure of 1 atm; CO2 concentration in bubbled gas 5%;
using 18 dm3 Benneck medium and illuminated by a Phillip Halogen Lamp 20W/12V/50Hz. As a result, culti-
vation of Chlorella using filtration treatment at optimum suction rate (σ) is successfully increasing biomass pro-
duction around 40% more than cultivation without filtration treatment.
Keywords: filtration treatment, Chlorella vulgaris Buitenzorg, bubble column photo bioreactor.
INTRODUCTION energy was kept constant, increasing photosyn-

thetic photon flux density PPFD (Ii) needs for
Nowadays, most became seriously envi- keeping maximum specific growth rate (Іµmax,opt)
ronmental problems was global warming effect (Falkowsky & Owens, 1980; Kaplan et al., 1980;
that related to increasing CO2 and other flue gas- Wijanarkoa et al., 2006; Wijanarko et al., 2008;
es in Earth's atmosphere level. This most In- Wijanarko & Dianursantib, 2009).
creased CO2 emission was caused by motor ve- One effort to overcome the effects of self-
hicles, industrial activity, logging and so on. shading is to use a filtration technique. Filtration
Various efforts have been made to overcome this technique is done by providing a filter device in
problem, one of which is done by using microal- the form of sponge (foam) into the photobioreac-
gae biological activity such Chlorella vulgaris tor. Results on previous studies suggested to op-
Buitenzorg that was shown its CO2 fixation abil- timizing suction rate of filtration technique for
ity through photosynthesis reaction. increase biomass production of Chlorella. Suc-
In addition, analysis of substance essential tion rate setting will affect to amount of cells that
content of this Chlorella strain, tend that this trapped in the filter and caused of culture bio-
strain have a potential purpose such its health mass density still constant during its cultivation
supplements potential, cosmetics and pharma- period.
ceuticals ingredients content, animal feed, chem-
ical products and environmentally friendly fuel MATERIALS AND METHODS
(Ohtaguchi & Wijanarko, 2002; Wijanarko et al.,
2004; Wijanarkoa et al., 2006; Wijanarkob et al., C. vulgaris Buitenzorg was supplied from
2006). This Chlorella strain have a high CO2 the Research Center of Fresh Water Fishery,
fixation ability, shown rapidly cultivated, and Depok Fishery Service, Indonesia. This strain is
adapted in wide temperature between tropical cultivated in a single 18 dm3 bubble column pho-
waters to the cold sea water and have high eco- tobioreactor containing Benneck medium at light
nomic value because it contains 59.8% protein intensity of 5 Klx (Fig. 1). The experiment was
content of 19 different amino acids; 11.6% fat; divided into two stages. The first was to deter-
2.8% chlorophyll; 3.6% water, 15 kinds of vita- mine the suction rate σμmax,opt that tend optimum
mins and 8 kinds of minerals (Wirosaputro, specific growth rate (μmax,opt) of C. vulgaris Bui-
2002). To minimize the shelf shading effect that tenzorg. The results from this stage were used
caused of the increasing micro alga density dur- for the next stage, then the second stage is the
ing its cultivation period and also to keep photon change of the suction rate for the maximum

growth rate (σμmax,opt) that is simultaneously ac- ty with or without cellular entrapped filtration
cording to biomass density (X) during cultiva- treatment was shown in Fig 3.
tion period.
OD600 measurement of cell suspensions was
performed for growth analysis. Calibration
curves guided a relation where a unit of OD600
corresponded to a cell dry weight concentration
of 0.71 g/l. CO2 concentration of input and out-
put gases (yCO2in, yCO2out) was analyzed by gas
chromatography (GC-TCD Shimadzu GC-8A).
Light intensity of the incident and transmitted
light were measured by Luxmeter (Luxtron LX-
103) and pH of the cultures was measured using
the pHmeter (Hanna Model 8314).
Fig 2. Correlation of suction rate that tend maximum

growth rate (σμmax,opt)) and C. vulgaris Buiten-
zorg’s culture biomass density (X)
1. CO2 storage bomb; 2. Air blower; 3. Electric switch; 4.

Light source; 5. CO2-trapped Erlenmeyer; 6. CO2 Erlen-
meyer discharge; 7. Photo-bioreactor; 8. Gas bubller; 9. T-
septum; 10. CO2 flowmeter; 11. Air flowmeter; 12. Suction
pump; 13. Cell trapped filter
Fig 1. Experimental apparatus
RESULTS AND DISCUSSION Fig 3. Cellular growth of C. vulgaris Buitenzorg in: A.

used cellular-entrapped filtration treatment; B. un-
In the first stage of the experiment, to obtain used cellular entrapped filtration treatment
suction rate σ that tend optimum specific growth
rate μmax,opt, Chlorella was cultivated in 4 varia- In the beginning period, cell growth in both
tions of culture biomass density Xc. Correlation treatments was similar cultivation trend. Howev-
result of suction rate that tend maximum growth er, after 60 h cultivation cause of cellular self
rate (σμmax,opt) as function of biomass density was shading phenomenon as consequence of rapid
developed and shown in Fig 2. This figure result increasing biomass density in medium culture in
is used as reference to next stage where the suc- unused cellular-entrapped filtration treatment,
tion rate σμmax,opt is set depend on culture culture increasing trend of total biomass density X was
biomass density of Xc for keeping its always different in both treatments. In cellular-
constant and will adjust using correlation curve entrapped filtration treatment, it shown that total
that shown in Fig 2 in case of accordance to its cellular biomass density was increasing to 5.48
in-situ biomass density Xc if it shown an in- g/dm3. However in unused cellular-entrapped
creasing during cultivation period. filtration, it tended an increasing to 3.83 g/dm3
Further step shown at culture biomass den- and it means that presence of cellular-entrapped
sity Xc 0.71 g/dm3, and suction set was set at its filtration was increase cellular growth around
optimum condition (σμmax,opt,) referred to previ- 40%. This result rather similar to previous result
ous results that shown in Fig 2 which was tend 3 that was tend an increasing of biomass density
l/min. Comparison result of total biomass densi- using cellular-entrapped filtration treatment (Wi-
janarko & Dianursantia, 2009).

Similar to above result, further analysis us- Hasselbalch’s equation (Wijanarko et al., 2004;
ing calculated incident growth rate, tend a simi- Wijanarkoa et al., 2006; Wijanarkob et al., 2006).
larity of cellular cultivation trend in both of
treatment at beginning step. Incident growth rate
μ, that is logarithmically proportional to incident
biomass density X and was calculated by follow-
ing logarithmic equation.
Result of incident growth rate during cultivation

in both treatments was shown in Fig 4.
Fig 5. Incident Bicarbonate ion concentration [HCO3-] of

C. vulgaris Buitenzorg in: A. used cellular-
entrapped filtration treatment; B. unused cellular
entrapped filtration treatment
Fig 4. Incident growth rate trend of C. vulgaris Buitenzorg

in: A. used cellular-entrapped filtration treatment;
B. unused cellular entrapped filtration treatment
Further observation in Fig 4, at the begin-

ning it was found a small differences in specific
growth rate (μ max). It was caused of biomass
density in cellular-entrapped treatment still low
and photosynthesis photon flux density for culti-
vation still enough and it make growth tend still
high for several time compare to another treat-
ment. Further result, compare to another treat-
ment, similar to trend of increasing of biomass
density trend, it could be understood incident
growth rate in cellular-entrapped filtration treat-
ment was remain still high and cultivation period
was longer. As additional result in it’s incident Fig 6. Incident specific CO2 transferred rate qCO2 of C.
growth rate decreasing trend in cellular- vulgaris Buitenzorg in: A. used cellular-entrapped
entrapped filtration treatment, it could be under- filtration treatment; B. unused cellular entrapped fil-
stood that cellular growth is only limited by nu- tration treatment
trient.
Bicarbonate ion concentration [HCO3-] trend Incident [HCO3-] in cellular-entrapped filtra-
was shown in Fig. 5 and determine the amount tion treatment showed higher than another treat-
diluted CO2 which was available and can be con- ment result, and it was caused by phenomenon of
sumed for micro algal’s metabolism. These ions specific CO2 transferred rate qCO2 from bubbling
are calculated from pH measurement using com- air enriched CO2 to culture medium that was
bination of Henry’s Law and the Hendersen- shown in Fig 6 and calculated by direct relation
of elimination function of CO2 concentration of

input and output gases (yCO2in - yCO2out) that was Kaplan, A., Badger, M.R. and Bery, J.A. 1980. Photosyn-
shown in following equation (Wijanarkoa et al., thesis and the intracellular inorganic carbon pool in
the bluegreen alga Anabaena variabilis: response to
2006; Wijanarkob et al., 2006; Wijanarko et al., external CO2 concentration. Planta. 149: 219-226.
2008).
Ohtaguchi, K. and Wijanarko, A. 2002. Elevation of the
efficiency of cyanobacterial carbon dioxide removal
by monoethanolamine solution. Technol. 8: 267-286.
Wijanarko and Dianursantib. 2009. Simulated flue gas fixa-
tion for large-scale biomass production of Chlorella
vulgaris Buitenzorg. Intl. J. Algae. 11 (4): 351-358.
Base on result in Fig 6 that was shown a-
verage specific CO2 transferred rate qCO2,av in Wijanarko, A. and Dianursantia. 2009. PPFD alteration and
cellular filtration for large scale biomass production
cellular-entrapped filtration treatment was 4.8 of Chlorella vulgaris Buitenzorg. J. Biosci. Bioeng.
times compare to another treatment, it could be 108: S117.
understood why an increasing of accumulated Wijanarko, A., Asami, K. and Ohtaguchi, K. 2004. The
bicarbonate ion concentration in culture medium kinetics of growth and the CO2 concentrating mecha-
[HCO3-] was happened and it pushed increasing nism of the filamentous cyanobacterium Anabaena
cellular growth that was tend by increasing total cylindrica in a bubble column. J. Chem. Eng. Japan.
biomass density and growth during cultivation 37: 1019-1025.
period. Wijanarko, A., Dianursanti, Sendjaya, A.Y., Hermansyah,
H., Witarto, A.B., Sofyan, B.T., Asami, K.,
Ohtaguchi, K., Soemantojo, R.W. and Song, S.K.
CONCLUSIONS 2008. Enhanced Chlorella vulgaris Buitenzorg
growth by photon flux density alteration in serial pho-
Cultivation of C. vulgaris Buitenzorg with tobioreactors. Biotechnol. Bioproc. Eng. 13: 476-482.
cellular-entrapped filtration treatment with suc- Wijanarkoa, A., Dianursanti, Gozan, M., Andika, S.M.K.,
tion flow rate was setting at σμmax,opt, could in- Widiastuti, P., Hermansyah, H., Witarto, A.B.,
crease cellular growth around 40% and tend a Asami, K., Soemantojo, R.W., Ohtaguchi, K. and
small differences in specific growth rate (μ max). Song, S.K. 2006. Enhancement of carbon dioxide fix-
ation by alteration of illumination during Chlorella
It occur cause of increasing of accumulated of vulgaris Buitenzorg’s growth. Biotechnol. Bioproc.
bicarbonate ion in medium culture [HCO3-] that Eng. 11: 484-488.
was happened by phenomenon of increasing CO2
Wijanarkob, A., Dianursanti, Heidi, Soemantojo, R.W. and
transferred rate qCO2,av during cultivation period. Ohtaguchi, K. 2006. Effect of light illumination alter-
ation on Chlorella vulgaris Buitenzorg’s CO2 fixation
REFERENCES in bubble column photobioreactor. Intl. J. Algae. 8:
(1): 53-60.
Falkowsky, P.G. and Owens, T.G. 1980. Light-shade adap- Wirosaputro, S. 2002. Chlorella untuk kesehatan global.
tation. Plant Physiol. 66: 592-595. Gajah Mada University Press. Yogyakarta.

Determination of Hydrodynamic Parameter in Bubble Column
Photobioreactor for Scale-Up Biomass Production
of Chlorella vulgaris Buitenzorg
Anondho Wijanarko, Putu Grahita Teja Kusuma, Dianursanti and Mohammad Nasikin
Bioprocess Technology Study Program, Faculty of Engineering, University of Indonesia

Kampus Baru UI-Depok, Depok 16424, Indonesia, e-mail: mail: anondho@che.ui.ac.id
(Correspondence to: Anondho Wijanarko)
ABSTRACT
Chlorella vulgaris Buitenzorg is an useful biomass product that was especially for supplement food and
health holistic drug, beside its photosynthetic capability for minimizing global warming effect in through to CO2
fixation. Investigating an optimum hydrodynamic factor, such as mass transfer coefficient (KLa) and also super-
ficial gas velocity (UG) is important for maximizing Chlorella biomass production using 40 l Benneck medium
in bubble column photobioreactor that was set at temperature of 29°C; pressure of 1 atm; CO2 concentration in
bubbled gas 5%; and illuminated by a Phillip Halogen Lamp 20W/12V/50Hz. As a result, a scale up process
based on similarity value of KLa at its optimum hydrodynamic factor tend an achieving higher biomass concen-
tration than similarity of UG value. It was concluded that similarity value of K La shown around 30.4% compare
to another similarity value and as a conclusion similarity value of K La is more acceptable for scale up Photo
bioreactor
Keywords: Chlorella vulgaris Buitenzorg, bubble column photobioreactor, hydrodynamic factor, KLa, iso UG.
INTRODUCTION a potential purpose such its health supplements

potential, cosmetics and pharmaceuticals ingre-
Nowadays global warming is the most seri- dients content, animal feed, chemical products
ously issue in global environmental problems. and environmentally friendly fuel (Ohtaguchi &
This problem was accelerated by rapid increas- Wijanarko, 2002; Wijanarko et al., 2004; Wi-
ing common industrial activity that was used janarkoa et al., 2006; Wijanarkob et al., 2006).
fossil fuel energy which was emitted CO2 and This Chlorella strain have a high CO2 fixa-
other gas pollution, as a logic consequence for tion ability, shown rapidly cultivated, and
rapid increasing in human population. As a adapted in wide temperature between tropical
common knowledge, CO2 gas in atmosphere is waters to the cold sea water and have high eco-
re-reflected amount of heat energy that escaped nomic value because it contains 59.8% protein
to surrounding, kept heat energy and make bio- content of 19 different amino acids; 11. 6% fat;
sphere condition warmly similar to condition at 2.8% chlorophyll; 3.6% water, 15 kinds of vita-
greenhouse. In greenhouse gas, CO2 gas has big- mins and 8 kinds of minerals (Wirosaputro,
gest contribution for earth temperature increases 2002).
cause of it amount is equal to 85% from green- The aim of this work is to increase biomass
house gas percentage total. production of C. vulgaris Buitenzorg in 40 dm3
Effort to reduce CO2 concentration in at- Photo bioreactor and focused in studying of
mosphere is the most important thing in solving hydrodynamic parameter and mass transfer
global warming issue. So many research activi- characteristic that was affected to its microbial
ties was done to reduce global warming issue, growth. Several kind of hydrodynamic paramater
one of them is by using Chlorella vulgaris Bui- that was known to elaborate in this work is
tenzorg to fixate CO2 gas in bubble column pho- overall mass transfer (KLa), residence time
to bioreactor. C. vulgaris Buitenzorg is a useful distribution (RTD) that was related to mixing
biomass product that was especially for supple- phenomenon, superficial gas velocity (UG), and
ment food and health holistic drug, beside its gas hold up (ε). This study was focused in
photosynthetic capability for minimizing global overall mass transfer (KLa) as general parameter
warming effect in through to CO2 fixation. In used to evaluate performance of photobioreactor.
addition, analysis of substance essential content Term overall mass transfer coefficient (KLa) is
of this Chlorella strain, tend that this strain have usually used to explain about volumetric mass

transfer coefficient in photobioreactor that has equal to 24.6 g/mol that was found from dry bi-
been influenced by several factors like agitation omass elemental analyze.
rate, sparger type, surfactants/antifoam agents, 44 dX
CUR  .
superficial gas velocity that was related to CO2  B dt
contained bubbled gas rate and temperature. In
Here, cellular biomass was concluded as formu-
this research, KLa parameter is determined to
lated chemical result as CH3.3N0.203O0.322P0.041.
superficial gas velocity UG that was related to
amount of CO2 supply that was used to
photosynthetic Chlorella growth.
Based on above mention, this middle scale
of bubble column photobioreactor was deter-
mined to found optimum KLa parameter that re-
lated to superficial gas velocity UG and could be
referenced for large scale production of Chlorel-
la biomass.
C. vulgaris Buitenzorg was supplied from

1. CO2 storage bomb; 2. Air blower; 3. Electric switch; 4.
the Research Center of Fresh Water Fishery, Light source; 5. CO2-trapped Erlenmeyer; 6. CO2 Erlen-
Depok Fishery Service, Indonesia. This strain is meyer discharge; 7. Photo-bioreactor; 8. Gas bubller; 9. T-
cultivated in a single 40 dm3 bubble column pho- septum; 10. CO2 flowmeter; 11. Air flowmeter; 12. Suction
to bioreactor containing Benneck medium at pump; 13. Cell trapped filter
light intensity of 5 Klx (Fig 1). Focus study in Fig 1. Experimental apparatus
this work is hydrodynamic parameter of mass
transfer coefficient (KLa). Standard experiment CO2 transfered rate CTR from bubbling air
was set at similar UG that shown as an optimum enriched CO2 to culture medium was calculated
parameter in 18 dm3 bubble column photo biore- by direct relation of elimination function of CO2
actor. Bubbling system was done using 4 sparger concentration of input and output gases (yCO2in -
tube and for giving air flowing capacity air flow yCO2out) that was shown in following equation
QG was set to 140 l/min. Setting rate of air flow (Wijanarkoa et al., 2006; Wijanarkob et al., 2006;
and CO2 was set using 2 part of Kofloc flowme- Wijanarko et al., 2008).
ters. Experiment apparatus was arranged accord-
ing to below schematic drawing that was shown
in Fig 1. 
CTR  yCO2 ,in  yCO2 ,out  QG   M CO 2  PT
V  RT
OD600Measurement of cell suspensions was
performed for growth analysis. Calibration RESULTS AND DISCUSSION
curves guided a relation where a unit of OD600
corresponded to a cell dry weight concentration Based on result experimental work in 18
of 0.71 g/l. CO2 concentration of input and out- dm3 bubble column photo bioreactor that was
put gases (yCO2in,yCO2out) was analyzed by gas tend optimum superficial velocity (UG) was
chromatography (GC-TCD Shimadzu GC-8A). found at 15.2 m/h, developing correlation be-
Light intensity of the incident and transmitted tween UG and mass transfer coefficient kLa was
light were measured by Luxmeter (Luxtron LX- done in Fig 2. The kLa value of optimum UG in
103) and pH of the cultures was measured using 18 dm3 bubble column photo bioreactor was
the pH- meter (Hanna Model 8314). found 0.303 min-1 and the UG in 40 dm3 bubble
Measurement mass transfer coefficient column photo bioreactor, based on result in Fig
(KLa) was done using dynamic method which is 2, is tend 12.2 m/h. Further cellular cultivation
done by DO meter (Doran, 2005). KLa meas- experiment work was done in 40 dm3 bubble
urement is done in both of 18 and 40 dm3 vol- column photo bioreactor which was UG was set
ume in order to get similar parameter at optimum at 12.2 m/h that was tend a similarity value to
superficial velocity UG. Intracellular uptake rate kLa of optimum UG in 18 dm3 bioreactor (it is
CO2 for cellular growth CUR (CO2 uptake rate) called as iso kLa - mode) and set at 15.2 m/h that
was determined by below equation and aB was

was tend optimum UG (it is also called iso UG –
mode).
0.6
0.5
40 dm3
K a CO (min )
-1
0.4
18 dm3
0.3
2
0.2
L
0.1 Fig 4. Incident growth rate at iso KLa – mode and iso UG
– mode condition
0
0 5 10 15 20 25
Reversible condition of bicarbonate concen-
U (m/h)
G tration [HCO3-] which along with increasing
Fig 2. Correlation graph between kLa and UG on both of
around 30% in iso kLa – mode tend higher than
18 and 40 dm3 bubble column photo bioreactor another mode (Fig 5). It found 4.16 mmol/dm3
that was around 60% up compare to iso UG –
Both of iso kLa – mode and iso UG – mode mode. This result was inline to the fact that cel-
experimental results was shown in Fig 3 and 4 lular growth in iso kLa – mode mare active than
that shown microalgal’s biomass production and another one that was shown with its biomass
incident growth rate. Although initial growth production result and also it cellular growth ra-
rate (µmax) in both iso kLa – mode and iso UG ther slightly.
mode were same, decreasing growth rate in iso
kLa – mode was rather slightly compare to de-
creasing trend of iso UG - mode growth rate. Bi-
omass production in iso kLa – mode tend better
than another mode and it produce biomass
around 30% up. As a temporary conclusion, it
could be understood that most hydrodynamic
parameter that was affected in biomass produc-
tion is kLa mass transfer rate between liquid to
gas.
Fig 5. [HCO3-] Incident bicarbonate ion at iso KLa and iso

UG
CO2 transferred rate at both of mode was

shown in Fig 6 and as comparison, CTR value at
iso KLa – mode is around 75% higher if it was
compared to another mode. This result explained
Fig 3. Biomass production at iso KLa – mode and iso UG – a cellular growth fact that at iso kLa – mode,
mode condition mass transfer rate of CO2 from bubbled gas to
culture medium was significant affected to ade-
quate CO2 supplying for cellular growth that was
initiated by Carbon Concentrating Mechanism

(CCM), an intercellular accumulation mecha- persed into cellular membrane to make CO2 di-
nism to kept CO2 and trapped in CO2 pool such luted species, such as HCO3- and H2CO3 that
vacuole before utilize in TCA cycle and in next were shown in value of d[HCO3-]/dt, and was
enzymatic reaction (Wijanarko et al., 2004). utilized to make metabolism substances such
Both of modeling carbon uptake rate CUR carbohydrate, fat, amino acid and so on using
and bicarbonate uptake rate d[HCO3-]/dt that TCA cycle and further next enzymatic reaction
were determined with differentiation result of that as shown in value of CUR, it could be
biomass production and incident bicarbonate as guessed that CO2 was absorbed into cellular
function of time was shown in Fig 7. It shown a membrane and accumulated in CO2 pool by
wide difference between CTR value and addi- CCM and also could be transformed to produce
tional result of CUR and d[HCO3-] /dt in both of unknown extracellular product that was dis-
iso UG - mode and iso kLa – mode. It was hap- persed into culture medium (Wijanarko et al.,
pened cause of the transferred CO2 from bub- 2004).
bled gas to culture medium except could be dis-
Fig 6. CO2 transferred rate CTR at iso KLa – mode and iso UG – mode condition
Fig 7. Modelling CUR and d[HCO3-] /dt at iso KLa – mode and iso UG – mode condition

CONCLUSIONS Wijanarko, A., Asami, K. and Ohtaguchi, K. 2004. The
kinetics of growth and the CO2 concentrating mecha-
nism of the filamentous cyanobacterium Anabaena
Comparison result of cultivation of C. vul- cylindrica in a bubble column. J. Chem. Eng. Japan.
garis Buitenzorg in both of iso UG - mode and 37: 1019-1025.
iso kLa – mode shown an difference around 30% Wijanarkob, A., Dianursanti, Heidi, Soemantojo, R.W. and
in biomass production. It could be understood Ohtaguchi, K. 2006. Effect of light illumination alter-
that Although initial growth rate (µmax) in both ation on Chlorella vulgaris Buitenzorg’s CO2 fixation
iso kLa – mode and iso UG mode were same, de- in bubble column photobioreactor. Intl. J. Algae. 8:
(1): 53-60.
creasing growth rate in iso kLa – mode was ra-
ther slightly compare to decreasing trend of iso Wijanarkoa, A., Dianursanti, Gozan, M., Andika, S.M.K.,
UG - mode growth rate and it could made a re- Widiastuti, P., Hermansyah, H., Witarto, A.B.,
Asami, K., Soemantojo, R.W., Ohtaguchi, K. and
versible condition of bicarbonate concentration Song, S.K. 2006. Enhancement of carbon dioxide fix-
[HCO3-] was around 30% in iso kLa – mode tend ation by alteration of illumination during Chlorella
higher than another mode and cause of at iso KLa vulgaris Buitenzorg’s growth. Biotechnol. Bioproc.
– mode, CTR value that was interpreted as trans- Eng. 11: 484-488.
ferred CO2 from bubbled gas to culture medium Wirosaputro, S. 2002. Chlorella untuk kesehatan global.
is around 75% higher if it was compared to an- Gajah Mada University Press. Yogyakarta.
other mode. Doran, P.M. 2005. Bioprocess engineering principles. Aca-
demic Press. London.
REFERENCES Wijanarko, A., Dianursanti, Sendjaya, A.Y., Hermansyah,
H., Witarto, A.B., Sofyan, B.T., Asami, K.,
Ohtaguchi, K., Soemantojo, R.W. and Song, S.K.
Ohtaguchi, K. and Wijanarko, A. 2002. Elevation of the 2008. Enhanced Chlorella vulgaris Buitenzorg
efficiency of cyanobacterial carbon dioxide removal growth by photon flux density alteration in serial
by monoethanolamine solution. Technol. 8: 267-286. photobioreactors. Biotechnol. Bioproc. Eng. 13:
476-482.

Microbial Conversion of Raw Glycerol to 1,3-Propanediol
by Clostridium butyricum NRRL B-1024
Wien Kusharyoto1, Martha Sari1, Nunik Sulistinah2 and Bambang Sunarko2
1
Cibinong Science Center, Jl. Raya Bogor Km. 46, Cibinong-Bogor 16911,
e-mail: wien.kyoto@gmail.com
2
Research Center for Biology, Indonesian Institute of Sciences (LIPI)
Cibinong Science Center, Jl. Raya Bogor Km. 46, Cibinong-Bogor 16911
(Correspondence to: Wien Kusharyoto)
ABSTRACT
Biodiesel (fatty acid methyl esters) has attracted considerable attention during the past decade as a renewa-
ble, biodegradable, and non-toxic fuel. Synthesis of biodiesel generates glycerol as the main by-product. Growth
inhibition of Clostridium butyricum NRRL B-1024 by commercial glycerol and raw glycerol from a palm oil-
based biodiesel production process was evaluated. C. butyricum NRRL B-1024 exhibited the same tolerance to
raw glycerol (87% w/v) and to commercial glycerol (87% w/v). Furthermore, 1,3-propanediol production from
commercial and raw glycerol, without any prior purification, was observed in batch and fed-batch cultures, on a
synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and
commercial glycerol as the sole carbon source. For both types of cultures, the conversion yield obtained was
around 0.6 mol of 1,3-propanediol formed per 1 mol of glycerol consumed. The highest 1,3-propanediol end-
concentration achieved during fed-batch cultures was 32–34 g/l, with productivities of 0.66–0.71 g/l/ h.
Keywords: 1,3-propanediol, biodiesel, Clostridium butyricum, palm oil, raw glycerol.
INTRODUCTION monomer to synthesize a new type of polyester

PTT, (polytrimethylene terephthalate).
Biodiesel (fatty acid methyl esters), which Several bacterial groups ferment glycerol
is derived from triglycerides by transesterifica- and produce 1,3-PD in significant quantities.
tion with short chain alcohols, has attracted con- They include species of Citrobacter, Klebsiella
siderable attention during the past few decades (Menzel et al., 1997; Ahrens et al., 1998), Clos-
as a renewable, biodegradable, and non-toxic tridium (Abbad-Andaloussi et al., 1995;
fuel. Synthesis of biodiesel generates a main by- Reimann et al., 1998; Biebl et al., 1999) and
product, glycerol, which represents 10% (w/v) of Lactobacillus (Schutz & Radler, 1984). Current-
the transesterification products. If biodiesel be- ly, Clostridium butyricum VPI 3266 is consid-
came a significant product of an oleochemical ered as probably the best natural 1,3-PD produc-
unit operation, capture of even a relatively small er, since production of 1,3-PD by this strain is
portion of the current non-renewable diesel mar- not a B12-vitamin dependent process (Saint
ket would result in a large increase in the amount Amans et al., 2001).
of glycerol available for the marketplace. With Recent studies on the prodiction of 1,3-PD
increasing supplies of glycerol will come a de- have been conducted using pure or commercial
crease in cost. Therefore, the oleochemical sec- glycerol, since in industrial or raw glycerol, the
tors will need to find ways to move this una- salts released from the transesterification of oils,
voidable co-product to market, and to seek new exert significant inhibitory effects on microbial
product introductions based on glycerol in order cell growth. Only few isolated C. butyricum
to better balance supply and demand. strains have been reported to grow on technical
Microbial conversion of glycerol to 1,3- glycerol (Petitdemange et al., 1995). However,
propanediol (1,3-PD) is particularly attractive in since 50% of the entire cost of the production of
that the process is relatively easy and does not 1,3-PD is due to the price of raw materials, raw
generate toxic by-products. 1,3-PD has numer- glycerol from biodiesel production processes
ous applications in polymers, cosmetics, foods, may be an interesting renewable carbon source
lubricants, and medicines. Industrial 1,3-PD pro- for the microbial conversion. Recently, several
duction has attracted attention as an important studies have shown the capability of some C.

butyricum strains to produce 1,3-PD from indus- % Inhibition = (1 – μi/μ0)  100
trial glycerol, however, there is still no report on where μi is the maximum specific growth rate in
the utilization of raw glycerol from palm oil- experiment i and μ0 is the maximum specific
based biodiesel production as carbon source for growth rate of the control experiment (no glyc-
1,3-PD production. erol added to the medium).
In this study, the ability of C. butyricum
NRRL B-1024 to produce 1,3-PD was investi- Batch fermentations without pH-regulation:
gated. Raw glycerol, the main by-product of the The cultures were carried out in 500 ml Schott
palm oil-based oleochemical industry, was used flasks containing 300 ml medium at 37°C. All
as sole carbon and energy source. The inhibitory media contained 50 g/l glycerol (commercial or
effects of commercial and raw glycerol on cell raw glycerol). Preculture was prepared by the
growth, as well as the formation of the major inoculation of a bacterial colony into 30 ml me-
metabolites of the strain (1,3-PD, acetate and dium, and incubated for 24 h at 37°C without
butyrate) were studied in anaerobic batch and agitation. Fermentation was initiated by the in-
fed-batch cultures. oculation of the preculture into the medium. In
order to ascertain the anaerobiosis during the
MATERIALS AND METHODS first fermentation steps of the culture (until 5 h
after inoculation), nitrogen gas was flushed into
Microorganism and growth medium: The bac- the medium at a rate of ± 0.05 l/h.
terium C. butyricum NRRL B-1024 was ob-
tained from ARS Culture Collection, US De- Fed-batch fermentations: The fed-batch cul-
partment of Agriculture, and maintained in Rein- tures were carried out with the aid of a 5 l biore-
forced Clostridial Medium (RCM, Oxoid) on actor (Eyela) filled with 2.25 l medium. The first
agar plates. Precultures and bioreactor cultures preculture was prepared by the inoculation of a
were carried out in M9-Medium containing (per bacterial colony into 25 ml medium, and incu-
liter): 50 g glycerol; 9.09 g KH2PO4; 0.535 g bated for 24 h at 37°C without agitation. This
NH4Cl; 0.123 g MgSO4.7H2O; 0.017 g preculture was inoculated into a second
CaSO4.2H2O; 0.01 g FeSO4.7H2O; 2.0 g yeast preculture with 225 ml medium. Fermentation
extract; 1.0 ml resazurine 0.1%; 0.25 g L- was initiated by the inoculation of the second
cysteine HCl, and 10 ml of trace elements solu- preculture into the fermentation medium (total
tion DSMZ 144. The pH-value of the medium volume = 2.5 l). Initial glycerol concentration
was adjusted to pH 7.0 by the addition of 5 M was 50 g/l, and additional glycerol was added
NaOH. The carbon sources used were commer- during the cultivation at a rate of 0.01 g/l.min.
cially available glycerol (Merck, purity 87% The agitation speed was 200 rpm, and the pH
w/v) and raw glycerol (PT. Sumi Asih, Jatimul- was adjusted to 7.0 ± 0.1 by automatic addition
ya-Bekasi) which was the main by-product of a of 5 M NaOH. The incubation temperature was
palm oil-based biodiesel production (purity 87% 37°C. An anaerobic environment in the bioreac-
w/v). tor was maintained by sparging with nitrogen
gas at a rate of ± 0.05 l/h.
Measurement of growth inhibition: Growth in-
hibition was measured in cultures performed in Analytical procedures: Cell concentration was
flasks containing 50 ml RCM with different measured turbidometrically at 660 nm. Glycerol
glycerol concentrations (0, 20, 40, 60 and 100 concentrations during fermentation were assayed
g/l), inoculated with 1 ml cells in early exponen- enzymatically using the enzymes glycerol kinase
tial growth phase, and incubated at 37°C. For and glycerol-3-phosphate oxidase. Reaction of
this study, commercial glycerol and raw glycerol hydrogen peroxide formed by the enzymatic re-
were tested. Growth was monitored every 2 h by actions with 4-aminoantipyrine and N-ethyl-N-
optical density (OD) measurement at 660 nm. (3-sulfopropyl)-m-anisidine in the presence of
The maximum specific growth rate of C. butyri- horseradish peroxidase resulted in a dye com-
cum NRRL B-1024, on the different types and pound quinonimine, which can be measured
concentrations of glycerol, was determined from spectrophotometrically at 540 nm. The determi-
the slope of the least square regression lines of nation of products (1,3-PD, acetic acid, butyric
the logarithm of OD vs time data. acid and lactic acid) was carried out with a Shi-
Inhibition (%) in each case was deter- madzu HPLC system equipped with a refractive
mined from the following equation: index detector. Separation was performed on a

Rezex ROA column (3007.8 mm; Phenomenex Batch fermentations: To study the effect of raw
Inc.) using the following operating conditions: glycerol on the production of 1,3-PD, the cul-
0.05 M sulphuric acid as mobile phase at a rate tures of C. butyricum NRRL B-1024 using
of 0.5 ml/min and column temperature of 60ºC. commercial glycerol and raw glycerol as the sole
carbon source, respectively, were performed in
RESULTS AND DISCUSSION batch fermentation without pH regulation. The
initial concentration of glycerol in the medium
Growth inhibition of C. butyricum by glycerol: was 50 g/l and the initial pH value of the medi-
The effect of raw glycerol (87% w/v) and of um was adjusted to 7.0. Samples for end-
commercial glycerol (87% w/v) on the growth of products analysis were taken 52 h after inocula-
C. butyricum NRRL B-1042 is depicted in Fig 1. tion.
No inhibitory effect was observed with 20 g/l of Fig 2. Cell growth and glycerol consumption by C. butyri-
cum NRRL B-1024 in batch fermentation using
any kind of glycerol. The percentage of growth
1.8 60
inhibition increased linearly with commercial
1.6
and raw glycerol concentrations between 20 and
Glycerol concentration [g . l ]
50
-1
1.4
100 g/l. Up to 100 g/l the percentages of growth
1.2
inhibition by commercial and raw glycerol were
OD at 660 nm
40
1.0
very similar.
0.8 30
80 0.6
0.4 20
OD (comm. glycerol)
0.2 OD (raw glycerol)
60 Concentration of comm. glycerol
Growth inhibition [%]
Concentration of raw glycerol 10

0.0
0 10 20 30 40 50
40
Culture [hours]
20
commercial and raw glycerol as carbon source, re-
Commercial glycerol
Raw glycerol
spectively
0
Cell density and glycerol consumption by
20 40 60 80 100
C. butyricum NRRL B-1024 are depicted in Fig
Glycerol concentration [g . l-1]
2. Based on the analysis by HPLC, the concen-
Fig 1. Growth inhibition of C. butyricum NRRL B-1024
tration of commercial glycerol left in the fermen-
by commercial and raw glycerol tation broth was 16.9 g/l which corresponded to
glycerol utilization of 66%, while the concen-
Previously, the inhibitory effect of increas- tration of raw glycerol left was 18.5 g/l or simi-
ing glycerol concentration on the growth of C. lar to glycerol utilization of 63%. The final con-
butyricum DSM 5431 (Petitdemange et al., centration of 1,3-PD obtained by the fermenta-
1995) and C. butyricum VPI 3266 (Gonzalez- tion of commercial glycerol was 16.9 g/l, with a
Pajuelo et al., 2004) has been observed as well. molar yield of 0.61 mol 1,3-PD formed for every
At 100 g/l glycerol, growth was inhibited by mol glycerol consumed. By using raw glycerol
around 60%, either using 100% commercial as the carbon source, the final concentration of
glycerol or 92% raw glycerol. This value is close 1,3-PD obtained was 15.8 g/l, which correspond-
to those obtained in our experiments using 87% ded to a molar yield of 0.60 mol 1,3-PD/mol gly-
commercial glycerol and 87% raw glycerol, re- cerol consumed (Tabel 1). Thus, the molar yields
spectively. It seems that C. butyricum NRRL B- of 1,3-PD in batch fermentation were almost
1024 has the same tolerance to raw and to com- equal between commercial and raw glycerol.
mercial glycerol when both have a similar grade
(at least 87%).
Table 1. Batch fermentation of commercial and raw glycerol by Clostridium butyricum NRRL B-1024 (M9-medium with-
out pH regulation, initial glycerol concentration 50 g / l, temperature 37°C)
Type of glycerol grade Glycerol comsumed End-concentration of 1,3-PD Molar yields
(w/v) (g / l ) (g / l) (mol 1,3-PD/mol glycerol)
Commercial (87%) 33.1 (66%) 16.9 0.61

Raw (87%) 31.5 (63%) 15.8 0.60
Fed-batch fermentations: The ability of C. bu- g/l acetate and 5.1 g/l butyrate were obtained in
tyricum NRRL B-1024 to produce 1,3-PD from continuous fermentation by C. butyricum VPI
commercial and raw glycerol, respectively, was 3266, leading to a yield of 0.65 mol 1,3-PD/mol
also shown in fed-batch fermentations. The ini- glycerol (Saint-Amans et al., 2001). Petitde-
tial concentration of glycerol in the medium was mange et al. (1995) also reported that an isolated
50 g/l and the pH value of the medium was strain, C. butyricum E5, could produce 58 g/l
maintained at pH 7.0 ± 0.1. During the fermenta- 1,3-PD from 109 g/l raw glycerol in fed-batch
tion, commercial and raw glycerol (80% w/v) culture. Another newly isolated strain, C. butyri-
was added into the culture at a rate of 0.01 cum F2b, was also shown to consume raw glyc-
g/l.min, respectively. In the fed batch fermenta- erol (65% grade) in a medium containing 1 g/l
tions (Fig 3) almost three times higher cell densi- yeast extract, and yielded around 0.66 mol 1,3-
ty could be obtained compared to batch fermen- PD/mol glycerol consumed in a continuous cul-
tations (Fig 2). ture fed with 90 g/l glycerol (Papanikolaou et
al., 2000). All of the results showed that the ef-
5 60 fect of raw glycerol on batch, fed-batch and con-
Glycerol concentration [g . l ] tinuous cultures was minimal, and it did not in-
4 50
-1
terfere with 1,3-PD production.

OD at 660 nm
3 40
Table 2. Fed-batch cultures of Clostridium butyricum
NRRL B-1024 on commercial and raw glycerol
30
2 (M9-medium, feeding with 80% glycerol, 37°C)
20
1 OD (comm. glycerol) Type of glycerol
OD (raw glycerol)
Concentration of comm. glycerol
10
Commercial Raw
Concentration of raw glycerol
0 glycerol glycerol
0 10 20 30 40 50 Glycerol total (g / l) 84.10 84.7
Culture [hours]
Glycerol consumed
68.40 (81%) 67.2 (79%)
(g / l)
Fig 3. Cell growth and glycerol consumption by C. butyri- End-product concentra-
cum NRRL B-1024 in fed-batch fermentation using tion (g / l)
commercial and raw glycerol as carbon source, re- 1,3-PD 33.9 31.7
spectively
Lactic acid 1.25 1.54
1,3-PD was the major fermentation end- Acetic acid 2.46 2.72
product, while acetic, butyric and lactic acid Butyric acid 12.6 11.5
were also generated. In every case, the glycerol Molar yields
utilization was similar, being 81% for commer- [mol 1,3-PD/mol glyc- 0.60 0.57
cial glycerol and 79% for raw glycerol. The final erol]
1,3-PD titres were almost equal, being 33.9 g/l Productivity (g / l / h ) 0.71 0.66
from commercial glycerol, and 31.7 g/l from raw
glycerol. In case of commercial glycerol the mo-
In the present work, 1,3-PD could be pro-
lar yield was 0.60 mol 1,3-PD/mol glycerol with
duced in anaerobic batch and fed-batch fermen-
a productivity of 0.77 g/l/h, whereas for raw
tations of raw glycerol from palm-oil based bio-
glycerol, the molar yield was 0.57 mol 1,3-
diesel production by the collection strain C. bu-
PD/mol glycerol with a productivity of 0.66
tyricum NRRL B-1024. No significant differ-
g/l/h. Thus, our results showed that there were
ences were observed with regards to 1,3-PD final
only slight differences in the molar yields of 1,3-
concentration, 1,3-PD yield and volumetric
PD and productivities for the cultures using
productivity, when fermentations of raw glycerol
commercial and raw glycerol. The fermentation
were compared to fermentations of commercial
patterns of C. butyricum NRRL B-1024 grown
glycerol. These facts reveal that raw glycerol
on commercial or raw glycerol were also similar
from palm-oil based biodiesel production is an
(Table 2).
interesting feedstock for biotechnological pro-
The results obtained in the present work are
duction of 1,3-PD. However, the economical
similar to those obtained previously with 30 g/l
viability of this process is dependent on raw
commercial glycerol, where 14 g/l 1,3-PD, 0.4
glycerol price and its availability.

ACKNOWLEDGEMENTS propanediol by Clostridium butyricum. Biores. Tech-
nol. 67: 123-128.
We would like to thank PT. Sumi Asih, Menzel, K., Zeng, A.P. and Deckwer, W.D. 1997. High
Jatimulya-Bekasi for providing us with raw concentration and productivity of 1,3-propanediol
from continuous fermentation of glycerol by Klebsiel-
glycerol samples, and Suri Handayani for tech- la pneumoniae. Enzyme Microbiol. Technol. 28: 82–
nical assistance. This work was supported by a 86.
grant from Competitive Research Program of
Mu, Y., Teng, H., Zhang, D.J., Wang, W. and Xiu, Z.L.
LIPI. 2006. Microbial production of 1,3-propanediol by
Klebsiella pneumoniae using crude glycerol from bi-
REFERENCES odiesel preparations. Biotechnol. Lett. 28: 1755–1759.
Papanikolaou, S., Ruiz-Sanchez, P., Pariset, B., Blanchard,
Abbad-Andaloussi, S., Manginot, C., Durr, C., Amine, J., F. and Fick, M. 2000. High production of 1,3-
Petitdemange, E. and Petitdemange, H. 1995. Isola- propanediol from industrial glycerol by a newly iso-
tion and characterization of Clostridium butyricum lated Clostridium butyricum strain. J. Biotechnol. 77:
DSM-5431 mutants with increased resistance to 1,3- 191–298.
propanediol and altered production of acids. Appl.
Environ. Microbiol. 61: 4413-4417. Petitdemange, E., Durr, C., Andaloussi, S.A. and Raval, G.
1995. Fermentation of raw glycerol to 1,3-
Ahrens, K., Menzel, K., Zeng, A.P. and Deckwer, W.D. propanediol by new strains of Clostridium butyricum.
1998. Kinetic, dynamic and pathway studies of J. Ind. Microbiol. 15: 498–502.
glycerol metabolism by Klebsiella pneumoniae in
anaerobic continuous culture. III. Enzymes and fluxes Reimann, A., Abbad-Andaloussi, S., Biebl, H. and Petitde-
of glycerol dissimilation and 1,3-propanediol for- mange, H. 1998. 1,3-Propanediol formation with
mation. Biotechnol. Bioeng. 59: 544–552. product tolerant mutants of Clostridium butyricum
DSM 5431 in continuous culture: productivity, carbon
Andrade, J.C. and Vasconcelos, I. 2003. Continuous cul- and electron flow. J. Appl. Microbiol. 84: 1125–1130.
tures of Clostridium acetobutylicum: culture stability
and low grade glycerol utilization. Biotechnol. Lett. Saint-Amans, S., Girbal, L., Andrade, J., Ahrens, K. and
25: 121–125. Soucaille, P. 2001. Regulation of carbon and electron
flow in Clostridium butyricum VPI 3266 grown on
Biebl, H., Menzel, K., Zeng, A.P. and Deckwer, W.D. glucose-glycerol mixtures. J. Bacteriol. 183: 1748–
1999. Microbial production of 1,3-propanediol. Appl. 1754.
Microbiol. Biotechnol. 52: 289-297.
Schutz, H. and Radler, F. 1984. Anaerobic reduction of
Gonzalez-Pajuelo, M., Andrade, J.C. and Vasconcelos, I. glycerol to 1,3-propanediol by Lactobacillus brevis
2004. Production of 1,3-propanediol by Clostridium and L. buchneri. Syst. Appl. Microbiol. 5: 169–178.
butyricum VPI 3266 using a synthetic medium and
raw glycerol. J. Ind. Microbiol. Biotechnol. 31: 442- Xu, Y.Y., Du, W., Liu, D.H. and Zeng, J. 2003. A novel
446. enzymatic route for biodiesel production from renew-
able oils in a solvent-free medium. Biotechnol. Lett.
Gonzalez-Pajuelo, M., Andrade, J.C. and Vasconcelos, I. 25: 1239–1241.
2005. Production of 1,3-propanediol by Clostridium
butyricum VPI 3266 in continuous cultures with high Zeng, A.P. 1996. Pathway and kinetic analysis of 1,3-
yield and productivity. J. Ind. Microbiol. Biotechnol. propanediol production from glycerol fermentation by
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Himmi, E.H, Bories, A. and Barbirato, F. 1999. Nutrient

requirements for glycerol conversion to 1,3-

Isolation and Rapid Characterization of Biosurfactant-Producing Mutants
of Soil Microbe Isolated From Oil Mining, Cepu
Swastika Praharyawan, Theresia Umi Harwati and Dwi Susilaningsih
Research Center for Biotechnology, Indonesian Institute of Sciences

Jl. Raya Bogor Km. 46 Cibinong 16911, Bogor, West Java, Indonesia,
e-mail: swastika.praharyawan@gmail.com
(Correspondence to: Swastika Praharyawan)
ABSTRACT
Surfactant in detergent that spilled out to the environment is house-hold waste mostly produced by their
daily activity. It is harmful for the environment and is difficult to be degraded by nature. Therefore, the re-
placement of surfactant with biosurfactant which is environmental friendly becomes urgent. We had successful-
ly isolated potential biosurfactant-producing microbes from soil sample in oil mining area of Cepu, Central Java.
By using ultraviolet light, mutagenesis was conducted and the mutants then were characterized comparing to the
wild type. This research was aiming at characterizing of potential mutant in producing biosurfactant that is val-
ued by the clear zone formation on the oil-surface agar media, by foaming formation and also by rapid quantifi-
cation using spectrophotometer compared to the wild type. Four potential mutants were obtained and showed
better performance compared to the wild type.
Keywords: biosurfactant, soil microbe, mutant, characterization, clear zone.
INTRODUCTION adhesive action against several pathogenic mi-

croorganisms etc (Singh & Cameotra, 2004; Das
Biosurfactants are produced as metabolic et al., 2008).
products or membrane components. These com- Despite possessing many commercially at-
pounds are lipopeptides, glycolipids, lipopoly- tractive properties and clear advantages com-
saccharides, neutral lipids and fatty acids or pared with synthetic surfactant, the production of
phospholipids (Margaritis et al., 1979; Cooper & biosurfactant on a commercial scale has not been
Zajic 1980; Rosenberg, 1982; Zajic & Steffens, realized because of their low yields and high
1984; Cooper 1986). Surfactants are important production costs – for such purposes, it is neces-
as they are used in many multiphase processes sary that they are produced and recovered profit-
(Layman, 1985). The features that make them ably on a large scale. One strategy can be adopt-
commercially promising alternatives to chemi- ed in order to make biosurfactant production
cally synthesized surfactants are their lower tox- cost-competitive which is development and use
icity, higher biodegradability and, hence, greater of overproducing mutant or recombinant strains
environmental compatibility, better foaming for enhanced biosurfactant yields.
properties (useful in mineral processing), and This research has focused on the isolation of
stable activity at extremes of pH, salinity and mutants which are presumed able in overproduc-
temperature (Kosaric, 1992; Banat et al., 2000). ing biosurfactant compared to the wild type. The
Unlike chemical surfactants, which are aim of the research is comparing the ability in
mostly derived from petroleum feedstock, these producing biosurfactant between the wild type
molecules can be produced by microbial fermen- and its mutants.
tation processes using cheaper agrobased sub-
strates and waste materials. In various industrial MATERIALS AND METHOD
processes, they are potentially useful surface-
active agents for emulsion polymerization, wet- Ultraviolet mutagenesis: The BT-38-CP isolate
ting, foaming, phase dispersion, emulsification was grown to logarithmic phase and then ap-
and de-emulsification. They have also been proximately 3000 cells were plated on nutrient
found to possess several properties of therapeutic agar plates. The cells were then UV radiated for
and biomedical importance: they have antibacte- 11 minutes to 14 minutes with ultraviolet light in
rial, antifungal and antiviral properties; they in- Clean Bench Model CC-60. The UV-irradiated
hibit fibrin clot formation; and they have anti-

cells were incubated at 37°C until colonies were 11 to 14 min irradiation showed activity in pro-
visible (Mulligan et al., 1989). ducing biosurfactant. The potential mutant then
sorted based on the clear zone diameter. Isolate
Screening Method: Each isolate was streaked that showed best performance in forming clear
onto nutrient agar containing crude-oil and then zone considered as potential biosurfactant-
incubated for 24 h at 37oC and pH 7. The plates producing mutant.
then were visually inspected for zones of clear- Four potential mutants were characterized
ing around the colonies, indicative of biosurfac- by applying several incubation conditions and
tant production. Potential mutants were sorted comparing to the wild type. The characterization
based on the diameter of clear zone (Morikawa of isolates in producing biosurfactant was evalu-
et al., 2000). ated based on its ability in forming clear zone on
the oil surface agar media. According to Mori-
Rapid Characterization of Biosurfactant Pro- kawa et al. (2000), the area displacement by a
ducing Mutant: Each isolate was streaked onto surfactant-containing metabolite is directly pro-
nutrient agar contain crude-oil and then incubat- portional to the concentration of the biosurfac-
ed for 48 h at varied temperature (37–60oC) and tant.
pH (6–10). The plates then were visually in- In order to compare the ability in producing
spected for zones of clearing around the colo- biosurfactant between wild type and its mutants,
nies, indicative of biosurfactant production. The pH of media and temperature condition variable
diameter of the clear zones depends on the con- were varied in the formation of clear zone. Ratio
centration of the biosurfactant. The zones of between clear zone diameter and colony diame-
clearing were scored by compare the diameter of ter were measured as main parameter determined
clear-zone and colony diameter. the ability of isolate to produce biosurfactant.
Rapid Comparison of Biosurfactant Concentra-

tion between Mutants and Wild Type: Crude
extract was acidified by using HCl 6 N until pH
2 in order to precipitate biosurfactant. Then, the
solutions were leaved in 4oC for 1 h to complete
precipitation. After 1 h, it was vortexed and bio-
surfactant then was assayed by spectrophotome-
ter at 600 nm measuring its turbidity
(Mukherjee, 2009).
Foaming and Emulsifying Properties: The

Fig 1. Clear zone formation at varied temperature
foam is produced by hand shaking of acidic
crude biosurfactant solution for 30 s. The stabil-
ity of the foam will be monitored by observing it
during 2 h. The ability of the biosurfactant to
emulsify crude oil is determined by measuring
its emulsification index (E24) (Abouseoud et al.,
2007).
The mutagenesis process of biosurfactant-

producing soil microbe BT-38 (wild type) was
conducted under Ultraviolet light radiation ac-
quired four potential biosurfactant-producing
mutants. The wild type was irradiated by ultravi-
olet light for 11 to 15 min in order to obtain mu-
tants isolate. Irradiation process for 15 min
caused the lost of the ability in producing biosur-
factant of mutant isolate indicated by the absent Fig 2. Clear zone formation at varied pH
of clear zone. While mutant isolate resulted from

The experiment showed that pH of media factant showed different result in extracting bio-
and temperature condition possessed significant surfactant on each sample.
effect to the isolates’ activity in producing bio-
surfactant. In the temperature of 60oC, mutant
isolate coded m-3 and m-4 showed better activi-
ty in producing biosurfactant compared to the
wild type, mutant m-1 and also m-2. This result
indicated that m-3 and m-4 were thermophilic
microorganisms that showed the ability in pro-
ducing biosurfactant in high temperature condi-
tion.
Like temperature, pH of media variable also
had influence on the isolates’ activity in produc-
ing biosurfactant. The experiment showed that
mutant isolates of m-2 and m-4 possessed better
activity in the biosurfactant production in wide aqua Tween 38-wt m-1 m-2 m-3 m-4
range of pH, 6 to 10, compared to the wild type Fig 4. Emulsification properties of biosurfactants, Tween,
and also the other mutants. As we can see m-4 and aqua
showed its superior ability among other isolates
in producing biosurfactant at varied condition of The emulsifying properties of biosurfactant
temperature and pH. produced by mutants were more effective in
emulsifying crude-oil compared to the wild type
(Fig 4). But, if biosurfactants were compared to
the chemical-based surfactant the result showed
that biosurfactants were less effective in emulsi-
fying crude oil.
CONCLUSIONS
m-1 m-2 m-3 m-4 38-wt m-1 m-2 m-3 m-4 38-wt
0H 2H
The Mutagenesis of soil microbe isolates
coded BT-38 resulted four potential mutants
Fig 3. Foaming properties of biosurfactants and its stability showed better performance in producing biosur-
factant compared to the wild type. Further re-
The foam property was also assayed in this search is needed to observe the stability of mu-
study. The result showed that mutant isolate m-1 tants’ ability in producing biosurfactant.
possessed best ability in producing foam among
other mutant isolates and also wild type. It was
REFERENCES
also showed that foaming formed by m-1 isolate
more stable compared to the others after it were
Abouseoud, Maachi, R. and Amrane, A. 2007.
leaved to stand for 2 hours. Foaming properties
Biosurfactant production from olive oil by
indicated the ability of producing biosurfactant
Pseudomonas fluorescens M. Communi-
in microorganism. The better its ability in pro-
cating Current Research and Educational
duce foam the higher concentration of biosurfac-
Topics and Trends in Applied Microbiolo-
tant produced will be. This result was supported
gy. 340-347.
as well by spectrophotometer assay of the turbid-
ity of acidic crude-extract solution as it is de- Banat, I.M., Makkar, R.S., and Cameotra, S.S.
scribed in the methodology. M-1 mutant isolate 2000. Applied Microbiol. Biotechnol. 53:
showed best ability in producing biosurfactant 495.
among other isolates, including the wild type. Cooper, D.G. 1986. Biosurfactants. Mirobiol.
This result is opposed to the prior result stated Sci. 3: 145-149.
that m-4 is the best mutant isolate in biosurfac-
tant production. It might be caused by the differ- Cooper, D.G. and Zajic, J.E. 1980. Surface ac-
ence of biosurfactant type among isolates. It tive compounds from microorganisms. Adv.
means that the isolation method of crude biosur- Appl. Microbiol. 26: 229-253.

Das, P., Mukherjee, S. and Sen, R. 2008. Anti- Mulligan, C.N., Chow, T.Y.K. and Gibbs, B.F.,
microbial potential of a lipopeptide biosur- 1989. Enhanced biosurfactant production by
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neya, M.J. 2004. Comparison of methods to
Morikawa, M., Hirata, Y. and Imanaka, T. 2000.
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A study on the structure–function relation-
microorganisms. J. Microbiol. Methods. 56:
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Studies on Marine Biosurfactant for Useful Biological Function
Dian Noverita Widyaningrum, DonnaFujie Rahaditha Utami and Dwi Susilaningsih

Jl. Raya Bogor Km.46 Cibinong 16911, Indonesia, e-mail: diannoverita@gmail.com
(Correspondence to: Dian Noverita Widyaningrum)
ABSTRACT
Biosurfactants are valuable microbial amphiphilic molecules with effective surface-active and biological
properties promising for industries and processes. These molecules could be used in cosmetic, pharmaceutical,
and food process as preservatives, emulsifier, detergent, etc. The quality of food that affected by chemical, bio-
chemical, physical, and microbiological processes, also their spoilages, the preservation processes are important.
Therefore, this study is emphasized on utilization of biosurfactant obtained from marine bacteria for vegetable
preservation. The methods are described as follow: 1. Dipping the vegetable (i.e. chili) into crude, whole cul-
tures and synthetic surfactant, 2. Incubation treated vegetables in room temperature within 8 days. Vegetable
spoilages were recognized by ranges of skin fading, vapor bad odor, colour and mucilage formation. The results
exhibited that biosurfactant crude of SR_DP.15 and SR_DP.16 could preserve chili better than the other treat-
ments. The chili was kept fresh during 8 days treatment. However, the others treatment was not significantly
prevent the spoilage. The further studies are needed.
Keywords: biosurfactant, preservation, spoilage.
INTRODUCTION a common cause of spoilage in all branches of

the food industry and the economic consequenc-
Surfactants are surface active agents with es can be serious (Dainty, 1996). Food spoilage
wide ranging properties including the lowering is defined as any change in the visual appear-
of surface and interfacial tensions of liquids ance, smell, or taste of a food product that makes
(Laith et al., -). Naturally occurring surface- it unacceptable to the consumer (Madigan et al.,
active compounds derived toxicity, inherent 2000).
good biodegradability (Madigan et al., 2000). Consumption of microbially contaminated
The uses of biosurfactants are in the food, cos- food can also cause serious infections or intoxi-
metics, and pharmaceutical industries, etc. cation. Infection happened if food swallowed
At present, the cost of production and insuf- contains microbe in number enough to be able to
ficient experience in applications limit the use of pain symptom (Mielmann, 2006), while intoxi-
bioemulsifiers. However, inasmuch as awareness cation as result of toxin produced by microor-
of water quality and environmental conservation ganism (Balia, -).
is increasing and demand for natural products is The spoilage potential of a microorganism
expanding, it appears inevitable that the high is the ability of a pure culture to produce the me-
quality, microbially produced from microorgan- tabolites that are associated with the spoilage of
ism are attracting attention in recent years be- a particular product. In general, several of the
cause they offer several advantages over chemi- organisms isolated from a food product will be
cal surfactant such as low bioemulsifiers will able to produce spoilage metabolites when al-
replace the currently used chemical emulsifiers lowed unlimited growth. It is crucial that quanti-
in many applications (Gautam & Tyagi, 2006). tative considerations are introduced, since the
Food is human basic need. Contamination spoilage activity of an organism lies in its quan-
of food (vegetable) cause disease at consumer. titative ability to produce spoilage metabolites.
Therefore, required food with good quality. These considerations in general, are the imple-
Microorganisms are capable of producing a mentation of a careful combination of microbi-
wide range of chemicals associated with their ology, sensory analysis and chemistry (Miel-
metabolic activities (metabolic by-products) giv- mann, 2006).
ing off-odours and off-flavours that are unac- The most important factors affecting the
ceptable or highly objectionable to the consumer growth of food spoilage bacteria, such as tem-
(Garbutt, 1997). Off-odours and off-flavours are perature, pH, water activity and sodium chloride

(Dwiari et al., 2008). Every microorganisms Vegetable spoilage were recognize by rang-
have range of pH and temperature. In the opti- es of skin fading, vapor bad odor and mucilage
mum pH and temperature, microorganisms can formation, optical density, and number of cells.
grow faster.
Preservation use to avoid danger of food RESULTS AND DISCUSSION
contamination. Preservation process of vegeta-
ble, such as coagulation, drying, salting, sugar Biosurfactant: Fig 1 and 2 showed oil degrada-
(Balia, -). Food dried has lower nutritional value tion by SR_DP.15 and SR_DP.16 bacteria. Fig 1
compared to its (the fresh material). Dying will refers to oil degradation with transparent zone,
reduce water content in food material so that while Fig 2 refers to oil degradation with mix oil
compounds content like protein, carbohydrate, and water. Bacteria produce biosurfactant to de-
fat, and mineral stayed in higher level concentra- grade ALCO. ALCO degraded by bacteria in 24
tion, however vitamins and dye becomes break- h. ALCO in medium as carbon source.
down or reduce. In draining process happened
change of color, skin fading, odor, and others.
There are two types of preservatives: (a).
Natural preservatives: salt, sugar, lemon juice,
vinegar, oil and spices. (b). Chemical preserva-
tives: potassium metabisulphate, citric acid and
sodium benzoate.

Fig 1. Plate assay
Preparation for collecting the cultures, synthet-

ic surfactant (salt benzoate): SR_DP.15 and
SR_DP.16 bacteria were inoculated into
precultures medium and incubated on shaker
incubation until ALCO (Arabian Light Crude
Oil) degraded. Precultures were inoculated into
culture medium and incubated on shaker incuba-
tion until ALCO was degraded. Culture for each
isolate prepares twice. Preculture and culture Control With bacteria
medium is 1/10 MB (Marine Broth) containing
ALCO 1000 ppm. MB per liter containing 3.74 g Fig 2. Fermentation
MB, 15.52 g NaCl, 1000 ml distilled water, and
pH 7.60. Materials were stirred. Precultures, cul- Microorganisms utilize a variety of organic
tures and synthetic surfactant were autoclaved compounds as the source of carbon and energy
for 15 min at 121oC. for their growth. When the carbon source is an
insoluble substrate like a hydrocarbon (CxHy)
Preparation for collecting the crude biosurfac- microorganisms facilitate their diffusion into the
tant: One of SR_DP.15 and SR_DP.16 cultures cell by producing a variety of substances, the
were sentrifuged at 8000 rpm for 20 min at 20oC. biosurfactants (Gautam & Tyagi, 2006). Biosur-
Supernatants used as crude biosurfactant. factants accumulate at interfaces, can form mi-
celles, lower the surface tension and thereby en-
Treatment and observation: Chili soaked into hance the solubility of poorly soluble com-
crude biosurfactant, whole culture, synthetic sur- pounds in water (Kuiper et al., 2004). Some bac-
factant, and chemical preservative for 5 h. teria excrete ionic surfactant, which emulsify
Chemical preservative used in this study was hydrocarbon substrates in the growth medium
sodium benzoate solution 1%. Treated chili in- (Gautam & Tyagi, 2006).
cubated in room temperature within 8 days. Ob- The quality and quantity of biosurfactant
servation was conducted at 0, 3, 6, and 8 days. produced are influenced by the nature of the car-
Chili broken and filtered to applies filter paper, bon substrate, the concentration of nitrogen,
then sentrifuged. Supernatant (crude biosurfac- phosphor, magnesium, ferric, and manganese
tant) applied to measure optical density and plate ions in the medium, and the culture conditions,
count. such as pH, temperature and agitation.

Salt: Salt is employed on large scale, especially of organic acids can delay food spoilage and of-
for meat, fish and vegetables. Salt has retained ten prevent multiplication of pathogens (Cliver,
its importance in food preservation to the present -).
day, although it is now used less as a preserva- Chili generates odor (Table 1) to start sixth
tive in its own right than in combination with days only at some treatment that is positive con-
other preservatives and preservation methods. trol (very bad odor), negative control (moderate)
Salt lowers the water activity of a system and and culture 15 (moderate). At eighth day, culture
thus renders conditions less favorable to micro- 15 increasingly stings (very bad odor). Based on
bial life. In addition, bacteria are more inclined observation of odor, culture 16, biosurfactant
to accumulate certain amino acids when the wa- crude 15 and 16 still be fresh (odorless). Off-
ter activity is lowered, thus salt inhibits their odours can be used to indicate the degree of
growth. Lastly, salt reduces the oxygen solubility spoilage.
in water. Hence, the quantity of oxygen available Chili have no same colour at the beginning.
to aerobic microorganisms in products contain- Based on observation (Table 2), color change at
ing high concentrations of salt is only a fraction sixth day to treatment of positive control, nega-
of that in substances with a low salt content tive control, culture 15, and biosurfactant crude
(Lee, 2004). 15, while at culture 16 and biosurfactant crude
Increasing the quantity of salt in the food 16 still be fresh.
changes its composition. Due to the presence of Data of optical density (OD) (Table 3) indi-
salt in the food, osmosis takes place. As a result, cates that average of OD at initial day is highest
water comes out of the food. When there is no or compared to other day. Bacteria in chili are es-
less water in the food, the microorganisms are timated to more and more along with increasing-
not able to grow and the food becomes safe. ly the duration holding time. But, there is other
factor of making value. OD that is measure and
Preservation: Factors used for food preservation chili colour. Ever greater of chilli measure,
are called „hurdles‟ and there are numerous hur- hence more and more chilli cell which be precip-
dles that have been applied for food preserva- itated into pelet and influences measurement.
tion. Potential hurdles for use in the preservation Chilli colour which still be fresh at initial day
of foods can be divided into physical, physico- also influences measurement. All of Optical
chemical, microbially derived and miscellaneous Density decrease up to eighth day. Culture 15
hurdles (Lee, 2004). Hurdles used for food had smallest OD. It could be said that culture 15
preservation influence the quality as well as the inhibit bacteria contamination better than nega-
safety of foods. The effects of hurdles on food tive control.
quality can be positive or negative, depending on All of chili still fresh at initial day but start
their intensity. mushy at third day and became wrinkle and solid
Every food is an ecosystem for the microor- at the sixth day up to eighth day with different
ganisms it contains. They don't “know” they are level (Table 4). Culture 15 shown best texture in
in food. Food can be contaminated with spoilage eighth day because level of its (the hardness) is
or pathogenic microorganisms. Fruit and vegeta- lowest (+), while negative control is worst be-
ble contamination problems can occur in the cause level of its (the hardness) is highest (+++).
growing environment. During growth the fruit or Number of cells culture 15 and 16, crude
vegetable can become contaminated from biosurfactant 15 and 16 decrease in every obser-
sources, such as soil, water, animals, birds, and vation that shown all of them inhibit microor-
insects. ganism growth better than positive control (Ta-
Consumers require high quality, preserva- ble 5). Beuchat & Hwang (1995) said sodium
tive-free, safe foods with an extended shelflife benzoate is effective in controlling yeast and
(Brul & Coote, 1999). A reason why consumers some bacteria as well as inhibiting the growth
are especially concerned about additives and and mycotoxin production by molds. But maxi-
contaminants is that these are not seen to be in- mal antimicrobial activity of sodium benzoate is
trinsic to the food but are considered as added achieved in low-pH while pH vegetable more
extras. than 5. Therefore, sodium benzoate is less com-
Preservatives are materials added to food patibly to preserves vegetable.
specifically to inhibit microbial growth (Garbutt,
1997). Sorbates, benzoates, and many other salts
Table 1. Odor indicator

Odor of Samples
Crude Crude
Day Control + Control - Culture 15 Culture 16
Biosurfactant 15 Biosurfactant 16
1 2 1 2 1 2 1 2 1 2 1 2
0 × × × × × × × × × × × ×
3 × × × × × × × × × × × ×
6 ++ × + × + × × × × × × ×
8 ++ × + × × ++ × × × × × ×
Note :
x:: odorless +: moderate ++: very bad odor
Table 2.Skin fading indicator

Ranges of Skin Fading of Samples
Crude Crude
Day Control + Control - Culture 15 Culture 16 Biosurfactant Biosurfac-
15 tant 16
1 2 1 2 1 2 1 2 1 2 1 2
0 × × × × × × × × × × × ×
3 × × × × × × × × × × × ×
6 brown, black × × blackness × × × × × blackness × ×
on the bottom on the top
8 × blackness × blackness blackness × blackness × × black × ×
on the top on the top on the top
Table 3. Density indicator

Optical Density of Samples
Day
Control + Control - Culture 15 Culture 16 Crude Biosurfactant 15 Crude Biosurfactant 16
0 2.817 2.853 2.659 2.603 2.817 2.709
3 2.389 2.482 2.128 1.565 2.108 2.329
6 1.669 1.855 1.846 1.567 1.684 2.013
8 0.614 0.755 0.044 1.149 1.130 1.211
Table 4. Skin texture indicator

SkinTexture of samples
Day
0 × × × × × ×
3 mushy ++ mushy ++ mushy + mushy + mushy ++ mushy +
6 dry, dry +++ dry, dry, dry, wrinkle + dry, wrinkle +
wrinkle ++ wrinkle + wrinkle +
8 wrinkle, solid +++ dry, wrin- wrinkle, dry, wrinkle, solid ++ dry, wrinkle, solid ++
solid ++ kle, solid + solid ++
Table 5. Number of cells indicator

Numbers of Cell (104) of Samples
Day
0 4.1 5.1 >300 20 10.1 18.3
3 1 8 31 - 12.5 3
6 >300 6.3 19 18 2.6 -
Crude biosurfactant SR_DP.15 is effective still in cell SR_DP16 which has not been secre-
preserve chili than its culture. Biosurfactant tion by cell.
yielded by SR_DP.15 entirely have been re- In this study, culture and crude biosurfac-
leased in extracellular to media. Biosurfactant in tant (without bacteria) were used to compare
culture not reduce bacterial contamination sig- ability of biosurfactant with and without bacte-
nificantly. Culture SR_DP.16 is effective pre- ria. Bacteria growth inhibition in chilli causes
serve chili than its biosurfactant crude. It is surfactant present noticeable bacteriostatic and
might be the isolate not fully releases biosurfac- biosidal properties thus they can act as multi tar-
tant extracellularly. Estimated that biosurfactant get agents against bacterial cells. In fact, surfac-

tants may exert toxic effects by causing mem- Dainty, R.H., 1996. Chemical/biochemical detection of
brane disruption leading to cellular lysis, by in- spoilage. In: Mielmann, A. 2006. Food spoilage char-
acteristics of Chryseobacterium species.
creasing membrane permeability causing metab-
olite leakage and by altering physical membrane Dwiari, S.R., Asadayanti, D.D., Nurhayati, Sofyaningsih,
M., Frida, S., Yudhanti, A.R. and Yoga, I.B.K.W.
structure or by disruption protein conformation 2008. Teknologi pangan. Jilid 1 untuk SMK. Jakarta:
thus interfering with important membrane func- Direktorat Pembinaan Sekolah Menengah Kejuruan,
tion (Pereira et al., 2007). Direktorat Jenderal Manajemen Pendidikan Dasar dan
Menengah, Departemen Pendidikan Nasional.
CONCLUSSIONS Garbutt, J. 1997. Essentials of food microbiology. In:
Mielmann, A. 2006. Food spoilage characteristics of
According to observation to OD, color, Chryseobacterium species.
odor, texture, and number of cells, crude biosur- Gautam, K.K. and Tyagi, V.K. 2006. Microbial surfactant:
factant SR_DP.15 and SR_DP.16 could preserve a review. J. Oleo. Sci. 55: 155-166.
chili better than the other treatments include Kuiper, I., Ellen, L., Lagendijk, R.P., Jeremy, P.D., Gerda,
chemical preservative (sodium benzoate 1%). E.M.L., Jane, E.T., Ben, J.J.L. and Guido, V.B. 2004.
Characterization of two Pseudomonas putida lipopep-
tide biosurfactants, putisolvin I and II, which inhibit
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Potential Marine Microbes to Produce Polysaccharide Degrading Enzyme
Apridah Cameliawati Djohan1, Ahmad Thontowi1 and Yopi1
1
Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI)
Jl. Raya Bogor Km. 46, Cibinong 16911, Bogor, West Java - Indonesia
Tel: 62-21-8754587; Fax: 62-21-8754588, e-mail : a_camelia707@yahoo.com
(Correspondence to: Apridah Cameliawati Djohan)
ABSTRACT
Indonesia is a country that has tropic marine condition which has plenty of tropical indigenous marine
microbes biodiversity. This research focus on identification potential marine microbe and further utilization of
enzimatic benefit for degrading polysaccharides compound. The identification process have been done by
several qualitative methods such as congo red and lodine solution for specific biomass as a begining
observation. The qualitative analysis showed there are potential microbes which can produce several types of
enzyme, 24 microbes producing cellulase, 28 microbes producing mannanase, 36 microbes producing agarase
and 28 microbes producing amylase. The crude enzyme product resulted by fermentation batch culture between
potential marine microbes and several biomass such as locust bean gum, carboxymethyl cellulose, agar and
starch. This method has intention to know the spesific enzyme which is produce by each microbe such as
mannanase, cellulase, agarase and amylase. The further research are quantitative analysis using Dinitrosalisilic
acid method and Thin layer Chromatography (TLC) analysis to know the spesific enzyme and their activities
which are produce by marine microbes related to variant biomass. The intention of this research is taking an
advantages from marine microbes enzyme for new application in biotechnology fields such as energy, functional
food, bioremediation and biodegrading polysaccharide.
Keywords: Polysaccharide, Marine Microbes, enzymes, Batch Culture.
INTRODUCTION cogen, or as a structural component, as in the

case of cellulose which is found in the cell walls
Polisaccharide is any of a group of poly- of plants. A single cellulose chain may contain
mers made from simple sugar molecules, or as many as 10,000 units of glucose. Polysaccha-
monosaccharides linked by glycosidic bonds. rides are part of the larger family of carbohy-
Polysaccharides are insoluble and may serve as a drates.
store of energy, as in the case of starch and gly-
Fig. 1. Polysaccharide

Fig. 2. Starch
Fig. 3. Cellulose
Fig. 4. Locust Bean Gum Fig. 5. agarose
Locust bean gum is a polysaccharide (a long from α and β-amylase were have function to
chain made of sugars) which combines the hydrolysis starch become maltose sugar.
sugars galactose and mannose. The main chain Amylase is an enzyme that breaks starch
consists of (1-4) linked beta-D mannose residues down into sugar. Enzyme α-amylase can hydro-
and the side chain (1-6) linked alpha-D lyze dietary starch into di- and trisaccharides. All
galactose. amylases are glycoside hydrolyses and act on α-
Enzyme is Substance that acts as a catalyst 1,4-glycosidic bonds. Agarase is an enzyme
in living organisms, regulating the rate at which found in agarolytic bacteria and is the first en-
life's chemical reactions proceed without being zyme in the agar catabolic pathway. It is respon-
altered in the process. Enzymes catalyze all sible for allowing them to use agar as their pri-
aspects of cell metabolism such as digestion of mary source of Carbon and enables their ability
food including proteins, carbohydrates, and fats to thrive in the ocean. Agarase are classified as
are broken down into smaller molecules; the either α-agarases or β-agarases based upon
conservation and transformation of chemical whether they degrade α or β linkages in agarose,
energy; and the construction of cellular materials breaking them into oligosaccharides. When se-
and components. Enzymes are proteins, many creted, α-agarases yield oligosaccharides with
enzymes are spesific to one substrate. Enzymes 3,6-anhydro-L-galactose at the reducing end
are classified by the type of reaction they whereas β-agarase result in D-galactose residues.
catalyze such as oxidation+reduction, transfer of Mannanases have been isolated from a number
a chemical group, hydrolysis, removal or of organisms, such as microorganisms, plants
addition of a chemical group, isomerization and and animals. There are some reports about
binding together of substrate units mannanase that can be useful in several process-
(polymerization). Cellulase are divided into two es in the food, feed as well as in the pulp and
types such as Endo+cellulase which is breaks paper industries (Z. Jiang et al., 2006). At least,
internal bonds to disrupt the crystalline structure there are two type of mannanase; exo-type and
of cellulose and expose individual cellulose endo-type mannanase based on the site of lysis
polysaccharide chains; Exo-cellulase cleaves 2- in the hydrolytic process; exo-acting mannanase
4 units from the ends of the exposed chains is able of removing one or more mannose from
produced by endocellulase, resulting in the the ends of polysaccharide chains while endo-
tertasaccharide or disaccharide such as type mannanase can randomly cleave bonds
cellobiose. Diastase enzyme is a recombinant within the chain.

MATERIALS AND METHODS Enzyme assays: enzyme activities were assayed
as follows: a reaction mixture containing 0.5 ml
Culture: marine bacteria from Pramuka island of 0.3% mannan in 20 mM acetate buffer as well
used in these experiments were identified using as 0.5 ml of enzyme solution in the same buffer
direct sampling method, bacteria sample have was incubated at 40°C for 30 min. The resulting
been collected from sea water. The medium for reducing power was determined by dinitrosali-
Sreening and purification bacteria was composed cylic acid (DNS) method (miller), using D-
with several polysaccharide substrates (0.5 % mannose as a standard. One unit of the man-
Starch, 0.5% CMC, 0,5% LBC and 0,2% Agar), nanase activity was defined as the amount of
0.075% peptone, 0.05% yeast extract and some enzyme liberating 1 µmol of reducing sugar per
minerals compound, pH was adjusted to 6.0, minute under the above condition.
(Mandels & Sternberg, 1976).
Thin layer chromatography: TLC was done on
Identification potential microbes: pure isolates Kieselgel 60 plates using n-butanol: Acetic acid:
were inoculated onto agar medium surface then water (2:1:1) as a solvent. Oligosaccharides were
incubate for 24 hours, medium was rich by carbon detected by heating the plates after spraying with
source from different polysaccharide substrates developed solution aniline hydrogen phthalate.
(0.5 % Starch, 0.5% CMC, 0,5% LBC and 0,2%
Agar), 0.075% peptone, 0.05% yeast extract and RESULTS AND DISCUSSION
some minerals compound (Mandels & Sternberg,
1976). Identification mannanase, cellulase and Screening and Identification: marine bacterian
agarase by bacteria using congo red dye for LBG, have been collected by direct sampling at
agar, and CMC medium. Identification amylase Pramuka island, In this primarly research result
production by bacteria in starch medium using 28 isolates are able to grow in CMC substrate,
iodine solution. 24 isolates in LBG, 36 isolates in agar and 28
isolates in starch. Screening activies showed at
Crude Enzymes Production: the most potential Fig. 6 and marine isolates collection at Fig. 7.
microbes were selected to produce spesific crude For further identification of potential isolates the
enzyme such as amylase, cellulase, mananase and congo red and iodine solution analysis were
agarase. They were inoculated into medium with done, which is result clear zone around the
spesific carbon sources (0.5 % Starch, 0.5% CMC, colonies showed at Fig. 8. The analysis had been
0,5% LBC and 0,2% Agar), 0.075% peptone, resulted 5 isolates potential producing cellulase,
0.05% yeast extract and some minerals compound 1 isolate producing mannanase, 2 isolates
(Mandels & Sternberg, 1976). Incubation process producing agarase and 2 isolates producing
have been doing for 24-48 hours. Crude enzyme amylase. The amount of bacteria which is able to
resulted after centifuge the medium in after degrade the polysaccharide substrates as their
harvest time. carbon source and potential product spesific
enzyme showed at table 1.
A B
C D
Fig. 6. Screening process using sea water direct sampling with several polysaccharide substrate (A) Agar, (B)
Locust Bean Gum, (C) Starch and (D) CMC.

A B C D
Fig. 7. Potential marine microbes which are able to produce agarase (A), cellulose (B), mannanase (C) and amylase (D).
Table 1. Screening and identification marine bacterian from pramuka island.

No. CMC PATI AGAR LBG
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 - - + -
31 - - + -
32 - - + -
33 - - + -
34 - - + -
35 - - ++ -
36 - - + -
Identification qualitative enzyme activities mannanase then for amylase using iodine
for marine bacterias in agar medium using congo solution was showed at Fig. 8.
red dye method for agarase, cellulase and

A B
Fig. 8. Qualitative analysis for enzyme activity using iodine solution (A), congo red (B).
Crude enzyme production: in this research only which is produced after 24 hours incubated in
for potential microbes which have ability to liquid medium for several substrates depend on
degrade polysaccharide substrate and result spesific enzyme that will be analyzed, the
spesific enzyme will be analyzed. Crude enzyme process was showed at Fig. 9.
A B
C D
Fig. 9. Crude enzymes production amylase (A), Mannanase (B), Agarase (C) and Cellulase (D).
Qualitative anaylsis using Thin Layer trisaccharide), and other sugar product. These
Chromatography: the qualitative analysis of analysis using TLC technique with standart such
reaction mixture, when incubating this crude as D-(+)-Glucose, D-(+)-Mannose, Mannotriose
enzymes with spesific substrate are releaze sugar and Mannopentaose, result was showed at Fig.
molecules such as glucose, mannose (di- or 10.

D-(+)-Glucose
D-(+)-Mannose
Manno-triose
di- or tri-saccharide
Manno-pentaose
Fig. 10. Qualitative anaylsis using thin layer chromatograpy method.
The layer resulted disaccharide and is produce from reaction between specific sub-
trisaccharide spot for each bacteria with different strate with crude enzyme from former enzyme
substrates, it was showed that each bacteria had production after centrifuge process. The
potential ability to degrade polysaccharide enzymes analysis showed that bacteria number 4
material become plain sugar molecules which has the highest amylase activity 0.3426 U/ml,
are can used as a functional food composisition bacteria number 3 has the highest mannanase
and other biotechnology application in science activity 0.8537 U/ml, bacteria number 4 has the
and industrial field. highest agarase activity 0.1167 U/ml and the last
is bacteria number 3 has the highest cellulase
Quantitative analysis using DNS method: the activity are not too significant for about 0.0676
analysis was showing the amount of sugar which U/ml.
A B
C D
Fig. 11. Crude enzymes production amylase (A), Mannanase (B), Agarase (C) and Cellulase (D).
In conclusion, Indonesian tropical marine bacteria which is has ability to produce spesific
condition had amazing nature potency not only enzyme such as amylase, cellulase, mannanase
the exotic and wonderful panoramic sight but and agarase. Further research will conducted
also the microorganisms biodiversity. This with genetic approachment dealing with
research showed the quality and quantity spesification for each potencial bacteria.
analysis of enzimatic process from several

REFERENCES Tamaru, Y., T. Araki, H. Amagoi & H. Mori. 1995.
Purification an Characterization of an Extracellular β-
1,4-mannanase from a marine bacterium, vibro sp.
Kagawa, M. & Z. Fujimoto, M. Momma, K. Takase & H.
Strain MA-138. Applied an enviromental
Mizumo. 2003. Crystal structure of baccilus substilis
microbiology. p. 4454-4458.
α-amylase in complex with acarbose. Journal of
Bacteriology. Dec 2003, p. 6981-6983. Yopi, D. Susilaningsih, A. Thontowi, A. Purnawan, A. C.
Djohan, Fahrurrozi & P. Lisdiyanti. 2007. Study on
Kensch, O. 2008. Mannanase engineering for fibre
Hemicellulytic Bacteria: Production of Oligosaccha-
degradation. Speciality Chemicals Magazine, p. 18-
rides from Palm Kernel Cake using Fermentation.
19.
Proceedings in Internasional Seminar “Advances in
Kurakake, M. & T. Komaki. 2001. Production of β- Biological Science:Contribution Towards a Better
mannanase and β-mannosidase from aspergillus Human Prosperity” Faculty of Biology, Gajah Mada
awamori K4 and their properties. Current Universuty, Yogyakarta-Indonesi. September 7-8,
Microbiology. 42: 377-380. 2007, p. 111-113.
Mandels, M. & D. Sternberg. 1976. Recent advances in
cellulase technology. Ferment Technol. 54:267-286.

Study of Factors Affecting the Lipase Production by Yarrowia Lipolytica
NRRL YB-423 in Submerged Fermentation
Asep Muhamad Ridwanuloh1, Eko Wahyu Putro1, Martha Sari1, Hariyatun1 and Wien
Kusharyoto1
1
Jl. Raya Bogor Km. 46 Cibinong, Bogor 16911, Tel. +62 21 8754587, Fax. +62 21 8754588
e-mail: am_ridwanulloh@yahoo.com
(Correspondence to: Asep Muhamad Ridwanuloh)
ABSTRACT
Lipase is an enzyme that presents numerous potentialities for biotechnological application. One of the
important applications of lipase is as biocatalyst in the enzymatic transesterification process for biodiesel
production from plant oils. Different factors affecting the lipase production by Yarrowia lipolytica NRRL YB-
423 in submerged fermentation have been studied. Lipase production was conducted in basal medium with
variation in plant oils, additional carbon and nitrogen sources, respectively. Fermentation was carried out at 30
o
C and 150 rpm, with initial pH 6 of the basal medium. The highest lipase activity produced in basal medium
containing olive oil was obtained after 3 days of culture. The highest lipase activity of 0.08 U/ml was obtained
using the basal medium containing palm oil, which was one-third as high as in the medium containing olive oil.
Further increase in lipase production by Y. lipolytica NRRL YB-423 could be obtained by the addition of carbon
sources other than plant oils and the use of organic nitrogen sources.
Keywords: lipase, Yarrowia lipolytica, submerged fermentation, biodiesel
INTRODUCTION as raw materials. Rapeseed esters are used in

Europe, and palm oil esters were evaluated in
Lipase enzymes has been greatly developed Malaysia as biodiesel. Soybean oil esters are
during the last years. It has been the most featured prominently as potential diesel fuel
important classes of industrial enzyme, since its alternatives, and there is a wide range of ongoing
utilization in the production of detergents, research in this area (Nelson, 1996).
cosmetics, bioenergy, pharmaceuticals, flavour Lipases was produced by animals, plants,
enhancers and foods (Corzo and Revah, 1999; and microorganisms. Lipases which are pro-
cancino et al., 2008). Lipases also have duced by microorganisms have gained special
capability to catalyze, hydrolysis and industrial attention due to their stability, selectiv-
transesterification of esters as well as the ity, and broad substrate specificity (Dutra et al.,
synthesis of esters and enantioselective 2008; Griebeler et al., 2009). A lot of microor-
properties (Treichel, 2010). Due to this function, ganisms are known as potential producers of ex-
lipases have been used as biocatalyst in biofuels tracellular lipases, including bacteria, yeast, and
such as biodiesel production. Biodiesel is a clean fungi (Abada, 2008). Fungal species are prefera-
burning alternative fuel, non petroleum based bly cultivated in solid-state fermentation (SSF),
diesel fuel consisting of short chain alkyl (me- while bacteria and yeast are cultivated in sub-
thyl or ethyl) esters, made by trans-etherification merged fermentation (SmF) (Dutra et al. 2008).
of vegetable oils or animal fats, which can be Y. lipolytica is one of the most extensively
used (alone or blended with conventional petro studied lipolytic yeast, and it has high capability
diesel) in unmodified diesel-engine vehicles. to produce Lipase. The Y. lipolytica is able to
Because of increasing environmental produce several lipases (extracellular, mem-
consciousness, the use of value-added products brane-bound, and intracellular activities), and its
from agricultural fats and oils as biofuels has lipase production depends on media composition
become important. This fact has been affected to and environmental conditions (Lopes et al.,
increasing utilization of lipases in biodiesel 2008). The present paper describes the study of
production. There are a lot of research and in- Factors Affecting the Lipase Production by Y.
dustrial application of biodiesel production using lipolytica NRRL YB-423 in Submerged
lipases, and a lot of fats and oils have been used Fermentation.

MATERIALS AND METHODS and addition of carbon sources were also tested
for their capacity to support the lipase
Microorganism: The yeast Y. lipolytica YB-423 production by this strain. Effect of incubation
was obtained from Agricultural Research times was also investigated to know affecting of
Service (ARS) Culture Collection, NCAUR, US their factors to support the lipase production.
Departement of Agriculture, and is used Effect of incubation times was investigated as
throughtout this study. the first factor to know the time which has max-
imum lipase activity. For investigation of incu-
Inoculum Preparation: Y. lipolytica was bation times, Y lipolytica was cultivated in basal
cultivated in 10 mL of basal medium containing medium containing olive oil for 5 days (Fig. 1).
olive oil 10 g, yeast extract 2.0 g, KH2PO4 0.5 g,
K2HPO4 0.5 g, MgSO4.7H2O 0.5 g, CaCl2.2H2O 0,0600
0.1 g and NaCl 0.1 g per liter solution with
initial pH of 6.0. The strain was incubated at 30 0,0500
Lipase activity (U/mL)

0
C and 150 rpm for 24 hours. 0,0400
Analysis of affecting factors in lipase
production: 1 mL of the inoculum of Y. 0,0300
lipolytica was added into 50 mL of the basal 0,0200

medium (pH 6.0) containing variation of lipid
sources, nitrogen sources and carbon sources in 0,0100
250 mL erlenmeyer flask. -

The culture was incubated for 5 days. 1 mL 24 48 72 96 120
of culture sample (in duplicate) was taken asep- Incubation Times (hours)
tically from the culture every 24 h and subjected
to determination of lipase activity to know effect Fig. 1. Effect of Incubation Time on Lipase Production
of incubation time. The soy bean, canola, palm,
and virgin coconut oils were added to the basal The optimal production of Lipase by Y.
lipase production medium instead of olive oil at lipolytica was obtained after 72 hours of culture;
the same volume, when the effects of carbon with enzyme activity is 0.053 U/mL. This result
sources were being studied. For investigation of was lower than lipase activity produced by other
nitrogen sources effects, similar concentrations strains of Y. lipolytica. Kamzolova et al. (2005)
of soy peptone, urea and NH4Cl compounds screened a huge number of Y. Lipolytica strains
were replaced instead of yeast extract. For and reported lipase activity ranging from 1.8-
investigation of additional carbon sources ef- 45.5 U/mL.
fects, similar concentrations of glycerol, sucrose, Variation of plant oils showed different
fructose, glucose, and maltose compounds were effect on lipase activity. The highest lipase
added to basal medium. activity (0.077 U/mL) was achieved in palm oil
medium and the lowest lipase activity (0.033
Determination of lipase activity: 0.8 mL of U/mL) was achieved in soy bean oil medium
substrate p-nitrophenyl-palmitate (4 mM) in 50 (Fig. 2). Lipase production using some plant and
mM sodium acetate buffer (pH 6.0) were mixed vegetables oils (sunflower oil, corn oil, olive
with 0.2 mL of enzyme extract. Substrate oil,etc.) and animal fats has been studied
hydrolisis was carried out for 15 min at 370C. (Papanikolaou et al., 2007; Papanikolaou, et al.,
The amount of released p-nitrophenol was 2002; Dominguez et al., 2003; Adamczak and
measured spectrophotometrically at 410 nm. One W. Bednarski, 2004).
unit (U) of lipase activity is defined as the
amount of enzyme that produces 1 μmol of
product per minute (Amaral, 2007).
Y. lipolytica NRRL YB-423 was cultivated

in basal medium containing various plant oils as
the sole carbon source, various nitrogen sources

0,0900 sources are used as cometabolism for utilization
0,0800 plant oil in other studies (Corzo and Revah,
1999; Kamzolova, et al., 2007). The result
0,0700
0,0600
makes the production more cost effective and
0,0500
also decreases the biomass.
0,0400
0,0300 0,1800
0,0200 0,1600
0,0100 0,1400

0,0000
0,1200
soy vco canola palm olive
Lipid Source 0,1000
0,0800
Fig. 2. Effect of Lipid Source on Lipase Production
0,0600
0,0400
Plant oils were applied as the sole carbon
source, because Y. lipolytica produces 0,0200
bioemulsifier (Amaral, 2006). The Y. lipolytica 0,0000
Glycerol Sucrose Glucose Fructose Maltose
degrade and oxidise very efficiently hydrophobic
Carbon Sources
substrates, such as fats, oils, alkanes and fatty
acids (Fickers et al., 2005; Papanikolaou et al., Fig. 4. Effect of Carbon Source on Lipase Production
2007).
Generally, addition of nitrogen sources re- In previous studies, investigators usually
sulted in increased lipase activity, but organic used only one or limited number of factor such
nitrogen sources in this research showed no as subtrate, nitrogen and carbon source. One
significant effect on lipase production among advantage of present study is testing more than
them. Addition of soy pepton as nitrogen source one factor and using more kind of plant oils,
to basal medium containing palm oil could small nitrogen and carbon source for knowing their
increase lipase activity to 0.12 U/mL. Organic effect to lipase production. Further study is still
nitrogen source provides nutritional needs necessary to reveal the optimal combination of
(amino acids, vitamins, etc.) for growth and the nutritional components for lipase production
culture supplements for extracellular lipase by Y. lipolytica in submerged fermentation.
production (Barth and Gaillardin, 1997).
CONCLUSION
0,1600
0,1400 Our results show that lipase can be
Lipase Activity (U/mL)
0,1200 produced by Y. lipolytica using renewable low-

0,1000 cost substrates such as plant oils. The maximum
0,0800 lipase were produced under palm oils, yeast
0,0600 extract and maltose effect. Our study suggests
0,0400 that reveal the optimal combination of the nutri-
0,0200 tional components for lipase production by Y.
0,0000 lipolytica in submerged fermentation is still
Yeast Soy Urea NH4Cl
extract peptone necessary.
Nitrogen Sources
REFERENCES
Fig. 3. Effect of Nitrogen Source on Lipase Production
A. Dominguez, M. Costas, M. A. Longo & A. Sanromán.
Additional carbon source such as sucrose 2003. A novel application of solid state culture:
and maltose showed significant effect on lipase production of lipases by Yarrowia lipolytica.
Biotechnology Letters, vol. 25, pp. 1225–1229.
production. Maltose showed the highest effect
for increasing the lipase activity among carbon Abada, E. A. E. 2008. Production and characterization of a
mesophilic lipase isolated from Bacillus
sources. The highest lipase activity of 0.15
stearothermophilus AB-1. Pakistan Journal of
U/mL was achieved in SmF using palm oils, Biological Sciences, 11, 1100–1106.
yeast extract and maltose. Additional carbon

Dutra, J. C. V., Terzi, S. C., Bevilaqua, J. V., Damaso, M. P. F. F. Amaral, A. P. R. de Almeida, T. Peixoto, M. H. M.
C. T., Couri, S., Langone, M. A. P., et al. 2008. Rocha-Leão, J. A. P. Coutinho, and M. A. Z. Coelho.
Lipase production in solidstate fermentation 2007. Beneficial effects of enhanced aeration using
monitoring biomass growth of Aspergillus niger using perfluorodecalin in Yarrowia lipolytica cultures for
digital image processing. Applied Biochemistry and lipase production. World Journal of Microbiology
Biotechnology, 147, 63–75. and Biotechnology, vol. 23, pp. 339–344.
G. Barth and C. Gaillardin. 1997. Physiology and genetics P. F. F. Amaral, J. M. da Silva, M. Lehocky, et al. 2006.
of the dimorphic fungus Yarrowia lipolytica. FEMS Production and characterization of a bioemulsifier
Microbiology Reviews, vol. 19, no. 4, pp. 219–237. from Yarrowia lipolytica. Process Biochemistry, vol.
41, no. 8, pp. 1894–1898.
G. Corzo and S. Revah. 1999. Production and
characteristics of the lipase from Yarrowia lipolytica P. Fickers, P. H. Benetti, Y. Wache, et al. 2005.
681. Bioresource Technology, vol. 70, no. 2, pp. 173– Hydrophobic substrate utilisation by the yeast
180. Yarrowia lipolytica, and its potential applications.
FEMS Yeast Research, vol. 5, pp. 527–543.
Griebeler, N., Polloni, A.E., Remonatto, D., Arbter, F.,
Vardanega, R., Cechet, J.L. et al. 2009. Isolation and S. Papanikolaou, I. Chevalot, M. Galiotou-Panayotou, M.
screening of lipaseproducing fungi with hydrolytic Komaitis, I. Marc, and G. Aggelis. 2007. Industrial
activity. Food and Bioprocess Technology derivative of tallow: a promising renewable substrate
doi:10.1007/s11947-008-0176-5. for microbial lipid, single-cell protein and lipase
production by Yarrowia lipolytica. Electronic
H. Treichel, D. de Oliveira, M.A. Mazutti, M.D. Luccio, J. Journal of Biotechnology, vol. 10, no. 3, pp. 425–435.
V. Oliveira. 2010. A review on microbial lipases
production. Food Bioprocess Technol 3:182–196. S. Papanikolaou, I. Chevalot, M. Komaitis, I. Marc, and G.
Aggelis. 2002. Single cell oil production by Yarrowia
L.A. Nelson, T. A. Foglia, and W. N. Marmer. 1996. lipolytica growing on an industrial derivative of
Lipase-Catalyzed Production of Biodiesel. JAOCS, animal fat in batch cultures. Applied Microbiology
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Yarrowia lipolytica by immobilization on biomass Yarrowia lipolytica yeast grown on animal and
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Lipase Production by Yarrowia lipolytica in Solid-State Fermentation
Utilizing Agroindustrial Residues as Substrate
Hariyatun1, Martha Sari1, Eko Wahyu Putro1, Asep Muhamad Ridwanuloh1
and Wien Kusharyoto1
1
Jl. Raya Bogor Km. 46 Cibinong Bogor 16911
Tel. +62 21 8754587, Fax. +62 21 8754588, e-mail: hariyatunsaksono@yahoo.com
(Correspondence to: Hariyatun)
Abstract
The potential use of lipases in a wide range of industries, including biodiesel industry, has been
proposed to increase process efficiency. The main hurdle in the application of lipase in the transesteri-
fication process for biodiesel production is the cost of lipase production. The enzyme is predominantly
produced by conventional submerged fermentation (SmF), a more expensive high technology process.
An economical alternative for enzyme production and application would be solid state fermentation
(SSF). Lipase production by Yarrowia lipolytica NRRL YB-423 in SSF using different substrates has
been studied. Agroindustrial residues, such as rice bran, wheat bran and tofu cake, as alternative cheap
and easy to obtain solid substrates were used alone or as combined substrate (1:1) in order to optimize
the lipase production. The SSF were performed in flasks at 30oC, with initial pH of 6.5 of the basal
medium added to SSF medium. The highest lipase activity is 1.46 U/g dry substrate was obtained after
7 days of culture with combined substrate (1:1) of tofu cake and wheat bran. Using combined sub-
strates (1:1) of tofu cake and wheat bran, the highest lipase activity is 2.38 U/g dry substrate was ob-
tained at 90% initial moisture after 9 days of culture.
Keywords: lipase, Yarrowia lipolytica, solid-state fermentation, agroindustrial residues
INTRODUCTION Castilho, 2000, Sharma et al., 2001; Castro et al.,

2004; Ranganathan et al., 2008; Tamalampudi et
The high potential applications of lipases al., 2008).
(triacylglycerol acylhydrolases), particularly Due to relatively expensive cost of lipase
those commercially significant produced by mi- production process in SmF, development of an
croorganism, as one of the leading enzymes (bio- economical alternative bioprocess of lipase pro-
catalysts) in wide range of industries have reduction by utilization of cheap and easy to obtain
cently become focus of a renewed interest in the agroindustrial residues in SSF could be the most
development of sources of lipases (Newmark, promising technique since many advantages are
1988; Harwood, 1989; Threichel et al., 2009). offered, such as less capital investment, impler
Lipases are hydrolytic enzymes that act in aque- techniques, high productivity, relatively easy
ous-organic interfaces, catalysing the cleavage of recovery of extracellular enzymes, low
ester bonds in triglycerides and producing glyc- wastewater output and higher lipase extract sta-
erol and free fatty acids. However, in environ- bility (Domínguez et al., 2003; Adinarayana et
ments with low water availability, lipases are al., 2004; Wolski et al., 2009). The technique of
able to catalyse esterification, interesterification SSF involves the growth and metabolism of mi-
and transesterifcation reaction, being thus very croorganisms in the absence or near absence of
versatile biocatalysts (Pandey et al., 1999; Shar- free water, employing a solid substrate or sup-
ma et al., 2001). Due to the versatile of the mo- port. SSF has proven to be an efficient way to
lecular structure and catalytic properties, these produce enzymes since it provides the microor-
enzyme have potential application in defferent ganisms with environmental conditions similar to
industrial sectors, such as biodiesel, food, cos- their natural habitat (Pandey et al., 1999). Pro-
metics, pharmaceutics, medicines, detergents, cesses using solid substrate, especially agroin-
emulsifier, cleaner, leather, pulp and paper, fine dustrial residues, are economically important for
chemicals, waste water treatment etc. (Freire & countries, such as Indonesia, which can provide

cheap and abundant raw materials to reduce pro- lipolytica was added into the sterilized substrate,
duction costs (Freire & Castilho, 2000; Damaso mixed thoroughly and incubated at 30 oC for a
et al., 2008). Due to their potential advantages, set of incubation time. Parameters of SSF exam-
utilization of agroindustrial residues provides ined were substrate or substrate combination,
alternative substrates and may help solving pol- incubation time and initial moisture content.
lution problems, which otherwise might be
caused by their disposal (Threicel et al., 2009). Optimization of Substrates: Different substrates
Yarrowia lipolytica has the extensive of agroindustrial residues were used alone, i.e.
lipolytic activity, can be versatile with respect to rice bran (R), wheat bran (W) and tofu cake (T);
its metabolic abilities and can be exploited for a or as combined substrate (1:1), i.e. rice bran +
variety of purposes (Bankar et al., 2009). This wheat bran (RW), rice bran + tofu cake (RT) and
research studied different basic parameters on wheat bran + tofu cake (WT), in order to opti-
lipase production by Y. lipolytica in SSF utilizing mize the lipase production. The initial moisture
the agroindustrial residues as substrate. content of 80% was adjusted and incubated for 7
days.
Effect of Incubation Time: The moisture content
Microorganism: The yeast Yarrowia lipolytica of the medium is adjusted to 80% to the optimum
NRRL YB-423 was obtained from Agricultural substrate. The lipase activity produced during
Research Service (ARS) Culture Collection, SSF is observed daily in a period of 10 days.
NCAUR, US Departement of Agriculture, and
was used throughout this study. Effect of Initial Moisture Content: The moisture
content of the medium is adjusted to 70, 80, 90
Maintenance of the yeast: The yeast Y. lipolyti- and 100%, respectively, by the addition of a cor-
ca is maintained in YPG-Agar Medium contain- responding volume of sterile deionized water to
ing (per liter distilled water): yeast extract 5.0 g, the optimum substrate. The lipase activity is de-
soy peptone 10.0 g, glucose 15.0 g and agar 15.0 termined after optimum days of culture.
g. The pH of the medium was adjusted to 6.5,
and the yeast culture was incubated at 30oC for Crude Enzymes Extraction: The fermented 1 g
48 h. Subculturing is carried out in every 2 of substrate is mixed thoroughly with 5 mL of 50
weeks, and the culture is stored at 4oC. mM phosphate buffer (pH 7.0), and incubated in
a rotary shaker for 60 min at 37oC and 150 rpm.
Inoculum Preparation: The yeast Y. lipolytica The crude enzyme extract is obtained by filtra-
was inoculated into 25 ml of YP Medium con- tion of the mixture and subsequent centrifugation
taining (per liter distilled water): soy pepton 5.0 at 4oC and 6000 rpm for 10 min. The supernatant
g, yeast extract 3.0 g and NaCl 3.0 g, and incu- from the centrifugation is subjected to the deter-
bated in an incubator shaker at 30oC and 150 rpm mination of lipase activity.
for 24 h (Oswal et al., 2002).
Determination of Lipase Activity: The substrate
SSF: Ten grams of substrate are weighted into a p-nitrophenyl-palmitate (4 mM) is dissolved in
250 mL erlenmeyer flask, and autoclaved at 50 mM sodium acetate buffer (pH 7) containing
121oC for 15 min. Further, 1 mL of sterile basal 4% Triton X-100. The reaction mixture compos-
medium (medium WK) is added. The composi- es of 0.95 mL of substrate solution and 0.05 mL
tion of medium WK (per 100 mL of distilled wa- of culture medium. Substrate hydrolysis is car-
ter) are urea 2.0, KH2PO4 1.0, MgSO4.7H2O 0.5, ried out for 15 min at 37oC and 150 rpm, and the
CaCl2.2H2O 0.13 g, NaCl 0.1 g and trace ele- reaction is stopped by the addition of 1 mL of
ments 100.0 μL, then adjusted to pH 6.5 with 6 acetone. The amount of released p-nitrophenol is
M HCl. Whereas, the trace elements composition measured spectrophotometrically at 410 nm. One
(per 100 mL of distilled water) are H3BO3 0.5 g, unit of lipase is defined as the amount of the en-
CuSO4.5H2O 0.04 g, FeCl3.4H2O 0.2 g, zyme liberating 1 μmol p-nitrophenol per minute.
ZnSO4.7H2O 0.4 g, MnSO4.H2O 0.4 g and 0.008
g/10 mL sterile filtered d-biotin 1 ml. RESULTS AND DISCUSSION
The selected initial moisture content for the
substrate was adjusted by the addition of sterile In SSF, the selection of a suitable solid sub-
deionized water. Two mL of the inoculum of Y. strate for the fermentation process is a critical

factor. Agroindustrial residues containing all the later stage, when nutrients are depleted, it reach-
components necessary for microbial develop- es its stationary phase and can start to produce
ment could be used as substrate to enhanced the secondary metabolites, thus resulting in a lower
production of lipases because rich in fatty acids, yield of enzyme (Özdemir et al., 2009). The
triacylglycerols and/or sugars as nutritive sub- highest lipase activity of those 3 phases after dai-
stances for the growth and metabolism of micro- ly observation during a period of 10 days was on
organism (Babu and Rao, 2007). The highest 9 days, which was longer than other strains of Y.
lipase activity among all the substrates (Fig. 1) lipolytica using mixed substrate of sugarcane
was obtained with combined substrates (1:1) of bagasse and wheat bran observed by Babu and
tofu cake and wheat bran (1.46 U/g dry sub- Rao (2007). In subsequent experiments, there-
strate), which was used as a substrate in later fore, 9 days was used as incubation time for the
studies. This lipase activity was lower than lipase production of lipase.
activity produced by other strains of Y. lipolytica
using mixed substrate of sugarcane bagasse and
wheat bran observed by Babu and Rao ( 2007).
The nature of the substrate is the most important
factor affecting fermentative processes. The
choice of the substrate depends upon several fac-
tors, mainly related to nutritive contents, cost and
availability (Threicel et al., 2009). Thus, process
optimization may involve the screening of sever-
al agroindustrial residues for microbial growth
and product formation. Confirming that com-
bined tofu cake and wheat bran were the most
suitable substrate possibly due to the higher con- Fig. 2. Effect of incubation time on lipase production.
tent of protein in tofu cake and carbohydrates in
wheat bran (Gomes, 1995 in Damaso et al., SSF is defined by the absence of free water
2008). component, however the substrate must possess
enough moisture. Moisture content of the sub-
strate plays a vital role to support the growth and
metabolism (enzymes biosynthesis and secretion)
of microorganism. The moisture level in SSF has
a great impact on the physical properties of the
solid particles (Mahanta et al., 2008). The neces-
sary moisture in SSF exists in the absorbed or
complex form within the solid matrix, which is
likely to be more advantageous for growth be-
cause of the possible efficient oxygen transfer
process. Higher moisture content can cause the
decreased porosity, change in particle structure,
Fig. 1. Substrate optimization on lipase production. development of stickiness, reduction in gas vol-
ume and decreased diffusion, thus limiting the
Incubation time depend on the characteris- oxygen transfer. Otherwise, lower moisture con-
tics of the culture, on growth rate and enzyme tent can cause the reduced solubility of nutrients
production. Fig. 2 showed a gradual increase in in substrate, low degree of swelling and high wa-
enzyme production and after which a gradual ter tension (Adinarayana et al., 2004; Babu and
decrease was observed in different 3 phase of Rao, 2007). Using combined substrates (1:1) of
increased lipase production during 10 days of tofu cake and wheat bran the highest lipase activ-
culture, i.e. at 2, 7 and 9 days. The reaction for ity after 9 days of culture was obtained at 90%
maximum enzyme production could be due to initial moisture, i.e. 2.38 U/g dry substrate (Fig.
the fact that the microorganism was in its expo- 3). This result was not in good agreement with it
nential phase. Moreover, the decrease in enzyme has been reported by Gangadharan (2006) in
yield of incubation may be the result of denatura- Özdemir et al. (2009) that moisture levels in SSF
tion or decomposition of lipase due to interaction processes vary between 30 and 85 %. However,
with other components in the medium. At the

it is evident that the enzyme activity is subject to Castro, H. F., A. A. Mendes & J. C. Santos. 2004. Modifi-
the water holding capacity of the substrate and cação de oleos e gorduras por biotransformação.
Quimica Nova. 27 (1): 14-156.
that it varies from substrate to substrate.
Damaso, M. C. T., M. A. Passianoto, S. C. de Freitas, D. M.
G. Freire, R. C. A. Lago & S. Couri. 2008. Utilization
of agroindustrial residues for lipase production by sol-
id-state fermentation. Brazilian J. Microbiol. 39: 676-
681.
Domínguez, A., M. Costas, M. A. Longo, & A. Sanromán.
2003. A novel application of solid state culture: pro-
duction of lipases by Yarrowia lipolytica. Biotechnol.
Letters. 25 (15): 1225-1229.
Freire, D.M.G. & L. R. Castilho. 2000. Lipases produzidas
por fermentacao submerse e em meio solido. Rev.
Bras. Farmacia. 81: 48-56.
Harwood, J. 1989. The versality of lipases for industrial
uses. Trends in Biochem. Sci. 14: 125-126.
Fig. 3. Effect of initial moisture content on lipase produc- Mahanta, N., A. Gupta & S. K. Khare. 2008. Production of
tion. protease and lipase by solvent tolerant Pseudomonas
aeruginosa PseA in solid-state fermentation using
These results encourage us for further opti- Jatropha curcas seed cake as substrate. Bioresour.
Technol. 99 (6): 1729-1735.
mize the other parameters for lipase production
in SSF utilizing economically available raw ma- Newmark, P. 1988. Two European Companies Market Li-
terials, with the purpose to enable its industrial pases. Bio/Technol. 6: 369.
applications. Oswal, N., P. M. Sarma, S.S. Zinjarde & A. Pant. 2002.
Palm oil mill effluent treatment by a tropical marine
yeast. Bioresour. Technol. 85: 35–37.
CONCLUSIONS
Özdemir, S., K. Güven, Z. Baysal & F. Uyar. 2009. Screen-
ing of various organic substrates and the development
The highest lipase activity was obtained of a suitable low-cost medium for a-amylase produc-
with combined substrate (1:1) of tofu cake and tion by B. subtilis. Food Technol. Biotechnol. 47 (4):
wheat bran. Moreover, with combined substrates 364–369.
(1:1) of tofu cake and wheat bran, the highest Pandey, A., S. Benjamin, C. R. Soccol, P. Nigam, N. Krieg-
lipase activity was obtained at incubation time of er & V. T. Soccol. 1999. The Real of Microbial Li-
9 days and initial moisture content of 90%. pases in Biotechnology. Biotechnol. Appl. Biochem.
29: 119-131.
ACKNOWLEDGEMENT Ranganathan, S.V., S. L. Narasimhan & L. Muthukumar.
2008. An overview of enzymatic production of bio-
diesel. Bioresour. Technol. 99 (10): 3975-3981.
This work was partially supported by a
grant from DIPA SEAMEO-BIOTROP 2009 to Sharma, R., Y. Chisti & U. C. Banerjee. 2001. Production,
WK. purification, characterization, and application of lipas-
es. Biotecnol. Adv. 19: 627-662.
REFERENCES Tamalampudi, S., M. R. Taklukder, S. Hama, T. Numatab,

A. Kondo & H. Fukuda. 2008. Enzymatic production
of biodiesel from Jatropha oil: a comparative study of
Adinarayana, A., K.V.V.S.N.B. Raju, M.I. Zargar, R.B.
immobilized-whole cell and commercial lipases as a
Devi, P.J. Lakshmi & P. Ellaiah. 2004. Optimization
biocatalyst. Biochem. Eng. J. 39 (1): 185-189.
of process parameters for production of lipase in sol-
id-state fermentation by newly isolated Aspergillus Threichel, H., D. de Oliveira, M.A. Mazzuti, M. Di Luccio
species. Indian J. Biotechnol. 3: 65-69. & J. V. Oliveira. 2009. A review on microbial lipases
production. Food Bioprocess Technol.
Babu, I.S. & G. H. Rao. 2007. Lipase production by Yar-
rowia lipolytica NCIM 3589 in solid state fermenta- Wolski, E., E. Menusi, D. Remonatto, R. Vardanega, F.
tion using mixed substrate. Research J. Microbiol. 2 Arbter, E. Rigo, J. Ninow, M. A. Mazutti, M. Di Luc-
(5): 469-474. cio, D. de Oliveira & H. Treichel. 2009. Partial char-
acterization of lipases produced by a newly isolated
Bankar, A.V., A. R. Kumar & S. S. Zinjarde. 2009. Envi-
Penicillium sp. in solid state and submerged fermenta-
ronmental and industrial applications of Yarrowia lip-
tion: A comparative study. LWT - Food Sci. Technol.
olytica. Appl. Microbiol. Biotechnol. 84 (5): 847-865.
42 (9). 1557-1560.

Palm Kernel Cake Biomass Fermentation Using Stirrer Fermentor
for Mannanase and Saccharides Production
Yopi1, Dwi Susilaningsih1, Awan Purnawan1, Heri Hermansyah2, Ahmad Thontowi1,
Apridah C. Djohan1, Swastika Praharyawan1 and Puspita Lisdiyanti1
1
Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jalan Raya Bogor Km.
46, Cibinong, Bogor, 16911, e-mail: yop_i@yahoo.com
2
Faculty of Chemical Engineering, Indonesia University, Depok, 16424.
(Correspondence to: Yopi)
ABSTRACT
Indigenous biomasses such as palm kernel cake contains of high heteromannan and useful to produce
mono-saccharides and oligo-saccharides which have potential roles as a component in functional food.
Fermentation process which is utilized palm kernel cake as a substrate has done by using stirrer fermentor scale
2 L. Degradation of palm kernel cake by Saccharopolyspora flava were showed that mono-saccharides and
oligo-saccharides have been produced during fermentation process, but the yield production of saccharides are
still low due to the complex structure of the polysaccharides in palm kernel. There are no significant changing
from both temperature and pH during the fermentation process. The palm kernel cake fermentation was done in
condition 2.5% substrate with 200 rpm in agitation for 4 days. Fermentation product for the first 24 hours are
monosaccharide and for the next 28 hours was produced oligosaccharides. The optimum oligosaccharide
production was occured at 44 hours fermentation, afterwards the oligosaccharide were degraded consecutively.
Final fermentation product are combination between monosaccharide and oligosaccharide.
INTRODUCTION Biomass waste from plantation industry,

agricultural and forest products in Indonesia
Mannan is a source of biomass after which is containing of mannan polysaccharides
cellulose and xylan are a lot of waste contained can be utilized for the production mannosa,
in palm oil, copra and coffee, which is currently oligosaccharides and other saccharide, especially
in motherland is not widely utilized. Mannan can waste from the production of palm oil, copra and
be hydrolized became mannosa and coffee. Oil palm is interesting commodity in
oligosaccharide and functioning as a prebiotic by Indonesian industry at the moment. Basically,
enzymes such as endo β-mannanase (1,4-β-D- the oil palm industry in Indonesia is still focused
mannan mannanohydrolase [EC 3.2.1.78]) and on the production of CPO. Still few industries
exo β-manosidase (β-D-mannanopyranoside are investing in derivative products such as palm
hydrolase [EC 3.2.1.25]) (Puls et al., 1993). kernel oil and utilization of waste from each
Ajimonoto General Foods has developed a stage of this CPO production. One of the wastes
patent for coffee products containing manno- from the palm oil industry is Palm kernel cake
oligosaccharides that is derived from Mannan is (PKC) which is a by-product from palm kernel
much contained in coffee grounds. Has been oil processing into palm kernel oil (PKO). All
reported functions of manno-oligosaccharides this time PKC was primarily intended as animal
from coffee grounds such as new prebiotic feed, almost half of the literature indicates that
compounds good for the growth of positive low-quality palm oil cake because of high crude
bacterial microflora in the human digestive fiber content and essential amino acid content is
system. In addition, the manno-oligosaccharide low. Therefore, initial recommendation on the
is predicted to have functions to boost the use of palm oil cake in animal feed is only
immune system and reduce the intensity of the around 10-25% (Jalaluddin, 2001).
binding of bacterial pathogens which are The main content of palm kernel cake
Salmonella enteriditis and Escherichia coli on Mannan is a polysaccharide, which is about 20 ~
the cell surface because of the similarity in 40%. Its texture is hard complicating the process
structure with the existing surface saccharide of utilization of palm kernel cake, the necessary
living cells (Sachslehner et al., 2000; Toeda et strength of microbes that can produce the
al., 2002). enzyme mannanase suitable for degradation of

Mannan on the palm oil cake. Research related oligosaccharide compounds formed will be
to the utilization of oil palm waste and microbial identified by Thin Layer Chromatography
applications mannolitik and mannanase enzymes (TLC).
produced have been done (Purwadaria, 1994,
1998). Whereas, regarding the production and Enzyme assays: supernatant of culture media
application of mannannase enzymes has been after centrifugation process were collected as
reported such as from Bacillus pumilus (Toshiro raw enzyme solution and analysis of the enzyme
et al., 1989), Caldocellum saccharolyticum activity using DNS methods. Enzyme activities
(Gibbs et al., 1991) and Streptomyces spp. were assayed as follows: a reaction mixture
In a preliminary study reported that containing 0.5 ml of 0.3% mannan in 20 mM
Saccharopolyspora flava bacteria can produce acetate buffer as well as 0.5 ml of enzyme
mannanase, cellulase and xylanase (Yopi et al., solution in the same buffer was incubated at
2008). Saccharopolyspora flava dominant 40°C for 30 min. The resulting reducing power
producing mannanase enzyme on a substrate of 1 was determined by dinitrosalicylic acid (DNS)
~ 2% palm kernel cake, whereas the consortium method (Miller, 1959), using D-mannose as a
of Saccharopolyspora flava and two kinds of standard. One unit of the mannanase activity was
Streptomycetes bacterias produce three enzymes defined as the amount of enzyme liberating 1
which is stable at substrate concentration of 4% µmol of reducing sugar per minute under the
palm kernel cake. above condition.
The paper reported the fermentation process
of palm kernel cake with Saccharopolyspora Protein Analysis: analysis of protein content was
flava using 2 Liter stirred fermentor scale and done by spectrophotometer readings at = 280
observe the mananase enzyme and nm as a standard solution of Bovine Serum
oligosaccharide products was produced during Albumin (BSA) is used with several different
the fermentation process. concentrations.
MATERIALS AND METHODS Analysis of Fermentation Products Using TLC:

thin layer chromatography was done on
Bacteria and substrate: Saccharopolyspora Kieselgel 60 plates using n-butanol: Acetic acid:
flava strain used in these experiments were water (2:1:1) as a solvent. Oligosaccharides were
identified from about 500 isolated bacteria detected by heating the plates after spraying with
belong to Biotechnology Culture Collection developed solution aniline hydrogen phthalate.
(BTCC) (Yopi et al. 1996). Palm kernel cake
was obtained from PT Indofeed (Bogor), which RESULTS AND DISCUSSION
is produced from plantations in Lampung.
Early stages of fermentation carried out by
Fermentation: fermentation process using airlift fermentor with 2% concentration of palm
stirred fermentor 2 L have been conducted at kernel cake as a substrate (Figure 1). During the
room temperature and incubated for 4 days. fermentation process in order to shuffle the
Preculture medium and production medium added anti-foam can run stably. Selama proses
contains of palm kernel cake substrate, yeast fermentasi ditambahkan anti foam agar
extract 0.05%, bacto pepton 0.075%, (NH4)2SO4 pengocokan dapat berjalan secara stabil.
0.14%, KH2PO4 0.2%, MgSO4.7H2O 0.03%, Reaction process at room temperature and occur
CO(NH4)2 0.03%, CaCl2 0.03%, FeSO4.7H2O naturally. There is no process of adding a
0.0005%, MnCl2.7H2O 0.00016%, ZnSO4.7H2O parameter and stabilizing. At this early stage
0.00014 %, and CoCl2 0.0002%. After the will only be viewed as a natural fermentation
fermentation process during the fourth day, crude process and analyze the saccharide products and
enzyme solution and the fermentation products mannanase enzymes produced during the
obtained from the fermentation solution process. The fermentation was conducted for
centrifuged at a speed of 6000 rpm for 10 five days and sampling was performed each day
minutes. Supernatant obtained was of samples to for continuous, microbial growth during
be analyzed enzyme activity, content protein and fermentation showed palm oil cake substrate
degradation process by S. flava.

Fig. 1. Fermentation PKC with stirrer fermentor.
The sample of the first day until the fifth that pH control during fermentation is not so
day of the analyzed protein content, pH, enzyme necessary, because the process of substrate
activity and concentration of microbial cells. hydrolysis reaction Mannan has been predicted
Result of pH analysis is in Table 1, show after on the first three days. This is in accordance with
four days of fermentation pH value decreased. the measurement of enzyme activity and cell
This result is similar to the results of flask concentration mannanase.
fermentation with a small size. This indicates
Table 1. Analysis of changes in pH during fermentation of the day 1 ~ 5.

No. Day pH measurement I pH measurement II
9:00 a.m. 3:00 p.m.
1 0 6.02 6.14
2 1 6.78 6.90
3 2 6.86 6.77
4 3 6.29 5.78
5 4 5.41 5.57
Growth of microbial cells during produce two types of mannanase enzyme, endo
fermentation begins to happen on the first day and exo type mannanases. Enzyme exo-type
increased continuously until the third day and mannanase in the early stages of fermentation is
then declined on the fourth day. Soluble protein necessary to cut the substrate in stages from the
content was the correlation with the existing end of the chain. Mannanse exo-type is produced
enzyme content did not change much until the by bacteria primarily on the first day of
third day, but increase sharply on the fourth day. fermentation. This is consistent with the results
These results are consistent with the mannanase of the fermentation product analysis by TLC
enzyme activity gradually increased and the which showed the main product at the beginning
highest on day four with an activities value 0154 of fermentation is a monosaccharide (Fig. 2).
U / ml (Table 2). S. flava predicted could

Table 2. Analysis of enzyme activity, protein content and concentrations microbial during fermentation.
Sampling Mannanase activity Protein Content Microbial Concentration

No
Day Time (U/ml) (mg/ml) (OD) 660)
1 0 9 a.m. 0.009 5.783 0.828
2 1 9 a.m. 0.005 5.976 0.883

3 p.m 0.006 5.145 1.048
3 2 9 a.m. 0.013 4.456 1.620
3 p.m 0.020 5.904 1.129
4 3 9 a.m. 0.070 5.759 2.360
3 p.m 0.057 5.493 2.661
5 4 9 a.m. 0.143 10.730 1.815
3 p.m 0.154 10.615 1.883
Monosaccharide in the early formation of oligosaccharide is then used by bacteria to

fermentation helps bacteria to grow more rapidly hydrolyzed become monosaccharide by
and this can induce the bacteria to produce a metabolic process. This is seen on the TLC
dominant endo type mannanase hydrolyze results showing on the fourth day starting
randomly. These results are consistent with the monosaccharide produced again. This interesting
analysis of fermentation products by TLC which phenomenon shows what happens during this
showed that the oligosaccharides consisting of fermentation process is the synergy of
various sizes began to form on the second day mannanase enzymes that have different substrate
onwards. Along with the fermentation time, specifically.
Fig. 2. Analysis of fermentation products from palm kernel cake with TLC.
The above results indicate that the process and oligosaccharides from PKC
oligosaccharide and enzyme can be produced by biomass.
using S.flava and PKC on the second and the
third day of fermentation process. At this stage ACKNOWLEDGEMENTS
the entire fermentation process is still done
manually and focuses on the analysis of The research was funded by Competitive
oligosaccharides are formed. For the next phase Research Grant 2006~2008 (Sub-Program of
will be optimized by changing the parameters of Product, Commodity and Technology)
fermentation temperature, agitation and Indonesian Institute of science (LIPI).
concentration of substrate and other parameters
in order to obtain various types of data that can
be used as material to determine the most
efficient parameters for enzyme production

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Haltrich. 2000. Hydrolysis of Isolated Coffee Mannan
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Characterization of three ß-Mannanase of an
Gibbs, M. D., D. J, Saul., E. Luthi & P. L. Bergquist. 1992. Alkalophilic Bacillus sp. Agric. Biol. Chem. 52(3):
The β-Mannanase from “Caldocellum 773-779
saccarolyticum” Is Part of a Multidomain Enzyme.
Apllied And Environmental Microbiology: 58:3864- Yopi, A. Thontowi, D. Susilaningsih & P. Lisdiyanti. 2006.
3867. Analisa mikroba mannolitik dari strain BTCC.
Prosiding Seminar Nasional Bioteknologi 2006. p
Jalaludin, S., Y. W. Ho, N. Abdullah & H. Kudo. 1991. 442-448.
Strategis for Animal Improvement In Shoutheast Asia.
In: Utilization of feed Resources in Relation to Yopi, D. Susilaningsih, A. Thontowi, A. Purnawan, A. C.
Utilization and Physiology of Ruminants in the Djohan, Fahrurrozi & P. Lisdiyanti. 2007. Study on
Tropics. Trop Agric. Res. Series 25 pp. 67-76. Hemicellulytic Bacteria: Production of
Oligosaccharides from Palm Kernel Cake using
Miller, G. L. 1959. Use of Dinitrocyclic Acid Reagent For Fermentation. Proceedings in Internasional Seminar
Determination of Reducing Sugar. Anal Chem. 31: “Advances in Biological Science:Contribution
426-428. Towards a Better Human Prosperity” Gajah Mada
Purawadaria, T., T. Haryati & J. Darma. 1994. Isolasi dan University. p 111-113.
seleksi kapang mesofilik penghasil mananase.
Majalah Jurnal dan Peternakan, Maret :26 -29.
Purwadaria, T., A. P Sinurat, T. Haryati, I. Sutikno & J.
Darma. 1998. Korelasi antara aktivitas enzim
mannanase dan sellulase terhadap kadar serat Lumpur
sawit hasil fermentasi dengan aspergillus niger.
Jurnal Ilmu Ternak dan Veteriner, Vol.3, No.4: 230-
236.

Mannolytic Activities of Aspergillus sp. BL5 Grown
on Different Carbon Substrates
Ahmad Thontowi1, Nanik Rahmani1 and Yopi1
1
Jalan Raya Bogor Km. 46, Cibinong, Bogor 16911, e-mail: athontowi@yahoo.com
(Correspondence to: Ahmad Thontowi)
ABSTRACT
The major constituents of hemicelluloses are 1,4--mannan. Mannan had been contained in several Indo-
nesian biomasses, such as palm kernel cake (PKC), konjac potato, coconut and coffee. They are a tropical agro-
industrial byproduct. A large number of agro-industrial residues have already been utilized in fermentative pro-
cesses to produce value added products. The production of mannanase by Aspegillus sp. BL5 from several
carbon sources were studied. The highest mannanase activity was showed in reaction between Aspergillus sp.
BL5 with PKC and residue of coconut substrates. The optimal result were obtained at 96 hours incubation
respectively in PKC and residue of coconut 1.3 and 1.2 U/ml.
Keywords: biomass, mannan, mannanase, Aspergillus
INTRODUCTION investment, and lower downstream processing

costs (Mitchell & Lonsane BK 1992). Environ-
Endo-1, 4--D-mannanase (EC. 3.2.1.78) is mental factors such as temperature, the moisture
a hemicellulytic enzyme that randomly content of the substrate and airflow significantly
hydrolyzes 1, 4--D-mannopyranosyl linkages affect SSF process, which in turn influences the
within the main chains of mannans and fermentation products (Pandey et al. 1999).
heteropolysaccharides such as galactomannans, Although several studies have been carried
glucomannans and galactoglucomannans out on the use of hemicellulosic compounds in
(Ademark et al. 1998). The manooligosaccha- the production of -mannanase via SmF process
rides released are further hydrolyzed by -D- (Lin & Chen 2004). Few attempts have been
mannosidase, -D-glucosidase and -D- made to produce -mannanase using agro-
galactosidase to produce mannose, glucose and industrial carbon sources in the SSF process
galactose (Burke & Cairney 1997). (Kurakake & Komaki 2001). On the other hand,
A variety of fungi are capable of producing most studies on b-mannanase production by fun-
mannan degrading enzymes. Aspergillus species gi under SSF have focused on the production of
are well known for their high potency in the pro- enzyme in shake flasks, and there has been much
duction of a wide range of microbial enzymes less work on the use of bioreactors for the pro-
(Gao et al. 2008). Among different Aspergillus duction of -mannanase in SSF (Magalha˜es &
species, Aspergillus niger has been shown to Milagres 2009).
produce highly active -mannanase in culture Despite having high practical potentialities,
(Ademark et al. 1998; van Zyl et al. 2009). the use of mannanase is still limited due to low
A large number of agro-industrial residues yields and high production costs. Based on man-
have already been utilized in fermentative pro- nanase applications, production of this enzyme
cesses to produce valueadded products. Among have important. In this paper, we were reported
these processes, solid substrate fermentation mannolytic activities of Aspergillus sp. BL5
(SSF) has been found to be an appropriate meth- rrown on different carbon substrates.
od for the utilization of agro-industrial by-
products in the production of microbial enzymes MATERIALS AND METHODS
(Pandey et al. 2001). The bioprocessing of agro-
industrial residues by SSF provides some eco- Microorganism: Aspergillus sp. BL5, used in
nomic and engineering advantages over sub- this study was obtained from the culture collec-
merged fermentation (SmF). These include high- tion of the Environmental Bioengineering Re-
er productivity per unit volume, lower capital search Group, Research Center for Biotechnolo-
gy, LIPI. The stock culture were maintained on a

mineral medium agar (MMA) slant including by HCl or NaOH. Submerged batch culture was
Locust Beam Gum (LBG) and routinely were carried out with 150 rpm agitation at 30 °C for
sub-cultured every 4 weeks. Inoculated plates six days. Sampling has been done every 24 hours
were incubated at 30oC for four days and the for determined the cell growth at 660 nm. The
spore suspension obtained was kept at 4oC. cells were removed by centrifugated at 12000
rpm in 4°C for 10 min to get crude enzyme
Chemicals and Substrates: LBG was purchased solution.
from Sigma Chemicals (St. Louis, MO). All oth-
er chemicals were of analytical grade. There are Determination of Enzyme Activity: Mannanase
eight of biomasses as a substrate source like was assayed by measuring the reducing sugars
LBG, palm kernel cake (PKC), hull of rice, sug- using dinitrosalicylic acid (DNS) method (Mil-
ar cane, grass, porang tuber, residue of coconut, ler, 1959). The mannanase activity was assayed
and suweg tuber were obtained from local manu- by using mixture contained 0.5 ml of 0.5% (w/v)
facture (PT. Ambico LtD. at Surabaya). Sub- LBG. The reaction mixture was maintained at
strates were ground to a particle size of 30 room temperature for 30 min. After incubation,
meshes and dried in an oven at 60⁰C for 48 h. 1.5 ml of DNS reagent was added and boiled for
5-15 min to release reducing sugars. One unit of
Screening of Mannolytic Bacteria: Mannanase enzyme activity (U) was defined as the amount
activity determination was conducted using the of enzyme liberating 1 mol of mannose per mi-
Congo Red method (Downie et al., 1994), with nute under the assay condition.
modifications, to obtain a better definition of the
activity zones. Screening for mannolytic bacteria RESULTS AND DISCUSSION
was performed on mineral salts agar medium
described by (Mandels and Sternberg, 1976) and Screening of Mannan-Degrading Bacterial
modified to contain (g/mL) 0.075 Peptone, 0.05 strains: The Aspergillus sp. BL5 was able to
Yeast extract, 0.14 (NH4)2 SO4, 0.2 KH2PO4, hydrolyze the galactomannan during growth on
0.03 MgSO47H2O, 0.03 CO(NH2)2, 0.03 CaCl2, several substrates in agar plates medium (Table
0.0005 FeSO4.7H2O, 0.00016 MnSO4.7H2O, 1). The clear zone was formed around
0.00014 ZnSO47H2O, 0.0002 CoCL2 and 2% Aspergillus sp. BL5 colonie in several substres
agar, pH was adjusted to 6 and sterilized by au- medium except on the husk and grass were not
toclaving at 121°C for 30 min. The agar plates formed. The further study in increasing
were inoculated by first dipping a sterile tooth mannanase activity by using all the substrates as
pick into an actively growing bacterial colony on a carbon sources for the mannanase production.
mineral salts agar media plus appropriate sub-
strate and incubated for 48-72 h at 35 ±1°C. Table 1. Mannolytic activity in the several carbon
Mannanase activity was detected on the cultures substrates by the Aspergillus sp. BL5
by staining the plates with congo red solution for Carbon Sources Mannolytic Avtivity
30 min. Galactomannan hydrolysis was observed Husk +
by the appearance of clearing zones around the PKC ++
Coconut +++
bacterial colony. The activity was calculated as Suweg +
the ratio of the diameter of the clearing zone to Porang ++
the diameter of the colony. LBG +
Sugar Cane +
Grass -
Production of Mannanase Enzyme using dif-
ferent Carbon Sources: Aspergillus sp. BL5 Note: (+) was indicated the clear zone area around the
was cultivated in variant medium which Aspergillus sp. BL5 colonie and (-) was not formed
containing of several substrates such as 3 g for
each PKC, hull of rice, sugar cane, grass, porang Mannanase Activity on Several Carbon
tuber, residue of coconut, and suweg tuber which Sources: Agro-industrial by product is available
are added with 0.075 g Peptone, 0.05 g Yeast in large amounts in Indonesia. They have been
extract, 0.14 g (NH4)2SO4, 0.2 g KH2PO4, 0.03 g used for several enzymes (Howard et al., 2003).
MgSO47H2O, 0.03 g CO(NH2)2, 0.03 g CaCl2, We evaluate the several substrates such as PKC,
0.0005 g FeSO4.7H2O, 0.00016 g MnSO4.7H2O, residue of coconut, LBG, suweg porang, potato
0.00014 g ZnSO47H2O, and 0.0002 g CoCL2 in 1 porang, grass, sugar cane, and husk. Figure 1
L medium. The medium pH was adjusted to 6.0 shows that several types of agro-industrial by

products were evaluated as substrates for man- cellulose or hemicelluloses (Mona & Amani,
nanase production by the Aspergillus sp BL5. 2008). Among these substrates, the PKC and
The Aspergillus sp. BL5 was grew well on vari- residue of coconut were found as the most suit-
ous substrates with significant differences in the able substrate for mannanase production. The
rate of enzyme production. The largest variation PKC and residue of coconut are potential.
in mannanase yield may be due to the nature of
biomasses.
1.0
from agro-industrial products which are used in biotechnologycal field. 2.0
Cell Growth CONTROL LBG PKC COCONUT
Mannanase Activity
Mannanase Activity (U/ml)

0.8
Cell Growth (OD 660 nm)
1.5
0.6
1.0
0.4
0.5
0.2
0.0 0.0
0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168
Time (Hours) Time (Hours) Time (Hours) Time (Hours)

1.0 2.0
PORANG SUWEG GRASS SUGAR CANE
Mannanase Activity (U/ml)

0.8
Cell Growth (OD 660 nm)
1.5
0.6
1.0
0.4
0.5
0.2
0.0 0.0
0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168
Time (Hours) Time (Hours) Time (Hours) Time (Hours)
Fig. 1. The Activity of the mannanase produced by Aspergillus sp. BL5 grown on various carbon sources. The cells were
growth on varian medium and incubated at 30oC for 6 days.
CONCLUSION rification and properties of a b-mannanase. J Biotech-

nol 63:199–210.
Burke RM, Cairney JWG. 1997. Carbohydrolase produc-
The highest mannanase activity was tion by the ericoid mycorrhizal fungus Hymenoscy-
showed in reaction between Aspergillus sp. BL5 phus ericae under solid-state fermentation conditions.
with PKC and residue of coconut substrates. Mycol Res 101:1135–1139.
The optimal result were obtained at 96 hours Downie, Bruce; Hilhorst, Henk W.M. and Bewley, J.
Derek. 1994. A new assay for quantifying endo-β-
incubation respectively in PKC and residue of mannanase activity using Congo red dye. Phytochem-
coconut 1.3 and 1.2 U/ml. istry, vol. 36, no. 4, p. 829-835.
Fisher SH, Sonenshein AL.1991. Control of carbon source
ACKNOWLEDGEMENTS and nitrogen metabolism in Bacillus subtilis. Ann.
Rev. Microbiol. 45: 107-135
Gao J, Weng H, Zhu D, Yuan M, Guan F, Xi Y. 2008. Pro-
The Research was funded by DIKTI reduction and characterization of cellulolytic enzymes
search grant and we grateful for their financial from the thermoacidophilic fungal Aspergillus terreus
and technical support. M11 under solid-state cultivation of corn stover. Bio-
resour Technol 99:7623–7629.
Howard RL, Abotsi E, Jansen Van Rensburg EL, Howard
REFERENCES S.2003.Lignocellulose biotechnology: issues of bio-
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T, Tjerneld F, Sta¨lbrand H. 1998. Softwood hemicel- Kurakake M, Komaki T. 2001. Production of b-mannanase
lulose-degrading enzymes from Aspergillus niger: pu- and -mannosidase from Aspergillus awamori K4 and
their properties. Curr Microbiol 42:377–380.

Lin TC, Chen C. 2004. Enhanced mannanase production by Mitchell DA, Lonsane BK. 1992. Definition, characteristics
submerged culture of Aspergillus niger NCH-189 us- and potential. In: Doelle HW, Mitchell DA, Rolz CE
ing defatted copra based media. Process Biochem (eds) Solid substrate cultivation. Elsevier, London, pp
39:1103–1109. 1–28.
Magalha˜es PO, Milagres AMF. 2009. Biochemical proper- Mona E. M. Mabrouk and Amani M. D. El Ahwany. 2008.
ties of a -mannanase and a b-xylanase produced by Production of -mannanase by Bacillus amylolequifa-
Ceriporiopsis subvermispora during biopulping con- ciens 10A1 cultured on potato peels. African Journal
ditions. Int Biodeterior Biodegrad 63:191–195. of Biotechnology Vol. 7 (8), pp. 1123-1128. Availa-
Magasanik B, Neidhardt FC.1987. Regulation of carbon ble online at http://www.academicjournals.org/AJB.
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Biology: Escherichia coli and Salmonella typimuri- Pandey A, Selvakumar P, Soccol CR, Nigam P. 1999. Sol-
am. Neidhardt FC (ed), Am. Soc. Microbi- id-state fermentation for the production of industrial
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Mandels M, Sternberg D.1976. Recent advances in Pandey A, Soccol CR, Rodriguez-Leon JA, Nigam P. 2001.
cellulase technology. Ferment Technol. 54: 267-286. Solid-state fermentation in biotechnology. Asiatech,
McTigue MA, Kelly CT, Fogarty WM, Doyle EM (1994). New Delhi.
Production studies on the alkaline amylase of three Ramesh MV, Lonsane BK.1987. Solid State fermentation
alkalophilic Bacillus sp.Biotechnol. Lett. 16:569-574. for production of alpha amylase by Bacillus mega-
McTigue MA, Kelly CT, Fogarty WM, Doyle EM.1994. terium 16 M. Biotechnol. Lett. 9:323-328.
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Miller, G.L.1959. Use of dinitrosalicylic acid reagent for 1,4--mannanase in A.niger. J Ind Microbiol Bio-
determination of reducing sugar. Anal. Chem. 31, technol 36:611–617.
426-428.

Appendices

Appendix 1. List of Participants
Ade Nena Nurhasanah

Research Center for Biotechnology-LIPI
Jl. Raya Bogor Km. 46 Cibinong Science Center, Cibinong, Bogor 16911
Tel.: 021-8754587, Fax.: 021-8754588
Ade Sumiardi
Program Study of Biology, Faculty of Mathematics and Science,
Mathla’ul Anwar University, Banten
Tel./Fax.: 0253-401773
E-mail: fmipaunma@gmail.com/adesumiardi@yahoo.com
Aditya Rinus P.
Kampus Baru UI Depok, Depok, West Java 16424
Tel.: 021-7863516/085669901340
Agung Marssada
Tel.: 021-7863516/081806137703
Agus Haryono
Polymer Chemistry Group, Research Center for Chemistry,
Indonesian Institute of Sciences (LIPI)
Kawasan Puspiptek Serpong, Tangerang Selatan 15314
Tel.: 62-21-756092/+62-21-7560549
E-mail: agus.haryono@lipi.go.id/haryonolipi@yahoo.com
Agustin Sri Mulyani
Balai Penelitian Bioteknologi Perkebunan Indonesia
Jl. Taman Kencana No. 1 Bogor
Ahmad Thontowi
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: athontowi@yahoo.com
Akhirta Atikana
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: akhirta@gmail.com
Akhmad Dicky Kurniawan
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: ahmad_dicky_kurniawan@yahoo.com
Alisin Febiyanti
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: alisin.febiyanti@lipi.go.id
Andi Halim
Department of Chemical Engineering, Faculty of Industrial Technology,
Bandung Institute of Technology
Jl. Ganesha No.10 Bandung 40132
E-mail: andi_85te@yahoo.co.id

Andri Fadillah Martin
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: andrifm@ymail.com
Andri Wardiana
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: awardian@yahoo.com
Angga Wijaya Holman Fasa
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: holmanfasa@yahoo.com
Anggia Prasetyoputri
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: anggia.prasetyoputri@lipi.go.id
Anky Zannati
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: anky_zannati@yahoo.com
Apriadi Situmorang
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: apriadi_situmorang@yahoo.co.id
Apridah Cameliawati Djohan
Jl. Raya Bogor Km. 46 Cibinong Science Center 16911
E-mail: a_camelia707@yahoo.com
Asep Bayu
Research Centre for Oceanography, Indonesian Institute of Sciences (LIPI)
Jl. Pasir Putih I Ancol Timur, North Jakarta
Tel.: 021) 64713850/ (021) 64711948
E-mail: asepbayu@yahoo.co.id
Asep Muhamad Ridwanuloh
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: am_ridwanulloh@yahoo.com
Asmini Budiani
Asrul M. Fuad
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: asrul.fuad_02@yahoo.co.id

B. Paul Naiola
Research Center for Biology-LIPI
Jl. Raya Bogor Km. 46 Cibinong Science Centre, Cibinong, Bogor 16911
E-mail: bpnaiola@yahoo.com
Bambang Prasetya
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: bambangpras@yahoo.com
Beom Soo Kim
Department of Chemical Engineering, Chungbuk National University
410 Seongbong-ro, Heungdeok-gu, Cheongju, Chungbuk 361-763, Korea
Tel. +82-43-261-2372 / +82-10-3132-2372
E-mail: bskim@chungbuk.ac.kr
Bernadetta Rina Hastilestari
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: b_rina_hl@yahoo.com
Betalini Widhi Hapsari
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: betalini_widhi@yahoo.com
Budi Satrio Maulana
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: budi.satrio.maulana@lipi.go.id
Budiasih Wahyuntari
Laboratoria Pengembangan Teknologi Industri Agro Biomedika (LAPTIAB),
Badan Pengkajian dan Penerapan Teknologi (BPPT)
Puspiptek, Serpong, Gedung 610, Tangerang
Tel.: 0811152394
E-mail: budiasih_solichin@yahoo.com/ budiasih@webmail.bppt.go.id
Byung-Gee Kim
School of Chemical and Biological Engineering, Seoul National University
599 Gwanangno, Gwanak-gu, Seoul 151-744, Korea
Tel.: +82-2-880-6774 / +82-10-3269-6774
E-mail: byungkim@snu.ac.kr
Byunggi Park
Soonchunhyang University
646 Shunchang Myon, Asan, Korea 336-745
Tel.: +82-10-3773-8751
E-mail: byunggi@sch.ac.kr
Cahya Ningrum
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: cn275@yahoo.com

Carla Frieda Pantouw
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: carla.frieda.pantouw@lipi.go.id
Chi Hyun Kim
Chosun University
375 Seosuk–dong, Dong-gu, Gwangju, Korea
Tel.: +82-62-230-6627 / +82-10-3666-6527
E-mail: ihlee@chosun.ac.kr
Cynthia Noviani
Tel.: 021-7863516
E-mail: nana@che.ui.ac.id/cnoviani@yahoo.com
Daechul Cho
Tel.: +82-41-530-1341 / +82-10-4343-5594
E-mail: daechul@sch.ac.kr
Deddy T. Nugroho
UPT BPP Biomaterial-LIPI
Deliana Dahnum
Research Centre for Chemistry-LIPI
Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten
Tel.: 021-7560929/021-7560549
E-mail: dhely_a@yahoo.com
Deritha Ellfy Rantau
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: deritha.ellfy.rantau@lipi.go.id
Dian Andriani
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: dea1401@yahoo.com
Diana Dewi
Balai Penelitian Bioteknologi BPPT
Gd. G30 Puspitek Serpong
Dian Fitria Agustiyanti
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: dianfitria_07@yahoo.com
Dian Noverita Widyaningrum
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: diannoverita@gmail.com

Dianursanti
Tel.: 021-7863516
Email: danti@che.ui.edu
Ditta Firmayanti
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: dittafirmayanti@yahoo.com
Dohoon Lee
Korea Institute of Industrial Technology
35-3, Hongcheon, Ipjangmyeon, Seobuk-gu, Cheonan, Chungnam 331-825, Korea
Tel.: +82-41-589-8349
E-mail: david@kitech.re.kr
Dwi Rahmat Aditya
Tel.: 021-7863516
E-mail: anondho@che.ui.ac.id
Dwi Susilaningsih
Tel.: 021-8754587/021-8754588
E-mail: dwisusilaningsih@yahoo.com.sg
Dwi Widyajayantie
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: wie_c2d7@yahoo.com
Dyah Retno Wulandari
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: dyahwulandari@yahoo.com
Edi Iswanto Wiloso
Research Center for Chemistry-LIPI
Puspiptek, Serpong, Tangerang 15314
Tel.: 021-756-0929/0816-774-601
E-mail: ediiswanto@yahoo.com/edi.iswanto.wiloso@lipi.go.id
Eko Wahyu Putro
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: ekowahyuputro@yahoo.com
Ekowati Chasanah
Research Center of Marine and Fisheries Product Processing and Biotechnology
Jl. KS. Tubun, Jakarta, Indonesia
E-mail: ekowati_ch@yahoo.com

Elis Sofianti
Department of Chemical Engineering, Faculty of Industrial Technology,
Bandung Institute of Technology
Gedung Labtek X, Jl. Ganesha 10 Bandung 40132
E-mail: tjandra@che.itb.ac.id
Ericko C. Utama
Magister of marine study, Department of Biology, Faculty of Mathematics and Science, University
of Indonesia
E-mail: e_ricko01@yahoo.com
Eris Septiana
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: eyis_jelek@yahoo.co.id
Euis Hermiati
UPT BPP Biomaterial LIPI
Eun Joo Jung
302-614, Gwanak-gu,Seoul 151-742 Korea
Tel.: +82-10-3228-8354
E-mail: plusyou0504@hanmail.net
Eunki Kim
National Research Laboratory of Bioactive Materials Laboratory, Department of Biological Engi-
neering, Inha University
253 Yonghyun, Inchon, Korea
Tel. +82-32-860-7514 / +82-11-9757-6490
E-mail: ekkim@inha.ac.kr
Evan Maulana
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: evan_131285@yahoo.com
Fadli Yusandi
Tel.: 021-7863516
Firman Adityo
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: adityo_firman@yahoo.co.id
Gio-bin Lim
The University of Suwon
Daelim Apt. 1-1202 Jamwon-dong, Seocho-gu, Seoul, Korea
Tel.: +82-10-7713-3169
E-mail: gblim@suwon.ac.kr

Go-Eun Kim
School of Biological Sciences and Technology, Chonnam National University
300 Yongbong-dong, Gwangju 500-757, Korea
Tel.: +82-62-530-1844
E-mail: dmkim@jnu.ac.kr/best6238@hanmail.net/ru1rura1ra@naver.com
Hariyatun
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: hariyatunsaksono@yahoo.com
Harnadiemas
Tel.: 021-7863516/085854894831
Hei Chan Lee
Department of Pharmaceutical Engineering, SunMoon University
# 100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea
Tel.: +82-41-530-2376/ +82-10-4234-2376
E-mail: heichan@sunmoon.ac.kr
Heri Hermansyah
Tel.: 021-7863516
E-mail: heri@chemeng.ui.ac.id
Heri Satria
Departement of Chemistry, University of Lampung
Bandar Lampung 35145 Indonesia
E-mail: satria_chemistry@unila.ac.id
Heru Darmawan
Tel.: 021-7863516
Hery Syapari
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: hery.syapari@lipi.go.id
Hesti Yuliastri
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: hesti.yuliastri@lipi.go.id
Hilda Farida
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: neng_hilda@yahoo.com
Hyo-Ihl Chang
College of Life Sciences and Biotechnology, Korea University
Anamdong 5 Sungbuk-Gu, Seoul 136-701, Korea
Tel.: +82-2-3290-3421
E-mail: hichang@korea.ac.kr

Hyung Joon Cha
Chemical Engineering, Pohang University of Science and Technology (POSTECH)
Pohang 790-784, Korea
Tel.: +82-54-279-2280 /+82-10-9690-3280
E-mail: hjcha@postech.ac.kr
In-Hyung Rhee
Tel.: +82-11-430-6180
E-mail: ihrhee@sch.ac.kr
In Hwa Lee
Chosun University
Tel.: +82-62-230-6627 / +82-10-3666-6527
Ira Djajanegara
Center of Bioindustrial BPPT
Gedung BPPT II Lt. 15, Jl. MH. Thamrin No. 8 Jakarta 10340
Tel.: 08158845256
E-mail: idjajanegara@yahoo.com
Ira Trisnawati
Tel.: 021-7863516/085286218603
Irma Kresnawati
Is Helianti
Center for Bioindustrial Technology, BPPT
Jl. MH. Thamrin No. 8 Jakarta 10340
Tel.: 021-7560536 ext. 124/08159633905
E-mail: ishelianti@yahoo.co.jp/ishelianti@webmail.bppt.go.id
Ishak Siregar
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: Isaac_cool_bgt@yahoo.co.id
Jae-Hak Kim
Department of Plant Biotechnology, Division of Biotechnology,
Kangwon National University
Chuncheon-City, Kangwon-Do, 200-703, Korea
Tel.: +82-33-250-6476 / +82-10-4943-4969
E-mail: hiwogkr@hanmail.net
Jaeyeong Song
School of Display and Chemical Engineering, Yeongnam University
303, Dae-dong, Gyeongsan-si, Gyeongsangbuk-do, Korea
Tel.: +82-53-810-3812 / +82-10-2442-0246
E-mail: jtlee@ynu.ac.kr
Jeongmin Kim
Tel.: +82-53-810-3812 / +82-10-3832-3797
E-mail: kjmnbb@nate.com

Jeyong Yoon
Tel.: +82-2-880-8927
E-mail: jeyong@snu.ac.kr
Jin Cheol Yoo
Department of Environmental Engineering, BK21 Team for Biohydrogen Production,
Chosun University
375 Seosuk-dong, Dong-gu, Gwangju, Korea
Tel.: +82-62-230-6380 /+82-10-2690-9885
E-mail: jcyu@chosun.ac.kr
Jintae Lee
Tel.: +82-53-810-3812 / +82-10-2442-0246
E-mail: jtlee@ynu.ac.kr
Joko Sulistyo
Research Centre for Biology-LIPI
Tel.: 021-8765066/021-8765062
E-mail: josulisty@yahoo.com
Jong Soo Kim
Sun Moon University
100 Galsan-ri, Tang Jeong Myon, Asan, Korea 336-708
Tel.: +82-10-6423-2381
E-mail: jskim@sunmoon.ac.kr
Jun Ho Choi
Chosun University
Tel.: +82-62-230-6627 / +82-10-3666-6527
Jungbae Kim
Department of Chemical and Biological Engineering, Korea University
Anam-Dong, Seongbuk-Gu, Seoul 136-701, Korea
Tel.: +82-10-8277-1133
E-mail: jbkim3@korea.ac.kr
Jung-Keug Park
Department of Biomedical Technology, Dongguk University
3-26, Pil-dong, Jung-gu, Seoul, Korea
Tel.: +82-11-9738-3365 / +82-2-2260-3302
E-mail: jkpark@dongguk.edu
Ju-Sang Kim
Marine Applied Microbes and Aquatic Organism Disease Control Laboratory,
Department of Aquatic Life Medicine, College of Ocean Science,
Jeju National University
Jeju 690-756, Korea
Tel.: +82-64-754-3473 / +82-10-9418-7405
E-mail: jusangi@naver.com
Keun Kim
The University of Suwon
San 2-2, Wau-ri, Bongdam-eup, Hwaseong-si, Gyeonggi-do 445-743, Korea
Tel.: +82-31-220-2344 / +82-10-8700-1438
E-mail: kkim@suwon.ac.kr

Khairul Anam
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: ka_anam@yahoo.com
Khoirur Rosyidin
Tel.: 021-8754587, Fax.: 021-8754588
Kim Changsu
Jeju University
Jecheon, Jeju, South Korea
Ki Seop Ahn
Baekseok Culture University
Anseo-Dong, Dong-Nam Gu, Cheonan, Korea 330-705
Tel.: +82-19-417-9648
Krishna Purnawan Chandra
Chemistry and Microbiology Laboratory, Department.Agricultural Product Technology,
Faculty Agriculture, Mulawarman University
Kampus UNMUL Gunung Kelua,
Jl. Tanah Grogot/Jl. Pasir balengkong 75119, Samarinda, Indonesia
Tel.: 0541-7773268/749352/085246736679
E-mail: kcandra_99@yahoo.com/candra@faperta.unmul.ac.id
Kyoun-Seon-Min
302-614, Gwanak-gu,Seoul 151-742 Korea
Tel.: +82-10-9958-0546
E-mail: min4605@snu.ac.kr
Lily MG
Research Centre for Oceanography-LIPI
Pasir Putih No.1 Ancol
Ludya Arica Bakti
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: ludya_arica@yahoo.co.id
Man Bock Gu
College of Life Sciences and Biotechnology, Korea University
Anam-dong, Seongbuk-Gu, Seoul 136-713, Korea
Tel.: +82-2-3290-3417 / +82-19-625-2440
E-mail: mbgu@korea.ac.kr
Maria Linawati
Tel.: 021-7863516/08561060133
Maria P. Omega
School of Biological Sciences, Faculty of Science, The University of Queensland
John Hines Building 5/517, St. Lucia, Australia, 4072
Tel.: +62-21-5373991/+61-423610727
E-mail: prihtamala_omega@yahoo.com

Marsiti Apriastini
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Martha Sari
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Maudhi Septian
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Merissa Bestari F.
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Mohamad Hidayat Syabana
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Mondya P.S.
Tel.: 021-7863516/085714261103
Monika Wijaya
Tel.: 021-7863516/085760528688
Moon-ki Park
Department of Herbal Pharmaceutical Engineering, Daegu Haany University
290 Yugok-dong, Gyeongsan, Gyeongbuk 712-715, Korea
Tel.: +82-10-2523-1420
E-mail: moonki@dhu.ac.kr
Moon-Soo Heo
Jeju 690-758, Korea
Tel.: +82-64-754-3473 / +82-10-3937-3473
E-mail: msheo@jejunu.ac.kr
Muhammad Kismurtono
Technical Implementation Unit for Development of Chemical Engineering Processes-LIPI
Desa Gading, Kec. Prayen, Kab. Gunung Kidul, Yogyakarta 55861
Tel.: 0274 (392570)/081578508760
E-mail: m_kismurtono@yahoo.co.id/BPPTKLIPI@yahoo.com

Muhammad Sidiq Habibi
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E-mail: ka_sidiq@yahoo.com
N. Sri Hartati
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: s_enny@hotmail.com
Namjun Cho
Korea University of Technology and Education
307 Gajeon, Beong-Cheon Myon
Cheonan, Korea 330-708
Tel.: +82-10-3420-1342
Neng Herawati
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: neng_herawati@yahoo.com/nengherawati@ymail.com
Niknik Nurhayati
Laboratoria Pengembangan Teknologi Industri Agro Biomedika (LAPTIAB),
Badan Pengkajian dan Penerapan Teknologi (BPPT)
Puspiptek, Serpong, Gedung 610, Tangerang
Tel.: 08121387463
E-mail: nik_nurhayati@yahoo.com
Nirwanto Honsono
Tel.: 021-7863516/081280896650
Ponco Widodo
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Puspita Lisdiyanti
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E-mail: p_lisdiyanti@yahoo.com
Putu Grahita Teja K.
Tel.: 021-7863516
Rakhman Sarwono
Research Centre for Chemistry-LIPI
Komp. PUSPIPTEK Blok VC/9, Serpong-Tangsel 15314
Tel.: 081382147221
E-mail: rach014@lipi.go.id

Ramasamy Harikrishnan
Jeju 690-757, Korea
Tel.: +82-64-754-3473 / +82-10-7654-3473
E-mail: rhari123@yahoo.com
Rani Meidianah
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E-mail: rani.meidianah@lipi.go.id
Reny Hayaty Z.
Tel.: 021-8754587, Fax.: 021-8754588
Republik Daudi P.
Tel.: 021-7863516/0219691429
Ria Millati
Food and Agricultural Product Technology Department, Gadjah Mada University Yogyakarta
E-mail: ria_millati@ugm.ac.id
Rohmatussolihat
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: rohmatussolihat@lipi.go.id
Roni Ridwan
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E-mail: rony_biotech@yahoo.com
Rudiyanto
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: rudidinasty@yahoo.com
Ryan Indra Mukti
Tel.: 021-7863516
E-mail: arbianti@chemeng.ui.ac.id
Sang-Chul Kim
Departement of Molecular science and Technology, Ajou university
San 5, Woncheon-Dong, Yeongtong-Gu, Suwon, Korea
Tel.: +82-31-219-2455 / +82-10-3698-6843
E-mail: warblaster@naver.com
Sangyong Kim
Korea Institute of Industrial Technology
35-3, Hongcheon, Ipjangmyeon, Seobuk-gu, Cheonan, Chungnam 331-825, Korea
Tel.: +82-41-589-8356
E-mail: sykim@kitech.re.kr

Sara Mutiara
Tel.: 021-7863516/08561134500
Seong Eun Bang
Chosun University
Tel.: +82-62-230-6649 / +82-10-5410-0579
E-mail: swkim@chosun.ac.kr
Seung Wook Kim
Department of Chemical and Biological Engineering, Korea University
1, Anam-dong, Sungbuk-ku, Seoul, 136-701, Korea
Tel.: +82-2-3290-3300 / +82-16-206-5702
E-mail: kimsw@korea.ac.kr
Shilfa Filayuri
Tel.: 021-7863516
E-mail: nana@che.ui.ac.id/shilfafilayuri@yahoo.co.id
Shin-Hae Hong
Department of Plant Biotechnology, Division of Biotechnology, Kangwon National University
Tel.: +82-33-250-6476 / +82-10-7241-9257
E-mail: sinhaeh@hotmail.com
Si Wouk Kim
Chosun University
Tel.: +82-62-230-6649 / +82-10-5410-0579
Siti Kurniawati
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: nina_smileus@yahoo.com
Soomin Kim
Tel.: +82-53-810-3812 / +82-10-3832-3797
E-mail: kjmnbb@nate.com
Soon-Kwan Hong
Tel.: +82-33-250-6476 / +82-10-2012-6476
E-mail: soonkwan@kangwon.ac.kr
Su Jin Lee
School of Life Sciences and Biotechnology, Korea University,
Anam dong 5 ga, Seongbuk-Gu, Seoul 136-701, Korea
Tel.: +82-10-5544-5035
E-mail: jju5035@korea.ac.kr

Suhandi
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: suhandi@lipi.go.id
Sung Ok Han
Laboratory of Industrial Microbiology and Bioenergy, College of Life Sciences and Biotechnology,
Life Science West Building Room # 208, Korea University
Anam-dong 5-ga, Sungbuk-gu,Seoul 136-701, Korea
Tel. +82-2-3290-3151 / +82-10-4619-2522
E-mail: samhan@korea.ac.kr
Sungmin Mun
#302-513, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea
Tel.: +82-2-880-8941
E-mail: msm527@snu.ac.kr
Sun Yan-Lin
Tel.: +82-33-250-6476 / +82-10-7537-0502
E-mail: laddiya@hotmail.com
Suzanne Ruth Billharz
University of California Berkeley
Jakarta
Tel.: 021-570-6585
E-mail: billharz@cbn.net.id
Swastika Praharyawan
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: swastika.praharyawan@gmail.com
Tai Hyun Park
Tel.: +82-2-880-8020 / +82-10-8882-8020
E-mail: thpark@snu.ac.kr
Tania Surya Utami
Tel.: 021-7863516
E-mail: nana@che.ui.ac.id
Tatsuki Wakayama
Global Environment and Energy Technology Consulting Section
Consultant, Technology Strategy Consulting Department, KRI (Kansai Research Institute), Inc.
Gotanda Icchoume East Bldg. 4F, 1-25-11, Higashi-Gotanda, Shinagawa,
Tokyo 141-0022, Japan
Tel.: +81-3-3444-3150, Fax.: +81-3-3444-3151
E-mail: wakayama@kri-inc.jp/tatsuki-wakayama@aist.go.jp
Tjandra Chrismadha
Research Centre for Limnology LIPI, Kompleks LIPI Cibinong
Jl. Raya Bogor Km 46 Cibinong 16911
Tel.: 021 8757071/021 8757076
E-mail: tjandra5660@yahoo.co.id

Toto Sugiato
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: toto.sugiarto@lipi.go.id
Tri Muji Ermayanti
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: tmermayanti@hotmail.com
Vincentia Esti Windiastri
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: vestiw@yahoo.com
Warda Tuharea
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: wardatuharea@gmail.com
Widyarani
Research Centre for Chemistry - Indonesian Institute of Sciences
Kampus LIPI Bandung, Jl. Cisitu-Sangkuriang, Bandung 40135
Tel.: 022-2503051
E-mail: widyarani@lipi.go.id
Wien Kusharyoto
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: wien.kyoto@gmail.com
Wulansih Dwi Astuti
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: wulan_nie@yahoo.com
Yana Rubiana
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: rubiyana39@yahoo.co.id
Yanni Sudiyani
Research Centre for Chemistry-Indonesian Institute of Sciences (LIPI)
Kawasan PUSPIPTEK Serpong, Tangerang 15314
Tel.: +62-21-7560090
E-mail: ysudiyani@yahoo.com
Yatri Hapsari
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: yatri0703@yahoo.com

Yeon Woo Ryu
Department of Molecular Science and Technology, Ajou University
5 Woncheondong, Youngtonggu, Suwon 443-749, Korea
Tel.: +82-31-219-2449 / +82-10-3333-4298
E-mail: ywryu@ajou.ac.kr
Yoon-Mo Koo
Department of Biological Engineering, Inha University
Incheon 402-751, Korea
Tel.: +82-32-860-7513
E-mail: ymkoo@inha.ac.kr
Yopi
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: yop_i@yahoo.com
Young Gyu Park
Daejin University
11-1 Sundandong, Pocheonsi, Kyungkido, 487-711, Korea
Tel.: +82-31-533-1970 / +82-10-3277-1989
E-mail: ypark@daejin.ac.kr
Yu Ri Park
Chosun University
Tel.: +82-62-230-6649 / +82-10-5410-0579
Yuliani
Department Agricultural Product Technology, Faculty Agriculture, Mulawarman University
Kampus UNMUL Gunung Kelua, Jl. Tanah Grogot/Jl. Pasir Balengkong 75119, Samarinda
Tel.: 0541-7773268/0852450514456
E-mail: kcandra@telkom.net
Yun Hee Choi
Chosun University
Tel.: +82-62-230-6380 /+82-10-2690-9885
E-mail: jcyu@chosun.ac.kr
Zainal Mustopa
Tel.: 021-8754587, Fax.: 021-8754588
E-mail: azmustopa@yahoo.com

Appendix 2. The Committee
Patron:
1. The Minister of Research and Technology

2. Chair of Indonesian Biotechnology Consortium
3. Chair of Indonesian Institute of Sciences
4. General Secretary of ASEAN Secretariat
5. The Minister of Energy and Mineral Resources
6. Korean Ambassador
Organizing Committee
Chair Prof. Dr. Ir. Bambang Prasetya

Vice Chair Dr. Dwi Susilaningsih, M.Pharm
Secretary I Dr. Tri Muji Ermayanti
Secretary II Cahya Ningrum, M.Si
Treasurer I Hery Syapari, S.Sos
Treasurer II Ditta Firmayanti, A.Md
Secretariate Eko Wahyu Putro, S.T
Hariyatun, S.Si
Budgetting Roni Ridwan, M.Si
Asep Muhamad Ridwanuloh, S.Si
Swastika Praharyawan, S.Si, Apt
Scientific Board Dr. Yopi
Dr. Puspita Lisdiyanti
Prof. Anondo Widjarnako
Dr. Hery Hermansyah
Symposium Dr. Asrul M. Fuad
Ahmad Thontowi, M.Si
Workshop A. Zainal Mustopa, M.Si
Dian Fitria Agustiyanti, S.T
Program Wulansih Dwi Astuti, S.Pt, M.Si
Vincentia Esti Windiastri, S.Si
Angga Wijaya Holman Fasa, S.H
Yatri Hapsari, S.Si
Akhirta Atikana, S.Si
Logistics Toto Sugiarto, BA
Apriadi Situmorang, S.P
Bernadetta Rina Hastilestari, S.Si
Publication Ludya Arica Bakti, S.Hum
Akhmad Dicky Kurniawan, A.Md
Refreshment Rani Meidianah
Suhandi

Appendix 3. List of Presentations
Invited Speakers
No. Name Title

1 Prof. Young Je Yoo Engineering Enzymes for Bioenergy and Biorefinery
New Technologies for 2nd Generation Biofuels and Biore-
fineries: Disintegration of Plant Cell Walls and Characteri-
2 Prof. Takashi Watanabe
zation of Surface Carbohydrates by Fluorescent-Labeled
Carbohydrate-Binding Modules (CBMs)
Prof. Klaus-D. Vorlop Integrated Bio- and Chemical Processes for the Conversion
3
of Renewables
4 Prof. Kazuhisa Miyamoto Microalgae- Key Resources for Biofuels
Separation of Woody Biomass Components and Utilization
5 Prof. Yasumitsu Uraki
of Separated Lignin
6 Prof. Jungbae Kim Nanobiocatalysis and its Potential Applications
7 Prof. Seung Koo Song The Korean Biofuel Policy
8 Prof. Don Hee Park The Status and Prospects of Bioenergy in Asia
Introduction of Asian Federation of Biotechnology
9 Prof. Jung Keug Park
(AFOB)
10 Prof. Tai Hyun Park Introduction of Asian Biotechnology Directory (ABD)
Assoc. Prof. Dr. Poonsuk
11 Biohydrogen as an Alternative Energy for Palm Oil Mill
Prasertsan
Conversion and Refining Process of Vegetable Oils to Fuel
12 Prof. Muhammad Nasikin
Based Downstream Products
Current Status and Development Perspective of Cellulose-
13 Prof. Dr. Ir. Bambang Prasetya
Ethanol in Indonesia
14 Dr. Aparat Mahakant TISTR Perspective on Biofuel from Microalgae
15 Dr. Siswa Setyahadi Enzymatically Process for Production of Biodiesel
Oral Presentations
No. Name Title

Oxygen-Tolerant Biological Hydrogen Production Using
1 Hyung Joon Cha
Recombinant Escherichia coli
The Challenges of Biofuel Implementation in Indonesia:
2 Muhammad Kismurtono
Environmental Prospective
Tropical Marine Photosynthetic Microbes for Alternative
3 Dwi Susilaningsih
Energies Resources
Evaluation of the Antimicrobial Efficacy and Treatment of
4 Jung-Keug Park
Tinea Pedis for SiO2-Ionized Loess
Specific and Selective Colorimetric Detection of
5 Man Bock Gu Antibiotics and Residual Drugs Using Unmodified Gold
Nanoparticles and ssDNA Aptamer
Protective Effect of the Natural Pigment on Naproxen In-
6 Hyo-Ihl Chang
duced Gastric Ulceration in Rat
In Searching the Possibilities to Produce Bioethanol (Gas-
7 B. Paul Naiola ohol) as New Renewable Energy from Lontar Palm (Bo-
rassus sundaicus L.) in East Nusa Tenggara, Indonesia

Ethanol Production from Oil Palm Empty Fruit Bunch Hy-
8 Ria Millati
drolyzate Using Saccharomyces cerevisiae
Two Stage Processes of Oil Palm Empty Fruit Bunch Fiber
9 Yanni Sudiyani
Kraft Pulp for Bioethanol
10 Sungmin Mun Bactericidal Activity and Mechanism of Supercritical N2O
Phycocyanin Production of Spirulina fusiformis Culture in
11 Tjandra Chrismadha
an Open Space Tubular Photobioreactor
12 Tania Surya Utami Using Compost as Biofilter to Reduce N2O Emission
Influence of 1,4-Dioxane in Enzymatic Process for Bio-
13 Krishna Purnawan Chandra diesel Production Using Crude Palm Oil (CPO) as Sub-
strate
Interesterification of Fried Palm Oil with Methyl Acetate
14 Ryan Indra Mukti
using Porcine pancreatic Lipase to Produce Biodiesel
Characteristics of Extracellular Enzymes from KLU 11.16
15 Ekowati Chasanah
Cultivated on Chitin Medium
Obtaining Xylooligosaccharides with Long Chains from
17 Is Helianti Corncobs Simultaneously with Endoxylanase Production
Using a Recombinant Strain Bacillus subtilis DB104
The Influence of Carbon Sources on Laccase Production by
18 Elis Sofianti White Rot Fungus Marasmius sp. in Solid State Fermenta-
tion
19 Sangyong Kim Production of Biochemicals by Fungal Fermentation
20 Yoon-Mo Koo Application of Ionic Liquids in Biorefinery Process
Control of Biodegradability of Polyurethane Foam Based
21 Agus Haryono on Palm Oil by Ratio of Soft Segment on the Polymer
Backbone
Dual Strategy for the Screening of Biocatalysts in the Pro-
22 Byung-Gee Kim
duction of Industrial Specialty Chemicals
23 Sung Ok Han Cellulosic Ethanol Production Using Cellulosomes
Provision of Superior Genotypes of Jatropha curcas for
24 N. Sri Hartati Biodiesel Production: Integrating Morphology and Yield
Variation with DNA-Based Marker
Poster Presentations
No. Name Title

Biological Production of Hydrogen and its Use for Fuel
P1 Tai Hyun Park
Cell
Scale up of Methanol Production by Methylosinus tricho-
P2
Si Wouk Kim sporium OB3b
P3 Pretreatment and Saccharification of Rice Straw
Microbial Production of Hydrogen from Biodiesel Byprod-
P4 Dohoon Lee
uct
Bioconversion to Ethanol of Rice Straw Pretreated Using
P5 Go-Eun Kim Hypochlorite-Hydrogen Peroxide and Followed Enzymatic
Saccharification
Construction of Yeast Strains Producing High Concentra-
P6 Keun Kim
tion of Ethanol from Tapioca

P7 Seung Wook Kim Biodiesel Production by Enzimatic Process
Methane Production from Swine Wastewater and Marine
P8 Jeongmin Kim
Macro Algae
Screening of ssDNA Aptamers for Alkylphenols by Using
P9 Su Jin Lee
a Novel Selection Method
Fabrication and Evaluation of Crosslinking Poly(Vinyl Al-
P10 Jun Ho Choi
cohol) Film for Biodegradability Control
LFG Landfill Gas Tablets Technology and Greenhouse
P11 Chi Hyun Kim
Management Research
Study of Carbon Dioxide Emissions through Embodied
P12 Young Gyu Park
Bioenergy of Renewable Biomass
The Production of Hydrogen Production in
P13 Maria Omega Chalymodomonas reinhardtii Induced by the Decrase of
Sulphur
Implementation Fuel of Methane from Biogas for Supply
P14 Muhammad Kismurtono
Energy in Small Industry (UKM-Tahu)
Comparison of Different Production Processes for Bioeth-
P15 Yanni Sudiyani
anol from Kraft Pulp of Oil Palm Empty Fruit Bunch Fiber
Preparation of Biodiesel from Kosambi-Oil of Schleichera
P16 Joko Sulistyo
oleosa Merr. by Application of Transesterification
Utilization of Lignin-Rich Biomass Waste from the Pro-
P17 Bambang Prasetya duction of Bagasse Bioethanol for Additive in Pilot Scale
Production of Fiber Based Composite
Potency of Indonesian Marine Microalgae (Chlorophyta)
P18 Hilda Farida
as Renewable Liquid Biofuel (Biodiesel) Producers
Environmental Factors Optimation in Photofermentation to
P19 M. Sidiq Habibi
Produce Biohydrogen by Sanur consortia
In situ Acid Transesterification: an Overview Simple
P20 Asep Bayu Method for Fatty Acids Methyl Esters (FAME) Preparation
from Marine Microalgae as a Biodiesel Feedstocks
Feasibility of Sorghum bicolor as Bioethanol Producer by
P21 Deliana Dahnum
Mass and Energy Balance Analysis
Uniresponse Kinetics Based on Ping Pong Bi Bi Mecha-
P22 Heri Hermansyah nism in Biodiesel Synthesis via non Alcohol Route in
Packed Bed Reactor
Updating Bioreactor Methods for Hydrogen Production
P23 Khairul Anam
Process Through Photo Fermentation
Isolation and Screening of Lignocellulolytic Actinomycetes
P24 Heri satria
from Rice Straw Waste
Screening for Natural Producers Capable of Producing 1,3-
P25 Dian Andriani
Propanediol from Glycerol
Pretreatment of Cellulosic Materials and Evaluation of
P26 Yeon Woo Ryu
their Hydrolysates Using Dilute Sulfuric Acid
P27 Eunki Kim Production of Anti-Inflammatory Sophorolipids
Production of Polyhydroxyalkanoates from Vegetable Oil
P28 Beom Soo Kim
by Ralstonia eutropha
Characteristics of Electricity Production by Metallic and
P29 In-Hyung Rhee
Carbon Anodes Immersed in an Estuarine Sediment
Preparation and Characterization of Antimicrobial Film for
P30 Jun Ho Choi
Poly(Vinyl Alcohol) and Silver Nitrate

Probiotic Effect in vivo of Zooshikella Strain JE-34 Against
P31 Streptococcus iniae Infections in Olive Flounder, Paralich-
thys olivaceus
Standardization of Prodigiosin-Like Pigment from
P32
Zooshikella sp. JE-34 for Fermentation Production
Diet Containing Probiotics and Herbal Extracts Improve on
P33 Growth Performance, Blood Biochemistry and Nonspecific
Immunity in Olive Flounder and Parrot Fish
Identification and Activation Characterization of Bioactive
P34 Compounds Produce Marine Actinomycetes Streptomyces
albogriseolus ACT-1
Optimal Growth Conditions and Activation Characteriza-
P35 tion of Bioactive Compounds Produce Marine Actinomy-
cetes Streptomyces microflavus ACT-18
Activation Characterization of Bioactive Compounds Pro-
P36 duce γ-Proteobacteria Oceanospirillales Zooshikella sp. JE-
Ju-Sang Kim/
34
Ramasamy Harikrishnan/
Investigate Effect of Feeding with Probiotics, Im-
Moon-Soo Heo
P37 munostimulants, Antibiotics, Vaccination and Chemother-
apy in Cirrhina mrigala against Apanomyces invadans
Use of Probiotics and Medicinal Herbs for the Control of
P38
Aeromonas hydrophila Infection in Carp
Antimicrobial Characteristics of Bacterial Secondary Me-
P39
tabolites from Phaeobacteria inhibens KJ-2
Optimum Growth Conditions for Production Antimicrobial
P40
Compound of Phaeobacter inhibens KJ-2
Characterization of Biosurfactant Substance Produced by
P41
Bacillus vallismortis BK6 Isolated from Jeot-kal
Cultural Characteristics for Production of Antibacterial
P42 Substance Produced by Bacillus vallismortis BK6 Isolated
from Jeot-kal
Antibacterial Activity and Free Radical Scavenging Activi-
P43 ty of Bacterial Metabolites Produced by Bacillus vallismor-
tis BK6 Isolated from Jeot-kal
The Translation Initiation Factor eIF1 Gene is Sensitive to
P44
Alkaline Stress in Leymus chinensis Trin.
The Relationship with Translation Initiation Factor eIF1
P45 Sun Yan-Lin
Gene Expression and Na+ Stress
Transformation of Leymus chinensis Trin. by Agrobacte-
P46
rium tumefaciens Infection of in vitro Cultured Callus
Efficient Production of Taraxacum coreanum by Means of
P47 Jae-Hak Kim
Tissue Culture for Creating a Green Tract of Land
High Affinity Peptides for the Recognition of the Acute
P48 Moon-ki Park Myocardial Infarction Biomarkers Troponin I Identified
Using Phage Display
Preparation of Hydrophilic Drug-Loaded Microparticles by
P49 Gio-bin Lim
Aerosol Solvent Extraction System (ASES)
Metabolic Engineering of Streptomyces venezuelae YJ028
P50 Hei Chan Lee
for Enhanced Production of Polyketides

Compost Maintains the Soil Properties and Supports the
P51 Rakhman Sarwono
Sustainable Agriculture
The Influence of Harvesting Period to Lipid Associated
P52 Tjandra Chrismadha Antioxidant Activity of Semicontinuously Grown Chlorel-
la vulgaris
The Cell Entrapment Method for Maintain the Maximum
P53 Dianursanti Growth Rate of Chlorella vulgaris Buitenzorg in Mid-
Scale Bubble Column Photobioreactor
Nitrous Oxide Biosorption in Biofiltration Process Using
P54 Cynthia Noviani
Cow-Manure Compost Based Medium
Performance Evaluation of Cow Manure Compost Based
P55 Shilfa Filayuri
Medium as Filter on Nitrous Oxide Biofiltration
Introduction Test Herbiside Activity of Bakteria Endofit
P56 Warda Tuharea Canarium hirsutum willd.var.hirsutum Plants Using Buta-
nol Solvents
The Ability of Bacterium Isolate from the Soil that Polluted
P57 by Used of Lubricant at PT. Krakatau Steel Cilegon to De-
Ade Sumiardi grade Hydrocarbon Compound
The Use of Zeolit as a Coagulant Adding Material for
P58
Treating Waste Water of Crude Palm Oil
Production and Characterization of Biosurfactant by Indig-
P59 Ericko C. Utama
enous Bacteria Lysobacter sp. Strain BT104
Enhancement of Chlorella sp.Biomass Production with
P60 Maudhi Septian Filtration in Circulating Media and Arrangement of Light-
ing Intensity
Determination of Nutrition Content from the Biomass of
P61 Ponco Widodo
Chlorella vulgaris Buitenzorg
Suction Rate Adjusment in Filtration Process of Media
P62 Heru Darmawan Culture Circulation for Increasing Biomass Production of
Determination of Hydrodynamic Parameter in Bubble Col-
P63 Putu Grahita Teja K. lumn Photobioreactor for Scale up Biomass Production of
Enhancement of Chlorella vulgaris Buitenzorg Biomass
P64 Fadli Yusandi Production in Medium Scale Bubble Column Photobiore-
actor with Arrangement of Air Superficial Velocity
Performance Evaluation of Mid-Scale Bubble Column
P65 Dwi Rahmat Aditya Photobioreactor for Chlorella vulgaris Buitenzorg Biomass
Production by Photon Flux Density Arrangement
Microbial Conversion of Raw Glycerol to 1,3-Propanediol
P66 Wien Kusharyoto
by Clostridium butyricum NRRL B-1024
Rapid Characterization of Biosurfactant-Producing Mutant
P67 Swastika Praharyawan
of Soil Microbe Isolated from Oil Mining, Cepu
Studies on Marine Biosurfactant for Useful Biological
P68 Dian Noverita Widyaningrum
Function
Potential Marine Microbes to Produce Polysaccharide De-
P69 Apridah Cameliawati Djohan
grading Enzyme
An Organic Solvent, Detergent and Oxidant Stable Metal-
P70 Jin Cheol Yoo
loprotease from Streptomyces sp. CS528
Screening and Cloning of Novel Deglycosylation Enzyme
P71 Byung-Gee Kim
from Rhizobium sp. GIN611

Electroenzymatic Synthesis of L-DOPA Using Tyrosinase-
P72 Kyoun-Seon-Min
Functionalized Composite Electrode
Optimization of Xylanase Production by Bacillus subtillis
P73 Budiasih Wahyuntari AQ 1 Using Corn Cobs, Liquid Tofu Waste and Response
Surface Methodology
Study of Factors Affecting the Lipase Production by Yar-
P74 Asep Muhamad Ridwanuloh
rowia lipolytica in Submerged Fermentation
Lipase Production by Yarrowia lipolytica in Solid-State
P75 Hariyatun Fermentation Utilizing Agroindustrial Residues as Sub-
strate
Optimizing the Palm Kernel Cake Biomass Fermentation
P76 Yopi
for Saccharides Production Using Stirrer Fermentor
Optimation of Production of Mannanase from Coconut and
P77 Ahmad Thontowi
Palm Kernel Cake by Aspergillus sp. BL5

Appendix 4. Index of Authors
Ade Sumiardi 247 Hariyatun 159, 289, 293

Ahmad Thontowi 281, 297, 303 Haznan Abimanyu 179
Alisin Febiyanti 241 Hendro Risdianto 125
Ambar Susilorukmi 69 Heri Hermansyah 109, 119, 185, 233,
Anatta Wahyu Budiman 185 297
Anondho Wijanarko 255, 259 Heri Satria 195
Aparat Mahakant 53 Heru Darmawan 255
Apridah Cameliawati Djohan 281, 297 Hilda Farida 69, 165
Arya Yudistira 185 Hong Shn-Hae 207
Asep Bayu 173 Hong Soon-Kwan 207, 213, 217, 223
Asep Muhamad Ridwanuloh 159, 289, 293 Hyung-Jin Song 201
Awan Purnawan 297 HyungSuk Kim 141
B. Paul Naiola 63, 81 Ines I.C. Atmosukarto 241
Bambang Prasetya 51, 69, 159 In-Hyoung Rhee 201
Bambang Sunarko 265 Jae-Hak Kim 223
Ben Hankamer 147 Jeyong Yoon 99
Byung-Gi Park 201 Joko Sulistyo 81, 155
Chakra Roy 69 Jong-Soo Kim 201
Claes Niklasson 89 Jung Keug Park 39
Cynthia Noviani 233 Jungbae Kim 33
Daechul Cho 201 Jung-in Kim 141
Deliana Dahnum 179 Kazuhisa Miyamoto 23
Delicia Yunita Rahman 69 Khairul Anam 69, 165, 191
Dian Noverita Widyaningrum 165, 275 Khoirun Nisa 155
Dianursanti 255, 259 Ki-Seob Ahn 201
Dicky Gustiawanto 165 Klaus-D. Vorlop 7
Don Hee Park 37 Krishna Purnawan Candra 117
Donna Fujie Rahaditha Utami 275 Lila Adriaty 109
Dwi Susilaningsih 69, 165, 169, 191, Maria Omega 147
271, 275, 297 Martha Sari 241, 265, 289, 293
Eko Wahyu Putro 159, 289, 293 Matthew Timmins 147
Elis Sofianti 125 Mey R. Widoretno 103
Elisabeth Titik Trihandayani 89 Mohammad J. Taherzadeh 89
Enny Sudarmonowati 131 Mohammad Nasikin 45, 109, 233, 255, 259
Fitriani 117 Moon Young Yoon 75
Gunawan 69 Muhammad Kismurtono 63, 81, 151

Muhammad Nur Cahyanto 89 Sun Yan-Lin 207, 213, 217
Muhammad Sidiq Habibi 69, 165, 169, 191 Sungmin Mun 99
N. Nurhidayat 81 Suraya 125
N. Sri Hartati 131 Swastika Praharyawan 271, 297
Nam-Jun Cho 201 Tai Hyun Park 41
Nanik Rahmani 303 Takashi Watanabe 5
Neti Yuliaty 241 Tami Idiyanti 179
Nunik Sulistinah 265 Tania Surya Utami 109, 119, 233
Peer Schenk 147 Teuku Beuna Bardant 81, 95
Poonsuk Prasertsan 43 Theresia Umi Harwati 69, 271
Puspita Lisdiyanti 297, Tjandra Chrismadha 103
Putu Grahita Teja Kusuma 259 Tjandra Setiadi 125
Rachma Wikandari 89 Toeti S. 69
Rakhman Sarwono 227 Tri Murningsih 81
Ria Millati 89 Wahyuni 131
Rita Arbianti 119, 185 Warda Tuharea 241
Roni Maryana 151 Wien Kusharyoto 265, 289, 293
Rusvirman Muchtar 247 Y. Cahyani 131
Ryan Indra Mukti 1 19 Yanni Sudiyani 95
S. Tursiloadi 81 Yasumitsu Uraki 25
Saenab Husain 69 Yayah Mardiati 103
Satriyo K.W. 151 Yopi 281, 297, 303
Seung Koo Song 35 Young Gyu Park 141
Seung Ug Hong 75 Young Je Yoo 3
Shin Young-Boum 213, 217, 223 Youn-Woo Lee 99
Siswa Setyahadi 55 Yuliani 117
Sok Chea 57 Zaenal Mustofa 69
Sudiyarmanto 179
Sukartin 117

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ISBN: 78-979-97789-9-4

Research Center for Biotechnology - LIPI, Indonesian Biotechnology Consortium (KBI),

for sustainable production of biofuel and industrial biochemicals

“Converging biorefinery to response climate change”

Research Center for Biotechnology-LIPI, Indonesian Biotechnology Consortium (KBI),

Proceedings ASEAN-Korea Symposium and Workshop on Biorefinery Technology,

Layouter : Research Center fo Biotechnology, LIPI

 Research Center for Biotechnology

for sustainable production of biofuel and industrial biochemicals

“Converging biorefinery to response climate change”

Foreword from Chairman of Organizing Committee......................................................................... xiii

Invited Speakers : ............................................................................................................................... 1

1. Engineering Enzymes for Bioenergy and Biorefinery (Abstract)

3. Biorefineries-Integrated Bio- and Chemical Processes for the Conversion of Renewables

4. Microalgae - Key Resources for Biofuels – (Abstract)

5. Separation of Woody Biomass Components and Utilization of Separated Lignin

6. Nanobiocatalysis and its Potential Applications (Abstract)

7. The Korean Biofuel Policy (Abstract)

8. The Status and Prospects of Bioenergy in Asia (Abstract)

9. AFOB (Introduction of Asian Federation of Biotechnology) (Abstract)

10. ABD (Introduction of Asian Biotechnology Directory) (Abstract)

11. Biohydrogen as an Alternative Energy for Palm Oil Mill (Abstract)

13. Current Status and Development Perspective of Cellulose-Ethanol in Indonesia (Abstract)

14. TISTR Perspective on Biofuel from Microalgae (Abstract)

Concurrent Session and Poster Presentations :.............................................................................. 59

1. The Challenges of Biofuel Implementation in Indonesia: Environmental Prospect (O03)

2. Sustainable Tropical Aquaculture: Microalgal Base Energies from Indonesia (O04)

7. Bactericidal Activity and Mechanism of Supercritical N2O (O11)

8. Phycocyanin Production of Spirulina fusiformis Culture in an Open Space Tubular Photo-

9. Using Compost as Biofilter to Reduce N2O Emission (O13)

B. Biorefinery ................................................................................................................................ 139

19. Potency of Indonesian Marine Microalgae (Chlorophyta) as Renewable Liquid Biofuel

20. Environmental Factors Optimization in Photofermentation to Produce Biohydrogen by

26. Characteristics of Electricity Production by Metallic and Carbon Anodes Immersed in an

29. Transformation of Leymus chinensis Trin. by Agrobacterium tumefaciens Infection of

36. Determination of Hydrodynamic Parameter in Bubble Collumn Photobioreactor for Scale

37. Microbial Conversion of Raw Glycerol to 1,3-Propanediol by Clostridium butyricum

40. Potential Marine Microbes to Produce Polysaccharide Degrading Enzyme (P69)

42. Lipase Production by Yarrowia lipolytica in Solid-State Fermentation Utilizing Agro-

Jakarta, February 18th, 2010

Prof. Dr. Bambang Prasetva

Ph.D. Lim, Giobin

Representatives from the ASEAN Secretariate,

Ladies and gentlemen,

Ladies and gentlemen,

Ladies and Gentlemen,

Ladies and gentlemen,

JAKARTA, 18 February 2010

The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 59

The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 61

INTRODUCTION permanent supply of national energy. The in-

The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 63

64 Jakarta, 18-20 February 2010

Liquid coal 0.0 80.5 80.5 ENVIRONMENTAL EFFECT

The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 65

66 Jakarta, 18-20 February 2010

The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 67

INTRODUCTION Like plants, microalgae use sunlight to pro-

The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 69

70 Jakarta, 18-20 February 2010

Table 1. Lipid producing microalgae from several environments in Indonesia.

The ASEAN-Korea Symposium & Workshop on Biorefinery Technology 71

Fig. 1. Starch or lipid accumulator microalgae

Fig. 2. Hydrocarbon depositor microalgae