Diversity of Cellulolytic,Oleaginous and Cellulooleginous Yeasts from Indonesia Resources

DIVERSITY OF CELLULOLYTIC, OLEAGINOUS AND
CELLULO-OLEAGINOUS YEASTS FROM INDONESIAN
RESOURCES

ATIT KANTI

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

STATEMENT OF ORIGINALITY1
I certify that Ph.D dissertation entitle Diversity of Cellulolytic, Oleaginous
and Cellulo-oleaginous Yeasts from Indonesian Resources is my original work
directed by supervisor committe has not been submitted to other university in any
form. Source of information used in this study are cited at the end of this
dissertation. By this I transfer the copyright to Graduate School of Bogor
Agricultural University.
Bogor, August 2014
Atit Kanti
NIM:G36110001

1

Copy right transfer of joint research collaboration with external agency of
IPB shall meet the MoU between parties.

RINGKASAN
ATIT KANTI. Diversity of Cellulolytic, Oleaginous and Cellulo-oleginous
Yeasts from Indonesian Resources. Supervised by NAMPIAH SUKARNO,
ENDANG SUKARA, LATIFAH K DARUSMAN and KYRIA BOUNDYMILLS.
Penelitian diversitas khamir yang diisolasi dari alam kebanyakan
dilakukan di daerah sub tropika, hanya sedikit yang dilakukan di daerah tropika.
Untuk mengungkapkan diversitas khamir di alam, perlu dilakukan isolasi khamir
yang berasal dari Indonesia. Pengetahuan tentang diversitas khamir di Indonesia
lebih banyak didapatkan dari makanan fermentasi tradisional.
Krisis energi yang menimpa dunia pada abad ke 20, memacu penelitian
pencarian sumber energi alternatif. Akhir-akhir ini biofuel diproduksi untuk
menanggulangi krisis energi minyak bumi. Akan tetapi masih ada silang pendapat
yang mempertanyakan biofuel sebagai energi alternatif karena sumber biofuel
yang juga merupakan sumber pangan. Biofuel yang menggunakan mikroba

diekplotasi dengan menggunakan single cell oils yang diproduksi oleh mikroba
pengakumulasi minyak seperti khamir dapat dijadikan sebagai alternatif unutuk
sumber energi baru yang mempunyai keunggulan diantaranya tidak memerlukan
tempat yang besar untuk proses produksi, dan dapat diproduksi dalam waktu yang
lebih singkat.
Penelitian ini bertujuan untuk mengungkapkan keragaman khamir yang
berasal dari berbagai macam sumber sampel, penemuan jenis baru, mempelajari
karakter fisiologi khamir untuk mendegradasi selulosa, dan kemampuan
mengakumulasi minyak. Penelitian ini juga mempelajari proses akumulasi minyak
yang dipengaruhi oleh berbagai macam sumber karbon.
Khamir diisolasi dari sumber sampel yang dikoleksi di daerah Sulawesi
Tenggara, Raja Ampat, Nusa Tenggara Timur, Bali dan Jawa Barat. Sampel yang
dikoleksi dari daerah Sulawesi Tenggara meliputi daun, seresah, tanah, insek,
insek larva, kayu lapuk dan jamur. Tanah merupakan sampel yang diambil dari
Raja Ampat. Nusa Tenggara Timur diwakili oleh sampel yang berasal dari tanah
dan seresah. Sampel yang berasal dari Jawa Barat meliputi daun dan bunga.
Material tanaman Piper betle danPiper nigrum merupakan sumber sampel yang
dikoleksi dari daerah Bali dan Gunung Salak. Identifikasi khamir dilakukan
dengan pengamatan karakter morfologi, fisiologi dan pendekatan molekular
dengan pemetaan daerah D1/D2 (26S) rDNA. Khamir yang berpotensi sebagai

jenis baru dikonfirmasi dengan identifikasi molekular pada daerah ITS1-5.8S
rDNA ITS5.
Empat ratus lima belas khamir diisolasi dari berbagai macam sumber
sampel. Dari Sulawesi Tenggara berhasil diisolasi 38 isolat dari Mekongga, dan
260 dari Papalia. Dua puluh tiga isolat diisolasi dari Raja Ampat, 15 isolat dari
Nusa Tenggara Timur, 47 isolat dari Bali, 24 isolat dari Gunung Salak dan 8 isolat
dari Cibodas.
Identifikasi dengan pendekatan molekular menunjukkan bahwa khamir yang
berhasil diisolasi termasuk ke dalam 40 marga dalam kelompok Ascomycota, dan

22 marga termasuk dalam kelompok Basidiomycota. Keragaman marga yang
paling tinggi sebanyak 32 marga ditemukan di Sulawesi Tenggara yang terdiri
dari 28 marga yang termasuk kelompok Ascomycota dan 4 marga termasuk ke
dalam kelompok Basidiomycota. Sembilan marga ditemukan dari sampel yang
berasal dari Raja Ampat yang terdiri atas 4 marga yang termasuk ke dalam
kelompok Ascomycota 5 marga yang termasuk ke dalam kelompok Basidiomycota.
Sampel dari material tanaman yang dikoleksi dari Bali menunjukkan bahwa hanya
kelompok khamir yang termasuk ke dalam kelompok Basidiomycota yang
berhasil diisolasi yang terdiri atas 4 marga. Tiga marga kelompok Ascomycota
merupakan khamir yang berasal dari sampel yang dikoleksi dari Cibodas. Khamir

paling banyak ditemukan pada 3 jenis sampel yaitu : daun, seresah, dan bagian
usus insek. Hasil ini menunjukkan bahwa Indonesia memiliki keragaman jenis
khamir yang tinggi.
Analisis homologi daerah D1/D2 menunjukkan 204 isolat memiliki nilai
substitusi > 1 % sehingga merupakan kandidat jenis baru. Sebagian besar kandidat
jenis baru berasal dari insek. Keragaman jenis khamir yang cukup tinggi pada
insek menunjukkan ada keterikatan antara khamir dan insek. Jenis-jenis baru yang
diisolasi termasuk kedalam Ascomycota (clade Yamadazyma, Wickerhamomyces,
Ogateae, Metschnikowia, Candida, Kodamaea) dan Basidiomycota (clade
Bulleromyces). Dua jenis baru diverifikasi secara lengkap sebagai Yamadazyma sp.
nov. yang termasuk dalam phylum Ascomycota , order Saccharomycetales,
family Debaryomycetaceae, diisolasi dari insek, sedangkanCiteromyces sp. nov.,
termasuk dalam ordeSaccharomycetales, family Wickerhamomycetaceaediisolasi
dari seresah.
Sebanyak 157 isolat selulolitik yang termasuk anggota Ascomycota dan
Basidiomycota diisolasi dari daun, seresah, insek usus, tanah, kayu lapuk, dan
bagian dari tanaman (batang, daun, bunga dan buah dari Piper betle danPiper
nigrum) mempunyai distribusi ekologi yang luas. Didapatkan lebih dari 10 isolat
merupakan kandidat jenis baru yang mempunyai kemampuan selulolitik.
Sporobolomyces poonsookiae Y08RA07, Rhodosporidium paludigenum

Y08RA29 and Cryptococcus flavescens Y08RA33 adalah jenis khamir yang
paling potential yang diisolasi dai Raja Ampat, Papua.
Dua ratus dua strain khamir mempunyai kemampuan mengakumulasi
lipid (oleaginous) yang diisolasi dari seresah, usus insek, kayu lapuk, dan sirih
( Piper betle L dan Piper nigrum L), yang dijumpai di Sulawesi Tenggara, Raja
Ampat, Nusa Tenggara Timur, Bali dan Jawa Barat. Khamir pengakumulasi
lipid dicirikan oleh sel yang menggambang pada medium gliserol 10 %, dan
memiliki gumpalan lipid di dalam sel. Candida adalah kelompok marga yang
paling dominan. Anggota dari Candida mengakumulasi lipid sekitar 20-40 %.
Marga lain yang mampu mengakumulasi lipid adalah Rhodosporidium,
Sporidiobolus, Metschinkowia dan Cryptococcus. Penelitian ini menunjukkan
khamir pengakumulasi lipid adalah polyphelitic.
Ditemukan sebanyak 78 isolat khamir yang mampu menghidrolisa
selulosa dan mengakumulasi lipid. Khamir disebut sebagai cellulo-oleaginous.
Candida merupakan marga utama khamir cellulo-oleaginous yang dapat diisolasi
dari berbagai sumber sampel seperti seresah, usus insek, kayu lapuk, dan sirih
(Piper betle L dan Piper nigrum L). Marga lain yang juga termasuk khamir

cellulo-oleaginous adalah Sporodiobolus, Aureobasidium, Rhodosporidium,
Pseudozyma, Debaryomyces, Pichia, dan Cryptococcus.

Beberapa sumber karbon seperti glukosa, gliserol, xilosa, dan CMC diuji
untuk media tumbuh khamir Candida intermedia PLE6DP6, Candida
orthosilopsis InaCC Y-302/Y09GS34, Candida oleophila InaCC Y306/Y09GS48), Crytococcus flavescent PL3DP6), (Cryptococcus humicola
PLE3DP9), (Cryptococcus luteolus InaCC Y-265/ Y10BS72), Yamadazyma aff.
mexicana PL1W2) menggunakan khamir Lipomyces starckeyii NBRC 10831
sebagai strain pembanding. Komposisi FAME yang dihasilkan dipengaruhi oleh
jenis sumber karbon. Berdasarkan komposisi FAME diketahui Candida
orthosilopsis InaCC Y-302/Y09GS34, Candida oleophila InaCC Y306/Y09GS48), Crytococcus flavescent PL3DP6) merupakan khamir cellulooleaginous yang potential untuk dikembangkan dalam penelitian biofuel dengan
menggunakan bahan limbah pertanian.
Kata kunci: khamir selulolitik, pengakumulasi lipid, selulo-oleaginos, biofuel.

SUMMARY
ATIT KANTI. Diversity of Cellulolytic, Oleaginous and Cellulo-oleginous
Yeasts from Indonesian Resources. Supervised by NAMPIAH SUKARNO,
ENDANG SUKARA, LATIFAH K DARUSMAN and KYRIA BOUNDYMILLS.
The studies of yeasts from natural habitats have been conducted
extensively in temperate regions, but very few studies conducted in tropical region.
At the microbial diversity level, further study is needed to verify the species
richness in Indonesian resources. The information about yeasts diversity in
Indonesia are mostly obtained from traditional fermented food.

The energy crisis that hit world since the 20th centuries triggers intensive
exploration of alternative energy resources. Recently, biofuel is produced to
address under supply of transportation fuel, but it is still suffering from social
controversy due to resource competition between food sufficiency particularly in
developing countries and huge energy demand due to rapid population growth.
Microbial based biofuel through exploiting Single Cell Oils (SCOs), popular as
microbial oil, produced by oleaginous yeasts, could offer an alternative for
renewable energy sources since consuming less space, and rapid biomass
production. This can be achieved by development of strains able to convert low
cost substrates, grow quickly to high density, and produce larger quantities of
neutral lipid, and development of improved harvesting. Microbial oils do have
some inherent advantages over plant oils: microbial biodiesel could be produced
year-round (given available feedstock).
The present study focus on isolation, identification of yeasts from
Indonesian resources, and proposal of new taxa as well as yeasts diversity study
and assessment of physiological character include analysis of hydrolyses
carboxymethyl-cellulose, lipid accumulation, and verification of lipogenesis of
selected strain using varying carbon sources. Source of microorganism were from
Indonesian natural resources obtained from Eastern and Western Part of Indonesia
includes South East Sulawesi, Papua, East Nusa Tenggara, Bali and West Java.

The natural resources were collected from varying sources include leaf surface,
leaf litter, soil, insect, insect larva, insect larva gut, insect tunnel, decay wood and
mushroom from South East Sulawesi; soil from Papua; soil and litter from East
Nusa Tenggara;litter and flower from West Java; and plant material of Piper betle
and Piper nigrum from Bali and Piper betle from West Java. The yeasts diversity
discussion is started in sequential from two hot spot biodiversity areas: Papalia
and Mekongga in South East Sulawesi, followed by the most pristine area Raja
Ampat, Papua and dry savana area in Kupang, East Nusa Tenggara, and ended
with plant associated yeasts in Jembrana, Bali represent low land and Gunung
Salak and Cibodas Botanical Garden, West Java for high land. Yeasts strains were
identified by morphology and by amplification of their D1/D2 domain of the end
of the large subunit (26S) rDNA. For the selected strains for proposal of new taxa,
the identifications were confirmed by sequencing of their ITS1-5.8S rDNA ITS5
regions.
In total 415 of yeasts strains were isolated from varying resources in
Indonesia. The yeasts isolated from Papalia South East Sulawesi were represented

by 260 strains, Mekonga area by 38 strains, Raja Ampat by 23 strains, Kupang by
15 strains, Bali 47 strains and West Java area by 24 strains from Gunung Salak
ecosystem, and 8 strains from Cibodas Botanical Garden.

Molecular identification revealed that the yeasts strains are taxonomically
diverse, 40 genera reside under the Ascomycota and 22 genera under
Basidiomycota. The most diverse genera (32 genera), include 28 genera under
Ascomycota and 4 genera under Basidiomycota, were occurred in Sulawesi.
While 9 genera (5 under Basidiomycota and 4 under Ascomycota ) were recorded
from Raja Ampat, and only 4 genera under Basidiomycota obtained in Bali, and 3
genera under Ascomycota obtained from Cibodas. Leaf, leaf litter and insect gut
were 3 most-richest yeasts sources. These results were implying enormous
diversity of yeasts in Indonesian resources. Insect gut is the new source of
microorganism used in this study. Soils were nutrient rich substrate, however less
divers of yeasts were isolated. This would be due to other microorganisms out
compete yeasts in soil ecosystem. More advance isolation and culturing technique
should be developed to obtain more divers soil yeasts.
Homology and phylogenetic analyses reveal that 204 yeasts strains showing
greater than 1% nucleotide substitution in the D1/D2 region of LSU rDNA, and
they are likely belong to different species. Most of the candidate for new taxa
were isolated from insect include larvae gut, insect larvae, and insect’s body
surface. High diversity of insect yeasts including novel taxa would imply that
insect live cycle is closely associated with yeasts. Novel taxa isolated from
varying Indonesian resources were phylogenetically diverse and distributed within

the Ascomycota
(clade Yamadazyma, Wickerhamomyces, Ogateae,
Metschnikowia, Candida, Kodamaea) and Basidiomycota (clade Bulleromyces).
Two novel species: Yamadazyma sp. nov. reside within phylum Ascomycota ,
order Saccharomycetales, family Debaryomycetaceae, representing yeasts
inhabiting insect, whileCiteromyces sp. nov., belonging to order
Saccharomycetales, family Wickerhamomycetaceae representing yeasts inhabiting
leaf litter are further described for complete proposal of new species.
Based on ability to hydrolyze carboxymethyl cellulose, 157 isolated strains
of Ascomycota and Basidiomycota inhabiting leaf, leaf litter, insect gut, soil,
decay wood, and part of plant (stem, leaf, flower and fruit of Piper betle and Piper
nigrum) were obtained implying that cellulolytic yeasts were polyphyletic. More
than 10 strains were candidate for novel species. Sporobolomyces poonsookiae
Y08RA07, Rhodosporidium paludigenum Y08RA29 and Cryptococcus flavescens
Y08RA33 are most potential cellulolytic yeasts isolated from Raja Ampat.
Two hundred two strains of oleaginous yeasts were isolated from leaf
litter, litter, insect gut, decay wood, Piper betle L and Piper nigrum L collected
in South East Sulawesi, Raja ampat, NTT, Bali and West Java. Our study proved
that natural resources were good source for isolating oleaginous yeasts. Most of
oleaginous yeasts were floating in preservation medium (glycerol 10 %) and with

intracellular lipid bodies. Candida was prominent genera of oleaginous yeasts,
and 15 strains could be novel species. The strain of this genus accumulate lipid
between 20-40 %. Other important genera were Rhodosporidium, Sporidiobolus,
Metschinkowia and Cryptococcus accumulate lipid from 20 to > 40 %. This study
reaffirm oleaginous yeasts are clearly polyphyletic.

Our interest was also obtaining yeasts that hydrolyze cellulose and
accumulate large amount of lipid. This group of yeasts are defined as cellulooleaginous. Based on this definition about 78 strains were included in this group.
Cellulo-oleaginous yeasts were mostly isolated from leaf, leaf litter, Piper betle,
insect larvae and insect tunel. We found several Candida were cellulo-oleaginous
yeasts, they were isolated from wide resources include leaf, leaf litter, Piper betle,
insect larvae and insect tunel implying that cellulo-oleaginous yeasts were
ecologically distributed in organic rich substrates. Sporodiobolus, Aureobasidium,
Rhodosporidium, Pseudozyma, Debaryomyces, Pichia, Cryptococcus are other
important yeasts that would be potentially explored for hydrolyzing
lignocellulose hydrolysate for biofuel production.
Several carbon sources were evaluated for lipogenesis character of
selected strains include Candida (Candida intermedia PLE6DP6, Candida
orthosilopsis InaCC Y-302/Y09GS34, Candida oleophila InaCC Y306/Y09GS48), and highest lipid accumulator (Crytococcus flavescent PL3DP6),
their relative (Cryptococcus humicola PLE3DP9),(Cryptococcus luteolus InaCC
Y-265/ Y10BS72) and strains candidate for novel species (Yamadazyma aff.
mexicana PL1W2) using Lipomyces starckeyii NBRC 10831 as a reference strain
for oleaginous yeasts. FAME species compositions were affected by C-source
feeding for lipid accumulation. Finally based on FAME species composition, we
confirm that selected oleaginous yeasts with CMC-ase activity are good candidate
for biofuel production using cheap materials such as agricultural wastes.
Key words: Indonesian resources, cellulolytic yeasts, oleaginous yeasts, taxonomy

© Copy Right IPB, 2014
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DIVERSITY OF CELLULOLYTIC, OLEAGINOUS AND
CELLULO-OLEAGINOUS YEASTS FROM INDONESIAN
RESOURCES

ATIT KANTI

A dissertation submitted to the Department of Biology and
the committee on graduate school of Bogor Agricultural
University in partial fulfillment of the requirements for
the degree of doctor of philosophy in microbiology

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

Examiner for internal examination

: Prof. Dr. Indrawati Gandjar
Dr. Gayuh Rahayu

Examiner for the external examination : Prof. dr. Dr. Retno Wahyuningsih
Prof. Dr. Lilis Nuraida, M.Sc

Dissertation Title
Name
NIM

: Diversity of Cellulolytic,Oleaginous and Cellulooleginous Yeasts from Indonesia Resources
: Atit Kanti
: G361100012
Approved by

Dr.Nampiah Sukarno
Principal Advisor

Prof. Dr. Latifah K Darusman
Advisor committe

Prof. Dr.Endang SukaraAdvisor
committe

Kyria Boundy-Mills, PhD.
Advisor committe
Signed by
Head of Microbiology Study
Pogram

Dean of Bogor Agricultural
Graduate School

Prof.Dr.Anja Meryandini, MS

Dr. Ir. Dahrul Syah, MSc Agr

Date of examination: 1st July 2014

Date of graduation:

ACKNOWLEDGEMENTS
First and foremost I want to thank God for blessing me to finish writing
Ph.D dissertation entitle: Diversity of Cellulolytic, Oleaginous and CelluloOleaginous Yeasts from Indonesian Resources. The research works were
started in September 2011.
I want to express my most sincerely gratitude to the advisor committee
Dr. Nampiah Sukarno, Prof. Dr. Latifah K Darusman of Bogor Agricultural
University, Prof. Dr. Endang Sukara of Indonesian Institute of Sciences and
Kyria Boundy- Mills, Ph.D of University California Davis, USA for all their
contributions of time, and ideas to make my Ph.D experience productive and
stimulating.
I would like to extent my gratitude to all members of InaCC for
laboratory assistance, and my colleagues that have contributed immensely to
my personal and professional time at Bogor Agricultural University during
Ph.D course.
I would like to expess my sincere gratefull to all the lecturers,
administrative staffs of post graduate program of Bogor Agricultural
University for their advisory, assistance and help during my study.
I gratefully acknowledge the funding sources that made my Ph.D work
possible. I was funded by the Indonesian Ministry of Science and Technology
through post graduate study grant. Material used in this study is a part of
research collaboration between University of California Davis and the
Government of the Republic of Indonesia, funded by Grant Number
U01TW008160 from the US National Institutes of Health Fogarty
International Center, the NIH Office of Dietary Supplements, the National
Science Foundation and the Department of Energy. This project was
supported by the USDA Agricultural Food Research Initiative of the National
Food and Agriculture, USDA, Grant #35621-04750.
I wish this study contribute to science and technology development
particularly on exploiting potential use of tropical microorganism.

Bogor, August 2014
Atit Kanti

CONTENT
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDICES

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viii

INTRODUCTION __________________________________________ 1
Overall objective ___________________________________________ 6
Specific objective ___________________________________________ 6
Scope of study _____________________________________________ 6
LITERATURE REVIEW _______________________________________ 7
MATERIALS AND METHODS ________________________________ 14
Research methodology ______________________________________ 14
Sampling sites and samples sources ____________________________ 15
Isolation procedure _________________________________________ 16
Analyses procedure ________________________________________ 19
RESULTS AND DISCUSSION_________________________________ 24
Conclusion _______________________________________________ 90
Recommendation __________________________________________ 90
REFERENCES ______________________________________________ 91
APPENDICES _____________________________________________ 110
BIOGRAPHY _____________________________________________ 111

LIST OF TABLES
1

List of samples collected as biotope for oleaginous and cellulolytic
yeasts studies

17

2

Parameter of the instrument GCMS for lipid species identification

21

3

Number of strains of yeasts isolated from varying soures: leaf surface,
leaf litter, soil, insect, insect larva, insect larva gut, insect tunel, decay
wood, fungal, and plant materials of Piper betle and Piper nigrum
collected from South East Sulawesi, Papua, East Nusa Tenggara, Bali
and West Java, Indonesia
Taxonomic placement of yeasts isolated from leaf surface, leaf litter,
and soil, in South East Sulawesi
Taxonomic placement of yeasts species isolated from insect larva gut in
South East Sulawesi
Taxonomic placement of yeasts species isolated from insect and insect
tunel, in South East Sulawesi, Indonesia
Taxonomic placement of yeasts species isolated from mushroom in
South East Sulawesi, Indonesia
Taxonomic placement of yeasts species isolated from decay wood,
decomposed wood, infected wood in South East Sulawesi, Indonesia
Taxonomic placement of yeasts isolated from soil from Raja Ampat,
Papua
Diversity of yeasts species isolated from leaf litter and soil in East Nusa
Tenggara, Indonesia
Diversity of yeasts species isolated from Piper betle and Piper nigrum
in Bali
Diversity of yeasts species isolated from Piper betle Gunung Salak,
Indonesia
Taxonomic placement of yeasts isolated from litter and flower in
Cibodas Botanical garden
The growth characteristics of the Yamadazyma sp.nov. strains are
compared with those of Y. mexicana and Y. philogaea
The growth characteristics of the Citeromyces sp.nov strains are
compared with those of C.matritensis NBRC 0954T
Diversity of cellulolytic yeasts isolated from various biotopes in South
East Sulawesi, Raja Ampat, NTT, Bali and Gunung Salak
Cellulolytic yeasts isolated from South East Sulawesi. Sample sources
were leaf-litter, litter, soil, decay wood, insect gut and soil
Cellulolytic yeasts isolated from South East Sulawesi. Sample sources
were insect, insect larva, insect tunel, insect frass, and mushroom
Cellulolytic yeasts isolated from NTT (East Nusa Tenggara), sample
types were litter and soil
Cellulolytic yeasts isolated from Piper betle and Piper nigrum from Bali
and Gunung Salak. Sample code started with character B and GS
indicate materials were obtained from Bali and Gunung Salak

25

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21
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23
24
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27
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31
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respectively. L, F, Fr and S represent leaf, flower, fruit and stem
respectively. Whereas PB and PN represent Piper betle and Piper
nigrum respectively
Cellulolytic yeasts isolated from soil in Raja Ampat Papua
Diversity of oleaginous yeasts isolated from secondary forest in South
East Sulawesi. Sample sources were leaf, leaf-litter, soil, decay wood
and gut insects
Diversity of oleaginous yeasts isolated from secondary forest in South
East Sulawesi. Sample sources insect, insect larva, mushroom, and gut
insects
Diversity of oleaginous yeasts from soil of Raja Ampat, Papua. Soil
from tropical rain forest was used as yeasts sources
Diversity of oleaginous yeasts from East Nusa Tenggara (NTT). Litter
from savanah was used as yeasts sources
Diversity of oleaginous yeasts from litter and flower collected in
Cibodas Botanical Garden
Diversity of oleaginous yeasts isolated from Piper bettle and Piper
nigrum. Sample sources were stem, leaf, flower and fruit leaf-litter, soil,
decay wood and gut insects
Number of genera, species and strains of oleaginous yeasts isolated from
Piper nigrum L and Piper betle L collected from Bali
Number of species and strains of oleaginous yeasts isolated from Piper
betle L collected from West Java
Number of species and strains of oleaginous yeasts isolated from Piper
betle L collected from West Java
Lipid composition of oleaginous yeasts grown on N-limited
Cellulo-oleaginous yeasts reside within Ascomycetous yeasts
Cellulo-oleaginous yeasts reside within Basidiomycetous yeasts
Fatty Acid Methyl Esther (FAME) compositition of total lipid procedure
from varying C-source
Summary of yeasts inhabiting varying sources

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LIST OF FIGURES
1
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3

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Research methodology for studying diversity of cellulo-oleaginous and
its physiological character
Flow chart of experimental works
Sampling sites for isolation of yeasts from Indonesian sources, sampling
was conducted in 2010, 2011, and 2012. Samples were collected from
soil, litter, plant materials, larvae of wood insect, leaf, stem, and fruit of
Piper nigrum and Piper betle
Number of strains from each location and sample source. (a) Papalia leaf
surface, (b) Mekongga leaf surface, (c) Papalia leaf litter, (d) Mekongga
leaf litter
Yeasts inhabiting Piper betle leaf in Bali
Yeasts inhabiting Piper betle stem in Bali
Yeasts inhabiting Piper betle leaf in Gunung Salak
Number of strains obtained from varying sources proposed as novel
taxa. Strain proposed as new taxa when strains showing greater than 1%
nucleotide substitution in the D1/D2 region of LSU rDNA
Phylogenetic placement of Wickerhamomyces clade and related species
determined from neighbor-joining analysis of D1/D2 LSU rRNA gene
sequences. Bootstrap values are from 1000 replicates
Phylogenetic placement of Ogataea clade and related species
determined from neighbor-joining analysis of D1/D2 LSU rRNA gene
sequences. Bootstrap values are from 1000 replicates
Phylogenetic placement of Metschnikowia and related species
determined from neighbor-joining analysis of D1/D2 LSU rRNA gene
sequences. Bootstrap values are from 1000 replicates
Phylogenetic placement of Kodamaea clade and related species
determined from neighbor-joining analysis of D1/D2 LSU rRNA gene
sequences. Bootstrap values are from 1000 replicates
Phylogenetic placement of Bulleromyces clade and related species
determined from neighbor-joining analysis of D1/D2 LSU rRNA gene
sequences. Bootstrap values are from 1000 replicates
Phylogenetic placement of Yamadazyma sp.nov among neighbouring
yeasts determined from neighbor-joining analysis of D1/D2 LSU rRNA
gene sequences. Bootstrap values are from 1000 replicates
Yamadazyma sp.nov. T1509RPC. Cells, after 3 days on Yeasts Malt
Extract Agar, at 25° C
Phylogenetic placement of Citeromyces sp.nov among neighbouring
yeasts determined from neighbor-joining analysis of D1/D2 LSU rRNA
gene sequences. Bootstrap values are from 1000 replicates
Citeromyces sp.nov. CLi04.N-2 (A), CLi04.N-3(B). Asci with
ascospores, after 19 days, Corn Meal agar, 25° C. Bar 5 µm
Scanning electron microscrops of Citeromyces sp.nov. (CLi04.N-2). On
Yeasts Malt Extract agar. Asci are persistent, thick-walled roughened

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23
24
25
26
27
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30

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32
33
34

35

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37

spherical ascospores after 16 days at 25°C
Scaning electron microscrops of Citeromyces sp.nov. (CLi04.N-3). On
Yeasts Malt Extract agar. Asci are persistent, thick-walled roughened
spherical ascospores after 16 days at 25°C
Morphological characters of growing colonies in PDA media after 5
days incubation
Clear
zone
formation
of
cellulolytic
Yeastscellulolytic
yeastsSporobolomyces poonsookiae Y08RA07 (A), Rhodosporidium
paludigenum Y08RA29 (B) and Cryptococcus flavescens Y08RA33 (C)
Profile of CMC-ase activity of tested isolated grown for 7 days in
media with CMC was the sole carbon sources
Profile of CMC-ase activity of Sporobolomyces poonsookiae
Y08RA07as affected by temperature and pH
Profile of CMC-ase activities of Rhodosporidium paludigenum
Y08RA29 during cultivation at various pH and temperature
CMC-ase activity profile of Cryptococcus flavescens Y08RA33 grown
in CMC used as the carbon sources at various pH and temperature
Cellulase activity of Sporobolomyces poonsookiae Y08RA07grown in
bamboo leaf, paper waste, and paddy straw
Cellulase activity of Rhodosporidium paludigenum Y08RA29 grown in
bamboo leaf, paper waste, and paddy straw
CMC-ase activity profile of Cryptococcus flavescens Y08RA33 grown
in bamboo leaf, paper waste, and paddy straw
Morphology of oleaginous yeasts: (A) floating cell on glycerol medium,
(B) intracellular lipid bodies (C and D)
Diversity of oleaginous yeasts isolated from Indonesian resources.
Sample sources: leaf litter; litter; decay wood; Piperbetle and
Pipernigrum,obtained from South East Sulawesi, Raja Ampat, NTT,
Bali and West Java, Indonesia
Number of strains accumulate lipid >30 % (total lipid/cell dry weight,
w/w)
Number of strain accumulate lipid greater than 20 % but less than 30 %
(total lipid/cell dry weight, w/w)
Strains accumulate Lipid > 40 % (total lipid/cell dry weight, w/w)
Morphology of oleaginous yeasts Candida intermedia PLE6DP6,
Candida orthosilopsis InaCC Y-302/Y09GS34, Candida oleophila
InaCC Y-306/Y09GS48) grown on N-limited with CMC as the sole Csource
CMC-ase activity and lipid accumulation of Candida intermedia
PLE6DP6, Candida orthosilopsis Y09GS34,
Candida oleophila
Y09GS48, Crytococcus flavescent PL3DP6, Cryptococcus humicola
PLE3DP9, Yamadazyma aff. mexicana PL1W2, and yeasts Lipomyces
starckeyii NBRC 10831 used as reference strains for oleaginous yeasts
Lipid accumulation by oleaginous yeasts at varying C-sources: (A)
glucose, (B) glycerol, (C) xylose and (D) CMC.
Metabolic pathways of lipid accumulation using glucose and acetate as
C-sources

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LIST OF APPENDICES
1
2
3
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5
6
7
8
9

Physiological characteristic of Yamadazyma sp. nov.
Physiological characteristic of Citeromyces sp. nov.
Guanine + Cytosine data of Citeromyces sp.nov.
Ubiquinone type of Citeromyces sp.nov.
Lipid compositition of Y09GS34 /InaCC Y302 analyzed by
GCMS
Published Paper 1: Cellulolytic yeasts isolated from Raja Ampat,
Indonesia
Published Paper 2: Indonesian oleaginous yeasts isolated from
Piper betle and Piper nigrum
Published Paper 3: Oleaginous yeasts with CMC-Ase activities
for biofuel production
Published Paper 4: Species diversity of yeasts inhabiting leaf
surfaces and leaf litter in secondary rain forest in South East
Sulawesi, Indonesia

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INTRODUCTION
Yeasts are unicellular fungi and include in two big groups of fungal taxa,
ascomycetous and basidiomycetous, which contain 149 genera and nearly 1500
species currently described. Taxonomist make clear distinction between yeasts
and those dimorphic filamentous fungi that often produce abundant yeasts-like
growth. Yeasts can be defined as fungi whose asexual growth predominantly
results from budding or fission, and which do not form their sexual states within
or upon a fruiting body (Kurtzman et al. 2011). For ascomycetous yeasts, this
distinction has been substantiated by molecular comparisons, which demonstrate
that budding and fission yeasts are phylogenetically distinct from one another.
One exception is the genus Eremascus, which has unenclosed asci, but budding
cells are not formed. A similar distinction can be made for basidiomycetous
yeasts, which are often phylogenetically separate from the mushrooms and other
taxa that form complex fruiting bodies. In summary, yeasts, are generally
characterized by budding or fission in ascomycetes and budding in
basidiomycetes as the primary means of asexual reproduction, and have sexual
states that are not enclosed in fruiting bodies (Kurtzman, 2001).
Reproductive system of yeasts, like fungi, is quite complex. They may
reproduce through asexual and sexual reproductive cycles. The most ubiquitous
mode of vegetative growth is through asexual reproduction by budding. The
asexual life cycle start with formation of a small bud (also known as a bleb), or
daughter cell, is formed on the parent cell. The nucleus of the parent cell splits
into a daughter nucleus and migrates into the daughter cell. The bud continues to
grow until it separates from the parent cell, forming a new cell. The daughter cell
produced during the budding process is generally smaller than the mother cell.
Some yeasts, including Schizosaccharomyces pombe, reproduce by fission instead
of budding, thereby creating two identically sized daughter cells. In general, under
high-stress conditions such as nutrient starvation, haploid cells will die; under the
same conditions, however, diploid cells can undergo sporulation, entering sexual
reproduction (meiosis) and producing a variety of haploid spores, which can go
on mating (conjugate), reforming the diploid cell.
The haploid fission yeasts Schizosaccharomyces pombe is a facultative
sexual microorganism that can undergo mating when nutrients are limiting.
Exposure of S. pombe to hydrogen peroxide, an agent that causes oxidative stress
leading to oxidative DNA damage, strongly induces mating and the formation of
meiotic spores. The budding yeasts Saccharomyces cerevisiae reproduces by
mitosis as diploid cells when nutrients are abundant, but when starved, this yeasts
undergoes meiosis to form haploid spores. Haploid cells may then reproduce
asexually by mitosis. In nature, mating of haploid cells to form diploid cells is
most often between members of the same clonal population and out-crossing is
uncommon. Analysis of the ancestry of natural S.cerevisiae strains led to the
conclusion that out-crossing occurs only about once every 50,000 cell divisions
(Kurtzman et al. 2011). These observations suggest that the possible long term
benefits of outcrossing (e.g. generation of diversity) are likely to be insufficient
for generally maintaining sex from one generation to the next. Rather, a short term
benefit, such as recombinational repair during meiosis, may be the key to the

2
maintenance of sex in S. cerevisiae. Some pucciniomycete yeasts, in particular
species of Sporidiobolus and Sporobolomyces, produce aerially dispersed,
asexual ballistoconidia.
Yeasts are chemoorganotrophic, as they use organic compounds as a
source of energy and do not require sunlight to grow. Carbon is obtained mostly
from hexose sugars, such as glucose and fructose, or disaccharides such
as sucrose and maltose. Some species can metabolize pentose sugars such as
ribose (Barnett et al. 2000) alcohols, and organic acids. Yarrowlia lipolytica
assimilate glycerol, and accumulate lipid when nitrogen is depleted (Sorger, 2002).
Some yeasts such as Trichosporon fermentans produce endoglucanase,
exoglucanase and β-glucosidase (Zhou et al. 2004). Yeasts having cellulolytic
characters and accumulating large amount of lipids are intensively studies for
biofuel production (Liang & Jiang, 2013).
Yeasts species either require oxygen for aerobic cellular
respiration (obligate aerobes) or are anaerobic, but also have aerobic methods of
energy production (facultative anaerobes). Unlike bacteria, no known yeasts
species grow only anaerobically (obligate anaerobes). Yeasts grow best in a
neutral or slightly acidic pH environment. With these physiological characters
yeasts have been a focus for many industrial product development (Zhang, 2011).
Yeasts may grown in many various temperature range for their best growth.
For example, Leucosporidium frigidum grows at - 2 to 20°C (28 to
68°F), Saccharomyces telluris at 5 to 35 °C (41 to 95°F), and Candida slooffi at
28 to 45 °C (82 to 113 °F). The cells can survive freezing under certain conditions,
however their viability are decreasing over time. Their ability to grow at wide
temperature ranges is other physiological advantages to use yeasts for many
research interest (Wilson & Talbot, 2009).
In general, yeasts are grown in the laboratory on solid growth media or in
liquid broths. Common media used for the cultivation of yeasts include potato
dextrose agar or potato dextrose broth, nutrient agar, yeasts peptone dextrose agar,
and yeasts mould agar or broth. Home brewers who cultivate yeasts frequently use
dried malt
extract and
agar
as
a
solid
growth
medium.
The antibiotic cycloheximide is sometimes added to yeasts growth media to
inhibit the growth of Saccharomyces yeasts and select for wild/indigenous yeasts
species.
The appearance of a white, thready yeasts, commonly known as kahm
yeasts, is often a by product of the lacto fermentation (or pickling) of certain
vegetables, usually the result of exposure to air. Although harmless, it can give
pickled vegetables a bad flavor and must be removed regularly during
fermentation Schoneck, 2002. Yeasts were isolated from Indonesian fermented
foods such as tape, brem, and dadih (Limyati & Juniar, 1998). The consistent
occurrence of yeasts in traditional fermented food would suggest yeasts having
untapped economic potentials.
Yeasts are ecologically distributed from terrestrial, marine, and fresh water
ecosystem (Fell et al. 2000). They have been isolated from numerous biotopes
including leaf surface, root, litter, flower, gill of marine fish, inscet gut, and event
from dessert soil. The occurance of yeasts in diverse biotopes, indicating its strong
physiological characters including its roles in mediating natural ecosystem
processes (Butinar et al. 2005), (Druvefors et al. 2002). During the past decades

3
soil yeasts ecology mostly comprised surveys conducted to determine yeasts
biodiversity in this habitat (Botha, 2006). It must be noted however, that the
functional diversity among soil microorganisms may outweigh the diversity
among taxonomic entities. Thus, unless the physiology of specific taxa playing a
key role in an ecosystem is known, the mere listing of microbial taxa occurring
within a particular habitat may be more of value to the taxonomist than to the
ecologist. Similarly, the potential of soil yeasts communities to affect ecosystem
function may be unrelated to yeasts species delineated using either selected
morphological and physiological tests, or analyses of taxonomic informative gene
sequences. It is well known that many yeasts species show either intraspecific
diversity or no difference when certain physiological traits such as assimilation of
specific carbon or nitrogen sources are compared (Kurtzman and Fell, 2006).
Characteristics that commonly occur among yeasts species frequently
encountered in soil are the ability to utilize L-arabinose, D-xylose and cellobiose
aerobically (Lian et al. 2013). These carbohydrates are the products of hydrolytic
enzymes, which originate from bacteria or moulds and act on lignocellulosic plant
material. Some soil yeasts are also found to assimilate intermediates of lignin
degradation i.e. ferulic acid, gallic acid, 4-hydroxybenzoic acid, protocatechuic
acid and vanillic acid (Sampaio, 2001).
The black yeasts or pigmented fungi mostly forms hyphal states
(Sterflinger, 2006). Typical representatives of this group are Aureobasidium,
Coniosporium and Exophiala, are known for the ability to grow oligotrophically
on mineral surfaces or to degrade recalcitrant materials such as complex aromatic
hydrocarbons as well as polymeric components of wood (Sterflinger, 2006). In
order to survive as saprotrophs in soils, containing either low nutrient
concentrations or recalcitrant woody materials as potential carbon sources the
non-filamentous budding yeasts, are unable to degrade these materials, therefore
to obtain nutrients they should perform other metabolism. A strategy that
microorganisms, including those in soil, frequently employ to overcome nutrient
limitation is to interact with other organisms and form symbioses (Van der
Heijden and Sanders, 2002).
Phylloplane yeasts may represent a life form that changes from one type of
ecological interaction to another as its habitat changes. For instance, epiphytes
residing on leaves of deciduous trees are joined by other fungi and become
saprophytes when the leaves drop and start to decay in the upper layer of the
forest floor. The subsequent process of nutrient recycling in the saprophytic
channel of the ecosystem is an important indirect role of many fungi that are
associated with leaves. Fungal decomposers are thought to drive the global carbon
cycle and phylloplane yeasts ultimately participate in that process in one way or
another. However, the role of phylloplane yeasts in soil processes may not be
significant because many only join the upper layers of soil and do not persist in
soil after leaves fall (Lanchane et al. 2011).
Yeasts are chemoorganotrophic organism occur in wide range ecosystem
type, and Indonesia consist of 42 ecosystem types and presumably harbor high
diversity of yeasts (Kanti et al. 2013). While ecological studies of yeasts from
natural habitats have been conducted extensively in temperate regions (Libkind et
al. 2008; Teoh et al. 2004; Wang & Bai, 2004), species richness data indicate a
need for additional studies in tropical ecosystems, particularly in Asia (Nakase et

4
al. 2005; Lee, 2009 a,b). Indonesia is a tropical nation comprised of over 17,000
islands, located at the intersection of multiple tectonic plates. It comprises five
main islands: Sumatra, Java, Borneo (known as "Kalimantan" in Indonesia),
Sulawesi, and Papua; two major archipelagos (Nusa Tenggara and the Maluku
Islands); and sixty smaller archipelagoes, and thus are rich in biodiversity, having
unique flora and fauna (ICBG, 2011), and presumably microbes as well (Kanti et
al. 2013). Rifai (1995) estimated Indonesia has more than 200,000 species of
fungi. However, little information on the species diversity of Indonesia indigenous
yeasts and yeasts-like fungi has been generated.
Studies of Indonesian yeasts primarily related to their role in fermented foods
(Abe et al. 2004; Kuriyama et al. 1997). Early studies of yeasts from natural
environments in Indonesia include that by Deinema in 1961, who found Candida
bogoriensis from the surface of leaves of the flowering shrubs Randia melleifera
(Rubiaceae) in Bogor. In recent years, studies had been performed to explore
yeasts diversity in Indonesia, (Nakase, 2005; Sjamsuridzal et al. 2010; Sudiana
and Rahmansyah 2002; Wellyzar et al. 2013).
Sulawesi is one of the five major islands of Indonesia, and is of geographic
and biological interest because it lies at the intersection of the Australian and
Southeast Asian tectonic plates, and having rich biodiversity occupying the
Wallace and Weber line. Sulawesi has high biodiversity, and reported to have
high biodiversity of flora (Cannon, 2005; Cannon, 2007) and fauna (Noyes, 2008;
Koch, 2011; Kimsey and Ohl, 2012). At the microbial diversity level, further
study is needed to verify the species richness of this area.
Other than Sulawesi, Raja Ampat, located on the north-western tip of
Papua, eastern Indonesia, is well known for island with high endemicity of plants
and animals. Located on East side of Walace line having tropical climate with wet
season throughout September to February, having calcareous soil. Intensive
survey on marine ecosystem showing high fish diversity representing more than
75 % of tropical fish are found in this marine ecosytem. Ecological surveys in the
Raja Ampat archipelago found 1320 species of coral reef fish (Allen et al. 2009)
and 553 species of scleracti- nian corals which is around 75% of the world’s total
(Veron et al. 2009). Though the important of this area as a biodiversity hot spot,
very few studies on microbiology, partcularly soil microbiology studies are
carried out in this area, and none dealt with diversity of oleaginous yeasts.
Crossing this island to the west side, still in the eastern part of the Wallace
line, East Nusa Tenggara Timur having dry climate with water scarcity. The soils
are dominated by litosol, rigosol, aluvial and mediteran, and having savanah
ecosystem are unique places that would harbour yeasts having unique
physiological characters. Again to the west side of the Wallace line, Bali island
has varying ecosystem type from dry in Eastern Part, and wet in the middle
island having Piperaceae plant used as traditional medicine (Aravind et al. 2010)
that would harbour oleaginous yeasts (Kanti et al. 2013). This phenolic rich plant
also found in Java, and has been used widely for medicinal herb on oral hygiene.
To obtain microorganisms from the last of submontain forest in West Java,
Gunung Salak and Cibodas Botanical Garden are selected as sampling site.
The energy crisis that hit the world since the 20th centuries triggers intensive
exploration of alternative energy resources. Recently, Biofuel is produced to
address under supply of transportation fuel, but it is still suffering from social

5
controversy due to resource competition between food and energy sufficiency.
Microbial based biofuel through exploiting Single Cell Oils (SCOs), popular as
microbial oil, produced by oleaginous yeasts, could offer an alternative for
renewable energy sources since consuming less space, and produce biomass
rapidly.
Due to the outstanding capacity of Saccharomyces cerevisiae and certain
other yeasts to produce ethanol and other organic products, yeasts will continue to
be developed as producers of ethanol and other fuels. This process can be
achieved by development of strains able to convert low cost substrates, grow
quickly to high density, and produce larger quantities of neutral lipid, and
development of improved harvesting and dewatering technologies.
Microbial oils do have some inherent advantages over plant oils: microbial
biodiesel could be produced year-round (given available feedstock), on land
unsuitable for agriculture, with production rates up to 100X that of plant oils in
liters/hectare/year (Atabani &Silitonga, 2012). Of the 33.000 known species of
algae (Brodie et al. 2007), at least forty are considered oleaginous (Griffiths and
Harrison, 2009), i.e. they have been demonstrated to accumulate over 20% lipid
by dry weight (Ratledge, 1979). Doubling times vary considerably, depending on
species and growth conditions, ranging from hours to days (Sheehan, 1998). Oil
and hydrocarbon content of oleaginous species can range from 20-60%, and up to
80% for exceptional genera such as Nannochloropsis, Schizochytrium and
Botryococcus (Chisti, 2007). Yeasts are more preferable used as biodiesel since
they multiply much faster than algae, and do not require light and large space.
Physiologically yeasts accumulate larger amount of lipid than algae (Sitepu et al.
2013). These critical findings reaffirming the urgency of understanding ecological
distribution, physiology and genetic chracter of oleaginous yeasts for viable
technological development of biofuel industry (Meng et al. 2011; Dai et al. 2007).
While oleaginous yeasts are having ability to use varying organic susbstrates
included organic acids, simple carbon sources: glucose, fructose, arabinose,
xylose and glycerol (Mussatto et al. 2008; Dai et al. 2007), the ability to hydrolize
complex carbohydrate included cellulose, hemicellose, major component of
agricultural waste woud be of benefit for reducing production cost of industrialscale biofuel (Choi et al. 2012).
Since several yeasts species are oleaginous and accumulate high
concentrations of lipids (Cohen and Ratledge 2005), they warrant evaluation as
components for industrial production of oils and biodiesel (Cohen and Ratledge
2005; Dai et al. 2007; Matsumoto et al. 2001; Ratledge 2002). As opposed to
culture in terrestrial water bodies, production of single-cell oils could be done in
fermentors or in specialized solar reactors, which would be more ecologically
sound than certain other technologies. A by-product of biodiesel is glycerol that
can accumulate in substantial quantities. Yarrowia lipolytica and other yeasts have
been evaluated for the fermentation of glycerol to higher-value products such as
the bulk chemical citric acid (Fickers et al. 2005). Study of cellulolytic and
oleaginous yeasts from natural habitat particularly tropical region have not been
intensively conducted, hence our study will fill this gap of knowledge.
Yeasts diversity from natural habitats have been conducted extensively in
temperate regions), species richness data indicated that there is a need for
additional studies in tropical ecosystems. Yeasts mediate hydrolyses of polymeric

6
substances include cellulose and produce intracellular metabolites including lipid
bodies which can be used as biofuel feeding material. This study will comprise:
a. Diversity of yeasts isolated from various unique habitats
b. The novel taxa of isolated yeasts
c. Physiological character of cellulolytic yeasts particularly on hydrolyses of
cellulose to produce fermentable substances
d. Unique physiological character of oleaginous yeasts especially on their ability
to use various carbon sources
Overall objective
To study yeasts diversity in Indonesian resources and verifying its
physiological characters especially on hydrolyses of cellulose and lipid
accumulation (lipogenesis) on various carbon sources, would be important bioresources for next generation biofuel research.

Specific objectives
The specific o