Microbiology Division, Research Center for Biology - LIPI

 

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  .:: Biosystematics and Culture Collection Research Group ::.

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Exploring, identifying, preserving and distributing Indonesian microorganism for basic taxonomic studies, biotechnology application, and other purposes

Indonesia is believed to be one of the mega-biodiversity of the world that includes biodiversity of microorganism. This is reasonable since Indonesia is blessed with very diverse ecosystems. Different ecosystems may contain different niche of microorganism. Indonesia is also inhabited by diverse ethnic groups with different cultures that  include unique traditional foods and medicines. Those unique foods or medicines may carry different microorganisms. Interaction with people from other nations has also enriched our traditions and along with it foreign microorganisms may also have enriched our micro-biodiversity.  Microorganism role in supporting civilization has been re-emphasized and received a great deal of attention from industry such as pharmaceutical, agricultural, biotechnological and environmental. Microorganism is not only used directly as fermentation agent, but it is the source of new chemicals, enzymes, genes and model system for scientific studies.

Microorganism as a treasure buried in the ecosystems must be explored, identified, characterized, and preserved and distributed for further uses. And it is the mandate of this research group to at least identify the collected microorganisms and preserve them and in turn distribute them. Collection of microorganism has been done for some times in Microbiology Division of Research Center for Biology, but it has been only recently that this task is going to be formalized and structured in the Microbiology Division as LIPI-MC (LIPI Microbial Collection).  LIPI-MC is intended to be a national microbial culture collection and as an ex situ conservation for microorganisms. Its facilities and methods of collection are being developed into a world-class standard. Our main tasks include: 1) developing protocols for microbial isolation and for cell procurement using selective media for specific microorganism , 2) establishing standardized microbial identification, taxonomic studies and preservation protocol, 3) providing authentic cultures for scientists, research institutes, and private companies pursuant to national regulation (National Law No.5 of 1994), international agreement (CBD article 9), The Bonn Guide Line, MOSAIC, and LIPI-MC material transfer agreement, 4) promoting and facilitating inventory of Indonesian microorganism which contribute to ex-situ conservation of Indonesian Biodiversity collected by LIPI and other research institutes, national and abroad.

Preservation technique is instrumental to culture collection and to it is dependent upon physiological characteristic of the microorganisms. This physiology is to some extent dependent upon the genotype, or the taxa of the microorganism. A preservation technique that works well with a certain taxa may not work with certain genotypes in those taxa. In this case, continuous evaluation and development of preservation protocols need to be developed to suit that purpose.  Some of our collections have been preserved with L-drying method and need to be evaluated.  Some isolates of yeast, Rhizobium and fungi that had been preserved with L-drying method for one year were tested for the viability in YEMA and PDA media, respectively. The culture then observed after 5 day incubation at their optimum temperature.
a. Only 21/25 yeast isolates assessed were viable indicating that s L-drying was incapable to maintained yeast cell viability. The yeast isolates that died upon viability test belonged to Basidiomycetes yeast which grew and preserved better on PDA medium.

b. From of 33 isolates of Rhizobium tested, only 5 isolates were viable, 22 isolates were contaminated by Bacillus sp., and the rest had low viability.  Low viability of these isolates could be due to low number of cells at the beginning of preservation.

c. From 35 isolates of fungi tested, 34 had good growth recovery, and 4 isolates did not grow back indicating low cell viability. In most cases freeze drying preservation is most suitable for fungi preservation.

Collection of microbes in our institution has been started in 1974 and we have ca. 1800 accession number isolated from local fermented foods, farmland, forest, horticultural plants, polluted areas, unique ecosystems such as hot water ecosystems, acidic soil, oil-polluted mud, etc. Only a small proportion of that collection has been identified at species level, most of them at genus or family level. In line with the establishment of LIPI-MC, identification of those unidentified accession is to be completed in five years. Our methods of identification based on morphological, physiological and genetic analysis dependent upon the microorganism. Fungi were identified based on their morphological characters, whereas bacteria through physiological and morphological analyses, and for yeast through morphology, physiology and genetic analyses. About 131 fungi isolates, 141 isolates of bacteria were identified at genus level. The fungi included the genus of Penicillium, Aspergillus, Trichoderma, etc. The identified bacteria belonged to Rhizobium, Pseudomonas, Enterobacter, and Bacillus. About 120 yeast accessions were identified at species level an included Pichia anomala, Rhodotorula minuta, Saccharomyces cerevisiae, etc. 

To enlarge our collection, this research group collaborates with other research group that collect microbes from certain ecological systems. Agricultural system  is an interesting option since its microbes might be specific  to agricultural system, and might be applicable for organic farming. To obtain species diversity of soil agrcicultural microorganism, several soil type including peat soil and mineral soil around rhizosphere of corn, banana, and sweet potato were selected as microbial sources. The soils were resuspended in NaCl solution and serially diluted to 105.  An aliquot of the diluted suspension was plated on agar medium specific for fungus, i.e., Rose Bengal Agar (RBA).  The cultures were incubated at 27 and 45oC. A total of 65 species of fungi were isolated from peat and mineral soil rhizospehre of corn, banana, and sweet potato. The highest fungi population was encontoured in rhizosphere of sweet potato incubated at 27oC, that is ca. 8.5x105, whereas the lowest population was observed on cultures plated with suspension derived from corn rhizospehre in peat soil, grown at 45oC, that is ca. 1.1x103.  The highest fungi diversity, 23 species, was obtained from rhizosphere of corn incubated at 27oC, and followed by 15 species in sweet potato, and 13 species from banana. There were 14 species of fungi grew at 45oC.  Acremoniella sp., Chloridium sp., Gliomastix murorum, Malbranchea pulchella, Thermomyces stellatus and  Torula sp. are major fungi recovered from this study and has been deposited in LIPI-MC.

Exploration and identification of lactic acid bacteria that were responsible for food fermentation was carried out from traditional foods collected from a market in Bengkulu and included thongchai, tempoyak, taoco, tape, brem, ketan hitam, lemma, fish fermentations, rebung besar, rebung asin. Food sample of 2 gram each was mixed with salt solution and serially diluted before cultured in GYP-CaCO3 medium. Preliminary selection of lactic acid bacteria was done by sub culturing the colony that formed clear zone. Morphological character of LAB was coccus, smooth, concave and white or yellow color. Other morphological characters were gram positive, catalase negative, rods. Physiological character of LAB was homofermentative, and growth temperature range 20oC- 45oC accession. The isolates culture character then use to become reference at the beginning of identification.
 
Preservation techniques employed in our culture collection with the conventional technique for short-term preservation in few years (left), and the modern technique for long-term preservation (right) with L-drying method and keep in ampoules Viable yeast (left), bacteria (center) and fungi (right) upon viability test from isolates that have been preserved by L Drying for 1 year. Not all of the preserved collection by this method is able to re-growth
   
Determine morphological characteristics of yeast (left is Saccharomyces sp.; and in the right is Pichia sp.). A solution for a simple identification purposes
   
Collection of microorganism and its storage system at LIPI-MC
*A is stored in agar slant (number of isolates)
B is stored in agar slant, and soaked in liquid paraffin C is stored with L-drying
   
Number of accession of collected microorganism at LIPI-MC
 
Detail of spore morphological characteristics of Aspergillus sp. (left) and Torula sp. (right). To differentiate among the genus in morphological spores shape. Lactic acid bacteria are isolated from colony that are forming clear zone on isolation medium (left). In microscopy appearance, those bacteria have rod morphology form (right).
   
 
PERSONNEL
RESEARCHERS
1 Dr. Iman Hidayat (Group Leader)
2 Ir. Suciatmih
3 Atit Kanti, M.Sc
4 Nilam Fadmaulidha Wulandari, S.Si. (Tugas Belajar/TB
5 Achirul Nditasari, M.Sc.
6 Sulistiani, M.Kes.
7 Muhammad Ilyas,S.Si.
8 Arif Nurkanto, S.Si.
9 Tri Ratna Sulistiyani, S.Si.
10 Agustinus Joko Nugroho,M.Si.


TECHNICIAN
1 Yeni Yuliani, S.P.
2 Mia Kusmiati, A.Md.
 
LABORATORIUM FACILITIES



  1. Microscop.
  2. Thermal cycler (PCR).
  3. GelDoc.
  4. Electrophoresis apparatus.
  5. Microplate reader.
  6. Spektrofotometer.
  7. Freeze Dryer.
  8. Rotary evaporator.
  9. Centrifuge.
  10. Sonicator.
  11. Safety Cabinet.
 
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Written by Staf Bidang Mikrobiologi
Last Update at 16-10-2012

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