Isolation and Screening of Microorganisms Nyoman S. Antara, Ph.D. Industrial Microbiology

Isolasi Mikroorganisme Mikroba di alam selalu tercampur Isolasi mikroba untuk produk yang spesifik Sumber mikroba Jenis Mikroba Produk spesifik: Metabolite products Enzyme Transformation compounds Biomass

Isolasi Bakteri Memisahkan bakteri dari campuran mikroba Kultur (spesies) tunggal untuk dipelajari lebih lanjut Dua metode konvensional isolasi bakteri: Metode cawan gores Metode cawan tuang

Cawan Gores

Cawan Tuang

Isolation of Lactic Acid Bacteria from Urutan GYP medium (per L) Glucose 10 g Yeast extraxt 10 g Peptone 5 g Beef extract 2 g Na-acetate 3H2O 2 g Tween 5% sol. 10 ml Salt sol.*　 5 ml Bromocresol purple 60 mg Agar 12 g CaCO3 5 g pH 6.8 *each ml contains: MgSO4 7H2O, 40 mg; MnSO4.4H2O, 2 mg; FeSO4.7H2O, 7 mg; NaCl, 2 mg Day 1 Day 2 Day 3 Day 5 GYP medium In isolation of LAB, the GYP medium was used. The composition of this medium is shown in this table, which compose of glucose, yeast extract and peptone as a major components. Bromocresol purple was used as indicator which give the specific yellow clear zone around LAB colony.　The LAB were isolated from urutan during fermentation naturally with distribution of 19 isolates from day 1, 22 isolates from day 2, 19 isolates from day 3, and 11 isolates from day 5 of fermentation. Total 71 isolates were isolated from natural urutan fermentation. Isolation From day 1 2 3 5 Total Number of isolates 19 22 19 11 71

Phenotypic characterization Gram staining and catalase test 71 isolates Cell morphology Gas production from glucose Utilization of sugars Growth at various temperatures Rod: 9 groups Cocci: 4 groups From Gram staining and catalase test, all of isolates were Gram-positive and lack of catalase. The isolates were grouped based on cell morphology, gas produce from glucose, fermentation of sugars, and growth on varying temperature. From this grouping it’s found 13 groups which consist of 9 groups were rod shape cell and 4 groups were cocci shape cell in paired and tetrad. The representative strains from the groups were then determined their lactic acid stereoisomers produced and sequence their whole of 16S rDNA. 13 representative isolates: Stereoisomers of lactic acid Sequence of 16S rDNA

Penapisan/Screening Seleksi mikroba tertentu Uji produksi Uji produktivitas Seleksi gen Rekayasa genetik

Isolasi Actinomycetes Actinomycetes : anggota family dari Actinomycetales Dieksploitasi untuk produksi antibiotik, enzim, dan senyawa komersial lainnya Hampir 5000 paten sudah dihasilkan dari klas actinomycetes Streptomyces merupakan genus yang paling banyak dieksploitasi Actinomycetes dapat ditemukan di mana-mana, tanah merupakan reservoar paling besar, juga diisolasi dari sedimen lumpur danau, sungai, dan juga dari fresh water dan marine environment

Selective Pretreatment Selection for Dry heat, 120oC for 1 h Micromonospora, Microbispora, and Streptosporangium Moist heat, 45oC for 2 h Rhodococcus Moist heat, 50oC for 10 min Elimination of spreading bacterial colonies on soil plate Moist heat, 70oC for 30 min Micromonospora

Selective Media Actinomycetes dapat survive dan tumbuh pada media dengan nutrisi sangat rendah Media dengan carbon-to-nitrogen ratio yang tinggi Kandungan sumber karbon dan nitrogen yang kompleks mengurangi tumbuhnya bakteri Ke dalam media harus ditambahkan cycloheximide (50 ug/ml) dan nystatin (50 ug/ml) untuk mencegah tumbuhnya fungi

Incubation Condition Suhu inkubasi 25 – 30oC Streptomyces diinkubasi selama 7 – 14 hari Micromonospora diinkubasi selama 14 – 21 hari Genera lain perlu tambahan waktu inkubasi 3 – 5 minggu Actinomycetes termofilik diinkubasi pada suhu 45 – 50oC, selama 2 – 5 hari, cawan di cek setiap hari

Preservation of Isolates Isolate Actinomycetes jangan disimpan dengan agar miring Lyofilisasi digunakan untuk preservasi dalam jangka waktu lama Sering digunakan cryoprotective: susu skim, beef serum, 12% sucrose broth steril Pembekuan juga bisa digunakan untuk mengawetkan Actinomycetes Viabilitas terbaik bila disimpan pada suhu -70oC Penggunaan suhu -20 – 50oC dapat bertahan 1 – 5 tahun

Isolation of Filamentous Fungi Materials Collecting Samples Media Basic Techniques Purifying Cultures Maintaining Cultures Identification

Filamentous Fungi Eucaryotic organism Occupy a wide range of habitats Oriental foods, soy sauce and tempe, and European cheeses Six general catagories of fungal products: Organic acids Pharmaceuticals Enzymes Insecticides Polysaccharides biomass Citric acid produced by A. niger