Mode of Fermentation; Batch, Fed Batch and Continuous Sri Kumalaningsih

Outline Overview Batch fermentation Fed batch fermentation Continuous fermentation

Outline Overview Batch fermentation Fed Batch culture Continuous culture Growth kinetics

Batch fermentation Most fermentations are batch processes Nutrients and the inoculum are added to the sterile fermenter and left to get on with it! Anti-foaming agent may be added. Once the desired amount of product is present in the fermenter the contents are drained off and the product is extracted. After emptying, the tank is cleaned & prepared for a new batch.

Product examples Food grade ethanol (cosmetics and pharmacy) Wine Kimchi Saurkraut Astaxantin

Outline Overview Batch fermentation Fed Batch culture Continuous culture Growth kinetics

INDUSTRIAL APPLICATION OF FED-BATCH Penicillin Glucose, phenyl acetic acid, ammonia source Cephalosporin Glucose, methionine Streptomycin Glucose, ammonia source Glutamic acid Urea, ethanol, (acetic acid) Amylase Carbon source Bakers Yeast Glucose Citric acid Glucose, ammonia

Continuous fermentation Some products are made by a continuous culture system. Sterile medium is added to the fermentation with a balancing withdrawal of broth for product extraction.

ADVANTAGES / DISADVANTAGES OF CC Uniformity of operation Process demands are constant i.e. continuous cycle of sterilisation, fermentation, harvesting, extraction Once in steady-state demands re process control are constant i.e. oxygen demand Disadvantages >> high cost Susceptibility to contamination Duration of run is longer  increased chance of contamination Strain degeneration arising from large number of generations

Set up for Continuous culture Fresh medium from reservoir Sterile air Flow-rate regulator Stirrer Culture Overflow Effluent

APPLICATION OF CONTINUOUS CULTURE INDUSTRY; Waste-treatment Single-cell protein Continuous beer production Continuous amino acids, organic acids production Continuous ethanol Continuous bakers yeast

TYPES OF CONTINUOUS CULTURE Method of control;  Chemostat - regulated by control of concentration of limiting nutrient  Turbidostat - regulated by biomass using optical density (photoelectric cell)  Biostat - regulated by systems monitoring biomass other than optical density (e.g CO2 production)

The development of growth in a chemostat Steady State Growth rate equals loss of cell biomass Cell Number Nutrient limitation causes decrease in m Population density increases mmax Inoculation Time in Hours

MODIFICATIONS OF BASIC CHEMOSTAT MULTI-STAGE Different environments or growth rates in the various reactors (e.g. 1st  biomass, 2nd  product) SINGLE STAGE WITH CELL RECYCLE Application in activated sludge waste-treatment Relationship between D and  different when recycle used. EFFECT OF FEEDBACK; 1. Increase biomass conc. in fermenter - lower in effluent 2. Decrease residual substrate 3. Maximise rate of product formation 4. Drift is increased - useful when substrate is dilute

Chemostats in series F1 SR X1 S1 V1 FO2 SR2 X2 S2 V2 F2

Growth Kinetics Involved by: Binary fision Growth Curve Spesific growth rate Substrate utilization Monod Equation

(1) The steps of binary fision

(2) Growth Curve Lag phase Log phase Stationary phase Death phase

The steps of growth 5 4 6 3 2 1

The steps of growth (1) (1) Fase permulaan Karakteristik: Terjadi adaptasi oleh mikroba yang diinokulasikan Mikroba memproduksi berbagai macam enzim dan zat antara yang berguna bagi pertumbuhan Sel-sel mikroba mulai membesar Akan tetapi belum terjadi pembelahan 1

The steps of growth (2) (2) Fase pertumbuhan yang dipercepat Karakteristik: Bakteri mulai membelah diri Akan tetapi waktu generasinya masih panjang Fase permulaan dan pertumbuhan yang dipercepat biasa disebut dengan lag-phase 2

The steps of growth (3) (3) Fase pertumbuhan logaritma Karakteristik: Pada fase ini kecepatan pembelahan paling tinggi Waktu generasinya pendek dan konstan Metabolisme terjadi paling pesat Keadaan terus berlangsung sampai salah satu nutrien habis Atau terjadi penimbunan racun akibat metabolisme yang menyebabkan terhambatnya pertumbuhan 3

The steps of growth (4) (4) Fase pertumbuhan yang mulai terhambat Karakteristik: Kecepatan pembelahan berkurang Sel yang mati bertambah banyak Terjadi karena makin berkurangnya nutrien dan bertambahnya racun Terdapat perubahan kondisi fermantasi (misal perubahan pH, kenaikan suhu) 4

The steps of growth (5) (5) Fase stasioner yang maksimum Karakteristik: Terjadi penurunan kadar nutrien dan penimbunan zat racun Kecepatan pembelahan berkurang Jumlah bakteri yang mati semakin meningkat Jumlah bakteri yang dihasilkan sama dengan jumlah bakteri yang mati sehingga jumlah bakteri konstan 5

The steps of growth (6) (6) Fase kematian yang dipercepat dan kematian logaritma Karakteristik: Disebut juga dengan fase menurun Kematian terus meningkat Kecepatan pembelahan menjadi nol Setelah sampai pada fase kematian logaritma kecepatan kematian mencapai maksimal dan jumlah sel menurun dengan cepat 6

Steps of growth

3. Specific growth rate ln X Waktu Slope =  X Waktu a b c d e f

2. Specific growth rate  (laju pertumbuhan spesifik pada setiap waktu pertumbuhan dapat ditentukan sebagai berikut : Keterangan : F = laju alir V = volume kultur  = laju kematian spesifik Akumulasi sel = pertumbuhan – pengeluaran – sel yang mati Untuk Kultur Fed Batch Untuk Kultur Curah (Batch) dx/dt=0 μ = D di mana D = F V

4. Substrate Utilization Laju Penggunaan Substrat Akumulasi Substrat = substrat masuk – substrat yang dikonsumsi untuk pertumbuhan – substrat yang dikonsumsi untuk sistesis produk – substrat yang dikonsumsi untuk perawatan – substrat keluar

4. Substrat Utilization Rate Laju Penggunaan Substrat F = Laju alir (l/jam) V = Volume kultur (l) S0 = [substrat yang masuk] (g/l) Yx/s = koefisien rendemen biomassa Yp/s = koefisien rendemen produk = laju pertumbuhan spesifik qp = laju pembentukan produk spesifik m = koefisien pemeliharaan

4. Specific Utilization Rate Untuk Kultur Curah/batch: Laju penggunaan substrat spesifik (qs) Untuk Kultur fed batch:

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