1. 1 FERMENTATION PROCESS Iman Rusmana Department of Biology FMIPA IPB 2 Fermentation Process  produces primary & secondary metabolites 3 Type : 1. Products directly produced from primary metabolites 2. Products produced from the same substrates but via differet pathway (not primary metabolic pathway) 3. Metabolism & product synthesis at different time

2. Total Productivity Product Sinthesis Filling  Prediction  optimalization of production process 2 Productivity & specific production rate [product] Productivity (P) = --------------------------- Time of fermentation 1. Time of fermentation 2. Time of Cleaning up & Fermentor setting up 3. Time of sterilisation 4. Time of lag phase Evaluation  Cost Efficiensi

3. Effect of fermentor setting up time : 1. Short Fermentation : 8 – 70 hours  time of setting up has significant effect to productivity 2. Long Fermentation: > 3 days  time of setting up has no significant effect to productivity Continue fermentation: D. K s P = D.X  P = D. Y s (S o - ----------- ) µ m - D Specific production rate (q p ) dP i /dt = q p. X P i = product concentration q p analog to dg µ  but no correlation Yield (Y s ) [biomassa] Y s = ------------------------- [substrate consumption] 3

4. Steps of Fermentation Process: 1. Storage of microbial inoculants 2. Re-culture of microbial inoculants 3. Preparation of starter inoculants 4. Fermentation (production) Storage of microbial inoculants  strain for production  long time use transfer periodicly Spontaneous Mutation Maintain as long as possible  stable / not change  Master culture (strain):  not so often to be cultivated (1x in 2 years) HOW ???  activity should be checked before used 3 general techniques  Working culture :  form master culture  purity and activity should be checked 4

5. 5 3 Techniques of culture storage: 1. Storage at low tempereture (2 – 6 o C)  easy methodes  not stable  slant agar  in refrigerator  high risk of contamination & mutation  re-culture : 2-4 bulan 2. Frozen storage (-18 C; - 80 C; or –196 o C)  - 196 C  liquid nitrogen  stepping of decreasing temp (1 C/min) or  + protective agent  not form ice cristal  for many years  95 % will not survive if  repeated thawing 3. Liofilization (freeze- drying)  + protective agent (skim milk or sukrosa)  long time/unlimited storage Re-culture of inoculant  use liquid or solid media Time of incubation depend on type of storage 1. Liofilization  4 – 10 days 2. froozen storage: - bakteria  4 –48 hours - Actinomycetes  1 – 5 days - fungi  1 – 7 days 2. low temperature storage: - bakteria  4 –24 hours - Actinomycetes  1 – 3 days - fungi  1 – 5 days

6. 6 Preparation of starter inoculants  should be enough for a big fermentor  not enough  delay of growth Genaral quantity of inocula : 1. Bakteria  0.1 – 3.0 % 2. Actinomycetes  5 – 10 % 3. Fungi  5 – 10 % 4. Spora suspention  1 – 5 x 10 5 /l Fermentation (production)  different size of fermentor  nutrient must be optimum Parameters must be controlled/monitored: 1. Temperature  optimum  temp > 1 C  yield will decrease up to 20 % 2. Aeration  dissolved O 2  0.25 – 1.0 vvm (vol of air/vol of media.min 4. Pressure  higher than air pressure (0.2 - 0.5 bar)  reducing risk of contamination 3. pH  Optimum growth  5.5 – 8.5 change  monitored & controlled (+ acid/basa)

7. 7 High density of Cell culture  Density related to productivity - Optimum of media  high ratio of C/N  inhibit growth - Toxic substances  eliminated - Dissolved O 2 :  Limiting factor : high density of cells  Optimum growth  need high dissolved O 2 ex: recombinant E. coli  0.0084 gram/lt at 25 o C (continue) Big air bubbles  low dissolved O 2 1. Sparging 2. High pressure of O 2 3. + chemical agents  Increase dissolved O 2 (ex. Perfluorocarbon) 4. Fermentor modification 5. Biology: cloning genes  identical molecules to haemoglobin from Vitreoscilla (Bailey dkk. 1990)  bind O 2  high [O 2 ] in cells Dissolved O 2

8. 3 Molecular biology agents 40 - 80Enzyme, antibiotics Back 8

9. 1. Products directly produced from primary metabolites Substrate A  Product Substrate A  B  C  Product - Growth - Substrate catabolisms - Product synthesis At the same time  Continue Ferm. - Single cell protein - Ethanol - Gluconic acid µ (specific growth rate) Specific substrate consumption rate Specific product synthesis rate Back 2 steps : 1. High growth & substrate consumption  no product synthesis 2. Low growth & product synthesis  high substrate consumption Back 9 2. Products produced from the same substrates but via different pathway (not primary metabolic pathway) Substrate A  B  C  Met. Primer (energi) D  E  product µ (specific growth rate) Specific substrate consumption rate Specific product synthesis rate

10. 10 3. Metabolism & product synthesis at different time  No product produced from primary catabolism step 1. Primer Met & growth & substrate consumption 2. Product Synthesis production  Vitamin  Antibiotics Back µ (specific growth rate) Specific substrate consumption rate Specific product synthesis rate