FERMENTATION OBJECTIVES

Identify useful products from microorganisms Explain the process of fermentation Describe aerobic & anaerobic fermentation Identify the microorganisms used and the main stages in the production of Penicillin, Cephalosporin, Erythromycin, Tetracycline, Rifamycin, Griseofulvin, Chloramphenicol by fermentation. Describe how Downstream processing is carried out to extract and purify the end-product of fermentation. FERMENTOR/ BIOREACTORS An apparatus that maintains optimal conditions for the growth of microorganisms, used in large-scale fermentation and in the commercial production of antibiotics and hormones. THE NEED FOR BIOREACTORS Small cultures originally were performed in dishes which could not create appreciable quantities of fermentation products (such as acetone, ethyl alcohol). These products had value if they could be produced in large volumes. To achieve this goal, the development of bioreactors was required, that could culture at least a few liters of cells and media. Eventually, bioreactors would be able to ferment thousands of liters at a time. CLASSIFICATION OF BIOREACTORS

Based on the agents used

Based on the process requirements

Those based on living cells

Those employing enzymes

DESIGN AND VARIOUS TYPES OF FERMENTERS Solid State there are various types of fermenters/ bioreactors 1. Fermacell (Laboratory) 2. Bubble Cap/ Airlift 3. Loop Recycle 4. Tower Bioreactors 5. Activated Sludge 6. Continuous Flow stirred tank 7. Packed Bed 8. Trickling Heat 9. Mist (Used for antibiotics) 10. Rotating Drum 11. Bubble Column 12. Commercial fermentation Plant 13. CSTR 14. Combinations and variations of CSTR and Airlift reactors 15. Membrane - two permeable membranes to deliver nutrients and export wastes and products from bacteria in a center tube 16. Cyclone column fermenter 17. Simple fermenters (batch and continuous) 18. Fed batch fermenter A TYPICAL FERMENTER a) b) c) d) e) f) An Agiator System An Oxygen Delivery System A Foam Control System A Temperature Control System A pH Control System Sampling Ports

g) A cleaning & Sterilization System h) A Sump & Dump Line For Emptying The Reactor.

FERMENTATION
Pasteurs definition: life without air, anaerobe red ox reactions in organisms New definition: a form of metabolism in which the end products could be further oxidized For example: a yeast cell obtains 2 molecules of ATP per molecule of glucose when it ferments it to ethanol Fermentation is often defined as a process where cells produce energy anerobically, or without oxygen. FERMENTATION PROCESS In general, fermentation involves the breaking down of complex organic substances into simpler ones.

Microbial or animal cells obtain energy through glycolysis, splitting a sugar molecule, and removing electrons in the process. The electrons are then passed to an organic molecule such as pyruvic acid. This results in the formation of waste products that are excreted from the cell. Waste products formed in this way include ethyl alcohol, butyl alcohol, lactic acid, and acetone. RANGES OF FERMENTATION PROCESS Microbial cell (Biomass)- Yeast Microbial enzymes- Glucose isomerase Microbial metabolites- Penicillin Food products- Cheese, yoghurt, vinegar, Vitamins, B12, riboflavin Transformation reactions- Steroid biotransformation

There are several major groups of fermentation

Trace elements (fe, Zn, Cu, co, Mo), Antifoaming agents (esters, fatty acids, fats, silicons, sulphonates, polypropylene glycol), buffers (CaCO3, Phosphates), Growth factors (thiamine, biotine, calcium panthothenate), Precursors (Phenyl ethylamine for benzyl penicillin, phenyl acetic acid for penicillin G), Inhibitors (sodium barbitol for rifampicin), Inducers (various enzymes), Chelators (used to precipitate metal ions such as EDTA, citric acid, polyphosphates.)

Fermentation :
Aerobic Adequate aeration Bioreactors- adequate supply of sterile air In addition, these fermenters may have a mechanism for stirring and mixing of the medium and cells Antibiotics, enzymes, vitamins. Anaerobic In anaerobic fermentation, a provision for aeration is usually not needed. Lactic acid, ethanol, wine DESIGN OF FERMENTATION PROCESS
Design of fermentation process depends on the type of final product i.e. on the basis of biproduct recovery. There are several vital and critical factors which are responsible for media designing of various fermentation process. Such asQuality of Water Quality Control of Raw Material Nutritional Requirements Sterilization process/ Practices Media Preparation System Geometry

a) b) c) d) e)

a) b) c) d) e) f) g) h)

Aeration Rate Intensity of Agitation Temperature Pressure pH & other parameters Dilution Rate Foaming Activity of Microorganisms

Quality of Water Demineralized water, high quality soft potable water (water obtained from reverse osmosis) Quality Control of Raw Material Salts, water or chemical constituents must be of high grade. Nutritional Requirements Energy source (glucose, Lactose, Starch, Sucrose), nitrogen sources, Inorganic and organic constituents. Sterilization process/ Practices Continuous sterilization during time of fermentation to kill bacteria continuously. Commonly membrane filtration is used in inlet and exit. Polypropylene cartridge is used in large scale fermenters. Media Preparation Media preparation for different fermenters depends on temperature, pH, dissolved O2 tension & , hormonal System Geometry When considering the design of vertical stirred vessels, the main variables in geometry are the height-to-diameter ratio, the number, type, dimensions and positions of impellers, the number and breadth of baffles, and the design and location of coils for heating and cooling. In relation to power input, Aeration Rate The flow rate can be set from 0-5 l/min Intensity of Agitation

To provide good mixing & thus increase mass transfer rate through bulk liquid & bubble boundary layer. It depends on number of impellers. Each impeller have 2-6 blades. Temperature 5-70C Pressure A compressor forces air into the reactor and produces 250kPa pressure. pH & other parameters It should be measured by pH electrodes and sensors. Dilution Rate Addition of acids & base and sample removal is made through capillaries Foaming During fermentation microorganisms produce detergent like molecules of proteins and long chain fatty acids. Antifoaming agents such as silicone oil are used to bind foam and preventing them from aggregating around bubbles. Antifoaming agents decreases oxygen transfer rate. a) Activity of Microorganisms The activity of the microorganism will itself have effects on the microenvironment, both directly and through effecting changes in the macro environment. The direct effects on the microenvironment will result from: (a) Consumption of substrates may cause deficiency (b) Output of products (c) Production of heat (d) Aggregation- may affect heat and mass transfer (e)The strain should have rapid growth (f) Genetically stable (g) Non toxic to human (h) Large cell size