Design of Fermenter Lecture

Basic functions of a fermenter for microbial or animal cell culture The vessel – capable of being operated aseptically for a number of days Adequate aeration and agitation – meet requirements of micro-organisms Power consumption should be as low as possible Temperature control and pH should be provided Vessel should be reliable in long-term operation and meet requirements of containment regulations. Aeration and agitations should not cause harm to the microorganisms

Basic functions of a fermenter for microbial or animal cell culture Sampling facilities should be provided Evaporation losses from fermenter should not be excessive Minimal use of labor in operation, harvesting, cleaning and maintenance Should have internal smooth surfaces Similar geometry of both smaller and larger vessels Similar geometry of vessels in plants to facilitate scale up

Aseptic operation and containment Aseptic operation involves protection against contamination Containment involves prevention of escape of viable cells from a fermenter or downstream equipment Lowest level hazard micro-organisms – require GILSP Good industrial large scale practices except some organism used in bacterial and viral vaccine production. Lowest level mo can be genetically or non-genetically engineered organisms.

Achievement and maintenance of aseptic conditions Sterilization of fermenter Sterilization of air supply and exhaust gas Aeration and agitation Addition of inoculum, nutrients and other supplements Sampling Foam control Monitoring and control of various parameters

Construction materials - Body Possible to use glass and/or stainless steel Glass vessel with a round or flat bottom and a top flanged carrying plate – autoclave Largest possible diameter-60cm Glass is useful because it gives smooth surfaces, which is non-toxic, corrosion proof and it is usually easy to examine interior of vessel

Construction materials - Body A glass cylinder with stainless-steel top and bottom plates More expensive (50%) Pilot-scale and industrial scale – stainless steel Chromium 10 -1 3% develops an effective film . The inclusion of nickel in high percent chromium steel enhances their resistance and improves engineering properties. The presence of Mo improves resistance of stainless steels to solutions of halogen salts and pitting by chloride ions in brine or sea water. Corrosion resistance can also be improved by tungsten, silicone and other elements

Construction materials - Body Aseptic seal – made between glass and glass, glass and metal or metal and metal joints between fermenter vessel and a detachable top or base plate Compressible gasket, a lip seal or an ‘O’ring With glass and metal, a seal can be made with compressible gasket, a lip seal or an O ring. With metal to metal joints only an O ring is suitable. This is placed in a groove machined in either the end plate, the fermenter body or both. This seal ensures that a good liquid and or gas-tight joint is maintained in spite of glass or metal expanding or contracting at different rates with changes in temperature during sterilization cycle or an incubation cycle.

Temperature control Provision of heat – fermenter in thermostatically controlled bath or by use of internal heating coils or by a silicone heating jacket through which water is circulated Silicone jacket consists of silicone rubber mats wrapped around the vessel Cooling surface/cooling water Heat will be produced by microbial activity and mechanical agitation and if this heat is not adequate then heat needs to be added or removed.

Aeration and Agitation Aeration – provide microorganisms in submerged culture with sufficient oxygen for metabolic requirements Agitation – uniform suspension of microbial cells in homogeneous nutrient medium Mechanical agitation is required in fungal and actinomycete fermentations

Structural components involved in aeration and agitation Agitator (impeller) Stirrer glands and bearings Baffles Aeration system (sparger)

Agitator (impeller) Achieve mixing objectives – bulk fluid and gas-phase mixing, air dispersion, oxygen transfer, heat transfer, suspension of solid particles and maintaining uniform environment throughout vessel contents Disc turbine could break up a fast air stream without itself becoming flooded in air bubbles

Baffles Four baffles incorporated into agitated vessels of all sizes to prevent vortex and to improve aeration efficiency Metal strips roughly one-tenth of vessel diameter and attached radially to the wall Minimizes microbial growth on baffles and fermenter walls.

Aeration system (sparger) Introduces air into liquid of fermenter Three basic types – porous sparger Orifice sparger – a perforated pipe Nozzle sparger – an open or partially closed pipe Combined sparger-agitator

Sterilization of the fermenter Designed – for steam sterilization under pressure Medium may be sterilized in vessel or separately and added aseptically Steam should be introduced through all entry and exit points except the air outlet from which steam should be allowed to leave

Sterilization of air supply Sterile air – large volumes in many aerobic fermentation processes Heat (expensive) and filtration

Sterilization of exhaust gas from a fermenter Sterilization of exhaust gas can be achieved by 0.2 μm filters on outlet pipe Aerosol formation may occur in fermenter and moisture and solid matter may then plug filter Filters – checked to ensure that no viable cells are escaping

Feed ports Addiiton of inoculum, nutrients and other supplements Sampling ports to test Additions of acid/alkali – silicone tubes pumped by peristaltic pumps after aseptic connection In larger fermenter nutrient reservoirs and associated piping- integral parts – can be sterilized with vessel Silicone tubes can be autoclaved separately.

Foam control Minimize foaming Excessive foaming – danger that filters become wet resulting in contamination Siphoning – loss of all or part of contents of fermenter

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