Production Vessel

Shaken culture technique History: (1)Began to emerge in the 1930s; (2)Developed as a major technique with the rise of the antibiotic industry Use: (1)Large-scale screening; (2)Yield improvement; (3)The initial stages of inoculum production Shaker: (1)Rotary shaking machine: most often used / flasks move in orbits of usually 50 mm at 200 to 250 rpm / culture moves smoothly around the inside of the flasks; (2)Reciprocating shaking machine: the culture is thrown back and forth, which may cause serious splashing

Vessels for shaken culture -1 Erlenmeyer flask: (1)The most often used; (2)25 to 50 mL of medium in 250- to 500-mL flask for those need relatively large amounts of oxygen; (3)Larger volumes of medium was used with a slower shaking rate for those need much less quantity of oxygen; (4)50-mL (wide mouth) flask may be used to allow greater numbers for screening

Vessels for shaken culture -2 Baffled flask: (1)For improving oxygen transfer; (2)Shaker speed may be increased to 400 rpm Sakaguchi flask: good oxygen transfer on reciprocating shaker Test tube: (1)25 X 150 mm or small vials; (2)Usually held up-right in racks or in a sloping position, e.g., 15 o to the vertical; (3)Can be packed together in large numbers in a minimum of space; (4)Not always as effective as small flask Micro-titer plate: new device for surface culture screening

Closure for shaken culture Cotton wool plug Reusable plugs (styrofoam bung) Plastic or metal closures for tubes Others

Practical problems involved in shaken culture Practical trials for the optimum volume of medium Careful placing of the flasks to improve balance Remaining the plug or closure dry Keep the stop period within a few minutes Antifoam agent, e.g., vegetable oils or polypropylene glycol 2000 (Dow Chemicals) (1 g/L or less)

Shaken culture vs. stirred fermenter culture (stirred-tank reactor, STR ) They tend to give different metabolic patterns. Uptake of oxygen: (1)Largely by direct absorption from the air in shaken culture; (2)Mainly by diffusion from the medium in stirred fermenter culture Shaken flasks give much better growth and production than would be expected from the rate of oxygen uptake indicated by the sulfite oxidation method. Mutants can be selected which respond well in both systems Chalk (CaCO 3 ) is usually added in the shaken culture to control pH, but it also has an effect on the growth pattern. This effect may extend to the particular type of CaCO 3 used.

Considerations in fermenter design, quality of construction, mode of operation, and the level of sophistication Production organism Optimal operating conditions required for target product formation Product value and the scale of production Overriding considerations are reliability and the need to minimize capital investment and running costs

Materials for construction of fermenter Traditional fermenters: open cylindrical or rectangular vessels made from wood or stone Pure culture fermenters: closed vessels / must withstand repeated sterilization and cleaning / constructed from non-toxic, corrosion-resistant materials (1) Glass and/or stainless steel for small vessels (2) Stainless steel with polished internal surfaces for pilot-scale and general production vessels (3) Mild steel lined with glass or plastic for very large fermenters in order to reduce the cost

Design rules for aseptic bioreactors -1 There is no direct contact between the sterile and non-sterile sections Any connections to the fermenters should be suitable for steam treatment / All associated pipelines transporting air, inoculum and nutrients need to be sterilizable Systems must be designed to allow aseptic inoculation, sampling and harvesting Every individual part should be easily maintained, cleaned and independently steam sterilizable, particularly valves

Design rules for aseptic bioreactors -2 Automated spray jets located within the vessels (cleaning-in-place, CIP) There should be no horizontal pipes or unnecessary joints and dead stagnant spaces Overlapping joints are unacceptable and flanged connections should be avoided as vibration and thermal expansion can result in loosening of the joints / Butt-welded joints with polished inner surfaces are preferred Pressure gauges and safety pressure valves in the form of a metal foil disc held in a holder set into the wall of the fermenter present a much lower contamination risk than spring-loaded valves

Design rules for aseptic bioreactors -3 Pumps should be avoided as they can be a major source of contamination: Centrifugal pumps generate high shear forces and are not suitable for pumping suspensions of shear- sensitive cells. Their seals are potential routes for contamination. Other pumps used include magnetically coupled, jet, and peristaltic pumps. Alternate methods of liquid transfer are gravity feeding or vessel pressurization.

Design rules for aseptic bioreactors -4 Sterilizable condenser to prevent evaporation Filter-sterilizing the exhaust gases to contain any aerosols generated within the fermenter Operation under positive pressures / Not be applicable to pathogens or certain recombinant DNA microorganisms

Basic types of bioreactors

Types of solid-substrate fermenters -1 Rotating drum fermenters: (1) A cylindrical vessel of around 100 L capacity mounted on its side onto rollers; (2) The drum is filled to only 30% capacity, otherwise mixing is inefficient Tray fermenters: (1) Used extensively for the production of fermented oriental foods and enzymes; (2) Substrates are spread onto each tray to a depth of only a few centimeters; (3) Trays are stacked in a chamber through which humidified air is circulated; (4) These systems require numerous trays and large volume incubation chambers

Types of solid-substrate fermenters -2 Bed systems: (1) As used in commercial koji production; (2) Consisting of a bed of substrate up to 1 m deep, through which humidified air is continuously forced from below Column bioreactors: (1) Consisting of a glass or plastic column, into which the solid substrate is loosely packed, surrounded by a jacket that provides a means of temperature control; (2) Used to produce organic acids, ethanol and biomass Fluidized bed reactor: (1) Provide continuous agitation with forced air to prevent adhesion and aggregation of substrate particles; (2) Particularly useful for biomass production for animal feed

References Calam, C. T Shake-flask fermentations. In Manual of Industrial Microbiology and Biotechnology (A. L. Demain and N. A. Solomon eds). American Society for Microbiology. M. J. Waites, N. L. Morgan, J. S. Rockey, and G. Higton Industrial Microbiology: An Introduction. Blackwell Science.

Fluidized bed reactor Sakaguchi flask STR