Process Development

Scale-up Laboratory flask: the first indication that a process of commercial interest is possible Laboratory fermenter: generally of glass and generally of liter size / to test variations in medium, temperature, pH, and so on inexpensively / examination of feeding strategies (batch, fed-batch, continuous, etc.) / selection of production system (stirred tank, airlift, packed bed, solid state, hollow fiber, etc.) Pilot plant: usually liter size / careful instrumentation and computer control is desirable, so that the conditions most similar to those in the laboratory fermenter can be obtained Commercial fermenter: generally 1,000-10, ,000 liters

Commercial fermenter Size of fermenter (1,000 liters) Product 1-20Diagnostic enzymes, substances for molecular biology 40-80Some enzymes, antibiotics Penicillin, aminoglycoside antibiotics, proteases, amylases, steroid transformations, amino acids Glutamic acid

Key factors that influence yield during scale-up process Inoculum propagation procedures: quality and quantity of inoculum Choice of medium: cheaper nutrient sources Sterilization protocols: degradation of heat-labile compounds Experience in larger fermenters: nutrient, temperature, pH and oxygen gradients; alter the generation of foam, shear forces and rate of removal of carbon dioxide

Microbiological considerations on process development - 1 Inoculum build-up: vegetative cells suspension / heat treatment (boiling for 90s for germination) of Clostridium sp. spores / non-toxic surface active agent for fungi or actinomyces spores / fragmented hyphae (Waring blender) of non- sporulating filaments Size of inoculum: % ( up to 20% for production tank) / fluctuation of environments for the cells such as change in pH value, increase in supply of nutrients, decrease of growth inhibitors, diffusion of essential cofactors out of cells, enzymes of primary metabolism must be adjusted to the new conditions / has an influence on the lag phase

Microbiological considerations on process development - 2 Stability of inoculum: log or stationary phase / supply essential nutrients to auxotroph to prevent back mutation / supply inducer for enzyme adaptation / preserve master culture / re-isolate, purify and test for production Contamination: microscopic observation and /or fermentation appearance, lysogenic phages (mutations result in decrease of production) and / or lytic phages (isolation of phage-resistant strains)

Microbiological considerations on process development - 3 Medium sterilization: crude, technical-grade nutrients in production medium / large volume and longer heating and cooling time / thermal degradation of essential nutrients and production of toxic substances / production sterile, not strictly sterile Foam control: antifoams e.g. silicones, polyoxyalkylene glycol, animal or vegetable oils / inhibitory effect / additional nutrients

Most important criteria used in scale- up studies on aerobic fermentations Constant volumetric oxygen transfer rate or constant dissolved oxygen level Constant power consumption per unit of broth or constant impeller tip speed Fermenter height-diameter aspect ratio ？

問題 工業生產規模的醱酵槽，其接種量 (size of inoculum) 常常達到 10~20% 。使用高接種量的 優缺點為何？ 種菌 (inoculum) 的特性對於主醱酵槽中產物的 生成有哪些影響？ 下列名詞的內涵： Antiforms, Polyoxyalkylene glycol, Production sterile