Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale

Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures. Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags. Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature. For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum. Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L. In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium. It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium. Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages. Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes.