4. Depending on the targeted biomass for preculture, nonindu-

cing carbon source (i.e., glycerol) needs to be adapted in the

medium composition; preculture volume(s) need to be

adjusted to the desired reactor size; i.e., a preculture consisting

of 8 g/L glycerol will result in a biomass concentration of

approximately 4 g/L, assuming a biomass substrate yield of

0.5 g/g. In case reactor volume consist of 1 L, the preculture

volume needs to be 100 mL to allow inoculation with 1/10th

of fermentation broth (see Note 3).

5. Reactors are adapted to batch culture conditions prior to inoc-

ulation: 37
C, 1400 rpm stirring, 2 vvm aeration, and a set pH

of 6.7. Media pH is set to desired value with 12.5% NH4OH

after sterilization.

6. Once set process parameters are achieved, dO2 probes are

calibrated for 100% dissolved oxygen.

7. After 20 h of cultivation, the preculture is quantified in its

optical density (OD600). Depending on the host strain and

the used amount of noninducing carbon source, the targeted

biomass should be achieved.

8. Batch media is inoculated with 10% Preculture; dO2 in cultiva-

tion must never be beneath 30% at all stages of the cultivation

! adjustment via PI controller is necessary.

9. End of batch phase is indicated by either a dO2 peak or a drop

in the residual CO2.

3.2.2

Continuous

Adaptation Phase

1. To get the cells in equilibrium state prior to induction, the

continuous cultivation is started; however, only noninducing

feed is applied at this phase (Fig. 3). Cell equilibrium state can

be obtained by a constant offgas signal in the biomass reactor.

Fig. 2 The process overview of the continuous cascaded cultivation on a time-dependent manner for seed

reactor (reactor 1) and recombinant production reactors (reactors 2 and 3); down-times consist of steam in

place (SIP) and cleaning in place (CIP) phases, whereas cultivation is separated in a batch phase, a continuous

adaptation phase, and a continuous induced cultivation phase

230

Julian Kopp and Oliver Spadiut