3.3.3

Product

Determination

Product determination is highly dependent on the given target

protein and subsequent analytic procedures; hence, no general

protocol can be given.

3.4

Calculation of

Flow and Substrate

Uptake Rates

3.4.1

Calculation of Flow

Rates

1. Dilution rate in the first stage needs to be calculated according

to chemostat principles rendering to Monod, Novick, and

Szilard with μ ¼ qs  YX/S ¼ D; if μ < Dcrit [5–7]; dilution

rates beyond Dcrit must be avoided.

2. Dilution rate is calculated via monitored feed volumes over

time (Eq. 1). Reactor volume must be kept constant. As feed-

ing rates are monitored gravimetrically and feed density is

determined, volumetric rates were calculated as follows:

DInd: Reactor ¼

_F indFeed þ _F biomass

V reactor

ð1Þ

Dind. Reactor . . . dilution rate in the reactor for protein expres-

sion (1/h).

Find. Feed . . . applied feeding rate of the induction feed (mL/h).

Fbiomass . . . applied biomass flow to the protein expression

reactor (mL/h).

Vreactor . . . volume in the protein expression reactor (L).

3.4.2

Calculation of

Substrate Uptake Rates

1. Feed composition in the induction reactor can be adjusted by:

(a)

The ratio of transferred cells to the amount of feed

solution.

(b)

The concentration in the induction feed.

2. Specific substrate uptake rate is calculated according to Eq. 2.

3. Real sugar uptake rates need to be used, evaluated by the

accumulated component subtracted from the fed substrate.

qs,c ¼ ^{cFeed  V Feed,in  cacc  V reactor}

Δt  X Δt

ð2Þ

qs,c . . . specific uptake rate of desired component (g/g/h).

cFeed . . . concentration of corresponding component in feed

(g/L).

VFeed,in . . . feed volume in reactor in timespan Δt (L).

cacc . . . concentration of the corresponding component which

accumulated in the reactor (HPLC measurement) (g/L).

Vreactor . . . volume in the protein expression reactor (L).

Δt . . . timespan for calculated uptake rate (h).

XΔt . . .

average biomass in the protein expression reactor

during the timespan Δt (g).

4. In case mixed feeds are used, a careful evaluation of the carbon

source to inducer ratio must be done prior to utilization.

Screening approaches therefore can be found in given citations

[35, 38].

A Guideline to Set Up Cascaded Continuous Cultivation with E. coli Bl21 (DE3)

235