Biomedical Engineering Reference
In-Depth Information
The rows represent envirome factors, the columns represent elementary modes,
and I
j,i
is the relative ''intensity'' of up- or downregulation of elementary cellular
function i by medium factor j.
5.2 Example: Metabolic Process Control of P. pastoris Cultures
We study here the optimization and control of a pilot 50-L fermentation of a
constitutive P. pastoris X33 strain expressing a single-chain antibody fragment;
for details see [
60
]. The reactor was inoculated at a starting volume of 15 L.
Cultivation temperature was controlled at 30 C, and pH was controlled at 5.0 with
addition of ammonium hydroxide 25 %, which was also the main nitrogen source
for the culture. The airflow rate was kept constant at 1,800 L/h throughout the
fermentation. Overhead pressure was controlled at 200 mbar. Glycerol feeding and
dissolved oxygen (DO) control was divided into three phases:
(i)
Glycerol batch phase—The reactor was operated initially in batch mode,
starting with a glycerol concentration of 40 g/L. DO drops very slowly and
remains close to saturation levels.
(ii)
Glycerol fed-batch phase—An exponential feeding program is initiated once
the concentration of biomass reaches the level of 18 g DCW/L. It is in this
phase that cell density increases significantly and DO decreases more rapidly.
Once the DO reaches 50 %, it is kept at that level by automatic closed-loop
control, manipulating the stirrer speed between 300 and 1,000 rpm.
(iii)
Oxygen transfer limitation phase—Once the stirrer speed reaches the maxi-
mum level of 1,000 rpm, DO decreases very rapidly and the glycerol feeding
program is aborted. From this point on, the DO is kept constant at a low level
(e.g., 3-5 %) by closed-loop manipulation of the glycerol feeding rate (DO-
stat feeding control strategy).
We have investigated how the calculated elementary modes in the example in
Sect. 2.4
correlate with the environmental parameters by applying the previously
described functional enviromics algorithm. Measured environmental parameters
comprised the temperature (T), pH, stirrer speed (Ni), pressure (Press), and the
concentrations of dissolved oxygen (DO), glycerol (Gly), biomass (X), product
(scFv), and inorganic compounds ([NH
4
+
], [K
+
], [Ca
2+
], [Mg
2+
], [S], and [P]).
From the concentrations of inorganic salts we have calculated the ionic strength
(IS) and the osmolarity (Osm). The measured rates were those of biomass (l),
glycerol (v
Gly
), oxygen (v
O2
), carbon dioxide (v
CO2
), and product (v
scFv
).
The initial number of elementary modes was 4,119, of which a small set of six
elementary modes were discriminated by functional enviromics (Fig.
5
b). The
total explained variance of measured fluxes was 81.4 %, and the correlation
between predicted and measured fluxes was acceptable (Fig.
5
c) given the high
level of noise in the measured rates.
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