Automated Measurement and Monitoring of Bioprocesses: Key Elements of the M3C Strategy - Measurement, Monitoring, Modelling, and Control of Bioprocesses - page 11

3.1.1 Optical Density
Various sensors that measure the turbidity of a suspension have been commercially
available for many years. Their major drawback is that they also ''see'' gas bubbles
and particles as well as cells, e.g., precipitates in the suspension or particulate
medium components. There are a few tricks reported to minimize the effect of gas
bubbles: one can capture a volume aliquot in the optical path from time to time, let
it degas through a vent hole, read the absorbance, and open the optical path again
for fresh suspension to enter (Foxboro Cerex). Older instruments sucked a sus-
pension aliquot out of the reactor and, after reading, pushed it back; however,
contamination problems occurred (Fundalux). One can also protect the optical path
from gas bubbles by surrounding this space (at the tip of the sensor) with a fine
grid, the mesh size of which permits cells to pass but gas bubbles to be excluded
(Komatsugawa). These sensors are also sensitive to the absorption of light due to
the color of the medium. To circumvent this effect, most instruments exploit light
sources (nowadays LEDs or lasers) with wavelengths in the near-infrared instead
of visible range. Some sensors determine the attenuation of light in the forward
direction (turbidimetry), while others measure light scattered at a 908 angle
(nephelometry) and others the reflected light. Some prototypes may also analyze a
combination of these. None of these sensors can discriminate between live and
active versus dead and inactive cells or abiotic particles. The control goal to keep
the optical density of a continuous culture constant is called a ''turbidostat''; i.e.,
the turbidity is forced to be pseudostatic.
3.1.2 Impedance (Permittivity)
Another type of sensor is based on the fact that all intact cells are enclosed by a
cytoplasmic membrane which functions somehow as an electrical insulator. If the
cells are exposed to an alternating electrical field, the charged species inside the
cells are attracted to one or the other side of the field, but they cannot leave
the cells. This mirrors an electrical capacitor, where the capacitance depends on
the size and the contents of the membrane-enclosed volume and the frequency of
the electrical field. Furthermore, the capacitance can be ''short-circuited'' by the
conductivity (like a parallel resistor) of the medium (i.e., the freely moving
charged species not enclosed by a membrane). The breakdown of the impedance
DC (also known as b-dispersion) provides an estimate of the cellular volume, and
the critical frequency at which this breakdown occurs provides an estimate of the
size of the cells. Nowadays, there are instruments on the market that can resolve
(or scan) a range of frequencies (from approximately 100 KHz to 20 MHz) and so
deliver dielectric spectroscopy information. This method usually works better with
large cells, for instance, CHO cell lines (e.g., [ 27 ]). It reaches its operating limits
when the conductivity of the medium is high (or rapidly changing); changes in
aeration also affect the signal. However, it estimates the intact biomass volume.
Sarrafzadeh et al. [ 28 , 29 ] were able to distinguish quite clearly between growing