How Cells Grow - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 552

present, the dry-weight measurement will be inaccurate. Typically, samples of culture broth
are centrifuged or filtered and washed with a buffer solution or water. The washed wet cell
mass is then dried at 80 C for 24 h; then dry cell weight is measured.
Bulk cell volume is used to rapidly but roughly estimate the cell concentration in a fermen-
tation broth (e.g. industrial antibiotic fermentations). Fermentation broth is centrifuged in
a tapered graduated tube under standard conditions (revolution per minute and time),
and the bulk volume of cells is measured.
Another rapid method is based on the absorption of light by suspended cells in sample
culture media. The intensity of the transmitted light is measured using a spectrometer.
Turbidity or optical density (OD) measurement of the culture medium provides a fast, inex-
pensive, and simple method of estimating cell density in the absence of other solids or light
absorbing compounds. The use of optical methods for medium containing hydrolysates of
woody biomass may not be ideal as plant cell fragments, and other particles can interfere
in the same were as microbial cells. The extent of light transmission in a sample chamber
is a function of cell density and the thickness of the chamber. Light transmission is modulated
by both absorption and scattering. Pigmented cells give different results than unpigmented
ones. Background absorption by components in the medium must be considered, particu-
larly if absorbing dissolved species are taken into cells. The medium should be essentially
particle free. Proper procedure entails using a wavelength that minimizes absorption by
medium components (600 e 700 nm wavelengths are often used), “blanking” against
medium, and the use of a calibration curve. The calibration curve relates OD to dry-weight
measurements. Such calibration curves can become nonlinear at high OD values (

> 0.3) and

depend to some extent on the physiological state of the cells.

11.1.2.2. Indirect Methods
In many fermentation processes, such as mold fermentations, direct methods cannot be
used. In such cases, indirect methods are used, which are based mainly on the measurement
of substrate consumption and/or product formation during the course of growth.
Intracellular components of cells such as RNA, DNA, and protein can be measured as indi-
rect measures of cell growth. During a batch growth cycle, the concentrations of these intra-
cellular components change with time. Concentration of RNA (RNA/cell weight) varies
significantly during a batch growth cycle; however, DNA and protein concentrations remain
fairly constant. Therefore, in a complex medium, DNA concentration can be used as
a measure of microbial growth. Cellular protein measurements can be achieved using
different methods. Total amino acids, Biuret, Lowry (folin reagent), and Kjeldahl nitrogen
measurements can be used for this purpose. Total amino acids and the Lowry method are
the most reliable. Recently, protein determination kits from several vendors have been devel-
oped for simple and rapid protein measurements. However, many media contain proteins as
substrates, which limits the usefulness of this approach.
The intracellular ATP concentration (g-ATP/g-cells) is approximately constant for a given
organism. Thus, the ATP concentration in a fermentation broth can be used as a measure of
biomass concentration. The method is based on luciferase activity, which catalyzes oxidation
of luciferin at the expense of oxygen and ATP with the emission of light.

luciferase

þ O 2 þ ATP light (11.1)

Luciferin !