12B702 -Bioprocess Engineering

UNIT- III
MONITORING OF BIOPROCESSES
1. INTRODUCTION
For most modern industrial production methods, process monitoring plays a
key role; bioprocess systems are no exception to this rule. Effective methods
of monitoring are required in order to develop, optimize, and maintain
biological reactors at maximum efficiently. An optimized process should lead
to streamlined performance, reductions in running and material costs, and
improvements in quality control. A number of bioprocess monitoring
instruments have been regular features of the industry for many years
(Fig.1), in particular, sensors capable of measuring physical parameters. The
challenge is to develop cost-effective devices capable of measuring a much
wider range of parameters, providing process operators with a deeper insight
into the process. Eventually effective monitoring will lead to better control

systems, providing cost and quality benefits.

Figure 1. Major areas of bioprocess monitoring.
Progress in monitoring, modeling and control of bioprocesses
The rapid development in biotechnology during the last few years was
enhanced by progress in genetic engineering. The successful application of
the recombinant micro-organisms and cells for industrial production of

12B702 -Bioprocess Engineering

therapeutically important proteins and in less extent of primary and

secondary metabolites impaired by the lack of suitable production processes.
An adaptation and improvement of the bioprocess engineering to the new
demands was necessary. Quickly turned out that beside improved medium
optimisation more efficient process monitoring is needed, which allows better
process modeling and closer process control. Especially, quick analysis
methods are needed, which allow immediate response to the changes in a
cultivation process. Highly selective in situ and on-line methods are
developed for process monitoring and applied in laboratories. In addition,
new analysis principles are discovered and miniaturization of instruments is
promoted.
However, some key variables, such as cell concentration and viability cannot
be monitored online. Therefore, they are identified by means of measured
data and suitable process model and the estimated state is used for state
estimation and process control.
2. OPERATING FEATURES
For any measuring technique, there are a number of criteria that the device
must satisfy if it is too accepted by commercial bioprocess operators (Fig. 2).
The following list of desirable characteristic is not exhaustive, but does cover
the main areas of concern.
Reliability
Reliability is a key issue for bioprocess monitoring equipment. Confidence in
the ability of an instrument to maintain its credibility in terms of performance
is a fundamental parameter. This encompasses a number of features relating
to the operation of the instrument, for example, ease of use, maintenance,
repair, and replacement. A successful monitoring instrument will have a low
failure rate; again, this is related to reliability.
Accuracy

12B702 -Bioprocess Engineering

Accuracy can be described as the relationship between the measured value

(by the instrument) and the actual value of a bioprocess variable. An
accurate instrument will achieve a low percentage error between these two
values. Generally this can be stated as
% error =(measured value - true value)/true value x 100%

Figure 2. Main criteria for a bioprocess monitoring instrument

Precision
Precision is a measure of instrument reproducibility, that is, the ability to
obtain the same value with repeated measurements of a process variable (at
a constant level). It follows that a precise instrument may not, necessarily,
be accurate. Therefore it is important to distinguish between these two
parameters.
Response time
The measurement of any process variable will entail a time delay between
change in the parameter and display of the measured value. This response
time should not be detrimental to the progress of the bioprocess, particularly
if the measurement is linked to a control action. Response times will be
affected not only by the type of instrument but also by the method of
measurement. Off-line sampling and measuring can involve a number of
time-consuming steps. In contrast, in situ devices can provide a real-time
measurement.

12B702 -Bioprocess Engineering

Calibration
In order to maintain the accuracy of a sensor it is (generally) desirable to
carry out a calibration step. This is carried out using set standards of
comparison. The effects of calibration can impinge on the process; in other
words, it may be difficult to carry out calibration of an in situ sensor, whereas
an on-line device could easily incorporate a calibration step(s) during routine
running.

Linearity
Under ideal conditions, the output signal from a sensor would be directly
proportional to the analyte concentration. However, this is not always the
case, and other models have to be used to reach a true value. Despite this
drawback, linearity is not an overriding prerequisite for a successful
monitoring instrument. Developments in modern software can be adapted to
compensate for nonlinearity.
Threshold and Sensitivity
Sensitivity is the magnitude of the output signal per unit change in the target
analyte concentration. The lowest level of detection is related to the
sensitivity and the signal-to-noise ratio. A number of factors influence the
sensitivity of a monitoring device, including sensor design, the operating
environment, periods of maintenance, and interfering noise levels. The
sensitivity will influence the dynamic range above which the device becomes
saturated; the highest level of detection will be the threshold limit for the
device.
3. METHODS OF MONITORING
In addition to the variety of monitoring devices available, a number of
methods (for carrying out the measurement exist (Fig. 3). Sampling and
sample handling is a vital issue, affecting both the accuracy and frequency of

12B702 -Bioprocess Engineering

measurements. Broadly, the main approaches to sampling can be described

in one of several ways.

Figure 3. Methods of monitoring.

Off-Line or At-Line Monitoring
Off-line monitoring involves taking a sample from the bioreactor and carrying
out the measurement at a different site, usually under laboratory conditions.
Off-line sampling can be detrimental in terms of cost, efficiency, and threat
to asepsis. Manually removing and measuring a sample requires technical
labor. The received signal will not be real time because of the delays. In
order to obtain the sample, the sterility of the bioreactor must be
maintained. Hence, provision must be made to achieve this. In some cases,
decentralized equipment has become available that allows measurements to
be made simply close to the production process (at-line), reducing some of
the delays associated with laboratory analysis.
In Situ or In-Line Monitoring
In situ sensors are placed directly in the bioprocess vessel. The use of in situ
sensors has long been established in the bioprocess industry, where dip-in
devices are used to monitor a number of parameters such as pH and
dissolved gas concentrations. A number of advantages are gained by
operating sensors in this fashion, including real-time monitoring (samplingrelated time delays are eliminated) with the sensors operating continuously
(any rapid change in the analyte concentration can be readily observed), and
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12B702 -Bioprocess Engineering

labor requirements and problems of contamination are both significantly

reduced. However, in situ sensors can have a number of drawbacks, for
example, the sensor system must be amenable to sterilization; if the lifetime
is short, replacement may be difficult (during a process run); the surface of
the sensor could become fouled by components of the growth medium,
affecting the signal output.
On-Line Monitoring
Methods

of

on-line

monitoring

involve

the

automatic

removal

and

measurement of sample or sample stream from the bioreactor. One example

of this has been the development (since the early 1980s) of flow injection
analysis (FIA). This is a liquid handling technique that has proved flexible in
adapting

to

most

chemical

and

biochemical

reaction

procedures,

representing an effective compromise between the desirability of in situ

monitoring and the technical ease of off-line measurements. The main
advantages of on-line methods include the following: sensor sterilization can
be readily accomplished, sample pretreatment (e.g., gassing, dilution,
removal of interferents), and sensor calibration can be built into the system.
The main disadvantages are a need for an effective and reliable sampling
system and the fact that the signal is discontinuous; frequency measuring
rate is determined by the limitation of the overall FIA arrangement. All of
these methods have their advantages and disadvantages. The choice of
approach adopted depends on a number of factors, not least of which will be
the availability of both sensor and system.
4. MEASUREMENT OF DIFFERENT PARAMETERS
Introduction
The widespread use of advanced control and process automation for
biochemical applications has been lagging as compared with industries such
as refining and petrochemicals whose feedstocks are relatively easy to
characterize

and

whose

chemistry

is

well

understood

and

whose

measurements are relatively straightforward. Biological processes are

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12B702 -Bioprocess Engineering

extraordinarily complex and subject to considerable variability. The reaction

kinetics cannot be completely determined in advance in a fermentation
process because of variations in the biological properties of the innoculant.
Therefore, information regarding the activity of the process must be
gathered as the fermentation progresses. Directly measuring all the
necessary

variables

which

characterize

and

govern

the

competing

biochemical reactions, even under optimum laboratory conditions, is not yet

achievable. Developing mathematical models, which can be utilized to infer
the biological processes underway from the measurements available,
although useful, is still not sufficiently accurate. Add to this the constraints
and compromises imposed by the manufacturing process and the task of
accurately predicting and controlling the behavior of biological production
processes is formidable indeed. The knowledge base in fermentation and
biotechnology has expanded at an explosive rate in the past twenty-five
years aided in part by the development of sophisticated measurement,
analysis and control technology. Much of this research and technology
development has progressed to the point where commercialization of many
of these products is currently underway.
Measurement Technology
Measurements are the key to understanding and therefore controlling any
process. As it relates to biochemical engineering, measurement technology
can be separated into three broad categories. These are biological, such as
cell growth rate, florescence, and protein synthesis rate; chemical, such as
glucose concentration, dissolved oxygen, pH and offgas concentrations of
CO2, O2, N2, ethanol, ammonia and various other organic substances; and
physical, such as temperature, level, pressure, flow rate and mass. The most
prevalent are the physical sensors while the most promising for the field of
biotechnology are the biological sensors.

Cell Mass Measurement

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12B702 -Bioprocess Engineering

The on-line direct measurement of cell mass concentration by using

optical density principles promises to dramatically improve the knowledge of
the metabolic processes underway within a bioreactor. This measurement is
most effective on spherical cells such as E. Coli. The measurement
technology is packaged in a sterilizable stainless steel probe which is
inserted directly into the bioreactor itself via a flange or quick-disconnect
mounting.
By comparing the mass over time, cell growth rate can be determined.
This measurement can be used in conjunction with metabolic models which
employ such physiological parameters as oxygen uptake rate (OUR), carbon
dioxide evolution rate (CER) and respiratory quotient (RQ) along with direct
measurements such as dissolved oxygen concentration, pH, temperature,
and off gas analysis to more precisely control nutrient addition, aeration rate
and agitation. Harvest time can be directly determined as can shifts in
metabolic pathways possibly indicating the production of an undesirable byproduct. Cell mass concentrations of up to 100 grams per liter are directly
measured using the optical density probe. In this probe, light of a specific
wavelength, created by laser diode or passing normal light through a
sapphire crystal, enters a sample chamber containing a representative
sample of the bioreactor broth and then passes to optical detection
electronics. The density is determined by measuring the amount of light
absorbed,

compensating

for

backscatter.

Another

technique

used

to

determine cell density is spectrophotometric titration which is a laboratory

procedure which employs the same basic principles as the probes discussed
above. This requires a sample to be withdrawn from the broth during reaction
and therefore exposes the batch to contamination.
Chemical Composition
The most widely used method for determining chemical composition is
chromatography. Several categories have been developed depending upon
the species being separated. These include gas chromatography and several
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12B702 -Bioprocess Engineering

varieties of liquid chromatography including low pressure (gel permeation)

and high pressure liquid chromatography and thin layer chromatography. The
basic principle behind these is the separation of the constituents traveling
through a porous, sorptive material such as a silica gel. The degree of
retardation of each molecular species is based on its particular affinity for
the sorbent. Proper selection of the sorbent is the most critical factor in
determining separation. Other environmental factors such as temperature
and pressure also play a key role.
The chemical basis for separation may include adsorption, covalent
bonding or pore size of the material. Gas chromatography is used for gases
and for liquids with relatively low boiling points. Since many of the
constituents in a biochemical reaction are of considerable molecular weight,
high pressure liquid chromatography is the most commonly used. Specialized
apparatus is needed for performing this analysis since chromtograph
pressures can range as high as 10,000 psi. Thin layer chromatography
requires no pressure but instead relies on the capillary action of a solvent
through a paper-like sheet of sorbent. Each constituent travels a different
distance and the constituents are thus separated. Analysis is done manually,
typically using various coloring or fluorescing reagents.
Gel permeation chromatography utilizes a sorbent bed and depends on
gravity to provide the driving force but usually requires a considerable time
to effect a separation. All of these analyses are typically performed in a
laboratory; therefore they require the removal of samples. As the reaction is
conducted in a sterile environment, special precautions and sample removal
procedures must be utilized to prevent contaminating the contents of the
reactor.
Oxygen
Generally, oxygen measurement falls into two main categories: dissolved
oxygen and exit gas analysis. For most aerobic fermentations, an accurate
determination of the dissolved oxygen concentration is of particular
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12B702 -Bioprocess Engineering

importance, for example, in maintaining the concentration above a specified

minimal level. In addition, oxygen transport processes can be determined
only by using an accurate method of measurement. The exit gas analysis for
oxygen, usually in conjunction with CO2 measurement, can be used to
determine the metabolic state of aerobic microorganisms and their oxygen
uptake.

Ex: Dissolved Oxygen Probes

Galvanic Electrodes
O2 Optodes.
Dissolved oxygen is one of the most important indicators in a fermentation or
bioreactor process. It determines the potential for growth. The measurement
of dissolved oxygen is made by a sterilizable probe inserted directly into the
aqueous solution of the reactor. Two principles of operation are used for this

10

12B702 -Bioprocess Engineering

measurement: the first is an electrochemical reaction while the second

employs an amperometric (polarographic) principle. The electrochemical
approach uses a sterilizable stainless steel probe with a cell face constructed
of a material which will enable oxygen to permeate across it and enter the
electrochemical chamber which contains two electrodes of dissimilar
reactants (forming the anode and cathode) immersed in a basic aqueous
solution. The entering oxygen initiates an oxidation-reduction reaction which
in turn produces an EMF which is amplified into a signal representing the
concentration of oxygen in the solution.
In the amperometric (polarographic) approach, oxygen again permeates
a diffusion barrier and encounters an electrochemical cell immersed in basic
aqueous solution. A potential difference of approximately 1.3 V is maintained
between the anode and cathode. As the oxygen encounters the cathode, an
electrochemical reaction occurs:

The hydroxyl ion then travels to the anode where it completes the
electrochemical reaction process:

The concentration of oxygen is directly proportional to the amount of

current passed through the cell.
Viscosity
Information on the rheology, or viscosity, can help in ensuring the
efficiency of a biological process. An example of this would be the
effectiveness of oxygen transfer throughout a medium and determining the
degree of branching of filamentous microorganisms. In addition, viscosity can
effect pumping, mass transfer, and mixing. Viscosity is the apparent shear
resistance between adjacent layers of liquids or gases moving at different
speeds. Typically, in fluids this is the result of molecular cohesion; rising
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12B702 -Bioprocess Engineering

temperatures lead to a decrease in viscosity. In contrast, viscosity increases

for gases under conditions of rising temperature. This results from an
increased measured molecular activity.

Ex: Cone and plate viscometer.

Coaxial cylinder rotary viscometer.
Impeller viscometer.
Exhaust Gas Analysis
Much can be learned from the exchange of gases in the metabolic process
such as O2, CO2, N2, and ethanol. Infact, most of the predictive analysis is
based upon such calculations as oxygen uptake rate, carbon dioxide
exchange rate or respiratory quotient. This information is best obtained by a
component material balance across the reactor. A key factor in determining
this is the analysis of the bioreactor off gas and the best method for
measuring this is with a mass spectrometer because of its high resolution.
Measurement of pH

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12B702 -Bioprocess Engineering

Metabolic processes are typically highly susceptible to even slight

changes in pH, and therefore, proper control of this parameter is critical.
Precise manipulation of pH can determine the relative yield of the desired
species over competing by-products. Deviations o as little as 0.2 to 0.3 may
adversely affect a batch in some cases. Like the cell mass probe and
dissolved oxygen probes described earlier, the pH probe is packaged in a
sterilizible inert casing with permeable electrode facings for direct insertion
into the bioreactor.

The measurement principle is the oxidation-reduction potential of the

hydrogen ion and the electrode materials are selected for that purpose.
Measuring pH, along with temperature, is one of the most common practices
in bioprocess monitoring. Correcting action can be taken, either manually or
automatically, to prevent unwanted increases or decreases in pH. The most
common form of pH sensor used for fermentation monitoring is based on the
electrode design introduced by Ingold in 1947.
Temperature
Maintaining optimum conditions for any bioprocess will invariably involve
monitoring and controlling the temperature of the broth. Precise temperature

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12B702 -Bioprocess Engineering

control and profiling are key factors in promoting biomass growth and
controlling yield.
Generally, bioprocesses are monitored over a temperature range of
between 0 0C and 100 0C (excluding the sterilization cycle). Furthermore, this
may require a control regime operating within a narrow range of
temperatures. A number of devices are available for obtaining an accurate
measurement of temperature conditions during a bioprocess operation,
based on a range of techniques.
Ex: Thermistors
Resistance Thermometers.
Thermocouples
Mercury-in-Glass Thermometers.
Bimetallic Thermometers.
Pressure
Many bioprocesses operate under conditions of overpressure. Monitoring
the magnitude of this pressure is an important factor, both in terms of safety
and optimization of the process. Industrial and laboratory fermenters are
designed to operate up to a safe working pressure. Increasing the applied
pressure above the upper limit can be dangerous. Furthermore, maintaining
a positive reactor head pressure can prevent contamination of the bioreactor.
In order to facilitate effective sterilization, fermenters need an accurate
pressure monitor. Pressure will also affect the solubility of gases (such as O2
and CO2). Several different approaches have been used in the development
of pressure gauges.

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12B702 -Bioprocess Engineering

Ex: Bourdon Tube Pressure Gauge.

Strain Gauges
Piezoelectric Manometers.
Diaphragm-Type and Pressure Bellows Sensors
Weight
Monitoring fluctuations in the weight of a vessel used for bioprocesses is
an effective way of measuring the contents and flow rate of additions. There
are a number of load cells available for this task. The principle of a load cell
is based on measuring the compressive strain that is placed on the device
(e.g., a solid or tubular steel cylinder) when under an axial load. Electrical
resistance strain gauges can be included in the device structure. A
proportional electrical resistance to the applied load can then be measured;
this varies in relation to changes in load. Obviously, a temperature
compensator must be included in the instrument to account for heat effects
on the resistors. These devices need to be rugged and have long-term

15

12B702 -Bioprocess Engineering

stability. For reasons of safety, it is also desirable for the device to be

accurate. A load cell designed to detect tensile forces can be used for weight
measurements of suspended vessels.

Fig: Schematic of the installation of a load cell.

Liquid Level Measurement
Liquid levels in a vessel can be determined using a number of techniques.
Determining the level can assist in formulating mass balances and
measuring nutrient additions. Hydrostatic pressure can be measured using a
single sensor positioned at the bottom of the vessel. Alternatively, two
sensors spaced at the top and bottom of the vessel (differential design) can
be employed. These sensors are electromechanical devices based on the
deformation of a spring; the resulting signal is displayed as an electrical
signal (capacitance or reactance).
Volumetric Flow Rate
Quite a number of technologies are available for measuring volumetric
flow rates. These include differential pressure transmitters, vortex meters
and magnetic flow meters. Each has its advantages and disadvantages. The
differential pressure transmitter is the most popular and has been in use the
longest. Its measurement principle is quite simple. Create a restriction in the
line with an orifice plate and measure the pressure drop across the

16

12B702 -Bioprocess Engineering

restriction. The measurement takes advantage of the physical relationship

between pressure drop and flow.
The primary disadvantages of the differential pressure producing flow
measurements are the permanent pressure drop caused by the restriction in
the line; sediment buildup behind the orifice plate (which could be a source
of bacterial buildup) and loss of accuracy over time as the edge of the plate
is worn by passing fluid and sediment.
Broth Level
As the broth in a fermenter or bioreactor becomes more viscous and is
subjected to agitation from sparging (the introduction of tiny sterilized air
bubbles at the bottom of the liquid) and from mixing by the impeller, it has a
tendency to foam. This can be a serious problem as the level may rise to the
point where it enters the exhaust gas lines clogging the ultrafilters and
possibly jeopardizing the sterile environment within the reactor. Various
antifoam strategies can be employed to correct this situation, however,
detection of the condition is first required.
Regulatory Control
Automatic regulatory control systems have been in use in the process
industries

for

over

fifty

years.

Utilizing

simple

feedback

principles,

measurements were driven toward their set points by manipulating a

controlled variable such as flow rate through actuators like throttling control
valves. Through successive refinements in first mechanical, then pneumatic,
then electronic and finally digital electronic systems, control theory and
practice has progressed to a highly sophisticated state.
Carbon Dioxide
For many bioprocesses, the measurement of CO2 is an important feature.
Increased levels of dissolved carbon dioxide can inhibit growth and reduce
the production of secondary metabolites.

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12B702 -Bioprocess Engineering

Redox Potential
Monitoring the redox potential of a bioprocess medium can provide
information about the equilibrium between oxidizing and reducing species
(electron acceptors and donors, respectively) present. Measurement of redox
potential is achieved using a combined metal-reference electrode system.
Mass Spectrometry
The use of on-line mass spectroscopy (MS) has developed since the first
report of Reus et al.

A wide range of gasses, both free and dissolved (e.g., CO2, O2, CH4), can
be measured. In addition, volatile organic compounds such as methanol,
ethanol, acetone, and simple organic acids can be monitored. The technique
is based on the rupture of molecules by a high-energy source into
corresponding ions.
Conductivity Sensors.
Conductivity sensors are particularly suited to monitoring levels of foam
(in a number of fermentations), where in excess it can cause problems. The
sensor consists of a stainless steel probe, insulated except for the tip. The
device can be used with noncorrosive conducting fluids. If the liquid or foam
level rises to contact with the tip, an electric current is passed through the
sensor; the foam acts as an electrolyte and the vessel as a ground. The
sensor can be coupled to a control device that dispenses antifoam to counter
the increased level.

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12B702 -Bioprocess Engineering

Capacitance Sensors.
Capacitance sensors operate by detecting changes in the relative dielectric
constant of the media, compared with air. Measurements are made by
detecting variations in electrical capacity brought about by changes in the
liquid level.
Acoustic Sensors.
Acoustic sensors operate via transducers that generate and detect ultrasonic
waves. Two formats are available: a single device that both transmits and
receives the signal, and two separate transducers with one function. By
monitoring the time it takes for the sound wave to travel, the liquid level can
be determined. With the single device the sound wave is directed onto the
liquid surface. When the level rises, the time delay is shortened.
Temperature Probes.
Liquid levels can be measured using a series of thermistors sited vertically
through the vessel. During operation, an electrical current is applied to the
sensors, raising their temperature above the ambient. When the liquid (or
foam) level reaches a sensor positioned lower down the vessel, the sensor
cools. This causes a temperature difference and, hence, a change in
electrical resistance. The resistance can be displayed as an output signal
indicating the liquid level.
Flow Measurement
Flow measurement is an important feature for many bioprocesses, for
both gases and liquids. These measurements are carried out on both the
influent and effluent streams.The most common form of gas and liquid
measurement is carried out using a variable area flow meter, or rotameter.

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12B702 -Bioprocess Engineering

Rotameter
This device consists of vertically mounted (usually conical) tube enclosing
a free-floating body that is able to move up (floating in the gas flow of
interest) and down a tapered bore running through the tube body. Typically,
the tube is constructed of glass or metal and the float is a ball or hollow
thimble shape. Because the position adopted by the flow rate is dependant
on both the gas flow and the viscosity of the medium, the instrument
requires careful calibration.
METHODS OF BIOMASS ESTIMATION
The monitoring of biomass concentration can be carried out using a
number of techniques. Conventionally, biomass concentration is measured
off-line using labor intensive, time-consuming methods such as dry weight
cell, plate or microscopic cell count, and measuring the optical density of
diluted samples. However, a number of more rapid methods have been
developed. Of particular interest is the development of real-time on-line
methods.
Generally, methods for determining biomass concentration can be divided
into two classifications: direct and indirect. The former is based on
determining the physical properties of the cell and its components. In

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contrast, indirect methods measure factors related to the cell and its activity
(e.g., respiration, electrochemical behavior, and nutrient fluctuation).
Direct Biomass Measurements
Some measure of the bacterial cell mass or numbers of a culture is almost
always used as the reference basis for measurements of cellular metabolic
activities, the types of morphological characters, or the amount of chemical
constituent; biomass and cell numbers are the two basic independent
parameters of bacterial growth. The methods for measuring biomass seem
obvious and straightforward, but in fact they are complicated if accuracy if
sought. Furthermore, the results may be expressed in different ways and, in
some of these ways, the values may be more relative than absolute.
Wet Weight
A nominal wet weight of bacterial cells originally in liquid suspension is
obtained by weighing a sample in a tared pan after separation and washing
the cells by filtration or centrifugation. In either case, however, diluent is
trapped in the interstitial (intercellular) space and contributes to the total
weight of the mass. The amount of interstitial diluent may be substantial. A
mass of close-packed, rigid spheres contains in its interstices 27% of space.
This is independent of sphere size. A mixture of sizes packs more densely,
and close-packed bacterial cells may contain an intersticial volume of 5 to 30
%, depending on their shape and amount of deformation. This is a problem
that is readily solved if the washing step can be carried out with pure water.
Simple weighing or just measuring the packed volume of cells can be an
excellent and rapid method with filamentous organisms or those that grow as
pellets. Then filter, wash and weigh or centrifuge, and measure the height of
the pellet. Both can be very rapid, and the procedures can be calibrated to
correct for the exogenous water or shapes of centrifuge tubes. Because the
particulate matter in the medium has different physical properties, it can be
possible to separate the cells from the particulates and estimate the cell

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12B702 -Bioprocess Engineering

volume directly. This correction could be established with radioactive or

fluorescent dextrans or other molecules that are too big to enter the cells.
Dry Weight
A nominal dry weight (solids content) of bacterial cells originally in a liquid
suspension is obtained by drying a measured wet weight or volume in an
oven at 80oC for 10 hrs to constant weight. The cells could be washed with
water (possibly extracting cell components), or (better) a correction could be
made for medium or diluent constituents that are dried along with the cells.
Separating the cells by filtration poses particular problems. More problems
arise if volatile components of the cells can be lost by oven drying, or if some
degradation

and

volatilization

occurs,

evidenced

by

discoloration

(particularly if a higher temperature is used). Some regain of moisture occurs

during the transferring and weighing process in room atmosphere, so this
should be done quickly within a fixed time for all replicate samples,
especially if the relative humidity is high. It is best, of course, to use tared
weighing vessels that can be sealed after drying.
The dry weight of cells may be expressed on a wet weight basis (grams of
solids per gram of wet cells) or on a wet volume basis (grams of solids per
cubic centimeter of wet cells or per cubic centimeter of cell suspension).
Because the drying can be a time-consuming process, and adequate
knowledge about the possibility of volatilization of some cell components is
not known, in the future,

adequate drying quickly at lower temperature

could be done in specially designed vacuum ovens. Such procedures could

also be calibrated for the residual water content. Often, a practical method is
to dry the cells in a microwave oven.
Indirect Biomass Measurements
Bioluminescence and Chemiluminescence: The use of bioluminescent
techniques is based on determining levels of adenosine triphosphate (ATP)
concentration. Generally, levels of ATP remain constant for living cells,

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12B702 -Bioprocess Engineering

decreasing when the cells die. ATP concentration can, therefore, be related to
biomass. A particular enzyme that has proved useful for this method is
luciferase, which catalyzes the following reaction:

By detecting the light produced by this reaction, ATP concentrations, and

hence, biomass, can be determined. This method can detect cell numbers as
low as 105 cells per ml; it can be automated, and the assay time is fairly
rapid. However, the method does suffer from several drawbacks, for
example, the extraction of ATP may be incomplete, there may be free ATP
present from other sources, and there maybe degradation of ATP by the
extraction reagents. Chemiluminescence is based on the detection of light
produced by the protein-catalyzed oxidation of luminol in the presence of
hydrogen peroxide.
Acoustic Resonance Densitometry. Acoustic resonance density is based
on determining the change in a resonant frequency that results from
changes in cell density. This is a noninvasive method that does not require
contact between the instrument and the culture medium. The method
incorporates an oscillatory circuit, amplifier, and test cell. Theoretically, the
fluid density of the sample can be calculated from the square of its
oscillation. Recent reports have described using this approach to monitor
cultures of hybridomas and human lymphoma cells. Further advantages for
this method include independence from flow rate and viscosity. However,
there are disadvantages including the need for a filtration system (to enable
the resonance density of the medium to be measured in the absence of
cells), with the inherent problems associated with such systems, including
poor sensitivity and problems caused by bubbles and particulate matter.

Capacitance, Conductivity, and Electrochemical Methods.

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The electrical properties of cells have been exploited in the development

of a number of techniques. Changes in media capacitance have been related
to biomass concentration (e.g., a decreasing capacitance coupled with an
increasing biomass concentration. In addition, it has been suggested that cell
viability is linked to capacitance measurements. Several in situ commercial
instruments are currently available for determining biomass concentration
based on capacitance measurements. The Biomass Monitor measures the
radio frequency conductance and capacitance of a cell suspension using a
constant

voltage,

four-terminal,

phase-sensitive

detector

system.

Furthermore, the probes can be inserted directly into bioreactors using a

standard 25- mm-diameter port. The probes are fully sterilizable and can be
cleaned in situ during operation.
Fluorescence.
Fluorescence is a characteristic possessed by a number of important
biological compounds including proteins, enzymes, and coenzymes. Simply
de- fined, it is the absorbance of light energy at a particular wavelength,
followed by reemission at a longer wavelength. After the passage of light
energy, the compound returns to its ground-state level. In the field of
bioprocess monitoring, fluorescence has been used, in particular, to monitor
reduced

nicotinamide

adenine

dinucleotide

(NADH)

and

reduced

nicotinamide adenine dinucleotide phosphate (NADPH) concentrations. These

compounds are irradiated at 340 nm and emit at 450 nm. Both compounds
are present in living organisms and are vital components of metabolic
processes. By monitoring the intensity of fluorescence at the characteristic
emission wavelength, it should be possible to calculate the total biomass
concentration. Unfortunately, the use of fluorescence as a marker for cell
concentration does suffer from a number of drawbacks including that the
fluorescence signal can originate from changes in metabolic state and not
from cell concentration levels, sensitivity may be affected by the presence of

24

12B702 -Bioprocess Engineering

compounds that can quench both the excitation and emission wavelengths,
and other compounds may fluoresce at the same wavelength.
Flow Cytometry.
Flow cytometry is a measuring technique based on the irradiation of a
sample solution (containing a cell population) with a suitable light source,
followed by monitoring of the scattered or absorbed light. In addition,
fluorescence can be used as the measuring parameter. This technique can be
used to ascertain a number of cellular features, such as the accumulation of
cellular components (e.g., DNA, RNA, and proteins), and cell dynamics (e.g.,
cell size distribution). Furthermore, flow cytometry can be used to
differentiate and quantify a range of species populations present in a mixed
medium.

Light Scattering and Turbidity.

Optical techniques based on either the scattering of light (nephelometry) or
the degree of transmitted light or optical density (turbidity) have been
developed for determining biomass. By monitoring the degree of light

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scattering using nephelometry, both cell numbers and mass can be

determined. This approach is particularly suited for bioprocesses that involve
low cell concentrations, where the background (compared) level is near zero.

Turbidity

measurement

can

be

used

for

both

online

and

off-line

determination of biomass. Light scattering by a turbid medium results from a

number of factors including particle size, shape, and number. Quantification
is based on the Lambert-Beer law
5. MICROBIAL CALORIMETRY
Introduction
Calorimetry is the science of measuring the heat of chemical reactions
or physical changes. Calorimetry involves the use of a calorimeter. Heat is a
universal and unavoidable by product of all biological phenomena, including
those that are exploited in biotechnology at technical scale. Any change in
growth rate, metabolism, biocatalytic activities and in other biological
phenomena occurring in technical reactors will invariably affect the rate at
which heat is released.
Yet heat effects in cellular cultures often go unnoticed when one is
working with conventional laboratory equipment because most of the heat
released by the culture is lost to the environment too quickly to give rise to a
perceivable temperature increase. This, however, is completely different at
large scale. As opposed to laboratory reactors, industrial size fermenters

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operate nearly adiabatically due to their much smaller surface to volume

ratio. Thus all the heat released by the culture must be removed by
appropriate cooling facilities.
Systematic monitoring of heat generation rates by the culture in largescale bioreactors would clearly be of considerable benefit for process
optimization and control. The information contained in this signal could be
used together with data on other relevant process parameters to obtain
quantitative on-line estimates of the activity, the metabolism and the state
of the culture.

One method of determining the energy exchange between the reaction

system and its environment is to conduct a calorimetric analysis. A
calorimeter is a thermally insulated container where a reaction system can
be performed and the energy exchange between the system and its
environment can be measured. The calorimeter and its contents are
considered the environment. The reaction system is a chemical or physical
process that occurs within the confines of the calorimeter.
Qsurr = Qcal + Qcontents
The Qcal can be determined if one knows the Heat Capacity of the
calorimeter. This Heat Capacity can be experimentally determined and is
expressed in Kilojoules / C degree. In order to determine the Q cal you multiply
27

12B702 -Bioprocess Engineering

the Heat Capacity of the calorimeter by the difference between the final and
initial temperature. For example if the Heat Capacity of a calorimeter was
determined to be 25.4 KJ/Celsius Degree, determine the Q cal if the initial
temperature during a calorimetric analysis was 30 C and the final
temperature was 50 C.
Qcal = Heat Capacity ( final temp - initial temp) = 25.4 Kj/C ( 50 - 30 C) =
508 Kj
Calorimetric Equipment
In order to make use of heat release measurements in bioprocess control
algorithms, suitable models must be available which relate the heat
evolution rate of the culture to other relevant process variables, such as
substrate consumption, growth rate or oxygen up-take. Numerous workers
have studied these relationships in calorimetric experiments at the
laboratory scale. The need for maintaining technically relevant, strictly
controlled culture conditions made it difficult to obtain meaningful results in
micro calorimeters.
Applications:

Heat as a quantitative indicator of cell metabolism

Control of fermentations by calorimetry

6. FLOW INJECTION ANALYSIS

The concept of flow injection analysis (FIA) was proposed in 1975 by
Ruzicka and Hansen. The fast and intensive development of the FIA
methodology was due to several factors essential for routine analytical
determinations, such as very limited sample consumption, the short analysis
time based on a transient signal measurement in a flow-through detector
and

an

on-line

carrying

out

difficult

operations

of

separation,

preconcentration or physicochemical conversion of analytes into detectable

species.
Principle of the FIA

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12B702 -Bioprocess Engineering

The three principles or cornerstones of FIA were identified by Ruzicka and

Hansen as sample injection, controlled dispersion of the injected sample
zone, and reproducible timing of the movement of the injected zone from the
injection point to the detector.

(C=carrier;

P=pump;

S=point

of

sample

injection;

RC=reaction

coil;

D=detector; W=waste)

Schematic diagram of the basic FI system

In the simplest form of FIA the sample is injected into a continuous flow of
reagent solution (carrier), dispersed, and transported to detector. Sample
dispersion is controlled through the suitable choice of the injected sample
volume, flow rate of carrier, length of the reaction coil, and diameter of the
tubing used. A schematic diagram of the basic FI system is shown in Figure

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12B702 -Bioprocess Engineering

Flow Injection Analysis (FIA), the first generation of FIA techniques, is

also probably the most widely utilized. In its simplest form, the sample zone
is injected into a flowing carrier stream of reagent. As the injected zone
moves downstream, the sample solution disperses into the reagent, causing
the product to form. A flow through detector placed downstream records the
desired physical parameter such as colorimetric absorbance or fluorescence.
The modern Flow Injection Analysis system usually consists of a high
quality multichannel peristaltic pump, an injection valve, a coiled reactor, a
detector such as a photometric flow cell, and an autosampler. Additional
components may include a flow through heater to increase the speed of
chemical reactions, columns for sample reduction, debubblers, and filters for
particulate removal.
The typical FIA flow rate is one milliliter per minute, typical sample
volume consumption is 100 microliters per sample, and typical sampling
frequency is two samples per minute. FIA assays usually result in sample
concentration accuracies of a few percent.
Sequential Injection Analysis
Sequential Injection Analysis (SIA) is the second generation approach to
FIA compatible assays. SIA usually consists of a single-channel high precision
bi-directional pump, a holding coil, a multiposition valve, and a flow through
detector. The system is initially filled with a carrier stream into which a zone
of sample and a zone of reagent(s) are sequentially aspirated into a holding
coil, forming a linear stack. These zones become overlapped due the
parabolic profile induced by differences between flow velocities of adjacent
streamlines. Flow reversals and flow acceleration further promote mixing.
The multi position valve is then switched to the detector position, and the
flow direction is reversed, propelling the sample/reagent zones through the
flow cell.

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12B702 -Bioprocess Engineering

The advantage of SIA over the more traditional flow injection analysis
(FIA) is that SIA typically consumes less than one-tenth the reagent and
produces far less waste an important feature when dealing with expensive
chemicals, hazardous reagents, or online/remote site applications. One
disadvantage of SIA is that it tends to run slower than FIA.
Online process monitoring using SIA is often an ideal solution. The
low reagent/sample consumption, waste production, and nearly hands-off
robustness make SIA the perfect choice. Example online applications include
fermentation monitoring of ammonia, glycerol, and glucose.

Or automated

sample dilutions prior to absorbance monitoring, perhaps many thousands

fold. Also remote site monitoring, where the system may run for days
without user intervention.
Biosensors
One possible answer to the problem of monitoring metabolites, both in
situ and on-line, may be solved in the near future by the use of biosensors.
Since their conception in the 1960s, these devices have generated
considerable interest. This has spread to a diverse range of fields including
clinical diagnostics, environmental protection, bioprocess monitoring, and
defence applications (56). In general the operation of a biosensor is

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characterized by three functional steps: recognition, physiochemical signal

generation, and signal processing. The biological component (e.g.,enzyme,
whole cell, antibody, and cell receptor) imparts a high degree of selectivity
on the biosensor. Coupled to the biological component; the transducer is
designed to respond to the changing physicochemical parameters caused by
the specific interaction of the biological component with the substrate. A
range of transducers have been used in the development of biosensors and
include the electrochemical (potentiometric and amperometric), optical,
calorimetric, piezoelectric, and thermometric . Most of these types have
been adopted in the development of biosensors intended for bioprocess
monitoring.

Enzyme-modified field effect transistors (FETs), whereby the biologically

active layer is positioned on top of the ion electrode membrane, have also
been used for bioprocess monitoring. The instrument can be used to
determine a range of metabolites (e.g., glucose, lactate, and ethanol).
Recently the company has introduced an on-line instrument. Indeed, on-line
biosensor systems have been demonstrated using a variety of biologicaltransducer systems . A wide range of metabolites have been monitored using
various bioprocess regimes . Commercially, a significant number of biosensor
devices are available for measuring a range of analytes. However, at
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12B702 -Bioprocess Engineering

present, the majority of these devices are aimed at the medical diagnostic
market. The goal is to develop cheap, reliable sensors that can operate
under a range of bioprocess conditions for extended periods with a minimum
of maintenance.

Nuclear Magnetic Resonance

Nuclear magnetic resonance is based on the detection of the response
from particular nuclei when exposed to a magnetic field and electromagnetic
radiation. Following absorption, the resonance frequency is shifted in a
characteristic response pattern, according to the environment of the sample
under detection. This approach can be used to determine a range of
intracellular factors such as ATP, ADP, sugar phosphate, and polyphosphate,
as well as pH. In these examples, concentrations of 31P are used to
characterize the compounds. This is an off-line monitoring system that is
currently expensive; hence, its use is primarily in the research field, not in
routine production environments.
Artificial Intelligence
The data obtained using the various measuring instruments already
discussed are invariably used to control and optimize the bioprocess being
carried out. Recent developments in the field of artificial intelligence have
led to investigations into the use of such systems for improving bioprocess
control, based on the received measurement output signals. This has
included the use of both knowledge-based expert systems (64,65) and neural
networks (e.g., 66,67) during bioprocess operation. A recent report (68)
described the successful use of a neural network as a tool for evaluating the
received measurement from an enzyme (penicillin-G amidase) pH-FET sensor
linked to a flow injection system. Undoubtedly, the adaptation of such
intelligent systems will develop over the coming years and will play an
important role in the precise control of bioprocess applications.

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12B702 -Bioprocess Engineering

Conclusion
Although a diverse range of monitoring equipment is available, only a
relatively narrow range of the more reliable instruments is used routinely in
practice. As culture techniques become more elaborate and high-valueadded products are produced, conventional methods will either prove
insufficient or require supplementation with a range of sensors able to
directly monitor key process parameters. Despite a clear demand for new
sensors (e.g., lactate, glutamine, and glutamate for animal cell culture), cell
cultivation represents only a modest market for analytical instrument
manufacturers. Hence, while progress is to be expected, it may be slower
than might be wished.
During the last 20 years, considerable developments have been obtained
in on-line and in situ process monitoring. Non-measurable variables are now
monitored by observers:state estimators and software sensors. For process
control, mathematical models and hybrid models are applied. The latter
cover mathematical models, literature and live data as well as expert
knowledge. Instead of looking for particular reactions, the network of
biochemical reactions is considered by metabolic flux analysis. The central
metabolism of micro-organisms and cells is well known. Also the biosynthesis
paths of secondary metabolites are well known. By metabolic flux analysis,
the quantitative fluxes can be determined. Most of their genes of the
metabolite network are identified and expressed. In spite of the fact that no
great improvement of the productivity could be obtained, because the
regulation of the metabolic network is still unknown. Metabolic engineering
will be possible, if the dynamic regulation the metabolic network is
determined. This is the aim the next 20 years.
8. COMPUTER BASED DATA ACQUISITION
Traditionally, measurements are done on stand alone instruments of
various types-oscilloscopes, multi meters, counters etc. However, the need
to record the measurements and process the collected data for visualization
34

12B702 -Bioprocess Engineering

has become increasingly important.

Data acquisition involves gathering

signals from measurement sources and digitizing the signal for storage,
analysis, and presentation on a personal computer (PC).
There are five components to be considered when building a basic DAQ
system (Figure 1):

Transducers and sensors

Signals

Signal conditioning

DAQ hardware

Driver and application software

Figure 1. Data Acquisition System

Data acquisition is the sampling of the real world to generate data that
can be manipulated by a computer. Sometimes abbreviated DAQ, data
acquisition typically involves acquisition of signals and waveforms and
processing the signals to obtain desired information. The components of data
acquisition

systems

include

appropriate

sensors

that

convert

any

measurement parameter to an electrical signal, which is acquired by data

acquisition hardware. Acquired data typically is displayed, analyzed, and
stored on a PC. This is achieved by using vendor supplied interactive control
software and hardware such as PowerLab, or custom displays and control can

35

12B702 -Bioprocess Engineering

be accomplished using a programming language such as experix, LabVIEW,

Visual Basic, or C. EPICS is used to build large scale data acquisition systems.
How Data is acquired
Transducers convert measurable physical phenomenon into electric signals.
Examples of tranducers include microphones for sound and photocells for
light.
Signals may be digital or analog depending on the tranducer used.
Signal conditioning may be necessary if the signal from the tranducer is
not suitable for the DAQ hardware to be used. The signal may be amplified or
deamplified, or may require filtering.
DAQ hardware is what usually interfaces between a the signal and a PC. It
could be in the form of modules that can be connected to the computer's
ports (parallel, serial, USB, etc...) or cards connected to slots (PCI, ISA) in the
mother board
Driver Software that usually comes with the DAQ hardware or from other
vendors, allows the operating system to recognize the DAQ hardware and
programs to access the signals being read by the DAQ hardware.
Transducers
Data acquisition begins with the physical phenomenon to be measured. This
physical phenomenon could be the temperature of a room, the intensity of a
light source, the pressure inside a chamber, the force applied to an object, or
many other things.
system

can

An effective DAQ
measure

all

of

these

different

phenomena.

A transducer is a device that converts a physical phenomenon into a

measurable electrical signal, such as voltage or current. The ability of a DAQ
system to measure different phenomena depends on the transducers to
convert the physical phenomena into signals measurable by the DAQ
hardware. Transducers are synonymous with sensors in DAQ systems. There
are specific transducers for many different applications, such as measuring
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12B702 -Bioprocess Engineering

temperature, pressure, or fluid flow. Figure 2 shows a short list of some

common transducers and the phenomena they can measure.

Figure 2. Phenomena and Existing Transducers

Data Acquisition Systems
Data acquisition systems, as the name implies, are products and/or
processes used to collect information to document or analyze some
phenomenon. In the simplest form, a technician logging the temperature of
an oven on a piece of paper is performing data acquisition. As technology
has progressed, this type of process has been simplified and made more
accurate, versatile, and reliable through electronic equipment. Equipment
ranges from simple recorders to sophisticated computer systems. Data
acquisition products serve as a focal point in a system, tying together a wide
variety of products, such as sensors that indicate temperature, flow, level, or
pressure.
Searching for the Right Data Acquisition Software
Remember when companies had fully staffed departments dedicated to
developing test systems and programs for you? Those days are gone. To stay
competitive in today's job market, you need to put your own test system
together. Choosing the most appropriate data acquisition software for your
application is critical to accomplishing this task.

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12B702 -Bioprocess Engineering

The first steps toward finding a data acquisition software package that fits
your application are understanding your current and future application
requirements and determining the types of tasks you would like to be able to
perform. These usually include one or more of the following:

Verifying signal connections.

Logging and streaming data to disk.

Monitoring real-time data.

Controlling a test or process.

Analyzing the acquired data.

Generating reports in a variety of graphical formats.

Designing turnkey applications that can be used by lesser-skilled

operators.

10. LABVIEW FOR MEASUREMENT AND DATA ANALYSIS

Thousands of engineers and scientists rely on LabVIEW for a variety of
applications: test and measurement, process control and automation,
monitoring and simulation. LabVIEW is the tool of choice due to its
unparalleled
capabilities,

connectivity
natural

to

instruments,

dataflow-based

powerful

graphical

data

programming

acquisition
interface,

scalability, and overall function completeness. One need that persists

regardless of the area of expertise is the fact that users must manipulate
data and measurements, and make decisions based on it.
Users generally start their work by acquiring data into an application or
program, because their tasks typically require interaction with physical
processes. In order to extract valuable information from that data, make
decisions on the process, and obtain results, the data needs to be
manipulated and analyzed. Unfortunately, combining analysis with data
acquisition and data presentation is not always a straightforward process.
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12B702 -Bioprocess Engineering

Application software packages typically address one component of the

application, but seldom address all aspects and needs to get to a complete
solution. LabVIEW was designed to address the requirements for a start-tofinish, fully-integrated solution, so that customers can seamlessly integrate
all phases of their application in a single environment.

Figure 1. LabVIEW Virtual Instrument Block Diagram

While there are many tools that independently address each of the
requirements, only LabVIEW combines all of them with the power of
graphical programming and state-of-the-art data acquisition hardware, using
the power of your PC. It is the combination of data acquisition, data analysis,
and presentation of results, that truly maximizes the power of Virtual
Instrumentation. A virtual instrument consists of an industry-standard
computer or workstation equipped with powerful application software, costeffective hardware such as plug-in boards, and driver software, which
together perform the functions of traditional instruments. This is why
applications and programs built with LabVIEW are referred to as VIs (virtual
instruments).
Computer based Data Acquisition Overview:
This overview will help you to understand the basics of data acquisition on a
computer.
Traditionally, measurements are done on stand alone instruments of various
types-oscilloscopes, multi meters, counters etc. However, the need to record
39

12B702 -Bioprocess Engineering

the measurements and process the collected data for visualization has
become increasingly important. There are several ways in which the data can
be exchanged between instruments and a computer. Many instruments have
a serial port which can exchange data to and from a computer or another
instrument. Use of GPIB interface board (General purpose Instrumentation
Bus) allows instruments to transfer data in a parallel format and gives each
instrument an identity among a network of instruments. All HP instruments in
the EE Undergraduate Laboratories and PCs are equipped with GPIB
interfaces. Another way to measure signals and transfer the data into a
computer is by using a Data Acquisition board. A typical commercial DAQ
card contains ADC and DAC that allows input and output of analog and digital
signals in addition to digital input/output channels. In the following overview
we will attempt to explain various aspects of a DAQ card and DAQ system
used in the EE Undergraduate Lab.
Sampling.
The data is acquired by an ADC using a process called sampling. Sampling a
analog signal involves taking a sample of the signal at discrete times. This
rate at which the signal is sampled is known as sampling frequency. The
process of sampling generates values of signal at time interval as shown in
following figure.

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12B702 -Bioprocess Engineering

The sampling frequency determines the quality of the analog signal that is
converted. Higher sampling frequency achieves better conversion of the
analog signals. The minimum sampling frequency required to represent the
signal should at least be twice the maximum frequency of the analog signal
under test (this is called the Nyquist rate). In the following figure an example
of sampling is shown. If the sampling frequency is equal or less then twice
the frequency of the input signal, a signal of lower frequency is generated
from such a process (this is called aliasing).

41