Monitoring of Bioprocess
Monitoring of Bioprocess
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
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
of
on-line
monitoring
involve
the
automatic
removal
and
to
most
chemical
and
biochemical
reaction
procedures,
and
whose
chemistry
is
well
understood
and
whose
variables
which
characterize
and
govern
the
competing
compensating
for
backscatter.
Another
technique
used
to
10
The hydroxyl ion then travels to the anode where it completes the
electrochemical reaction process:
12
13
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.
14
15
16
for
over
fifty
years.
Utilizing
simple
feedback
principles,
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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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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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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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and
volatilization
occurs,
evidenced
by
discoloration
22
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:
voltage,
four-terminal,
phase-sensitive
detector
system.
nicotinamide
adenine
dinucleotide
(NADH)
and
reduced
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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.
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Turbidity
measurement
can
be
used
for
both
online
and
off-line
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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:
an
on-line
carrying
out
difficult
operations
of
separation,
28
(C=carrier;
P=pump;
S=point
of
sample
injection;
RC=reaction
coil;
D=detector; W=waste)
29
30
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
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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.
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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
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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):
Signals
Signal conditioning
DAQ hardware
systems
include
appropriate
sensors
that
convert
any
35
can
An effective DAQ
measure
all
of
these
different
phenomena.
37
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:
operators.
connectivity
natural
to
instruments,
dataflow-based
powerful
graphical
data
programming
acquisition
interface,
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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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).
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