INTRODUCTION:

Optimal cell growth and metabolite production, as well as biocatalyst activity, is

achieved only through a narrow range of environmental conditions. Accordingly, inorder to
develop, optimize and assure the most efficient biological reactor operation, it is crucial that
the state of the cell or enzyme environment be monitored and controlled. Monitoring a
fermentation process may need basic knowledge of the bioprocesses, and the running
conditions should be recorded.

There are many types of bioreactor used in bioprocesses such as the continuous stirred
tank reactor (CSTR), plug flow column, bubble column reactor, packed bed bioreactor,
trickle bed bioreactor, tower fermenter, air lift bioreactor, and immobilized bioreactor. The
most common form is the CSTR which operates under aerobic condition and is the
concentration is uniform everywhere. A standard stirred-tank bioreactor is normally supplied
with a means of measuring temperature, agitator speed, pH, the incoming air-flow rate and
dissolved oxygen concentration. This basic instrumentation can be complemented with
several other sensors capable of determining the pressure drop, dissolved CO2 concentration,
biomass, off-gas analysis, foam control, power and torque, and liquid flow feeding. Most of
these measurements are followed continuously, on-line.

BIOPROCESSES INSTRUMENTATION FOR MEASUREMENTS

 Temperature
 Gas Flowrate
 Liquid Flowrate
 Off-Gas Analysis
 pH
 Foam
 Dissolved Oxygen
 Fig. 1. Instrumentation control for CSTR

The figure above shows the usual instrumentation for a bioreactor with an agitating
motor and all control units. Measurements and control of the fermentation conditions are very
important for bioprocess control as they provide knowledge and hence a better understanding
of the operation. The recorded data can be used to improve the process. Controlling operating
conditions is important for maintaining viable cells, and it makes the interpretation of
fermentation data easier.

Bioreactor Controlling Probes

In bioprocess plant instrumentation and process control, variables are very important.
Reading and processing the information about the biosystem and monitoring the cells are the
major aims of the process instrumentation. Controlling pH, measuring the dissolved oxygen
in the fermentation broth and controlling foam are all considered major parameters and
necessary biological information for large-scale operations. The main objective is operate a
bioreactor without any problems. To do so we need to be familiar with all the controlling
facilities and process instrumentation. Application of biosensors in the bioreactors is very
common, so it is good to know how the controlling unit operates.
Characteristics of Bioreactor Sensors

The sensor in a bioreactor provides knowledge and information on the state of the
process and also supplies suitable operational data for the process variables. Some of the
physical and chemical effects on the bioreactor have to be translated to electrical signals,
which can be amplified and then displayed on a monitor or recorder and used as an input
signal for a controlling unit.

Temperature-measuring Devices

1.) THERMOCOUPLES
 A thermocouple is a sensor used to measure temperature. It is the most widely
used temperature sensors since they are cheap, provide rapid response, and
may be used to measure both high and low temperatures. They are less
accurate (± 0.5 ºC to ± 2.2 ºC) than other sensors however, and must be
calibrated periodically.
 Thermocouples consist of two wires of different metals that are welded at one
end. Connecting the free terminals to a transducer closes the electric circuit. If
a temperature difference exists between the free terminals and the welded end,
an electric current will flow through the circuit.

2.) RESISTANCE TEMPERATURE DETECTOR (RTD)

 Resistance thermometers also known as Resistance Temperature Detector
(RTD) is a popular device as it provides more precise temperature
measurement (± 0.1 ºC) than thermocouples, and does not need periodic
calibration.
 These devices are more expensive and are suitable over a narrower
temperature range than thermocouples, though. Its operating principle is based
on the change in electric resistance metals experience with temperature.
Platinum RTDs (Pt 100) are widely used.

Gas-Flow Rate
Gas flow rate is measured by a floating meter or a mass flow meter that can measure flow
rate independent of pressure and temperature effects (Katoh et. al., 2015). In view of the
measurement by flow meters, the flow rate is controlled manually or automatically by
controlling a flow control valve. Gas-flow rate is utilized to calculate the oxygen uptake rate,
the RQ (respiratory quotient) and the CO 2 evolution rate. Biogas-generation rate is measure
by a volumetric flow meter in anaerobic digestion (methane fermentation) to observe
methane generation.
Gas-Flow Measurement Device

1. Differential Pressure Producers

Pérez-Correa and Agosin (n.d.)

believed that differential pressure
producers are frequently found in industrial
applications given their reliability and ease
of use. Their accuracy ranges from ± 0.8
percent to ± 5 percent. The basic principle
is that any obstruction (concentric orifice,
Venturi, Pitot tube, etc.) in the fluid stream
generates a pressure loss that is related to
the flowrate of the stream.The main
limitation of such devices is that the cost in
energy from the loss of pressure caused can
be considerable. Further, a differential
pressure transducer is needed to be able to Figure 2. An example of a differential pressure
include the measurement in a digital producer.
control system.

2. Turbine Anemometer
In a turbine anemometer, a rotating device is set in the way of the fluid, where its
rotational speed is relative to the fluid velocity and gives exact flow measurements over wide
ranges.

Figure 3. An example of a turbine anemometer

3. Thermal Anemometer
Thermal anemometers can likewise be utilized
quantify air speed effectively, in which a thin wire
immersed in the fluid is heated by an electric current.
The velocity of the fluid is related to the heat
dissipated from the wire.If anemometers are used for

Figure 4. Typical example of a thermal

anemometer
flow rate measurements, full calibration is required since the velocity given relies upon the
anemometer’s transversal position within the duct.

Liquid-Flow Rate
According to Katohet. al. (2015),liquid flow rate is measured when a medium is fed into a
bioreactor in continuous and fed-batch operation. The flow rate of cooling water is also
monitored in industrial bioprocessing plants.

Liquid-Flow Measurement Device

1. Turbine Meter

Turbine meters are small electric turbines that

generate an electromotive force (emf), which is
proportional to the mean fluid velocity. These
instruments are reliable, precise and can be used at
different measurement ranges, although they are
expensive and may only be used with clean liquids. Figure 5. Typical example of a turbine meter

2. Magnetic Sensors

Magnetic sensors are suited for dirty liquids since they are non-invasive, but are very
expensive. In a magnetic flowmeter, a magnetic field is generated and channelled into the
liquid flowing through the pipe. Following Faraday’s Law, flow of a conductive liquid
through the magnetic field will cause a voltage signal to be sensed by electrodes located on
the flow tube walls. When the fluid moves faster, more voltage is generated. Faraday’s Law
states that the voltage generated is proportional to the movement of the flowing liquid. The
electronic transmitter processes the voltage signal to determine liquid flow.

Figure 6. An example of magnetic sensor

 3. Doppler Sensors

Doppler sensors, which are less expensive than

magnetic sensors, can also be used to measure flow
rates of dirty liquids. At no flow conditions, the
frequencies of an ultrasonic wave transmitted into a
pipe and its reflections from the fluid are the same.
Under flowing conditions, the frequency of the
reflected wave is different due to the Doppler Effect.
When the fluid moves faster, the frequency shift
increases linearly. The transmitter processes signals Figure 7. An example of magnetic sensor
from the transmitted wave and its reflections to
determine the flow rate.

4. Radioactive sensors

Radioactive sensors are based on the tracking of radioactive trace species. These are very
precise instruments and applicable to any kind of fluid, but they are very expensive and
dealing with radioactive species is difficult.

Off Gas Analysis

CO2 evolution from a bioreactor is closely related to the physiological state and the
activity of microorganisms in a bioreactor because CO 2 evolves as a result of catabolism and
respiration by microorganisms or cells. Therefore, it is helpful to measure the content of
CO2/O2 in the exhaust gas in order to understand the physiological climate of a bioreactor.

Off Gas Analysis Instruments

1. Gas Chromatography

The sampling air must be dried and the flowrate regulated before entering the instrument.
The main advantages of GC are that many compounds can be monitored with the same
instrument over a wide range of values.In gas chromatography, the components of a sample
are dissolved in a solvent and vaporized in order to separate the analytes by distributing the
sample between two phases: a stationary phase and a mobile phase. The mobile phase is a
chemically inert gas that serves to carry the molecules of the analyte through the heated
column. Gas chromatography is one of the sole forms of chromatography that does not utilize
the mobile phase for interacting with the analyte. The stationary phase is either a solid
adsorbant, termed gas-solid chromatography (GSC), or a liquid on an inert support, termed
gas-liquid chromatography (GLC).
 Figure 8. Schematic Diagram of gas chromatograph
2. Gas Analysers

Gas analyzers, on the other hand, are more precise and provide a faster response (few
seconds) than gas chromatography. Some of such instruments are:

a. Paramagnetic analyzers – available for CO2 and O2, are very precise, do not require
periodic calibration, present low interference to other gases and have long lifetimes,
but they are expensive.
b. Infrared instruments – available for CO2 only, are precise and have a long lifetime,
though they are expensive and need occasional calibration.
c. Electrochemical analyzers – only for O2, are low cost instruments that provide good
precision, however a fuel cell must be changed periodically (between 6 months and 2
years).

pH

Control of pH is usually a major factor as many fermentations yield products that can
alter the pH of the growth media. The pH has a major effect on cell growth and product
formation by influencing the breakdown of substrates and transport of both substrate and
product through the cell wall. In fine chemicals, organic acids, amino acids and antibiotic
fermentations, even a small change in the pH can cause a large fall in the productivity. Also,
in animal-cell fermentations, the pH is strictly affected by the cell density. Fermentation
media often contain buffering salts, usually phosphates, but their capacity to control pH can
be exceeded and addition of acid or alkali may be required. The pH can be maintained at the
desired value by their automatic addition in response to changes recorded by the pH
electrode. pH Electrode isa sensing element that consists of a pair of electrodes in contact
with the liquid sample, and a temperature compensation device. One of the electrodes is used
as a reference, while the other is sensitive to the pH of the sample.
 Figure 9. Instrumentation for a pH contro system in a bioreactor
Retrieved from: Najafpour, G. (2007). Biochemical Engineering and Biotechnology. Amsterdam, The
Netherlands: Elsevier

Foam

In a gas and liquid system, when gas is introduced into a culture medium, bubbles
areformed. The bubbles rise rapidly through the medium and dispersion of the bubbles
occursat surface, forming froth. The froth collapses by coalescence, but in most cases
thefermentation broth is viscous so this coalescence may be reduced to form stable froth. Any
compounds in the broth, such as proteins, that reduce the surface tension may influence foam
formation. The appearance of foam is a very undesirable phenomenon, since, in the course of
its appearance, there is a risk to lose an essential part of the fermentation broth. During
fermentation, foaming may occur suddenly. Some foams are easy to destroy and can be
removed by foam breaker; others are quite stable and are relatively hard to remove from the
top of the bioreactor. The suppression of foam is usually accomplished by mechanical
agitation such as a foam breaker. The mechanical devices operate on the centre of the shaft.
They are generally blades or disks that are mounted on the same agitator shaft. Chemical
antifoams are used to regulate the foam and prevent any foaming on the surface of broth. The
chemical anti-foams are expensive and minimal amounts must be used. Use of chemical anti-
foam may complicate the microbial fermentation process, and some may act as an inhibitor.
Therefore they have to be regulated to eliminate any side effect on the bioprocess.

There are two main types of foam detectors that are being used in bioprocess; they
work by detecting either changes in electrical capacitance or changes in electrical resistance.

The capacitance foam detector is made of two electrodes, which are installed in the
bioreactor. They measure the capacitance of the air space above the normal working liquid
level in the bioreactor. If foaming occurs, the air space capacitance is reduced. Detection is
by a change in the magnitude of a small alternating electrical current, which is applied
between the two electrodes. The change in electrical current is converted to an output signal
from the detectors; the change in current is directly proportional to the amount of foam
formed.

The foam detector based on the resistance method acts on the conductivity of the
probe. The length of the probe is coated with some electrically insulating material, leaving
the tip of the probe exposed to the media. As the foam builds up, it contacts the tip of the
probe, thus completing an electrical circuit and producing an output signal. In the
fermentation medium, as the tip of the probe contacts the generated foam, an electrical circuit
is generated, and an output signal is produced for foam detection.

Figure 10. Foam sensing and control

Retrieved from: http://portal.unimap.edu.

The use of a chemical agent as an anti-foam is affected by an on–off algorithm with

variable dosing time and time delay. If the presence of foam is detected, then the controller
first activates a delay timer. This type of foam controller works with some delay and variable
dosing time. If at the end of the delay period the foam is still present, then the dosing pump is
activated and chemical agent is added to the bioreactor. If the foam is still detected at the end
of this period, the combined system of delay and dosing is reactivated. With this method of
controller, addition of any unnecessary anti-foam is prevented.

Dissolved Oxygen

Dissolved oxygen refers to the level of free, non-compound oxygen present in water
or other liquids. The dissolved oxygen content of the fermentation broth is also an important
fermentation parameter, affecting cell growth and product formation. The rate of oxygen
supply to the cell is often limited because the solubility of oxygen in fermentation is low.
Unfortunately, measurement and control of dissolved oxygen under bioreactor conditions is a
challenging problem. The low solubility of oxygen makes the measurements very difficult.
There are several methods to determine the concentration of dissolved oxygen. Most DO
probes use a membrane to separate the point of measurement from the broth. Three main
methods are used: (1) the tubing method; (2) use of mass spectrometer probes; and (3)
electrochemical detectors, which is also the common method of measuring dissolved oxygen
used in bioreactors.
 Two types of detector are commercially available: galvanic and polarographic
detectors. Both use membranes to separate electrochemical cell components from the broth.
The membrane must be permeable only to oxygen and not to any other chemicals, which
might interfere with the measurement. Oxygen diffuses from the broth, across the permeable
membrane to the electrochemical cell of the detector, where it is reduced at the cathode to
produce a measurable current or voltage, which is proportional to the rate of arrival of oxygen
at the cathode. It is important to note that the measurement rate of oxygen arrival at the
cathode depends on the rate of arrival at the outer membrane surface, the rate of transfer
across the membrane, and the rate of transport from the inner surface of the membrane to the
cathode. The rate of arrival at the cathode is proportional to the rate of diffusion of oxygen
across the membrane. The rate of diffusion is also proportional to the overall concentration
driving force for oxygen mass transfer. We assume the oxygen concentration at the inner
surface of the membrane is efficiently reduced to zero. The rate of diffusion is thus
proportional to the oxygen concentration in the liquid only. The electrical signal produced by
the probe is directly proportional to the dissolved oxygen concentration of the liquid. The
probe has to be calibrated for accurate measurements.

In the galvanic detector, the electrochemical detector consists of a noble metal like
silver (Ag) or platinum (Pt), and a base metal such as lead (Pb) or tin (Sn), which acts as
anode. The well-defined galvanic detector is immersed in the electrolyte solution. Various
electrolyte solutions can be used, but commonly they may be a buffered lead acetate, sodium
acetate and acetic acid mixture. The chemical reaction in the cathode with electrons generated
in the anode may generate a measurable electrical voltage, which is a detectable signal for
measurements of DO. The lead is the anode in the electrolyte solution, which is oxidized. The
series of chemical reactions occurring in the cathode and anode is:

Retrieved from: Najafpour, G. (2007). Biochemical Engineering and Biotechnology. Amsterdam, The
Netherlands: Elsevier

Polarographic electrodes are different from the galvanic type. In this type of electrode
the external negative base voltage is applied between the cathode (Au or Pt) and the anode
(Ag/ AgCl) so that oxygen is reduced at the cathode according to the sequential reactions
stated below. The following reaction takes place at cathode and anode respectively:

Retrieved from: Najafpour, G. (2007). Biochemical Engineering and

Biotechnology. Amsterdam, The Netherlands: Elsevier
An electrical output for a polarographic detector is produced according to the reactions
above. Again, various electrolytes can be used for this type of detector, but they are usually
based on KCl or AgCl with additional high molecular mass compounds, which are added to
prevent the loss of electrolyte during sterilisation.

Figure 11. Example of Galvanic and Polarographic detectors

Retrieved from: http://portal.unimap.edu

The difference between the polarographic and the galvanic detector is that
polarographic detectors need to warm up to polarize the electrodes before calibrating,
otherwise wrong readings will occur if measurements are made when the amount of time
required has not been attained while galvanic detectors does not need any warm up time.