1. Huzairy Hassan School of Bioprocess Engineering UniMAP

2. BIOREACTOR INSTRUMENTATION & CONTROL

3. Controlled parameters including pH, temperature, pressure, dissolved oxygen, etc in almost all bioreactors are necessary to maintain the optimal conditions for product formation in the complex environment of bioreactors. Some probes (e.g., pH and dissolved oxygen) enter the reactor through the penetrations in the fermenter shell. - Increase the probability of contamination. - Thus, the probes themselves must also be sterilizable, preferably with steam.

5. CategorySensorPossible control function PhysicalTemperature Pressure Agitator shaft power Rpm Foam Weight Flow rate Heat / cool Foam control Change flow rate ChemicalpH Redox Oxygen Exit-gas analysis Medium analysis Acid or alkali addition, carbon source feed rate Additives to change redox potential Change feed rate Change in medium composition Table 2: Process sensors and their possible control functions

6. Figure 1 shows usual instrumentation for a bioreactor (CSTR) with an agitating motor and all control units. The vessel is jacketed for cooling and heating, with a separate side unit of a temperature-controlled bath. Steam is used for sterilization and elimination of contaminants.

7. Figure 1: instrumentation control for continuous CSTR bioreactor.

8. The typical examples or instrumentation control in the bioreactor: 1) Flow rates of gases (inlet air and the exhaust gas) - flow meter for measuring flow rates, such as rotameter, which provides a visual readout or is fitted with a transducer to give an electrical output. 2) Thermal mass flow meters - gas flows through a heated section of tubing and the temperature differences across this heated section are directly related to the mass flow rate.

9. 3) Flow rate of liquid - The use of normal rotameter may cause some error, so a level sensor is used. - As the liquid level reaches the probe, the conductivity of the media surrounding the probe changes, so monitoring is based on the conductance of the liquid level. - Such capacitance probes or conductance probes are used to detect foam on the surface of bioreactor.

10. - Some of the physical and chemical effects on the bioreactor have to be translated to electrical signals, which can be amplified and the displayed on a monitor or recorder and used as an input signal for a controlling unit. - The response of most of the process follows a signoidal S-shaped curve. - The dynamics given by the instrument response signal comprise several processes taking place in series. - For example, a dynamic aeration experiment: 1) The transfer of oxygen from the air into the liquid causes reduction in rate of mass transfer due to the reduction in concentration gradient as the existing driving forces.

11. 2) The rate of change of the O 2 concentration in the gas phase is determined by the magnitude of the time constant, which depends on the gas volume and the mass transfer coefficient, K L a. - The time-varying liquid-phase concentration is registered by the dissolved O 2 electrode and is transmitted as an output signal. -Again, the signal is affected by the electrode time constant.  thus, the process dynamics are determined by the combination of the time constant for gas phase, liquid phase and the electrode dynamics.

12. Figure 2: Computer simulation of a dynamic aeration experiment with a slow DO electrode. - The y-axis shows the response fraction with respect to time. -The gas phase response is typically first-order, and the liquid phase shows some lag or delay on the signal. - The electrode response is much more delayed for a slow-acting electrode.

13. - Most fermenters operate around 30 – 36 ºC, but certain fermentation may require control of T in a range of 0.5 ºC. - To maintain bioreactor T within the limited range, the system may require regulation of heating and cooling by the control system. -Heat is generated in the fermenter by dissipation of power, resulting in an agitated system; heat is also generated by the exothermic biochemical reactions, at which the fermentation vessel requires cooling.

14. Figure 3: A fermenter with a temperature-controlled heating jacket

15. Methods of Measurement 1) Mercury-in-Glass Thermometers - may be used directly in small bench fermenters but its fragility restricts its use. - in larger fermenter, it’s necessary to insert it into a thermometer pocket in the vessel, which introduces a time lag in recording the vessel temperature. - can be used solely for indication, not for automatic control or recording.

16. 2) Bimetallic Thermometers - consists of a bimetallic helical coil surrounded by a protecting tube or well. - the coil winds or unwinds with changes in T and causes movement of a fixed pointer. - a pen can be fitted to the pointer so that the T changes can be monitored on a chart. - Advantage: less subject to breakage - Disadvantage: cost slightly more, less accurate, & limited to local indication.

17. 3) Pressure Bulb Thermometers - is a pressure gauge connected by small-bore tubing, which may be up to 60m in length, to the detecting bulb (12 x 125 mm). - the whole system is gastight and filled with an appropriate gas or liquid under pressure (2800 - 8000 kN m -2 ). - the movement of the free end of the receiving element can be used to operate a pen on a chart recorder or an electrical / pneumatic control.

18. 4) Thermocouples - a current can flow through a circuit consisting of wires of 2 dissimilar metals which had the junction of the wires maintained at different temperature. - the current produced can be measured on a calibrated instrument or recorder and is a measure of point T at a joint. - therefore, by holding the T constant at all junctions, except one within a given circuit, it is possible to measure T as a function of the hot-junction T with reference to cold-junction T. - cheap and simple to use but have low resolution due to cold-junction requirement.

19. 5) Thermistors - Semiconductors, which exhibit a change in electrical conductivity with T. - main characteristic: large change in resistance with a small T change. - T reading is obtained with a Wheatstone bridge or simpler or more complex circuit depending on the application. - relatively cheap, proved to be stable, give reproducible readings, and can be sited remotely from the read-out point. - Very sensitive and inexpensive, give highly nonlinear output. - Modern transistorised, integrated circuits combines the features, often display more linear output.

20. - To avoid contamination, the thermometers are usually fitted into thin-walled stainless-steel pockets, which project into the bioreactor. - The pockets are filled with a heat-conducting liquid to provide good contact and to speed the instrument response. - The changes in the electrical resistance of the electrical wires to the thermometer can be quite large, with their resistance being affected by changes in the ambient T. - These effect can be eliminated by using separate wires to supply and sense of current. - Platinum resistance thermometers (as standard): have advantage of high accuracy of resistance detectors, high stability and that the linear output signal can be obtained in normal T ranges.

21. - The high T steam for sterilization may require separate instrumentation within a T range of 50 – 150 ºC. -The energy balance for the bioreactor (batch wise): where M is the total mass of the reactor for batch system is constant in kg, C p is the specific heat in kJ.kg -1.K -1, T is the temperature of the fermentation in K and t is time in s. The term µX is the rate of cell growth in kg.m -3.s -1,V is the working volume of the bioreactor in m 3, and –ΔH x is the exothermic heat generated inside the fermenter in kJ.kg -1 cell. Under steady-state conditions of controlled T, dT/dt = 0, the rate of heat accumulation is zero and the rate of heat production, which is related to the rate of cell growth is equal to the heat transfer by the jacketed system. The rate of heat transfer is the mean T gradient, [T - T j ], multiplied by overall heat transfer coefficient (U) in kJ.m -2.s -1.K -1. T j is the temperature of the coolant in the jacket in Kelvin (K).

22. - The rate of O2 supply to the cell is often limited because the solubility of O2 in fermentation is low, thus make the measurement and control of dissolved O2 (DO) is difficult. - Most DO probes use a membrane to separate the point of measurement from the broth; all probes require calibration before use.

23. Methods of measurement include: 1) Tubing methods - an inert gas flows through a coil of permeable silicon rubber tubing, which is immersed in the bioreactor. - O2 diffuses from the broth, through the wall of the tubing and into the flow of inert gas passing through the tube. - the concentration gradient exists because of the diffusion of O2 into the inert gas. - then, the concentration of O2 gas in the inert gas is measured at the outlet of the coil by an oxygen gas analyzer. - has a relatively slow rate of response, of the order of several minutes. - Advantage: simple and in situ sterilization is easily carried out.

24. 2) DO Electrodes / Electrochemical Detectors - Use membrane to separate electrochemical cell components from the broth. - The membrane must be permeable only to O2 and not to any other chemicals. Sequence step:  O2 diffuses from the broth across the membrane  to the electrochemical cell of detector, where it is reduced at the cathode to produce a measurable current or voltage, which is proportional to the rate of arrival of O2 at the cathode.  The measurement of the rate of O2 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 O2 across membrane. The rate of diffusion is also proportional to the overall concentration driving force for O2 mass transfer.  We assume the O2 concentration at the inner surface of membrane is efficiently reduced to zero. The rate of diffusion is thus proportional to the O2 concentration in the liquid only.   the electrical signal produced by the probe is directly proportional to the dissolved O2 concentration of the liquid.

25. a) Galvanic Electrodes - In small fermenters (1 dm 3 ) - have lead anode, silver cathode and employ/immerse in KOH, Chloride, bicarbonate, or acetate as an electrolyte. - the sensing tip of the electrode is a Teflon, polyethylene or polystyrene membrane which allows passage of the gas phase so that an equilibrium is established between the gas phases inside and outside the electrode. Cathode:O 2 + 2H 2 O + 4e - 4OH - Anode:Pb Pb 2+ + 2e - Overall:O 2 + 2Pb + 2H 2 O 2Pb(OH)

26. - because of the slow movement of O2 across the membrane, this type of electrode has a slow response of the order of 60 s to achieve a 90% reading of the true value (or 50 s for 98 % response). - suitable for monitoring very slow changes in O2 concentration. - Chosen because of the compact size and relatively low cost. - Disadvantages: - very sensitive to temperature fluctuations, and should be compensated for T using a thermistor circuit. - have limited life because of corrosion of the anode i.e. lead anode in the electrolyte solution is oxidized.

27. b) Polarographic Electrodes - Commonly used in pilot and production fermenters, needing instrument ports of 19 or 25 mm diameter. - Have silver anodes which are negatively polarized with respect to reference cathodes of platinum or gold, using aqueous KCl as the electrolyte. - Different from galvanic where the external negative base voltage is applied between the cathode and anode so that O2 is reduced at the cathode. Cathode:O 2 + 2H 2 O + 2e - H 2 O 2 + 2OH - H 2 O 2 + 2e - 2OH - Anode:Ag + Cl - AgCl + e - Overall: 4Ag + O 2 + 2H 2 O + 4Cl - 4AgCl + 4OH -

28. - Response times of 0.05 to 15 seconds to achieve a 95% reading. - Very precise may be both pressure and temperature compensated. - Although may initially cost 600% more than galvanic, the maintenance costs are considerably lower as only the membrane should need replacing.

29. - The pH has 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 batch culture, the pH of an actively growing culture will not remain constant for very long, where it changes with the metabolic products of microorganisms. - Rapid changes in pH can be reduced by the careful design of media, particularly in the choice of carbon and nitrogen sources, and also in the incorporation of buffers or by batch feeding. or by addition of appropriate quantities of ammonia or NaOH if too acidic, or sulfuric acid if the change is to an alkaline condition.

31. - Now, pH measurement is carried out using a combined glass reference electrode (can withstand repeated sterilization at T 121 ºC and pressure 138 kN/m 2 ). - The electrodes may be silver or silver chloride with KCl as an electrolyte. - Occasionally, calomel or mercury electrodes are used. - The electrode is connected via leads to a pH meter/controller. - The electrodes should be able to withstand autoclaving conditions or capable of being withdrawn aseptically, once the fermentation units are to be sterilized.

32. - In the case of the On/Off controller, the controller is set to a predetermined pH value. - When a signal actuates a relay, a pinch valve is opened or a pump started, and acid or alkali is pumped into the fermenter for a short time which is governed by a process timer (0 to 5 seconds) - The addition cycle is followed by a mixing cycle which is governed by another process timer (0 to 60 s) during which time no further acid or alkali can be added. - At the end of the mixing cycle, another pH reading will indicate whether or not there has been adequate correction of the pH drift. – In small volumes the likelihood of overshoot is minimal.

33. Pressure Measurement & Control - Pressure will be raised during steam-sterilization cycle. - The correct pressure in different components should be maintained by regulatory valves controlled by associated pressure gauges. - Safety valves should be incorporated at various suitable places in all vessels and pipe layouts which are likely to be operated under pressure. - The valves should be set to release the pressure as soon as it increases markedly above a specified working pressure.

35. Carbon Dioxide Electrodes - The measurement of dissolved CO 2 is possible with an electrode since a pH or voltage change can be detected as the gas goes into solution. - For example; an electrode consisting of a combined pH electrode with a bicarbonate buffer (pH 5.0) surrounding the bulb and ceramic plug, with the solution being retained by a polytetrafluoroethylene ( PTFE) membrane held by an O-ring. - But, this electrode is not steam sterilizable.

36. - Modification: immobilize a thin layer of aqueous bicarbonate over the glass sensing membrane of the pH electrode, which in turn is covered by a steel reinforced silicone membrane and an O- ring. - It is possible to calibrate the electrode in situ using gas mixtures of known CO 2 partial pressure and check it with buffer solution. This can be steam sterilized.

37. - In a gas and liquid system, when gas is introduced into a culture medium, bubbles are formed. - The bubbles rise rapidly through the medium and dispersion of the bubbles occurs at surface, forming froth. - The froth collapses by coalescence, but in most cases, the fermentation 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 stability of preventing bubbles coalescing depends on the film elasticity, which is increased by the presence of peptides, proteins and soaps. - In contrast, the presence of alcohols and fatty acids will make the foam unstable….. Why??

39. The problems due to foaming include; loss of broth, clogging of the exhaust gas system and possible contamination, that is due to wetting of the gas filters. How to prevent? 1) Some foams are easy to destroy and can be removed by foam breaker. - Others are quite stable and hard to remove from the top of bioreactor  mechanical agitation (foam breaker) - mechanical devices operate at the center of the shaft, which are generally blades or disks, that are mounted on the same agitator shaft. - advantage: does not require expensive chemical to be added to the fermentation broth.

40. 2) Chemical Anti-foam - expensive and minimal amount should be used. - may complicate the microbial fermentation process, and some may act as an inhibitor. - therefore, need to be regulated to eliminate any side effects on the process. - Usually based on silicon and act by reducing the interfacial tension of the broth. - the addition of anti-foam may require the detection of foam. 3) Ultrasonic waves – additional means of destroying foam.

41. Foam detectors:

42. Evaporation Control - Aerobic cultures are continuously sparged with air, however, most components of air are inert and leave directly through the exhaust gas line. - If air entering the fermenter is dry, water is continually stripped from the medium and leaves the system as vapor. - Evaporation problem is more pronounced in air-driven reactors because the gas flow rates required for good mixing and mass transfer are generally higher than in stirred bioreactors.

43. Example of problem: Acetobacter sp. are used to produce acetic acid from ethanol in a highly aerobic process requiring large quantity of air. For stirred tank reactors which operate at air flow rates between 0.5 and 1.0 vvm (volume of gas per volume of liquid per minute) that from a starting alcohol concentration of 5%, 30 – 50% of the substrate is lost within 48 hours due to evaporation !!. - How to overcome: 1) Air sparged into the fermenters may be pre-humidified by bubbling through columns of water outside the fermenter. - humid air has less capacity for evaporation than dry air. 2) Fermenters are equipped with water-cooled condensers to return to the broth any vapors carried by the exit gas.

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