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Fermentation under control

Off-gas analysis as a real-time indicator for vital process parameters
Fermentation under control

Biotechnological processes have to be as standardised as possible to guarantee optimum product yields. Online off-gas analysis is an integral part of this standardisation because vital process parameters – such as biomass development, substrate consumption and product formation – are monitored and calculated. Fast and direct intervention in the process at critical stages is also enabled by this method.

Karin Koller

Successful fermentations of microorganisms and cell cultures are possible only with highly complex measurement and control technology. Optimum culture conditions must be created, the pH be maintained and foam formation repressed. Since most fermentations are aerobic, the oxygen supply must be ensured at all times. Microorganisms can only utilise dissolved gases. Oxygen therefore has to be transferred from the gas bubble to the culture broth. The oxygen transfer rate (OTR) is dependent on the volumetric oxygen transfer coefficient (kLa) and the saturation concentration in the liquid phase. kLa is influenced by media viscosity and the amount of mixing. Surface active substances such as antifoam agents reduce kLa, while salts increase it by reducing the bubble size. At the same time, high salt concentrations lead to a lower oxygen solubility in the medium.
Owing to these interactions, the DO (dissolved oxygen) controller is not solely responsible for regulating the oxygen supply. Various control loops such as the agitator, pressure, gas flow, gas mixing station, media dosage and antifoam are also directly or indirectly involved. Oxygen undersupply of the cells immediately leads to oxygen limitation. Cell growth becomes linear instead of exponential, the product formation rate is significantly lowered and products of the anaerobic metabolism are formed. All this has to be avoided absolutely through optimum interaction of all relevant measurement systems with the correction elements involved.
Adequate oxygen and nutrient supply of the cells only leads to optimum cell growth if any metabolic toxins that have formed and carbon dioxide can be sufficiently removed from the cells. An accumulation of CO2 within the cells inhibits growth and leads to reduced product formation.
The highest priority for industrial fermentations is the product yield. High growth rates do not necessarily correspond to high product formation rates in every process. In many cases the cells are initially cultivated under optimum growth conditions. After a critical biomass has been formed, the cell metabolism is switched to obtain high product yields via secondary metabolism. To succeed with this technique, it is vital to determine the exact time of the change. The metabolic switch is induced by repressors or inducers, by limiting one or more media components, by altering the culture conditions or by supplying a different substrate. In addition, culture conditions are often altered to repress undesired by-products and facilitate downstream processing.
Offline versus online
The determination of the exact time for the start of production proper is often associated with time-consuming offline analysis. Relevant factors such as the concentration of media components or metabolites are not measurable with online probes. Offline analysis has the advantage of determining exactly the parameter needed to control the fermentation course. Nevertheless, this advantage is offset by several severe disadvantages, of which the contamination risk during sampling is only one. Since offline analysis is very laborious, a lot of working time must be invested and the results are only available after a significant time delay. This delay and the frequency of reasonable sampling create a risk of missing important developments in the fermentation. Online analysis can avoid this problem. One suitable method, or several methods combined, can provide precise information about the course of various relevant fermentation parameters once they have been calibrated and/or subjected to mathematical modelling.
Facets of off-gas analysis
Meaningfully applied and with suitable equipment, off-gas analysis is a very powerful tool for monitoring and controlling a process continuously. This method measures oxygen and carbon dioxide concentrations in the exhaust air of the reactor. If the amount of oxygen inserted into the fermenter is quantified, e.g. by means of gas flow control, off-gas analysis provides information about the oxygen uptake rate (OUR) of the cultivated cells, which is in turn an indicator for cell growth. The carbon dioxide production rate (CPR) is measured simultaneously in fermentations of microorganisms. In cell cultures the status of the bicarbonate buffer and the CO2 input from pH control must be considered when calculating the CPR.
The respiration quotient (RQ) can then be derived from this data. RQ is the ratio of formed CO2 and consumed O2. When an aerobic organism metabolises glucose, six molecules of CO2 are formed during respi-ration from every molecule of glucose with six molecules of O2. In this case RQ equals 1 and remains constant as long as the organism consumes glucose. Every change of RQ indicates significant changes in the fermentation course. The causes of this change can be various: the substrate is limiting or not utilised anymore, the oxygen supply is insufficient and anaerobic metabolism has started, or the organism is ready to consume the substrate for production in the secondary metabolism.
Depending on the type of fermentation and the manufactured product, measures can be taken to influence the fermentation course. The secondary substrate for product induction is added in the fermentation medium described above. Substrate is dosed in a fed-batch process. If a limiting cofactor is necessary for substrate consumption, it should be dosed. If the organism switches to anaerobic metabolism, additional steps must be taken to provide sufficient amounts of oxygen by means of a cascade function with an agitator, pressure or gas flow controllers.
Off-gas analysis is suitable not only for monitoring biomass, growth rate, substrate consumption and thus product formation, but also for checking DO probe functionality, upscaling experiments or documenting sterility tests.
Components for gas analysis
Bioengineering supplies a variety of modular components for gas analysis. Their combination to form a gas analyser is dependent on the size of the plant, the type of process and the culture parameters to be monitored. CO2 and O2 analysis are either united in one unit or conducted separately in two analysing devices. In both cases the CO2 concentration is determined by the infrared method, where infrared radiation emitted by the measurement device is weakened by the characteristic absorption spectrum of the gas and then detected.
In combination analysers, the oxygen concentration is measured chemo-electrically according to the Clark method by applying voltage between a silver anode and a platinum cathode. The current created by reduction of the diffusing oxygen is proportional to the partial pressure of the oxygen gas. In devices with separate oxygen analysis the oxygen concentration is determined paramagnetically. This method exploits the paramagnetic properties of oxygen molecules as a result of electronic movement and measures their strength in a magnetic field. The 8-channel gas processing unit simultaneously measures the exhaust air properties of up to eight fermenters. In smaller plants the 4-channel gas processing unit is used. Manual or automatic operating mode can be selected on all devices.
All measurement results are converted into standard electrical signals which are available for further processing. Data transfer is not confined to simple acquisition for process documentation, but can also be used to calculate factors correlating with the measured gas content. If the gas analyser is embedded in the control software of the plant, direct process intervention is pos-sible based on the generated and transmitted data. A step sequence program enables all available controllers to react to the process values of the gas analyser. A defined value must be exceeded as a condition of starting an action. Alternatively, Bioengineering’s control software calculates the curve progression of the process value and triggers an action as soon as the inflection point of the curve is reached. Depending on the process, these actions might be dosage of media components or correction agents, support of oxygen supply, control of continuous or fed-batch processes or harvesting of the culture broth.
The applications described above show how a seemingly unspectacular online measurement method, such as off-gas analysis in bioreactors, can become an effective tool for holistic process control when embedded in the plant’s measurement and control system and meaningfully correlated to critical process values. It is just as true for bioengineering as for any other process that every value determined in real time and interlinked with other parameters raises the process standardisation rate.
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