November 15, 2008 (Vol. 28, No. 20)
Embedding Methodology in the Control System of a Plant Increases Its Utility
Biotech processes should be standardized as much as possible to produce optimum product yields. On-line off-gas analysis is an integral part of standardization since it allows vital process parameters—such as biomass development, substrate consumption, or product formation—to be monitored. This methodology also allows fast and direct intervention into critical process stages.
Successful fermentation of microorganisms and cell cultures is possible only with the implementation of highly complex measurement and control technology. Optimum culture conditions must be created, the pH has to be maintained, and foam formation must be repressed. Finally, oxygen supply is critical since most fermentations are aerobic.
Because microorganisms can only utilize dissolved gases, oxygen 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 like antifoam agents decrease kLa, while salts increase it by lowering the bubble size. At the same time, high salt concentrations lead to a lower oxygen solubility in the medium.
Dissolved Oxygen Control
As a result of these interactions, the dissolved oxygen (DO) controller is not solely responsible for control of the oxygen supply, the agitator, pressure, gas flow, gas mixing station, media dosage, and antifoam control loops have roles as well. Undersupply of oxygen to the cells leads to oxygen limitation, which results in linear instead of exponential cell growth, significantly lower product-formation rates, and formation of products related to anaerobic metabolism. Optimizing interactions between all of the measurement and control systems is therefore crucial.
Adequate oxygen and nutrient supply leads to optimum cell growth, but only if metabolic toxins and carbon dioxide are removed from the cells. Accumulation of CO2 within the cell inhibits growth and leads to reduced yield.
Product yield is the highest priority in industrial fermentation operations. High growth rates do not necessarily correspond to high product formation rates, however. In many cases, cells are initially cultivated under optimum growth conditions. After a critical biomass has been formed, cell metabolism is switched to produce high yields via secondary metabolism. In order for this technique to succeed, the optimal time for the metabolic switch must be determined.
This metabolic switch is induced by repressors or inducers, limitation of one or more media components, alteration of culture conditions, or by supply of a different substrate. Culture conditions are also often altered to repress undesired byproducts and to facilitate downstream processing.
Off-Line versus On-Line
Time-consuming off-line analysis is often used to determine when to start high-yield production. Off-line analysis is able to ascertain the exact parameters needed to control the fermentation course, including concentration of media components. Unfortunately, this advantage is compromised by several disadvantages, including the contamination risk during sampling, the laborious nature of the procedure, and the time delay.
On-line analysis avoids these problems. One method or the combination of several methods can provide precise information about relevant fermentation parameters. Off-gas analysis is an on-line tool that can be used to monitor and control a process continuously. It measures oxygen and carbon dioxide concentrations in the exhaust air of the reactor. If the amount of oxygen inserted into the fermentor is quantified via gas-flow control, off-gas analysis provides information about the oxygen uptake rate (OUR) of the cultivated cells. Simultaneously, the carbon dioxide production rate (CPR) can be measured for microorganisms. In cell cultures, however, the status of the bicarbonate buffer and the CO2 input from pH-control must be considered for the calculation of CPR.
From this data, the respiration quotient (RQ) can be calculated. RQ is the ratio of formed CO2 to consumed O2. When an aerobic organism metabolizes glucose, six molecules of O2 and six molecules of CO2 are formed during respiration.
In this case, RQ equals one and remains constant as long as the organism consumes glucose. Each change in RQ indicates significant changes in the fermentation course. There are many reasons for this change including the substrate is limiting or not utilized anymore, 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. A secondary substrate for product induction can be added in the fermentation. In a fed-batch process, a substrate can be dosed; if a limiting cofactor is necessary for substrate consumption, it must be dosed now. If the organism switches to anaerobic metabolism, additional steps must be taken to provide sufficient amounts of oxygen by cascade function with agitator, pressure, or gas-flow controllers.
Off-gas analysis often used to monitor biomass, growth rate, substrate consumption, and thus product formation, however, it can also be used for DO-probe functionality, upscaling experiments, or for documenting sterility tests.
Bioengineering supplies a variety of modular components for gas analysis. Which components are combined to create a gas analyzer 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 combined in one unit or conducted separately in two analyzing 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 detected.
In combination analyzers, oxygen concentration is measured chemoelectrically, according to the Clark method—with a silver anode and a platinum cathode to which voltage is applied. The current created by the reduction of diffusing oxygen is proportional to the partial pressure of the oxygen gas. In devices with separate oxygen analysis, the oxygen concentration is determined paramagnetically (e.i., the paramagnetic properties of oxygen molecules are exploited). These properties are created by electronic movement and measured in a magnetic field.
The eight-channel gas-processing unit simultaneously measures the exhaust air properties of up to eight fermentors. In smaller plants, a four-channel gas processing unit is used. Manual or automatic operation modes are options in all devices.
All measurement results are converted into standardized electrical signals, which are available for further processing. Data transfer is not confined to data acquisition for process documentation, but can also be used for calculation of factors correlating with the measured gas content. If the gas analyzer is embedded in the control software of the plant, direct process intervention based on the generated, and transmitted data is possible. A step sequence program enables correlation of all available controllers to the process values of the gas analyzer. In order for an action to be initiated, a defined value must be exceeded.
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 has been reached. Depending on the process, the action might be a dosage of media components or correction agents, support of oxygen supply, control of continuous or fed-batch processes, or harvest of the culture broth.
Off-gas analysis, a seemingly unspectacular on-line measurement method, can become an effective tool for process control if embedded in the measurement and control system of a plant and sensibly correlated to critical process values. For every process value determined in real-time and interlinked with other parameters, the standardization rate of the process is raised.
Karin Koller (engineering@bioengineering. ch) is scientific assistant at Bioengineering. Web: www.bioengineering.ch.