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.