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It is a well known problem that the difference in ionic strength between media and the internal buffer of the pH sensor leads to a pH drifting effect during an experiment. This means the measured pH value differs slightly from the pH value in the liquid. During longer experiments we recommend to crosscheck the pH value in the bioreactor with an external pH meter and adapt the pH calibration parameter in the control software once a day.

Screenshot taken from video

Adjusting the pH value during the run prevents a pH shift

Temperature plays an important role with regards to sample and buffer pH as well as the electrode's characteristics. The temperature dependency of buffers is fully known and is shown on the packing. Even if the pH control compensates the influence of temperature on the sensor sensitivity, no compensation can be made for the pH shifts caused by altered reference potentials. If possible, samples, buffers, and sensors should all have the same temperature.

Temperature dependency of an ideal pH electrode

Dissolved oxygen (DO) is a relative unit for measuring the oxygen concentration that is dissolved in a given aquatic environment corresponding to the ambient air pressure. The medium composition has an impact on the solubility of oxygen, which means that 100 % DO in one medium can reflect a total different oxygen concentration than 100 % DO in another medium. Furthermore process parameters like the temperature or pressure can also impact the solubility, therefore it is always neccesary to calibrate the 100 % DO under process conditions and completed oxygen saturation.

According to the working principle of a Clark electrode, the measured signal is directly proportional to the concentration of oxygen. In an aquatic environment completely desaturated with oxygen, the current (zero setpoint) should not exceed 3 nA. In ambient air, the calibration slope should be between 50 nA and 100 nA.

The only biological systems actually producing oxygen and thus showing a negative OTR are phototrophic cells such as plant cells or algae. In all other cases, either the calibration is wrong or the boundary conditions for the calculations are incorrect. A possible cause for this result is the humidity diluting the inlet's dry air and generating reduced oxygen concentrations at the outlet. Humidity compensation through the exhaust analyzer prevents this effect. The settings need to be adjusted carefully before each process run.

Gas will pick up water vapor while passing through a bioreactor. This effect might have to be compensated in order to prevent false OTR readings.

Single components of PTFE are resistant against corrosion. However they are not free of soiling and deposition. To elongate their life time, clean them with ethanol. Rinse all components several times with demineralized water (DI) afterwards. Optionally use 70 - 80 % ethanol to disinfect. PTFE parts can also be cleaned by using a laboratory washer.

PTFE components such as feed lines can be rinsed with ethanol.

Clean metal spargers immediately after each use with a soft brush. Then rinse with plenty of distilled water. We recommend regular cleaning with 1 M sodium hydroxide (NaOH) solution, followed by rinsing with demineralized water (DI). Wear protective clothing (laboratory coat, protective gloves, and goggles) during and after cleaning. Leave the sparger standing in 1 M NaOH for 30 – 60 minutes or longer. Additionally, connect compressed air for cleaning of the pores. Alternatively use acetone. Then flush with 70 % ethanol to remove acetone. Afterwards flush with plenty of distilled water. Always avoid cleaning agents containing tensides, as an easy removal by rinsing is not possible.

For hard-to-clean surfaces, place them into an ultrasonic bath for 15 – 20 minutes. Repeat if necessary.

Metal spargers should be cleaned regularly using sodium hydroxide or acetone.

Fouling and calcination can contaminate stainless steel components. Before starting the cleaning procedure, remove non-stainless steel components such as O-rings and use an ultrasonic bath to clean the stainless steel parts (10 minutes at 70 °C). Use detergents like P3-Ultrasil 53 or Tickopur RW77. Please contact us if you require more information. In order to remove calcination, use an ultrasonic bath containing a weak organic acid like acidic acid or citric acid. Wear protective clothing (laboratory coat, protective gloves, and goggles) during and after the procedure. Rinse all components at least three times with demineralized water (DI) after ultrasonic cleaning. Optionally use 70 % ethanol to disinfect. Avoid solutions containing chloride like hydrochloric acid.

Foam can be generated in all kinds of growth media, especially in protein-rich media. Agitation and gas flow increase the amount of foam in the bioreactor.

The most effective way of preventing or getting rid of foam, is to use an anti-foaming agent. The most frequently used antifoams are either oil-based or silicone-based. It is very much dependent on the strain used and the experimental conditions chosen, which one works best for your process. Silicone-based antifoams may be problematic during downstream processing, for example in (ultra) filtration and column filtration setups; oil-based antifoams generally do not have this disadvantage. However, oil-based antifoams may be consumed by the cell, resulting in less anti-foaming effect over time and the need for further additions.

For microbial processes, liquid acids and bases are used. If acid is required, 2M H3PO4 or 2M H2SO4 can be used. In every case, do not use HCl as it is harmful for steel components. If base is required, 2M NaOH or 2M KOH can be used as well as 10 – 15 % NH4OH. We recommended to check the chemical resistance of the pump tubing used with the supplier. For example, using high concentrations of acid or base with silicone tubing may lead to leaking pump tubing and possible damage to the controller. Other types of tubing such as Marprene are more suitable for higher concentrations of acid and base.

For cell culture processes, CO2 gas is used in the control algorithms (3-Gas or 4-Gas) as the acid component. As base, 8 – 10 % bicarbonate buffer is used. Other types of liquid base or even liquid acid can be considered, but as animal cell lines are generally more susceptible compared to microbial cells, care should be taken whenever changing the chemicals in use.

Acid/base solutions suitable for cell culture and microbial fermentation, respectively (not complete)

Gel-filled pH sensors might have reached the end of their lifetime after they have been used for various runs and have been autoclaved regularly. If dirt or protein residue is present on the glass surface, you can remove  these with either water and a soft cloth or with a pepsin/trypsin cleaning solution. Nevertheless we highly recommend to purchase a replacement sensor.

For DO sensors, we recommend to replace the electrolyte before a new calibration is performed as well as to check the membrane covering the glass electrode tip. If a sensor membrane is damaged, e.g. a small hole in the membrane surface or a tear in the membrane, it requires replacement. The old membrane cannot be used anymore. Spare membranes and electrolyte are therefore highly recommended to have on  at hand. Details on how to replace the membrane and refresh the electrolyte can be found in the user manual of the DO sensor.

DO membrane kit, including electrolyte and membrane bodies
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