RavenBiotech Inc.- On line methanol monitoring and control in Pichia pastoris fermentation - FAQ

Does the system control an external pump?

Yes, a user selectable jumper located on the circuit board allows a TTL or dry contact signal to be output to an external pump which will accept either one as a start/stop.

What type of pump is best suited for the control of methanol in Pichia pastoris fermentations?

Generally, peristaltic pumps are used for feeding nutrients or other additives to a fermentor. Pumps of this type which offer variable speed are best suited for nutrient addition and allow the addition rate to be regulated during the different phases of fermentation.

How can the probe be adapted to my headplate port of choice?

The standard probe is 12 mm outside diameter and approximately 40 cm in length. This design allows simple installation into a compression type port adapter. If your fermentor does not have this type of port adapter available, optional custom probe adaptation to your specific port and bioreactor configuration is available.

What type of air supply is required for operation?

A clean, dry, well regulated source of industrial air, such as that supplied from a compressed air cylinder is most appropriate and is readily available to most facilities. Air from other sources such as air compressors, etc. may not be oil free and well regulated, leading to contamination and variable response. Since the Methanol Monitoring and Control System only consumes approximately 15 ml per minute, an average size 4 foot tall cylinder should last many weeks in continuous operation.

Do other factors such as pH, agitator speed, etc. affect system response?

Factors such as pH (3-9), agitator speed (100-1200 rpm), air sparge rate, supplemental oxygen addition rate and antifoam addition do not have an effect on the system's response or stability, therefore allowing trouble free control of methanol concentration throughout the fermentation. This is critical when using varying air flow, agitator speed and on-demand supplemental oxygen addition to control the dissolved oxygen level.

What factors do have an effect on the stability and response?

This system can be affected by factors such as humidity and temperature. The fermentor temperature has a marked effect on the mass transport of organic volatiles across
the probe membrane. This has not been a problem since most fermentor systems maintain a steady temperature very close to the setpoint,. However, because of the influence of temperature on the output of the sensor, the calibration must be done at the operating temperature. Other combustible organic vapours such as other alcohols acetone, etc. will interfere since the sensor is not methanol specific. This is not a problem in aerobic cultures since interfering species are normally absent. It can be an advantage, if, for example, ethanol is produced eg under oxygen limitation. This could be repressive. Since Pichia pastoris will utilize ethanol as a carbon source, allowing consumption of the ethanol prior to induction with methanol can be monitored. Another infrequent source of interference can be produced by overheating of a complex media additive such as yeast extract, which in turn may produce organic volatiles as degradation products.

How often does the probe membrane need to be replaced?

Under normal operating conditions and proper cleaning and storage, the probe membrane should last 4 to 6 fermentations before replacement may be necessary. Membrane replacement is fast and simple.