Standard Operating Procedure (SOP)

Standard Operating Procedure (SOP)
Produce TWO standard operating procedures (SOPs) for the fermenters found in the Pilot Scale Cat 2 laboratory.

1)One SOP for the 15L fermenter.
2)One SOP for the 100L fermenter.

Documents are provided with the operating instructions for both fermenters. You will need to read the documents carefully and then rewrite them using the correct SOP template to produce concise and easy to follow SOPs.
The following provides instructions for setting up both the 15L and 100L fermenters found in the Pilot Scale Category 2 laboratory at the University. The fermenters are used to grow volumes of bacteria producing specific enzymes of use in the pharmaceutical and diagnostic market. The 15L fermenter is partly automated, whereas the 100L fermenter requires manual operation.
The fermenters require the following services, cold water, steam, compressed air. An example of the media used is supplied in a separate document. Alkali and acid is used for pH adjustment where necessary.

Operation of 15 L fermenter
Sterilisation cycle
Power up the fermenter and turn on the water, steam and air supplies. Start the control computer and allow the program to finish loading before starting up the data logging computer. Follow the procedure sheet entitled ‘Computer control of fermenters and data logging’ for data monitoring, data comparison and data logging instructions. NOTE: Currently the 100 L fermenter cannot be data logged at the same time as the 15 L fermenter. The connection of the 100 L fermenter to the back of the 15 L fermenter always causes the ‘loss’ of the 15 L fermenter, i.e. the computer will see the 100 L fermenter but will no longer be able to see the 15 L fermenter until such time as the 100 L fermenter is disconnected. Attach the lead onto the oxygen probe (ensure that the membrane is kept wet at all times), and leave enough time for it to polarise. Attach the lead and calibrate the pH probe, then attach it, along with the oxygen probe, to the fermenter. Ensure that the harvest valve is closed, along with all other ports (apart from the addition port, which is obviously used to fill the fermenter with culture medium). Ensure that a new septum is added to the inoculation port. Check that the manual air outlet valve, and the other two valves on the air outlet line are open. Check that the manual steam valve on the bottom/harvest port is closed. Next, fill the fermenter to the working volume with culture medium (the working volume is 15 L, but the volume of the inoculum to be added must be taken into account so that the fermenter is not over-filled). NOTE: Although 15 L is the working volume used, this is not the maximum working volume, in fact it is the smallest possible working volume. Close the addition port. Start the agitation, temperature and air control. With regard to the agitation, first select ‘On-Off’ control, then when the stirrer is heard to speed up, select ‘PID’ control (this is to overcome a program design flaw). In order to overcome another design flaw, the temperature set-point needs to be set to 70°C immediately prior to starting the sterilisation sequence. Once the cooling part of the sterilisation cycle is active, this will enable the air to start passing though the outlet filter at 80°C, at which point the temperature set-point can be re-set to the required growth temperature. Select Steril, then Temp and check that the sterilisation temperature is set to 122°C, the sterilisation time is set to 15 min and that the drain time is set to 1 min. Select Sterilise and then select OK. When the temperature reaches >100°C, check that the dissolved oxygen reads 0% and calibrate as necessary. When the temperature reaches the sterilisation temperature (122°C) open the manual valve to steam sterilise the harvest/drain port. Check that the relevant parts of the fermenter are at high temperature by use of a wash bottle. When the cooling stage is initiated, close the manual valve for the steam sterilisation of the harvest/drain valve. Upon cooling to just below 80°C, re-set the temperature set-point to that required for growth (as described above – vi). Check that all parameters are at their appropriate set-points and control modes. When all parameters have settled, re-calibrate the dissolved oxygen probe to 100% as necessary.

Inoculation and growth cycle
Check that all parameters are at their appropriate set-points and control modes. Set up the peristaltic pump and remove the first flask from the incubator. Attach the piping to the peristaltic pump and attach the inoculation needle to the fermenter using aseptic techniques. Re-set the elapsed fermentation time (EFT) on the Biocommand and then transfer the contents of the flask at a speed of 40%. Retrieve the second flask from the incubator, attach its piping to the peristaltic pump and swap over the inoculation needles, again using aseptic techniques. Transfer the contents of the flask at a speed of 40%. Remove the inoculation needle from the fermenter and close up the inoculation port. Clean out the flasks, piping and inoculation needles and put away the peristaltic pump.

Shut down
The steam supply should be turned off and the water supply should be turned off. The fermenter should be cleaned by flushing with hot water and then remove and store the pH probe in3M KCl and the dissolved oxygen probe in distilled water. Switch off the air at the control computer. Finally, turn off air supply.
Maintenance
The solenoid valves get blocked with fine particulate matter from time to time, and so need to be pulled apart and cleaned.
The DO probe needs its electrolyte replaced from time to time, and may also need its membrane replaced if this has been damaged.

Operation of 100 L fermenter
Sterilisation cycle
Ensure that the data cable is disconnected from the 15 L fermenter, but only if the 15 L fermenter is (or is going to be) running and its data being logged. Follow the procedure sheet entitled ‘Computer control of fermenters and data logging’ for data monitoring, data comparison and data logging instructions. NOTE: Currently the 100 L fermenter cannot be data logged at the same time as the 15 L fermenter. The connection of the 100 L fermenter to the back of the 15 L fermenter always causes the ‘loss’ of the 15 L fermenter, i.e. the computer will see the 100 L fermenter but will no longer be able to see the 15 L fermenter until the 100 L fermenter is disconnected. Turn ‘red switch’ on control box to ON (check that the power is ‘on’ at the wall) and switch on the ML-4100 controller.
The cold water supply needs to be turned on and also the air supply and steam supply should be turned on. Turn on the cold water supply. The lead should be attached onto the oxygen probe (ensure that the membrane is kept wet at all times), and left enough time for it to polarise. Next, attach the lead and calibrate the pH probe, then insert it, along with the oxygen probe, into available ports on the fermenter. Ensure that the harvest and sampling valves are closed.
Fill the fermenter to the working volume with culture medium (the working volume is 100 L, but the volume of the inoculum to be added must be taken into account so that the fermenter is not over-filled). Close the addition port. NOTE: the o-ring on the plug needs to be lubricated every time with polypropylene glycol G2000. Check that all valves are shut (but do not adjust valves No. 1, 10, 11 & 14). Ensure that ‘sterilise’ operating mode is selected on control box. Set desired sterilising temperature (usually 122°C). Ensure that ‘recirculation pump’ is switched ‘off’ on the control box. ‘Condenser steam’ valve no. 1 and ‘condenser water’ valve no. 11 should already be set, allowing condensate to pass through and lubricate the bottom seals. NOTE: Condensate lubrication is required at ALL times when the stirrer is activated.
It is important to check that condensate is passing through the seal by examining the sight glass, and ensure that the pressure between the seals does not exceed 15 psig (~1 bar). Ensure that the agitation is set to ‘manual’ on the control box and 0 speed. Press ‘start’ (green button) and gently increase the agitation to 300 rpm. Set the temperature to ‘On-Off’ control on the ML-4100 controller. When ready, open ‘jacket condensate’ valve no. 2, then open ‘exhaust condensate’ valve no. 3
When the temperature has risen to 65 C open condensate’ valve no. 6 and sparger steam’ valve no. 5 to sterilise the inlet air filter. When 100°C is reached check that the dissolved oxygen probe is reading 0% and calibrate the probe as necessary.
Sterilise the ports that do not open directly into the fermenter when 100°C is reached and then the remaining two at ~115°C. Hold the fermenter at the sterilisation temperature for the required length of time (5 – 20 minutes depending on the fermentation).
NOTE: Open and close all valves slowly.

 

Rapid cooling cycle
Close the steam valves on all the ports and reset the temperature to the desired growth temperature on the ML-4100 controller. The ‘exhaust condensate’ valve No. 3 needs to be closed along with ‘jacket condensate’ valve No. 2, ‘sparger steam’ valve No. 5, ‘inlet filter condensate’ valve No. 6.
The ‘air bypass’ valve No. 8 should be opened, (the regulator should be pre-set to 20 psig in order to maintain head pressure in the vessel during cooling). Next, open ‘waste water’ valve No. 12 and ‘rapid cooling’ valve No. 9. ‘Control cooling’ valve No. 10 should be already set (open)
When the vessel temperature falls below 90°C close ‘air bypass’ valve No. 8, but keep an eye on the pressure loss in the vessel and top-up as necessary to ensure that the pressure stays above 5 psig. When the vessel temperature reaches 5°C above the desired growth temperature: Close ‘rapid cooling’ valve No. 9. ‘recirculation water’ valve No 13 should be opened, ‘Heat exchange system’ valve No. 14 should already be set (open). Switch ‘recirculating pump’ to ‘on’ on the control box. Switch to ‘growth’ on the control box. Open ‘exhaust condenser water’ valve No. 22 and switch the agitation to ‘auto’ on the control box.. On the ML-4100 controller, set the temperature to ‘PID’ control. Open ‘vessel exhaust’ valve No. 15 slowly at the same time as opening ‘main air’ No 16.
Adjust air flowmeter (valve No. 16) to give desired air flow rate (set-point is usually 10 L/min). When all parameters have settled, re-calibrate the dissolved oxygen probe to 100% as necessary. Ensure that the dissolved oxygen is set to PID control on the ML-4100 controller.

Inoculation and growth cycle
Check that all parameters are at their appropriate set-points and control modes.
Ensure that ‘vessel exhaust’ valve No. 15, ‘recirculation water’ valve No. 13, ‘waste water’ valve No. 12, ‘heat exchange steam’ valve No. 14, ‘exhaust condenser water’ valve No 22 are open, and that steam sterilisation valves on all ports are closed.

Inoculation:

Briefly steam the bottom ports on the 15 L and 100 L fermenters. Attach the solid transfer pipe to the bottom ports of the 15 L and 100 L fermenters. Ensuring that the valve connected directly to the steam trap is open, open the steam valve on the bottom port of the 15 L fermenter until most of the condensate has been expelled, then close it. Then open the steam valve on the bottom port of the 100 L fermenter until most of the condensate has been expelled and then re-open the valve on the 15 L fermenter. Steam the transfer line for 10 minutes.
Close both steam valves and then close the valve connected directly to the steam trap. Open the 100 L fermenter’s drainage valve and close the manual air outlet valve on the 15 L fermenter. Once a pressure of ~5 psig has built up open the 15 L fermenter’s drainage valve to start the inoculation of the 100 L fermenter. Turn off the temperature control.
When the culture has cleared the bottom blade on the 15 L fermenter, switch off all parameters except the air.
When all the culture has been transferred, close the 100 L fermenter’s drainage valve, open the 15 L fermenter’s manual air outlet valve, and when the pressure has been released, open one of the head plate ports. Remove the transfer line from the 100 L fermenter, close up the bottom port and then steam sterilise the bottom port.

Harvest and shut down
Connect up the flexible hose from the drainage port to the filtration rig. Open the 100 L fermenter’s drainage valve and then close ‘main air’ valve No. 16 to start the transfer. If needed, increase ‘main air’ valve No. 15.
When the bottom blade is cleared, press the ‘red button’ on control box to stop the stirrer. Set the sirrer to ‘manual’ on the control box. Turn the control modes for the temperature and dissolved oxygen to ‘off’ on the ML-4100 controller. When all contents have been transferred, re-open ‘main air’ valve No. 16 and open up the main top port of the fermenter. Turn ‘recirculation pump’ on control box to ‘off’. Open ‘jacket drain’ valve No. 4. Close ‘recirculation water’ valve No. 13 and close ‘waste water’ valve No. 12. Close ‘exhaust condenser water’ valve No 22 and finally close ‘jacket drain’ valve No. 4 after leaving sufficient time for draining. Then turn off the steam supply and turn off the water supply. All that remains is to clean out the vessel and with hot water and then remove and store the pH probe in 3M KCl and the dissolved oxygen probe in distilled water. Switch off ‘main air’ valve No 15 and turn off the air supply.
Maintenance
The backup air compressor, that is used for both the 100 L and 15 L fermenters, has a non-return valve that fails to operate on a fairly regular basis. It is obvious when this occurs since the compressor will be heard to be discharging air all the time when the compressor pumps are not operating. The non-return valve needs to be removed and one of three actions undertaken.
i) The rubber seal needs cleaning of any debris
ii) The rubber seal needs turning over so that a new surface is used
iii) The rubber seal needs replacing
At the moment, the rubber seal is made from white silicon (this is not the original seal, nor necessarily the original type of material), and it is possible that this is a little too soft for this application. It may be worth considering a harder, rubber like material for the production of new seals. It may also be worth checking with the manual/manufacturer as to the original seal material and the cost of original replacements.

 

 

 

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