STANDARD OPERATING PROCEDURE Fermentor, 15-L Model: NLF-22 Manufacturer: Bioengineering AG Location: Fermentation Facility, 1621 Food Sciences Building Publication Date: 01/22/2015 Iowa State University does not discriminate on the basis of race, color, age, ethnicity, religion, national origin, pregnancy, sexual orientation, gender identity, genetic information, sex, marital status, disability, or status as a U.S. veteran. Inquiries regarding non-discrimination policies may be directed to Office of Equal Opportunity, 3350 Beardshear Hall, 515 Morrill Road, Ames, Iowa 50011, Tel. 515 294-7612, email eooffice@iastate.edu.
Description and Uses The Bioengineering NLF-22 Fermentor can be used for batch, fed-batch or continuous culture with automated control of ph, dissolved oxygen (DO), agitation, and temperature. It has a maximum volume of 15-L and a maximum operating temperature of 150 C. Power Specifications Motor: Bioengineering NLF-22 Voltage/Amperage: 220V/30A Speed/Frequency: Variable speed control@50-60 Hz. Maximum Operating Volume: 15-L Maximum Operating Temperature: 150 C For further information on power specifications, please see the Operating Manual located in the Fermentation Facility, 1621 Food Sciences Building. Potential Hazards and Safety Precautions High Voltage/Possible Electric Shock (220-V) Make sure that the wall outlet receptacle is properly wired and grounded, and matches the instrument s power cord and plug. Do not touch the power cord or plug if hands or feet are wet, or if standing on a wet/damp surface, as severe electrical shock or death may result. Excessive Weight/Possible Damage to Hands or Fingers when Moving or Repositioning Parts Certain parts of the fermentor, such as the upper lid, are very heavy. Use extreme caution when handling or moving these parts during fermentor assembly/disassembly. If available, wear shoes with steel caps when using the fermentor. Combustibility and Explosion/Possible Physical Damage or Injury Do not use the fermentor with materials capable of developing flammable or explosive vapors. Hot Surfaces/Possible Burns to Hands and/or Extremeties The fermentor is made of stainless steel and has a jacket that is filled with steam under pressure during the sterilization-in-place (SIP) process. As a result, the outer surfaces of the fermentor become HOT enough to cause serious skin burns if the fermentor is touched. Use extreme caution when working near the fermentor during sterilization. 2
Biohazard/Possible Infection from use of Pathogenic Microbes Normal operation involves the use of many types of microorganisms, all of which can be opportunistic pathogens. Occasional use may also involve working with genetically modified microbes. Use extreme caution and good sterile techniques when working with these organisms. After use, autoclave the fermentor to kill any remaining microbes. Required Personal Protective Equipment Lab Coat Hair Net Safety Glasses or Goggles No Open-toed or Open-heeled Shoes Insulated Rubber Gloves Steel-toed Shoes are recommended Required Training *Denotes courses offered online Machine & Site-Specific Training Fire Safety & Extinguisher Training* Laboratory Safety: Core Concepts* Laboratory Safety: Spill Procedures Bloodborne Pathogens and Sharps Safety* Operation Operation: Start-up 1. Be sure to complete all the required training listed above, including machine and site-specific training by the Fermentation Facility manager. 2. The Operating Manual provides a more complete description of the Bioengineering NLF-22 Fermentor. Be sure to read the manual before operating the fermentor to become familiar with its correct operation and individual component parts. 3. Be sure the drain port is closed. 4. Install the temperature probe (located on the side of the fermentor). 5. Install any other probes, if needed, such as the ph or O 2 probe. Note: If the ph probe is to be used, it must first be calibrated. Refer to Section VII: Calibration of the ph Probe in the Operating Manual. 3
6. Add media to the fermentor. 7. Install the top plate by performing the following steps: a. First, apply a small amount of silicone grease to the O-ring on the underside of the plate. b. If the fermentor is to be aerated, be sure that one of the openings in the top plate aligns correctly with the conical opening of the sparge tube in the fermentor. c. Insert the eight (8) screws in the plate. Tighten by hand, then with the T-shaped Allen wrench that is available. Tighten screws in a crosswise manner. Note: The top plate is heavy. Be sure to get a firm grip on the plate when attaching it to the fermentor. Be sure to wear close-toed shoes or (preferably) safety shoes with steel caps, if available. 8. Attach fittings to the top plate as follows: a. Light b. Pressure gauge c. Pressure relief valve d. Condenser with exhaust air filter (EAF). Note: Open the valve on the EAF 3-4 turns before starting the sterilization process. e. If the fermentor is to be sparged with air or oxygen, sterilize the air filter separately in advance using the autoclave. Also, there are several sizes of needles for introducing additives to the fermentor. Note: Be sure to use the large bore (~8mm) needle for sparging. The other needles have a narrower bore. If the wrong needle is used, there will be a gap between the needle and the sparge tube. This will result in most of the air not going into the fermentation broth, but lost into the headspace instead. f. Check to make sure all head-plate ports are either sealed with a blind plug or appropriate fitting. Make sure all lower ports are secured properly with blind plugs or securely fastened probes. Note: Failure to complete this step can result in serious physical injury! 9. Open the valve on the Sample/Drain-Port Steam Supply. This will send steam to the sample port to sterilize it. 10. Open the steam supply. This is valve with a green handle near the floor on the north wall of the Fermentation Facility (1621 Food Sciences Building), behind the 100-L fermentor. 11. Open the cold water supply. This is located near the floor on the north wall of the Fermentation Facility. Water is delivered to the fermentor via a ~¾" diameter green hose. 4
Operation: Sterilization 1. Turn on the temperature/ph control box (this is the grey box on the left in Figure 1). 2. Turn the Agitation Control knob fully counterclockwise. This is located on the agitation control box (the grey box on the right in Figure 1 above). Turn ON the power to the agitation control box by twisting the RED power knob to the right. Turn the Agitation Control knob clockwise to start the agitator. Note: During sterilization, the agitator should be set to and kept at ~300 rpm. The agitation ensures that the fermentor contents are heated uniformly. 3. Turn the circulation pump to ON by using the toggle switch on agitation control box. 4. Set the parameters for the sterilization at 121 C as follows: Figure 1 a. Two temperature set points must be entered. Press the arrow on the far left side of the temperature display. Then, press the arrow on the far right side of the display to select the desired set point. Use the UP and DOWN triangular arrows to enter the temperature. Save the setting by pressing the arrow on the far right side of the display. b. Repeat this process for the second set point. For sterilization, set both set points at 121 C. c. When both set points have been entered, press the arrow on the far left side of the display. d. Set the water and steam toggle switches to the Controller (DOWN) positions. 5. While the fermentor is heating up, the valve on the exhaust air filter (EAF) should be in the open position (~3-4 turns counter-clockwise). If the valve is closed, open it carefully. Note: Do not stand in front of the outlet, as condensate from the last run may be present in the EAF. If so, hot water and/ or steam may gush out of the EAF and cause burns! Stand off to the side while opening the valve. 6. As the fermentor and contents approach the sterilization temperature (121 C), steam will begin to exit the EAF. When this happens, wait 30-60 seconds, then fully close the EAF valve. This seals the fermentor and allows the internal pressure to rise to 1 atmosphere (~1 bar) for sterilization to occur. 7. Below the temperature display are two toggle switches to control the flow of water and steam to the fermentor. The middle position is OFF. Flip both switches DOWN to the Controller position. This will send steam and cooling water to the fermentor. With the water and steam control toggle switches in the Controller position, the heating process is under automatic control by the control box. The steam and cooling water control valves perform a balancing act to keep the temperature at 121 C. The solenoid switches in these valves open and close as needed to send steam or cooling water to the fermentor to maintain the set-point temperature. 8. The fermentor should be held at 121 C and ~1 bar for 20 minutes to sterilize the contents. With steam flowing to the fermentor, the temperature eventually reaches 121 C. At this point, start timing the sterilization. Note: The temperature control is not perfect. As a result, temperature swings of 1-1.5 C above and below the set point will occur. Lower temperature swings below 121 C occur more frequently. If this happens, flip the steam control switch up from Controller to Manual. 5
This will send more steam to the fermentor to raise the temperature, which may go as high as 121.5-122 C. At this point, flip the steam control switch back to Controller. If the temperature again drops below 121 C, repeat this process as often as necessary over the 20-minute sterilization period. 9. Place the HOT sign on top of the fermentor to warn against touching the vessel. 10. During sterilization, steam will be sent to the sample port, and can be seen exiting the sampling tube. When this occurs, turn the knob on the sample port valve until it is open ~½ turn. This will allow steam to continue to flow to the sample port, but avoids the use of excess steam. The steam sterilizes the sample port to prevent contamination of the fermentor contents during sampling. Operation: Fermentation 1. After 20 min (or about 1 min before the end of the sterilization time), change the set-point temperature on the control box to the desired fermentation temperature. This will send cooling water to the fermentor to lower the temperature. Note: The steam and cold water should be left open during the fermentation to maintain the desired temperature in the vessel. Keeping the steam supply open also ensures that steam continually flows to the sampling port to prevent contamination during sampling. 2. When the fermentation temperature reaches ~105 C, open the valve on the EAF about 3-4 turns. This prevents a vacuum from forming in the vessel as the contents cool. Note: Do not stand in front of the outlet, as steam or hot water will be forcibly ejected when the valve is opened and may cause injury! 3. If the organism to be grown in the fermentor produces a surfactant (e.g., B. subtilis) that is to be collected and causes the media to foam, then close the EAF valve completely once the fermentation temperature has been reached. Note: If this is not done, the foam will exit the fermentor and be lost. 4. The cooling water will lower the temperature of the fermentor and its contents fairly quickly (~30 min). However, the temperature of the vessel above the level of the water jacket will remain hot for several hours. Note: Do not touch this part of the fermentor, especially the top plate, for at least 4 hours after the desired culture temperature has been reached. 5. When the fermentor has cooled, ancillary equipment may be attached to the fermentor as follows: a. Air inlet filter (if aeration or sparging is required, the filter should be sterilized separately in advance using the autoclave); b. Sterile tubing for adding sterile medium supplements and/or inocula to the fermentor; c. Cooling water lines to the condenser; d. Other equipment deemed necessary for the fermentation. NOTE: The attachment of tubing, etc. (a and b above) requires removal of one or more stainlesssteel (SS) plugs from the fermentor cover, then insertion of a sterile large-bore needle through the septa beneath the plugs. Prior to insertion of the needle(s), squirt several milliliters of 70% ethanol, methanol, or isopropyl alcohol on the septa to minimize possible contamination. 6
6. When the fermentor reaches the desired temperature, set the operating parameters as follows: a. Agitation (stirring): Turn ON the agitation control box. b. Turn the agitation control knob clockwise to the desired stirring speed; c. If required, open the air supply and, using the rotameter, d. set the air flow to the desired rate (l/min). e. If the culture is to be aerated, the air filter and connecting tubing must be separately sterilized in advance using the autoclave and then, once sterilized, attached to the fermentor. 7. Inoculate the fermentor as follows: a. A pre-culture of the organism of choice should be prepared in advance. To transfer this to the fermentor, prepare an appropriately sized glass or plastic bottle. This must have a screw cap with a siphon that reaches to the bottom of the bottle. b. If needed, also add a stir bar to keep the culture suspended. c. To the upper (outer) end of the siphon, attach a length of rubber tubing ~3 feet long. Attach an oversize-bore needle d. to the tubing. Cover the needle with a metal sleeve or aluminum foil, then sterilize the bottle, tubing and needle. e. Aseptically transfer a pre-determined volume of the pre-culture to the sterile bottle, along with any other fermentation additives, e.g., media supplements, etc. Place the bottle on a stir plate to mix the contents and keep the cells in suspension. f. Insert the needle through one of the septa on the fermentor s top plate. g. Attach the tubing to a peristaltic pump. Turn on the pump to deliver the inoculum, etc., into the fermentor. h. When this is done, remove the needle from the septum and cover the septum with a stainlesssteel plug. 8. Monitor the fermentation as follows: a. Samples can be periodically taken from the fermentor to measure growth, substrate utilization, product formation, etc. b. The sample/drain port at the bottom of the fermentor is constantly heated by steam, so it is HOT! Note: You must turn OFF the steam to the drain port ~10 minutes prior to sampling. If you must draw a sample before the 10-minute cooling period ends, always wear waterproof-insulated gloves before proceeding with the following steps. c. Open the valve by slowly lifting up valve lever. d. Allow ~25 ml of fermentor contents to drain into a waste container. This clears the dead volume from the drain tube. e. Collect the sample for monitoring. 9. Close the drain valve and re-open the steam supply to the sampling port. Then, autoclave the contents of the waste container. 7
Operation: Harvest and Shutdown 1. Shut off the main steam supply valve (green handle, along the north wall of the Fermentation Facility). 2. Shut off the main water supply valve (yellow handle, along the north wall of the Fermentation Facility). 3. Move the Steam and Water toggle switches to the OFF position. 4. Shut off main power switch to the temperature/ph control box. 5. Shut off the circulation pump. 6. Turn the Agitation Control knob fully counterclockwise to stop the agitator. 7. Shut off the main power to the agitation control box. 8. Reduce the airflow to 1-2 LPM. Note: The flow of air is necessary to help push the contents from the fermentor. 9. Open the exhaust valve if necessary. 10. Close the drain-port steam valve. 11. Remove the drain-port hose. Then, place a container under drain port and slowly open (press down) the drain-port valve to collect the fermentation broth. After collection is complete, close the drainport valve. 12. Completely shut off the airflow. 13. Remove the pressure gauge from the vessel head plate and place a funnel in the resulting hole. 14. Pour tap water into the funnel until the fermentor is about half full. 15. Remove the funnel and replace the pressure gauge. 16. Sterilize the fermentor and its contents (see Sterilization procedure listed earlier) to kill any organisms remaining in the fermentor. 17. When the sterilization is complete and the fermentor has cooled, drain and dispose of the fermentor contents. 18. Report any operational difficulties to the Fermentation Facility manager and initiate clean-up procedures. Clean-up Procedures 1. After the fermentor has been harvested, remove all apparati (air filter, light, pressure gauge, etc.) from the top of the fermentor. 2. With the available T-shaped, 6-mm hexagonal Allen wrench, remove the top plate. 3. Fill the fermentor about halfway with warm tap water. 4. Add a non-abrasive detergent (such as Joy or Ivory Liquid) and turn on the agitator (~300 rpm) for a few minutes. 8
5. After the allotted time, turn OFF the agitator. Then, scrub the inside of the fermentor with a brush or sponge. 6. Turn ON the agitator again and drain the fermentor. When drained, turn OFF the agitator. 7. Rinse the inside of the fermentor with distilled water. Then, drain again. 8. Clean the head plate, light, probes, etc. in the sink with non-abrasive detergents. Then, rinse with distilled water. 9. Fill the fermentor with tap water. 10. Replace the cover plate and any plugs, pressure gauge, etc. so the fermentor is sealed. 11. Clean up the surrounding work area. 12. Initiate check-out procedures by having the fermentor and surrounding work area inspected by the Fermentation Facility manager. Machine Care and Maintenance The fermentor must be properly cleaned and inspected after each use. All final inspections are performed by the Fermentation Facility manager. All maintenance should be performed by the Fermentation Facility manager or a trained service technician. Accessories T-shaped, 6-mm hexagonal Allen wrench 9