CHEMICAL ENGINEERING LABORATORY CHEG 4137W/4139W Bioreactor Objective: The laboratory has acquired a bioreactor for the purpose of growing cell cultures. In the initial attempt to grow E. coli, the bacteria were successfully grown in a flask. However, transferring these bacteria to the reactor resulted in a lengthy lag phase, and no significant amount of bacteria was grown in an 8hour reaction time. After some analysis, it was determined that the test conditions that most likely diminished bacterial growth are as follows: Incubation time: Impeller mixing speed: Incubation/reaction temperature: Glucose loading in growth media:

17 hours 200 RPM 37 °C 0.4% by mass

The incubation time has been identified as the most likely cause of test failure, and will be adjusted to 15 hours for your test. Your goal is to determine how to manipulate the other variables to encourage bacterial growth. You will measure the bacterial density over time and construct a bacterial growth curve. You will also use a numerical method to estimate the glucose concentration in your reactor as time progresses. Due to the lengthy nature of a single reactor run, you will likely only achieve one complete reactor run during the duration of your lab. As such, it is important to communicate with other groups performing this experiment to prevent tests of repeated conditions. Ultimately, you will need to determine the conditions for optimum bacterial growth. Major Topics Covered: Kinetics, Biochemistry, Numerical Methods Theory: In this experiment, you will be determining some of the kinetic parameters for the growth of E. coli. Some relevant equations include:

dX = µX dt

dS −µ X = dt YX S

µ=

µ MAX S KS + S

Where X = biomass concentration, S = substrate concentration, YX/S = maintenance coefficient (~0.5 for this experiment), µ = growth parameter, Ks = Monod constant, and µMAX = the maximum growth rate parameter. You will be measuring the biomass optically using cell density in a UV-Vis spectrometer at 600 nm. Determining other parameters will be the focus of this experiment. For relevant information, see the references at the end of this document. Safety Precautions: E. coli is a biological agent, so the following safety precautions MUST be followed when working with it. Failure to observe these precautions will result in expulsion from the lab. 1. Gloves, goggles, and lab coats must be worn at all times when working with the bioreactor. 2. If you wish to walk away from the reactor bench, first remove and dispose of your gloves, then

3. 4. 5. 6.

7.

wash your hands with antibacterial soap. DO NOT wear your gloves around the lab or when working in the communal computer space. Always throw out used gloves in the Biological Waste Container; do not try to conserve gloves by using them multiple times. Any glassware and cuvettes used in this experiment must first be sanitized with a 1% solution of bleach, then washed out with soap and water. It is okay to leave glassware sitting overnight as long as it has been bleached first. Place only contaminated glassware in the wash basin. Anything that enters the wash basin should immediately be considered contaminated. Before and after working with the bacteria, spray down the countertop with a 1% bleach solution, then with DI water, then with a 70% ethanol solution. When discarding samples, pour some 70% ethanol solution into your spare plastic bottle. Empty all samples into the bottle, spraying the cuvettes, centrifuge tubes, etc. out with ethanol. This waste solution may be poured down the drain at the completion of your experiment. The bottle must be washed with soap and water after. ALWAYS wash your hands with antibacterial soap after removing your gloves.

Available Variables: Temperature, Glucose Concentration, Mixing Speed Procedure: See Bioreactor Operations Method Due to the lengthy set up of this experiment, the following schedule is recommended: Lab Period 1 • Prepare and autoclave media and glassware • Clean bioreactor and prep for autoclave Lab Period 2 • Finish media preparation • Autoclave bioreactor Lab Period 3 • Remove reactor from autoclave • Hook reactor up to control station The day before Lab Period 4 • Prepare media flasks and inoculate with bacteria • Leave in incubator-shaker for 15 hours Lab Period 4 (start as early as possible, i.e. 8-10 AM) • Pump fresh media into reactor • Seed reactor with contents of inoculated flask • Sample reactor every 30 minutes for 5 to 6 hours until it reaches a stable condition for two points. • Sterilize reactor and contaminated flasks with bleach Lab Period 5 • Finish cleaning reactor and glassware • Data analysis/redo time

Lab Period 6 • Data analysis/redo time Analysis: Your analysis must include: 1. A determination of the Monod constant, Ks, and the max growth parameter, µmax for your selected conditions. Report: Describe the design of your experiments and the results obtained, including an error analysis. Provide thoughtful and quantitative discussion of results. Be sure to include your bacterial growth curve (you may want to consider a semilog plot for this) and glucose concentration curve. Report all appropriate biokinetic parameters. Express any discrepancies between observed and predicted results in terms of quantified experimental uncertainties or limitations of the correlations or computational software used. Pro Tips: 1. The bioreactor is a very expensive piece of equipment. Take the following precautions to avoid damaging or breaking it: • When moving the reactor vessel away from the bench, always transport it on a cart. • When autoclaving the reactor, always tighten the lid on loosely so the glass has room to expand as it heats. • When reassembling the bioreactor, the impeller motor should gently slide onto the impeller. Do NOT try to force it. You may need to readjust the position of the impeller to get a good fit. 2. Never tightly cap anything you autoclave. Either apply the caps loosely or cover open holes with aluminum foil. 3. Only glass or metal materials are autoclavable. The only plastic materials that can be autoclaved are the 1 L media bottles, the Tygon tubes on the bioreactor, and the pipet tips. Do not put any other plastic laboratory equipment in the autoclave. 4. You may autoclave glassware many days in advance. It will remain sterile as long as the aluminum foil cover is not disturbed. 5. Use the thermally reactive autoclave tape on items to ensure the autoclave is functioning as intended. 6. When leaving the lab with anything that may be biologically active, travel in pairs. One partner will carry anything with bacteria, and the other partner will open doors for the first to minimize contamination. Use the basin to carry flasks that contain bacteria. 7. It is wise to prepare more than one inoculation flask at a time. In the event one of your bacteria samples does not grow, you will have back-ups. 8. When performing the inoculation, make sure the outside of your flasks and the rubber mat in the incubator/shaker are clean and dry. Do not exceed 200 RPM. 9. Do not turn on the bioreactor unless water is flowing from the faucet. Do not try to run the reactor unless the reactor is on the base and connected to the water lines. 10. When running the bioreactor, be sure to keep all open ports capped with foil unless taking samples. 11. When preparing diluted samples, always use fresh pipet tips. Be careful not to insert a pipet tip contaminated with bacteria into your fresh media, as this will force you to discard the media

solution. References:

1. Shuler, M.L., Kargi, F., Bioprocess Engineering: Basic Concepts. 2nd Ed., Prentice Hall, New York, (2001).

Bioreactor Operations Method Step 1: Begin Media Preparation Sterilize your working area with ethanol and wash all media bottles and glassware with soap and water. Mass the following quantities of salts: 1. 32 g Na2HPO4.7H2O 2. 7.5 g KH2PO4 3. 1.25 g NaCl 4. 2.5 g NH4Cl Dissolve the salts in 500 mL distilled water in a 1 L autoclave-safe plastic bottle. Label the bottle appropriately with label tape and a marker. Pour 900 mL distilled water into a second 1 L autoclave-safe plastic bottle. Add 6.3 g glucose to the water and mix. This amount of glucose will result in a 0.4% by weight glucose solution when the media is fully prepared. Label the bottle appropriately with label tape and a marker. Add approximately 500 mL distilled water to a third 1 L autoclave-safe plastic bottle. Label the bottle appropriately with label tape and a marker. Apply autoclave tape to the following: 12. The three plastic media bottles (loosely capped) 13. At least two empty plastic media bottles 14. Three 125 mL erlenmyer flasks (capped with aluminum foil) 15. At least one 250 mL erlenmyer flask (capped with aluminum foil) 16. Two 100 mL graduated cylinder (capped with aluminum foil) 17. Box of 100 mL pipet tips Place the above in the autoclave and run the sterilization sequence. This sequence will take approximately 2-4 hours. Step 2: Sterilize Bioreactor • Disconnect the impeller motor and place it on the mount on top of the reactor. • Disconnect the water lines on the reactor base and the condenser tube. • Carefully place the reactor on a cart and move it to the sink. • Undo the screws in the reactor lid, being careful not to let the support ring fall quickly. Using hot water and soap, scrub the inside of the bioreactor and any surfaces on the lid that will be contained within the reactor. Do not forget to unscrew the vent port at the top and clean inside. • After rinsing off the soap, reattach the lid. Screw the lid on loosely (the end of the screw should be even with the bottom surface of the support ring). Cover all open holes on the bioreactor with aluminum foil. This includes the holes at the end of the sampling tubes. You do not have to cover the cooling water ports in the base and condenser tube with foil. • When the autoclave is done sterilizing the media bottles and glassware from step 1, place the bioreactor on a cart and transport it to the autoclave. Remove your media and glassware wearing protective thermal gloves, carefully place the reactor into the autoclave, and start the sterilization sequence. Again, this should take 2-4 hours.

Step 3: Complete Media Preparation (In the Hood) 1. Sterilize your working area with 1% bleach, then DI water, and 70% ethanol. 2. Using the now-sterilized 100 mL graduated cylinder, measure 140 mL of the salt solution into each of the empty sterilized solution bottles. 3. Using the same graduated cylinder, measure 400 mL of the glucose solution into each of the spare solution bottles. 4. Using some of the sterilized water and a sterilized flask, prepare a 160 mL solution of 17.5 mM MgSO4 heptahydrate and a 160 mL solution of 0.7 mM CaCl2 dihydrate. Minimize the time that flasks are exposed to atmosphere. 5. Add 80 mL of each stock solution to each of the solution bottles. Now each bottle should contain a 700 mL solution that is 20% salt solution by volume, 0.4% glucose by mass, 2 mM MgSO4, and 0.1 mM CaCl2. You will have 1400 mL of media total for this experiment. 6. Label these solutions with your group members' names and date. Place the completed media bottles into a refrigerator until they are needed. Step 4: Assemble Reactor • Once the reactor has been sterilized in the autoclave, remove it using thermally protective gloves and place it onto a cart. • Once the reactor is at room temperature, tighten the screws on the lid evenly. Move in an Xshape pattern, then go around clockwise to make sure everything is screwed on tightly and evenly. Make sure the cooling water ports for the reactor and the condenser are aligned. • Carefully place the reactor back on the base. The reactor should sit firmly on the small pegs on the base. Reconnect the water lines to their appropriate ports. • Place the impeller motor back onto the reactor. It should sit firmly atop the reactor. You may need to manipulate the impeller manually to get a good fit. You should not try to force the motor onto the reactor. • Place the thermocouple into the sheath in the reactor. Some water can also be added to the port to facilitate heat transfer. • Turn on the faucet and turn on the bioreactor. Using the controls, manually manipulate the peristaltic pump controls to loop the feed tubing through it. Be sure to turn the faucet and reactor off if you are not performing the reaction that day. Step 5: Prepare Bacteria This step MUST be completed about 15 hours before you begin your reactor run. This step will require at least two people in your group to work the day before your lab period. This step should take no more than an hour. • Place one of your 700 mL media solutions, your three sterilized 125 mL flasks, a sterilized 100 mL graduated cylinder, and your sterilized pipet tips into the provided basin. Bring these materials to the designated biosafety hood. Do not transport materials using the cart during this step. It is recommended that you have a partner with you for this step as well. • Have one partner pour 60 mL of the media into the two flasks. If the media is cold, gently heat it until the temperature is 37 °C. • Have the other partner inoculate each of the flasks with 1 mL of E. coli using a micropipette and the sterilized tips. Draw the E. coli into the tip slowly to avoid sucking in bubbles. • Have the partner who inoculated the flasks cover them with foil and place everything back in the basin. Return to the bioreactor, having the partner who did not touch the E. coli open doors for the other partner to minimize contamination. • Place the three flasks into the incubator/shaker, making sure the flasks are clean and dry on the

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outside. Set the temperature to 37 °C and the mixing to 200 RPM. Note the time. Return the media to the refrigerator and pour a mixture of bleach and water into the basin to sterilize the contents.

Step 6: Run the Reactor • Measure 1045 mL of media and meter this amount into the reactor using the peristaltic pump. • Set the desired mixing rate and temperature using the control panel. Move the cursor around the screen with the arrow buttons. Press “Adjust” to make selections. Use the number pad to input exact values. Use “Enter” to confirm your selections. • Fill two 50 mL centrifuge tubes with media. Cap and save for later. • Fill the contaminated basin with 10% bleach solution and pour some ethanol into a spare bottle. • Pipet 1000 uL of fresh media from the centrifuge tubes into a clean cuvette. Use this blank to zero the spectrophotometer at 600 nm. • In the fume hood, extract a sample from your inoculated flask using the micropipet. You will need about 200 uL. Add the 200 uL of bacteria and 800 uL fresh media into a clean cuvette. Measure the absorbance at 600 nm in the spectrophotometer. You may need to retake this reading until the absorbance is between 0.1 and 0.25. Empty the samples into the bottle of ethanol, then place all contaminated glassware into the tub of bleach. Turn off the incubator and allow to cool slightly. Wipe down the interior of the incubator with 1% bleach solution, then DI water, then ethanol. • Once the media in the reactor is at temperature, pour 55 mL of your inoculated bacteria into the reactor carefully. • Extract about 2 mL from the reactor using a syringe and place it in a microcentrifuge tube. Cap the tube tightly and move to the hood. Put 1000 uL into a clean cuvette and measure the absorbance at 600 nm. Again, empty the contents into the ethanol bottle and place all contaminated equipment into the tub of bleach. Wipe down the spectrophotometer area with a paper towel that has been wet with ethanol. • Every 30 minutes, repeat step 8. If your readings on the spectrophotometer begin to get high (i.e. above 0.25), you will need to dilute the next sample you take with fresh media. Be sure to note the composition of each sample (i.e. how much sample and how much is fresh media). • Once your measurements are stabilized for an hour, the reaction has completed. Take your final samples, then pour about 100 mL of bleach into the reactor. You may leave the bleach solution mixing in the reactor overnight. Discard the contents of your waste container and place it in the disinfection basin overnight. • Before you leave lab when your reaction is complete, clean the countertop and the hood with 1% bleach solution, then DI water, then ethanol. Step 8: Clean Up • Empty the basin of bleach solution and refill with soap and water. Leave your glassware to soak. • Disconnect the cooling water tubes and impeller motor as in Step 2. Place some paper towels down on the top of the cart and move the bioreactor on top of it. • Spray down the countertop and control station surface with 1% bleach solution, then distilled water, then ethanol. • Empty the contents of the bioreactor into a sink, and scrub all surfaces with soap and water. This includes the inside of the reactor, the impeller and tubes inserted into the reactor, and all exterior surfaces. • Throw out the paper towels and sterilize the top surface of the cart with ethanol. Once the

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reactor is clean, place it gently back on the cart and return it to the control base. Clean the rest of your glassware and cuvettes with soap and water. Leave glassware and bottles on the drying rack to dry. Place the clean cuvettes on a paper towel on the counter.