BIOTECH 2B03 Lab 5.docx

Experiment 5 – Anaerobic Fermentation of Yeast Purpose To study batch growth yields for yeast growing on glucose as the single carbon source substrate under anaerobic conditions. Introduction *The basis of biotechnology is often fermentation biochemistry, as yeasts, molds, or bacteria must be cultur...

Experiment 5 – Anaerobic Fermentation of Yeast Purpose To study batch growth yields for yeast growing on glucose as the single carbon source substrate under anaerobic conditions. Introduction *The basis of biotechnology is often fermentation biochemistry, as yeasts, molds, or bacteria must be cultured in order to yield a high titre of the product desired. Using the fermentation technology, large genetic engineering, pharmaceutical, agricultural, food and diagnostics companies are synthesizing their products in huge quantities, heretofore unavailable without tremendous costs involved. Fermentation is one of the oldest known biological processes, dating back thousands of years. Fermentation involves the use of microorganisms to carry out an enzyme catalyzed transformation of sugars (organic matter) to ethanol. Yeast is one of the first microbial species involved in the fermentation process. The fermentation of glucose occurs primarily when the glucose concentration is high or when oxygen is not available under anaerobic conditions. The stoichiometry of this reaction is: C6H12O6 → 2C2H5OH + 2CO2 + e- As the yeast consumes sugar, the microbial growth rate decreases and falls to zero. The oxidation of glucose predominates in low glucose concentration below 50 mg/L in aerobic cultures. The stoichiometry of this reaction is: C6H12O6 + 6O2 → 6CO2 + 6H2O + e- Anaerobic fermentation may be carried out in a bioreactor which is sealed to the atmosphere and continuously stirred. The pH and temperature are monitored and controlled for optimum yeast growth. Yeast cell biomass concentration can be determined indirectly by measuring the optical density of a culture sample. Ethanol concentration is determined using chemical assay or using a refractometer. Reference Industrial Microbiology, An Introduction Waites, Morgan, Rockey and Higton Preparation Videos Please watch all associated videos on A2L prior to the start of your lab session Preparation Questions List three general uses for industrial fermentation ethanol. Define the terms “commercially sterile” and “axenic” with respect to fermentation. Explain the term Refractive Index (n) of liquids, and describe how the values may be measured experimentally. Reagents 1 M NaOH solution Malt extract Yeast extract (Refrigerate) Bactopeptone Glucose, sucrose, fructose, molasses, grapes/grape juice, other fruit/juice, grains(?) NaCl Yeast (Refrigerate) Formaldehyde solution 37% Hypochlorite bleach solution Anaerobic Start-Up Procedure: Week One Procedure Preparation of Media In a 500ml capped Erlenmeyer flask, mix: 1.5g malt extract 1.5g yeast extract 2.5g Bactopeptone 250ml dH2O (alternative: also add 5g NaCl) In a 500ml capped Erlenmeyer flask, mix: 200g glucose (alternative: use other sugars or different concentrations – 100gr, 50gr) bring up to 250ml with dH2O Place aluminum foil on top of the flask and screw on the cap. There should be 2” of foil on the outside of the flask. Unscrew the cap a quarter turn. If screw caps are not available, use a sponge plug, taped into neck of flask, then covered with aluminum foil. Label each flask with a felt tip pen. Autoclave the mixtures on the liquid setting (approximately 45 minutes). Store the two mixtures until week two in refrigerator. Label samples. Store in refrigerator. Preparing the Bioreactor WEAR GLOVES, GOGGLES AND LAB COAT Remove the lid and attached hosing from the bioreactor, and dump any contents down the drain. Remove the Dissolved Oxygen probe (DO Probe) from the bioreactor. DO NOT EXPOSE THE DO PROBE TO BLEACH Spray the inside of the reactor as well as the stirrer, sparger, sample port, etc. with bleach, and rinse thoroughly with dH2O. Replace the lid. Wipe the DO probe with a mild solution of formaldehyde (formalin). Do not re-insert the DO probe into the bioreactor. Store the reactor until week two. Running the Bioreactor: Week Two Safety Considerations Wear lab coat, gloves and safety goggles. Never start the temperature control program on the bioreactor control panel without first inserting the temperature probe into the bioreactor. Always make sure that the stirrer motor is safely secured to the bioreactor. Fermentation Procedure Remove the lid of the reactor. Rinse the stirrer, sparger, sample port, etc. and inside of the reactor thoroughly with dH2O, until there is no longer any residual odour of bleach in the reactor. Rinse the DO probe with dH2O Empty the contents of the two flasks containing the media from week one into the bioreactor. Top up the bioreactor to the 3L mark with dH2O (to about 3cm below the metal flange). Take a 20ml aliquot from the bioreactor vessel with the syringe. Put it in a 30ml capped test tube. Store in the fridge. This will be used as the blank. Add 3g of yeast to the bioreactor vessel. Place the bioreactor top on the vessel. Tighten down the knurled nuts on the top in a criss-cross pattern. Insert the temperature, pH level and DO probes into the reactor. Attach the thermal jacket to the reactor. Attach the 0.1 M NaOH supply hose to one of the input ports for acid or alkali. Close the other two inputs with a stop cock to keep the reaction anaerobic. The reactor must be kept sealed to the atmosphere Close the sparger port with a stop cock. Set up a water gas trap using a side armed Erlenmeyer flask connected to the CO2 vent Connect the level control hosing to the sample port. Level control should be above the surface of the liquid. Turn on the biocontroller. Set the following set points on the controller: Temp: 30°C (alternative: use 25°C or 40°C) pH: 3.0 Stirrer: 550rpm Samples are withdrawn from the sample port using a syringe and collection jar, so that the reactor is not opened to the atmosphere Make a note of the dissolved oxygen (DO) and pH every five minutes for the first half hour or until it levels off. Note how long each takes to level off and at what value it levels off. Collect 5ml and 20ml samples starting with the beginning of the lab period, and every ~24 hours over the next 3 days. SAMPLING PROCEDURE FOR THE BIOREACTOR Insert the syringe in the end of the sampling port tube. Open the tube clamp. Drawback the plunger to the 20ml mark. Then depress the plunger reintroducing broth back into the bioreactor. Repeat step 3 two more times (this clears the tube of any unreacted broth). Pull the plunger back to the 25ml mark and close the tube clamp disconnect the hose from the syringe and inject the sample into a 30ml capped test tube. (Take another aliquot of about 5ml and inject it into the test tube.) Be sure that the tube clamp is closed tightly after all sampling (keep the reaction anaerobic). You are now ready to start the procedures for the optical density and ethanol content. Please split these tasks between the group members. After you have taken your final day‘s sample, thoroughly clean the bioreactor as described above in “Preparing the bioreactor”. Determining Ethanol, Glucose and Yeast Cell Concentration Biomass Content: Using the CECIL 2011 Visible Spectrophotometer: Set the wavelength to 600nm. Turn on and allow the instrument to warm up for 20 minutes. Be sure to select a Visible cuvette; it will have a V on the bottom. Your blank must be clear (do not shake) there may be sediment on the bottom. If it is not clear place it into a centrifuge until the top portion is clear. Take the cuvette and place 3.8ml of distilled water into it, and then add 200ul of the blank into the cuvette, mixing completely. Insert the cuvette it into the cell holder, close the lid then press the zero button to set the absorbance to zero or (100% transmission) for the blank. Remove the cuvette. Prepare a known dilution of the fermentation broth in the same cuvette; a 20X dilution is a good starting point. Withdraw 200μl of broth sample from the test tube using the micropipette. Re-suspend well before withdrawing. Add the 200μl of broth into the cuvette. Add 3.8ml dH20. Re-suspend the sample completely. Measure the absorbance of the sample at 600nm. Absorbance values in the range of 0 to 1 are acceptable. If the absorbance is greater than 1, dilute the sample further. Record the dilutions. Record the yeast cell absorbance in Table 1. Clean the cuvette and place it upside down in the tray. Glucose: Use glucose test strips on the undiluted sample; follow instructions on box. Record the value. Ethanol: Separate ethanol using the micro-distillation apparatus. RAPID DISTILLATION - START UP Turn on the condenser cooling water at the sink; line up the marks on the valve. Rinse out still pot by adding a 20ml aliquot of distilled water to the addition funnel, open the addition funnel valve and let it drain into the still pot, close the valve. Open up the aspirator valve until all of the water from the still pot has drained out, then close the aspirator valve. Add your 20ml sample to the addition funnel, open the valve and let it drain into the still pot, then close the addition funnel valve. Place your receiving flask (25ml Erlenmeyer) under the condenser outlet. Make sure the water reservoir is 2/3 full; this is approximately at the height of the label on the water reservoir. If it is low, slowly add water using the water reservoir valve. Turn on the heater switch, and then set the heater control to 9. The water will start to boil in about 5 minutes. After 10 ml of distillate has been collected, cork the flask and shut down the apparatus. RAPID DISTILLATION - SHUT DOWN Turn off the heater switch; return the heater control to the zero setting. Add a 20ml aliquot of distilled water to the still pot and rinse using steps 2 and 3 from start up procedure. Repeat the rinsing procedure two more times; be sure to leave the last 20ml aliquot in the still pot and close both valves. Turn off the condenser cooling water valve at the sink. USING THE ETHYL ALCOHOL REFRACTOMETER Use the hand held American Optical Refractometer. Record the Refractive index (R.I.) of the solution. Record the ethanol concentration in Table 1 using the CRC Handbook for conversion of R.I. to % ethanol or use the Atago Digital Refractometer (see supplier manual for instructions). Fill a small beaker with distilled water and get two pasture pipettes (one for the blank, one for the sample distillate). Turn on the refractometer. Clean the lens with a Kim-wipe. Place 2 or 3 drops of distilled water on the lens; press the zero button. When the readout stops blinking and reads zero, wipe off the lens, and using the other pasture pipette take up some of your sample distillate from the stoppered flask. Place 2 or 3 drops on the lens. Take 2 or 3 readings. Clean off the lens; turn off the instrument and place it back in the box. Table 1: Yeast and Ethanol Concentration Data Initial Measurement 24 Hours 48 Hours 72 Hours % Ethanol [Glucose] Yeast (Abs) pH Reading Report Note: A formal lab report is required for this lab. You may hand in one report for per lab group. Make sure to reproduce one formal set of ‘Results/Observations’ in word-processed form. Also, include photocopies/scans of all individual ‘Original Data’ and put your name on the top of each scan/photocopy. Plot your and class results of % ethanol concentration, yeast cell optical density and glucose assay verses time on one graph for each measurement. Analyze each of the data based on different variables studied by different groups for % ethanol yield, yeast biomass and glucose assay and figure out whether the results obtained followed the expected path or not. If the results obtained not as expected, then what you think should have been with each variable tested in this experiment and why? You should explain and discuss all of that in your discussion.  Post Laboratory Questions Write out the chemical equation for fermentation of your particular sugar or starch substrate. If not starting with glucose, what extra enzyme(s) must yeast use to breakdown your substrate? Do the time and concentration data for glucose, ethanol and yeast follow an expected path? Explain. In particular, observe whether the ethanol readings are increasing or decreasing with time, and explain your observations. HINT: Look at the reaction equation for anaerobic fermentation. What change(s) do the refractometer readings represent over time in the fermentation broth? Is there a limit to the % ethanol that can be obtained using this method? If so, explain why i.e. what microbial properties and bioreactor conditions/parameters affect this limit? The yeast cell concentration is measured indirectly by measuring the optical density of the culture. Suggest a method to determine the actual yeast concentration. Compare the pH at 0 hrs (day 0) to 24 hrs (day 1). How did it change? Why did the pO and pH change? Did it change significantly from day 1 to day 3? If they did change rapidly at the beginning of the reaction and then stop changing? Why or why not? Be specific.