CHEN 4800/5800: Bioprocess Engineering, Spring 1996
University of Colorado-Boulder,Chemical Engineering Dept.
Lab Exercise 2: Yeast Fermentation
In this laboratory exercise, we will study the growth characteristics of the yeast Saccharomyces cerevisiae in batch and continuous cultures. A lab-scale (5 liter) fermentor will be used to study batch growth kinetics of the yeast growing on glucose as the single carbon substrate provided in the presence of oxygen. A second fermentor will contain glucose and glycerol as two different carbon substrates for the yeast to utilize in the presence of oxygen. A third lab-scale fermentor will be used to observe the sustained metabolic oscillations that may occur in a continuous yeast fermentation at intermediate dilution rates. You will help set up the fermentations, learn how some of the culture conditions can be monitored and controlled by computer, take samples from the fermentors, measure the cell mass concentration (through optical density) and concentrations of glucose, ethanol, and glycerol to investigate the kinetics of cell growth, and the different patterns of multiple substrate utilization.
Saccharomyces cerevisiae uses the following three major pathways for growth on glucose:
1) The fermentation of glucose, which occurs primarily when the glucose concentration is high or when oxygen is not available. The cells attain a maximum specific growth rate of about 0.45 hr-1 with a low biomass yield of 0.15 g dry mass per gram glucose consumed and a high respiratory quotient (the ratio of CO2 production rate to the O2 consumption rate) and a low energy yield of only about 2 ATP per mole of glucose metabolized. The stoichiometry of this reaction is
C6H12O6 --------------> 2C2H5OH + 2CO2 +
where represents chemical energy utilized in the growth processes.
2) The oxidation of glucose, which predominates at glucose concentrations below 50 mg/l in aerobic cultures. The cells attain a maximum specific growth rate of only about 0.25 hr-1 with a biomass yield of about 0.5 g dry mass per gram glucose consumed, a respiratory quotient of about 1, and a high energy yield of 16-28 ATP per mole of glucose metabolized. The stoichiometry of this reaction is:
C6H12O6 + 6O2 --------------> 6CO2 + 6H2O +
3) The oxidation of ethanol, which predominates when fermentative substrates are not available or in very limited supply. The cells attain a maximum specific growth rate of about 0.2 hr-1 with a high biomass yield of about 0.6-0.7 g dry mass per gram ethanol consumed, a low respiratory quotient of about 0.7, and an energy yield of about 6-11 ATP per mole of ethanol metabolized. The stoichiometry of this reaction is:
C2H5OH + 3O2 --------------> 2CO2 + 3H2O +
Utilization of glycerol, a non-fermentable carbon source, by Saccharomyces cerevisiae is is repressed by glucose. After the depletion of any faster growth-supporting substrate, the enzymes necessary for the utilization of glycerol are induced, and an exponential growth phase on glycerol will follow a diauxic lag phase.
The temperature of the water bath surrounding the fermentors will be controlled at 30°C, and the impellers inside each fermentor will be operated at 400 rpm. The pH will be monitored and controlled at pH 5, the optimum for yeast growth. One of the two batch fermentors will have glucose as the only initial carbon source at a concentration of 5 g/l. The other batch fermentor will have two initial carbon sources - glucose at 1 g/l and glycerol at 4 g/l.
The continuous culture will begin as a batch culture. After the cells grow to sufficient concentration in the mid-exponential phase, the feed inlet and outlet pumps will be switched on to start the continuous culture, at an intermediate dilution rate of about 0.15 hr1. Oxygen will be sparged into this fermentor at constant rate of 10 l/min.
Introduction to Lab Procedures
You will monitor the growth characteristics of the yeast in the batch fermentors in three ways - by measuring the concentration of cells, by determining the concentration of the carbon substrates glucose and/or glycerol, and by assaying for the amount of ethanol present at regular intervals throughout the batch culture. Yeast cell concentration can be determined indirectly by measuring the optical density (absorbance) of a culture sample. You will take a sample of the culture medium from the fermentor and read its absorbance using a spectrophotometer. Up to a certain cell density, the concentration of yeast cells (gdw/l) in the sample is proportional to the absorbance reading on the spectrophotometer. The calibration curve correlating cell concentration with absorbance deviates from a linear correlation at high cell densities. Because of this, it's a good idea to dilute any of your high OD samples (that may be on the non-linear portion of the curve) by a known dilution factor to confirm that the measured OD values fall on the linear portion.
You'll use an automatic glucose analyzer to determine the glucose concentration in the culture samples and estimate how much glucose the cells have consumed. The glucose analyzer determines the amount of glucose in your samples according to the following reactions:
A membrane in the analyzer contains the glucose oxidase enzyme, and the analyzer senses the electron flow generated by the H2O2 when it is oxidized at the platinum anode. The current generated is proportional to the glucose concentration in the culture sample and the analyzer has been calibrated to give the glucose concentration directly.
The ethanol concentration is determined using a simple and quick chemical assay. This assay is based on the following reaction:
The amount of NADH produced in this reaction is proportional to the amount of ethanol added as a substrate. This quantity of NADH is determined spectrophotometrically at 340 nm. The Ethanol Assay Reagent that you will use for this assay contains NAD and Alcohol Dehydrogenase.
The concentration of glycerol in the cell-free culture medium will be analyzed by high performance liquid chromatography. The HPLC method will also provide measurements of glucose and ethanol in the liquid, in addition to glycerol.
The dissolved oxygen concentration in the culture medium is monitored in situ by a galvanic probe containing a silver anode and a lead electrode in an acetate electrolyte. The dissolved oxygen from the culture medium permeates the membrane and initiates an electrochemical reaction. The current generated is proportional to the dissolved oxygen concentration in the culture medium and is calibrated with the maximum solubility of oxygen in the culture medium when sparged with pure oxygen. The changes in the dissolved oxygen concentration with fermentation time in each fermentor is recorded and plotted directly on the computer screen. The oxygen is controlled by computer activation of valves which allow either pure oxygen or pure nitrogen to be bubbled into the fermentor. The "per" value indicated on the computer monitor is the percentage of time that the valve is open to pure oxygen. The remaining percentage of the time, the valve is open to pure nitrogen. You can regulate the flow rate of these gases to the fermentor using the rotameter on the front of the fermentation control apparatus.
2) Experimental Procedures
A) Determining Cell Concentration
1) Zeroing the spectrophotometer. Set the wavelength to 630 nm. Using the knob on the left, set the reading to 0% transmission when the chamber is empty; using the knob on the right, set the reading to 100% transmission when the chamber contains a test tube with about 4 ml of pure medium.
2) Determining cell concentration. First flush out the sample tube for your group's fermentor by taking an 8-10 ml sample which you will then discard. Take another 8-10 ml sample from your group's fermentor and gently mix. Take about 4 ml from your sample tube and transfer it to a glass test tube. Clean the outside of the test tube with ethanol, insert it into the spectrophotometer, and record the absorbance reading. If the absorbance reading is greater than 0.25, a typical limit of linear correlation between the absorbance and cell mass concentration, dilute the sample with a known amount of pure medium, and measure the absorbance again to check if the absorbance reading is on the linear portion of the calibration curve. Record the time you take the sample along with the absorbance reading in the linear range as well as the dilution factor.
B) Glucose measurement. Take your undiluted yeast sample tube and insert the thin plastic sample tubing from the glucose analyzer into it. If the analyzer is in the "Standby" mode, hit the "Run" key; after the machine has calibrated, hit the "Sample" key. If the analyzer is not in the "Standby" mode, just hit the "Sample" key. The glucose concentration in the medium from your sample will print out in about one minute. Record this value.
C) Ethanol assay
1) Take 0.5 ml from your undiluted sample, pipet it into an eppendorf tube, and spin at 14,000 rpm for 5 minutes.
2) Add 2 ml of Ethanol Assay Reagent to each of three cuvettes.
3) Add 10 µl of pure medium to the first cuvette; this is your Blank. Cover with Parafilm and mix gently.
4) Add 10 µl of the Ethanol Standard (.08% w/v) to the second cuvette; this is your Standard. Cover with Parafilm and mix gently.
5) Add 10 µl of sample (from the supernatant from step a) above) to the third cuvette; this is your Sample. Cover with Parafilm and mix gently.
6) Incubate cuvettes at room temperature for 10 minutes.
7) Clean the outside of the cuvettes, and then read the absorbance of each at 340 nm; use the Blank to zero the spectrophotometer. If the Sample absorbance is > 1.700, dilute the supernatant from your sample 1:4 and perform the assay again using this diluted supernatant. Don't redo the Blank and Standard.
8) Calculate the ethanol concentration for your sample using the following formula:
Record this value.
D) HPLC Assay
The protocol for this new assay is currently under final development and testing, and will be provided on the day of the experiment.
3) Report - Due March 7, 1996
A) Draw a graph of:
a) logarithm of cell concentration vs. time
b) glucose/glycerol concentration vs. time
c) ethanol concentration vs. time
for the two batch fermentors.
B) Determine the specific growth rate and the yield coefficient (gram dry weight of cells produced per gram of carbon source consumed) for each growth phase in the two batch fermentors. (The calibration between the absorbance reading and the dry cell mass concentration of the yeast cells will be performed at the end of the batch cultures and provided in the next class period).
C) Interpret these two graphs in light of the background information on yeast metabolic pathways and the "cybernetic" principle that cells choose to grow at the fastest possible rate. Specifically, discuss why the cell mass, glucose, and ethanol concentration profiles look as they do for each batch fermentor.
D) Briefly discuss the mechanisms of metabolic competition between the three pathways for the sustained oscillations that you may have observed in the continuous fermentation.