Background - Objective - Equipment - Emphasis - Prelab - Hints - UV/Vis Operation
This is a new experiment. You and the faculty will all be learning together. Because growing organisms typically takes more time than mixing chemicals, the amount of data any one team can collect in their two lab periods is limited. All teams will share their results for the design project. Each team will have a slightly different focus to their experiments. I expect that there will be give and take and there will be changes made as the quarter continues. This is actually a real research project. I don’t know the answer you should get.
Background (Return to top)
Micro-organisms are widely used to produce specialty chemicals and to eliminate persistent waste chemicals. Often, the reactor conditions that are ideal for production of the desired chemical or for elimination of the unwanted chemical are not the same as those to maximize growth of the micro-organism itself. Frequently, micro-organisms are kept and grown in one reactor where growth conditions are optimized, and then a sample from that reactor is used to inoculate the other reactor, in which chemical production or elimination will take place. In this way, a pure strain of micro-organism is maintained and used in a long series of batch or semibatch operations. Our company is trying to grow and maintain a specialized strain of baker’s yeast. It is our job to plan a series of experiments that will determine a mathematical equation to describe the rate of yeast growth and define the optimum conditions for yeast growth. In this case, “optimum” means the maximum ratio of yeast cell mass produced per dollar spent on yeast nutrient solution ingredients. We will call this ratio the yield. Yeast nutrient solution includes D-glucose (dextrose), peptone, and yeast extract dissolved in distilled water. The standard composition of the nutrient solution is 20 g/L glucose, 10 g/L yeast extract, 20 g/L peptone. Because our focus is the production of yeast, not the production of alcohol, the yeast will be grown aerobically.
Dr. Crist will provide you with copies of two papers that may or may not be useful:
· X. Li, X. Chen, and M. Hardin, “Investigation into the Propagation of Baker’s Yeast”, Chemical Engineering Education, pp. 196-199, summer 2004.
· M. Shuler, N. Mufti, M. Donaldson, R. Taticek, “A Bioreactor Experiment for the Senior Laboratory” Chemical Engineering Education, pp. 24-28, winter 1994.
You should also consult other references to learn about typical kinetic expressions to describe the growth of micro-organisms and the factors that growth rates and yields depend on. You should also learn why the nutrient solution contains each of those ingredients, so that you will be able to speculate on what effect each of them might have on the growth rate and yield.
Team #1 will investigate the effects of glucose concentration and yeast extract concentration on yeast cell growth at 25 ºC and 35 ºC. Team #2 will investigate the effects of yeast extract concentration and peptone concentration at 25 ºC and 35 ºC. Team #3 will investigate the effects of glucose concentration and peptone concentration at 25 ºC and 35 ºC. Team #4 will investigate the effects of glucose concentration and peptone concentration at 30 ºC and 40 ºC. Each team should perform four simultaneous experiments at one temperature in one lab period, and four simultaneous experiments at the other temperature in the next lab period. Each set of experiments should be designed to help you identify important factors and interactions between/among factors.
Also, each team should, for each experiment, measure the glucose concentration in the nutrient solution and the glucose concentration in each reaction flask after the yeast cell mass has reached the plateau. I want to know what mass of cells was produced (at the plateau stage) per mass of glucose consumed for each flask.
You may perform additional experiments. It would be simple to run a room-temperature experiment on the stirrer plate at the same time that the flasks were in the shaker bath.
Design Objective (Return to top)
Rather than designing a process, you will design a series of experiments. Based on the experimental results from this quarter from all teams, each of you will specify the experiments that you would do if you had four additional laboratory periods. These experiments should be aimed at determining a mathematical equation to describe the rate of yeast growth and defining the optimum conditions for yeast growth. In this case, “optimum” means the maximum ratio of yeast cell mass produced per dollar spent on yeast nutrient solution ingredients.
Equipment and Supplies (Return to top)
You have all of the usual equipment and supplies in the analytical room of the UO Lab available to you. In addition, there were some special items that can be used with this experiment.
· There is a shaker bath that can hold up to 4 flasks at constant temperature and agitate them constantly.
· There is a CO2 meter to measure gas phase CO2 concentrations.
· There is a glucose meter of the type used by diabetics for home monitoring. You may use up to 25 of the test strips for your experiment, which means you may make up to 25 glucose measurements. Each test strip can only be used once. The useful range of the glucose meter appears to be about 30 – 300 mg/dL. You may have to dilute your samples to stay in this range. For the glucose testing, dilute with distilled water.
· There is a stirrer plate.
· There are some stoppers that have been drilled so that you can plug a flask and bubble through air.
· There is a UV/Vis spectrometer and there are a number of cuvettes, which should be THOROUGHLY rinsed after you use them each period. This link for the spectrometer goes to the procedure for the kinetics experiment. Since you are already familiar with the operation of the spectrometer from the kinetics experiment last quarter, it should be a simple matter for you to adapt the procedure to make measurements of individual samples. In theory, the amount of light scattered by the sample (and therefore not reaching the detector), which the spectrometer will record as absorbance, should be proportional to the amount of yeast in the sample. In practice, the relationship between cell mass and “absorbance” is probably only linear over a fairly narrow range. Keep the absorbance below 1.0, and preferably below 0.5, by diluting the sample when necessary. You will have to develop and evaluate the calibration curve if you want to use absorbance as a surrogate for cell mass. For the UV/Vis measurements, dilute samples with nutrient solution. The nutrient solution should also be used for the blank.
Emphasis (Return to top)
The emphasis in this project is on the experimental design. Make sure that the experiments and data analysis you plan are designed to identify which factors have important effects and whether there are interactions between them. Make sure that the way you present your results makes any effects and/or interactions clear.
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Prelab Guidelines (Return to top)
These are in addition to the standard guidelines in the syllabus.
In your
introduction, give a general introduction
to cell growth kinetics, including the typical pattern of cell growth
and the
typical equations used to describe it.
Also, discuss the role that each of the nutrients probably plays. Use this information to develop your expected
data and results. The most important
parts of your prelab are the procedure
(which will
probably be modified significantly as the experiment progresses), the
text
matrix, and the data analysis sections.
..
Hints (Return to top)
· The lag phase lasted about 1 hour in test runs in December 2004 with approximately 1 g of yeast in approximately 150 ml nutrient solution.
· I recommend quantifying the amount of yeast as dry mass.
· Pieces of filter paper weigh about 1 g each. Don’t forget to weigh before you filter.
· The glucose meter seems to work best in the range of roughly 30 – 300 mg/dL. Variation of ± 5 % in readings does not seem unusual. You may have to dilute samples to keep the concentration in the working range.
· Fill the flasks only about half or two-thirds full, in case of foaming.
· Remember that you are not really measuring absorbance with the spectrometer; it is really scattering. The relationship between cell mass and “absorbance” will probably be linear only over a relatively small range. Dilute your samples to keep the “absorbance” below 1, and preferably below 0.5.
· It is normal for the nutrient solution to be yellow.
· Make fresh nutrient solution at the start of each lab period. Do not try to store it, even overnight.
· Be sure to tell Jim a day in advance what temperature you will want for the shaker bath. mailto:caesar@bobcat.ent.ohiou.edu
· Send mail to Dr. Crist: crist@ohio.edu.
· Return
to top
of the yeast fermentation handout.
(Last modified on 01/06/07)
Prepared by Dr. Valerie Young 2006