EXPERIMENT NO. 9

BATCH SUBMERGED FERMENTATION

OF BAKER'S YEAST IN A SHAKER FLASK

Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
ENCH485

Table of Contents

Objectives

To study the batch growth kinetics of a submerged culture.

Introduction

The proper procedure for a batch fermentation is first to inoculate a small flask of nutrient broth with a pure culture from a Petri dish, a culture tube (containing liquid nutrient), or a slant tube (containing solid gel). The inoculated flask is constantly agitated in a temperature controlled flask shaker. A small amount of the culture in the original flask is pipetted out during the exponential growth phase, or log phase, and is used to inoculate the next flask. This process is repeated a few times to ensure that the culture is acclimated before it is employed to study the fermentation kinetics. A similar process of repeated inoculation is carried out in the fermentation industry to build up enough inoculum needed to seed a larger fermentor. To reduce the shock resulting from a drastic change in the growth environment, the composition of the media used in preparing the inoculum should optimally be identical as that used in the main process.

When working with a pure culture, one must operate under the assumption that contaminating microorganisms are present everywhere in the open environment, a fact demonstrated in our previous experiment. It is important to know intuitively when sterile tools or glassware must be used and when sterilization is not necessary. This requires the ability to distinguish clearly the sterile side from the nonsterile side. In this experiment, the interior of the shaker flask is the sterile portion of the system. Anything that is that part of the system and anything that ever comes in direct contact with that part of the system must be sterile. Thus, the nutrient in the shaker flask before inoculation must be sterile, which in turn requires that the reservoir storing the filtered nutrient is sterile and that the entire process of dispensing the nutrient from the medium jar to the shaker flask is carried out aseptically. In addition, items that enter the shaker flask such as the cotton plug, inoculation loop, sampling pipets, and even air must all be sterile.

Most practical industrial fermentation processes are based on complex media because of the cost and the choice of the nutrients and the ease of nutrient preparation. For example, complex media for yeast fermentation can be easily prepared in a lab by following the same recipe as that used in the YPG agar, minus the agar: 5g/l yeast extract, 10g/l Peptone, and 5g/l glucose. However, the use of complex media is discouraged in the fundamental studies of fermentation kinetics because of the possibility of variations in the nutrient composition from run to run. For example, the exact content of a yeast extract preparation is not known, and its nutritional quality may vary from batch to batch. On the other hand, a defined medium can be reproduced time after time to ensure the reproducibility of biochemical experiments. The disadvantage of a defined medium is that there is always the possibility of missing some important growth factors. The formulation of a defined medium is often a tedious process of trial and error. However, a well formulated defined medium can support the healthy growth and maintenance of cells as effectively as, or sometimes superior to, a complex one. A defined medium will be used in this experiment.

List of Reagents and Instruments

A. Equipment

B. Reagents

Procedures

  1. Nutrient Preparation (Defined Medium):
    The overall nutrient composition is shown in Table 1. To facilitate nutrient preparation and to minimize the chance of making the fatal mistake of omitting one or two trace components, two concentrated (100 fold) stock solutions can be made according to the formula given in Table 2 for minerals and Table 3 for vitamins.
    • Mix the concentrated mineral stock solution according to Table 2. Adjust the quantity according to the need. Bottled solutions can be stored on a shelf for more than six months. A yellowish color may develop upon prolonged storage; however, yellowing of the solution seems to impart no harmful effect.
    • Mix the concentrated vitamin stock solution according to Table 3. Keep the stock solution in the dark at 4ºC. Discard the solution if mold growth can be visually detected. The storage life of the stock solution is approximately 2-3 months.
    • To obtain a working nutrient solution, add to 750 ml of stirred deionized water the components of Table 4 in the order listed. Precipitates may form if the order is not strictly followed. KOH pellets are used to neutralize the phosphoric acid and bring the pH close to the desired value. Add water to 1 liter. Finally, adjust the pH to the desired value by dropwise adding 1N KOH solution. (A pH value of 5.00 will be used in this experiment.)
  2. Nutrient Sterilization:
    Next, the prepared nutrient must be sterilized. Usually, this is done by autoclaving. However, autoclaving is not a practical sterilization method for the formulation used in this experiment. First, the heat of autoclaving will caramel the sugar and darken the nutrient to a brown color. Secondly, vitamins will be destroyed by the heat. Furthermore, the loss of liquid due to boiling during the autoclaving process will change the concentration of various nutrient components, including the rate limiting carbon source. Evaporation loss is especially severe when ethanol is the designated carbon source.

    Instead, membrane filtration will be used to sterilize the nutrient in this experiment. This can be accomplished by drawing the nutrient from a mixing jar and forcing it through an in-line filter (0.2 µm pore size) either by gravity or with a peristaltic pump. See Figure 1. The sterilized medium is fed into an autoclaved nutrient jar with a rubber stopper fitted with a filtered vent and a hooded sampling port. Do not overfill the nutrient jar, for the nutrient will be forced out of the venting filter and wet it. A wet venting filter must be aseptically replaced with another dry sterile one. Otherwise, the wet filter will support the unwarranted proliferation of a wide range of microorganisms which will soon destroy the filter membrane and enter into the nutrient jar and contaminate the broth. For the same reason, soon after the filling process is completed, clamp the tubing at position "A" as indicated in Figure 1, disconnect the in-line nutrient filtration unit, and wash the residual nutrient from the newly exposed part of the tubing. If the nutrient filter is to be reused, wash and autoclave it before it is destroyed by the microbial growth either due to clogging or breaking of the membrane.

    The hooded sampling port consists of a tubing reaching into the bottom of the nutrient jar and a rubber bulb on the side. Normally, a sampling bottle is attached to the sampling port to keep its tip airtight and sterile. When the rubber bulb is squeezed, the air in the sampling bottle is forced into the nutrient jar. When the bulb is released, nutrient equal to the volume of the displaced air is sucked up the sampling tube and is collected in the sampling bottle. A sterile medium bottle with the same cap thread size may be attached to the sampling port in place of a sampling bottle if fermentation is to be conducted directly in the medium bottle or if it is more convenient to store the media in smaller bottles, each holding enough for one or two shaker flasks. The content from the media bottles may later be poured into shaker flasks as needed. Alternatively, a sterilized flask may be placed under the exposed sampling tube. Applying pressure to the vent will force liquid out from the jar into the flask. Flame both the sampling port and the mouth of the sterile sampling bottle before screwing on the sampling bottle.

    Although the turbidity in the nutrient jar may be due to the precipitation of some of the nutrient components, it is almost always due to the presence of contaminants. At the first sign of contamination, either totally kill the contaminants by autoclaving or reduce the viability by adding bleach solutions. Discard the contents only after sanitization.

    The setup in Figure 1 is useful for preparing a relatively large volume of sterile nutrient. A simpler setup consisting of a vented filtration flask as shown in Figure 2 may be desired if the quantity needed is not too large.

  3. Shaker Flask Preparation:
    • Make cotton-gauze plugs to fit the mouth of 250ml shaker flasks.
    • Plug the flask and cover the plug with a piece of aluminum foil before autoclaving. The aluminum foil will prevent dust from directly settling on the cotton plug while standing on the shelf waiting to be used. This is generally the case where many flasks are simultaneously autoclaved for later use.
    • After autoclaving the flasks, cool them to room temperature.
    • Pour sterile nutrient into the flasks aseptically.
  4. Inoculum Preparation:
    • Find a single isolated colony of yeast on the Petri dish from which the culture is to be transferred.
    • Following the aseptic plate streaking techniques introduced in the previous weeks, lift a small loopful of the creamy culture off the agar plate. Dip the loop into a 250 ml flask containing 100 ml of 5.0g/l glucose. Swirl the loop in the nutrient solution to dislodge the selected culture from the loop.
    • Flame the neck of the flask and the cotton plug before inserting the plug back on the flask. Also, flame the loop to kill the residual microorganisms.
    • Place the flask in a temperature controlled shaker at 37ºC. The exponential growth phase will last from 2 to 24 hours after inoculation. The exact time and duration depend on the physiological condition of the inoculum. The instructor will provide an exponentially growing culture.
  5. Shaker Flask Inoculation:
    • Follow the same procedure as Step 1 to prepare nutrient solutions with the following carbon sources: 
           ----------------------------
     Run   Carbon Source  Weight
     ----------------------------
      A      Ethanol      5.0 g/l
      B      Glucose      5.0 g/l
      C      Sucrose      5.0 g/l
      D      Glucose      2.5 g/l
             Sucrose      2 5 g/l
     ----------------------------
    • Follow the same procedure as Step 2 to sterilize the nutrient through filtration.
    • Follow the same procedure as Step 3 to autoclave 1000 ml flasks and transfer 500 ml of the nutrient into each of them.
    • Inoculate the flask with 10 ml of suspended actively growing yeast culture obtained at the end of Step 4 with a sterile 10ml pipet.
  6. Batch Fermentation Monitoring:
    Remove the flask from the shaker and draw a sample at 90-120 minute intervals. The fermentation should last for approximately 24-36 hours before the culture enters the stationary phase. See Notes 1-2.
    • Although a 10 ml sample is more than adequate to analyze for optical density, glucose/sucrose concentration, and ethanol concentration, an extra 10 ml will ensure the availability of a sufficient quantity of sample broth, should the quantitative analysis fail repeatedly. Immediately after taking a 20 ml aliquot with a sterile pipet, flame the neck of the flask and place the plug back in the mouth.
    • Record the pH of the sample just taken.
    • Save a drop of the sample on a slide for microscopic examination later.
    • Initially, when the cell density is still low, the optical density of the sample can be measured without dilution with water. Perform this step quickly with a spectrophotometer at 550 nm. Then, filter out the cells from the sample. After the optical density is over 0.5, save 1 ml of the sample by pipetting it into a test tube for optical density measurement. Force the remaining sample through a filter.
    • The clear filtrate is collected in a tightly capped sampling vial for later analysis. Freezing the filtrate will better preserve the existing condition.
    • If a 1 ml sample is saved for the optical density measurement, dilute the sample with 5 ml of water. Record the optical density.
    • Clean the filter unit, test tubes, and pipet. Return the flask back to the shaker and get ready for the next sampling.
  7. Dry Cell Weight Measurement:
    Terminate the experiment when the stationary phase is reached. Obtain a calibration curve for the cell concentration in g/l as a function of the optical density.
    • Measure the volume of the remaining culture.
    • Weight an empty aluminum weighing pan or a sheet of dried filter paper stored in a desiccator.
    • Separate the cells from the broth either by centrifugation or by filtration.
    • After drying the cell paste in an oven set at 100ºC for 24 hours, measure the weight of the weighing pan or the filter paper.
  8. Quantitative Analysis:
    After the experiment is concluded, for each sample, measure the glucose concentration with the DNS reagent, the sucrose concentration according to the accompanying     Supplement D, and the ethanol concentration according to the accompanying Supplement E or a gas chromatograph.

Notes

  1. Because of the limited rate of microbial growth, the experiment will continue day and night. Each student will be assigned a period of time during which he is responsible for overseeing the experiment, including sample taking. Thus, more than ever, it is necessary that the group cooperate closely to complete the experiment. All the runs will be performed side by side.
  2. Because the entire sampling sequence must be carried out quickly, make sure that everything is in order before taking a sample.

Discussions

Yeast has been in use since the beginning of human civilization. It is still a very versatile microorganism widely used in a wide range of fermentation industries. For example, the carbon dioxide released as a result of carbohydrate metabolism is used to raise dough in baking; the ethanol produced supports a multi-billion dollar alcoholic beverage industry; single cell protein is used to supplement animal feed. It is also currently the most widely used microorganism for ethanol production as the alternative energy source, Zymomonas mobilis being the other potentially dominant one. Although fermentation was practiced even before recorded history, the fact that microorganisms were responsible for the leavening and brewing actions was not realized until the last century.

Questions

  1. For each run, calculate and plot the cell biomass concentration, glucose concentration, ethanol concentration, and pH as a function of time. Identify the major phases in a batch fermentation: lag, exponential, stationary, and death. How many growth phases are there? How many lag phases are there?
  2. Speculate on the existence of lag phases.
  3. Microorganisms are "picky eaters." Which carbon source does the yeast prefer the most? Which the least? Comment on the biological reason of the observed preference on the carbon source as related to the survival of the microorganism.
  4. What kinds of plots are needed to find the parameters of the Monod cell growth model, i.e. the maximum specific growth rate and the Michaelis-Menten constant? Report the parameters observed in this experiment for baker's yeast.
  5. Did the pH change during the course of the fermentation? If so, what was the cause of the change in the pH? Did the change in the pH coincide with the different batch growth phases?
  6. Identify the major elements of life, i.e. nutrient requirement, in a typical yeast cell. List the amount of each element in a typical yeast cell. Of these essential elements, which are represented in our synthetic medium formulation? What chemicals are used to provide each of these elements in our experiment? From the relative ratios of the elements present in the media, identify the limiting substrate. (Is the carbon source indeed the limiting substrate as we have implicitly assumed in applying the Monod model? Is every thing else in excess? If so, by how much?)
  7. Which nutrient components are vitamins, and why are they provided in the media? (Obviously they are not included to derive trace elements.)
  8. What are the roles of EDTA and phthalic acid?
  9. Comment on ways to improve the experiment.

References

  1. Oura, Erkki, Effect of aeration intensity on the biochemical composition of baker's yeast. I. Factors Affecting the type of metabolism, Biotech. Bioeng. 16, 1197, 1974.

Media Composition

     Composition of Defined Medium for Baker's Yeast
     --------------------------------------------
     Compound                       Concentration
     --------------------------------------------
     MgCl2*6H2O                     0.52     g/l
     (NH4)2SO4                     12.0      g/l
     H3PO4 (85%)                    1.6     ml/l
     KCl                            0.12     g/l
     CaCl2*2H2O                     0.2      g/l
     NaCl                           0.06     g/l
     MnSO4*H2O                      0.024    g/l
     CaSO4*5H2O                     0.0005   g/l
     H3BO3                          0.0005   g/l
     Na2MoO4*2H2O                   0.002    g/l
     NiCl                           0.0025  mg/l
     ZnSO4*7H2O                     0.012    g/l
     CoSO4*7H2O                     0.0023  mg/l
     KI                             0.0001   g/l
     FeSO4(NH4)2SO4*6H2O           0.035    g/l
     myo-Inositol                   0.125    g/l
     Pyridoxine-HCl (Vitamin B6)    0.00625  g/l
     Ca-n-Pantothenate              0.00625  g/l
     Thiamine-HCl (Vitamin B1)      0.005    g/l
     Nicotinic Acid                 0.005    g/l
     D-Biotin (Vitamin H)           0.000125 g/l
     Carbon Source (e.g. Glucose)   0-50     g/l
     EDTA                           0.1      g/l
     --------------------------------------------





     Mineral Stock Solution (100X)
     -----------------------------
     Compound       Weight-Volume
     -----------------------------
     H3PO4 (85%)     160.   ml
     KCl              12.00  g
     CaCl2*2H2O       20.00  g
     NaCl              6.00  g
     MnSO4*H2O         2.40  g
     CaSO4*5H2O        0.05  g
     H3BO3             0.05  g
     Na2MoO4*2H2O      0.20  g
     NiCl              0.25 mg
     ZnSO4*7H2O        1.20  g
     CoSO4*7H2O        0.23 mg
     KI                0.01  g
     -----------------------------
     Add water to 1 liter





     Vitamin Stock Solution (100X)
     --------------------------------
     Compound  Weight-Volume
     --------------------------------
     myo-Inositol          12.5    g
     Pyridoxine-HCl         0.625  g
     Ca-n-Pantothenate      0.625  g
     Thiamine-HCl           0.5    g
     Nicotinic Acid         0.5    g
     D-Biotin               0.0125 g
     --------------------------------
     Add water to 1 liter





     Normal Strength Working Nutrient Solution
     -----------------------------------------------
     Compound                         Weight-Volume
     -----------------------------------------------
     Phthalic acid, monopotassium salt   0.20   g
     MgCl2*6H2O                          0.52   g
     EDTA                                0.1    g
     (NH4)2SO4                          12.00   g
     Mineral Stock Solution             10.    ml
     FeSO4(NH4)2SO4*6H2O                 0.035  g
     Vitamin Stock Solution             10.    ml
     Carbon Source (e.g. Glucose)        0-50   g
     KOH (for pH=5.0)                    1.62   g
     -----------------------------------------------
     Add water to 1 liter
     Adjust pH to 5.00 with 1N KOH & 1N HCl solutions

Sampling Procedures

  1. Seek help from the T.A. if you are unsure of your duties.
  2. Record the time.
  3. Open the flask shaker door. The shaking action should stop when the door is open.
  4. Pump the syringe twice to let the dead volume in the sampling tube recirculate back into the flask, becauseit is the residual broth from the previous sampling and is not representative of the prevailing condition in the shaker flask.
  5. Withdraw approximately 7 ml of sample in the syringe, and disconnect the syringe from the rubber stopper by twisting it counterclockwise, not by pulling it. Do not contaminate the syringe tip by touching it. Inject the sample into a small beaker. Twist the syringe back onto the rubber stopper and collect another 7 ml of sample. At the end of sampling, twist the syringe back onto the rubber stopper the way you found it for the next person. Close the shaker door. A total of 15-20 ml should be enough to carry out all the required analysis.
  6. Save a drop of the sample on a slide glass. Visually inspect for the presence/absense of contaminants under a microscope at the end of the procedure.
  7. Measure and record the pH.
  8. Measure the optical density at 550nm. Dilute just enough sample with water as needed (probably 1 ml of sample with 5 ml of water) to keep the reading below 0.500; keep the remainder undiluted, because doing so will make the subsequent glucose/ethanol analysis very difficult. Report your O.D. Example: if you read an absorbance of 0.123 after diluting 1~ml of sample with 5~ml of water and if the absorbance of the supernatant from the next step, i.e., the background, was 0.214, you should report 
    O.D. = 0.123 X 6 - 0.214 = 0.524
  9. Centrifuge the undiluted sample at 10,000 rpm for 5 minutes to collect the supernatant. Avoid resuspending cells back into the supernatant.
  10. Measure the absorbance of the clear supernatant, and include this information in your calculation of the cell optical density above. Your supernatant should be colorless; however, this is not always true if other media, e.g., YPG formulation used to prepare plates last week, were used.
  11. Save the supernatant in a clean properly labelled sampling bottle and store it in the freezer. This frozen cell-free sample will be thawed and quantitatively analyzed for glucose and ethanol during the next class.
  12. Repeat the procedure for other flasks.
  13. Turn off microscope light. Turn off spectrophotometer. Clean up all glassware you have used before you leave.

Data Forms

                    SHAKER FLASK A (5 g/l Ethanol)

     -----------------------------------------------------------------
      Nominal Time   Name     Sample #    Actual Time    pH     O.D.
     -----------------------------------------------------------------
      3/24 12am   Inoculation   A1
            6am                 A2
            9am                 A3
           12pm                 A4
            3pm                 A5
            6pm                 A6
            9pm                 A7
      ----------------------------------------------------------------
      3/25 12am                 A8
            3am                 A9
            6am                 A10
            9am                 A11
           12pm                 A12
            3pm                 A13
            6pm                 A14
     -----------------------------------------------------------------





                    SHAKER FLASK B (5 g/l Glucose)

     -----------------------------------------------------------------
      Nominal Time   Name     Sample #    Actual Time    pH     O.D.
     -----------------------------------------------------------------
      3/24 12am   Inoculation   B1
            6am                 B2
            9am                 B3
           12pm                 B4
            3pm                 B5
            6pm                 B6
            9pm                 B7
      ----------------------------------------------------------------
      3/25 12am                 B8
            3am                 B9
            6am                 B10
            9am                 B11
           12pm                 B12
            3pm                 B13
            6pm                 B14
     -----------------------------------------------------------------





                    SHAKER FLASK C (5 g/l Sucrose)

     -----------------------------------------------------------------
      Nominal Time   Name     Sample #    Actual Time    pH     O.D.
     -----------------------------------------------------------------
      3/24 12am   Inoculation   C1
            6am                 C2
            9am                 C3
           12pm                 C4
            3pm                 C5
            6pm                 C6
            9pm                 C7
      ----------------------------------------------------------------
      3/25 12am                 C8
            3am                 C9
            6am                 C10
            9am                 C11
           12pm                 C12
            3pm                 C13
            6pm                 C14
     -----------------------------------------------------------------





             SHAKER FLASK D (5 g/l Sucrose + 5 g/l Glucose)

     -----------------------------------------------------------------
      Nominal Time   Name     Sample #    Actual Time    pH     O.D.
     -----------------------------------------------------------------
      3/24 12am   Inoculation   D1
            6am                 D2
            9am                 D3
           12pm                 D4
            3pm                 D5
            6pm                 D6
            9pm                 D7
      ----------------------------------------------------------------
      3/25 12am                 D8
            3am                 D9
            6am                 D10
            9am                 D11
           12pm                 D12
            3pm                 D13
            6pm                 D14
     -----------------------------------------------------------------

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Cheese Production from Milk
Batch Submerged Fermentation of Baker's Yeast in a Shaker Flask
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Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
301-405-1910 (voice)
301-314-9126 (FAX)
e-mail: nsw@umd.edu