Objectives

Introduction

Due to the constraint in the available equipment to carry out the immobilization procedure aseptically, the experiment will be conducted without autoclaving. The immobilized cell reactor will be employed to convert glucose into ethanol anaerobically. The reasons for choosing this system of microorganism and product are many folds. First, the anaerobic condition will eliminate the need for aeration, which causes many technical problems. Secondly, the lack of oxygen will prevent the uncontrolled growth of aerobic contaminants in an unsterilized fermentor. The presence of high levels of ethanol should also discourage most microorganisms from taking over the fermentor. To reduce further the chance of contamination by bacteria, the pH of the fermentor will be kept low; a value of 4.0 should drastically slow down the growth of most bacteria but only slightly affect the yeast's ethanol producing capacity.

The production of ethanol in an immobilized bioreactor is a relatively well studied process. As high as 95% of the theoretical yield of alcohol based on glucose (8.5 % ethanol from 14% glucose) has been reported. A high space velocity, defined as the volume of nutrient feed per hour per gel volume, of 0.4-0.5 hr^-1 is commonly used to maximize the ethanol productivity. An ethanol productivity of 20 g/l-hr can be achieved.

Both the steady state response and the transient approach to the steady state will be studied in this experiment .

List of Reagents and Instruments

A. Equipment

• Beakers
• Graduated cylinder
• Balance
• Pipets
• Magnetic stirrer
• Syringe & needle
• Spectrophotometer with flow through cell
• pH probe and controller
• Microcomputer with data acquisition capabilities

B. Reagents

• Growth medium, see the recipe in Experiment No. 9.
• Alginic acid, sodium salt
• CaCl₂
• Yeast culture
• NH₄OH
• Reagents for glucose analysis
• Reagents for ethanol analysis (or a GC)

Procedures

1. Immobilized Cell Preparation:
   • Dissolve 9 g of sodium alginate in 300 ml of growth medium, following the same procedure adopted in enzyme immobilization to avoid clump formation. Stir until all sodium alginate is completely dissolved. The final solution contains 3% alginate by weight. See Note 1.
   • Thoroughly suspend about 250 g of wet cells in the alginate solution prepared in the previous step. Let air bubbles escape. See Note 2.
   • Drip the yeast-alginate mixture from a height of 20 cm into 1000 ml of crosslinking solution. (The crosslinking solution is prepared by adding an additional 0.05M of CaCl₂ to the growth media. The calcium crosslinking solution is agitated on a magnetic stirrer. Gel formation can be achieved at room temperature as soon as the sodium alginate drops come in direct contact with the calcium solution. Relatively small alginate beads are preferred to minimize the mass transfer resistance. A diameter of 0.5-2 mm can be readily achieved with a syringe and a needle. The beads should fully harden in 1-2 hours. Note that the concentration of the CaCl₂ is about one fourth of the strength used for enzyme immobilization.
   • Wash the beads with a fresh calcium crosslinking solution.
2. Immobilized Cell Reactor Construction:
   • Construct an immobilized cell reactor with a 500ml Erlenmeyer flask fitted as shown in Figure 1. Place the hardened beads in the flask and seal it with a rubber stopper with appropriate hose connections.
   • Make all necessary connections. Start the experiment by filling the flask with the growth media (100g/l glucose) to the working volume of 350ml.
3. Immobilized Cell Reactor:
   Then following sequence of events will be monitored both on-line and off-line. The responsibilities of on-line data acquisition and off-line sample collection and analysis will be shared by the entire class; the exact assignment will be determined in class. A microcomputer will be programmed to take data on the glucose concentration and the rate of NH₄OH addition needed to maintain the pH at 4.0. The off-line samples will be analyzed for the optical density (for free cell concentration), glucose concentration, and ethanol concentration. Furthermore, the liquid and gas flow rate will be measured with a graduated cylinder as indicated in Figure 2.
   • The reactor will be operated in a batch manner until no more glucose is utilized. This can be detected with the leveling off in the glucose concentration.
   • Substrate feeding will then commence at the rate of 0.4/hr. Record the substrate flow rate. The approach to the first steady-state during the start-up will be followed.
   • Various parameters (nitrogen consumption rate, carbon dioxide evolution rate, glucose concentration, ethanol concentration, and free cell level) at the high steady state are recorded.
   • Decrease the substrate feeding rate to 0.2 /hr Measure the substrate flow rate and follow the transient approach to the new low steady state.
   • Repeat part 2c) for the new steady state.
4. If time permits, continue shifting the flow rate and obtain more information on steady states. Continue operating the bioreactor until noticeable deterioration in the performance is detected due to gel swelling, cell death, or severe contamination.

Notes

1. To avoid the premature gel formation, the phosphate concentration in the medium must be adjusted to less than 100µM.
2. Because cell growth can break the bead and is generally considered undesirable beyond what is needed to compensate for the endogenous decay, the cells used for immobilization ideally should have just entered the stationary phase. An equivalent amount of dried cell culture may also be used in lieu of wet cell paste. The actual cell loading may be varied according to the substrate concentration in the feed and the desired product levels. The ratio of wet weight to dry weight is approximately 4 for most cells.

Discussions

The lower microorganisms (bacteria, yeasts, and fungi) can be rather easily immobilized with a number of methods: entrapment, ion exchange adsorption, porous ceramics, and even covalent bonding. In terms of dollar values, chemicals of plant origin account for the lion's share of the market. Some examples of plant extracts are drugs, flavors, and perfumes. Despite the recent surge in research activities in animal cell culture throughout the country, few applications actually exist beyond the production of monoclonal antibodies. Immobilized insect tissues have been used in pesticide research and has a potentially quite large commercial market in agriculture.

Most of the principles involved in enzyme immobilization are directly applicable to cell immobilization. Covalent bonding, affinity bonding, physical adsorption, and entrapment in synthetic and natural polymer matrices. The most popular and practical immobilization technique deals with cell recycle with an ultrafiltration membrane or a hollow fiber cartridge. Although this process is not ordinarily viewed as cell immobilization at all, it is functionally equivalent, the cell recycle devices effectively retaining the catalysts in a bioreactor and accomplishing the same objective as cell immobilization.

An immobilized cell bioreactor is well suited for those cells whose growth phases and product formation phases are uncoupled. Cell biomass and primary metabolites are growth associated products, but secondary metabolites such as antibiotics and various enzymes are produced during the stationary phase. The uncoupling of the phases means that productive cells cannot compete with the nonproductive cells in a continuously operated suspension fermentor because the productive cells spend the nutritional and energy resources producing chemicals in quantities far above the amount necessary for their survival, instead of reproducing themselves to propagate further. On the contrary, cell growth in an immobilized cell reactor must be severely limited if gel swelling or breakage is to be avoided. However, once the cells are immobilized, the cell viability must be concomitantly sustained over a long period of time. Thus, immobilization is advantageous for sustaining slowly growing cells, especially plant tissues. In summary, one wishes to keep the immobilized cells alive without multiplying.

Questions

1. Considering the reaction stoichiometry, calculate the theoretical yield of ethanol from glucose. What is the steady-state productivity of the bioreactor? Would you conclude that the productivity of an immobilized cell reactor is higher than a continuously operated suspension type?
2. Plot the response of the reactor (glucose concentration, productivity, etc.) as a function of time after a shift-up in the substrate flow rate. Was there any delay/lag in the response? How long did it take to reach the steady state? Repeat for a shift-down in the flow rate.
3. If immobilization procedure can be carried out aseptically, how long do you expect an immobilized cell reactor to operate between down times? What are some of the reasons for terminating a continuous fermentor.
4. Comment on ways to improve the experiment.

References

1. Mattiasson, Bo Immobilized Cells and Organelles, Volume I and II, CRC Press, 1983.
2. Venkatsubramanian, K., Immobilized Microbial Cells, in ACS Symposium Series, 106, American Chemical Society, Washington, D.C., 1979.
3. Nagashima, M., Azuma, M., and Noguchi, S., Technology developments in biomass alcohol production in Japan: continuous alcohol production with immobilized microbial cells, Ann. N.Y. Acad. Sci., 413, 457, 1983.

Forms

                              IMMOBILIZED CELL FERMENTATION
                                       DATA SHEET
-------------------------------------------------------------------------------------------------
  ----------------Nominal Values---------          --------------Measured Values---------------
-------------------------------------------------------------------------------------------------
Dilution Rate  Time Constant  Flow Rate     Time     Liquid      Gas      O.D.   Ethanol   Sugar
    D              T=1/D           F               Flow Rate  Flow Rate           Conc.    Conc.
  (hr-1)           (hr)        (ml/min)              (ml/min)  (ml/min)  (A.U.)   (g/l)    (g/l)
-------------------------------------------------------------------------------------------------
   0.10           10.0           0.67      Mon  6pm
   0.30            3.3           2.00      Wed  0am
   0.50            2.0           3.33      Wed 10am
   0.75            1.5           5.00      Wed  4pm
   1.00            1.0           6.67      Wed  9pm
   0.50            2.0           3.33      Thu  0am
   0.30            3.3           2.00      Thu  9am
   Quit                                    Thu  7pm
-------------------------------------------------------------------------------------------------

1. Measure the liquid flow rate.
2. Measure the gas flow rate.
3. Collect a vial of sample from the exit.
4. Measure the O.D. of the sample.
5. Centrifuge and freeze the supernatant.
6. Change the nutrient flow rate by adjusting the pump speed knob.
7. Measure the new liquid flow rate to ensure that it is reasonably close to the nominal value.
8. Analyze the off-line sample for sugar and ethanol concentrations. (To be done on next Monday.)