Hie all,
Iam working on microencapsulation technology. I am trying to use sodium alginate as a material to immobilise the cells. I tried using 4% sodium alginate along with 1.5% Cacl2 solution but couldn't achieve any satisfactory results. What could have possibly gone wrong? How can I troubleshoot this?
Dear Surajvanshikumar,
You may use the following procedure:
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.
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.
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.
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.
http://www.eng.umd.edu/~nsw/ench485/lab11.htm
Hoping this will be helpful,
Rafik
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