EXPERIMENT 2
IMMOBILIZED CELL REACTOR FOR ETHANOL PRODUCTION
1.0
OBJECTIVES
1.1
To understand the principles of cell immobilization by using gel
entrapment method in alginate gel
1.2 To analyze the effect of immobilization on etahnol production
2.0
COURSE OUTCOMES
CO1 – Ability to recognize, compare and draw the schematic diagram for
specific types of bioreactors by applying the knowledge of science and
engineering.
3.0
INTRODUCTION
An immobilized cell are cells attached to an inert, insoluble material such as
calcium alginate (produced by reacting a mixture of sodium alginate solution
and cells with calcium chloride). This can provide increased resistance to
changes in conditions such as pH or temperature. The immobilization of cells
has proven particularly valuable, because it allowed cells to be easily reused
multiple times for the same reaction with longer half-lives and less degradation
and has provided a straightforward method of controlling reaction rate as well
as reaction start and stop time. It has also helped to prevent the contamination
of the substrate with enzyme/protein or other compounds, which decreases
purification costs. These benefits of immobilized cells have made them highly
applicable to a range of evolving biotechnologies.
4.0
MATERIALS AND EQUIPMENTS
4.1 1.0% and 3.0% (w/v) sodium alginate solution
4.2 0.2 M calcium chloride solution
4.3 20 g/L glucose solution
4.4 Saccharomyces cerevisiae grown for 24 hours
4.5 Distilled water
4.6 Flasks and column reactor
4.7 Pipettes and beakers
4.8 Hot plate and magnetic stirrer
4.9 Retort stand
4.10 Syringes
4.11 Spectrophotometer
4.12 Gas Chromatography
4.13 DNS
5.0
PROCEDURES
5.1 Preparation of immobilized enzyme beads
5.1.1
Mix 1 ml of yeast cell broth with 9 ml of 3% (w/v) sodium
alginate solution
5.1.2
Drip the polymer solution prepared in step 5.1.1 from a height
of approximately 20 cm into the stirred calcium chloride
(CaCl2) solution with a syringe at room temperature.
5.1.3
Leave the beads in the calcium solution to cure for 1 hour.
5.1.4
Filter the formed beads and wash thoroughly with distilled
water.
5.1.5
Dry the beads using filter paper (Whatman no.1) followed by
exposure to the open air for 1 hour before use.
5.2
6.0
Determination of ethanol produced from glucose in batch/continuous
reactor.
5.2.1
Transfer all of the gel beads prepared in Part 5.1 in a beaker
(for batch reaction) or a column (for continuos reaction). (You
will be comparing the results between batch and continuos
reactors later).
5.2.2
Add 10 ml of glucose solution in the beakers or column
containing the beads. The glucose and ethanol concentration
at this time is to be determined.
5.2.3
For batch reaction, shake the flasks at 250 rpm. For continuos
reaction, pump the glucose solution at 2 ml/hr.
5.2.4
Measure ethanol concentration every 30 min.
RESULT
6.1
Record your result into a table below and then determine the ethanol
production for each cycle from the graph based on your maltose
standard curve .
Time
Glucose
(g/L)
Ethanol
(g/L)
0
30 min
60 min
90 min
120 min
7.0
DISCUSSION
Discuss in detail the obtained results by relating them with theory which you
have learned in class. Compare the ethanol production between batch and
continuos reactors.
8.0
CONCLUSION
Draw some conclusions based on the experimental procedures done and the
results obtained in this experiment.
9.0
QUESTIONS
9.1
By comparing the ethanol production between batch and continuos
reaction, which gives highest production and why?
9.2
Give suggestions to improve the usability of the immobilized enzyme.
9.3
State your observations on the beads size before and after reaction.