Experimental Procedures

1. The appropriate volume of YPG medium was prepared, which consists of:

YPG medium
Component Mass (g/L)
Glucose 20
Peptone 20
Yeast extract 10
Commercial antifoam 0.5 mL

2. The medium was then distributed into two Erlenmeyer flask (each 500 mL Erlenmeyer
flask containing 200 mL of YPG medium). The pH was adjusted to 4.5 by adding 2N NaOH
or 2N HCl.
3. The medium is sterilized for 3 hours along with the universal bottles.
4. After autoclaving, let the medium cool, then the medium was inoculated with 20 mL of
yeast cell suspension.
5. The culture was incubated at 30°C for 24 hours and 190 rpm rotational speed.
6. 10 mL sample was taken for every 6 hour interval starting from 0 hour to 42 hour into the
universal bottles.
Procedures to Analyze the sample obtained

i. Absorbance
1. The absorbance of the samples for each time interval was determined by using a
UV-VIS Spectrophotometer at wavelength 600 nm.
2. The reading of the absorption for each sample was recorded.
3. A calibration curve was plotted.

ii. Dry weight determination

1. Two dry clean microcentrifuge tubes (1.5 mL volume size) were carefully and
precisely weighed.
2. 5 mL culture sample was carefully mixed and 1.5 mL was accurately pipetted into
each tube.
3. The tubes were centrifudged at 3000 rpm for 10 minutes.
4. The supernatant was carefully decanted and the cell pellet was resuspend in
approximately 1.5 mL saline (0.9 % NaCl) and the tubes were re-centrifudged.
5. The supernatant was decanted and the tubes were placed in 90°C oven for 20 hours.
6. The tubes were removed from the oven and immediately placed in a dessicator
which contain a drying agent until cool.
7. The tubes were re-weighed.
8. CDW (X) was calculated by using this formula:

wt.tube and dried cells (g)−wt. of tube (g)

X (g.L-1) = X 103
sample volume (mL)

Then the correlation between the absorbance and CDW value can be developed.
This standard curve was used in the latter experiments.
iii. Ethanol determination

Part I
1. Four 150 mL or 250 mL beakers are labelled 1 to 4.
2. By using a 10 mL graduated cylinder, 5.0 mL of 0.25 M potassium dichromate
(K2Cr2O7) solution was measured and put into each beaker.
3. 1 drop of 0.1 M silver nitrate (AgNO3) was added into each beaker. The beaker was
then swirled immediately after the addition.
4. By using a burette, 5.0 mL OF 6 M sulfuric acid (H2SO4) was added into each
beaker. The beaker was swirled immediately after the addition.
5. Distilled water and alcohol were added into each beakers as indicated below:

Beaker # Alcohol Alcohol Water

Concentration
1 0% None 20 drops
(BLANK)
2 2.5 % 5 drops of 10 % alcohol solution 15 drops
3 5.0 % 10 drops of 10 % alcohol 10 drops
solution
4 10.0 % 20 drops of 10 % alcohol None
solution

6. The chemical reaction was allowed to proceed for 5 minutes. The beakers were
swirled several times during the five minutes.
7. By using the graduated cylinder, the contents of each beaker were diluted by adding
39.0 mL of distilled water into each beaker. Each beaker was stirred with a clean
stir rod.
8. On the spectronic 20, the wavelength was set to 560 nm.
9. With the cover of the sample holder closed, and no cuvette in place, the left knob
(the zero control) was turned until 0 % transmittance was read.
10. Using BLANK solution (beaker #1), the cuvette was filled until two-thirds full.
Then, it was placed in the sample holder and the instrument was set, using the right
knob (light control) to 100 % transmittance.
11. Then, the BLANK was removed and saved to adjust the transmittance before taking
every sample reading.
12. In another cuvette, it was filled two-thirds full with the contents from beaker #2 and
the absorbance was read. After taking the reading, the contents of the cuvette were
poured into a waste container.
13. Step 12 was repeated for beakers #3 and #4.
14. A calibration curve was created. The absorbance values were taken and the
corresponding alcohol concentrations was plotted. The line of the best fit was
drawn, going through the 0/0 point of the x and y axis.

Part II

1. A scenario was given, telling about the police giving a sample of the clear liquid
and asking to analyze it, in which it turns out to be unknown. The analysis is to
show whether the clear liquid is an alcohol or not.
2. The number of the unknown sample was written on the data sheet and the beaker
was labelled with the unknown number.
3. Steps 2 to 4 from Part I was repeated.
4. 20 drops of the unknown sample and no distilled water were added into the beaker
labelled unknown.
5. Steps 6 to 12 from Part I were repeated.
6. The alcohol concentration in the unknown sample was determined by using the
absorbance value from samples and the calibration plot from Part I.
iv. Glucose determination
1. The reducing sugar was determined by using DNS (3,5 dinitrosalicylic acid)
method.
2. The DNS reagent was prepared as follows:

5 g DNS was dissolved in 400 mL H2O. then, 8 g of NaOH was added followed by
300 g of potassium sodium tartrate (Rochelle salt). The volume was made up to 1
L with distilled H2O.

3. The following test tubes were prepared:

Tubes no.

Addition 1 2 3 4 5 6 7 8 9

1 mL of - 0 6 12 18 24 30 36 42
Samples
with
different
hour
interval
(hrs)
DNS 2 2 2 2 2 2 2 2 2
reagent
(mL)

4. The reaction mixture was boiled for 10 min and allowed to cool down. Then, 10
mL of distilled water was added.
5. The absorbance was read at 750 nm using tube 1 as reference.
6. The standard curve for the samples was prepared.