CHEMICAL AND BIOMOLECULAR ENGINEERING LAB

CH3802

Fermentation

Location: N1.2-B2-41

Name: Stanley Johnson Joel Smith

Matric Number: U2120799L
Group: LAM36
Date of experiment: 16/10/2023
 Yeast Fermentation
Abstract

Yeast as a unicellular fungus plays a crucial role in the fermentation process to produce ethanol.
Ethanol is an eco-friendly alternative solution to fossil fuels and its demands are growing rapidly. The
choice of growth conditions, aerobic or anaerobic will impact the metabolic pathways a lot. As
described below, yeast converts sugar into ethanol and carbon dioxide under anaerobic conditions.

This experiment will investigate the factors influencing the efficiency of ethanol production by yeast.
The yeast cells progress through various growth phases known as lag phase, exponential phase,
deceleration phase, stationary phase, and death phase.

To optimize the fermentation process for ethanol, we could (1) use a high-sugar feed, (2) use a
flocculating yeast for easier separation and (3) integrate advanced sensors for precise fermenter
monitoring and control. There is also 2 ways in which ethanol production is increased. The first
method is through repeated batch fermentation using flocculating yeast which encourages yeast cell
separation leading to increased biomass and increased glucose conversion. The 2nd method is by
adding a nitrogen source which promotes higher biomass while maintaining yeast viability in glucose.

1
 Objectives

• To understand and analyze the process of yeast fermentation and characterize its efficiency
through cell growth, glucose consumption and ethanol production at various temperatures.
• To comprehend and understand how the different equipments can be used to measure
glucose and ethanol content.
• Determine how the temperature affects the rate of fermentation and what is the optimal range
of temperature for fermentation.

Results

Cell concentration (% weight) versus time, t

[Our group worked on T = 35 ̊C ]

Cell concentration table

Cell Actual cell

Time(hrs) OD reading concentration Dilution concentration
1 0.1199 -0.00064 10 -0.006377776
2 0.141 0.000601 10 0.006007234
3 0.1461 0.000921 10 0.009208245
4 0.1763 0.002993 10 0.02992799
5 0.1925 0.004236 10 0.042357332

Sample calculation:

Based on eqn(3),

Where b0 = - 0.0055, b1 = 0.0282, b2 = 0.0812 , b3 = 0.182

At t = 1 hr,

2
Ccell = -0.0055 + (0.0282 x 0.1199) + (0.0812 x 0.11992) + (0.182 x 0.11993)

= -0.0006377776

However , this concentration is for the diluted sample. Therefore following the dilution law,

Ccell = - 0.0006377776 x 10 = - 0.006377776

Cell concentration table for T = 30 ̊C

Time(hrs) OD reading Ccell Dilution Actual Ccell

1 0.0986 -0.001755594 10 -0.017555943
2 0.1598 0.001822566 10 0.018225664
3 0.1912 0.004132445 10 0.041324445
4 0.2147 0.006098766 10 0.060987657
5 0.306 0.015947219 10 0.159472193

Cell concentration table for T = 25 ̊C

Time(hrs) OD reading Ccell Dilution Actual Ccell

1 0.131 -3.17424E-06 10 -3.17424E-05
2 0.133 0.000115127 10 0.001151267
3 0.145 0.00085108 10 0.008510798
4 0.151 0.001236258 10 0.012362583
5 0.213 0.005949337 10 0.059493375

3
Figure 1. Graph of cell concentration against time ( 35 ̊𝐶 )

Figure 2. Graph of cell concentration against time ( 30 ̊𝐶 )

Figure 3. Graph of cell concentration against time ( 25 ̊𝐶 )

4
pH and ORP (mV), versus time, t

25 30 35
Time ORP(mV) pH ORP(mV) pH ORP(mV) pH
0 16.88 6.08 49.34 5.998 67.35 5.953
5 19.49 6.068 42.12 5.971 63.88 5.883
10 18.3 6.051 31.23 5.942 57.25 5.855
15 15.05 6.03 27.84 5.91 47.51 5.817
20 14.78 6.006 22.63 5.876 44.25 5.777
25 12.6 5.983 10.95 5.842 36.93 5.735
30 8.425 5.955 1.625 5.809 16.94 5.696
35 5.738 5.932 -18.23 5.779 -17.78 5.657
40 1.625 5.907 -39.84 5.751 -38.34 5.625
45 -8.212 5.882 -49.09 5.724 -52.45 5.591
50 -20.06 5.858 -55.63 5.698 -58.85 5.56
55 -32.38 5.836 -64.44 5.671 -63.88 5.533
60 -43.94 5.814 -66.1 5.646 -69.85 5.506
65 -49.71 5.794 -68.53 5.624 -72.6 5.481
70 -54.13 5.775 -74.81 5.601 -74.38 5.455
75 -60.14 5.754 -75.75 5.581 -78.56 5.433
80 -62.53 5.739 -77.55 5.562 -80.97 5.409
85 -64.88 5.722 -83.18 5.54 -82.75 5.384
90 -69.4 5.703 -84.32 5.519 -87.08 5.361
95 -70.79 5.685 -85.9 5.499 -88.76 5.338
100 -72.31 5.669 -93.68 5.442 -90.62 5.316
105 -76.54 5.653 -98.61 5.422 -94.67 5.293
110 -77.26 5.635 -97.5 5.403 -96.56 5.273
115 -78.49 5.62 -97.1 5.384 -97.31 5.252
120 -82.38 5.605 -101 5.366 -98.12 5.233
125 -82.61 5.59 -101.4 5.347 -98.5 5.21
130 -83.09 5.575 -101.9 5.33 -97.63 5.189
135 -86.74 5.56 -103.4 5.313 -97.63 5.17
140 -87.14 5.545 -106.9 5.295 -99.26 5.148
145 -87.86 5.533 -107.1 5.276 -98.87 5.13
150 -91.4 5.518 -108.6 5.257 -99.25 5.111
155 -92.18 5.504 -111.9 5.237 -101.2 5.089
160 -92.75 5.488 -112.4 5.218 -101.4 5.067
165 -95.49 5.476 -113.9 5.2 -102 5.047
170 -96.99 5.464 -114.8 5.181 -104.1 5.028
175 -97.68 5.45 -114.3 5.163 -103.9 5.006
180 -100.2 5.435 -115.3 5.144 -104.1 4.989
185 -100.8 5.424 -117.2 5.126 -103.8 4.966
190 -100.3 5.411 -117.3 5.108 -104.4 4.948
195 -101.7 5.4 -117.4 5.089 -106.5 4.927
200 -102.1 5.388 -120.3 5.071 -108.6 4.907

5
205 -102.2 5.375 -120.3 5.053 -108.5 4.887
210 -104.2 5.361 -120.5 5.034 -109.4 4.867
215 -105.3 5.348 -123.3 5.018 -112.8 4.849
220 -105.1 5.337 -123.2 4.999 -112.6 4.829
225 -105.6 5.324 -124 4.983 -112.7 4.808
230 -108.2 5.309 -116.4 4.964 -115.5 4.789
235 -108.4 5.297 -114.9 4.949 -115.1 4.768
240 -108.9 5.284 -116 4.932 -114.4 4.751
245 -110.8 5.271 -118.7 4.914 -111 4.731
250 -109.9 5.26 -118.4 4.897 -109.7 4.711
255 -109.8 5.248 -119.6 4.88 -108.7 4.693
260 -111.6 5.233 -123.4 4.864 -108.8 4.673
265 -111.3 5.222 -124.1 4.848 -107.3 4.654
270 -111.5 5.214 -126.4 4.83 -105.2 4.636
275 -113.9 5.199 -129.2 4.813 -104 4.618
280 -113.3 5.186 -130 4.797 -99.53 4.599
285 -113.3 5.175 -130.8 4.78 -94.31 4.582
290 -115.6 5.161 -63.14 4.76 -89.15 4.565
295 -115 5.149 -61.04 4.745 -83.21 4.547
300 -114.6 5.135 -66.39 4.728 -72.13 4.533

Graph of ORP and pH against time( 25 C

̊ )
40
20
0
-20
-40
ORP
-60
pH
-80
-100
-120
-140
105
0
15
30
45
60
75
90

120
135
150
165
180
195
210
225
240
255
270
285
300

Figure 4. Graph of ORP and pH against time (25 ̊C)

6
 60
Graph of ORP and pH against time( 30 ̊c)
40

20

-20 ORP

-40 pH

-60

-80

-100

-120

-140

110
0
10
20
30
40
50
60
70
80
90
100

120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
Figure 5. Graph of ORP and pH against time (30 ̊C)

Graph of ORP and pH against time( 35 C

̊ )
100

50

0
ORP
-50 pH

-100

-150
105
0
15
30
45
60
75
90

120
135
150
165
180
195
210
225
240
255
270
285
300

Figure 6. Graph of ORP and pH against time (35 ̊C)

7
Sugar content (°Plato) versus time, t

Temperature 35 C̊
Glucose(%wt) Sugar( P
̊ lato) Sugar( P̊ lato)
Time Refractometer DMA 35 Alex 500
0 6.4 5.8 5.7
1 6.3 5.7 5.6
2 6.1 5.5 5.46
3 6.1 5.3 5.29
4 5.9 5.1 4.93
5 5.7 4.9 4.8

Temperature 30 C̊
Glucose(%wt) Sugar( P
̊ lato) Sugar( P
̊ lato)
Time Refractometer DMA 35 Alex 500
0 7.6 6.6 6.4
1 7.4 6.2 6.12
2 7.1 5.9 5.88
3 7.2 5.9 5.81
4 7 5.7 5.58
5 6.6 5.4 5.29

Temperature 25 C ̊
Glucose(%wt) Sugar ( P
̊ lato) Sugar ( P
̊ lato)
Time Refractometer DMA 35 Alex 500
0 6.8 6.2 6.2
1 6.6 6 5.98
2 6.6 6.1 6.01
3 6.4 5.8 5.72
4 6.4 5.8 5.65
5 6.4 5.6 5.57

8
 Graph of sugar content against time 25 C
̊
8
7
6
5
4
3
2
1
0
0 1 2 3 4 5 6

Refractometer DMA 35 Alex 500

Figure 7. Graph of sugar content against time (25 ̊C)

Graph of sugar content against time 30 C

̊

8
7
6
5
4
3
2
1
0
0 1 2 3 4 5 6

Refractometer DMA 35 Alex 500

Figure 8. Graph of sugar content against time (30 ̊C)

9
 Graph of sugar content against time 35 C
̊

7
6
5
4
3
2
1
0
0 1 2 3 4 5 6

Refractometer DMA 35 Alex 500

Figure 9. Graph of sugar content against time (35 ̊C)

Measured and calculated alcohol content, %v/v versus time, t

25 ̊C
Density (g/cm^3) OG-PG f Alcohol Content
Time (hr) Calculated Measured
DMA 35 Alex 500 DMA 35 Alex 500 DMA 35 Alex 500
DMA 35 Alex 500 Alex 500
0 1.0215 1.0224 0 0 0.1243 0.1243 0.0000 0.0000
1 1.0208 1.0217 0.702 0.702 0.1244 0.1244 0.0873 0.0873 1.62
2 1.0209 1.0219 0.6017 0.501 0.1244 0.1244 0.0749 0.0623 1.68
3 1.0196 1.0207 1.905 1.705 0.1245 0.1245 0.2372 0.2123 1.6
4 1.0194 1.0204 2.106 2.005 0.1245 0.1245 0.2622 0.2496 1.86
5 1.0189 1.0201 2.607 2.307 0.1246 0.1245 0.3248 0.2872 1.88

30 ̊C

Density (g/cm^3) OG-PG f Alcohol Content

Time (hr) Calculated Measured
DMA 35 Alex 500 DMA 35 Alex 500 DMA 35 Alex 500
DMA 35 Alex 500 Alex 500
0 1.0215 1.0224 0 0 0.1243 0.1243 0.0000 0.0000
1 1.0214 1.0223 0.100437 0.100437 0.1243 0.1243 0.0125 0.0125 0.76
2 1.0202 1.0212 1.30568 1.205243 0.1244 0.1244 0.1625 0.1500 0.72
3 1.0201 1.021 1.406117 1.406117 0.1244 0.1244 0.1750 0.1750 0.82
4 1.019 1.0201 2.510923 2.310049 0.1246 0.1245 0.3127 0.2877 0.96
5 1.0179 1.019 3.615728 3.414855 0.1247 0.1246 0.4507 0.4256 1.24

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35 ̊C

Density (g/cm^3) OG-PG f Alcohol Content

Time (hr) Calculated Measured
DMA 35 Alex 500 DMA 35 Alex 500 DMA 35 Alex 500
DMA 35 Alex 500 Alex 500
0 1.0201 1.0213 0 0 0.1243 0.1243 0.0000 0.0000
1 1.0191 1.0202 1.004369 1.104806 0.1244 0.1244 0.1249 0.1374 0.2
2 1.018 1.0197 2.109175 1.60699 0.1245 0.1245 0.2626 0.2000 0.34
3 1.0174 1.019 2.711796 2.310049 0.1246 0.1245 0.3378 0.2877 0.42
4 1.0165 1.0175 3.615728 3.816602 0.1247 0.1247 0.4507 0.4759 0.56
5 1.0156 1.0165 4.519661 4.820971 0.1248 0.1248 0.5638 0.6016 0.66

Sample Calculations (25 ̊C at T = 1 hr)

Density of water = 0.99713 g/cm3

1.0215
Original density (OG) ( T =0) = 𝑥 1000 = 1024.4
0.99713

1.0208
Present density (PG) (T =1) = 0.99713 𝑥 1000 = 1023.7

OG – PG = 1024.4 – 1023.7 = 0. 702

F = 0.0001(0.702) + 0.1243 = 0.1244

Calculated alcohol: A(%) = f x (OG – PG) = 0.1244 x 0.702 = 0.0873

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12
Discussion

In the graph of cell concentration (i), label the lag, exponential, deceleration, stationary and/or death
phase. Which growth phases were experienced for the different subgroup during the experiment?
Explain how you determine each growth phase from your data and graph.

13
 Temperature Time period for Lag Phase Change in Concentration
25℃ 0h – 5h 0.0595
30℃ 0h – 5h 0.176
35℃ 0h – 5h 0.0487

In figures 1 to 3, the graph for cell concentration against time was drawn and the relevant data is
organized in the table above. The initial cell concentration for all three temperatures were calculated
to be negative and then increased to positive values over time. This is due to the fact that there was no
cell death initially and thus the sample was clear leading to the negative initial value.

For all the temperatures, the cell concentration was concluded to be in the lag phase throughout the 5
hours of the experiment. For all the graphs plotted, we generally see increase in cell concentration
over time. For temperature of 25℃ , the concentration increased slightly by 0.0595. At temperature
of 30℃ , it increased by 0.176 and for temperature of 35℃ , it increase by 0.0487 only. However,
small fluctuations were seen in concentration for 25℃ and this could be attributed to the temperature
setting which could not have been optimum for yeast.

It is deduced to be undergoing lag phase as there is only a marginal increase in the cell concentration
and this could be due to the increase in the cell mass. As the growth medium was inoculated with the
yeast , the yeast cells adapted by reorganizing their molecular structure which leads to an increased
cell mass. Therefore during the 5 hours, the cells were still in the lag phase.

Moreover as shown in the figure above, after the lag phase comes the exponential phase where the
amount of cells increases rapidly with their mass. During this phase, there will be a time period where
the cells will grow at the same rate. Following this is the next phase known as the deceleration phase,
where the conditions and nutrients for cell growth will decrease which leads to the growth rate
decreasing. There will be a decreasing rate till the stationary phase where there is no more cell growth
as there is no cell division occurring. The amount of cells remains constant till the last phase which is
the death phase. In the death phase, the amount of cells decrease as they use up the nutrient which
leads to depletion and therefore no conditions for the cells to survive.

In this experiment, we have only observed the lag phase and if time permits, we would be able to see
all the phases involved.

14
Discuss about the change in pH and ORP with time, emphasizing on the different metabolic
processes which could result in such a trend.

The graphs of ORP and pH against time was plotted for the three temperatures. The experiment was
conducted for 300 minutes and pH and ORP values were taken in intervals of 5 minutes.

For all three temperatures, a decrease in the pH was observed and there seems to be a linear
relationship between the pH level and time. For temperature at 25℃, the pH dropped from 6.08 to
5.13 and at 30℃, it dropped from 5.998 to 4.728 and lastly at 35℃, the pH dropped from 5.953 to
4.533. The drop in pH levels shows that the solution at the end is more acidic than the initial solution.

In this experiment, as the yeast grows anaerobically where the glucose is converted from glucose to
ethanol and carbon dioxide. Therefore, as more carbon dioxide is produced which is acidic in nature
which leads to the drop in pH level.

Oxidation-reduction potential (ORP) shows the solution's ability to transport electrons. A lower ORP
value shows that there is less oxidizing agent which is more anaerobic whereas higher ORP value
shows more oxidizing agent which is more aerobic.

For temperatures 25℃ and 35℃, the ORP value decreased with time and for 30℃ it decreased
initially until nearing the end where its value spiked before dropping again. For 25℃ , the ORP
decreased by 11.745mV while for 30℃, the overall drop was 115.73mV with a general decrease from
t =0 to t = 280 min and increase till t = 300 min. At 35℃, the drop was 139.48mV.

As the glucose gets oxidised into carbon dioxide, the oxidizing agent gets depleted fast which led to
the drop in the ORP values. As there is relatively high oxygen content compared to carbon dioxide,
the glucose gets oxidized to pyruvate followed by citric acid cycle when pyruvate reacts to give
energy and carbon dioxide. Since there were also high levels of oxygen and therefore oxidizing
agents , the ORP levels were high initially and as oxygen gets depleted over time the ORP levels also
decrease.

15
Yeast grows anaerobically by fermentation and NAD+ (oxidizing agent) is regenerated from NADH
as pyruvate is converted to carbon dioxide and ethanol. The NAD+ could have regenerated quickly
which lead to increase in oxidizing agent , thus the ORP value increased for the temperature of 30℃.

Compare and comment on the results obtained in Exp. 5 and 6. Which equipment and which
method of determining the sugar and alcohol content is more accurate and why? Using the data
from the equipment you deem more accurate, discuss the respective profiles of graphs (iii) and
(iv) with respect to the information provided on yeast metabolic pathways.

For this experiment, we used three equipments, refractometer, DMA 35 and Alex 500 to measure the
sugar content in the samples. Figures 7 to 9 show how the sugar content changes over time.

We used ̊Plato measurement which is equivalent to 1g of sugar per 100g of solution [1]. As such no
conversions is required for these results.

A common decreasing trend is observed for glucose concentration across all three devices and
temperatures. This is supported by the fermentation theory where glucose is supposed to be oxidized
and decrease.

Despite the general decreasing trend, there was a slight increase from t = 2hr and t = 3hr for
temperatures 30℃ and 35℃. This could be due to the fact that there could be inhomogeneous
mixture which lead to the inaccuracies of the sugar content.

Sugar content
For sugar content, we could see the DMA 35 and Alex 500 generally have similar results and the
refractometer results are usually higher. Therefore, DMA 35 and Alex 500 have consistency and as
they measured similar results, they are preferred over the refractometer. On top of that, Alex 500 can
measure up to 2 decimals while DMA 35 only measure to 1 decimal place, which places Alex 500 as
the most accurate equipment out of all three.

16
Alcohol Content

The alcohol content is measured from the Alex 500 equipment and this is the direct measured alcohol
content. The content can also be calculated from the density results obtained from the DMA 35 and
Alex 500 device.

For all three temperatures, the calculated alcohol content from both the DMA 35 and Alex 500 were
very similar and followed the same trend throughout the experiment. Since they followed the same
trend, there is no preference for better equipment. For the temperature of 35℃, however, the
calculated Alex 500 values differed slightly yet followed the same trend.

Another noticeable trend is that the measured alcohol content was always higher than the calculated
values for all three temperatures. The calculated alcohol content was calculated using an empirical
method which uses density values measured by both equipments. As such, based on the formula
given , the accuracy of the calculated value depends heavily on the initial density value at t = 0 hr.
Therefore if the mixture is not mixed properly initially, this leads to a deviation of density value from
the actual value which affects the final calculated alcohol content.

Therefore, this shows that it is much more accurate to directly measure the alcohol content from Alex
500 rather than calculating from density measurements which leads to more inaccuracies.

To conclude, it is best to use the Alex 500 equipment to measure the sugar and the alcohol content.

17
As you can see from the graph above, for all three temperatures the sugar content followed the same
trend of decreasing with time. At T = 30℃, the sugar content dropped the most followed by T =35℃,
and T = 25℃. However, for the measured alcohol content with time , we see an opposite trend of
increasing with time. The alcohol content increased most for T = 30℃ followed by T =35℃, and T =
25℃.

18
Initially, the yeast grows aerobically with oxygen being present in the reactor and thus the glucose
was converted to carbon dioxide and energy.

The oxygen level gets depleted as the reaction progresses and making the yeast to grow anaerobically.
This would result in glucose being converted to ethanol and carbon dioxide.

The glucose gets converted to pyruvate through the glycolysis reaction and the conversion og glucose
has led to its depletion which is evident in the graph above as the sugar content decreases with time.

As the system reaches anaerobic growth, the glucose continues to be converted to pyruvate but in this
case, where there was absence of oxygen, the pyruvate gets converted to ethanol. Through
decarboxylation, pyruvate decarboxylase catalyzed the conversion of pyruvate to ethanal. This
produces a molecule of carbon dioxide and ethanal gets reduced to ethanol by NADH and H+ , giving
a molecule of NAD+ in the end. Therefore, as time progresses, more alcohol is formed which is
supported by the graph above.

It is also observed that initially from t = 0 hr and t = 1 hr, the change sin sugar and alcohol content
was minimal, and it is because that the yeast cells are just introduced to the lag phase. The cells has to
restructure their molecular arrangement due to the new environment which led to a slight increase of
the cells. This is why very little glucose was converted and low amount of ethanol was produced
initially.

Looking across temperature, the glucose conversion and alcohol production increased with
temperature. This is in line with the theory that as temperature increases, the rate of reaction increases
as kinetic energy increases.

Using the data from all three temperature groups, explain how the chosen temperature led to
different results and describe how the graphs vary. Support with literature, how fermentation

19
rates vary with temperature and discuss what are the possible improvements that can be done
to optimize this batch fermentation process.

From the graphs, it is evident that the rate of reaction increased with time as glucose levels dropped
and the ethanol concentration increased. The glucose drop was largest for the temperature of 30℃
followed by T = 35℃ and T = 25℃. The alcohol content increased with time and the greatest
increase was observed at T = 35℃ followed by T = 25℃ and T = 30℃. The optimal fermentation
temperature for yeast is found to be between 28℃ and 33℃ [2].

For this experiment, the operating temperatures were around and within the optimal range. This is
crucial as fermentation is an enzyme-catalysed reaction and small temperature changes causes big
changes. As temperature increases, the rate of fermentation also increases. The heat energy increases
and thus kinetic energy of enzymes also increases which causes the enzymes to move around more
rapidly. With the increased movement, the likelihood of effective collision between substrate and
enzymes also increases, leading to the increased rate of formation of products [3].

However, at lower temperatures the yeast would be less sensitive to the high alcohol concentration
and the lag phase of fermentation will be longer. This slows down the rate of fermentation. At higher
than optimal temperatures, the enzyme function fails and thus stopping the fermentation process.

To improve the fermentation process, there are many methods available. One of the possible method
is utilizing pressure to optimize the process. Increase in pressure conditions can help the yeast to
build tolerance towards the high ethanol concentration. Since the yeast will not be stressed as easily
under high concentration, this allows for more ethanol to be formed. Therefore, this increases the
production of alcohol, increasing the efficiency of the fermentation process.

The optimal pH range for yeast is pH 4 – pH 6. To maintain a stable pH range for efficiency, we
could use a buffering agent. The buffering agent could absorb excess H+ generated from the
conversion of NADH. This could prevent the pH level dropping too low and maintain the pH range
for fermentations. Possible buffers to use could be carbonate/phosphate buffers.

20
In this experiment we have looked at the different methods/measurements which can be used to
monitor growth characteristics of yeast and the extent of fermentation in a fermentation process.
What are advantages and disadvantages of these methods?

For this experiment, the measurements used were cell concentration, sugar and ethanol content, pH and
lastly ORP. These measurements investigated the growth of yeast with respect to time and temperature.

Cell Concentration

Cell concentration is used to figure the different phases of the fermentation process and also predict the
end point of fermentation. The advantages of this measurement is that we could use the concentration
to know when the yeast has entered a certain phase such as the exponential phase. It is also a clear
indicator and comparison method to tell which is the optimal setting for fermentation by showing us
the greatest increase in the cell concentration.

The disadvantage is that when we use cell concentration, there will also be dead cells inside the system
which will be included in the concentration. This makes it hard for us to distinguish between the death
and stationary phase and also to determine if the cells are alive.

Sugar content

The sugar content tells us when the yeast has been used to produce ethanol. The rate of sugar decreasing
is proportional to the amount of yeast in the mixture.

The advantage of using this measurement is that it tells us the precise time when the glucose is broken
down into ethanol by yeast. It also helps us to calculate the amount of ethanol produced as the ratio of
glucose to ethanol is 1:1.

However, the disadvantage is that glucose is also used for the aerobic reaction of yeast. As the glucose
is turned into carbon dioxide and ethanol, it is difficult for us to determine the amount of ethanol
produced for either the aerobic or anaerobic reaction.

21
Alcohol

The alcohol measurement is taken to check the quality of the fermentation process. It is used specially
in the fermentation process to determine the purity of the alcohol.

The advantages of this measurement is that the amount of alcohol is directly measured which gives us
access to information such as sugar depletion rate, yeast growth rate. This could be realised when the
alcohol concentration decreases, it tells us lesser glucose is being converted which means glucose is
the mixture is depleting. Thus this tells us when the system requires the additional glucose for
conversion.

The disadvantage is that we had to use the Alex 500 machines to take the alcohol measurement which
is expensive to obtain and well care has to be always taken to ensure accuracy.

ORP

ORP helps to measure the rate of glucose being oxidized and tell us the amount of oxidizing agent
present in the system. Lower ORP values indicate lower presence of oxidizing agent which is anaerobic
conditions while higher ORP indicates higher presence of oxidizing agent which is aerobic conditions.

Oxygen plays a crucial role in fermentation as it helps to promote the proliferation of yeast cells . The
oxygen supply can be regulated to optimize the fermentation process. It is also important to ensure there
is always glucose in the system as oxygen without glucose would cause the yeast to break ethanol into
acetic acid turning the quality of the product down.

The advantages of ORP is that we are able to determine what conditions the yeast is in whether
anaerobic condition or aerobic condition. The yeast culture’s metabolic pathway could be well
determined at any time using the ORP values.

The disadvantage is that ORP is not determined by one significant factor but rather influenced by
several factors which makes it difficult to determine the fermentation rate from ORP alone.

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pH

pH level shows us how acidic or basic the mixture is which then determines how efficient the
fermentation process is. The optimal range of pH which is pH 4-6 would tell us that there is essential
chemical compounds that increase the fermentation process.

As the yeast converts glucose into ethanol and carbon dioxide , producing more carbon dioxide which
is acidic in nature. This results in a lower pH value of the system and thus by the pH level we can
estimate the rate of carbon dioxide formation and know what phase the system is in especially the
exponential phase.

The advantage of pH measurement is that it can tell us how healthy the yeast is and help us to detect
which phase the system is in. On top of that, it helps to ensure the flavor of the end product is stable,
especially in the beer industry.

The disadvantage is that the pH equipment used is much more expensive than a simple pH strip test.

Suggest a few scenarios in which each specific method can be used

In this experiment, to measure the sugar content we used the pocket refractometer. The pocket
refractometer could also be used in other scenarios. One of it could be to measure the sygar
concentration in samples during beer brewing. This helps to ensure the consistency of the beer
production. We could also use it to measure tap water’s salinity to ensure it is suitable for everyday
drinking.

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We also used DMA 35 to measure the glucose concentration and density . DMA 35 is convenient and
portable to use so we could use it on-site for measurements instead of moving the test samples from
one place to another. It could help to detect sample densities regularly in beer brewing to ensure
accuracy. On top of that , it could be useful in the battery industry where it could estimate charge density
in lead batteries.

Lastly, Alex 500 equipment was used to measure glucose content , density and alcohol content. Since
the Alex 500, gives us 3 parameters of a sample, we could use it in beverage industries where at one
time several parameters could be tested with efficiency. Moreover, the effects of flavoring on beverage
such as glucose content could be monitored closely. The rate of fermentation in beer industry could be
monitored and determined on site instead of outsourcing to third parties.

Conlusion

This whole experiment has given us a better understanding of the fermentation process and how it
happens. By utilizing measurement parameters such as pH and ORP the growth of the cells were
monitored. The cell concentration was determined by spectrometer while sugar content was obtained
from refractometer, DMA 35 and Alex 500. The alcohol content was also obtained from DMA 35 and
Alex 500. This experiment was conducted in 3 different temperature of T = 35℃ ,T = 25℃ and T =
30℃ and their effect on fermentation was studied.

From the experiment, it is derived that with a higher temperature, there would be a higher fermentation
rate and this is evident from the increasing sugar consumption and alcohol production as temperature
increases.

As the optimal range for fermentation is 28℃ - 33℃, and this experiment is conducted only at T =
35℃ ,T = 25℃ and T = 30℃ , which are around the optimal range, it gives us insufficient evidence to
determine the optimal fermentation temperature. However, we were unable to pinpoint the exact
optimal temperature as it may lie in between the temperature conditions we experimented. Therefore,
if we could conduct in intervals of 1℃ around the range, it would be easier to read the data and
determine the optimal temperature.

These results could help as a guide in fermentation processes such as beer brewing to determine the
optimal conditions.

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Reference

1. Plato gravity scale. (n.d.). Oxford Reference.

https://doi.org/10.1093/acref/9780195367133.013.0911

2. Zhao, Y., & Ran, W. (2021, January 1). Anaerobic fermentation process for biomethane
production from vegetable waste. https://doi.org/10.1016/b978-0-12-821763-4.00001-6

3. Kucharczyk, K., & Tuszyński, T. (2018, May 29). The effect of temperature on fermentation
and beer volatiles at an industrial scale. Journal of the Institute of Brewing, 124(3), 230–235.
https://doi.org/10.1002/jib.491

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