Aerobic and Anaerobic Fermentation

With Varying Stirring Rates

Tom Hewitt
Jason Morris
Stephanie Pratt
Katie Snell

Carnegie Mellon University

Department of Chemical Engineering
May 14, 2001
Abstract

This experiment tested the effect of stirring rate on aerobic and anaerobic
fermentation by measuring the components of the following carbon mass balance:

Cglucose consumed = CCO2 + Ccell mass - Cinitial cell mass + Cethanol

We chose to vary the stirring rate because mixing is an essential feature of batch
reactions. The results show that a higher stirring rate accelerates the fermentation process.
In aerobic fermentation, the rate of glucose consumption increased by 190% and the rate
of ethanol production increased by 170% when the stirring rate was changed from 150
rpm to 600 rpm. The effect was less apparent for anaerobic fermentation, in which the
rate of glucose consumption increased by only 64% and the rate of ethanol production
increased by 50%. We were unable to complete the above carbon mass balance due to
equipment failures and unreliable cell mass data, however, the carbon we were unable to
account for generally increased over time, corresponding to the expected carbon in cell
mass.
Table of Contents

Introduction..………………..………………………………………………….1

Background and Theory.……………………………………………………….2

Procedure/Apparatus………………………………………………….…..……3

Results and Discussion……………………………….….….…………………5

Conclusion…………………………………………………………………….12

References……………………………………………………………………..13

Appendices…………………………………………………………………….14
Introduction

This experiment was commissioned by Robert Tilton and Matthew Cline of the
Chemical Engineering Department at Carnegie Mellon University. The goal of this
experiment was to determine the effect of stirring rate on cell growth in both aerobic and
anaerobic metabolic processes. We chose to study stirring rate because agitation plays an
essential role in fermentation. Several quantities associated with fermentation were
measured in order to determine the effect of stirring rate, including carbon dioxide
production, glucose consumption, change in cell mass, and ethanol production. By
balancing the sources of carbon, we can evaluate the accuracy of our assumption that
sugar, cell mass, carbon dioxide, and ethanol are the only carbon-containing compounds
involved in fermentation.
The process of fermentation has many industrial applications. The earliest
products of fermentation were ethanol, acetic acid and lactic acid, later expanded to
include the manufacture of antibiotics and steroids in the pharmaceutical industry.1
Current research involves finding a role for fermentation in the fossil fuel industry.2
The next section briefly outlines the fundamental theory behind fermentation. An
explanation of the equipment and methods used in our experiment follows. Next, we
present our experimental results and discuss the conclusions drawn from that data. The
last section discusses recommendations for future experiments.

1
Hahn – 1968.
2
Mills – 1999.

1
Background and Theory

Fermentation is a metabolic pathway by which cells catabolize, or break down, a

carbon source to produce energy. The cells consume carbohydrates, leading to an
increase in cell mass by replication and the production of carbon dioxide and other
organic compounds. To track the consumption and production of various carbon products
we analyzed the following carbon balance:

Cglucose consumed = CCO2 + Ccell mass - Cinitial cell mass + Cethanol

The calculation of carbon for the components in the above equation is relatively
simple and self-explanatory, except for the cell mass. To obtain these experimental
values, we estimated that 70% of the dry cell mass is water by weight and that
approximately half of the remaining mass is carbon.3 Thus, the experimental dry cell
mass is multiplied by 0.15 to calculate the mass of carbon in the cell mass.
There are four distinct regions in the growth cycle of yeast undergoing
fermentation, as detailed in Keri Mills’ Background Information on Fermentation
Processes and Fermentor Operations (1999). The first of these is the lag phase, where
the microbes adjust to their environment and begin to consume food. Although cell mass
may increase, the number of cells remains the same. In our experiment, we minimized the
lag phase by placing the yeast in the fermentor the night before we ran the experiment.
Then the cells enter the exponential growth phase. In this stage, the yeast
consumes food and multiplies to form new cells at a high rate. Here, the production of
carbon dioxide rapidly rises and the concentration of glucose decreases rapidly.
Next, in the stationary phase, the net growth rate is zero, meaning the growth rate
is equal to the death rate. The carbon dioxide production levels off and glucose
consumption also slows.
During the decline phase, the death rate exceeds the growth rate. This is due either
to exhaustion of nutrients or to buildup of byproducts. In this period, both the sugar
concentration and carbon dioxide production approach zero.
As with any other chemical reaction, stirring rate may affect the rate of the
fermentation reactions. The yeast cells need to be in contact with sugar and oxygen (in
aerobic fermentation) in order to react. The yeast cells are incapable of swimming, so if
just suspended in the fermentor broth, they will quickly exhaust the surrounding
resources and smother in their own waste products.3

3
Steel – 1958.

2
Procedure/Apparatus

In the experiment we used a 7.5L New Brunswick Fermentor to perform the

fermentation process. Each week a different trial was run consisting of either aerobic or
anaerobic conditions and varying stirring rate. The four different trials are listed below.

Week 1: aerobic – 150 rpm stirring rate

Week 2: aerobic – 600 rpm stirring rate
Week 3: anaerobic – 150 rpm stirring rate
Week 4: anaerobic – 600 rpm stirring rate

In order to conduct the experiment under aerobic and anaerobic conditions

different gases were bubbled through the fermentor. For the aerobic case CO2 was used
and for the anaerobic case nitrogen was used. Each gas was bubbled through the
fermentor at a rate of 1.0 standard cubic feet per hour (SCFH). The same recipe was used
for each of the trials. The night before the experiment the baker’s yeast was added to the
fermentor in 3L of distilled water and the environment was heated to 35°C. This allowed
the yeast to “wake up” which would produce better results. The next day the rest of the
ingredients were mixed in 1L of distilled water and added to the fermentor. A list of the
ingredients are shown in the following table:

Table 1: Fermentor Broth Recipe

Broth Contents Amount
Distilled Water 4.0 L
(NH4)2SO4 1.9 g
Mg SO4 • 7H2O 0.3 g
KCl 0.06 g
KHPO4 0.05 g
Fe(II) SO4 • 7 H2O 0.03 g
Yeast Extract 5.0 g
Baker’s Yeast 37.0 g
Glucose 16.0 g

Once the rest of the ingredients were added to the fermentor, LabView software
was used to record the amount of CO2 that was generated by the system. The CO2 was
measured using a Lira Model 3000 detector. The method of calibration of the CO2
detector can be found in the old reports.4
Samples were taken from the fermentor once every 10 minutes for the first hour
and then once every 30 minutes for the duration of the trial. The samples were analyzed
in order to determine the production of ethanol, increase in cell mass, and amount of
glucose consumed by the yeast. In order to determine the increase in cell mass 14 mL of
the sample was placed in a preweighed vial and then centrifuged to separate the cell mass

3
from the liquid. The vial was then placed in a vacuum oven and allowed to dry
overnight. Once the vial reached a constant weight the original weight of the vial was
subtracted and this difference was the amount of cell mass in the system at that particular
time. To determine the production of ethanol by the system, a sample was filtered and
introduced into a gas chromatography unit. Unfortunately, the GC could not adequately
separate the peaks in the water and ethanol mixture. Therefore, the sample had to be
analyzed using UV-Vis. A kit was ordered to accurately detect the amount of ethanol in
the sample. The sugar content of the sample was also determined using
spectrophotometric analysis. A set of standards ranging from 0.4 g/L to 5.0 g/L was used
to construct a calibration curve. A 15 µL sample was mixed with 3 mL of a trinder
solution. The mixture was analyzed using the spectrophotometer over a 12-minute period
at a wavelength of 505 nm. The trinder solution caused a kinetic reaction with the
glucose so that it could be absorbance tested.

4
Results and Discussion

One aspect of fermentation studied in this experiment was the effect of stirring
rate. Figure 1 shows the data for grams of carbon in sugars versus time for the first two
weeks. The trials were both done aerobically with one stirring rate of 150 rpm and one at
600 rpm.

Carbon in sugar - weeks 1 and 2 (aerobic)

7
grams carbon in sugars

6
5
4 150 rpm
3 600 rpm
2
1
0
0 5000 10000 15000 20000
Time (s)

Figure 1: Carbon in sugar vs. time. Aerobic runs, one at 150 rpm and one at 600 rpm.

As expected, the amount of sugar decreased over time. The increase in stirring rate
caused the sugar to be used up more quickly. In the low stirring rate, all the carbon was
gone at approximately 20000 seconds, whereas it was gone after 7000 seconds in the high
stirring rate. This translates into a 190% increase in rate of sugar consumption for the
higher stirring rate.
Since the sugar gets consumed much faster, we would expect something else to be
produced much faster with a high stirring rate. Figure 2 shows the carbon in ethanol for
the first two weeks. The maximum amount of ethanol was produced at 10000 seconds
for 600 rpm and 27000 seconds for 150 rpm, meaning there was a 170% rate increase.
This corresponds well to the increase in rate of sugar consumption. It should also be
noted that the amount of carbon in ethanol began to decrease towards the end of these
runs. This characteristic is discussed in team 2’s report.

5
 Carbon in ethanol - weeks 1 and 2

3.5
3
grams C in ethanol

2.5
2 150 rpm
1.5 600 rpm
1
0.5
0
0 10000 20000 30000 40000 50000 60000
time (s)

Figure 2: Carbon in ethanol vs. time. Aerobic runs, one at

150 rpm and one at 600 rpm.

The data from the third and fourth weeks can be analyzed in the same way as
weeks 1 and 2. These anaerobic runs were also performed at 150 rpm and 600 rpm. The
sugar data is shown in Figure 3.

Carbon in sugar - weeks 3 and 4 (anaerobic)

7
grams carbon in sugars

6
5
4 150 rpm
3 600 rpm
2
1
0
0 5000 10000 15000
Time (s)

Figure 3: Carbon in sugar vs. time. Anaerobic runs, one at

150 rpm and one at 600 rpm.

The carbon reached zero at 8800 seconds for 600 rpm and at 14400 seconds for
150 rpm, translating into a 64% rate increase with a higher stirring rate. This means that

6
the rate of ethanol production should also increase at a similar rate in weeks 3 and 4.
This data is shown in Figure 4.

Carbon in ethanol - weeks 3 and 4 (anaerobic)

3.5
y = 0.0003x
3 2
R = 0.9018
grams C in ethanol

2.5
2 150 rpm
y = 0.0002x
1.5 2
600 rpm
R = 0.9939
1
0.5
0
0 5000 10000 15000
time (s)

Figure 4: Carbon in ethanol vs. time. Anaerobic runs, one at

150 rpm and one at 600 rpm.

When this data is fit to a linear trendline, slopes are obtained that show a rate
increase of 50%, which is comparable to the sugars. All of this data collectively shows
that stirring rate does increase the rate at which the fermentation process takes place.
However, the stirring rate has a much smaller effect in the anaerobic case compared to
the aerobic case. This is discussed in team 2’s report.
In order to close the mass balance, the data for carbon dioxide and cell mass must
also be analyzed. The carbon dioxide data obtained during week one showed some
irregularities, as shown in Figure 5.

%CO2 vs. time - Week 1

4.5
4
3.5
3
%CO2

2.5
2
1.5
1
0.5
0
0 10000 20000 30000 40000 50000 60000
time (s)

Figure 5: %CO2 vs. time for week 1. Aerobic, stirred at 150 rpm.

7
The CO2 detector got wet, causing the sharp peaks on the graph. For our purposes of
closing the carbon balance, Figure 5 was modified to eliminate these false peaks.

Modified %CO2 vs. time - week 1

2.5

1.5
%CO2

0.5

0
0 10000 20000 30000 40000 50000 60000
time (s)

Figure 6: Modified %CO2 for week 1. Aerobic, stirred at 150 rpm.

Figure 6 shows a graph that is more representative of the results expected from looking at
previous reports and our results from week four. The data from week 4 shows a strange
bump at around 2000 seconds, but the data from team 2 also shows this bump.

% CO2 vs. time - week 4

7
6
5
CO2 (%)

4 team 2
3 team 9
2
1
0
0 5000 10000 15000 20000
time (s)

Figure 7: %CO2 vs. time for week 4. Anaerobic, stirred at 600 rpm.

From Figure 7, it appears that team 2’s run went more quickly than ours; this is because
an error was made in preparing our yeast and they were not heated a day ahead of time as
planned. Therefore, team 2’s data for CO2, alcohols, and sugars is used in all of our

8
analysis on week 4, since their yeast was prepared in the same manner as our first three
runs.
Due to difficulties with the CO2 detector, no data was obtained for the second or
third weeks. The data from weeks 1 and 4 can be used to close the carbon balance as will
be described later.
The cell mass data obtained from the fourth week is shown in Figure 8.

Carbon in Cell Mass - week 4

8
7
6
5
grams C

4
3
2
1
0
0 2000 4000 6000 8000 10000 12000 14000
time (s)

Figure 8: Carbon in cell mass for week 4 vs. time. Anaerobic, stirred at 600 rpm.

The cell mass varies significantly with time, although it shows no trend. Four samples
were taken at the first time (2100 seconds) to test the reliability of the data. As seen in
the figure, each of the four samples gave very different cell masses, so we concluded that
the cell mass data is unreliable. The small weights that were measured are prone to error,
since the balances used are not accurate to more than 1 or 2 decimal places. Future
groups should look into different methods for measuring cell mass, such as optical
density.
The measured quantities of CO2, alcohols, and sugars were used to generate a plot
of the known carbon in the system over time. We have assumed that carbon will be
found in only four forms: sugars, CO2, ethanol, and cell mass, and the sum of these
components should be constant over time. Since the first three of these values have been
measured, the remainder will be the projected cell mass in the system. Plotting these
values over time for week 1, we obtain Figure 9.

9
 7

4
CO2
Mass C (g)

3
Sugar
Ethanol
2
Other
1

0
0 10000 20000 30000 40000 50000 60000
-1

-2
Tim e (sec)

Figure 9: Carbon vs. time for week 1. Aerobic, stirred at 150 rpm.

From this plot, with the exception of the end point, the theoretical cell mass
increases over time, as expected. At the first time interval, the sum of the carbon in the
sugars, ethanol, and CO2 is greater than the starting value of carbon, suggesting that
carbon has been generated, which is impossible. Since this trial was run at a lower
stirring rate, and the measurement time was close to the start time, the tank may not have
been well mixed. Therefore, the sample drawn may have contained a high concentration
of sugar, which was unrepresentative of the entire tank. The remaining data, with the
exception of the last measurement, strongly supports the belief that cell mass increases
over time. The final data point, however, leads us to believe that cell mass is not the only
other source of carbon. Since this value decreases, and the cell mass is expected to
increase over time, we would assume that there is another source of carbon which
decreases as the trial approaches its end point.
As discussed previously, analysis of the anaerobic trial at 600 rpm will be done
using the data obtained by Team 2. We once again plot these values over time to predict
how cell mass should change over time.

10
 7

4
CO2
Mass C (g)

Sugar
3
Ethanol
Other
2

0
0 2000 4000 6000 8000 10000 12000

-1
Time (sec)

Figure 10: Carbon vs. time for week 4. Anaerobic, stirred at 600 rpm.

From Figure 10, once again the sum of the carbon in sugars, CO2, and ethanol
generally decreases over time, meaning that the remainder (mostly cell mass) increases.
The ethanol data taken between 3000 seconds and 7000 seconds is not in line with the
other ethanol points, making the projected cell mass (other) curve irregular over that time
interval. This “other” curve also decreases above 6000 seconds as the sugars are
completely consumed, similarly to the results of week 1. If the balance of the carbon in
the system was cell mass, we would not expect this decrease to occur, and this drop in
unmeasured carbon supports the belief that there is some other carbon source that
decreases at the end of the process.
As seen in both Figure 9 and Figure 10, the cell mass in the system does not
exceed 3 grams of carbon at any time. This is another indication that the cell mass data
obtained in this experiment is inaccurate. With a more accurate means of calculating the
cell mass in the system, the amount of carbon from sources other than sugars, CO2,
ethanol, and cell mass could be determined.

11
Conclusion

From the data obtained in this experiment, we found that stirring rate has a
significant effect on the rate of sugar consumption and ethanol production. In the aerobic
trials, increasing the stirring rate from 150 rpm to 600 rpm yielded a 190% rate increase
in sugar consumption. In addition, increasing the stirring rate from 150 rpm to 600 rpm
in the anaerobic trials led to a 64% increase in the rate of sugar consumption. This
indicates that the availability of the sugar to the yeast is a limiting factor for the reaction.
The mass balance suggests that sugar, CO2, ethanol, and cell mass are not the only
significant sources of carbon in the system. With a better measurement of cell mass, it
would be possible to determine exactly how much of the carbon is represented by other
sources, but from the decrease in the expected cell mass curve in this experiment, it can
be seen that other components cannot be neglected. Future experiments should include a
different method for cell mass measurement in order to close the carbon balance
accurately. Testing could also be done for organic acids, which may be a source of
carbon in the system. Research should be done on the decreasing amount of ethanol at
the end of the process that was found in this experiment. More stirring rates could also
be tested to determine at what point increased stirring no longer accelerates the
fermentation process and if higher stirring rates kill the yeast cells.

12
References

Appel, J.; Dorn, V. et. al. Aerobic Batch Fermentation of Saccharomyces cerevisiae.
March 24, 2000.

Hahn, Peter A. Chemicals from Fermentation. Doubleday & Company, Inc., New York.
1968.

Mills, Keri. Background Information on Fermentation Processes and Fermentor

Operations. 1999.

Steel, R. ed. Biochemical Engineering: Unit Processes in Fermentation. The MacMillan

Company, New York. 1958.

13
List of Appendices (all files separately enclosed)

Appendix A: CO2 Data

--CO2-Week1-Team9.xls
--CO2-Week4-Team9.xls
--CO2-Week4-Team2.xls

Appendix B: Cell Mass Data

--Cell mass-Team9.xls

Appendix C: Alcohol Data

--Alcohols-Team9.xls
--Alcohols-Team2.xls

Appendix D: Sugar Data

--Sugars-Team9.xls
--Sugars-Team2.xls

14