ChE 414

Chemical Engineering Laboratory II

Formal Report

Experiment 1
Fermentation: Kinetics of Yeast Growth

Conducted by: Kathlene Jacobson

Email: Kathlene.jacobson@usask.ca
Lab Partner: Stuart Houle

Date Performed: November 6, 2007

Date Due: November 20, 2007
“Free” Late Days Used: 3

Submitted to: Dr. Mehdi Nemati

Department of Chemical Engineering
University of Saskatchewan

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ABSTRACT

Fermentation is a well-known process and is used for the production of ethanol having
many applications such as fuels, beers, wines, and other alcoholic drinks. Fermentation involves
the growth of a microorganism on an organic substrate. The end products of fermentation include
the microorganisms as well as reduced or oxidized organic compounds. Sugars such as glucose
are a good substrate for microorganisms to grow on because they yield both oxidizable and
reducible intermediates. The type of microorganism and substrate, and environmental factors
such as temperature and pH determines the end products of fermentation.

The purpose of this experiment was to determine and examine the growth parameters and
patterns of fermenting yeast. Furthermore, the data collected in this lab was then used to
determine a ‘scaled-up’ fermenter to be used in large-scale industry. In this experiment
Saccharomyces cerevisiae, commonly known as baker’s yeast, was grown in a bioreactor with
specified conditions for the production of ethanol. The reactor was maintained at a temperature
of 26oC and had an initial glucose and yeast concentration of 40 g/L and 4.0 g/L, respectively.
Samples were taken during the fermentation process and were analyzed for yeast, glucose, and
ethanol concentration. Ethanol and cell yield factors were found to be 0.398 and 0.297,
respectively, and the maximum specific growth rate was 0.165 hr-1.

Using the kinetic information determined by the experiment and given parameters, a
1000L batch reactor was designed. It was determine it would take 31.1 hours for the fermentation
to complete (includes yeast growth lag-time – 2 hours and fermenter preparation – 7 hours). The
final ethanol concentration would be 43.81 g/l, or 43.81 kg of ethanol produced. Assuming 50
weeks of operation, 270 batches could be produced each year, producing a total of 11840 kg of
ethanol annually.

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TABLE OF CONTENTS

Abstract…………………………………………………………………………………..…………

Table of Contents…………………………………………………………………………………...

Nomenclature……………………………………………………………………………………….

Introduction…………………………………………………………………………………………

Literature Review…………………………………………………………………………………...

Apparatus…………………………………………………………………………………………...

Procedures…………………………………………………………………………………………..

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

13

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Conclusions…………………………………………………………………………………………

18

Recommendations…………………………………………………………………………………..

19

References…………………………………………………………………………………………..

21

Appendices………………………………………………………………………………………….

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Appendix A – Raw and Calculated Fermentation Data…………………..………………..

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Appendix B – Graphs………………………………….…………………………………...
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Appendix C – Sample Calculations………………………………………………………...
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NOMENCLATURE

Symbol Definition Units

 Molar absorptivity
µ Specific Growth Rate h-
µM Maximum Specific Growth Rate h-
KS Monod or Saturation Constant g/l
l Path Length cm
OD Optical Density
P Product Concentration g/l
S Substrate Concentration g/l
So Initial Substrate Concentration g/l
t Time hr
X Cell Mass Concentration g/l
Xo Initial Cell Mass Concentration g/l
YP Product Yield Factor g/g
YX Cell Yield Factor g/g

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INTRODUCTION
The fermentation experiment was designed to examine the biological process of

Saccharomyces cerevisiae (yeast) growth on glucose and to determine the kinetics of this

process. Fermentation is a biological process defined as the anaerobic conversion of sugar to

products such as carbon dioxide, and alcohol (ethanol in our case). The sugar acts as the organic

substrate for the yeast and allows the microorganisms to grow and yield both oxidizable and

reducible intermediates. The microorganisms in this case being yeast, grows by metabolizing the

sugars. This is commonly done by one of two processes: lactic acid and alcoholic fermentation

processes. When the sugar is consumed and alcohol and carbon dioxide are produced, adenosine

triphosphate (ATP) is also produced. The ATP formed is a vestibule of energy that is used for the

growth and reproduction of the yeast cells. The condition under which this lab was performed

was a nearly anaerobic environment, forcing the cells to consume more sugar and thus increasing

the production of ethanol.

LITERATURE REVIEW

FERMENTATION

Fermentation is the process of releasing energy from organic compounds by producing

alcohol or lactic acid in the absence of oxygen. In the catabolic reaction of fermentation which

produce energy, or ATP, the organic compounds serve as the primary electron donor and the

ultimate electron acceptor. If oxygen is present for this process, it acts as an electron acceptor

and aerobic respiration occurs in the place of fermentation. The primary difference between

fermentation and respiration is: In fermentation, the process occurs in the absence of external

electron acceptors and by contrast, in respiration, molecular oxygen or some other electron

acceptor must be present.

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 Fermentation is commonly carried out utilizing glucose as its organic substrate. “A

common biochemical pathway for the fermentation of glucose is glycolysis, also named the

Embden-Meyerhor pathway” (Madigan and Martinko 2006, pg. 118). Several other types of

fermentations are known and the microorganism and substrate used; as well as environmental

factors including temperature and pH determine the products. Main products of fermentation

include ethanol, lactic acid, carbon dioxide, and hydrogen. Production of ethanol the most

prevalent and widespread, utilizing the fermentation process for the production of beers, wines,

and most recently, fuels.

YEAST

The microorganism used in this experiment is Saccharomyces cerevisiae, or more commonly,

baker’s yeast. Figure 1 below is a picture of the yeast cells used taken digitally with microscopic

magnification during the lab time. Yeasts are single celled organisms (eukaryotes) and are

considered a main group of the fungi phylogeny. Yeasts are typically 5 – 10 micrometers in

diameter, are spherical, cylindrical or oval and can reproduce asexually or sexually. S.

Cerevisiae is a type of asexual yeast that reproduces from a process called ‘budding’. Budding

occurs when a small bud cell forms on the cell, which gradually enlarges and separates from the

mother cells. Budding is capable of occurring in either anaerobic or aerobic conditions. S.

Cerevisiae is the yeast that is exploited most to produce ethanol.

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 FIGURE 1: Microscope photograph during experimental run of Saccharomyces cerevisiae

CELL GROWTH

When culturing of microorganisms by inoculating them onto new growth media, they pass

through a number of growth phases, which can be seen below in figure 2 and applies to the

multiplication and death of yeast cells.

FIGURE 2: Growth curve of microorganisms

(http://faculty.ircc.edu/faculty/tfischer/images/bacterial%20growth%20curve.jpg)

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The growth curve describes the entire life cycle. The lag phase compensates for the period of

time the microbial population needs when inoculated into fresh media to start growing. During

this phase, cells grow slowly and there is little cell mass generated. Once the cells have had time

to adapt and obtain proper nutrients, growth accelerates at a constant maximum where each cell

divides into two cells continuously. This is called the exponential phase and is commonly where

the healthiest cells are found. As the culture of cells continues to grow and multiply, nutrients in

the media become consumed and the cells excrete waste products. Both of these actions result in

growth limitation ceasing exponential growth. This causes the microbial population to reach a

stationary phase. In this phase, there is no net decrease or increase in cell number. Inevitably the

stationary phase is followed by the death phase where the cells begin to die off due to the lack of

food (nutrients) or due to the buildup of their own excretions that produce a toxic environment.

The growth phase can be modeled by a relationship relating yeast growth and cell mass:

(Eqn 1)

Where: t = time (hr)

 = specific growth rate (hr-1)
X = cell mass concentration (g/L)

The specific growth rate, μ, can be found using the Monod equation as a function of substrate
concentration:

(Eqn 2)

Where: M = maximum specific growth rate (hr-1)

Ks = Monod or saturation constant (g/L)

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 S = substrate concentration (g/L)

So = initial substrate concentration (g/L)

When the saturation constant is very small compared to the value of substrate

concentration (S >> Ks), the saturation constant becomes negligible, and equation 2 above can be

rewritten as; g = M when the cell growth is in the exponential phase. Equation three below is

obtained by integrating equation two.

(Eqn 3)

Where: Xo = cell concentration at time = 0 (inoculation)

Equation three is useful in the determination of the maximum specific growth rate. Upon

plotting ln(X/Xo) versus time for the log phase of growth, a straight line is produced, and the

maximum specific growth rate is obtained from the slope of the graph (slope = M).

According to both the lab manual and the ChE 461 text, Bioprocess Engineering, if the

production of product and the decrease of substrate are constant over the batch run, both product

and cell yields can be determined by the following equations:

(Eqn 4)

(Eqn 5)

ABSORBANCE AND THE BEER-LAMBERT LAW

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 The absorbance, or optical density, of an object quantifies how much light is absorbed by

it. This information can then be used to find other properties of the object through the Beer-

Lambert law. The Beer-Lambert law relates absorbance, A, of a sample to its concentration, c,

path length, l, and molar absorptivty,  (A = lc) Therefore, the concentration of yeast can be

found by measuring the absorbance against known standards or by use of a standard curve.

Absorbance, for the purpose of this experiment, can be found using a spectrophotometer at a

wavelength of 640 nm.

APPARATUS

Throughout the course of the experiment, seven pieces of equipment were used.

The most crucial piece of equipment was, of course, the fermenter. For the purpose of this

experimenter a VIRTIS Omni-Culture Plus bench-top fermenter was employed and was operated

on batch mode. The fermenter can be seen in figure 3 below and consists of a 2500 ml glass jar

with a working volume of 2000 ml in the batch mode. The control box attached to the fermenter

allowed data to be collected regularly for RPM (mechanical agitation speed of the impeller),

temperature and pH. Samples can be withdrawn from the vessel from a sampler atop the glass

container. Temperature was maintained at an approximate constant of 26 C by using a LAUDA

water bath.

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 Figure 3: VIRTIS Omni-Culture Plus bench-top Fermenter

Analysis of the samples was carried out using a spectrometer, gas chromatograph,

centrifuge, glucose analyzer, and microscope. The spectrophotometer used in the experiment was

a MANDEL UV mini 1240 spectrophotometer. It was used to estimate the cell concentration of

a sample of the microbial suspension from the fermenter.

A centrifuge was used to separate the yeast from the mixture taken directly from the

fermenter. The centrifuge used was HETTICH ZENTRIFUGEN Universal 32 R centrifuge. The

centrifuge is capable of separating heavier mass by creating a sediment. This occurs when the

centrifuge spins and applies a force that is perpendicular to the axis of the sample being

centrifuged. After the mixture had been centrifuged, both gas chromatography and glucose

analysis could be carried out.

The gas chromatograph used was of model MANDEL GC-2014 gas chromatographer.

This piece of equipment is fed a gaseous analyte that carried by a mobile phase (also gas) and

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then based on its traveled path, it analyze. In this experiment, the analyte is originally liquid and

the gas chromatograph vaporizes it.

The glucose analyzer used was an YSI 2700 SELECT biochemistry Analyzer. Once the

centrifuged sample is placed in the correct sample port, the analyzer draws a sample from a vial

pumps it into the analyzer. Once the analysis was complete, the glucose concentration, in grams

per liter, is displayed on the display and printed out.

The final piece of equipment used was the LEICA optical microscope that was used along

with a digital camera to capture an image of the yeast cells in the fermentation mixture at 400

times magnification.

In Summary, the seven pieces of equipment used were:

 VIRTIS Omni-Culture Plus bench-top Fermenter

 LAUDA Water Bath
 MANDEL UV mini 1240 spectrophotometer
 MANDEL GC-2014 gas chromatographer
 YSI 2700 SELECT Biochemistry Analyzer
 HETTICH ZENTRIFUGEN Universal 32 R centrifuge
 LEICA optical microscope

EXPERIMENTAL PROCEDURES

The primary portion of this experiment was dedicated to the actual batch fermentation reaction.

When arriving to perform the experiment, the broth media (excluding glucose and yeast) had

already been prepared, heated to 26 C with the water bath, and agitation had been started and set

to 400 RPM. The Broth media contained;

 1500 ml sterile reversed osmosis water
 NH4Cl – 1.32 g/l
 MgSO4.7H2O – 0.11 g/l
 CaCl2.H2O – 0.08 g/l
 K2HPO4 – 2.0 g/l

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The following procedure was carried out starting a 10 am.

i. 60 grams of glucose was weighed out in a weigh boat and added to the fermenter
ii. A 4-5mL sample was taken in order to examine the “pre-inoculation” conditions

and to calibrate the spectrometer. The spectrophotometer is calibrated at 640 nm

by filling a cuvette with the yeast-less broth and zeroing the absorbance.
iii. 12 grams (8g/L) of yeast (Saccharomyces cervisiae) is measured and quickly

added to the fermenter. The time was recorded and the experiment is left for

approximately 2 hours
iv. After 2 hours, another sample (about 4-5 ml) was taken and the optical density

was read immediately. Again, the time was recorded and the experiment was left

until the scheduled lab time approximately one hour later.

v. At approximately 1:30pm, samples (4-5mL) were taken;
i. Every 10 minutes for the next 1 hour (pH and temperature recorded)
ii. Every 30 minutes for following 1 hour (pH and temperature recorded)
iii. Every 20 minutes for following 40 minutes (pH and temperature recorded)
iv. The next morning (pH and temperature recorded)
After every sample was taken, each was immediately tested to determine its optical

density using the spectrometer, and then after being centrifuged, the sample is analyzed

for glucose content and ethanol concentration.

vi. For each sample taken, 1 ml of the sample is diluted by reverse osmosis water

by a factor of 1/50 or 1/100 in a volumetric flask. The diluted mixture was then

transferred to a cuvette analyzed by the spectrophotometer in order to record the

absorbance.
vii. The remainder of the sample (3-4 ml) was then poured into a plastic centrifuge

tube and placed in the centrifuge along with a tube of water to balance the

apparatus for 1 minute

viii. 1.5 ml of the decanted liquid is drawn from the top of the centrifuge tube using

a plastic pipette and put into a glass vial (vial not capped) and placed into the

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 turntable of the glucose analyzer for analysis. Once analysis is complete, the

glucose concentration is recorded

ix. After the glucose analysis, the vial is capped and inserted into the injector

assembly of the gas chromatographer and tested for ethanol. The results are

printed out and recorded.

x. Part of a sample is smeared onto a microscope slide, viewed under the microscope

and photographed with a digital camera mounted on the microscope

xi. Once the final sample is taken and analyzed, the fermenter is shutdown, emptied,

and cleaned. All other equipment used is also cleaned.

RESULTS AND DISCUSSION

Through the use of the Monod equation and similar applications, many parameters

associated with yeast fermentation were found and examined. Some of these parameters

included product yield factor, cell yield factor, and growth rates. Furthermore, analyzing the

separate growth phases (the exponential phase in particular) made finding such parameters an

easier goal.

When analyzing the raw and calculated data tabulated in Table 1 in Appendix A, it can be

observed that once the broth was inoculated with yeast, the concentration of yeast cells and

ethanol increase while the concentration of glucose decreased with time. At time zero

(inoculation) the yeast concentration was 6.93 g/l, the ethanol concentration was 0 g/l, and the

glucose concentration was 40 g/l. At the end of the experiment, the yeast concentration was 15.1

g/l, the ethanol concentration was 23.1 g/l, and the glucose concentration was 0.009 g/l. Once the

yeast was introduced into the fermenter, it began to metabolize the sugar, reproduce, and produce

ethanol. Besides just ethanol being produced, carbon dioxide was also produced. This value was

not measured directly but is evident due to the decrease in pH as the experiment was carried out.

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Initially, the pH of the fermentation broth was 7.0, which dropped to 4.3 by the end of the

experiment. This indicates an acidic mixture, caused by the addition carbon dioxide dissolved in

solution creating carboxylic groups.

In Figure 4 below, the concentration of yeast was plotted as a function of time. As stated

in the procedure, samples were taken periodically and the optical density was measured. From

the optical density, the yeast concentration was measured throughout the experiment by taking

samples in intervals. The yeast cell concentration could then be determined using correlations

design for different dilutions:

X = 41.889*(OD-0.1509) for 50 ml dilution

X = (77.336*OD)+0.0115 for 100 ml dilutions

25.00

20.00
Yeast Concentration (g/L)

15.00

10.00

5.00

0.00
0 5 10 15 20 25
Time (hr)

Figure 4: Yeast Growth Throughout the Duration of the Batch Run

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From this graph, it can be seen that the cell growth doesn’t follow the typical s-curve pattern that

was described in the literature review. Both the lag and exponential phase present on the graph

are exhibited in the shape expected, which is beneficial because most calculations needed for the

purpose of this lab require the data exclusively from the exponential phase. However, the

stationary phase contains a significant drop in cell concentration instead of demonstrating

constant growth that is expected for the portion of this graph. This non-conformity is due to a

drop in the measured optical density. The decrease in cells is unexpected and should not have

occurred. Because the pH and temperature had mostly stabilized at the point of the cell

concentration drop, the cells should have continued their growth phase as expected. If the pH or

temperature had changed, the cell death could have been accounted for because the conditions

would no longer have been optimum for normal growth patterns.

The first two hours of the experiment accounts for the lag-phase, and the exponential

phase occurs between 2 and 4 hours. The information collected for the exponential phase can be

used to calculate to determine the maximum specific growth rate, μ M. This was accomplished by

plotting ln(X/Xo) versus time which can be seen in Figure 5 below. Once the data was plotted, it

portrayed a straight line with the slope of the equation equaling μ M, which is 0.165 hr-1. The R2

value of 0.855 shows that the linear fit is not as close as is desired. Again, the data strays from

what is expected from the literature review.

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 1.200

1.180

1.160
y = 0.1645x + 0.4786
2
R = 0.8555
1.140

1.120
Series1
Linear (Series1)
ln(X/Xo)
1.100

1.080

1.060

1.040

1.020
2.00 2.50 3.00 3.50 4.00 4.50 5.00
time (hr)

Figure 5: Determination of Maximum Specific Growth Rate

From the literature review, it was stated that the cell and product yields could be calculated

using equations (4) and (5). These results and calculations are tabulated in Table 2 below. The product

yield factor at the end of the experiment was found to be 0.577 with an average of 0.556 and the cell

yield factor at the end of the experiment was 0.204 (a significant decrease from 0.680 that occurred

after two hours) with an average of 0.472. The theoretical yield of ethanol is 51.1 % by weight (based on

2 moles of ethanol produced for one mole of sugar), whereas the experiment produced a 55.6% yield of

ethanol. Giving a percent difference of 8.8% between the theoretical and experimental values.

Table 2: Cell and Ethanol Yield for Fermentation

Sample time ∆t (hour) Yp (g/g) Yx (g/g)
Pre 10:20 0
1 12:07 1.78
2 1:36 3.26 0.4952 0.209
3 1:46 3.43 0.5175 0.682
4 1:56 3.59 0.5318 0.635
5 2:06 3.76 0.5414 0.680

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 6 2:16 3.93 0.6570 0.670
7 2:26 4.10 0.5772 0.632
8 2:36 4.26 0.5548 0.609
9 3:06 4.76 0.5439 0.231
10 3:36 5.26 0.5587 0.171
11 4:06 5.76 0.5603 0.203
12 9:27 AM 23.12 0.5766 0.204

The value of the saturation constant, KS, is difficult to calculate with the data obtained in

this experiment. In the literature review, it was stated that during the exponential phase, K S<<S,

and therefore was considered negligible. Since we were concerned with calculating the

maximum specific growth rate, the majority of samples were taken during the exponential phase,

and none were taken during the death phase (although this could be argued since my particular

data shows significant cells death where the stationary phase should be occurring). Since K S is

dependent on the substrate concentration, S, samples should have been taken during the death

phase where the substrate concentration was small and did not outweigh the value of KS in order

to determine its value.

All of the information obtained from this experiment was used to design of large-scale

fermenter for producing mass quantities of ethanol (calculations can be seen sample calculations

in the section dedicated to fermenter design). In the design of a 1000 L fermenter with 90 g/l

glucose and 1.0 g/l yeast inoculation concentration, it would take 31.1 hours for the fermentation

to complete (includes yeast growth lag-time and fermenter preparation). The final ethanol

concentration would be 43.81 g/l, or 43.81 kg of ethanol produced. Assuming 50 weeks of

operation, 270 batches could be produced each year, producing a total of 11840 kg of ethanol

annually. The tabulated results can be seen below in Table 3.

Table 3: Scale-Up Fermenter Design Results

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Xconversion X (g/L) P (g/L) t (hours) Methanol tcycle (hours) Methanol/t (g/h) Methanol kg
11840.50
0.876 38.22 43.811 22.08 43810.85 31.08 1409.58

CONCLUSIONS

1. The growth curve of Saccharomyces cervisiae, shown in Figure 4, does not successfully

depicts cell growth and its behavior through the various growth phases. Both the lag phase

(observed between 0 and 2 hours) and the exponential phases (observed between 2 and 4

hours) are displayed as expected. However, the stationary phase observed after the 4 th hour

does not demonstrate a balance between the cell growth and cell death. There is instead a

noticeable change in cell growth, a depletion of cells.

2. The growth phase differential model provides a value for the maximum specific growth

rate, max, of 0.165 hr-1. This was found using the data from the exponential phase with 86%

accuracy
3. The cell yield factor was found to be and 0.204 at the end of the experiment with an

average value of 0.472 over the course of the experiment. This low value for the end of the

experiment again depicts cell death during what should have been the stationary phase.
4. The ethanol or product yield factor was found to be 0.577, with an average value of 0.556.
5. The value of the Monod constant, KS, is difficult to determine in this experiment due to its

relationship with substrate concentration and the assumption that K S is much smaller than

the substrate concentration during the exponential phase when the majority of the data was

recorded.
6. A 1000 L fermenter with an initial glucose concentration of 90 g/l and a yeast inoculation

of 1.0 g/l would take 31.1 hours to ferment, yielding in a final ethanol concentration of

43.81 g/l (43.81 kg of ethanol produced). The amount of ethanol that could be produced

per year is 11840 kg (270 batches per year over 50 weeks of operation).

RECOMMENDATIONS

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1. The overall process could be improved in order to permit less oxygen to enter the system.

2. The yeast used in this lab is also exposed to oxygen which causes the yield to drop. Using

fresh Saccharomyces cervisiae could produce higher ethanol yields, higher cell yields,

and a reduction in cell growth lag-time.

3. The pH of the fermentation media (broth) should have been adjusted to 4.0 - 4.5 prior to

the inoculation of the yeast to prevent the growth of unwanted organisms during

fermentation, such as bacteria. This would then reduce lag time and increase ethanol and

cell yields.
4. More frequent sampling throughout the experiment could have resulted in more accurate

results and therefore, better represented graphs. In addition if more sample had been

taken, the saturation constant could have been calculated.

5. The accuracy of the analytical equipment (gas chromatograph, glucose analyzer, and

spectrophotometer) should have been checked with standards frequently to ensure

reliable data. Furthermore, each experimental sample should have been tested more than

once to ensure reproducibility.

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REFERENCES

Biochemistry 212 Class Notes. Taken semester II 2007.

"Chemical Engineering 414.2 Laboratory Manual", Department of Chemical Engineering,

University of Saskatchewan, 2007

Madigan and Martinko. 2006. Brock’s Biology of Microorganisms. Upper Saddle River, NJ :

Prentice Hall

Shuler, M.L., & Kargi, F. (2002). Bioprocess Engineering, Basic Concepts. Upper Saddle River,
NJ: Prentice Hall, Inc.

APPENDIX A:
RAW AND CALCULATED DATA
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Table 2: Cell and Ethanol Yield for Fermentation
Sample time ∆t (hour) Yp (g/g) Yx (g/g)
Pre 10:20 0
Pre 12:07 1.78
1 1:36 3.26 0.4952 0.209
2 1:46 3.43 0.5175 0.682
3 1:56 3.59 0.5318 0.635
4 2:06 3.76 0.5414 0.680
5 2:16 3.93 0.6570 0.670
6 2:26 4.10 0.5772 0.632
7 2:36 4.26 0.5548 0.609
8 3:06 4.76 0.5439 0.231
9 3:36 5.26 0.5587 0.171
10 4:06 5.76 0.5603 0.203

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 11 9:27 AM 23.12 0.5766 0.204

Yp(avg) Yx(avg)
0.5559 0.472

Table 3: Scale-Up Fermenter Design Results

Xconversion X (g/L) P (g/L) t (hours) Methanol tcycle (hours) Methanol/t (g/h) Methanol kg
11840.50
0.876 38.22 43.811 22.08 43810.85 31.08 1409.58

APPENDIX B:
GRAPHS

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24
 APPENDIX C:
SAMPLE AND DESIGN

CALCULATIONS

SAMPLE CALCULATIONS

Calculation of mass of glucose

Mass of Glucose = So*V

Where: So = 40 g/L
V = 1.5L
Mass of Glucose = (40g/L)*(1.5L) = 60g

Calculation of mass of yeast extract for inoculation

Mass of Yeast = Xo*V

Where: Xo = 4.0 g/L
Mass of Yeast = (4.0 g/L)*(1.5 L) = 6.0g/L

Calculation of yeast or cell mass concentration, X, for a 1/50 dilution for sample 2:

X = 41.889*OD -0.1509
Where: OD = 0.177
X = 41.889(0.177) – 0.1509 = 7.30 g/L

Calculation of yeast concentration, X, for a 1/100 dilution for sample 6:

X = 77.336*OD + 0.0115

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 Where: OD = 0.283
X = 77.336(0.283) + 0.0115 = 21.9

Calculation of ethanol or product concentration, P, for sample 6:

P = (Volume Percent Ethanol)*10

Where: Volume Percent Ethanol = 1.368
P = 1.368*10 = 13.68 g/L

Calculation of Product Yield Factor for sample 12 (final sample):

Where: P = 23.06 g/L for ethanol

S0 = 40g/L
S = 0.009 g/L
P
YP  = (23.06 g/L)/(40g/L – 0.009g/L) = 0.577
(S 0  S )

Calculation of Cell Yield Factor for sample 12 (final sample):

(X  X 0 )
YX 
(S 0  S )
Where: X = 15.1 g/L
Xo = 4.0 g/L

(X  X 0 )
YX  = (15.1 g/L - 4.0 g/L)/(40 g/L - 0.009 g/L) = 0.203
(S 0  S )

Determination of Maximum Specific Growth Rate, μM

From Appendix B, Figure B2, the equation of the linear trend line is:

26
 ln(X/Xo) = 0.1645x + 0.4786

Therefore, μM=0.165 h-.

DESIGN CALCULATIONS

Calculation of final yeast concentration, X

Where: Yx = 0.472 (average)

So = 90 g/L
S = 0.0g/L
Xo = 1.0g/L
X = 0.203 * (90 g/L – 0.0 g/L) + 1.0 g/L = 38.22 g/L

Calculation of final ethanol concentration, P

Where: Yp = 0.577 (average)

P = 0.577 * (90 g/L – 0.0 g/L) = 43.81 g/L

Calculation of final mass of ethanol

Where: P = 43.81 g/L

V = 1000 L

Mass of ethanol = (43.81 g/L * 1000L) = 43811 g

Calculation of time for each fermentation batch

27
 Where: X = 38.22 g/L
tPREPARATION = 7 hours
tLAG-PHASE = 2 Hours

t = [ln(38.22/1)]/(0.165 hr-1) = 22.08 hours

tTOTAL = 22.08 + 2+ 7 = 31.1 hours

Calculation of the number of fermentation batches produced per year

Batches/year = 50 weeks/year * 7days/week * 24 hours/day * 1/31.1 hours

Batches/year = 270

Calculation of the mass of ethanol produced per year

Where: batches/year = 270

Mass of EtOH = 43810 g

Mass of Ethanol/year = 270 * 43810 = 11840 kg

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