ChE 135 - Process Engineering Laboratory Formal Report

The Effect of Glucose Concentration on the Rate of Baker’s

Yeast Fermentation and The Determination of Its Rate Law.
Camille Margaret S. Alvarillo​ 1​, Maria Crispina B. Buensuceso​1​, Ysabel Marie C. Gonzales​1​, Jaron Nicolas T. Uy
1​
​University of the Philippines Diliman, Quezon City

Abstract. The objective of the experiment was to determine the effect of glucose concentration on the rate of
the reaction, as well as to determine the rate law and its parameters through integral and differential analysis.
This was done through a fermentation kinetics setup using yeast and glucose solutions, where the level of water
was measured through time to determine the amount of CO​2 produced and in turn, the amount of glucose
consumed in the system. It was observed that the volume of water decreased with time due to the production of
CO​2​. However, because of systematic errors and the use of inactive yeast, the data gathered from the
experiment was not enough to provide valid and firm conclusions. More trials must be done, and strict
adherence to the procedure must be done in order to meet the objectives of this experiment.
Keywords:​ fermentation; reaction kinetics; Michaelis-Menten; enzymes

1. ​Introduction also increased (Wojciechowski & Rice, 2003). In

The respiration of baker’s yeast is one way
to produce carbon dioxide which may occur
aerobically or anaerobically. Aerobic respiration is
the breakdown of glucose to produce energy in the
presence of oxygen. On the other hand, the
production of energy may also occur anaerobically
where in glucose is broken down without the use
of oxygen. This process is also known as
fermentation. Both anaerobic (Equation 1) and
aerobic (Equation 2) respiration are expressed in
the reactions below.
C​6​H​12​O​6​ → 2 C​2​H​5​OH + 2 CO​2 +
​ 2 ATP   analysis in this enzymatic reaction experiment is
C​6​H​12​O​6​ + 6 O​2​ → 6 CO​2​ + 6 H​2​O +  (2)
(16-18)ATP 
order to fully understand and characterize the rate
of the reaction and how different parameters affect
it, its rate law and order of the reaction is
determined experimentally. This can be done
through method of initial rates, fractional-life
methods, integral analysis, and differential
analysis.
A rate law is a differential equation which
expresses the change in concentration of a reactant
as a function of time. For this study, only the
differential and integral methods will be used. One
way to best model kinetic data for differential
(1)
through the Michaelis-Menten plots. This plot
relates the velocity of the reaction as a function of
the concentration of the substrate.

The respiration of baker’s yeast is affected Considering the reaction:

by several factors which include temperature and
concentration of glucose present in the system. E + S ↔ E S → E + P    (3)

When the temperature and the concentration of

glucose are increased, the rate of the reaction is
ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) - Fermentation
Kinetics

wherein the enzyme, E, first reacts with the The above mentioned linearizations provide a
substrate, S, to form a complex, ES, which later on more convenient way to determine the Michaelis
reacts to form products, P, and free enzymes, the constant, K​M​, and V​max​, the maximum velocity
rate of this reaction is given by achieved by the system.

V max [S]
The objective of this experiment is to
(4)
v= K M + [S]    perform glucose fermentation in order to
determine the kinetic model of the enzymatic
where V​max is the maximum velocity achieved reaction and how its concentration affects the rate
by the system of the reaction. This experiment also aims to
distinguish whether the fermentation is aerobic or
K​M is the Michaelis constant, which is anaerobic, and to determine kinetic parameters
the substrate concentration at 50% of using integral and differential analysis.
V​max
This experiment will only focus on the
[S] is the concentration of the substrate
effect of glucose concentration to the fermentation
(Michaelis, n.d.).
reaction and is limited to the determination of
kinetic parameters and modeling of the data
According to Valence (2017), the obtained. The effect of the yeast concentration, as
following express the rates of the reaction, from well as the formulation of a reaction mechanism
zero to second order, in both differential and will no longer be considered.
integral form.

Table 1: Equations for Differential and Integral

2. ​Materials and Methodology
Method The experiment began with the assembling
of the fermentation set up as seen in Figure 1
Differential Integrated obtained from the Process Engineering Laboratory
Order
Form Form Manual (“Process Engineering Laboratory
rA = d[A]
=− k [A] = [A]o − kt
Manual,” 2018). An aspirator was used to adjust
Zeroth dt
the water level to 50 ml.  
d[A] ln [A] = ln [A]o − k t  
First rA = dt
=− k [A]

d[A] 2 1 1
Second rA = dt = − k [A] [A]
= [A]o
+ kt

Furthermore, from the rate expression

established by Michaelis and Menten, the
Lineweaver-Burk (Equation 5), Eadie-Hofstee Figure 1: Fermentation Kinetics Setup
(Equation 6), and Hanes-Wolf (Equation 7)
linearizations were developed:
The following solutions found in Table 2 were
1 1 KM (5) prepared for the experiment.
rA = V max + V max [S]

V
rA = V max − K M [S] (6)

[S] KM 1 (7)
rA = V max + V max [S]
ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) - Fermentation
Kinetics

Table 2. Solution Preparation n = PV (8)

RT

Concentration
Afterwards,  the  concentration  of  glucose 
Glucose Solution 0.5% w/v in  the  solution  was  obtained.  This  was  done  by 
determining  the  total  amount  of  glucose present in 
Yeast Solution 1% w/v the  solution  and  dividing  it  by  the  total  solution 
HOAc-NaOAc Buffer 0.4 M volume.  The  total  amount  of  glucose is defined by 
the  initial  amount  of  glucose  present  in  the  0.5% 
glucose  solution  minus  the  amount  of  glucose 
The yeast solution was prepared with warm
consumed due to the formation of CO​2​.  
distilled water, and was left aside for 30 minutes in
 
order for the yeast cells to re-suspend.
Once  all  the  concentrations were obtained, 
the  integral  and  differential  analysis, as well as the 
The experimentation proper involved the
determination  of  the  ​Michaelis-Menten constants
measurement of the reaction rate. A water bath
were performed.
was first heated, and its temperature was kept
constant at 30°C. The glucose solution was
Based on the glucose concentration of the
warmed in the water bath. Then, 12.5 mL of yeast
system, it was found that as more CO​2 was​ formed
solution and 12.5 mL of the buffer solution were
and more glucose was consumed, a decrease in the
added simultaneously to the the Buchner flask, and
volume of water was observed. This was because
the magnetic stirrer set to a speed of 500 rpm. 25
CO​2​ displaced the water present in the system.
mL of the glucose solution was then added to the
flask, and it was covered immediately by a rubber
Moreover, based from a study made by a
stopper. The speed of the magnetic stirrer was
Microbiology Laboratory in the University of
adjusted to 70 rpm.
Manchester, the more glucose present there is, the
faster the reaction will be because the yeast will
The progress of the reaction was
become more active. However, the trend will only
determined by recording the initial volume of the
last up to a certain point, because excessive
mixture in the Buchner flask, and by taking the
glucose concentrations can slow down and/or
volume of the water in the burette every 30
affect negatively the rate of yeast fermentation.
seconds until no significant change was observed.  
This may be because the water potential in the
solution is no longer enough to allow yeast growth.
3.​ ​Results and Discussion In addition, even though there is excess glucose
Based  on  the  gathered  kinetic  data,  one  present in the solution, there may no longer be any
can  conclude  that  the  production  of  CO​2  was  the  sugar available for yeast growth (​Arroyo-López,
result  of  an  anaerobic  reaction  simply  because  the  et. al., 2009)​. Furthermore, the enzymes present in
reacting  system  was  a  closed  system.  This  means  yeast served as catalysts for fermentation to occur.
that  air,  which  is  the  source  of  oxygen,  is  not  These enzymes break down glucose to make it
directly  supplied  in  the  reaction;  thus  an  aerobic  viable for fermentation, also generally making the
reaction would not have been feasible.  reaction faster, but in certain mediums, enzymes
The  analysis of the kinetic data began with  can become denatured. When enzymes become
the  calculation  of  moles  of  CO​2  formed  based  on  denatured, they become inactive and can no longer
the  change  in  volume  of  water  observed  given  a  function. This can be caused by wrong levels of
certain  time  interval.  With  the  assumption  of  an  pH, and for this experiment, the solution pH must
ideal  gas  system  at  atmospheric  pressure,  the  not be too acidic and should just be at around 4-5.
number  of  moles  of  CO​2    formed  was  found 
through Equation 8.
The  integral  analysis  was  done  by 
linearizing  the  concentration  of  glucose,  [A]  and 
ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) - Fermentation
Kinetics

time  based  on  the  assumed  order of reaction of the  Table 3: Integral Analysis Calculated Parameters
system,  as  expressed  in  Table  1.  Figures  2,  3,  and 
4  present  the  linearized  data  for  the  zeroth  order,  Order k (M s​-1​) [A]​expt (M)
​ % Error
first  order,  and  second  order  reactions,  4.087 x 10​-7 0.0278 0.2945%
Zeroth
respectively.  Table  3  presents  the  calculated 
parameters,  such  as  the  initial  glucose  First 1.474 x 10​-5 0.0278 0.2945%
concentration,  as  well  as  the  rate  constant,  k.  It 
Second 5.314 x 10​-4 0.0278 0.2945%
also  compares  the  experimental  glucose 
concentration with the theoretical of 0.0277 M.    
From  the  graphs,  it  may  be  seen  that  the 
data  obtained  does  not  fit  any  of  the  theoretical 
reaction  orders  well,  with  an  R​2  of  only  0.6.  In 
fact,  the  concentration  of  A  was  only  recorded  to 
change  once,  so  despite  the  relatively  small 
relative  error  in  terms  of  concentration  for  each 
reaction  order,  the  data  is  not  sufficient  for 
analysis.  There was no appreciable fermentation of 
the  yeast  that  occured  so  no  conclusions  in  terms 
  of  the  order  of  the  reaction  can  be  made  and 
further trials are needed.  
Figure 2: Zeroth Order Reaction 
There  was  also  an  attempt  to  analyze  the 
gathered  data  through  the  differential  analysis 
method. ​However, due to some systematic errors, a 
change  in  the  volume  of  the  H​2​O  was  not 
significantly  observed,  which  indicated  the 
absence  in  the  production  of  CO​2​.  This  may  have 
been  caused  by  the  absence  of  the  fermentation 
reaction  given  that  the  yeast  used  in  the  system 
was  no  longer  functioning properly. Therefore, the 
differential  analysis  method  results  to  an  error  in 
  calculations,  as  seen  in  Appendix  B  and  C. 
Because  of  this,  no  constant  parameters  for  the 
Figure 3: First Order Reaction 
Michaelis-Menten  linearizations  can  be  calculated 
as well.  
Differential  rate  laws  give  the  rate  of 
reaction  on  a  more  molecular  level,  since  the  rate 
is  a  function  of  the  change  of concentration over a 
period  of  time.  With  this,  in  order  to  perform  a 
differential  analysis,  the  order  of  reaction  must 
first  be  assumed  before  performing  any 
calculation.  Numerical  differentiation  must also be 
performed  in  order  to  solve  for  the  reaction  rate 
  order and kinetic constant (Washington University, 
n.d.).  However,  this  method  yields  poor  accuracy 
Figure 4: Second Order Reaction
because  ideally,  rate  laws  cannot  be  determined 
just  from overall balanced chemical reactions, they 
must  be  experimentally  determined.  On  the  other 
hand,  integrated  rate  laws  are  often  used  to 
determine  the  rate  constant  and  order  of  reaction 
from  experimental  data.  For  the  integral  method, 
ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) - Fermentation
Kinetics

though,  the  order  also  has  to  be  assumed  initially.  Additionally,  it  is  suggested  that  a 
However,  until  the  curve  fit  is  linear,  the  order  of  preliminary  test  on  yeast  activity  is  performed  to 
the  reaction  must  be  increased  in  order to improve  ensure  that  it  is  active.  This  may  be  done  by 
the  accuracy  of  the  rate  constant.  A  good  adding  highly  concentrated  glucose  solutions  to  a 
advantage  of  the  integral  method,  though,  is  that  sample  of  the  dissolved  yeast  and  checking  for 
the  integration  process  smoothes  out  experimental  bubbling.  If  it  does  not  bubble,  a  new  batch  of 
errors,  which  improves  the  accuracy  of  obtaining  yeast must be tested and used. 
the rate constant (Reyes, n.d.).    
In  terms of other systematic errors present, 
5. References
it  is  important  to  note  that  the  experimental set-up 
avoided  other  factors  that  could possibly affect the  Arroyo-López, F. N., Orlić, S., Querol, A., &
Barrio, E. (2009). Effects of temperature,
fermentation  reaction,  like  ​temperature and pH.
pH and sugar concentration on the growth
These were, as much as possible, kept constant. It parameters of Saccharomyces cerevisiae, S.
was necessary to do so because temperature and kudriavzevii and their interspecific hybrid.
pH ar3e great drivers of enzyme reactions as these International journal of food microbiology​,
can also speed up or slow down the fermentation 131​(2), 120-127.
process. As previously mentioned, the wrong level
of pH can cause the enzymes to become denatured. Michaelis-Menten Kinetics and Briggs-Haldane
Temperature can do that as well; the temperature Kinetics. (n.d.). Retrieved from
must not be too high for fermentation to proceed. https://depts.washington.edu/wmatkins/kine
tics/michaelis-menten.htm
Also, since glucose concentration was the variable
in observation, it was necessary to keep these Process Engineering Laboratory Manual. (2018).
variables constant so that the effects of these other Department of Chemical Engineering.
variables would not interfere with that of
concentration. Reyes, A. (n.d.). Differential and Integrated Rate
Laws. Retrieved from
http://laney.edu/abraham-reyes/wp-content/
4. Conclusions and uploads/sites/229/2015/08/Differential-and-
Recommendations Integrated-Rate-Laws1.pdf
The  objective  of  this  experiment  was  to 
Vallance, C. (2017). Reaction Kinetics.
determine  the  effect  of  glucose  concentration  on 
the  rate of the reaction. It also aimed to determined 
Washington University (n.d.). Determining Rate
the  rate  law  of  the fermentation reaction. Based on 
Laws. Retrieved from
previous  studies,  it  was  found  that  the  more 
https://classes.engineering.wustl.edu/che50
glucose is present in the solution, the faster the rate 
3/ChE 505 Chapter 10N.doc
of  the  reaction  will  be.  However,  the  experiment 
remains  inconclusive  due  to  procedural  errors  and 
Wojciechowski, B., & Rice, N. (2003).
the use of non-living yeast. 
Experimental methods in kinetic studies.
 
Elsevier 
It  is  recommended  that  the  buffer  solution 
prepared  is  to  the  correct  pH  level,  as  too-low  pH 
levels  may  kill  the  yeast,  and  nullify  the 
experiment.  Other  precautionary  measures  include 
ensuring that the yeast is suspended in the solution. 
One  way  to  do  so is to prepare fresh yeast solution 
for  every  trial.  Furthermore,  more  trials  should  be 
done  to verify results. For the yeast to be used, it is 
recommended  that  it  is  stored  in  a  freezer  to 
prevent spoilage.  
 
 ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) -
Fermentation Kinetics

Appendix A: Raw Data 

 
Trial 1 Trial 2 Trial 3 Trial 4
Vol. H​2​O Vol. H​2​O Vol. H​2​O Vol. H​2​O
Time (s) (mL) Time (s) (mL) Time (s) (mL) Time (s) (mL)
0 39.4 0 38.9 0 39.5 0 39
30 39.3 30 38.9 30 39.4 30 39
60 39.3 60 38.8 60 39.3 60 39
90 39.2 90 38.8 90 39.3 90 38.9
120 39.2 120 38.8 120 39.3 120 38.9
150 39.1 150 38.8 150 39.2 150 38.9
180 39.1 180 38.8 180 39.2 180 38.9
210 39 210 38.8 210 39.2 210 38.9
240 38.9 240 38.8 240 39.2 240 38.9
270 38.9 270 38.8 270 39.2 270 38.9
300 38.9 300 39.2 300 38.9
330 38.9 330 39.2 330 38.9
360 38.9 360 39.2 360 38.9
390 38.9 390 38.9
 
NOTE:  Due  to  the  fact  that  the  buffer  used  from  Trials  1  to  3  was  too  acidic,  only  data  from Trial 4 
was analyzed in this study.  
 
 
 ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) -
Fermentation Kinetics

Appendix B: Sample Calculations

Initial Moles of Glucose (0.5 w/v% glucose solution):

0.25 g glucose
mole of glucose = 180.156 g glucose = 0.001387686227 mol glucose

Integral Analysis

Sample Data:
Time (s) Vol. of H​2​O (ml)

1 60 39.0

2 90 38.9

Volume of CO​2​ released

V​2​ - V​1​ = 39 ml - 38.9 ml = 0.1 ml

Moles of CO​2
1L
PV (1 atm)(0.1 ml)( 1000 ml )
RT = L atm
(0.082057 mol )(298.15 K)
= 0.000004087422687 mol CO2
K

Concentration of Glucose, [A]

0.001387686227 mol glucose − (0.000004087422687 mol CO2 )/2 mol
[A] = 0.05
= 0.02771285032 L

Differential Analysis

Sample Data:
Time (s) Vol. of H​2​O (mL)

1 60 39.0

2 90 38.9

3 120 38.9

Instantaneous Rate (via Central Difference)

d[A] An+1 −An−1

dt = tn+1 −tn−1 = 38.9−39
120−60 =− 6.812371146 x 10−7

NOTE: For the other data points, there would be a division error because the concentrations
of A would not change with respect to time.
 ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) -
Fermentation Kinetics

Appendix C: Numerical Data Tables

Integral Analysis
Vol. H​2​O CO​2​ Released
Time (s) (mL) (mL) mol CO​2 A (mol/L) ln(A) 1/A
0 39 0 0 0.02775372455 -3.58438523 36.0312
30 39 0 0 0.02775372455 -3.58438523 36.0312
60 39 0 0 0.02775372455 -3.58438523 36.0312
90 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
120 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
150 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
180 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
210 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
240 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
270 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
300 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
330 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
360 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
390 38.9 0.1 0.000004087422687 0.02771285032 -3.585859063 36.08434312
 ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) -
Fermentation Kinetics

Differential Analysis
CO​2
Time Vol. H​2​O Released
(s) (mL) (mL) mol CO​2 A (mol/L) dA/dt ln(-dA/dt) 1/(-dA/dt)
0 39 0 0 0.02775372455 0 #NUM! #DIV/0!
30 39 0 0 0.02775372455 0 #NUM! #DIV/0!
60 39 0 0 0.02775372455 -0.0000006812 -14.19935541 -1467917.673
0.0000040874226
90 38.9 0.1 87 0.02771285032 -0.0000006812 -14.19935541 -1467917.673
0.0000040874226
120 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
150 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
180 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
210 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
240 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
270 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
300 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
330 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
360 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
0.0000040874226
390 38.9 0.1 87 0.02771285032 0 #NUM! #DIV/0!
 ChE 135 - (Alvarillo, C.M.S.; Buensuceso, M.C.B.; Gonzales, Y.M.C.; Uy, J.N.T.) -
Fermentation Kinetics

Linearization of Michaelis-Menten Equation

CO​2
Time Vol. H​2​O Released
(s) (mL) (mL) mol CO​2 A (mol/L) dA/dt (-dA/dt)/A 1/(dA/dt) A/(-dA/dt)
0 39 0 0 0.027753 0 0 #DIV/0! #DIV/0!
30 39 0 0 0.027753 0 0 #DIV/0! #DIV/0!
60 39 0 0 0.027753 -0.00000022 0.0000081 4403753.019 -122220.548
90 38.9 0.1 0.000004087 0.02774 -0.00000022 0.0000081 4403753.019 -122160.548
120 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
150 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
180 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
210 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
240 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
270 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
300 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
330 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
360 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!
390 38.9 0.1 0.000004087 0.02774 0 0 #DIV/0! #DIV/0!