ChE 135 Process Engineering Laboratory Formal Report Group

Kinetic Modelling of Anaerobic Glucose Respiration

Ronald Eldrick D. Guico1, Grace Iris L. Miranda1, Nathaniel M. Saporsantos
1 University of the Philippines Diliman, Quezon City

Abstract. In this experiment, the kinetic model of glucose fermentation by Baker’s Yeast was determined and
analyzed. Three runs of varying initial glucose concentrations at pH 5 were performed. The kinetics of the
reaction followed the Michaelis-Menten equation satisfactorily with average R2 values of 0.988, 0.938, and
0.974 for runs 1, 2, and 3, respectively. The rate was found to increase with increasing values of the kinetic
parameter k. In addition, the rate decreases as the Michaelis-Menten constant M increases.
Keywords: yeast; glucose; fermentation; kinetic modelling; Michaelis-Menten

1 Introduction

The equations and derivations included throughout the paper contain letters and sysmbols where :

rA – rate of disappearance of A M – Michaelis-Menten Constant

CA – concentration of A CE – concentration of enzyme
CA0 – initial concentration of A CR – concentration of products
k’ – rate constant CE0 – initial concentration of enzyme
P – pressure Cx – concentration of intermediate ngas – moles of gas
V – Colume T – temperature
R – gas constant t – time
k – constant parameter for Michaelis-Menten expression
k1 – rate constant for forward step in equilibrium step of M-M mechanism
k2 – rate constant for backward step in equilibrium step of M-M mechanism
k3 – rate constant for last step in M-M mechanism

Respiration is a chemical process in which an be found in cells in which a multistep process

organic compound is broken down to produce
energy. Fermentation, or anaerobic respiration, is a
type of respiration in which the compound is
transformed in the absence of oxygen producing
ethanol, carbon dioxide and energy in the form of
Adenosine Triphosphate (ATP). This process takes
place in yeast wherein an enzyme catalyzes the
production of alcohol and carbon dioxide. The
balanced reaction of the fermentation of glucose is
shown in equation (1).
(1)
On the other hand, aerobic respiration produces
energy in the presence of oxygen. This reaction may
converts the substrate into energy (Campbell et al.).
Water is produced in exchange for the formation of
alcohol. The aerobic respiration of glucose is shown
in equation (2).
C6H12O6 + 6O2  6H2O + 6CO2(g) + (16-18)ATP (2)
It can be seen that the total number of gaseous
molecules does not change as the substrate is
converted to products in aerobic respiration. Thus,
one way to determine the mechanism by which
respiration occurs is by noticing a change in the
amount of gas. This can be done by observing a
change in either pressure or volume.
 ChE 135 – Voltes III – Kinetic Modelling of Anaerobic Glucose Respiration
To fully understand a reaction, a kinetic analysis
must be done. The kinetics of a reaction may be
analyzed using the integral and differential methods.
The integral method of analysis is done by
comparing an equation derived from an integrated
rate law to the experimental data (Levenspiel). If the
data doesn’t fit the expression, another equation is
guessed and tested. Consider the reaction in
equation (3).
(3)
A general nth-order rate expression in terms of
the disappearance of A may be written as equation
(4).
(4)
Integrating the equation results in equation (6).
(5)
(6)
If the reaction is first order, n = 1, and equation
(4) becomes equation (7) upon integration.
(7)
The example above only applies to 1st and nth-
order reactions. Equations of a different form are
derived in a similar manner. A plot of C A vs. t from
experimental data is then compared to the equation
chosen. If the two are not in agreement with each
other, another equation is chosen. The integral
method is recommended when the rate expressions
are relatively simple, or when the data points are
scattered. It can only be used to test mechanisms
with a particular rate expression. It becomes tedious
when the rate expression is complex or when a rate
equation is to be developed to fit experimental data.
The differential method of analysis makes use of the
data directly in that the rates at specific values of
time are calculated (Levenspiel). This is then
compared to a known rate expression of the form
given in equation (8) or equation (9).
(8)
(9)
To obtain the rate at the ith data point for N data
points, forward, central, and backward differences
can be used (Kreyszig). Central difference is used
for data points in between the initial and final data
points to reduce the error. The rates are given by
equations (10) – (12).
(10)
(11)
(12)
A plot of vs CA is then compared to
equation (8). If possible, linearization of the
equation should be done to produce smaller errors as
compared to those produced by a general curve. The
vs CA data are then modified to fit the
linearized form of the equation. One advantage of
using the differential method is that it can be used
for rate expressions that are complex and difficult to
integrate. However, this method is inaccurate when
only a few data points are obtained from
experiment.
In 1913, Michaelis and Menten published a
paper about a proposed mechanism by which some
enzyme-catalyzed reactions occur. From previous
studies of enzyme-catalyzed reactions, it was
observed that some of these reactions have a rate
proportional to the initial enzyme concentration,
CE0. At high concentrations of the reactant, the rate
becomes independent of the reactant concentration.
In addition, at low reactant concentrations, CA, the
rate becomes first order with respect to the reactant
A. From these observations, they proposed the
mechanism shown in equations (13) – (15) .The
elementary steps are given in equations (13) to (14)
while the overall reaction is shown in equation (15).
(13)
(14)
(15)
To obtain the rate expression for this
mechanism, the following assumptions are made.
(16)
(17)
Equation (16) is a mass balance equation on
the enzyme. Equation (17) pertains to the steady-
state approximation which implies that the
intermediate X is consumed immediately after it is
 ChE 135 – Voltes III – Kinetic Modelling of Anaerobic Glucose Respiration
formed. The rate expression for each component is
written and shown in equations (18) to (19)
(18)
(19)
Eliminating CE from equation (19) using
equation (16) gives equation (20).
(20)
(21)
Substituting equation (20) into equation (19)
results in equation (22)
(22)
The constant M in equation (22) is known as
the Michaelis Constant. Its typical value ranges
from 10-1 to 10-5 M (Chaplin). The rate expression is
consistent with the observations made in previous
kinetic studies. The rate is directly proportional to
the initial enzyme concentration CE0 , which is found
in the numerator of equation (22). At low reactant
concentrations, eq. (22) reduces to equation (23).
(23)
Equation (23) shows the first order dependence
on the reactant concentration of the reaction at low
CA. Finally, at high reactant concentrations,
equation (22) becomes equation (24) and becomes
independent of the reactant concentration.
(24)
To perform an integral method of analysis
using the Michaelis-Menten expression, equation
(22) is rearranged and integrated. The result is
shown in equation (26)
(25)
(26)
To determine the value of the constants,
equation (26) is rearranged to equation (27).
(27)
If the initial enzyme concentration is unknown,
the constants k3 and CE0 can be expressed as one
constant giving equation (28).
(28)
A plot of vs can then be used
to compare the Michaelis-Menten expression to the
data obtained from experiment and to obtain the
constants M and k.
To perform a differential analysis on the M-M
mechanism, equation (22) may be rearranged to
equation (29), (30), or (31).
(29)
(30)
(31)
A plot of vs , , or
can be used to compare the data
to the M-M expression, and to obtain the constant
parameters M and k.
In this experiment, the kinetic model of the
enzymatic reaction through glucose fermentation is
determined using the integral and differential
methods. Various rate expressions and the
Michaelis-Menten expression are tested. In addition,
the kinetic parameters are also calculated using
linearized plots of the kinetic model. The
dependence of the model on reactant concentration
is then analyzed. The volume reading is converted
to moles of gas produced using the ideal gas
equation (eq. 32).
(32)
The assumption that the gas formed behaves as
an ideal gas is valid as the compressibility factor of
CO2 at the experimental conditions is almost equal
to unity (Van Ness et al.). The amount of glucose
that reacted is then calculated using either equation
 ChE 135 – Voltes III – Kinetic Modelling of Anaerobic Glucose Respiration
(33) for anaerobic respiration or (34) for aerobic
respiration.
(mol glucose reacted) = ngas (33)
(mol glucose reacted) = ngas (34)
Finally, the concentration of the reactant is
calculated using equation (35) assuming that the
reaction volume is constant at 50 mL.
(35)
2 Materials and Methodology
The fermentation experiment was performed in
three separate trials with the same amount of
reagents used and similar procedure followed.
2.1 Set-up assembly
A fermentation set-up was assembled by connecting
an inverted burette to the Buchner flask suspended
in a water bath using a rubber tubing. The burette
was submerged in a basin filled with tap water and
was fixed by an iron stand. In order to have accurate
readings on the burette, it was ensured that it was
upright and perpendicular to the basin. The water
level was adjusted and was maintained by securing
the rubber tubing with a metal clip, with an
assurance of no gas leakage.
2.2 Solution preparation
A glucose solution was prepared by weighing the
required amount of anhydrous glucose and
dissolving it in distilled water. A separate yeast
solution was prepared by weighing yeast and
dissolving to distilled water preheated at 50oC,
allowed to sit for 20 to 30 minutes. Appropriate
amount of solid NaCH3COO•3H2O was weighed
and dissolved in 99.7% acetic acid and diluted to
produce a 0.4M acetic acid-acetate buffer with pH
5.
2.3 Reaction rate measurement
The glucose solution was preheated in the water
bath at 30oC. The yeast solution and the buffer were
poured in the Buchner flask and was also preheated
to the same temperature as that of the glucose, with
the magnetic stirrer speed set to 500 rpm. The
glucose solution was added and the stirrer speed was
adjusted to 1150 rpm for the first trial, and was
maintained at 500 rpm for the last two trials. The
volume of the water in the burette was recorded
after the removal of the metal clip.
2.5 Reference formatting
Use automatic inline citation in APA style. A
sample is presented here (Muñoz, López-Mesas, &
Valiente, 2012). You may also use other citation
tools such as EndNote or Mendeley for automatic
citations (Grases, Prieto, Gomila, Sanchis, & Costa-
Bauzá, 2009).
3 Results and Discussion
3.1 Nature of the reaction
In all the trials in the experiment, the level of water
inside the inverted burette decreases over time,
which means gas is generated in the reaction.
Anaerobic respiration has a net gain of 2 moles CO2
per mole glucose, whereas aerobic respiration yields
no additional gas molecules, hence the reaction
occurring in the experiment is predominantly
anaerobic.
3.2 pH of solution
Due to the nature of enzymes present in the yeast
that facilitates the reaction, the optimal pH of the
system is 4-6.8 (Tabah et al.). In the fermentation
process however, the pH of the system increases due
to the large amount of CO2 produced in the reaction,
which forms H2CO3 when dissolved in water.
The acetate buffer solution is added to mitigate
the decrease in pH, having the pH range of 3.7-5.6
(Dawson et al.), which is within the optimal pH for
fermentation.
3.3 Yeast Activation
Active Dry Yeast was used in the experiment, and in
order for the reaction to proceed, the yeast must be
first activated from its incubated state (Zarei et al.).
This is why the yeast solution was heated to 50 OC,
and allowed to re-suspend for 20 minutes. Usually a
pinch of sugar is added to know the state of the
yeast. If bubbles form, the yeast is activated.
3.4 Kinetic modelling
The volume of the gas generated is measured by the
volume of H2O displaced by the gas. This is then
used to calculate the moles of glucose reacted and
concentration of glucose in the system using
equations (33) & (35). The data is then fitted to the
 ChE 135 – Voltes III – Kinetic Modelling of Anaerobic Glucose Respiration

Michaelis-Menten expression using both Integral
and Differential analysis where the kinetic
parameters M and k are found. The k parameter
corresponds to the maximum rate of reaction while
M is the concentration of substrate, in this case
glucose, required to achieve half the maximum rate
of reaction. M is also associated with the affinity of
the substrate to the enzyme (Dahziel). Eq. (23) also
shows the effect of M and k to the actual rate of
reaction.
3.4.1 Integral Analysis
Equation (28) is used to find the kinetic parameters
M and k in trials of increasing initial glucose
concentration:
The value of k corresponds to the maximum rate
of reaction and generally directly proportional to the
rate of reaction. The trend of k shows no linear
relationship with the initial concentration of
glucose. This either means k reaches a maximum
somewhere between 0.125% w/v and 0.5% w/v, or
errors of the third trial vastly skewed the data. This
is supported by the R2 values, as the R2 value of the
third trial is the lowest among the trials.
3.4.2 Differential Analysis
Equations (29), (30), and (31) were used to find the
kinetic parameters M and k in increasing initial
glucose concentration:

Figure 2. Differential Analysis using Eq. (29). Blue

(0.125% w/v), Orange (0.25%, w/v), Gray (0.5% w/v)

Figure 1. Integral Analysis. Blue (0.125% w/v), Orange

(0.25%, w/v), Gray (0.5% w/v)
The Michaelis-Menten kinetic parameters were
found using linear regression:

Table 1. Kinetic Parameters from Integral Analysis

Glucose
M
concentration k (mol/L*s) R2
(mol/L)
(w/v)
0.125% 0.012863 2.27E-06 0.99148

0.250% 0.06406 6.86E-06 0.990563

Figure 3. Differential Analysis using Eq. (30). Blue
0.500% 0.122877 1.07E-06 0.970558 (0.125% w/v), Orange (0.25%, w/v), Gray (0.5% w/v)

The high R2 values of the fitted data shows that

the values are consistent. Since the value of M is
inversely proportional to the affinity of the
substrate, i.e. glucose, to the enzyme in the yeast, as
initial concentration of glucose increases, the
affinity of the substrate to the enzyme decreases.
This generally means lower rate of reaction.
 ChE 135 – Voltes III – Kinetic Modelling of Anaerobic Glucose Respiration

0.125% w/v and 0.500% w/v. The k parameter is

directly proportional to the rate of reaction as shown
in Eq. (23).
The non-linear relationship between the k
parameter and the glucose concentration and the
contradicting effects of k and M parameters on the
rate of reaction makes it difficult to ascertain the
relationship between the rate of reaction and the
glucose concentration.

4 Conclusion and
Figure 4. Differential Analysis using Eq. (31). Blue Recommendation
(0.125% w/v), Orange (0.25%, w/v), Gray (0.5% w/v)
Integral and Differential analysis of the
Michaelis-Menten expression show that the M
The Michaelis-Menten kinetic parameters were
parameter increases as glucose concentration
found using linear regression:
increases, which means that substrate affinity
towards the enzyme decreases with substrate
concentration.
Table 2. Kinetic Parameters from Differential Analysis
Glucose
The k parameter is shown to have a non-linear
Differential relationship with glucose concentration, having a
contration M (mol/L) k (mol/L*s) R2
analysis
(%w/v) maximum between the data points. This can either
0.125% 0.005188 9.09E-06 0.99372 be caused by errors in the experiment or the
using Eq.
0.250% 0.040052 3.47E-05 0.971867 complicated relationship between the maximum rate
(29)
0.500% 0.051818 2.5E-06 0.969669 of reaction that cannot be modelled in the
Michaelis-Menten expression. The contradicting
0.125% 0.005044 8.62E-06 0.970183
using Eq. effects of the k and M parameters on the rate of
0.250% 0.034039 2.7E-05 0.80496 reaction makes it difficult to categorize the exact
(30)
0.500% 0.051753 2.48E-06 0.957802 relationship between the concentration of the
0.125% 0.004672 7.31E-06 0.99821 substrate and the rate of reaction. Possible causes of
using Eq.
0.250% 2.62E-02 1.66E-05 0.985358
error can be improper sealing of clamps and
(31) stoppers, difficult reading of burette, and unstable
0.500% 0.050843 2.23E-06 0.999905
temperature while reaction is occurring.
It is recommended to repeat the experiment with
Based on the R2 values, the most accurate multiple replicates per concentration in order to
equation used for differential analysis is equation ensure the accuracy of data, and to use other models
(31), and the least accurate is equation (30). Using besides the Michaelis-Menten expression, like the
Eq. (31) is also convenient, as the slope and the y- Monod model that might better fit the system.
intercept of the linear fit already corresponds the M
and k parameters respectively. The most
conservative values are also found using equation References
(31).
Campbell, M. K., Farrell, S. O., & McDougal, O. M.
M is shown to increase as initial glucose
(2018). Biochemistry. Boston, MA: Cengage
concentration increases, whereas k reaches a
Learning.
maximum between 0.125% w/v and 0.500 w/v.
Chaplin, M. (2014, August 6). Simple Kinetics of
Enzyme Action. Retrieved February 05, 2018,
3.5 Effect of Substrate Concentration
from http://www1.lsbu.ac.uk/water/enztech/
kinetic s.html
Both Integral and Differential analysis show that M
Kreyszig, E. (2006). Advanced Engineering
increases as the initial concentration of substrate, i.e.
Mathematics. Hoboken, NJ: Wiley.
glucose, increases. This means as concentration of
Levenspiel, O. (1972). Chemical Reaction
substrate increases, the affinity of the substrate to
Engineering. 3rd ed. New York, London:
the enzyme decreases, and overall decreases the rate
Wiley.
of reaction.
Smith, J. M., C., V. N., Abbott, M. M., & Swihart,
The k parameter however increases to a certain
M. T. (2018). Introduction to Chemical
point then decreases, making a maximum between
 ChE 135 – Voltes III – Kinetic Modelling of Anaerobic Glucose Respiration

Engineering Thermodynamics. New York:

McGraw-Hill Education.
Tabah, B., Pulidindi, I. N., & Gedanken, A. (2015).
Study on Fermentation Kinetics for
Accelerated Production of Bioethanol from
Glucose, Sucrose and Molasses. Journal of
Bioprocessing & Biotechniques 5: 232.
Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.;
Jones, K. M. (1986) Data for Biochemical
Research; 3rd ed., Oxford Science Publ.
Zarei, O., Dastmalchi, S., & Hamzeh-Mivehroud,
M. (2016). A Simple and Rapid Protocol for
Producing Yeast Extract from Saccharomyces
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Culture Media. Iranian Journal of
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Dalziel, K. (1962) Physical Significance of
Michaelis Constants. Nature 196, 1203-1205.
 ChE 135 – Voltes III – Kinetic Modelling of Anaerobic Glucose Respiration

Appendix: Sample calculations

First data point on First Trial: