Fermentation

Kinetics of Yeast Growth

and Production
Introduction
Fermentation can be defined as an energy yielding process where yeast
converts organic molecules (such as sugar) into energy, carbon dioxide
or/and ethanol depending on the respiration pathway.
Yeast can respire in anaerobically and aerobically.
However, yeast gets more energy from aerobic respiration, but in the
absence of oxygen it can continue to respire anaerobically, though it does
not get as much energy from the substrate. Yeast produces ethanol when it
respires anaerobically and ultimately the ethanol will kill the yeast (find out
why is yeast continue to produce ethanol even the last is an inhibitor).
C
6
H
12
0
6
2 CH
3
CH
2
OH + 2 CO
2
+ 2 ATP
C
6
H
12
0
6
+ 6O
2
6CO
2
+ 6H
2
O + 16-18 APT
When the feed substrate to the reactor is not
monosaccharide e.g. sucrose (C
12
H
22
O
11
), yeast
enzyme cause glycosidic bond to break in a process
called hydrolysis
Industrial and Commercial Applications



Food Industry
~ Beer
~ Bread
~ Cheese
~ Wine
~ Yogurt

Pharmaceutical Industry
~ Insulin
~ Vaccine Adjuvants

Energy
~ Fuel Ethanol


Objective
To find the kinetics of the system by using
Nonlinear Regression (guess for k
s
and
m
)

The Sum of the Least Squares and the
Lineweaver-Burk Plot methods in order to
determine the parameters
m
and k
s
To determine the yield coefficient and to
project min. and max. amount yeast cell mass,
carbon dioxide and ethanol produced
Experimental Set Up
Apparatus

Bioreactor
pH meter
Sampling
device
Mixer
Temperature
sensor
YSI 2700
Biochemistry
Analyzer
pH probe
D-oxygen
probe
Experimental: Procedure
Using Biochemistry Analyzer and Spectrophotometer
to measure and make calibration curves for sugar and
yeast cell concentrations
Reactant initial concentration
dextrose/or sucrose 25 g/L
yeast 3 g/L
volume reactant solution 2 L
Initial conditions & assumptions
Initial Conditions
2 L of solution
50 g sugar
pH around 5.0
Temperature around 28-30C
Assumptions
the bioreactor content is
well mixed and has a constant medium volume at a certain
initial conditions
Temperature is constant
pH maintained at optimal pH of 3.00
All reactants or nutrients present in excess except for sugar
substrate.
Theory
In ideal fermentation process in which the growing cells are
consuming the substrate (sugars), and producing more cells
according to the following scheme.




rsx = rate of substrate consumption
rx = rate of cell growth
s = substrate concentration
x = cell concentration
P = ethanol concentration (in anaerobic case)


r
x
Cells (x)
P
Cells (x)
r
sx
Theory





The plot showing the trends for yeast cell growth over time


r
x

dC
x
dt


r
x

dx
dt
x

B
i
o
m
a
s
s

Theory continue
Yeast Growth occurs in 4 stages

Lag phase, yeast mature and acclimate to environment (no growth occurs)
The exponential growth section, the rate of reaction follows first order kinetics


During the deceleration phase, a large number of parameters, each with saturation effects,
have an effect on the kinetics of yeast growth (such as substrate and waste concentrations)


The growth rate is ruled by the limiting substrate concentration (sugar)


The final equation, often referred to as the Monod equation, looks very similar to the
Michaelis-Menten equation.



Stationary phase, no growth occurs due to high waste concentration or compleate substrate
consuming

x r
dt
dx
x

x r
s x

) (

+

s s
s
m s
s k
s

) (
k
s
= the Monod constant (g/L)

m
= a maximum specific growth reaction rate (min
-1
)

r
x

dx
dt

m
x
S
K
s
+ S

O
K
o
+ O

P
K
p
+ P





...

r
x

dx
dt

m
x
S
K
s
+ S






Lineweaver-Burk Rearrangement

+

s s
s
m s
s k
s

) (
m m
s
m
s
s
s
k
s
s k

1 1 1
) (
+
+

Nonlinear Regression
1. Define Model

2. Solve for R
predicted
(dx/dt)
(calculate dx/dt from the polynomial equation fitted
to the curve x(t)

3. Make initial guess for k
s
and
m


(
m
is the max. specific growth rate can be achieved
when S >> k
s

ks is saturation constant or the value of limiting
substrate conc. S at which
s
equal to the half of
m
4. Minimize (R-R
predicted
)
2
using solver function in
Excel by varying k
s
and
m

+

s s
s
m x
s k
s
x
dt
dx
r
Yield Coefficient Determination



Ratio of cell or Ethanol concentration to substrate concentration.
Knowing Y
x/s
will give you an idea for how much additional
yeast cell mass, on average, is produced for a given amount of
sugar substrate consumed.
As well allowed you to calculate a lower bound on the
experimental stoichiometric coefficient, , and therefore to
calculate ranges for ethanol and CO
2
production.
(Yeast Cell) + C6H12O6 (CO2 + CH3CH2OH) + (Yeast Cells)

s s
x x
ds
dx
Y
o
o
s
x

Y
p
s

dP
ds

P P
0
s
0
s
Error in Lineweaver-Burk
Parameters
Error in k
s
and
m
relative to error in slope and y-intercept of
linear fit

Random Error in y values:


STDEV of slope:


STDEV of y-intercept:

( )
( ) 2

n
y y
s
i i
x
y
( )

2
x x
s
s
i
x
y
b
( )

2
2
x x n
x
s s
i
i
x
y a
Lower Bound on
(stoichiometric coefficient)
(Yeast Cell) + C
6
H
12
O
6
(CO
2
+ CH
3
CH
2
OH) + (Yeast Cells)

Where, theoretically, = 2.


Assume all yeast generated is attributable only to sugar
complete consumption

Conservation of mass requires that the remaining product be
equimolar amounts CO
2
and ethanol