KC31003 Bioprocess Principles

Group 2
Group Member:
TAI WEI KEONG (BK09110063)
SEAH YONG UNG (BK09110144)
RUSSELL ROSS SULAU (BK09110156)
SCOTT BIONDI R. VALINTINUS (BK09110151)
CLARICE V. BINJINOL (BK09110005)
TAN YIT ZEN (BK09110015)
 Question 1
The growth rate of E. coli be expressed by Monod
kinetics with the parameters of µmax = 0.935 hr-
1 and Ks = 0.71 g/L (Monod, 1949). Assume that

the cell yield YX/S is 0.6 g dry cells per g

substrate. If CX0 is 1 g/L and CS1= 10 g/L when
the cells start to grow exponentially, at t0 = 0,
show how ln Cx, Cx, Cs, dlnCx /dt, and dCx /dt
change with respect to time.
 Given data
• µmax = 0.935 hr-1
• Ks = 0.71 g/L
• YX/S = 0.6 g
• CX0 = 1 g/L
• Cs1= 10 g/L
Rate of microbial growth is characterized by the
net specific growth rate
Equation (1) Equation (2)

Equation (3)

Net specific gross specific rate of loss

growth rate growth rate of cell mass

0, cells start to grow exponentially at

 Equation (4)

We assume that a single chemical species, S is a

growth –rate limiting. This kinetics can be
described by the Monod equation.

Equation (5) Equation (6)

Based on our question, we assume

Equation (7)

Equation (8) Equation (9)

Equation (10)
 SAMPLE CALCULATION
• Cx increased at a step size of 0.1.
• Cs data is calculated by using following
equation:
Equation (11)

• The value of t is calculated from the

integration with the values of Cs and Cx for
each iteration made is calculated based on
the previous 2 assumptions that made.
Cx ln Cx Cs t dlnCx/dt dCx/dt
1.0 0.0000 10.0000 0.0000 0.8720 0.9149
1.1 0.0953 9.8333 0.1093 0.8699 0.9998
1.2 0.1823 9.6667 0.2093 0.8676 1.0839
1.3 0.2624 9.5000 0.3016 0.8651 1.1674
1.4 0.3365 9.3333 0.3872 0.8624 1.2500
1.5 0.4055 9.1667 0.4672 0.8595 1.3317
1.6 0.4700 9.0000 0.5423 0.8563 1.4124
1.7 0.5306 8.8333 0.6131 0.8529 1.4922
: : : : : :
6.4 1.8563 1.0000 3.3949 0.0496 0.3199
6.5 1.8718 0.8333 3.7076 0.0332 0.2173
6.6 1.8871 0.6667 4.1677 0.0199 0.1324
6.7 1.9021 0.5000 4.9231 0.0099 0.0669
6.8 1.9169 0.3333 6.4171 0.0033 0.0225
6.9 1.9315 0.1667 10.8661 - -
 Cx and Cs versus time
12

10

8
Cx and Cs

6
Cx
CS

0
0 2 4 6 8 10 12
time
 lnCx versus time
10.0000

1.0000
0.0000 2.0000 4.0000 6.0000 8.0000 10.0000 12.0000
ln Cx

0.1000

0.0100
Time
 dlnCx/dt versus time
1.0000
0.0000 1.0000 2.0000 3.0000 4.0000 5.0000 6.0000 7.0000

0.1000
dlnCx/dt

0.0100

0.0010
Time
 dCx/dt versus time
3

2.5

2
dCx/dt

1.5

0.5

0
0 1 2 3 4 5 6 7
time
 Question 2
Immobilized cell or enzyme technology for
formation of biocatalyst has been valuable
tool in many bioprocesses. Among the
technologies, entrapment of biological
materials within calcium alginate gel beads
is the most commonly used.
a) Explain why calcium alginate
has been popular as
immobilization material. Suggest
alternative materials suitable for
immobilization.
Reasons:
1) The hydrogel formation occurs under
extremely mild conditions
2) Does not depend on the formation of
more permanent covalent bonds between
polymer chain.
3) It is biocompatible & has good
mechanical strength for many different
applications.
 Alternative material
1) Polyacrylamide
-Non-ionic
-Properties of the enzymes are only
minimally modified in the presence of the
gel matrix.
-Diffusion of the charged substrate &
products is not affected.
2) Chitosan

- Obtained from alkaline hydrolysis of

chitin.
- It is very biocompatible
-Has been used in many applications
including drug delivery system.
- Disadvantage:
 Limited solubility in water
 Require dilute acidic solution for
dissolution.
b) Describe at least two purposes for
using immobilization system in such
fashion and give example on how the
each purposed is serve.
 Purposes
1)Cells last longer
-Ordinary cells (only 1-2days) but immobilized cells
can last for 30days.
• How?
a) cells suspended into solution of sodium alginate.
b)Added to a calcium chloride solution.
c)Packed into column & a solution to be converted is
fed into the top of the column
d)Allowed to flow through the bed of beads contain
immobilized biocatalyst in the cells.
e)Conversion takes place & product comes out t the
bottom.
-Example: immobilized yeast cells
2. Retain the protein while allowing
penetration of substrate
-Example: calcium alginate
-Widely used due to the mild condition
-Result in minimum denaturation of the biocatalyst.
-Can be formed in extremely mild conditions which
ensure that enzyme activity yields over 80% can
be achieved.
-Entrapment in insoluble calcium alginate
gel:rapid,nontoxic,inexpensive,& versatile
methods.
c) Describe at least three methods
for large-scale production of calcium
alginate beads and state the pros
and cons of each method.
 Method I
Preparation of Calcium Alginate Microgel
Beads in an Electrodispersion Reactor
Using an Internal Source of Calcium
Carbonate Nanoparticles
• Pulsed electric fields are utilized to
atomize aqueous mixtures of sodium
alginate and CaCO3 nanoparticles
(dispersed phase) from a nozzle into an
immiscible, insulating second liquid
(continuous phase) containing a soluble
organic acid.
 Advantages
• Alginate molecules are stabilized by the
electrostatic force in a gel
• The coalescence of the emulsified drops
containing sodium alginate and CaCl2 is
capable of producing micrometer-sized
calcium alginate (Ca-alginate) gel beads.
• The scale-up potential of this process is
almost unlimited.
• The technique is suitable for biological or
food applications.
• The characteristic highly porous structure of
Ca-alginate particles
 Disadvantages
• The random process of droplet
coagulation vary widely in size and shape
• This processing involves harsh conditions
that can denature biological compounds.
• The difficulty in using dispersion/external
gelation techniques with ionic
polysaccharide is that the calcium source
(CaC12) is insoluble in the oil phase.
 Method II
Production of calcium alginate beads
by using Sound Wave Induced
Vibration Method
• A new process for the immobilization of
calcium alginate, which employs a vibration
of membrane induced by sound waves from a
horn-type speaker, is developed to produce
uniformly-sized beads of predictable
diameter.
• Frequency of sound wave and production
rate are varied to find the optimal control
ranges for the bead production.
• The controllable bead size range is 1.50-3.50
mm
• The experimental production rates are 0-12
dm3 h-1 by a single nozzle.
 Advantages
• Alternative to conventional continuous
bead production methods
• Low apparatus cost
• More easier to set up compare to other
methods
• Better control of bead size and shape
 Disadvantages
• Speed of sound varies with temperature
• Difficult to accurately measure the exact
arrival time of the wave front at different
sensors
 Method III
Production of alginate beads by
emulsification/internal gelation
• Alginate microspheres were produced by
emulsification/internal gelation of alginate sol
dispersed within vegetable oil.
• Gelification was initiated within the alginate
solution by a reduction in pH (7.5 to 6.5),
releasing calcium from an insoluble complex.
• Smooth, spherical beads with the narrowest
size dispersion were obtained when using
low-guluronic-acid and low-viscosity alginate
and a carbonate complex as the calcium
vector.
• Diameters ranging from 50 gm to 1000 tm
were obtained with standard deviations
ranging from 35% to 45% of the mean.
 Advantages
• The resultant broad size distribution.
Producing small-diameter alginate beads
(200-1000 pm), potentially on a large scale
(m3/h).
• Enhanced rates of bioconversion and to avoid
bead rupture resulting from the accumulated
fermentation gas. Ionic polysaccharide is that
the calcium source.
• By controlling the conditions under which the
water-in-oil dispersion is produced, the bead
size can be controlled from an few
micrometers to millimetres in diameter.
 Disadvantages
• Its cost is high in processing.
• Emulsion is unstable.
• The emulsion tends to separate into a
watery layer and an oily layer.
d) Describe one industrial application
for each immobilized cell and enzyme
system with the aid of PID diagram.
Application of immobilized cell
in Beer brewing industry
• Main advantages of using immobilised cells
for production of beer are:
1- Enhanced fermentation productivity due to
higher biomass densities
2- Improved cell stability
3- Easier implementation of continuous
operation
4- Improved operational control and flexibility
5- Facilitated cell recovery and reuse
6- Simplified downstream processing.
(Source: Shreve, R. N., & T.Austin, G. (1984). Shreve's Chemical Process Industries. New
York: McGraw-Hill.)
• Immobilisation cell system is applied in
the fermenter.
• Kirin 3 stages bioreactor system.
(Source: Kirin's Three-Stage Bioreactor. (n.d.). Retrieved May 10, 2012, from
SpringerImages: http://www.springerimages.com/Images/Chemistry/1-
10.1007_s11947-010-0435-0-10)
Application of Immobilized Cell
in Bioreactor
• Enzyme-immobilized membrane
bioreactors (EMBR) is a combination of
product separation process with enzymatic
catalysis in continuous processes.
• The selective membrane are use to
separate the enzymes from the reaction
products. EMBR have been widely used in
the field of fine chemicals synthesis.
(Source: Fang, Y., Huang, X.-j., Chen, P.-C., & Xu, Z.-K. (2011). Polymer
materials for enzyme immobilization and their application in bioreactors.)
e)Describe the problems that
envisaged when using immobilized
cell/enzyme technology.
1)Immobilized enzymes
- Often adversely affects the stability and activity of
the enzymes.
- Used for a prolonged period of operation
- Present large problems in diffusion of the
substrate to reach the enzyme.
- Another problem:
Leakage of enzyme
Based on the physical occlusion of enzyme
molecules within an enclosed gel structure 
diffusion of enzyme molecules to the surrounding
medium is limited.
Any use of enzymes as catalysts requires
consideration of their operational life
2) Immobilized cell
- May cause extra diffusion limitations
- Main problem: mass transfer resistance
imposed by the fact that the substrate
has to diffuse to the reaction site and
inhibitory or toxic products must be
removed to the environment.
- Depend on the relative rates of
bioconversion and diffusion.
 Question 3
• A by-product of fermentation process has shown
to be inhibitory to cell growth. From the data
below:
– A) Obtain the kinetic parameters µm, ks.
– B) Determine the type of inhibition by the inhibitor.
Justify your answer and what do you think of the data
accuracy.
– C) Determine inhibition constant, kp. What can u do
to improve the reliability of the value?
 Specific growth Specific growth
By (g/L) Glucose (g/L) rate µ (hr-1) By (g/L) Glucose (g/L) rate µ (hr-1)

0.00 3.00 0.15 25.00 3.00 0.07

4.00 0.17 4.00 0.07

5.00 0.19 5.00 0.09

8.00 0.25 8.00 0.10

12.00 0.26 12.00 0.15

16.00 0.29 20.00 0.18

20.00 0.41 3.00 0.05

20.00 3.00 0.08 4.00 0.05

32.00
4.00 0.10 5.00 0.07

5.00 0.11 8.00 0.07

6.00 0.11 12.00 0.09

10.00 0.17 20.00 0.11

20.00 0.23
 Solution 3a
• Table shows the cell growth with same
substrate concentration parameter at the
• Conditions => different by-product
concentration which caused inhibition to the
cell growth
• By-product concentration increased from 0 to
32g/L.
• Double reciprocal or Lineweaver-Burke (LWB)
plot is used to obtain ks and µmax.
Calculated value for 1/[s] and 1/[µ]
By-product Specific growth
Glucose (g/L) 1/[S] 1/µ
(g/L) rate µ (hr-1)
0.00 3.00 0.3333 0.15 6.6667

4.00 0.2500 0.17 5.8824

5.00 0.2000 0.19 5.2632

8.00 0.1250 0.25 4.0000

12.00 0.0833 0.26 3.8462

16.00 0.0625 0.29 3.4483

20.00 0.0500 0.41 2.4390

20.00 3.00 0.3333 0.08 12.5000

4.00 0.2500 0.10 10.0000

5.00 0.2000 0.11 9.0909

6.00 0.1667 0.11 9.0909

10.00 0.1000 0.17 5.8824

20.00 0.0500 0.23 4.3478

By-product Specific growth
Glucose (g/L) 1/[S] 1/µ
(g/L) rate µ (hr-1)

25.00 3.00 0.3333 0.07 14.2857

4.00 0.2500 0.07 14.2857

5.00 0.2000 0.09 11.1111

8.00 0.1667 0.10 10.0000

12.00 0.1000 0.15 6.6667

20.00 0.0500 0.18 5.5556

32.00
3.00 0.3333 0.05 20.0000

4.00 0.2500 0.05 20.0000

5.00 0.2000 0.07 14.2857

8.00 0.1667 0.07 14.2857

12.00 0.1000 0.09 11.1111

20.00 0.0500 0.11 9.0909

Equation
 Graph of 1/[µ] versus 1/[s]
25.0000

y = 39.386x + 7.9577
20.0000

y = 32.705x + 4.6395
15.0000

[I]=0.00 g/L
[I]=20.00 g/L
[I]=25.00 g/L
y = 28.183x + 3.3185
1/[µ]

10.0000 [I]=32.00 g/L

Linear ([I]=0.00 g/L)
Linear ([I]=20.00 g/L)
Linear ([I]=25.00 g/L)
Linear ([I]=32.00 g/L)
5.0000

y = 13.556x + 2.3682

0.0000
-0.2000 -0.1000 0.0000 0.1000 0.2000 0.3000 0.4000

-5.0000
1/[s]
 Tabulated value of µ and k max s

Concentration Y intercept = µmax, hr-1 X intercept = - Ks

g/L 1/µmax 1/Ks
0.00 2.3682 0.4223 -0.1747 5.72
20.00 3.3185 0.3013 -0.1177 8.49
25.00 4.6395 0.2155 -0.1416 7.05
32.00 7.0887 0.1411 -0.1686 5.93
 Solution 3b
• LWB plot and calculations in part A suggests that
the inhibition type could be noncompetitive
because value of µmax decreased proportionally
with inhibitor concentration (varying µmax).
• KS values from 5.72 to 8.49 considered small
differences
• Slope increment with increased inhibition
concentration means that it is a non competitive
• Uncompetitive would have the same slope
• Accuracy of data may not be good. It doesn’t
show that it is a 100 % non competitive
inhibitor.
• LWB plot, plotting like is just taken best line of
graph and not through exact points.
• Experimental data might not be 100%
accurate.
• May show uncompetitive type
• Reliability of inhibition constant can be
improved through second LWB plot taken the
best line of graph.
• Inhibition constant = x-intercept
• Non-competitive type hence Ks should be
taken as average to improve Kp value
reliability.
Determination of K s,average
Derivation for non-competitive
equation
 Kp determination
• µmax,app is the growth rate , µmax with the
influence of inhibition which is µmax calculated
at part A with different concentration of
inhibitor.
• Graph is plotted of each inverse apparent
specific growth rate against concentration of
inhibitor which is secondary LWB plot to find
value of inhibition constant, Kp.
 1/µmax,app
[I], g/L µmax,app (hr-1)
or 1/[V'max]

0.00 0.4223 2.368

20.00 0.3013 3.319

25.00 0.2155 4.640

32.00 0.1411 7.087

 Graph of 1/[V'max] vs [I]
8

y = 0.1305x + 1.8419

4
1/[V'max]

0
-15 -10 -5 0 5 10 15 20 25 30 35

-1
[I], g/L
Kp determination
THANK YOU