CH6610

Chemical Reactor Engineering

Assoc. Prof. Dr. Vu Dinh Tien
Faculty of Chemical Engineering - SCLS
Topic 7 - Theory
Bioreactions and Bioreactors

Fogler, Chapter 9
• Enzymes
• Michaelis-Menten Equation
• Lineweaver-Burk Equation
• Enzyme Inhibition
• Microbial Growth Kinetics – Monod Model
• Analysis of Bioreactors

Slide 2
Introduction
• Enzymes: Protein catalyst that execute complex biochemical
reactions- all synthetic and degradative reactions in living organisms!
• Increases the rate of reaction without undergoing permanent
chemical change – not used up (consumed) by the reaction
• Substrate: the reactant that the enzyme acts on

• Goals of this lecture:

• Predict rates of enzyme-catalyzed reactions
• Determine effect of chemical inhibitors on reaction rate
• Develop mathematical expression based on fundamental steps of
reaction
• Apply model to cell growth
Slide 3
Michaelis-Menten (M-M) Equation
Vmax: maximum reaction
rate, further increases in
substrate, S, no longer
[rP] increase the reaction
velocity, v

v = reaction velocity = rP = -rS

Km = substrate concentration where reaction velocity v = Vmax/2
[S] = substrate concentration [P]: product concentration
Empirically found the V C
v = max S
Michaelis-Menten equation: K m + CS
Where Vmax depends on the amount of enzyme

The University of Adelaide Slide 5

Rate Equation for Enzymatic Reaction
S
v = rP = V max Goal: derive this experimentally determined reaction rate
Km + S
𝑘1 𝑘2 E: enzyme S: substrate
𝐸 + 𝑆 ⇄ 𝐸𝑆 𝐸+𝑃 ES: enzyme-substrate complex
𝑘−1
dCP
rate of product formation : v = rP = = k 2CES
dt
We cannot measure CES, so we need to get CES in terms of species we can
measure. Start by writing the rate equation for CES :
dCES
= k1CSCE − ( k −1 + k 2 ) CES
dt
The free enzyme concentration CE is also difficult to measure. Use the
mass balance to get CE in terms of CES and CE0.
CE = CE0 − CES where CE0 = CE,t =0
Substitute into rate eq for CE:
dCES
→ = k1CS ( CE0 − CES ) − ( k −1 + k 2 ) CES
dt
Slide 6
CES in Measurable Quantities
dCES
→ = k1CS ( CE0 − CES ) − ( k −1 + k 2 ) CES
dt
𝑑𝐶𝐸𝑆
Pseudo-steady state assumption: ES is a reactive intermediate, so =0
𝑑𝑡
dCES
= 0 = k1CS ( CE0 − CES ) − ( k −1 + k 2 ) CES Now solve for CES
dt
Multiply out and rearrange → k −1CES + k 2CES = k1CSCE0 − k1CSCES

Bring CES to left side of equation → k −1CES + k 2CES + k1CSCES = k1CSCE0

Factor out CES → CES ( k −1 + k 2 + k1CS ) = k1CSCE0

k1CSCE0
Divide by quantity in bracket → CES =
k −1 + k 2 + k1CS
CSCE0
→ CES = Plug this expression
Divide top & bottom by k1 k −1 + k 2 + C
S for CES into dCP/dt
k1
Slide 7
Derivation of the M-M Equation
𝑘1 𝑘2 E: enzyme S: substrate
𝐸 + 𝑆 ⇄ 𝐸𝑆 𝐸+𝑃 ES: enzyme-substrate complex
𝑘−1
dCP
rate of product formation : v = rP = = k 2CES
dt
CSCE0 Plug this expression
→ CES =
k −1 + k 2 + C for CES into dCP/dt
S
k1
dCP k 2CE0 CS Compare to
rP = = = =
V maxCS
dt k −1 + k 2 + C experimentally v rP
S K m + CS
k1 observed rate eq:
Vmax = k 2CE0 When CS>>Km, then:
Vmax occurs when enzyme is fully rP = −rs  Vmax
saturated with S (in ES form) When CS<<Km, then:
+ V maxCS
K m = k −1 k 2 rP = −rS =
k1 Km
Slide 8
Complications with Measuring
Rates with the M-M Equation
• In practice, Vmax can be
difficult to estimate
using the MM
equation.
• Various different
values of Vmax were
reported.
• Since a solution with
infinite concentration
of substrate is
impossible to make, a
different equation was
needed.

Slide 9
Lineweaver-Burk Equation
Lineweaver & Burk V maxCS 1 Km + CS
inverted the MM rP = → =
equation Km + CS rP V maxCS

1  Km   1  1
→ =   +
rp  V max   CS  V max
y= (m ) ( x ) + b
By plotting 1/V vs 1/CS,
a linear plot is obtained:
Slope = Km/Vmax
y-intercept = 1/Vmax
x-intercept= -1/Km

Slide 10
 3. Noncompetitive (mixed)
Inhibition
Noncompetitive inhibition No inhibition

v= rP =
( Vmax (1 + CI KI ) ) CS
vs rp =
V maxCS
CS + K m K m + CS
Vmax observed w/
V
noncompetitive Vmax,app = max substrate and inhibitor bind
inhibitor CI
1+ different sites
KI

Vmax, app < Vmax 1 Km  1  1

=   +
Km, app = Km rP Vm,app  CS  Vm,app

Slide 14
Kinetics of Microbial Growth
(Batch or Semi-Batch)
Region 1: Lag phase
microbes are adjusting to
the new substrate
Region 2: Exponential
growth phase
microbes have acclimated
to the conditions
Region 3:
Stationary phase
limiting substrate or
oxygen limits the growth
rate
Region 4: Death phase
substrate supply is
exhausted
Slide 16
Quantifying Growth Kinetics
• Relationship of the specific growth rate to substrate concentration
exhibits the form of saturation kinetics
• Assume a single chemical species, S, is growth-rate limiting
• Apply Michaelis-Menten kinetics to cellular system
𝜇max 𝐶𝑆
→ called the Monod equation 𝑟𝑔 = 𝐶𝐶
𝐾𝑠 + 𝐶𝑆
• mmax is the maximum specific growth rate when CS >>Ks
• CS is the substrate concentration
• CC is the cell concentration
• Ks is the saturation constant or half-velocity constant. Equals the
rate-limiting substrate concentration, S, when the specific growth
rate is ½ the maximum
• Semi-empirical, experimental data fits to equation, assumes that a
single enzymatic reaction, and therefore substrate conversion by that
enzyme, limits the growth-rate
Slide 17
 Mass Balance on Cell Growth with
Products
Overall balance for cells growing on carbohydrate
with products:
CHmOn + a O2 + b NH3 → c CHaObNd + d CHxOyNz + e H2O + f CO2

Carbohydrate Nitrogen Cell material Product

(can be any source (biomass)
organic material)
Individual elemental balances:
1) Carbon: 1=c+d+f
2) Hydrogen: m + 3b = ca + dx + 2e
3) Oxygen: n + 2a = cb + dy + e + 2f
4) Nitrogen: b = cd + dz
Slide 19
Yield Coefficients

Cell yield for DC cell mass formed

YC/S = −
substrate: DS substrate consumed

Cell yield DC cell mass formed

YC/O2 = −
for O2: DO2 oxygen consumed

Product yield DP product mass formed

for substrate:
YP / S = −
DS substrate consumed

Slide 20
CRE Algorithm for Batch Bioreactors
1) Mass Balances
Accumulation = [In] - [Out] + [Growth] - [Death]
dCC
V = v0CC 0 − v0CC + Vrg − Vrd
dt
Let D = v0 / V (Dilution rate)

(1) dCC
= D(CC 0 − CC ) + rg − rd
dt
dCS
Similarly (2) = D(CS 0 − CS ) + rS
dt

Slide 21
CRE Algorithm for Batch Bioreactors

2) Rate Laws:
 mmax CS 
(3) rg = kOBS  CC
 K S + CS  Empirical formulae to account for
inhibition of the products on the
 CP  n Reaction (eg. Alcohol production
(4) kOBS = 1 − *  from glucose)
 CP 
Cp*= Product concentration at which all metabolism ceases

Slide 22
CRE Algorithm for Batch Bioreactors
3) Stoichiometry
A) Yield Coefficients
1
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑛𝑒𝑤 𝑐𝑒𝑙𝑙𝑠 𝑓𝑜𝑟𝑚𝑒𝑑 𝑌𝑆Τ𝐶 =
𝑌𝐶Τ𝑆 = 𝑌𝐶Τ𝑆
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑡𝑜 𝑝𝑟𝑜𝑑𝑢𝑐𝑒 𝑛𝑒𝑤 𝑐𝑒𝑙𝑙𝑠

𝑚𝑎𝑠𝑠 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑓𝑜𝑟𝑚𝑒𝑑

𝑌𝑃Τ𝑆 =
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 𝑡𝑜 𝑓𝑜𝑟𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡

B) Maintenance
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 𝑓𝑜𝑟 𝑚𝑎𝑖𝑛𝑡𝑒𝑛𝑎𝑛𝑐𝑒
𝑚=
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 × 𝑡𝑖𝑚𝑒

− rS = rgYS C + rPYS P + mCC

Slide 23
CRE Algorithm for Batch Bioreactors

3) Stoichiometry
Rate of Substrate Consumption
−𝑟𝑆 = 𝑟𝑔 𝑌𝑆Τ𝐶 + 𝑟𝑃 𝑌𝑆Τ𝑃 + 𝑚𝐶𝐶
• Often we can’t separate substrate consumption used for cell
growth from that used for product formations during
exponenial growth – ie product is produced even during
growth phase.
• In this case we lump the substrate consumed into the stoich
coefficient 𝑌𝑆Τ𝐶

−𝑟𝑆 = 𝑟𝑔 𝑌𝑆Τ𝐶 + 𝑚𝐶𝐶

(grams of substrate consumed)

𝑌𝑆Τ𝐶 =
(grams of cells produced)

Slide 24
CRE Algorithm for CSTR Bioreactors
(Chemostats)
= 0 − v0CC + (rg − rd )V
dCC
V (cells)
dt
dCS
V = v0CS 0 − v0CS + rSV (substrate)
dt
1 v0
D= = (dilution rate)
 V
(1) = − DCC + (rg − rd )
dCC
dt
(2) dCS
= D(CS 0 − CS ) + rS
dt
(3) dC P
= DCP − rP (product)
dt
Slide 26
CRE Algorithm for CSTR Bioreactors
(Chemostats)
2) Rate Laws:
mmax CS
(4 ) rg = CC KOBS
k S + CS
 CSCP  n
mmax
(54 ) KrgOBS= = 1 − C* CKOBS
k S + CSCP 
(6) rP = YP /C rg CP  n

(5) KOBS = 1 − * 
3) Stoichiometry
(7) rS = −YS /C rgC−PmC  C
((68)) rrDP == YkPD/C
CCrg
(7) rS = −YS /C rg − mCC
(8) rD = kDCC

Slide 27
CRE Algorithm for CSTR Bioreactors
(Chemostats)
4) Combine:
1. Steady State - Neglect Death Rate and Cell Maintenance
2. Cell: From Eq (1) 0 = −𝐷𝐶𝐶 + 𝑟𝑔
m maxCS
DCC = rg = CC = mCC
K S + CS
m maxCS
D=m=
K S + CS
DK S
CS =
m max − D
Slide 29
CRE Algorithm for CSTR Bioreactors
(Chemostats)

3. Substrate: From Eq (2)

0 = 𝐷 𝐶𝑆0 − 𝐶𝑆 + 𝑟𝑆

DCS 0 − CS  = −rS = YS / C rg = YS / C DCC

 DK S 
CC = YC S CS 0 − CS  = YC S CS 0 − 
 m max − D 

Slide 30
Chemostat Maximum Product Flow Rate
DCC From cell Mass balance
m maxCS
DCC = rg = CC = mCC
K S + CS
𝑑𝐷𝐶𝐶
=0
𝑑𝐷
when
 KS 
D max prod 
= m max 1 − 
K S + CS0 
 
D
Dmaxprod DW

Slide 32
Topic 7 – Theory: Summary

• From this lecture you should be able to:

o describe and analyse enzymatic reactions and the
different types of inhibition as displayed on a
Lineweaver–Burk plot.
o explain the stages of cell growth and how the Monod
equation for cell growth is coupled with mass balances
on the substrate, cells, and product to obtain the
concentration-time trajectories in a batch reactor.
o apply the growth laws and balance equations to a
chemostat (CSTR) to predict the maximum product flow
rate and the wash-out rate.

Slide 33