CHAPTER 3

STOICHIOMETRY OF MICROBIAL
GROWTH AND PRODUCT
FORMATION

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 1
A
CH
 TOPIC OUTCOMES
To understand the concept of stoichiometric.

To calculate the stoichiometric coefficient for cell growth.

To calculate the yield coefficient of a biochemical reaction.

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 2
A
CH
 CONTENTS

3.1 Introduction of stoichiometric

3.2 Stoichiometric calculations

3.3 Biomass and product yield

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 3
A
CH
 INTRODUCTION
The law of conservation of mass has been used to
determine unknown quantities entering and leaving
bioprocess

For mass balance with reaction, the stoichiometry of

conversion must be known before the mass balance can be
solved

When cell growth occurs, cells are a product of reaction

and must be presented in reaction equation

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 4
A
CH
 Cell growth and product formation are complex processes
reflecting the overall kinetics and stoichiometry of the
thousands of intracellular reaction that can be observed within a
cell

Metabolic stoichiometry has many applications in

bioprocessing

It can be used to compare theoretical and actual product

yields, check the consistency of experimental data, and
formulate medium

In chemical and biochemical reaction, atoms & molecules

rearrange to form new groups

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 5
A
CH
 Mass and molar relationship between the reactants
consumed and products formed can be determined using
stoichiometric calculations

During chemical and biochemical reactions, these 2

quantities are conserved:

i) total mass
i.e total mass of reactant = total mass of product
180 g glucose produces 92 g ethanol and 88 g CO2

ii) no. of atoms of each element

i.e the no. of C,H,O,N in reactants = no. of C,H,O,N
respectively in products

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 6
A
CH
 Note that, there is no corresponding law of conservation of
moles

moles of reactants ≠ moles of products

i.e 1 molecule glucose breaks down to give 2 molecules of

ethanol and 2 molecules of CO2

C6 H12O6 ® 2C2 H 6O + 2CO2

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 7
A
CH
Example 3.1 Stoichiometry of amino acid synthesis

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 8
A
CH
 2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 9
A
CH
 Stoichiometric Calculations

[product]
[substrate]

[cellular
material]

Material balance on
biological reaction
2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 10
A
CH
 A typical cellular composition can be represented as:

CHαOβNδ

One mole of biological material is defined as the amount

containing 1 gram atom of carbon.

All atoms of carbon, hydrogen, oxygen, nitrogen and other

elements consumed during growth are incorporated into
new cells or excreted as products

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 11
A
CH
 Consider the following simplified biological conversion in
which no extracellular products other than H2O and CO2 are
produced

CH m On + aO2 + bNH 3 ® cCH a Ob N d + dH 2 O + eCO 2

Where CHmOn represents 1 mole of carbohydrate and

CHαOβNδ stands for 1 mole of cellular material

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 12
A
CH
 Simple elemental balances on C, H, O, N yield the following
equations:

CH m On + aO2 + bNH 3 ® cCH a Ob N d + dH 2 O + eCO 2

(3.1)

C: 1 = c + e
H: m + 3b = cα + 2d
O: n + 2a = cβ + d + 2e (3.2)

N: b = cδ

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 13
A
CH
 The respiratory quotient (RQ) is

molesCO2 produced e
RQ = = (3.3)
molesO2 consumed a

Equations 3.2 and 3.3 constitute 5 equations for 5

unknowns a, b, c,d, and e.

With a measured value of RQ, these equations can be solved

to determine the stoichiometric coefficient

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 14
A
CH
 Substrate Biomass
CHmOn c CHαOβNδ

a O2 CELL d H2O

b NH3 e CO2
Nitrogen source

Fig 3.1 Conversion of substrate, oxygen and nitrogen for cell growth

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 15
A
CH
 Bacteria tend to have slightly higher nitrogen contents
(11-14%) than fungi (6.3-9.0%)

When composition analysis is not available we can use

the following general formula:

CH1.8O0.5N0.2

Compositions of several species in terms of 4 elements

are listed in Table 3.1

The formulae in Table 3.1 refer to dry biomass and are

based on 1 C atom.

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 16
A
CH
Table 3.1 Elemental composition and degree of reduction for selected organism (Doran, 2003)

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 17
A
CH
 Example 3.2 Stoichiometric coefficients for cell growth

Production of single-cell protein from hexadecane is described by the

following reaction equation:

C16 H 34 + aO2 + bNH 3 ® cCH1.66O0.27 N 0.20 + dCO2 + eH 2 O

Where CH1.66O0.27N0.20 represents the biomass. If RQ=0.43

Determine the stoichiometric coefficients.

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 18
A
CH
C16 H 34 + aO2 + bNH 3 ® cCH1.66O0.27 N 0.20 + dCO2 + eH 2 O
Solution:

C balance : 16 = c + d (1)
H balance : 34 + 3b = 1.66c + 2e (2)
O balance : 2a = 0.27c + 2d + e (3)
N balance : b = 0.20c (4)
RQ : 0.43 = d/a (5)

Solve this set of simultaneous equations. Since b is already written

simply as function of c in (4), therefore try express the other
variables solely in terms of c.
2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 19
A
CH
 From (1):

d = 16 – c (6)

From (5):
a = d/0.43 = 2.326 d (7)

Combining (6) and (7) gives an expression for a in terms of c;

a = 2.326 (16 – c)
a = 37.22 – 2.326c (8)

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 20
A
CH
 Substituting (4) into (2) gives:
34 + 3(0.20c) = 1.66c + 2e
34 = 1.06c + 2e
e = 17 – 0.53c (9)

Substituting (8), (6) and (9) into (3) gives:

2(37.22 – 2.326c) = 0.27c + 2(16-c) + (17 – 0.53c)
25.44 = 2.39c
c = 10.64

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 21
A
CH
 Using this result for c in (8), (4), (6) and (9) gives:
a = 12.48
b = 2.13
d = 5.37
e = 11.36

Check that these coefficient values satisfy Eqs (1) – (5)

The complete reaction equation is:

C16 H 34 + 12.5O2 + 2.13NH 3 ® 10.6CH 1.66O0.27 N 0.20 + 5.37CO2 + 11.4H 2 O

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 22
A
CH
EXERCISE 1
aC5H10O5 + bO2 + cNH4OH →
CH1.66N0.13O0.40 + dCO2 + eH2O

A yeast is growing aerobically on arabinose & ammonium

hydroxide according to above reaction. RQ = 1.4. Write the
stoichiometric equation.

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 23
A
CH
 Exercise 2
Assume that experimental measurements for a certain organism have shown that
cells can convert two-thirds (wt/wt) of the substrate carbon (alkane or glucose) to
biomass.

Calculate the stoichiometric coefficients for the following biological reactions:

Hexadecane;

C16 H 34 + aO2 + bNH 3 ® c(C4.4 H 7.3 N 0.86 O1.2 ) + dH 2 O + eCO2

Glucose;
C6 H12O6 + aO2 + bNH 3 ® c(C4.4 H 7.3 N 0.86O1.2 ) + dH 2O + eCO2

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 24
A
CH
 Electron Valence
No. of available electron – calculated from the valence
of various elements:

C: 4
H: 1
O: -2
N: -3
P: 5
S: 6
2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 25
A
CH
 2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 26
A
CH
 Degree of reduction (DOR), γ – defined as the number of
equivalents of available electrons in that quantity of material
containing 1 g atom carbon

For example: substrate (CwHxOyNz )

No. of electron = 4w + x – 2y – 3z

the DOR, γS = [(4w + x – 2y – 3z)/w]

Another example: Ethanol (C2H5OH)

No. of electron = 2(4) + 5(1) + 1(-2) + 1(1) = 12

γ = 12/2 = 6
2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 27
A
CH
Consider the aerobic production of a single extracellular product

CH mOn + aO2 + bNH 3 ® cCH a Ob Nd + dCH x Oy N z + eH 2O + fCO2

The degrees of reduction of substrate, biomass, and product :

g s = 4 + m - 2n
g b = 4 + a - 2 b - 3d
g p = 4 + x - 2 y - 3z

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 28
A
CH
 Biomass Yield

g cells produced
YX/S =
g substrate consumed

Product yield

g product formed
YP/S =
g substrate consumed

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 29
A
CH
 Example 3.4: Yield coefficient

Calculate the yield coefficient YX/S and YX/O2 for both reactions

Hexadecane;

C16 H 34 + 11.07O2 + bNH 3 ® 2.42(C4.4 H 7.3 N 0.86O1.2 ) + dH 2O + eCO2

Glucose;

C6 H12O6 + 1.473O2 + bNH 3 ® 0.909 (C4, 4 H 7.3 N 0.86O1.2 ) + dH 2O + eCO2

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 30
A
CH
 For hexadecane ;

2.42 (MW) biomass

YX/S =
(MW) substrate

2.42 (91.34)
YX/S =
226

= 0.98 g cells/g substrate

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 31
A
CH
 For hexadecane;

2.42 (MW) biomass

YX/O2 =
11.07 (MW) oxygen

2.42 (91.34)
YX/O2 =
11.07 (32)

= 0.624 g cells/g oxygen

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 32
A
CH
 For glucose ;

0.909 (91.34)
YX/S = = 0.461 g cells/g substrate
180

0.909 (91.34)
= 1.761 g cells/g oxygen
YX/O2 =
1.473 (32)

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 33
A
CH
EXAMPLE 3.5
Determine the yield coefficients YX/S and YP/S.

Time (h) Substrate Cell concentration Product

concentration (g/L) (g/L) concentration
(g/L)
0 100.0 1.0 1.5
2 97.0 2.4 8.5
4 80.4 6.1 20.0
8 76.9 10.5 34.0
10 48.1 26.0 43.0
12 30.6 43.0 46.5

DX DP
YX / S = - YP / S = -
DS DS
43.0 - 1.0 46.5 - 1.5
=- =-
30.6 - 100 30.6 - 100
= 0.61gcells / gsubstrate = 0.65 gproducts / gsubstrate

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 34
A
CH
 Thank you

2
ER
P T BSB3123 BIOPROCESS TECHNOLOGY 35
A
CH