BTE 2523: Biochemical Engineering Fundamental

Lecture 3: Chapter 4

Stoichiometry of growth and product formation,
growth stoichiometry,
material balance calculations,
balances on reactive process,
combustion reactions,
additional considerations on chemical and biological
processes.
Balance on a Cell
Typical Inputs into Biological Systems
Carbon Source (Glucose,amino acid)
Nitrogen Source, NH4Cl, (NH4)2SO4,
amino acids, proteins
Oxygen Source, Air (O2)
Hydrogen Source
Phosphate Source, PO4
-3
Typical Outputs in Biological
Systems
Biomass
Products
Metabolites
Products and metabolites are
easy to write a chemical formula
How to write a formula for cells?
Stoichiometry of Growth and Product Formation
A mass balance that involves a reaction the stoichiometry of the conversion needs to
be known before the mass balance can be solved
Firstly, need to show how equations for growth and product formulation are
formulated
Elemental Balances and Growth
In any fermentation, biotransformation or animal cell culture all the atoms of carbon,
hydrogen, oxygen, nitrogen and the other elements can be accounted for
In terms of growth, the substrate provide these elements and they are modified to cells
and products
The simplest case is when the only extra-cellular products are CO2 and water. Under
such circumstances the following generalised equations can be written:

CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O
Where is the formula of the main carbohydrate source
is the formula of the main nitrogen source
is the formula for ash free biomass

a-e are the stoichiometric coefficients based on 1 mole of substrate
CwHxOyNz
HgOhNi
cCHON
An absence of such compounds as NADH and ATP from the equation since
they are not exchanged with the environment.
Other compounds such as minerals and vitamins often added to medium
their contribution can be neglected due to such a small quantity
the biomass contains only the C, H, O and N elements, since these make up
over 90% of the cells, Table 1 illustrates how these elements vary amongst
different species
Table 1: Typical composition of
organic and inorganic
components of microorganisms
Table 2: The elemental composition of E.coli, one of the
most widely used organisms in the biotechnology industry.
Clearly the ash free component of
E.coli cells, the components
containing C, O, N and H amount to
92% of the cell dry weight. It is
common to merge all the other
components into the ash content
for mass balance calculations
Table 3: The elemental formula for various organisms
Table 4: The elemental composition as a function of
culture conditions
Shows the effect of culture
conditions on elemental formula of a
range of microorganisms
The stoichiometry balance:
CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O
Is not complete unless the stoichiometric coeffiecients a-e are known
if the formula for the biomass is known from tables 3 and 4, these can be
evaluated by solution of the sinultaneous equations given below:
C balance : w = c +d
H balance : x + bg = c +2e
O balance : y + 2a + bh = c +2d + e
N balance : z + bi = c
However, only four equations and five unknowns so additional information is
required. Various parameters can be utilized to overcome this problem. These
include:
(i) Respiratory quotients
(ii) Yield of biomass on substrate
RQ = d/a =
Moles CO2 produced
Moles O2 consumed
Yxs=
kg biomass
Kg substrate
=
c (MW cells)
(MW substrate)
However, a large parameters affect the yield, including medium composition, pH,
temperature, electron acceptor. The yield figure would be based on the mass of
biomass including the ash content and so correction for this has to be made
Product stoichiometry
CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O + fCjHkOlNm
If in addition to the biomass an extracellular product can be formed with a
composition CjHkOlNm. The mass balance can then be rewritten:
With the additional stoichiometric coefficient f, another unknown.
The product yield, experimentally determined is utilised, defined as:
YPS=
g product formed
g substrate consumed
=
f (MW product)
(substrate)
the relationship does not hold of product formation is linked to growth, secondary
metabolite production or biotransformation. In such cases, independent equation
must be used to describe growth and product formation.
Example: Stoichiometric coefficients for cell growth
Electron balances: Degree of reduction
Available electron refers to the number of electrons available for transfer oxygen on
combustion of a substance to CO2, H2O and nitrogen-containing compounds

The number of available electrons found in organic material is calculated from the
valence of the various elements: 4 for C, 1 for H, -2 for O, 5 for P and 6 for S.

The number of available electrons for N depends on the reference state: -3 if
ammonia, 0 for molecular N2 and 5 for nitrate

The degree of reduction, can be defined as the number of equivalents of available
electrons in that quantity of material containing 1g atom carbon. For substrate
CwHxOyNz, the number of available electrons is (4w + x 2y 3z).

the degree of reduction for the substrate, s = (4w + x 2y 3z)/w

degree of reduction for CO2, H2O and NH3 is zero


Electron available for transfer to oxygen are conserved during metabolism.

in a balanced growth equation, number of available electrons is conserved by virtue
of the fact that the amounts of each chemical element are conserved. Applying this
principle to this equation:


With ammonia as nitrogen source, the available-electron balance is:

wS + 4a = cb

Where s and b are the degrees of reduction of substrate and biomass, respectively



CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O
Theoretical Oxygen Demand
Oxygen is often limiting substrate in aerobic fermentations and the demand is
represented by the stoichiometric coefficient a in the stoichiometric equations
In relation to degrees of reduction, the oxygen requirements is related to the
electrons available for transfer to oxygen and oxygen demand can be derived from an
electron balance
if product synthesis occurs as in the equation:



CwHxOyNz + aO2 + bHgOhNi cCHON + dCO2 + eH2O + fCjHkOlNm
the electron balance is : ws 4a = cB fjP (this equation can be used to calculate oxygen demand)
Where P is the degrees of reduction of the product
Maximum Possible Yield
For the example given above the electron balances:



ws 4a = cB fjP
can be written as a fractional allocation:



the expression can be used to calculate the upper bounds of the yields. If is
defined as the fraction of available electrons in the substrate transferred to biomass:



in the absence of product formation, all available electrons would be used for
biomass synthesis and B =1. under such conditions, the maximum values of the
stoichiometric coefficients c is:
S
P
S
B
S
w
fj
w
c
w
a

4
1
substrate biomass product

s
B
B
w
c

B
S
w
c

max
Cmax can be converted to a biomass yield with mass units using the yield expression
given. Therefore, even if the stoichiometry of growth is unknown, we can calculate an
upper limit for biomass yield from the molecular formulae for substrate and product.
if the composition of the cells is unknown B, can be taken as 4.2 corresponding to
an average biomass formula CH1.8O0.5N0.2.

similarly the maximum product yield in the absence of biomass synthesis given by:
P
S
j
w
f

max

The chemical reaction equation for the aerobic growth of a
microorganism on glucose and ammonia is as following:


using the information given below, determine the total amount
(in kg) of glucose, ammonia and oxygen that are required to
achieve a biomass concentration of 20 kg (dry cell mass)/m
3
in
a 15 m
3
fermenter can be calculated.

Biomass contains 6.4% (w/w) ash, Atomic weight: H = 1, C = 12, N = 14, O = 16,

aC6H12O6 + bO2 + cNH3 dC6H10O3N3 + eCO2 + fH2O
Problem:
Example 4.8 Product yield and oxygen demand

The chemical reaction equation for respiration of glucose is:

C
6
H
12
0
6
+60
2
6C0
2
+6H
2
0.
Candida utilis cells convert glucose to CO
2
and H
2
0 during
growth. The cell composition is CH
1.84
0
0.55
N
0.2
plus 5%
ash. Yield of biomass from substrate is 0.5 g/g . Ammonia is
used as nitrogen source.
a) What is the oxygen demand with growth compared to that
without?
b) C. utilis is also able to grow with ethanol as substrate,
producing cells of the same composition as above. On a mass
basis, how does the maximum possible biomass yield from
ethanol compare with the maximum possible yield from
glucose?

Combustion Reactions
Combustion the rapid reaction of a fuel with oxygen that produces CO2,
H2O and frequently CO and SO2
the significance of these reactions lies in the tremendous quantities of
heat they release used to produce steam to drive the turbines that
generate most of the worlds electrical power
most of the fuel used in power plant combustion furnaces either coal
(carbon some hydrogen and sulfur and various noncombustible materials),
fuels, oil, gaseous fuel (such as natural gas, primarily methane) or liquefied
petroleum gas usually propane and/or butane:
a combustion reaction in which CO is formed from a hydrocarbon referred
to as partial combustion or incomplete combustion of hydrocarbon
example



Complete combustion of propane
C + O2
CO2
C3H8 + 5O2 3CO2 + 4H2O
C3H8 + 7/2O2 3CO + 4H2O
CS2 + 3O2 CO2 + 2SO2
Complete combustion of carbon
partial combustion of propane
Complete combustion of carbon
disulfide
Air has the following molar composition
N2 78.03%
O2 20.99%
Ar 0.94%
N2 He, Ne, Kr, Xe 78.03%
CO2 0.03%
100.00%
Average MW = 29.0
Composition on a wet basis denote the component mole fractions of a
gas that contains water
composition on a dry basis denotes the component mole fractions of the
same gas without the water
the product gas that leaves a combustion chamber referred to as the
stack gas or flue gas
when the flow rate of a gas in a stack is measured, it is the total flow rate of
the gas including water
Example : Composition on wet and dry bases
1. Wet basis Dry basis
A stack gas contains 60 mole% N2, 15% CO2, 10% O2 and the balance H2O.
Calculate the molar composition of the gas on a dry basis

2. Dry basis Wet basis
An Orsat analysis (a technique for stack gas analysis) yield the following dry
basis composition:

N2 65%
CO2 14%
CO 11%
O2 10%
A humidity measurement shows that the mole fraction of H2O in the stack gas
is 0.07. Calculate the stack gas composition on a wet basis

Theoretical and Excess Air
If two reactants participate in a reaction and one is considerably more
expensive than the other, the usual practice is to feed the less expensive
reactant in excess of the valuable one
this has the effect of increasing the conversion of the valuable reactant at
the expense of the cost of the excess reactant and adding pumping cost
the extreme case of an inexpensive reactant is air which is free
theoretical oxygen: the moles (batch) or molar flow rate (continuous) of O2
needed for complete combustion of all of the fuel fed to the reactor,
assuming that all carbon in the fuel is oxidized to CO2 and all hydrogen is
oxidized to H2O
theoretical air: the quantity of air that contains the theoretical oxygen
excess air: the amount by which the air fed to the reactor exceeds the
theoretical air
Percent excess air :


Example: if 50% excess air is supplied,

(moles air)fed (moles air) theoretical
X 100%
(moles air) theoretical
(moles air) fed =
1.5 (moles air) theoretical
Example : Theoretical and excess air
One hundred mol per hour of butane (C4H10) and 5000 mol per hour of air
are fed into a combustion reactor. Calculate the percent excess air.
Material Balances on Combustion Reactors
The procedure for writing and solving material balances for a combustion
reactor is essentially same as that for any other reactive system
there are some additional points:
When you draw and label the flowchart, remember to include the N2 at
both the inlet and outlet of the reactor, and unreacted fuel and O2 as well as
combustion products (CO2, H2O, CO at the outlet
If there is any percent excess air, the O2 actually fed may be calculated by
multiplying the theoretical O2 determined from the fuel feed rate and
composition and the reaction stoichiometery for complete combustion by
(1 + fractional excess air)



Example : Combustion of Ethane
Ethane (C2H6) is burned with 50% excess air. The percentage conversion of the
ethane is 90% of the ethane burned, 25% reacts to form CO and the balance
for form CO2. Calculate the composition of the flue gas and the ratio of water
to dry flue gas
Summary of Chapter 4
At the end of Chapter 4 you should:
(i) understand the terms: system, surroundings, boundary and
process in thermodynamics
(ii) be able to identify open and clossed systems, and batch,
semi-batch, fed-batch and continuous processes
(in) understand the difference between steady state and
equilibrium
(iv) be able to write appropriate equations for conservation of
mass for processes with and without reaction;
(v) be able to solve simple mass-balance problems with and
without reaction; and
(vi) be able to apply stoichiometric principles for macroscopic
analysis of cell growth and product formation.