L05 - Metabolic Stoichiometry 2021

CHE4171 Biochemical Engineering
Lecture 5
Metabolic Stoichiometry
A/Prof. Edward Ooi Chien Wei

Learning objectives
 Introduction to growth stoichiometry
 Stoichiometric calculations
– elemental balances
– degree of reduction
 Theoretical prediction of yield coefficients

Why study growth stoichiometry?
 Bioreaction is a complex process reflecting overall kinetics and stoichiometry of thousands of intracellular
reactions.
 Although bioreaction is complex, the stoichiometry of conversion of substrates into products and cellular
materials can be represented by a simple ‘pseudochemical’ equation.
 From stoichiometry, engineers can

– compare potential substrates in terms of biomass yield, product yield, evolution of heat etc.
– know how close to its thermodynamic limit a system is operating
 Example of stoichiometric-balanced chemical equations: N2 + 3H2 → 2NH3 2H2 + O2 → 2H2O

Definition of growth yield
 Yield coefficients are defined based on the amount of consumption of

another material.
Typical cell growth curve
Stoichiometric balance of a simple bioreaction
Reactants Products
Cell
(Inputs) e.g. E. coli (Outputs)
System boundary Recall Mass Balance in Level 2

Growth stoichiometry
Considering an aerobic cell growth:
Carbon source + Nitrogen source + O2 → Cell biomass + CO2 + H2O
e.g. C6H12O6 e.g. NH3
What am I ‘made of’?
So, what is the ‘chemical formula’ for cells?

Cell composition
Dry weight of cells consists of: From elemental analysis, we can estimate
Element E. coli Yeast the ‘chemical formula’ of cells:
C 50% 50%
O 20% 34%
CHaObNd
N 14% 8%
H 8% 6% In general,
P 3% 1%
C varies from 46 - 50%
S 1% <1%
K 1% <1%
H: 6 - 7%
Na 1% <1% O: 29 - 35%
Other <1% <1%
N: 8 - 14%
‘Chemical formula’ of cells
When composition analysis for a

particular microbe is not available, use:
CH1.8O 0.5 N 0.2
‘Molecular weight’ of cells = 24.6

A generalized stoichiometry for bioreaction
Example of Example of Oxygen source Biomass Product Carbon

carbon source nitrogen source (for aerobic cells) (e.g. ethanol, lactic acid) dioxide Water
1 C6H12O6 + aNH3 + bO2 → αCH1.8O0.5N0.2 + βCHxOyNz + γCO2 + δH2O
Normalized to 1
Stoichiometric coefficients
mole of carbon
source compound
Note: a, b, α, β, γ, δ, x, y, z depend on the type of cell involved.
Determination of theoretical bioreaction yields from stoichiometry
Consider aerobic growth of yeast on ethanol given as:
C2H5OH  0.153NH3  1.851O2  1.03CH1.704O0.408N0.149  0.97CO2  2.346H2O
Calculate Theoretical Yields : YX / E ,YX / O2 ,YX / NH3

Recall from previous lecture: Yield Coefficient

Mass Cells Pr oduced dX
YX / S  
Mass Substrate Consumed dS
Yield coefficients can also be defined for other species (e.g. product, O2, etc.)

MWcells = 22.32 g/mol;
MWethanol = 46 g/mol;
MWNH3 = 17 g/mol;
Solution
g cell / g ethanol
g cell / g NH3
Recall: mass = mole × MW
Note: yield based on ethanol = 0.5;

If YX/E << 0.5 medium formulation, i.e., inadequate for good growth

MWcells = 22.32 g/mol;
MWethanol = 46 g/mol
Solution
Growth yield based on consumption of oxygen
g cell / g O2
Recall: mass = mole × MW

Balancing a bioreaction equation - Determination of stoichiometric coefficient
C6H12O6  aNH3  bO2  aCH1.8O0.5N0.2  bCHxOyNz  CO2  dH2O
How can we determine these coefficients?

 Perform mass balance
– six unknowns
– require six independent equations
– chemical reaction - atoms conserved (Atomic or Elemental Balance)
4 Atom Balances:
C: 6 = a + b +  To solve this problem,
H: 12 + 3a = 1.8a + xb + 2d we require 2 more
equations
O: 6 + 2b = 0.5a + yb + 2 + d
N: a = 0.2 a + zb
Develop additional equation using RQ: Respiratory Coefficient
Cancelled, if no extra-cellular product formed
C6H12O6  aNH3  bO2 aCH1.8O0.5N0.2  bCHx OyNz  CO2  dH2O
 RQ = Moles [CO2/O2]= /b
 Only applicable for aerobic cells. RQ is readily measured.
 If nature of extra-cellular product is known (x, y, z):
– can calculate a, b,  & d from RQ + one other measurement.

 If no extra-cellular product:
– need only RQ {or other} measurement for stoichiometry. The term CHxOyNz is cancelled.
Develop additional equation using RQ: Respiratory Coefficient
 Example: E. coli: RQ measured as 1.175. what are a, b, α, γ & δ?
Atom balances :
C : 6  a  ; H : 12  3a  1.8a  2d; N : a  0.2a; O : 6  2b  0.5a  2  d
RQ :  /b  =0.85
1.175
 1 1 0 0 0  a   6 
1.8 0 2 3 0     12 
Solve using Matrix, thus becoming
     a mathematical problem!
0.2 0 0 1 0   d    0  & can solve for a, b, a,  & d
    
 0 1 0 0 1.175
0.85   a   0 
0.5 2 1 0 2   b   6 
aa = 4.2; b b =0.36;
0.9; aα
1.28;  1.79; d γ4.48;
= 4.49;  δ4.925.
= 1.51; = 3.31
Additional equations using Yields Coefficients
For example, if there is an extra-cellular product
 Assuming x, y, z is known, one more unknown is present, b, thus we need one more equation
g product formed b  MW product)

 Since YP S  
g substrate consumed (MW substrate)
If you can determine YP/S experimentally, you can use the above equation to find b
(Similarly, you can use Yx/s)
Alternatively …..
Develop additional equation using ‘Degree of Reduction’
 Applied when more independent equations are required

– e.g. for complex reactions with extracellular product formation.
 Electron balance
– Shows how the available electrons from substrates are distributed in reaction.
 The available electrons are those that would be transferred to oxygen upon oxidation of
a compound to CO2, H2O and NH3 (or N2, depends on the reference state).
 The number of available electrons found in organic matter is calculated from the valence
of the composed elements.
Number of available electron for key elements
The available electrons are those that would be transferred to oxygen upon oxidation
of a compound to CO2, H2O and NH3 (or N2, depends on the reference state)
Element Valence Number of available electron (∑ charge)

C 4 +4 (donate electrons)
H 1 +1 (donate electron)
N 5 -3 (reference state NH3; accept electrons)
0 (reference state N2 ; no electron transfer)
O 6 -2 (accept electrons)
P 5 +5 (donate electrons)
S 6 +6 (donate electrons)
Calculation of degree of reduction ()
Electron balance of compound
 = S (mole × available electrons) / mole of atomic C
i to j
Element Number of available electron

Example
C +4
• CO 2 = [(1)(4) + (2)(-2)] / 1= 0

H +1
N -3 (ref. state NH3)
• C H 6 12O6 = [6(4) + 12(1) + 6(-2)] / 6 = 4

O
0 (ref. state N2)
-2
• C H OH = [2(4) + 6(1) + (1)(-2)] / 2 = 6

2 5
P +5
S +6
Degree of reduction () of bioreaction
C6H12O6  aNH3  bO2 aCH1.8O0.5N0.2  b CHx OyNz  CO2  dH2O

Substrate Biomass Product
Substrate : s = (6×4 + 12×1 – 6×2) / 6 = 4

Biomass : b = (4 + 1.8 – 3×0.2 – 2×0.5) / 1 = 4.2
Product : p = (4 + x – 2y – 3z) / 1
Note: the values of  for

NH3 = – 3 + 3 = 0,
Thus, we are able to get one more equation based on Electron Balance:
O2 = –2×2 = – 4,
CO2 = [4 + (–2×2)]/1 = 0, 1×4 + 0 + (-4)b = 4.2a + b(4 + x – 2y – 3z) + 0 + 0
H 2O = 2 – 2 = 0
Equation is further simplified if product (x, y, z) is known
Summary
 Elemental balances
 Respiratory coefficient
 Electron balance and degree of reduction
 Theoretical prediction of yield coefficients

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CHE4171 Biochemical Engineering

A/Prof. Edward Ooi Chien Wei

 Introduction to growth stoichiometry

 Theoretical prediction of yield coefficients

 From stoichiometry, engineers can

 Example of stoichiometric-balanced chemical equations: N2 + 3H2 → 2NH3 2H2 + O2 → 2H2O

 Yield coefficients are defined based on the amount of consumption of

System boundary Recall Mass Balance in Level 2

What am I ‘made of’?

So, what is the ‘chemical formula’ for cells?

When composition analysis for a

CH1.8O 0.5 N 0.2

‘Molecular weight’ of cells = 24.6

Example of Example of Oxygen source Biomass Product Carbon

1 C6H12O6 + aNH3 + bO2 → αCH1.8O0.5N0.2 + βCHxOyNz + γCO2 + δH2O

Consider aerobic growth of yeast on ethanol given as:

C2H5OH  0.153NH3  1.851O2  1.03CH1.704O0.408N0.149  0.97CO2  2.346H2O

Calculate Theoretical Yields : YX / E ,YX / O2 ,YX / NH3

C2H5OH  0.153NH3  1.851O2  1.03CH1.704O0.408N0.149  0.97CO2  2.346H2O

Recall from previous lecture: Yield Coefficient

C2H5OH  0.153NH3  1.851O2  1.03CH1.704O0.408N0.149  0.97CO2  2.346H2O

Recall: mass = mole × MW

Note: yield based on ethanol = 0.5;

C2H5OH  0.153NH3  1.851O2  1.03CH1.704O0.408N0.149  0.97CO2  2.346H2O

Growth yield based on consumption of oxygen

Recall: mass = mole × MW

C6H12O6  aNH3  bO2  aCH1.8O0.5N0.2  bCHxOyNz  CO2  dH2O

How can we determine these coefficients?

C6H12O6  aNH3  bO2 aCH1.8O0.5N0.2  bCHx OyNz  CO2  dH2O

 RQ = Moles [CO2/O2]= /b

 Only applicable for aerobic cells. RQ is readily measured.

 If nature of extra-cellular product is known (x, y, z):

– can calculate a, b,  & d from RQ + one other measurement.

C6H12O6  aNH3  bO2 aCH1.8O0.5N0.2  bCHx OyNz  CO2  dH2O

 Example: E. coli: RQ measured as 1.175. what are a, b, α, γ & δ?

g product formed b  MW product)

 Applied when more independent equations are required

Element Valence Number of available electron (∑ charge)

Element Number of available electron

• CO 2 = [(1)(4) + (2)(-2)] / 1= 0

• C H 6 12O6 = [6(4) + 12(1) + 6(-2)] / 6 = 4

• C H OH = [2(4) + 6(1) + (1)(-2)] / 2 = 6

C6H12O6  aNH3  bO2 aCH1.8O0.5N0.2  b CHx OyNz  CO2  dH2O

Substrate : s = (6×4 + 12×1 – 6×2) / 6 = 4

Note: the values of  for

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