Stoichiometry of Microbial

Growth and Product Formation

 Stoichiometry of Growth and Product
Formation
• Mass balances are essential in the design of
biological processes and systems.
• It means establishing the quantity of material input
to the system (eg. nutrients and O2) and the
resulting material outputs (eg. biomass, CO2, and
other extracellular products).
• The first step in mass balance for biological systems
is writing the balanced stoichiometric reaction for
the overall reaction occurring in the system.
 Stoichiometry of Growth and Product
Formation

Substrate
Dry biomass
CwHxOyNz
c CHαO β Nδ

a O2
Cell d CO2

Nitrogen source
e H2 O
b HgOhNi
 Stoichiometry of Growth and Product
Formation
• Cell growth reactions obey the law of
conservation of mass.
• Carbon, hydrogen, nitrogen and other elements
consumed are either incorporated to new cells or
excreted as products.
Assumptions:
• Only compounds taken up or produced in
significant quantities are considered.
• Only extracellular products are CO2 and H2O.
 Stoichiometry of Growth and Product
Formation

Substrate
Dry biomass
CwHxOyNz
c CHαO β Nδ

a O2
Cell d CO2

Nitrogen source
e H2 O
b HgOhNi

Reaction for aerobic cell growth:

CwHxOyNz + aO2 + bHgOhNi cCHαOβNδ + dCO2 + eH2O
 Stoichiometry of Growth and Product
Formation
CwHxOyNz + aO2 + bHgOhNi cCHαOβNδ + dCO2 + eH2O

• CwHxOyN  substrate
eg. Glucose: w = 6, x = 12, y = 6, z = 0
• HgOhNi  nitrogen source
eg. NH3 (g = 3, i = 1), NO3- (h = 3, i = 1)
• CαHβOγNδ  dry biomass
• a, b, c, d and e are stoichiometric coefficients
 Stoichiometry of Growth and Product
Formation
• The general formula for biomass is CHαOβNδ .
• Microorganisms such as Escherichia coli contain a
wide range of elements. However, 90 – 95% of
biomass consists of C, H, O, and N.
• The formula for dry biomass are based on one C
atom.
• For different cells and conditions, CH1.8O0.5N0.2 can be
used as a general formula. The average molecular
weight of a dry biomass can be assumed to be 24.6.
Stoichiometry of Growth and Product
Formation
 Stoichiometry of Growth and Product
Formation
CwHxOyNz + aO2 + bHgOhNi cCHαOβNδ + dCO2 + eH2O
Elemental balances
The stoichiometric coefficients a, b, c, d, and e can be determined
from elemental balances.
C balance: w = c + d
H balance: x + bg = cα + 2e
O balance: y + 2a + bh = cβ + 2d + e
N balance: z + bi = cδ

Only four equations can be made using elemental balances. An

additional equation is required!
 Stoichiometry of Growth and Product
Formation
•Respiratory
  coefficient (RQ)

- measured experimentally with a respirometer

Common RQ various for metabolism of
a) Carbohydrates: RQ ~ 1
b) Lipids: RQ ~ 0.7
c) Proteins: RQ ~ 0.8
 Stoichiometry of Growth and Product
Formation
Aerobic growth of Saccharomyces cerevisiae on ethanol is
simply described by the following overall reaction:
C2H5OH + aO2 + bNH3  CH1.704N0.149O0.408 + dCO2 + eH2O

Determine the stoichiometric coefficients where RQ = 0.66.

 Stoichiometry of Growth and Product
Formation
• Elemental balances provide no insight to the
energetics and proton-electron balances in biological
reactions
Electron balances
Available electron – number of electrons available for
transfer to oxygen on combustion of a substance to
CO2, H2O and nitrogen-containing compounds such as
NH3.
Available electrons for C (+4), H(+1), O(-2), P(+5), S(+6)
 Stoichiometry of Growth and Product
Formation
• The number of available electrons for N depends on
the reference state: NH3 (-3), N2 (0), and NO3-(+5).
• The reference state for cell growth is the same as the
nitrogen source in the medium.
Example: If ammonia is used as nitrogen source, the
available N will be assumed to be -3; zero if nitrogen is
used and 5 for nitrate
 Stoichiometry of Growth and Product
Formation
• Degree
  of reduction γ – number of equivalents of
available electrons per gram of atom C.
• Degree of reduction for a substrate γs
For general substrate, CwHxOyNz where NH3 is the N-
source.

Ex. Methane (CH4): 1 atom C

Glucose (C6H12O6): 6 atom C

 Stoichiometry of Growth and Product
Formation
• Degree
  of reduction for a biomass γb
For general biomass CHαOβNδ , where NH3 is the N-
source.

Exercise: Find degree of reduction for general biomass

CH1.8O0.5N0.2.
 Stoichiometry of Growth and Product
Formation
• The
  degree of reduction of CO2, H2O and NH3 is zero.
• In a balanced growth equation, number of available
electrons is conserved.
• For a cell growth reaction with ammonia as nitrogen-
source, the available-electron balance is
CwHxOyNz + aO2 + NH3 cCHαOβNδ + dCO2 + eH2O
 Stoichiometry of Growth and Product
Formation
•Biomass
  yield YX/S

• used to determine the stoichiometric coefficient c

 Stoichiometry of Growth and Product
Formation
•Cell  growth with product formation
Extracellular product: CjHkOlNm
CwHxOyNz + aO2 + NH3 cCHαOβNδ + dCO2 + eH2O + fCjHkOlNm

f  stoichiometric coefficient for product

• Product yield from substrate YP/S

 Stoichiometry of Growth and Product
Formation
• Degree
  of reduction for product γp
For general product CjHkOlNm , where NH3 is the N-
source.
 Stoichiometry of Growth and Product
Formation
•  
Theoretical Oxygen Demand
• Oxygen is often the limiting substrate in aerobic cellular
growth.
• Oxygen demand is represented by the stoichiometric
coefficient a.
CwHxOyNz + aO2 + NH3 cCHαOβNδ + dCO2 + eH2O + fCjHkOlNm

This equation allows evaluation of a without requiring the

quantities of NH3, CO2 and H2O involved.
 Stoichiometry of Growth and Product
Formation
•  

The fractional allocation of available electrons in the

substrate is shown by the following:

This relationship can be used to obtain the upper

bounds for the yields of biomass and product from
substrate.
 Stoichiometry of Growth and Product
Formation
•  
Fraction of available electrons transferred from substrate
to O2

Fraction of available electrons transferred from substrate

to biomass

Fraction of available electrons transferred from substrate

to extracellular product
 Stoichiometry of Growth and Product
Formation
•  
 Stoichiometry of Growth and Product
Formation
•Aerobic
  growth of Saccharomyces cerevisiae on
ethanol is simply described by the following overall
reaction:
C2H5OH + aO2 + bNH3  CH1.704N0.149O0.408 + dCO2 + eH2O

a) Determine the stoichiometric coefficients where RQ

= 0.66.
b) Determine the biomass yield coefficient, YX/S and
oxygen yield coefficient,
 Stoichiometry of Growth and Product
Formation
•Exercise:
  Aerobic degradation of benzoic acid by a
mixed culture of microorganisms can be represented by
the following reaction.
C6H12COOH + aO2 + bNH3  cC5H7NO2 + dH2O + eCO2
(substrate) (bacteria)
a) Determine a, b, c, d, and e if RQ = 0.9
b) Determine the yield coefficients YX/S and
c) Determine the degree of reduction for the substrate
and the bacteria.
 Problem Set 1
• The
  growth of S. cerevisiae on glucose under
anaerobic conditions can be described by the
following overall reaction:
C6H12O6 + βNH3  0.59 CH1.7O0.2O0.45 (biomass) + 0.43 C3H8O3
+ 1.54 CO2 + 1.3 C2H5OH + 0.036 H2O

a) Determine the biomass yield coefficient YX/S

b) Determine the product yield coefficient YEtOH/S,
c) Determine the coefficient β.
 Problem Set 2
• Biological denitrification of nitrate-containing
waste waters can be described by the
following overall reaction.
aCH3OH + NO3- + H+  bC5H7NO2 + cN2 + dCO2 + eH2O

a) Determine a, b, c, d, and e if YX/S = 0.5 g X/g N.

b) Determine the degree of reduction of bacteria and
methanol.