STOICHIOMETRY OF MICROBIAL GROWTH AND PRODUCT FORMATION

INTRODUCTION
Cell growth and product formation are complex processes reflecting the overall kinetics
and stoichiometry of the thousands of intracellular reactions that can be observed within a cell.
Also, we may need to know how close to its thermodynamic limit a system is operating.
Thermodynamic limit –the limit for a large number N of particles where the volume is
taken to grow in proportion with the number of particles.
If a system is close to its thermodynamic limit, it would be unwise to improve production
through mutation or genetic engineering. When cells growth occurs, cells are a product of reaction
and must be represented in reaction equation. Although the cell is complex, the stoichiometry of
conversion of substrates into products and cellular materials is often represented by a simple
pseudo-chemical equation. When cell growth occurs, cells are a product of reaction and must be
represented in the reaction equation.
OTHER DEFINITIONS

Overall Growth Yield Coefficient (𝒀𝑴

𝑿⁄𝑺 ) – maximum yield of cell mass per unit mass of
substrate consumed when no maintenance is considered.
ATP Yield Coefficient (𝒀𝑿⁄𝑨𝑻𝑷 ) – represents the amount of biomass synthesized per mole
of ATP generated.
Relationship between the two: (𝑌𝑋⁄𝑆 = 𝑌𝑋⁄𝐴𝑇𝑃 𝑁) as N = moles ATP/mol substrate

Regularities
𝑌𝑋𝑀⁄𝑆 ≥ 10.5 g dry wt/mol ATP
26.95 kcal/g equivalent of available electrons transferred to oxygen (coefficient of variation of
4%)
4.291 g equivalent of available electrons per quantity of biomass containing 1 g atom carbon
0.462 g carbon in biomass per gram of dry biomass
𝑌𝑋⁄𝑒 − = 3.14 ± 0.11 g dry wt/g equivalent of electrons

STOICHIOMETRIC CALCULATIONS
A material balance on biological reactions can easily be written when the compositions of
substrates, products, and cellular material are known. The law of conservation of mass has been
used to determined unknown quantities entering or leaving bioprocess. Usually, electron–proton
balances are required in addition to elemental balances to determine the stoichiometric coefficients
in bioreactions. All carbon, hydrogen, oxygen, nitrogen and other elements consumed during
growth are incorporated into new cells or excreted as products. Confining to those compounds
taken up or produced in significant quantity, if the only extracellular products formed are 𝐶𝑂2 and
𝐻2 𝑂, we can write the following equation for aerobic cell growth:
 𝐶𝑤 𝐻𝑥 𝑂𝑦 𝑁𝑧 + 𝑎𝑂2 + 𝑏𝐻𝑔 𝑂ℎ 𝑁𝑖 → 𝑐𝐶𝐻𝛼 𝑂𝛽 𝑁𝛿 + 𝑑𝐶𝑂2 + 𝑒𝐻2 𝑂

where: 𝐶𝑤 𝐻𝑥 𝑂𝑦 𝑁𝑧 is the chemical formula for the substrate

𝑏𝐻𝑔 𝑂ℎ 𝑁𝑖 is the chemical formula of the nitrogen source
𝑐𝐶𝐻𝛼 𝑂𝛽 𝑁𝛿 is the chemical ‘formula’ for the dry biomass
a, b, c, d, and e are stoichiometric coefficient

Figure 1 – Conversion of Substrate, Oxygen, and Nitrogen for Cell Growth

The figure above shows the macroscopic view of metabolism; it ignores the detailed
structure of the system and consider only those components which has net interchange in the
environment, including ATP and NADH. Vitamins and minerals taken up during metabolism is
neglected as it is consumed in very little amount and does not contribute to the stoichiometry and
energetics of reaction. Though this macroscopic view is simple in its approach, it provides a
powerful tool for thermodynamic analysis.

Table 1 – Elemental Composition for Various Microorganisms

Bacteria tend to have slightly higher nitrogen contents (11-14%) than fungi (6.3-9.0%). For
particular species, cell composition depends on the culture conditions and substrate utilized.
However, the results are remarkably similar for different cells and condition; 𝐶𝐻1.8 𝑂0.5 𝑁0.2 can be
used as general formula when composition analysis is not available. The ‘average molecular
weight’ of the biomass feed is 24.6, although 5-10% residual ash is often added into account for
those elements not included in the formula. Coefficients a, b, c, d, and e are obtained and evaluated
using normal procedures for balancing equations and is govern by the set of equations below.
C Balance: w=c+d
H Balance: x + bg = cα + 2e
O Balance: y + 2a + bh = cβ + 2d + e
N Balance: z + bi = cδ
Another additional information is needed to complete the five unknowns in the equation,
and is represented by the parameter respiratory quotient (RQ):
𝑚𝑜𝑙𝑒𝑠 𝐶𝑂2 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 𝑑
𝑅𝑄 = =
𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑂2 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 𝑎
EXAMPLE:
Production of single-cell protein from hexadecane is described by the following reaction equation:
C16H34 + aO2 + bNH3 --> cCHl.66O0.27N0.20 + dCO2 + eH2O
where CHl.66O0.27N0.20 represents the biomass. If RQ= 0.43, determine the stoichiometric
coefficients.
Solution:
C balance: 16 = c + d
H balance: 34 + 3b = 1.66c + 2e
O balance: 2a = 0.27c + 2d + e
N balance: b = 0.20c
RQ: 0.43 = d/a.
We must solve this set of simultaneous equations. Solution can be achieved in many different
ways; usually it is a good idea to express each variable as a function of only one other variable, b
is already written simply as a function of c in (4); let us try expressing the other variables solely in
terms of c. From (1):
d = 16 – c
From (5):
d
a= =2.326d
0.43
Combining (6) and (7) gives an expression for a in terms of c only:
 a = 2.326(16 - c)
a =37.22 - 2.326c
Substituting (4) into (2) gives:
34 + 3(0.20c) = 1.66c + 2e
34 = 1.06c + 2e
e = 17-0.53c
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
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:
C16H34 + 12.48O2 + 2.13NH3 --> 10.64CHl.66O0.27N0.20 + 5.37CO2 + 11.36H2O

DEGREE OF REDUCTION
Elemental balances provide no insight into the energetics of the reaction. Consequently,
the concept of degree of reduction has been developed and used for proton–electron balances in
bioreactions. Available electrons refer to the number or electrons available for transfer to oxygen
in combustion of a substance to 𝐶𝑂2, 𝐻2 𝑂 and nitrogen-containing compounds.
Degree of reduction γ – number of equivalents of available electrons per gram
atom C
The degree of reduction of any element in a compound is equal to the valence of this
element. Therefore, for substrate 𝐶𝑤 𝐻𝑥 𝑂𝑦 𝑁𝑧 , the number of available atoms is 4w + x – 2y – 3z
and the degree of reduction for the substrate, 𝛄𝑆 , is (4w + x – 2y – 3z)/w. Degree of reduction for
𝐶𝑂2, 𝐻2 𝑂, and 𝑁𝐻3 is zero. The following are examples of how to calculate the degree of reduction
for substrates.
Methane (𝐶𝐻4 ): 4w + x – 2y – 3, 1(4) + 4(1) = 8 γ = 8/1 = 8
 Glucose (𝐶6 𝐻12 𝑂6): 4w + x – 2y – 3 6(4) + 12(1) + 6(-2) = 24 γ = 24/6 = 4
Ethanol (𝐶2 𝐻5 𝑂𝐻): 4w + x – 2y – 3 2(4) + 6(1) + 1(-2) = 12 γ = 12/2 = 6
The degrees of reduction of biomass is 𝛾𝑏 = 4𝑤 + 𝛼 − 2𝛽 − 3𝛿.
In a balanced growth equation, number of available electrons is conserved by the virtue
of the fact that amounts of each chemical element are conserved. The available-electron balance
equation, as ammonia as nitrogen source, is:
𝑤γ𝑠 − 4𝑎 = 𝑐γ𝐵
where: γ𝑠 and γ𝐵 are the degrees of reduction for substrate and product respectively.
The available-electron balance is independent of the complete set of the elemental
balances; if the stoichiometric equation is balanced in terms of each element including H and O,
the electron balance is implicitly satisfied.

Table 2 - Degree of Reduction and Weight of One Carbon Equivalent of One Mole
of Some Substrates and Biomass

BIOMASS YIELD
As cells grow there is, as a general approximation, a linear relationship between the
amount of biomass produced and the amount of substrate consumed. This relationship is
expressed quantitively using biomass yield, 𝒀𝑿𝑺 .
𝑔 𝑐𝑒𝑙𝑙𝑠 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑
𝒀𝑿𝑺 =
𝑔 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑
Large number of factors influences biomass yield, including medium composition, nature
of carbon and nitrogen sources, pH and temperature. Biomass is greater in aerobic than in
aerobic cultures; choice of electron acceptor e.g 𝑂2, nitrate or sulfate, can also have a significant
effect.
When 𝒀𝑿𝑺 is constant throughout growth, its experimentally determined value can be
used to determine the stoichiometric coefficient c expressed in terms of:
 𝑐(𝑀𝑊𝑐𝑒𝑙𝑙𝑠)
𝒀𝑿𝑺 =
𝑀𝑊 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
where MW is molecular weight
‘MW cells’ means biomass formula weight plus any residual ash
However, before applying measured values of 𝒀𝑿𝑺 to evaluate c, we must be sure that the
experimental culture system is well represented by the stoichiometric equation. For example, we
must be sure that substrate is not used to synthesize extracellular products other than 𝐶𝑂2 and 𝐻2 𝑂.
One complication with real cultures is that some fraction of substrate consumed is always used for
maintenance activities such as maintenance of membrane potential and internal pH, turnover of
cellular components and cell motility. These metabolic functions require substrate but do not
necessarily produce cell biomass, 𝐶𝑂2 and 𝐻2 𝑂. For the time being, we will assume that available
values for biomass yield reflect substrate consumption for growth only.

EXAMPLE:
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.
a. Calculate the stoichiometric coefficients for the following biological reactions:
Hexadecane: C16H34 + aO2 + bNH3 --> c(C4.4H7.3N0.86O1.2) + dH2O + e CO2
Glucose: C6H12O6 + aO2 + bNH3 --> c(C4.4H7.3N0.86O1.2) + dH2O + e CO2
b. Calculate the yield coefficients YX/S (g dw cell/g substrate), YX/O2 (g dw cell/g O2) for both
reactions. Comment on the differences.

Solution
a. For hexadecane,
amount of carbon in 1 mole of substrate = 16(12) = 192 g
amount of carbon converted to biomass = 192(2/3) = 128 g
Then,
128 = c(4.4)(12)
c = 2.42.
amount of carbon converted to CO2 = 192 - 128 = 64 g
64 = e (12)
e = 5.33
The nitrogen balance yields
14b = c(0.86)(14)
b = (2.42)(0.86)
b = 2.085
The hydrogen balance is
34(1) + 3b = 7.3c + 2d
d = 12.43
The oxygen balance yields
 2a(16) 1.2c(16) 2e(16) d(16)
a 12.427

For glucose,
amount of carbon in 1 mole of substrate 72 g
amount of carbon converted to biomass 72(2/3) 48 g
Then,
48 4.4c(12)
c 0.909.
amount of carbon converted to CO2 72 48 24 g
24 12e
e 2
The nitrogen balance yields
14b 0.86c(14)
b 0.782
The hydrogen balance is
12 3b 7.3c 2d
d 3.854
The oxygen balance yields
6(16) 2(16)a 1.2(16)c 2(16)e 16d
a 1.473

b. For hexadecane,
2.42 (𝑀𝑊)𝑏𝑖𝑜𝑚𝑎𝑠𝑠
𝑌𝑥 =
𝑠 (𝑀𝑊)𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
2.42 (91.34)
𝑌𝑥/𝑠 = = 0.98 𝑔 𝑑𝑤 𝑐𝑒𝑙𝑙𝑠/𝑔 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
226

2.42 (𝑀𝑊)𝑏𝑖𝑜𝑚𝑎𝑠𝑠
𝑌𝑥 =
𝑂2 12.43(𝑀𝑊)𝑂2
2.42 (91.34)
𝑌𝑥 = = 0.557 𝑔 𝑑𝑤 𝑐𝑒𝑙𝑙𝑠/𝑔 𝑂2
𝑂2 12.43 (32)

For glucose,
0.909 (91.34)
𝑌𝑥/𝑠 = = 0.461 𝑔 𝑑𝑤 𝑐𝑒𝑙𝑙𝑠/𝑔 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒
180

0.909 (91.34)
𝑌𝑥 = = 1.76 𝑔 𝑑𝑤 𝑐𝑒𝑙𝑙𝑠/𝑔 𝑂2
𝑂2 1.473 (32)

The growth yield on more reduced substrate (hexadecane) is higher than that on partially
oxidized substrate (glucose), assuming that two-thirds of all the entering carbon is incorporated in
cellular structures. However, the oxygen yield on glucose is higher than that on the hexadecane,
since glucose is partially oxidized.

PRODUCT STOICHIOMETRY
Consider formation of an extracellular product 𝐶𝑗 𝐻𝑘 𝑂𝑙 𝑁𝑚 during growth. Then the
chemical equation for the whole process will be:
𝐶𝑤 𝐻𝑥 𝑂𝑦 𝑁𝑧 + 𝑎𝑂2 + 𝑏𝐻𝑔 𝑂ℎ 𝑁𝑖 → 𝑐𝐶𝐻𝛼 𝑂𝛽 𝑁𝛿 + 𝑑𝐶𝑂2 + 𝑒𝐻2 𝑂 + 𝑓𝐶𝑗 𝐻𝑘 𝑂𝑙 𝑁𝑚

Where f is the stoichiometric coefficient of the product. This is usually provided as another
experimentally determined yield coefficient, the product yield from substrate, 𝒀𝑷𝑺 .
𝑔 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑠 𝑓𝑜𝑟𝑚𝑒𝑑 𝑓(𝑀𝑊 𝑝𝑟𝑜𝑑𝑢𝑐𝑡)
𝑌𝑃𝑆 = =
𝑔 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 (𝑀𝑊 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒)
The same with 𝒀𝑿𝑺 , we must be sure that the experimental system used to measure YPS
does not hold if product formation is not directly linked with growth. In these cases, independent
reaction equations must be used to describe growth and product synthesis.
THEORETICAL OXYGEN DEMAND
Oxygen demand is an important parameter in bioprocessing as oxygen is often the limiting
substrate in aerobic fermentations. Oxygen demand is represented by the stoichiometric coefficient
a. Oxygen requirement is related directly to the electrons available for transfer to oxygen; the
oxygen demand can therefore be derived from an appropriate electron balance. When product
synthesis occurs, the electron balance is:
𝑤γ𝑆 − 4𝑎 = 𝑐γ𝐵 + 𝑓𝑗γ𝑃
where γ𝑃 is the degree of reduction of the product. Rearranging gives:
1
𝑎 = (𝑤γ𝑆 − 𝑐γ𝐵 − 𝑓𝑗γ𝑃 )
4
It means that if we know which organism(γ𝐵 ) substrate (𝑤 𝑎𝑛𝑑 𝛾𝑆 ) and product
(𝑗 𝑎𝑛𝑑 γ𝑃 ) are involved in cell culture, and the yields of biomass (c) and product (f), we can
quickly calculate the oxygen demand. Of course we could also determine a by solving for all the
stoichiometric coefficients.
MAXIMUM POSSIBLE YIELD
The fractional allocation of available electrons in the substrate can be written as:
4𝑎 𝑐γ𝐵 𝑓𝑗γ𝑃
1= + +
𝑤γ𝑆 𝑤γ𝑆 𝑤γ𝑆
 The first term on the right-hand side is the fraction of available electrons transferred from
substrate to oxygen, the second term is the fraction of available electrons transferred to biomass,
and the third term is the fraction of available electrons transferred to product. This relationship can
be used to obtain upper bounds for the yields of biomass and product from substrate.
Let us define CB as the fraction of available electrons in the substrate transferred to
biomass:
𝑐γ𝐵
𝜁𝐵 =
𝑤γ𝑆
In the absence of product formation, if all available electrons were used for biomass
synthesis, 𝜻𝑩 would equal unity. Under these conditions, the maximum value of the
stoichiometric coefficient c is:
𝑤γ𝑆
𝑐𝑚𝑎𝑥 =
γ𝐵
𝑐𝑚𝑎𝑥 can be converted to a biomass yield with mass units using 𝑌𝑋𝑆 , as it is equal to γ𝐵 .
Therefore, even if we do not know the stoichiometry of growth, we can quickly calculate an upper
limit for biomass yield from the molecular formula for substrate and product. If the composition
of the cells is unknown, γ𝐵 can be taken as 4.2 corresponding to the average biomass formula
𝐶𝐻1.8 𝑂0.5 𝑁0.2. Maximum biomass yield can be expressed in terms of mass (𝑌𝑋𝑆,𝑚𝑎𝑥 ) or as number
of C atoms in the biomass per substrate C atom consumed (𝑐𝑚𝑎𝑥 /𝑤 ). These quantities are
sometimes known as thermodynamic maximum biomass yields.
Likewise, the maximum possible product yield in the absence of biomass synthesis can be
determined,
𝑤γ𝑆
𝑓𝑚𝑎𝑥 =
𝑗γ𝑃