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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

Cell Growth Measurement


Growth – orderly increased of all chemical components
Balaced Growth – growth during which a doubling of the biomass is accompanied by a doubling
of all measurable properties of the population such as protein, DNA, RNA and intracellular water.
Cell growth can be determined by:
- Cell number
- Cell mass
- Cell activity
MEASUREMENT OF CELL NUMBER
Microscopic Counts - the number of cells in a population can be measured under a microscope
by counting cells placed in special counting chambers.
Two types of Chambers:
1. HEMOCYTOMETER – blood cell counting chamber for use with microorganisms of 3
µm in diameter or larger
2. PETROFF-HAUSSER COUNTING CHAMBER – for used primarily with bacteria
Direct-Counting Method
ADVANTAGES DISADVANTAGES
1. Minimal equipment is required 1. Dead cells cannot usually be
2. Results are obtained rapidly distinguished from live cells
3. Morphological characteristics of the 2. The method is not suitable for cell
organisms can be observed suspensions of low density
3. Small cells are difficult to see under the
microscope and can be missed when
counting
4. The actual counting procedure is
tiresome and may cause considerable
eyestrain
5. It is not suitable for highly flocculating
cells such as mycelium

Viable Plate Count


Viable cell – one that is able to divide and form a colony
Two ways of performing plate count
1. Spread Plate Method – a volume of no larger than 0.1 ml is spread over the agar surface
2. Pour Plate Method – sample is mixed with melted agar and poured into a sterile plate
Coulter Counter
- To avoid tedium of direct microscopic counting
- Using this, cell size can also be measured
- Cannot ditinguish between cells and any impure particles
- Difficult to use with microorganisms in chains and is useless with mycelial organisms
MEASUREMENT OF CELL MASS
Cell Dry Weight
- can be measured directly by taking an aliquot of cell suspension and centrifuging it
- most direct approach for the quantitative measurement of cell mass and is probably the
most reliable and reproducible
- can only be used with dense cell suspensions
Turbidity
- based on the fact that small particles scatter light proportionally, within certain limits,
to their concentration
- when a beam of light is passed through a suspension of organisms, the reduction in the
amount of light transmitted as a consequence of scattering is thus a measure of cell
density. Such measurements are usually made in spectrophotometer, which reads in
Absorbency (A) units.
Absorbency – logarithm of the ratio of intensity of light striking the suspension (Io) to that
transmitted by the suspension (I)
𝐼𝑜
A = 𝑙𝑜𝑔 𝐼

Indirect Methods
The indirect methods for measuring cell mass are based on the overallstoichiometry for
growth and product formation, which may be written in the general form:

The change of the cell mass can be monitored indirectly by measuring the following:1
1. Nutrient Consumption
2. Product Formation
3. Cell Components
4. Heat Evolution
5. Viscosity

Effect of Environmental Conditions on Microbial Growth


Temperature - important factor affecting the performance of cells
Groups of Organisms
1. Psychrophiles- (Topt < 20°C)
2. Mesophiles - (Topt = from 20° to 50°C)
3. Thermophiles - (Topt > 50° C)
As the temperature is increased toward optimal growth temperature, the growth rate
approximately doubles for every 10°C increase in temperature. Above the optimal temperature
range, the growth rate decreases and thermal death may occur.
Temperature also affects product formation. However, the temperature optimum for growth
and product formation may be different. The yield coefficient is also affected by temperature. In
some cases, such as single-cell protein production, temperature optimization to maximize the yield
coefficient is critical. When temperature is increased above the optimum temperature, the
maintenance requirements of cells increase resulting in a decrease in the yield coefficient.
Temperature also may affect the rate-limiting step in a fermentation process. At high
temperatures, the rate of bioreaction might become higher than the diffusion rate, and diffusion
would then become the rate-limiting step.
Hydrogen-ion concentration (pH) affects the activity of enzymes and therefore the microbial
growth rate. The optimal pH for growth may be different from that for product formation.
Generally, the acceptable pH range varies about the optimum by ±1 to 2 pH units. Different
organisms have different pH optima: the pH optimum for many bacteria ranges from pH = 3 to 8;
for yeast, pH = 3 to 6; for molds, pH = 3 to 7; for plant cells, pH = 5 to 6; and for animal cells, pH
= 6.5 to 7.5. Many organisms have mechanisms to maintain intracellular pH at a relatively constant
level in the presence of fluctuations in environmental pH. When pH differs from the optimal value,
the maintenance-energy requirements increase. One consequence of different pH optima is that the
pH of the medium can be used to select one organism over another.
Dissolved oxygen (DO)- is an important substrate in aerobic fermentations and may be a
limiting substrate, since oxygen gas is sparingly soluble in water. At high cell concentrations, the
rate of oxygen consumption may exceed the rate of oxygen supply, leading to oxygen limitations.
When oxygen is the rate-limiting factor, specific growth rate varies with dissolved-oxygen
concentration according to saturation kinetics; below a critical concentration, growth or respiration
approaches a first-order rate dependence on the dissolved-oxygen concentration.
Above a critical oxygen concentration, the growth rate becomes independent of the dissolved-
oxygen concentration. Oxygen is a growth-rate-limiting factor when the DO level is below the
critical DO concentration.
Dissolved carbon dioxide (DCO2) concentration may have a profound effect on
performance of organisms. Very high DCO2 concentrations may be toxic to some cells. On the
other hand, cells require a certain DCO2 level for proper metabolic functions. The dissolved carbon
dioxide concentration can be controlled by changing the CO2 content of the air supply and the
agitation speed.
High substrate concentrations that are significantly above stoichiometric requirements are
inhibitory to cellular functions. Inhibitory levels of substrates vary depending on the type of cells
and substrate.
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:
(1) C balance: 16 = c + d
(2) H balance: 34 + 3b = 1.66c + 2e
(3) O balance: 2a = 0.27c + 2d + e
(4) N balance: b = 0.20c
(5) 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 C balance,
d = 16 – c
From the RQ,
d
a= =2.326d
0.43
Combining the new formed equations 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
EXAMPLE:
The chemical reaction equation for respiration of glucose is:
C6H12O6 + 6O2  6CO2 + 6H2O.
Candida utilis cells convert glucose to CO2 and H2O during growth. The cell composition is
CH1.84O0.55N0.2 plus 5% ash. Yield of biomass from substrate is 0.5 g g- 1. Ammonia is used as
nitrogen source.
(a) What is the oxygen demand with growth compared to that without?
(b) C. utilisis 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?
Solution:
Molecular weights: glucose = 180; ethanol = 46
MW of biomass is (25.44 + ash); since ash accounts for 5% of the total weight, 95% of the total =
25.44. Therefore, MW of biomass is equal to
25.44
𝑀𝑊 = = 26.78
0.95
If given γS for glucose is 4.00; γS for ethanol is 6.00,
γB =[4(1)+1(1.84)-2(0.55)-3(0.2)]=4.14

For glucose w = 6; for ethanol w = 2.


a. YXS == 0.5 g g-1 Converting this mass yield to a molar yield:
0.5 g biomass 180 g glucose 1 gmol biomass gmol biomass
YXS = ( )( )( ) = 3.36 =c
g glucose 1 gmol glucose 26.78 g biomass gmol glucose

For oxygen demand, in the absence of product formation:


1
a = [6(4)-(3.36)(4.14)] = 2.52
4
Therefore, the oxygen demand for glucose respiration with growth is 2.5 gmol O2 per gmol glucose
consumed. By comparison with the chemical reaction equation for respiration, this is only about
42% that required in the absence of growth.
b. Using the data above, the maximum possible yield for glucose is
6(4.00)
cmax = =5.80
4.14
Converting this to a mass basis:
5.80 g biomass 1 gmol glucose 26.78 g biomass g biomass
YXS,max = ( )( )( ) = 0.86
gmol glucose 180 g glucose 1 gmol biomass g glucose
For ethanol,
2(6.00)
cmax = =2.90
4.14
Converting this to a mass basis:
2.90 gmol biomass 1 gmol ethanol 26.78 g biomass g biomass
YXS,max = ( )( )( ) = 1.69
gmol glucose 46 g ethanol 1 gmol biomass g ethanol

Therefore, on a mass basis, the maximum possible amount of biomass produced per gram ethanol
consumed is roughly twice that per gram glucose consumed.