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.
OTHER DEFINITIONS

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

Y MX / 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
Y X / e ¿ = 3.14 ± 0.11 g dry wt/g equivalent of electrons
−¿

GROWTH OF BACTERIAL CULTURES

Bacterial Division: Occurs mainly by binary fission. A few bacterial species reproduce by
budding.
Generation Time: Time required for a cell to divide, and its population to double.
Generation time varies considerably:
 E. coli divides every 20 minutes.
 Most bacteria divide every 1 to 3 hours. Some bacteria require over 24 hours to
divide
 Figure 1.4. Binary Fission

BACTERIAL GROWTH PHASES:

A. Lag Phase
 Period of adjustment to new conditions.
 Little or no cell division occurs, population size doesn’t increase.
 Phase of intense metabolic activity, in which individual organisms grow in size.
 May last from one hour to several days.
B. Log Phase
 cells begin to divide and generation time reaches a constant minimum
 Cells are at highest metabolic activity.
 Cells are most susceptible to adverse environmental factors at this stage.
i. Radiation
ii. Antibiotics
C. Stationary Phase
 Population size begins to stabilize.
 Number of cells produced = Number of cells dying
 Overall cell number does not increase.
 Cell division begins to slow down.
 Factors that slow down microbial growth:
 Accumulation of toxic waste materials
 Acidic pH of media
 Limited nutrients
 Insufficient oxygen supply
D. Death or Decline Phase
 Population size begins to decrease.
 Number of cells dying > Number of cells produced
 Cell number decreases at a logarithmic rate.
 Cells lose their ability to divide.
  A few cells may remain alive for a long period of time.

Figure 1.5. Logarithmic Representation

of Bacterial Growth

CELL GROWTH MEASUREMENT

Growth – orderly increased of all chemical components
Balanced 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
1. Minimal equipment is required
2. Results are obtained rapidly
3. Morphological characteristics of the
organisms can be observed
DISADVANTAGES
1. Dead cells cannot usually be
distinguished from live cells
2. The method is not suitable for cell
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 distinguish 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 overall stoichiometry 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. 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.

MICROBIAL GROWTH AND PRODUCT FORMATION

Cell growth usually refers to cell proliferation, the rapid increase in cell numbers that
occurs through repeated cell division. Cell growth can also refer to the enlargement of cell
volume, which can take place in the absence of cell division. Microbial growth is quantified by
increase in the macromolecular and chemical constituents of the cell and growth pattern of each
microbe is unique. Cell growth and cell division are inseparable for microbes as bacteria divide
by binary fission, yeast cells by budding and viruses divide intracellularly.
REQUIREMENTS FOR GROWTH
I. Physical Requirements
1. Temperature: Microbes are loosely classified into several groups based on their
preferred temperature ranges.
A. Psychrophiles – “cold-loving”, also known as cryophiles, are extremophilic
organisms that are capable of growth and reproduction in low temperatures,
ranging from −20 °C to +10 °C. They are found in places that are permanently
cold, such as the polar regions and the deep sea. Its cell membranes have high
levels of fatty acids which remain fluid at colder temperatures.
Examples: Arthrobacter spp, Psychrobacter spp, Halomonas spp,
Pseudomonas, sphingomonas
a) True Psychrophiles - sensitive to temperatures over 20°C. Optimum
growth at 15°C or below. Found in very cold environments (North pole,
ocean depths). Seldom cause disease or food spoilage.
b) Psychrotrophs - Optimum growth at 20 to 30°C. Responsible for most
low temperature food spoilage
B. Mesophiles - “middle loving”, organism that grows best in moderate
temperature, neither too hot nor too cold, with an optimum growth range from
20 to 45 °C (68 to 113 °F). The term is mainly applied to microorganisms and
include most pathogens and common spoilage organisms.
C. Thermophiles – “heat loving”, optimum growth between 50 to 60°C.
Adapted to live in sunlit soil, compost piles, and hot springs. Some
thermophiles form extremely heat resistant endospores.
 Extreme Thermophiles (Hyperthermophiles): Optimum growth at
80°C or higher. Archaebacteria. Most live in volcanic and ocean vents.

Figure 1.1. Growth Rates of

Bacteria Growth at Different
Temperatures

2. pH
 Most bacteria prefer neutral pH (6.5-7.5).
 Molds and yeast grow in wider pH range, but prefer pH between 5 and 6. 4
  Acidity inhibits most microbial growth and is used frequently for food
preservation (e.g.: pickling).
 Alkalinity inhibits microbial growth, but not commonly used for food
preservation.
 Acidic products of bacterial metabolism interfere with growth. Buffers can be
used to stabilize pH

Organisms can be classified as:

A. Acidophiles: “Acid loving”
 Grow at very low pH (0.1 to 5.4)
 Lactobacillus produces lactic acid, tolerates mild acidity.
B. Neutrophiles:
 Grow at pH 5.4 to 8.5. I
 Includes most human pathogens.
C. Alkaliphiles: “Alkali loving”
 Grow at alkaline or high pH (7 to 12 or higher)
 Vibrio cholerae and Alcaligenes faecalis optimal pH 9.
 Soil bacterium Agrobacterium grows at pH 12.

3. Osmotic Pressure: Cells are 80 to 90% water.

A. Hypertonic solutions: High osmotic pressure removes water from cell,
causing shrinkage of cell membrane (plasmolysis).
Used to control spoilage and microbial growth
 Sugar in jelly
 Salt on meat
B. Hypotonic solutions: Low osmotic pressure causes water to enter the cell. In
most cases cell wall prevents excessive entry of water. Microbe may lyse or
burst if cell wall is weak.
C. Isotonic solutions:  when two solutions, separated by a semipermeable
membrane, have equal concentrations of solutes and water.

Figure 1.2. Isotonic Versus

Hypertonic Solution
Plasmolysis
 Figure 1.3. Effects of Osmosis on Bacterial Cells

II. Chemical Requirements

4. Carbon: Makes up 50% of dry weight of cell.
 Structural backbone of all organic compounds.
 Chemoheterotrophs: Obtain carbon from their energy source: lipids,
proteins, and carbohydrates.
 Chemoautotrophs and Photoautotrophs: Obtain carbon from carbon
dioxide.

5. Nitrogen, Sulfur and Phosphorus

A. Nitrogen: Makes up 14% of dry cell weight. Used to form amino acids, DNA,
and RNA.
Sources of nitrogen:
 Protein: Most bacteria
 Ammonium : Found in organic matter
 Nitrogen gas (N2 ): Obtain N directly from atmosphere. Important
nitrogen fixing bacteria, live free in soil or associated with
legumes (peas, beans, alfalfa, clover, etc.).
 Legume cultivation is used to fertilize soil naturally.
 Nitrates: Salts that dissociate to give NO3
B. Sulfur: Used to form proteins and some vitamins (thiamin and biotin).
Sources of sulfur:
 Protein: Most bacteria
  Hydrogen sulfide
 Sulfates: Salts that dissociate to give SO42-
C. Phosphorus: Used to form DNA, RNA, ATP, and phospholipids.
Sources: Mainly inorganic phosphate salts and buffers
6. Other Elements: Potassium, magnesium, and calcium are often required as enzyme
cofactors. Calcium is required for cell wall synthesis in Gram positive bacteria.
7. Trace Elements: Many are used as enzyme cofactors.
Commonly found in tap water:
 Iron
 Copper
 Molybdenum
 Zinc
8. Oxygen: Organisms that use molecular oxygen (O2), produce more energy from
nutrients than anaerobes; can classify microorganism based on their oxygen
requirements

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 CO 2 and H 2 O , we can write the following equation for aerobic cell growth:

C w H x O y N z + aO2 +b H g Oh N i → cC H α O β N δ +dC O2 +e H 2 O

where: C w H x O y N z is the chemical formula for the substrate

b H g Oh N i is the chemical formula of the nitrogen source
cC H α O β N δ 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; CH 1.8 O0.5 N 0.2 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 C O 2 produced d
RQ= =
moles of O 2 consumed a
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 CO 2, H 2 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 C w H x O y N z , the number of available atoms is 4w + x – 2y – 3z
and the degree of reduction for the substrate, γ S , is (4w + x – 2y – 3z)/w. Degree of reduction for
CO 2, H 2 O , and NH 3 is zero. The following are examples of how to calculate the degree of
reduction for substrates.
Methane (CH 4): 4w + x – 2y – 3, 1(4) + 4(1) = 8 γ =
8/1 = 8
Glucose (C 6 H 12 O6 ): 4w + x – 2y – 3 6(4) + 12(1) + 6(-2) = 24 γ = 24/6 = 4
Ethanol (C 2 H 5 OH ): 4w + x – 2y – 3 2(4) + 6(1) + 1(-2) = 12 γ =
12/2 = 6
The degrees of reduction of biomass is γ b=(4 w+ α−2 β−3)/w .
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:
w γ s−4 a=c γ B

where: γ s and γ B 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, Y XS.
g cells produced
Y XS=
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 Y XS is constant throughout growth, its experimentally determined value can be
used to determine the stoichiometric coefficient c expressed in terms of:
c ( MWcells)
Y XS=
MW of substrate
where: MW is molecular weight
‘MW cells’ means biomass formula weight plus any residual ash
However, before applying measured values ofY XS 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 CO 2 and
H 2 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, CO 2 and H 2 O . For the time being, we will assume
that available values for biomass yield reflect substrate consumption for growth only.

PRODUCT STOICHIOMETRY
Consider formation of an extracellular product C j H k O l N m during growth. Then the
chemical equation for the whole process will be:
C w H x O y N z + aO 2 +b H g O h N i → cC H α O β N δ +dC O 2 +e H 2 O+f C j H k O l N m

Where f is the stoichiometric coefficient of the product. This is usually provided as

another experimentally determined yield coefficient, the product yield from substrate, Y PS.
 g products formed f ( MW product )
Y PS= =
g substrate consumed (MW substrate )

The same with Y XS, 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:
w γ S−4 a=c γ B +fj γ P

where γ P is the degree of reduction of the product. Rearranging gives:

1
a= (w γ S−c γ B −fj γ P )
4
It means that if we know which organism( γ ¿¿ B)¿ substrate ( w ¿ γ S) and product ( j∧γ P )
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:

4 a c γ B fj γ P
1= + +
w γ S w γ S w γS

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 ζ B as the fraction of available electrons in the substrate transferred to
biomass:
cγB
ζ B=
w γS
 In the absence of product formation, if all available electrons were used for biomass
synthesis, ζ B would equal unity. Under these conditions, the maximum value of the
stoichiometric coefficient c is:
w γS
c max =
γB
c max can be converted to a biomass yield with mass units using Y XS, as it is equal to γ B .
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, γ B can be taken as 4.2 corresponding to the average
biomass formula CH 1.8 O0.5 N 0.2. Maximum biomass yield can be expressed in terms of mass
(Y ¿¿ XS ,max)¿ or as number of C atoms in the biomass per substrate C atom consumed
(c ¿¿ max¿ ¿¿ 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,
w γS
f max =
j γP