UNIT III

METABOLIC STOICHIOMETRY
AND ENERGETICS

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 STOICHIOMETRIC OF CELL GROWTH
STOICHIOMETRY OF PRODUCT FORMATION

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 Growth Stoichiometry
•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.
•Atoms of carbon, nitrogen, hydrogen, oxygen and the other
elements are rearranged in the metabolic processes of the cell,
but the total amounts of each of these elements incorporated
into cell material is equal to the amounts received from the
envrionment.
•Further, the amount of some metabolic product formed or the
amount of heat released by cell growth is often proportional to
the amount of consumption of some substrate or the amount of
formation of another product.
.

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• The complexity of the reactions can be represented by
rigorous material balance constraints as well as approximate,
empirical stoichiometric considerations
• These stoichiometric considerations have broad implications
in biochemical technology ranging from growth medium
formulation to computer control and cooling requirements in
bioreactors.
• Also, as in reactor analysis in general, knowledge of
stoichiometric relationships is critical in formulating
bioreactor material balances and making most effective and
systematic use of reaction kinetics.

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 Medium Formulation and Yield Factors
• Cell growth involves consumption of substrates which provide
energy and raw materials required for the synthesis of
additional cell mass.
• Medium formulation is actually more complicated for the
following reasons
(i) Some substrate elements are released in products not
assimilated into cell material
(ii) Rate limitations as well as stoichiometric limitations must be
considered
(iii) Specific nutrients may be limiting or specific products may be
inhibitory due to the metabolic properties of a particular cell
strain.

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• It is necessary to consider both stoichiometry and kinetics
when formulating a strategy for simplifying the stoichiometric
representation of cell growth and product formation.
• The number of nutrient components in the medium is
typically very large, and we cannot in practice include all of
them in our considerations either of process stoichiometry or
of process kinetics.
• The rational simplification of the description of the system
based upon the identification of certain key, limiting
compounds or elements.
• The stoichiometric relationship is also potentially useful for
bioreactor monitoring: measurement of substrate
concentration implies a corresponding estimate of the cell
concentration if initial values of both quantities are known.

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 Product Formation Stoichiometry
• A variety of metabolic end products is released into the growth
medium or accumulated intracellularly.
• The pertinent stoichiometries for product formation may be
classified usefully into the following four classes; the first three
correspond to the fermentations classification formulated by
Elmer L. Gaden, Jr. in 1995.
1. The main product appears as a result of primary energy
metabolism. Eg. Ethanol production during anaerobic growth of
yeast
2. The main product arises indirectly from energetic metabolism Eg.
Citric acid formation during aerobic mold cultivation
3. The product is a secondary metabolite. Eg. Penicillin production
in aerated mold culture
4. Biotransformation. The product is obtained from substrate
through one or more reactions catalyzed by enzymes in the cells.
Eg. Steroid hydroxylation 9
• Class 1 processes have a relatively simple stoichiometric
description.
• Product appears in relatively constant proportions as cell mass
accumulates and substrate is consumed.
• Here, the processes of substrate utilization, cell mass synthesis,
and product formation are linked as in a simple, single chemical
reaction.

CHmOn + aO2 + bNH3 -------- cCHαOβNδ + dH20 + eCO2 + f CHxOy Nz

• For class 2 situations the simple stoichiometry does not apply.

Product formation is not necessarily proportional to substrate
utilization or cell mass increase.
• Representation of the stoichiometry in such a case requires an
indepentent reaction equation for product formation.

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• The stoichiometric descriptions appropriate for classes 3 and
4 cases depend on the particular substrates and products
involved.
• Product formation in these cases is typically completely
uncoupled from cell growth.
• Secondary metabolite accumulation is dictated by kinetic
regulation and activity of the cells
• Examination of product formation stoichiometry offers
several useful insights for engineering analysis.
• One application is estimation of upper bound for product
yields.
• These numbers can be very useful in preliminary economic
analysis .

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ELEMENTAL BALANCE, DEGREE OF REDUCTION
SUBSTRATE AND BIOMASS

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 Elemental Balances
• Cell growth and metabolic activities are similarly described as
a simple chemical reaction.
• It is also necessary to establish a definite formula for dry cell
matter.
• The elemental composition of certain strains of
microorganism is defined by an empirical formula CHαOβNδ
• The general biochemical reaction for biomass production is
based on consumption of organic substrate as shown below.
• Substrate oxidation is simplified in the following biochemical
oxidation
CHmOn + aO2 + bNH3 -------- cCHαOβNδ + dH20 + eCO2
•
• CHmOn - 1 mole of carbohydrate
• CHαOβNδ - 1 mole of cellular material
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Elemental balances on C,H,O and N yield the following equation

C: 1=c+e
H: m+3b=c+2d 1
O: n+2a=c+2d
N: b=c

Respiratory quotient, 2

Equation 1 and 2 constitute five equations for 5 unknowns a, b, c, d, e.

With the measured value of RQ these equations can be solved to
determine the stoichiometry coefficients.
• Degree of Reduction
Degree of reduction is equal to valency of element.

• The degree of reduction of some key elements

• C=4; H=1; N=-3; O=-2; P=5 And S=6

• The degree of reduction of any element in a compound is equal to the

valence of the element

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Examples for calculating the degree of reduction

Methane (CH4): 1(4) + 4(1) =8; γ=8/1=8

Glucose (C6H12O6): 6(4) +12(1) +6(-2) =24; γ=24/6=4
Ethanol (C2H5OH): 2(4) +6(1) +1(-2) =12; γ=12/2=6

High degree of reduction indicates a low degree of oxidation.

Consider the aerobic process of single extracellular product

CHmOn+aO2+bNH3-------- cCHαOβNδ+dCHxOyNz+eH20+fCO2

The degree of reduction of substrate, biomass and product are

γs=4+m-2n
γb=4+α-2β-3δ
γp=4+x-2y-3z

Degree of reduction of CO2, NH3, and H2O is 0.

 ELECTRON BALANCE
YIELD COEFFICIENT OF BIOMASS AND PRODUCT
FORMATION

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 Electron Balance
CwHxOyNz + aO2 + bHgOhNi ---- cHαOβNδ + dH2O + eCO2 + fCjHkOlNm

Electron Balance : wγs – 4a = cγb + fjγp

a = (wγs - cγb - fjγp)/4

Maximum Possible Yield 4a/ wγs + cγb/ wγs + fjγp/ wγs = 1

ξ + ξ b + ξp = 1

Maximum Biomass Yield ξb = 1

ξ b = cγb/ wγs
cmax = wγs/γb

Maximum Product Yield ξp = 1

ξp = fjγp/ wγs
fmax = wγs/jγp
An energy balance for aerobic growth is
Qocγb+Qodγp= Qoγs-Qo4a
biomass product substrate oxygen

Where Qo—heat evolved per equivalent of available electrons transferred

to oxygen

If Qo is constant, cγb+dγp= γs-4a

1= cγb/γs +dγp/γs+4a/γs
1= ξb+ ξp+ ξ

Where,
ξ is the fraction of available electrons in the organic substrate that is
transferred to oxygen,
ξb is the fraction of available electrons incorporated into the biomass
ξp is the fraction of available electrons incorporated into the extracellular
product.
 Yield Coefficients
Yield coefficients based on other substrates or product formation may be defined as:
Yield coefficient Yx/s= -∆X/∆S

Apparent Yield coefficient Yx/o2 = -∆X/∆O2

Observed Yield coefficient Yp/s= -∆P/∆S

Theoretical Yield coefficient Y x/s = Yx/ATP N

Where Yx/ATP = Moles of Biomass produced / Moles of ATP generated

And N = Moles of ATP produced / gram of substrate consumed.

Maintenance coefficient

For continuous culture, M (based on substrate) = kd/ Ym x/s

Where kd is the first order rate constant.

Overall Yield Coefficient Ym x/s = maximum biomass produced per gram of substrate
consumed when maintenance is not considered.

Ykcal = Moles of Biomass produced / Kcal of heat evolved in fermentation 20

• True or Stoichiometric or theoretical yield = (total mass or mole of product formed)
/(mass or moles of reactant used to form that particular product)
• Observed or apparent yield = (mass or moles of product present) /(total mass or
moles of reactant consumed)
• YX/S = mass or moles of biomass produced per unit mass or moles of substrate
consumed .
• YP/S = mass or moles of product formed per unit mass or moles of substrate
consumed .
• YP/X = mass or moles of product formed per unit mass or moles of biomass.
• YX/O = mass or moles of biomass formed per unit mass or moles of oxygen
consumed .
• YCO2/S = mass or moles of CO2 formed per unit mass or moles of substrate
consumed .
• RQ = moles of CO2 formed per mole of O2 consumed applied only for balanced
growth .
• For continuous culture, M (based on substrate) = kd/ Ym x/s
Where kd is the first order rate constant
• Overall Yield Coefficient Ym x/s = maximum biomass produced per gram of substrate
consumed when maintenance is not considered

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 Problems
1. Assume that experimental measurement for a certain organism
has shown that cells can convert 2/3 w/w of the substrate carbon
(alkane or glucose) to biomass.

a) Calculate stoichiometric coefficient for the following biochemical

reaction:

Hexadecane:
C16H34+aO2+bNH3--------------- cC4.4H7.3N0.86O1.2+dH2O+eCO2

Glucose:
C6H12O6+aO2+bNH3--------------- cC4.4H7.3N0.86O1.2+dH2O+eCO2

b) Calculate yield coefficient Y x/s and Y x/o2 for both reactions.

Solution:
Hexadecane reaction:

C-balance : Amt. of carbon in 1 mole of substrate = 16x12=192g.

Amt. of carbon converted in biomass = 192 x 2/3 = 128g.
Amt. of carbon in biomass = 12x4.4c=52.8g.
128g=52.8c g
c=128/52.8=2.424
Amt. of carbon converted to CO2 = 192-128=64g
12xe=64
e=5.33g

N-balance : b=0.86c
b=2.0846

H-balance : 34+3b=7.3c+2d
d=11.27

O-balance : 2a=1.2c+d+2e
a=12.42
a=12.42; b=2.0846; c=2.424; d=11.27; e=5.33

Y x/s = (c x Molecular wt. of biomass)/Molecular wt. of substrate

Molecular wt. of biomass = 4.4(12)+7.3+0.86(14)+1.2(16) = 91.34g

Molecular wt. of substrate = 16(12)+34 = 226g

Y x/s = 2.424 x 91.34 / 226

=0.9796 g of biomass/g of substrate.

Y x/o2 = (c x Molecular wt. of biomass)/(a x Molecular wt. of oxygen)

Y x/o2 = (2.424 x 91.34) / (12.42 x 32)
= 0.5566 g of biomass/g of O2
2. The growth of Saccharomyces cerevisiae on glucose under anaerobic
conditions can be described by the following overall reaction,

C6H12O6 + βNH3 0.59CH1.74N0.2O0.45 + 0.43C6H8O3 + 0.036H2O + 1.54CO2 +

1.3C2H5OH

a) Determine Y x/s, Y ethanol/s, Y CO2/S, Y C6H8O3/s

b) Determine the coefficient β

3. Aerobic growth of bacteria on ethanol described by the following overall

reaction:
C2H5OH + aO2+bNH3--------------- cC5H7NO2+dH2O+eCO2

a) Determine coefficients a,b,c,d and e where RQ =0.66

b) Determine Y x/s and Y x/o2
4. Aerobic degradation of benzoic acid by a mixed culture of MO can be
described by the following overall reaction:

C6H5COOH + aO2+bNH3--------------- cC5H7NO2 + dH2O + eCO2

a) Determine coefficients a,b,c,d and e where RQ=0.9

b) Determine Y x/s and Y x/o2
c) Determine the degree of reduction of substrate and bacteria.

Solution :
γsubstrate = [(7x4)+(6x1)+(2x-2)]/7 = 4.286

γbacteria = γbiomass = [(5x4)+(7x1)+(1x-3)+(2x-2)]/5 = 4

 5. Aerobic degradation of an organic compound by a mixed culture of MO
in waste water can be represented by the following overall reaction:
C3H6O3 + aO2+bNH3--------------- cC5H7NO2 + dH2O + eCO2

a)Determine coefficients a,b,c,d and e if Y x/s = 0.4 g of dry wt. cells/g of

substrate

b)Determine Y x/o2 ,Y x/s

c)Determine the degree of reduction of substrate and bacteria.

d)Determine RQ of the organism.

 6. Biological denitrification of nitrate containing waste water can be
described by:

NO3- + aCH3OH + H+ --- bC5H7NO2 + dH2O + eCO2

a)Determine coefficients a,b,c,d and e if Y x/s = 0.5 g of dry wt.cells/g of N

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

ENERGETIC ANALYSIS OF MICROBIAL GROWTH AND
PRODUCT FORMATION
OXYGEN TRANSFER IN AEROBIC CULTURE

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 Energetic analysis of microbial growth and product
formation
• Microorganisms carry out oxidation-reduction reactions in
order to obtain energy for growth and cell maintenance.
• The amount of energy released per electron equivalent on an
electron donor oxidized varies considerably from reaction to
reaction
• Cell maintenance has energy requirements for activities such
as cell movement and repair of cellular proteins that decay
because of normal resource recycling or through interactions
with toxic compounds.
• When cells grow rapidly in the presence of nonlimiting
concentrations of all factors required for growth, cells make
the maximum investment of energy for synthesis.
• However, when an essential factor, such as the
electron-donor substrate, is limited in concentration, then a
larger portion of 30
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• of the energy obtained from substrate oxidation must be
used for cell maintenance.
• The net yield of cells decreases with a decrease in the rate of
substrate utilization.

• The net yield becomes zero when the energy supplied

through substrate utilization is just equal to m, the
maintenance energy.

• Under these conditions, all energy released is used just to

maintain the integrity of the cells.
• If the substrate available for the microorganisms decreases
further, then food is insufficient to maintain the
microorganisms, and they go into net decay. 32
• Conversely, when substrate and all other required factors are
unlimited in amount, the rate of substrate utilization will be at
its maximum, and the net yield, Yn, will approach the true yield,
Y.
• Over the years , many approaches have been taken to describe
how the true yield relates to the energy released from electron
–donor oxidation.
• Although no unified approach for relating growth to reaction
energetics is yet widely accepted, a method that is based on
electron equivalents and that differentiates between the
energy portion of an overall biological reaction and the
synthesis portion, has proven useful.

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OXYGEN TRANSFER IN AEROBIC CULTURES

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 Oxygen Transfer in aerobic culture
OTR (Oxygen Transfer Rate) = No2=kLa(C*-CL)
Where,
kL is the oxygen transfer coefficient (cm/h)
a is the gas-liquid interfacial area (cm2/cm3)
kLa is the volumetric oxygen transfer coefficient (h-1)
C* is saturated DO concentration (mg/l)
CL is the actual DO concentration in the broth (mg/l)
No2 is the rate of oxygen transfer (mg O2/l.h)

Oxygen Uptake Rate

Where
qo2 is the specific rate of oxygen consumption (mg O2/g dw cells.h),

YX/o2 is the yield coefficient on oxygen (g dw cells/g O2),

X is cell concentration (g dw cells/l).

When oxygen transfer is the rate-limiting step, the rate of oxygen
consumption is equal to the rate of oxygen transfer. If the maintenance
requirement of O2 is negligible compared to growth, then

µgX/ (YX/o2) = kLa(C*-CL)

or
dX/dt= (YX/o2) kLa(C*-CL)
 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 in Eqs (1) and (2).
•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 as represented by Eq.
(2), the electron balance is (Eq. 3):

(1)

(2)

(3)

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• where fp is the stoichiometric coefficient for the product. Product synthesis
introduces one extra unknown stoichiometric coefficient to the equation;
thus, an additional relationship between coefficients is required. This is
usually provided as another experimentally-determined yield coefficient,
the product yield from the substrate, YPS:

• As mentioned above with regard to biomass yields, we must be sure that the
experimental system used to measure YPS conforms to Eq. (2). Eq. (2) does not hold if
product formation is not directly linked with growth; accordingly it cannot be applied
for secondary-metabolite production such as penicillin fermentation, or for
biotransformations such as steroid hydroxylation which involve only a small number
of enzymes in cells. In these cases, independent reaction equations must be used to
describe growth and product synthesis.

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• where γp is the degree of reduction of the product. Rearranging gives:
(4)

• Eq. (4) is a very useful equation. It means that if we know which organism (γ B)'
substrate (wand 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 of Eq. (2)) allows more rapid evaluation and does not require that the
quantities of NH3, CO2 and H20 involved in the reaction be known.

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 • From Eqn. 3, The fractional allocation of available electrons in the substrate can be
written as:
(5)

• In Eq. (5), 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 the product.
• This relationship can be used to obtain upper bounds for the yields of biomass and
product from the substrate.

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 Problem:
7. Candida utilis cells convert glucose to CO2 and H2O during growth, the cell
composition is CH1.84O0.55N0.2 + 5% ash. Biomass yield of X from substrate =
0.5g of cells/g of substrate. Ammonia is used as nitrogen source.

C6H12O6 + 6O2 -- 6H2O + 6CO2

a)Find O2 demand.

b)Candida utilis is also able to grow with ethanol as substrate producing cells of
the same composition as above. On a mass basis, how does the max. possible
biomass yield from ethanol compared with the max. possible biomass yield
from glucose?

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HEAT EVOLUTION IN AEROBIC CULTURE

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 Heat Generation by Microbial Growth

• About 40% to 50% of the energy stored in a carbon and

energy source is converted to biological energy (ATP) during
aerobic metabolism, and the rest of the energy is released as
heat.
• For actively growing cells, the maintenance requirement is
low, and heat evolution is directly related to growth.
• The heat generated during microbial growth can be calculated
using the heat of combustion of the substrate and of cellular
material.
• A schematic of an enthalpy balance for microbial utilization of
substrate is presented in the following figure.
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 The heat of combustion of the substrate is equal to the sum of metabolic
heat and the heat of combustion of the cellular material

where,

is the heat of combustion of the substrate (kJ/g substrate),

Yx/s is the substrate yield coefficient(g cell/g substrate),

is the heat of combustion of cells(kJ/g cells),and

1/YH is the metabolic heat evolved per gram of cell mass produced(kJ/g
cells).
and can be determined from the combustion of substrate and cells.
• The total rate of heat evolution in a batch fermentation is
• QGr = VLµnet x 1/YH
• Where VL is the liquid volume (I) and X is the cell
concentration (g/l).
• In aerobic fermentations, the rate of metabolic heat evolution
can roughly be correlated to the rate of oxygen uptake, since
oxygen is the final electron acceptor.
• QGr = 0.12QO2
• Here QGr is in units of kcal/h,while QO2 is in millimoles of O2/h.
• Metabolic heat released during fermentation can be removed
by circulating cooling water through cooling coil or cooling
jacket in the fermenter.
• Often, temperature control is an important limitation on
reactor design. The ability to estimate heat-removal
requirements is essential to proper reactor design.
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THERMODYNAMIC EFFICIENCY OF
CELL GROWTH

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 Thermodynamic efficiency of growth
• Energy limited growth - Calculating from the energy balance of an
elementary microbial Division
• In many cases, growth is limited by the amount of energy available. The
threshold level of resources necessary to trigger division then corresponds
to a threshold level of energy
• This threshold level can be estimated using different Gibbs energy balance
methods.
• We use the Gibbs energy dissipation method to determine the growth
stoichiometry.
• For more clarity, the approach is illustrated with the example of E. coli
growing aerobically on glucose
• The catabolism is associated with a Gibbs energy variation, denoted

• a and b being stoichiometric coefficients

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 References
• Shuler. M.L., Kargi. F., “Bioprocess Engineering: Basic Concepts”, 2ndEdition.
Pearson, 2002
• Stanbury.P.F., Whitaker.A., Hall.S.J., “Principles of Fermentation
Technology”, 2nd Edition, Butterworth– Heinemann, 1995.
• Doran. P. M., “Bioprocess Engineering Principles”, Academic press, 1995
• http://etabu.com/m/tutorials/view/METABOLIC-STOICHIOMETRY-AND-E
NERGETICS
• https://hal.inria.fr/hal-00825781v3/document

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