ChEn 5751 -Fall 2008

Stoichiometry of Bioprocesses
• Outline:
– Stoichiometry and energetics of chemical reactions
ChEn 5751 – Stoichiometry and energetics of biochemical reactions
– Reactions, coupled reactions, reaction networks,
Stoichiometry and Energetics of biopolymer synthesis, biomass synthesis
Biochemical Reactions – Biomass and product composition
– Stoichiometryy of biomass and product
p synthesis
y
– Yield coefficient
– Theoretical maximum
Prof. Wei-Shou Hu
– Anaerobic processor

Materials Balance in Biological First Step in Materials Balance-

Systems Define the System to Balance
• Stoichiometric Equation for a biochemical reaction involving C, H, N, • In most cases, we balance on a bioreactor or a cell, a
O:
reactor ion, or a set of reactions
• Balance on a bioreactor:
∑α C i ai H bi N ci Odi = 0 ( ΔH ro ),( ΔG o ),( ΔG 'o ) – Batch process:
t=0 Process t=tf
Inputs Outputs
• Characteristics of biochemical reactions: – Fedbatch process:
– Most biological reactions (almost all) occur in aqueous solution
Inputs
– The Gibb’s free energy change is of more interest to us, but not the heat
of reaction.
reaction t=0 tt=0
0…ttf t=tf
– Reactions are feasible only the corresponding enzymes are present Inputs Process Outputs feed stream is added during process
– Coupling of reactions: many reactions require cosubstrates or cofactors,
which are largely “recycled” or “regenerated” by another reaction or
reactions. Those reactions are thus “coupled”. - Continuous Process:
Inputs Outputs feed stream is added and removed continuously
Process
- Rate of accumulation = rate of inputs – rate of output + rate of generation –
rate of consumption

Prof. Wei-Shou Hu 1
ChEn 5751 -Fall 2008

What systems we do balance on? Balance on a Cell

Autocatalytic
Inputs Outputs
Physical system
Carbon source
Reactor
newly
Cell Nitrogen source generated
biomass

Other nutrients Cell

Abstract
A sett off reactions
ti
Energy source Metabolites
Reaction network (CO2, H2O, etc)
(eg. hv, H2, Fe2+)
Often products
also
carbon
source

Balance on a Biochemical Reaction Ethanol Production by Yeast Saccharomyces cerevisiae

or a Biochemical Reaction System Glucose → → ethanol, carbon dixodie

Whatt should
Wh h ld bbe b
balanced?
l d? • Macroscopically,
M i ll one mole l off glucose
l entering
t i
• Elements (C, N, O, H, P, S, etc.) the cell, may give rise to two moles of ethanol
and two moles of CO2
• Charge (if charges are not balanced, this will
• Macroscopic balance is thus:
create electropotential)
c6H12O6 → 2 C2H5OH + 2 CO2
The reactions are “physiologically feasible, i.e. the
enzyme system exists to carry the reaction out • Biochemically the result is the net reactions in
• Example: Production of ethanol glycolysis, converting glucose to pyruvate, and
in ethanol fermentation reaction

Prof. Wei-Shou Hu 2
ChEn 5751 -Fall 2008

Balance on reaction : glycolysis

ATP Overall Reaction of Glycolysis
ADP Pyruvate + NADH → Ethanol + CO2 + NAD++C2H5OH
Inputs Outputs
CHO
HCOH O
ATP ADP HOCOH OC
+ 2ADP3-3 +
+2N
2NAD+ +
+2P
2Pi3-3 2 + 2ATP4- + 2NADH+2H2O
HCOH CO
HCOH CH3

CH2OH pyruvate

Glycolysis 2ATP → 2ADP + 2Pi + Work + Heat

Ethanol Fermentation
NAD+ NADH+H+
ADP Pyruvate + NADH → Ethanol + CO2 + NAD+
ATP
Macroscopic balance (balance on the cell):
glucose → 2Ethanol + 2CO2
Biochemical reaction balance (balance on reactions):
glucose + 2ADP + 2Pi → 2Ethanol + 2CO2 + 2ATP
Where’s the difference come from?
ADP ATP Why intracellular NADH/NAD+ is balanced, but not ATP/ADP?

Typical Inputs into Biological Typical Outputs in Biological

Systems Systems
Source of Materials: • Biomass
• Products
• Carbon Source, Glucose C6H12O6, amino acid{ CN • Metabolites
• Nitrogen Source, NH4Cl, (NH4),SO4, amino acids, proteins – Products and metabolites are easy to write a
chemical formula
• Oxygen Source, Air (O ) 2
– How to write a formula for cells?
• Hydrogen Source
• Phosphate Source, PO4-3

Prof. Wei-Shou Hu 3
ChEn 5751 -Fall 2008

Cell Composition
Elemental Composition of Bacterial Chemical composition of cells
Cell and Yeast % of Dry Mass
• Most abundant component is water, often not
Average
g Composition
p of a Cell included in biomass formulation
Mass (pg/cell) % of Dry Mass • Consisted of carbon compounds, proteins,
Animal Cell Bacterium Animal Cell Bacterium carbohydrates, lipids, nucleic acids, and non-
Wet Weight 3500 1.5
carbon compounds, electrolytes,
Dry Weight 600 0.3 100 100 • major elemental components: C, N, H, O, P, S
Protein 250 0.17 41 55
yp
• Typical cellular formula:
Carbohydrate 150 0.015 20 5
Lipid 120 0.015 1.5 5
– Bacterium CH1.6N0.20O0.27 to CH2N0.26O0.45
DNA 10 0.015 1.5 5 – Yeast CH1.64N0.16O0.52P0.01S0.005
RNA 25 0.075 2.5 25 – Animal cells CH1.98N0.26O0.49
Water 1.2 500 400
Volume 4 X 10-9 cm3 1.5 X 10-12 cm3

Biomass Formula Yield Coefficient on Biomass

Example:
Yield on biomass =(mass of biomass
Element C H N O Ash produced)/(mass of substrate consumed)
% of Dry Weight 53.0 7.3 12.0 19.0 8
Atomic Weight of the Element 12 1 14 16
• Many different way of expressing yield; biomass
No. of mole per 100 g 4.42 7.3 0.857 1.19
may be wet biomass or dry biomass; may be
Normalize to C 1 1.60 0.194 0.269 expressed as total mass or total carbon mass
• Yield is not dimensionless, example of units: g
dry biomass/g glucose; g dry biomass/mole
CH1.66, N0.194, O0.269, constitute
tit t 0.92
0 92 off mass glucose
l
Formula weight= (12 + 1 x 1.66 +14 x 0.194 + 16 x 0.26)/(1-ash fraction) • The value of yield is dependent on the units
= 20.8 chosen for the expression

Prof. Wei-Shou Hu 4
ChEn 5751 -Fall 2008

Yield Coefficient on Biomass

• Is affected by the type of substrate
Yield on biomass = mass of biomass produced
Dry Biomass
glucose
E coli growing on different carbon sources
mass off substrate
b t t consumedd
succinate

slope = yield

Substrate consumed

• Is affected by the medium, growth condition (yield is higher at optimal

conditions, i.e., optimal pH, temperature, osmolality, nutrient composition
It is not dimensionless. The units can be g cells/g
Aerobic
Yeast grown aerobically and Anaerobically
glucose. The value is dependent on the units
Dry Biomass
chosen for the expression.
Anaerobic

Substrate consumed

Stoichiometric Equation for

Yield coefficient of biomass
Biomass Formation
• Determined experimentally
C6H12O6+
• The efficiency differs from organisms,
∑ i Cα i H β i Nγ i Oδ i + aO2 + bNH 3 → cCα ' H β ' Nγ 'Oδ ' + dCO2 + eH 2 O
growth conditions, and substrates

Inputs Outputs
Subsrates consumed → biomass produced+ (CO2 + water) produced

Can write four balance equations on elements carbon, nitrogen,

hydrogen, and oxygen
There are four unknown

Prof. Wei-Shou Hu 5
ChEn 5751 -Fall 2008

Examples of Different Forms of

Yield Coefficients
Units Example
Biomass Equation-undetermined
Yp/s Yield of product based Kg product/kg substrate, Yethanol/glucose
on substrate mole product/mole
Experimental observation substrate
Yx/s Yield of biomass based Kg biomass/kg substrate Ybiomass/glucose
on substrate

Yx/ATP Yield of biomass based Kg bimass/mole ATP Ybiomass/glucose

on ATP (i(i.e.,
e ATP
generated from
substrate consumed)
Yx/o Yield of biomass mased Kg biomass/kg oxygen, kg
on oxygen biomass/mole oxygen

The basic forms of yield coefficient Theoretical Yield of a Product

Δx • Knowing the theoretical maximum efficiency that
Yx / s = a raw material can be converted to a product is
Δs important in process design and innovation. The
return on the investment of improving the
Δt dx production yield is higher if the yield is
Yx / s = substantially below the theoretical maximum
x ds
concentration

than if the yield is already approaching the

}
dx/dt
xt 2 x : dry biomass maximum To estimate such a maximum one
maximum.
ds/dt
s Yx / s =
∫xt 1
d
dx s : substrate
needs to consider both material and energetic
st 2 aspects. Largely one needs to consult the
time
∫st 1
ds biochemical pathways of the organism.

Prof. Wei-Shou Hu 6
ChEn 5751 -Fall 2008

Products that are converted directly from the Consider carbon utilization (carbon source is the substrate)
raw materials in an energetically favorable Aerobic Process
(i) Without product (other than CO2, H2O) formation
fashion
ΔX
The producer confers a biochemical pathway to convert the raw materials to the product with a ΔS = ΔSx energy
net generation of chemical energy.
O2 Δ CO2 + Δ H2O
Example: Ethanol production by yeast yeast, one mole glucose can be converted to two moles of
ethanol, and generates two moles of ATP. The reaction in thus energetically feasible,
and the theoretical maximum is (2x46/180=) 0.51 Kg ethanol/Kg glucose. The true (ii) With product formation partition ΔS into ΔSx and ΔSp,
yield in process is lower because some glucose will be diverted to make biomass. The estimate ΔSp from the theoretical maximum
basic way of calculating the theoretical yield of such products is to tabulate the pathway
and examine its energetic conversion.
e.g. production of 20g glutamate and 10 g cells
Example #2: Production of glutamic acid from glucose and ammonia in corynebacterium
glutamican encompasses the following pathways. from 100 g glucose. Y p/s, max = 0.8
Glucose + 2ADP + 2Pi → 2 Pyruvate + 2 ATP
Pyruvate + NAD + → acetylCoA + CO2+NADH
Pyruvate + CO2 + ATP + H2Ooxaliacetate +ADP + Pi
ΔSp= 20/0.8 =25 (theoretical yield 0.8 g glutamate/g glucose)
Acetyl CoA + oxaliacetate + NAD+ → α-ketoglutarate + CO2 + NADH 100g
α-ketoglutarate + NADPH + NH3 →glutamate +NADP + + H2O ΔSx= 100-25=75g, Δχ=10
Net reaction is:
Glucose + NH3 + ADP + Pi+ NADPH + 2NAD + → glutamate +CO2 + ATP + 2NADH + NADP + so Yx/s= 10g cell/ 75 g glucose
Overall, it is energetically feasible since a net production of 1 ATP and 1 NADH. So, the
theoretical maximum conversion based on carbon is
5/6 (mole carbon in glutamate/mole carbon in glucose), or on a mass basis (143/180=) 0.79

Products which require energetic

Anaerobic process contribution from the catabolism of raw
Substrate consumed for biomass and organic products are coupled. materials.
Production of organic solvent is the way energy is generated. • Some products from biosynthetic pathways require the
catabolism of substrate to make the reaction
energetically
ti ll ffavorable.
bl IIn thi
this case, ffrom th
the
ΔX
biochemical pathways one derives the biosynthetic
ΔS=ΔSx energy reaction equation:
ΔCO2, ΔP (organic solvent) • The energy is supplied by catabolism of the substrate,
assuming catabolism is primarily derived from
substrate:
• The maximum yield is the combination of the two
reactions that gives a net production of ATP equivalent
Except in the case organic solvents are produced after cell growth.
The energy generated can be considered as used for maintenance.
is zero. For first appropriation, one can assume one
mole NADH is equivalent to three moles of ATP. In all
such calculation we assume that ∆Go for each reaction
is already negative.

Prof. Wei-Shou Hu 7