Quantification of Microbial Rates

Consumption or Production

Micro-organism

N-source Biomass
Substrate Product
O2 Heat

H2O H+ HCO3-

#1_Rates Marc Deront (Sirous Ebrahimi) 1

 Fundamentals of microbial growth stoichiometry

Composition of biomass
Anabolism
Maintenance
Catabolism
Coupled anabolism + Maintenance / catabolism
Microbial growth stoichiometry using conservation principles
Degree of reduction
Stoichiometric calculations in undefined chemical systems

#2_Stoichio Marc Deront (Sirous Ebrahimi) 2

 Biomass Composition
Molecules Elements
Proteins 30-60% C 40-50%
Carbohydrates 5-30% H 7-10%
Lipids 5-10% O 20-30%
DNA 1% N 5-10%
RNA 5-15% P 1- 3%
Ash (P, K+, Mg2+, etc) Ash 3-10%

Typical biomass composition formula

(CHON :49% 7% 33% 11%)
C1H1.8O0.5N0.2P0.01
MW =112+1.81+0.516+0.214 =24.6 MW =24.6 gr/C-mole

1 kg dry biomass contains e.g. 5% ash 950 gr organic matter

= 950 gr / (24.6 gr/C-mole)
= 38.62 C-mole biomass
#2_Stoichio Marc Deront (Sirous Ebrahimi) 3
 Anabolism
From C-source (e.g. glucose) + () of all reactions
Amino acids Proteins
Sugars Carbohydrates
Fatty acids Lipids
Nucleotides DNA RNA
______________________________
gives the Anabolic reaction

(..) C-source + (..) N-source + (..) P-source + (..) Energy

1 C1H1.8O0.5N0.2P0.01 + (..) H2O + (..) H+ + (..) HCO3-
Note: No electron
acceptor is required,
For Anabolism, Thermodynamic Energy is needed only energy!

for biomass growth and synthesis.

#2_Stoichio Marc Deront (Sirous Ebrahimi) 4

 Maintenance

Micro-organism

(Self) Restoration of : Pump out

Leakage processes Protein
Degradation processes

degraded Leakage of
ions, etc.
Thermodynamic Energy is required
for Maintenance. Energy

Membrane

#2_Stoichio Marc Deront (Sirous Ebrahimi) 5

 Catabolism
Catabolism generates the energy required for Anabolism & Maintenance
Catabolic reaction consists of a red/ox reaction, between 2 couples
electron donor couple
electron acceptor couple

1 glucose + (..) O2 (..) HCO3 +(..) H+ + (..) H2O

e_donor couple: glucose / HCO3(soluble CO2)
Oxidation: C Oxid. nb (0) (+4)
e_acceptor couple: O2 / H2O
C6H12O6 + 6 O2 6 CO2 + 6 H2O + Energy

Reduction: O Oxid. nb (0) (-2)

1 glucose (..) ethanol + (..) HCO3 +(..) H+ + (..) H2O

e_donor couple: glucose / HCO3
e_acceptor couple: HCO3 / ethanol Gibbs Energy (kJ) produced
GCat.reaction
#2_Stoichio Marc Deront (Sirous Ebrahimi) 6
 Anabolism/Catabolism coupling
Growth system definition

electron donor (D) Biomass (X)

+ C1H1.8O0.5N0.2
electron acceptor (A) Energetically
Coupled
Catabolism Anabolism
oxidized donor C-source
+ N-source
reduced acceptor
Maintenance
H2O
HCO3
H+

Catabolism provides energy to Anabolism and Maintenance

Note: C-source and electron donor are often the same compound (but not always)

#2_Stoichio Marc Deront (Sirous Ebrahimi) 7

 Coupled Anabolism/Catabolism
C-source (Anabolism) and electron-donor (Catabolism) are often the same (e.g. organic
substrate)

Only a fraction of the substrate, used as C-source, ends in biomass.

The other part is catabolized as electron donor to provide Energy for Anabolism and
Maintenance requirements

Concept of Yield produced biomass (X)

of Biomass on Substrate YSX YSX =
consumed substrate (S)

YSX is the result of anabolic/catabolic coupling

#2_Stoichio Marc Deront (Sirous Ebrahimi) 8

 Global growth equation
As Catabolism provides energy to Anabolism and Maintenance
YSX is the result of
anabolic/catabolic coupling
The global growth equation is the result of the catabolic reaction
(Energy) and anabolic reaction (Biomass).

1
Substrate + (..) electron acceptor + (..) N-source(e.g.NH4+)
YSX
+ (..) H2O + (..) HCO3 + (..) H+ + 1 C1H1.8O0.5N0.2
+ (..) oxidized substrate + (..) reduced acceptor

Note: A + B C + D A B + C +D (= 0)

#2_Stoichio Marc Deront (Sirous Ebrahimi) 9

 Stoichiometry of general growth equation
1
Substrate + (..) electron acceptor + (..) N-source(e.g.NH4+)
YSX
+ (..) H2O + (..) HCO3 + (..) H+ + 1 C1H1.8O0.5N0.2
+ (..) oxidized substrate + (..) reduced acceptor

N-source, and Biomass are always present with H2O, HCO3-, H+ are partner
elements (from medium)
Substrate often acts as electron-donor and C-source
Only substrate and electron-acceptor are case specific
Most often only 1 measured yield is available e.g. biomass yield on substrate YSX
YSX unit [C-mole of biomass /mole consumed substrat ]

What about the stoichiometric coefficients ?

#2_Stoichio Marc Deront (Sirous Ebrahimi) 10
 Stoichiometry of global growth equation
1
Substrate + (..) electron acceptor + (..) N-source(e.g.NH4+)
YSX
+ (..) H2O + (..) HCO3 + (..) H+ + 1 C1H1.8O0.5N0.2
+ (..) oxidized substrate + (..) reduced acceptor

By convention Biomass stoichiometric coefficient is always 1

Often catabolic reaction end products are H2O, CO2 which are taken into account
through partner elements
If YSX unit [C-mole of biomass / mole substrate] is available
Only 5 coefficients are required to determine the global growth equation
With Conservation principles - Balances
These coefficients can be determined over atomic element (C, H, O and N)
balances and charge (+, ) balance
#2_Stoichio Marc Deront (Sirous Ebrahimi) 11
 Example global growth stoichiometry
Aerobic growth on oxalate 1/3
Establish the global growth equation for aerobic growth of Pseudomonas oxalaticus on oxalate
(C2O42-, 88 g/mole), and NH4+ as N-source. The measured biomass yield is 0.0506 gram TSS
biomass per gram oxalate (C2O42 ). Biomass has 5% ash.
C-source: Oxalate
N-source for biomass: NH4+
Catabolism:
Electron donor couple: Oxalate / CO2
Electron acceptor couple: O2 / H2 O

1- Convert Biomass yield YSX 0.0506 [gTSS. gOxal.-1] in [C-mole X / mole oxalate]
Note: 1 C_mole X = 24.6 g Biomass [VSS]

0.95 gVSS 1 C-mole X 88 g oxal.

YSX = 0.0506 [gTSS/g oxal.]
gTSS 24.6 gVSS mole oxal.
C-mole X
YSX = 0.172 Observed growth yield of Biomass over substrate
mole oxal.

#2_Stoichio Marc Deront (Sirous Ebrahimi) 12

 Example global growth stoichiometry
Aerobic growth on oxalate 2/3
2- Set up the global stoichiometric
growth equation

f C2O42+ a NH4++ b H+ + c O2 + d H2O + 1 C1H1.8O0.5N0.2 + e HCO3-

a. Use YSX value -1 -1 mole oxal.
f = = = - 5.815
to calculate f coefficient: Ysx 0.172 C-mole X
b. How many unknown coeff. and how many conservation equations?
5 unknowns (a, b, c, d, e)
5 conservation balances (C, H, O, N, charge)
c. Set up the conservation balances C 2.f + 1 + e = 0
H 4.a + b +2.d + 1.8 + e = 0
O 4.f + 2.c + d + 0.5 + 3.e = 0
N a + 0.2 = 0
d. Solve for a, e, b, d, c (with f = -5.815) Charge -2.f +a + b e = 0

- 5.81 C2O42 - 0.2 NH4+ - 1.86 O2 - 0.8 H+ 5.41 H2O + 1 C1H1.8O0.5N0.2 + 10.63 HCO3-

#2_Stoichio Marc Deront (Sirous Ebrahimi) 13

 Example global growth stoichiometry
Aerobic growth on oxalate 3/3
Global Growth reaction

- 5.81 C2O42 - 0.2 NH4+ - 1.86 O2 - 0.8 H+ 5.41 H2O + 1 C1H1.8O0.5N0.2 + 10.63 HCO3-

Catabolic reaction 1 C2O42 0.5 O2 H2O + 2 HCO3 (for 1 mole oxalate)

to provide Energy
Catabolism of growth 3.71 C2O42 1.86 O2 3.71 H2O + 7.43 HCO3
consumes: 1.86 mole O2
and 3.71 mole Oxal.

Anabolic reaction = Global Growth reaction Catabolic reaction

For substrate : 5.81 Global_Growth - 3.71 Catabolisme 2.1 mole available/used for Anabolism
Note: No electron
2 + +
- 2.1 C2O4 - 0.2NH4 - 0.8 H -1.7 H2O + 1 C1H1.8O0.5N0.2 + 3.2 HCO3 acceptor is required,
only energy!

3.71 2.1
Catabolism fraction: = 64% Anabolism fraction: = 36%
5.81 5.81
#2_Stoichio Marc Deront (Sirous Ebrahimi) 14
 Example global growth stoichiometry
Anaerobic growth on glucose 1/3
Establish the global growth reaction for anaerobic growth on glucose C6H1206 with
ethanol C2H60 as product and NH4+ as N-source. The measured biomass yield is
YSX = 0.123 g VSS biomass / g glucose
C-source: Glucose
N-source for biomass: NH4+
Catabolism:
Electron donor couple: Glucose / CO2
Electron acceptor couple: CO2 / Ethanol

1- Convert Biomass yield YSX 0.123[gVSS. gGlucose.-1] in [C-mole X / mole Glucose]

Note: 1 mole Glucose = 180 g
1 C-mole X 180 g Glucose
YSX = 0.123 [gVSS/g glucose]
24.6 gVSS mole Glucose.
C-mole X
YSX = 0.9 Observed growth yield of Biomass over substrate
mole Glucose
#2_Stoichio Marc Deront (Sirous Ebrahimi) 15
 Example global growth stoichiometry
Anaerobic growth on glucose 2/3
2- Set up the global stoichiometric growth equation

f C6H12O6+ a NH4++ b H+ + c H2O + d C2H6O+ e HCO3- + 1 C1H1.8O0.5N0.2

-1 -1 mole Glucose
a. Use YSX value f = = = - 1.11
to calculate f coefficient: Ysx 0.9 C-mole X
b. How many unknown coeff. and how many conservation equations?
5 unknowns (a, b, c, d, e)
5 conservation balances (C, H, O, N, charge)
c. Set up the conservation balances C 6.f + 2.d + e + 1 = 0
H 12.f + 4.a + b + 2.c + 6.d + e + 1.8 = 0
O 6.f + c + d + 3.e + 0.5 = 0
N a + 0.2 = 0
d. Solve for a, e, b, d, c (with f = -1.11) Charge a + b e = 0

-1.11 C6H12O6 -0.2 NH4++ 2.12 H+ -1.47 H2O +1.87 C2H6O +1.92 HCO3- + 1 C1H1.8O0.5N0.2

#2_Stoichio Marc Deront (Sirous Ebrahimi) 16

 Example global growth stoichiometry
Anaerobic growth on glucose 3/3
Global Growth reaction
-1.11 C6H12O6 - 0.2 NH4++ 2.12 H+ -1.47 H2O +1.87 C2H6O +1.92 HCO3- + 1 C1H1.8O0.5N0.2

Catabolic reaction 1C6H12O6 + 2C2H6O + 2HCO3- + 2H+ 2H2O (for 1mole glucose)
to provide Energy
Catabolism of growth 0.93C6H12O6 +1.87C2H6O +1.87 HCO3- +1.87 H+ 1.87 H2O
consumes: 0.93 mole Glucose
produces: 1.87 mole Ethanol.

Anabolic reaction = Global Growth reaction Catabolic reaction

For substrate : 1.11 Global_Growth 0.93 Catabolisme 0.18 mole available/used for Anabolism

- 0.18 C6H12O6 - 0.2 NH4+ + 1 C1H1.8O0.5N0.2 + 0.05 HCO3- + 0.25 H+ +0.40 H2O

0.93 0.18
Catabolism fraction: = 84% Anabolism fraction: = 16%
1.11 1.11
#2_Stoichio Marc Deront (Sirous Ebrahimi) 17
 Stoichiometry of general growth equation
1
Substrate + (..) electron acceptor + (..) N-source(e.g.NH4+)
YSX
+ (..) H2O + (..) HCO3 + (..) H+ + 1 C1H1.8O0.5N0.2
+ (..) oxidized substrate + (..) reduced acceptor

Energetic coupling of Catabolism / Anabolism determine YSX Biomass yield

over substrate [C_mole X / mole S]
If YSX is known coefficients are given from C, H, O, N and Charges conservative
balances !

As Global Growth = Anabolism + Catabolism

Catabolic and Anabolic substrate fractions are given by the electron acceptor
stoichiometry of Global Growth Reaction
#2_Stoichio Marc Deront (Sirous Ebrahimi) 18