Microbiología y bioquímica del tratamiento

anaerobio de aguas residuales

Curso Internacional:
Diseño, operación y mantenimiento
de reactores anaerobios de flujo ascendente
Mérida, México, 10-13 noviembre 2008

Natural Anaerobic Environments

20% O2 AIR 20% O2

Interface
8 ppm O2

8 ppm O2
Water

0 ppm O2

- organic matter + organic matter

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Anaerobic biodegradation (digestion)

Basic generic equation that represents the complex

process of anaerobic digestion:

Anaerobic
microorganisms

Organic
pollution Biogas
CH4 + CO2
Methane + Carbon dioxide

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 History of anaerobic microbiology
• Volta (1776): discovery of CH4 in swamp-gas

• Early microbiologists: Béchamp (1868), Popoff (1875)

• Microbiology of methane bacteria:

– Söhngen (1906) Methanothrix soehngenii (in defined mix) (renamed Methanosaeta
soehngenii, Patel)
– Schnellen (1947): first pure cultures (Methanosarcina barkeri, Methanobacterium
formicium)

• Bryant (1967) very important discovery:

– Methanobacterium Omelianski (fermenting EtOH) exist of 2 bacteria !!
– EtOH =>Acetate + H2 (not directly to CH4 !!)

• Description of the new kingdom of Archeabacteria

– methane bacteria
– sulphate reducera
– halophilic bacteria,etc.

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Classification of methanogens
living organisms

eukaryotes prokaryotes

archaebacteria eubacteria

methanogens extreme halophiles thermoacidophiles

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Carbon cycle
(CH2O)n
Photosynthesis: Respiration:
Algae Organic compounds plants, animals,
Green -plants microorganisms
Cyanobacteria

Methane-oxidizing
bacteria
Aerobic
CH4 (CO2)
Anaerobic
Methanogenic
bacteria Phototrophic sedimentation
bacteria Anaerobic respiration
(Methyl
compounds) Fermentation

Organic compounds

(CH2O)n

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 Comparison Aerobic - Anaerobic

Characteristic Aerobic Anaerobic

Reaction C6H12O6 + 6O2 → 6CO2 + 6H2O C6H12O6 → 3CO2 + 3CH4

Energy release ΔG°’ = -2840 kJ/mol glucose ΔG°’ = -393 kJ/mol glucose

Carbon balance 50% → CO2 95% → CH4 + CO2 (= biogas)

50% → biomassa 5% → biomassa

Energy balance 60% → biomassa 90% retained in CH4

40% → heat production 5% → biomassa
5% → heat production
Biomass production Fast growth of biomass, Slow growth of biomass
Resulting in a sewage sludge problem

Energy input for Yes No

aeration

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Anaerobic versus Aerobic treatment

Heat loss
10
0k
gC
OD
Influent
+
AEROBIC
Aeration
(100 kWh) Effluent,
2-10 kg COD

Sludge, 30-60 kg
Biogas
35 m3 (285 kWh)

Influent

100 kg COD ANAEROBIC

Effluent,
10-20 kg COD

Sludge, 5 kg

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Anaerobic Conversion of Organic Matter

Organic Polymers
proteins carbohydrates lipids
Hydrolysis
Mono- and oligomers
amino acids, sugars, fatty acids
Acidogenesis

Volatile Fatty Acids

Lactate
Ethanol
Acetogenesis

H2 / CO2 Acetate

Methanogenesis
CH4 / CO2

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 Anaerobic Conversion of Organic Matter

The anaerobic biodegradation process can be divided in

seven sub-processes:

1. Hydrolysis of proteins, carbohydrates, and lipids

2. Fermentation of aminoacids and sugars

3. Anaerobic oxidation of long chain fatty acids and alcohols

4. Anaerobic oxidation of intermediates (fatty acids, no acetate)

5. Production of acetate from CO2 and H2 (homoacetogenesis)

6. Conversion of acetate to CH4 (acetoclastic methanogenesis)

7. Production of CH4 from the reduction of CO2 and H2

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Hydrolysis

• Polymeric compounds are converted into their monomer

or dimeric components.

• The hydrolysis is performed by extra-cellular enzymes.

• Slow process (generally rate limiting): dS/dt = -Kh•S

• Retention time and particle size are rate determining

parameters

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Hydrolysis

• Optimum pH = 6

• Cellulose/hemicellulose degradation depends on lignin

fraction

• Hydrolysis of fats hardly proceeds < 15-20°C (rate limiting

step)

• (Product) inhibition by:

– LCFA
– NH3
– amino acids
– H2 ?

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 Hydrolytic enzymes

Long Chain Fatty Acids (LCFA)

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Hydrolysis - Enzymes

• Hydrolysis of suspended solids is a surface-related

process

• The more specific surface, the faster the process

• Individual enzymes work at constant rate (constant T, pH)

• Number of enzymes determines the total rate

From Dr. Wendy Sanders

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Hydrolysis – Surface related process

Enzymes

Particle of
substrate

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 Hydrolysis – Surface related process

More enzymes
“attack” the
substrate

Particle
breakdown
or “lysis”
Rate
increases

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Hydrolysis – Mechanism 1

Enzyme

Bacteria

Substrate

Product
Loss of
Overproduction of products
enzymes

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Hydrolysis – Mechanism 2

Bacteria

Product

Substrate

Enzyme

Bacteria approach the

substrate before
producing enzymes

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 Hydrolysis of carbohydrates

• Cellulose is hydrolysed by cellulase (mixture of exo-

glucanases, endo-glucanases and cellobiases)

• The hydrolysis of starch is performed by a mixture of

amylases that is able to hydrolyse the α -1,4 bonds and
the α -1,6 bonds of the amylose and amylopectin.

From Dr. Wendy Sanders

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Hydrolysis of proteins

• Proteinases (Peptidases + Proteases)

• Protein → polypeptides → peptides → amino acids

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Hydrolysis of lipids

• Most lipids in waste(water) are present as

triacylglycerides

From Dr. Wendy Sanders

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 Bio-degradation of cellulitic matter versus lignin content

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Digestible part of wood

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Acidogenesis / Fermentation - Sugars

• Release of protons (H+) and reaction products (proton acceptors)

• H2 formation (catalyzed by the enzyme hydrogenase)

• Performed by a very large group of bacteria (about 1% of all bacteria

facultative fermenters)

C12H22O11 + 9 H2O → 4 CH3COO- + 4 HCO3- + 8 H+ + 8 H2

C12H22O11 + 5 H2O → 2 CH3CH2CH2COO- + 4 HCO3- + 6 H+ + 4 H2

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 Acidogenesis / Fermentation - Sugars

End products depend on circumstances, e.g.:

• Glucose fermentation in a two-step system

– more reduced products like ethanol, lactate, propionate, butyrate,
CO2 and H2

• Glucose fermentation in a one-step system

– acetate, H2 and CO2

• Production of acids proceeds up to pH = 4

(product inhibition)

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Acidogenesis / Fermentation - Proteins

• Organically bound N (amino acids) is released as NH4+

(stickland reaction: oxidation-reduction)

Alanine: CH3CHNH2COO- + 3 H2O → CH3COO- + HCO3- + NH4+ + 2 H2

Glycine: 2 CH2NH2COO- + 2 H2 → 2 CH3COO- + 2 NH3

alanine + glycine + 3 H2O → 3 acetate + 2 NH3 + NH4+ + HCO3-

(2 NH3 + 2 H2O + 2 CO2 → 2 NH4+ + 2 HCO3-)

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Acidogenesis / Fermentation – Long Chain Fatty Acids

• Anaerobic degradation of LCFA proceeds via β-oxidation

CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-COO-

• Palmitic acid:
CH3-(CH2)14-COO- + 14 H2O → 8 CH3COO- + 7 H+ + 14 H2

• With uneven numbers: acetate + propionate are formed:

CH3-(CH2)14-CH2COO- + 14 H2O → 7 CH3COO- + CH3CH2COO- 7 H+
+ 14 H2

• Unsaturated LCFA are firstly hydrogenated before degradation

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 Gibb's Free energy

[C]c ⋅ [D]d
aA + bB ⇔ cC + dD ΔG' = ΔG'0 +RT ln
[ A ]a ⋅ [B]b

• Δ G‘ = Actual Gibb's free energy change [kJ/mole]

• Δ G‘0 = Standard Gibb’s free energy change [kJ/mole]

under standard conditions (pH = 7, T = 25°C, p = 1 atm., the activity of all
compounds present in solution is 1 mole/kg

• R=gas constant (8.28 J)

• T=absolute temperature [K]

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Acetogenesis (Acetate formation)

• Conversion of fermentation products into acetic acid, CO2, and H2

• Mainly formation of propionic acid, butyric acid and ethanol

propionate- + 3H2O → acetate- + HCO3- + H+ + 3H2 Δ G0’ = + 76.1 kJ/mole

butyrate- + 2H2O → 2 acetate- + H+ + 2H2 Δ G0’ = + 48.1 kJ/mole

ethanol + 2H2O → acetate-

+ H+
+ 2H2 Δ G0’ = + 9.6 kJ/mole
_______________________________________________________________________________________________

4 H2 + CO2 → CH4 + 2H2O Δ G0’ = -138.9 kJ/mole

Need for syntrophic associations !!!

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Methanogenesis

• Acetoclastic methanogenesis:

CH3COOH → CH4 + CO2

• Autotrophic methanogenesis:

CO2 + H2 → CH4 + CO2

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 Methanogenesis
→ Substrates
ΔG0 (kJ/mole CH4)
4H2 + CO2 => CH4 + 2H2O -130.4
4HCOOH => CH4 + 3CO2 + 2H2O -119.5
4CO + 2H2O => CH4 + 3CO2 -185.5
4CH3OH => 3CH4 + CO2 + 2H2O -103.0
CH3OH + H2 => CH4 + H2O -112.5
4CH3NH3 + 2 H2O => 3CH4 + CO2 + 4NH4+ - 74.0
+
2(CH3) 2NH2 + 2H2O => 9CH4 + 3CO2 + 4NH4 - 74.0
CH3COOH => CH4 + CO2 - 32.5
Most important substrates: hydrogen and acetate
furthermore: formate, carbon monoxyde, methanol and methylamines

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Impact of P(H2) on thermodynamics

[C ]c ⋅ [ D ]d
ΔG ' = ΔG '0 + RT ln
ΔG’ (kJ/mole) [ A]a ⋅ [ B ]b
-100
propionate- + 3H2O → acetate- + HCO3- + H+ + 3H2

-50
Reaction butyrate- + 2H2O → 2 acetate- + H+ + 2H2
possible
Methanogenic niche
0
Reaction
impossible
4 H2 + CO2 → CH4 + 2H2O
50

2 4 6 8
-log PH2 (atm)
High H2 Low H2
pressure pressure
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Parallel processes

• Sulphate reduction:

Organic matter + SO42- → HS- + H2O + 3HCO3-

8H2 + 2SO42- → H2S + HS- + 5H2O + 3OH-

8H + SO42- Æ H2S + 2H2O + 2OH-

• Denitrification:

Organic matter + NO3- → N2 + H2O

10H + 2H+ + 2NO3- Æ N2 + 6H2O

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 Take-home message (1)

• Anaerobic microbial conversion differs from aerobic

• Anaerobic digestion is a complex process

• Ultimate COD removal via production of CH4

• Anaerobic bacteria have a narrow substrate spectrum:

complex consortia are needed for complete COD removal

• Environmental factors affect the process

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Take-home messages (2)

• Anaerobic bacteria form bacterial aggregates (anaerobic

granular sludge).

• Different bacterial groups are involved

• Granulation was a crucial discovery for the development

of anaerobic systems

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