ANAEROBIC

DIGESTION
FUNDAMENTALS I

Dr. CRISTINA CAVINATO

LECTURE 1

Summer School on Biogas Technology Renewable Energy

Production and Environmental Benefit, 12-17 August 2013
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 INTRODUCTION: a brief history

Jan Baptist Van Helmont

(Brussels, 12 January 1580 – 30 December 1644)
He recorded that decaying organic material produced flammable
gases.

In November 1776, Alessandro Volta performed his classic

experiment disturbing the sediment of a shallow lake,
collecting the gas and showing that this gas was flammable.

He resolved that there was a direct connection between how

much organic material was used and how much gas the
material produced.

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION: a brief history

John Dalton and Humphrey Davy

during 1804–1808

They established that this combustible gas

was methane

Antoine Bèchamp, in 1868, reported that the formation of methane

during the decomposition of organic matter was through a
microbiological process.

The development of microbiology as a science led to research by A.M. Buswell and others in
the 1930s to identify anaerobic bacteria and the conditions that promote methane
production.

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION

•First plant built in 1859 at Leper colony in India

•Used in 1897 to power streetlights in Britain

•Thousands of ‘backyard’ digesters throughout China,

India other Asian countries

•Most sewage treatment works in Europe stabilize their

sludge using AD

•Increasingly being used in Europe to manage municipal

waste and to create heat and power

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 INTRODUCTION

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION

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 INTRODUCTION

Anaerobic Digestion consists of a

series of bacterial events that
convert organic compounds to
methane, carbon dioxide, and new
bacterial cells. These events are
commonly considered as a three-
stage process.

(Ahring, 2003)

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION

Different consortia of microorganisms with different function in the anaerobic digestion

process are needed.

Three major groups of microorganisms have been identified with different functions in
the overall degradation process:

The hydrolyzing and fermenting microorganisms

The obligate hydrogen-producing acetogenic bacteria

Two groups of methanogenic Archaea

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION

The hydrolyzing and fermenting microorganisms

Are responsible for the initial

attack on polymers and
monomers found in the waste
material and produce mainly
acetate and hydrogen, but also
varying amounts of volatile fatty
acids (VFA) such as propionate
and butyrate as well as some
alcohols.

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION

The obligate hydrogen-producing

acetogenic bacteria

convert propionate
and butyrate into acetate and
hydrogen.

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION

Two groups of methanogenic

Archaea

produce methane from

acetate or hydrogen,
respectively.

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ANAEROBIC DIGESTION FUNDAMENTALS I
 INTRODUCTION

AD is a ‘series’ process,
disruption of one part of
the process disrupts the
whole process.

Process rate proceeds at

the rate of the slowest
step.

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 MICROBIOLOGY OF AD

1 Hydrolytic bacterIa

2 Fermentative bacteria

3 Acetogenic becteria

4 Methanogenic archaea

5 Sulphate reducing bacteria

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 MICROBIOLOGY OF AD

Enzymes used in AD

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Hydrolytic organisms

- Act by secreting extra cellular enzymes which break the bonds of polymeric
substances producing shorter chain compounds

- Attach the surface of the substrate using a secreted ‘sticky’ extracellular polymeric
substances (EPS)

- Fast growth rates but hydrolysis can be rate limiting in highly cellulosic substances

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 MICROBIOLOGY OF AD

Hydrolytic organisms
- The fastest stage in anaerobic systems

- Rarely a rate limiting step

- Biological reaction can be:

- Intracellular, performed by intracellular enzymes

- Extracellular, performed by extracellular enzymes

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REACTIONS

- Carbohydrates sugars, alchools

- Cellulose glucose, cellobiose

- Lignin degraded very slowly

- Proteins Aminoacids, peptides

- Fats Fatty acids, glycerol

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Hydrolytic enzyme

- Example: Fat hydrolysis by lipase

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Hydrolysis of cellulose

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Hydrolysis of proteins

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Lignine The three common monolignols:

paracoumaryl alcohol (1), coniferyl alcohol (2)
and sinapyl alcohol (3)

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 MICROBIOLOGY OF AD

Fermentative bacteria (acidogenic microorganisms)

REACTIONS:

- Sugars Fatty Acids (succinate, acetate,

proprionate, lactate, formate), carbon
dioxide, hydrogen

- Amino Acids Fatty Acids,

ammonia, sulphides, carbon
dioxide, hydrogen

- Glycerol acetate, cabon dioxide

- Alchools Fatty Acids, carbon dioxide

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Fermentative bacteria (acidogenic microorganisms)

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Type of fermentation

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Acetogenic microorganism

- Acetogenic bacteria produce acetic acid, hydrogen and carbon dioxide from
fermentation products

- Fall into two main groups:

- Hydrogen producing acetogens

- Homoacetogens

- Slow growth rates

- Sensitive to physical and chemical conditions (temperature, pH, hydrogen partial

pressure)

- Work in synergy with methanogenic microorganisms-interspecies hydrogen transfer

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 MICROBIOLOGY OF AD

Hydrogen producing acetogens

- Butyrate

CH3CH2CH2COOH + 4H2O  CH3COOH + 2CO2 + 6H2

- Proprionate

CH3CH2COOH + 2H2O  CH3COOH + CO2 + 3H2

- Propanol

CH3CH2CH2OH + 3H2O  CH3COOH + CO2 + 5H2

Homoacetogens

4H2 + 2CO2  CH3COOH + 2H2O

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 MICROBIOLOGY OF AD

Methanogens
- Present in natural habitats (sediments, digestive systems) and are
responsible for the production of methane from a wide variety of
methylated compounds

- Slow growth rates

- Sensitive to physical conditions (pH, temperature) and can be

inhibited by many compounds

- Main methanogens in anaerobic digesters fall into two main groups

- Acetoclastic-acetate degrading

- Hydrogenotrophic-hydrogen utilising

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Methanogens

Acetoclastic methanogens

CH3COOH  CH4 + CO2

- 2/3 of methane produced by this route
- Slowest growth rate and most sensitive organisms

Hydrogenotrophic methanogens

4H2 + CO2  CH4 + 2H2O

- 1/3 of methane produced by this route

- Higher growth rate and less sensitive organisms

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Acetoclastic methanogens

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Carbon flow in anaerobic environments with active methanogens

Only between 20 and 30% of the carbon is transformed into intermediary products
before these are metabolized to methane and carbon dioxide

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Carbon flow in anaerobic environments without active methanogens

(basic mechanism of a two phase approach to produce VFA and Hydrogen)

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

The rate and efficiency of the anaerobic digestion process is controlled by:

· The type of waste being digested,

· Process temperature,
· The presence of toxic materials,
· The pH and alkalinity,
· The hydraulic retention time,
· The rate of digester loading,

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

- Competition between different organisms for the same substrate

- Synergies between different groups of organisms

- Physical/Chemicals factors:

- feedstock composition
- feedstock structure
- pH
- pH equilibriums
- nutrients (trace elements)
- inhibition (VFA, ammonia)
- liquid/gas transfer etc. (H2 partial pressure)

- Chemical reactions are catalyzed by biological enzymes (biochemical

process).
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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

Each group of organisms have an pH optima for maximum rate of

reaction

- methanogens: pH7-8 optimal, pH 6.5-8.5 operational

- fermentation: pH 5-7 optimal
- hydrolysis: pH 5-7 optimal

Optimal pH gives highest rate related

Deviation from optimum value could be:

- Introduced with the influent

- Consequence by excess production and accumulation of acidic or basic
conversion products such as VFA or Ammonia.

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ANAEROBIC DIGESTION FUNDAMENTALS I
 MICROBIOLOGY OF AD

FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

Equilibria

Ionic equilibriums can have a large effect on the AD process since undissociated (non-
ionic) forms can pas through cell membranes and cause inhibition

CH4
+
CO2 <-> H2CO3 <-> H+ + HCO3- <-> H+ + CO3--
+
Organic substrate +H2O NH3 <-> NH4+ + OH-
+
R COOH <-> H+ + RCOO-
+
H2S <-> H+ + HS- <-> H+ + S--

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ANAEROBIC DIGESTION FUNDAMENTALS I
 MICROBIOLOGY OF AD

FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

Eg Ammonia NH3 + H2O  NH4+ + OH-

Free ammonia (non ionic) is more

inhibitive to the AD process than
the ammonium ion.

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 MICROBIOLOGY OF AD

FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

Acidic   Alkaline Relationship

Organic substrate

Fatty acid CO2, H2O, NH3, H2S

CH3COOH <-> CH3COO- + H+
pKa= 4.76

H2CO3 <-> NH4+ + HCO3-

Bicarbonate alkalinity

Acetic acid and hydrogen sulphide are both more inhibitive at lower pH since the
non ionic forms is prevalent.

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

Acetic acid could be inhibitive at lower pH since the non ionic forms is prevalent.

CH3COOH <-> CH3COO- + H+

pKa= 4.76

Non ionic form of acetate is able to pass through the membrane. At low pH (<5)
the non ionic form is prevalent. This could cause an overload of acetic acid inside
the cell.

At higher pH value (>8) acetic acid is in his ionic form, and it is unable to pass the
membrane causing an accumulation outside the cell.

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 MICROBIOLOGY OF AD

FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

• Is mainly present in protein (also Urea)

• In the AD nitrogen is converted to ammonia

• Ammonia is known to be beneficial at low concentrations ( about 200 mg/l)

but can be inhibitive at high concentration:

- complex inhibition mechanism

- ammonia is antagonistic/synergistic with other substances

• Anaerobic digesters can become acclimatized to high ammonia

concentrations:

- due to a shift in the internal mechanism of methanogens?

- a shift in the dominant species in the digester?

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

• Hydrogen producers: acidogens and acetogens, thermodynamics

unfavourable at high hydrogen concentration.

• Hydrogen consumers: hydrogenotrophic methanogens and homoacetogens,

require hydrogen as substrate to produce methane and acetate.

• Hydrogen plays an important intermediary role during acetogenesis, as the

reaction will only occur if the hydrogen partial pressure is low enough to
thermodynamically allow the conversion of all the acids. Such lowering of the
partial pressure is carried out by hydrogen scavenging bacteria, thus the
hydrogen concentration of a digester is an indicator of its health.

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

• Interspecies hydrogen transfer: a symbiotic

relationship between methanogenic and
acetogenic/acidogenic anaerobic
microorganisms

• Hydrogen consumers are constantly supplied

with substrate

• Hydrogen producers have hydrogen removed

from solution allowing them to continue to
metabolize their substrates

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

• Sulphur is present in all biological materials especially those containing high

concentration of protein.

• Sulfates present in the feed material are reduced to hydrogen sulphide by sulfate
reducing bacteria (SRB)

• SRB compete with methanogenic organisms for the same substrates in order to
reduce sulphur:

- H2 + SO4 2-  H2S + H2O

- CH3COOH + SO4 2-  H2S + CO2 + H2O

• SRB reduce the total biogas production from AD

• Sulphid is toxic to many organisms (200-1500 mg/l)

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

Competition between SRB and methanogens

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

• The presence of sulfates causes numerous disadvantages to the commercial

anaerobic digestion process

• Quality of biogas is lower due to hydrogen sulphide (H2S) content:

- hydrogen sulphide (H2S) has a strong unpleasant odour

- hydrogen sulphide is corrosive to machinery

• Biogas must me cleaned before use

• Metals can be precipitated from

digestate as sulphides
(leading to nutrient deficiencies)
causing failure of the process

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FACTORS THAT CAN AFFECT THIS COMPLEX SYSTEM:

• AD has low nutrient requirements

• Nutrient are often inhibitive in high concentrations
• Usually feedstock is nutrient sufficient
• In nutrient limited systems supplementation can have a positive impact
• Nutrient limitations can be caused by precipitation of sulphide.
Element Enzyme Anaerobic microorganism
Selenium Formate hydrognase Acetogenic bacteria
Glycine reductase Several clostridia
Hydrogenase Methanococcus vanielii
Nicotin acid hydroxylase Clostridium barkeri
Xanthine dehydrogenase Some clostridia
Tungsten Formate dehydrogenase Acetogenic bacteria
Nickel Carbon monoxide dehydrogenase Some clostridia

hydrogenase Methanobacterium, desulfovibrio gigas

Methyl reductase Methanogenic bacteria

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 CONCLUSIONS

When biogas yields of your AD reactor in not so satisfactory,

Take care of who is working together to produce it and try to understand

what is the inhibition factor that make them

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