Reviews in Environmental Science and Bio/Technology 3: 117129, 2004.

2004 Kluwer Academic Publishers. Printed in the Netherlands.

117

Assessment of the anaerobic biodegradability of macropollutants

Irini Angelidaki1; * & Wendy Sanders2
1

Environment & Recourses DTU, Building 115, 2800 Lyngby, Technical University of Denmark; 2 Wageningen
Agricultural University, Subdepartment of Environmental Technology, P.O. Box 8129, 6700 EV Wageningen
(*author for correspondence, phone: +45-45251429; fax: +45-45932850; e-mail: ria@er.dtu.dk)

Key words: anaerobic, biodegradation assays, hydrolysis, macropollutants, methane potential

Abstract
A variety of test procedures for determination of anaerobic biodegradability have been reported. This
paper reviews the methods developed for determination of anaerobic biodegradability of macro-pollutants.
Main focus is paid to the nal mineralization of organic compounds and the methane potential of compounds. Hydrolysis of complex substrates is also discussed. Furthermore, factors important for anaerobic
biodegradation are shortly discussed.

1. Introduction
Anaerobic degradation or digestion can be
dened as a biological conversion process without
external electron acceptor such as oxygen as in
aerobic processes or nitrate/sulphate as in anoxic
processes. In the anaerobic process organic carbon
is converted by subsequent oxidations and reductions to its most oxidized state (CO2 ), and its most
reduced state (CH4 ). A wide range of microorganisms catalyze the process in the absence of
oxygen (McInerney et al. 1980). The main products of the process are carbon dioxide and methane, but minor quantities (usually less than 1% of
the total gas volume) of other products such as
nitrogen, nitrogen oxides, hydrogen, ammonia,
hydrogen sulphide and other volatile compounds
are also generated (McInerney et al. 1980). The
mixture of gaseous products is termed biogas and
the anaerobic degradation process is often also
termed the biogas process. As the result of the
removal of carbon, organic bound non-carbon
compounds are released to their soluble inorganic
form.
With the increasing application of the anaerobic digestion process there is an urgent need to
review methods for estimation of the biodegradability and methane potential of wastes used in for
anaerobic digestion.

A substance or a compound is biodegradable

if it can be decomposed by the action of microorganisms. Microorganisms can use this compound as energy source and as carbon
source. A compound can be mineralized i.e. converted besides to new microbial biomass to the
end carbon products i.e. to carbon dioxide
and methane. In some cases complete mineralization is not seen and the compound can be
involved in microbial metabolism with only transformation (also called primary biodegradation)
of the compound to intermediates, but without total conversion to end products. An
organic compound can be processed during anaerobic degradation through metabolism (the compound is supplying energy and
carbon source for the micro-organisms) or by cometabolism (the compound is converted only at
the presence of another, usually easily degradable organic compound such as glucose, ethanol
etc.), that is supplying micro-organisms with
energy and carbon for their cell mass built up).
In this paper biodegradability with reference
to macro-pollutants only, will be treated,
since
micro-pollutants
do
not
generate
enough biogas in order to determine
biodegradation through biogas production
and there are also uncertainties related to
adsorption.

118
2. Some general considerations concerning
/inuencing anaerobic biodegradation
There are several physical, chemical and physiological factors in the environment that aect biodegradation of organic compounds, such as
availability of the compounds, the availability of
electron donors and acceptors, oxygen concentration, temperature, pH, moisture, salinity, sorption
of chemicals to particulate material, concentration
of the chemicals. Dierent factors might have
dierent inuence according the specic characteristics of the compound.
2.1. Redox conditions
One of the major factors governing biodegradation is the nature and availability of electron acceptors. From a thermodynamic point of view,
oxygen is the most favourable electron acceptor.
Under anoxic conditions, the biodegradation will
often depend on the availability of electron acceptors, such as nitrate, iron, sulphate or carbon
dioxide. In truly anaerobic conditions there is absence of inorganic electron acceptors other than
CO2 , and small amount of energy is gained by
consecutive oxidations reductions of the organic
matter, or CO2 is used as electron acceptor. The
energy released in a redox process as a result of
electron transfer from one compound to another,
is used for the maintenance and growth of the
microbial population in the environment.
2.2. Temperature
Temperature aects survival and growth of
microorganisms and it also inuences their metabolic activities. In general, higher temperatures
that do not kill microorganisms result in higher
metabolic activities. Temperature is the most
important variable in controlling the rate of
microbial metabolism in anaerobic environments
(Westermann et al. 1989).
Anaerobic digestion is applied under three
dierent temperature ranges, i.e. the mesophilic
(2540 C), the thermophilic (4560 C) and the
psychrophilic (<20 C). In general, the overall
process rates double for every 1 C increase in
operating temperature up to an optimum temperature 60 C (Harmon et al. 1993). The optimum
temperature for growth usually is close to the

upper limit of its range. A further increase of the

temperature beyond its optimum temperature can
result in a sharp decrease of the growth rate (Chen
& Hashimoto 1978).
The eect of temperature on anaerobic degradation is theoretically only inuencing the degradation rates and not the ultimate biodegradability
of a component. However, the biodegradation
rates can be so slow that the achievable biogas
potential appears to be lower than at optimal
conditions. Kaparaju and Rintala (2003) have
found methane potential to be 2.4, 12.6 and 15.7%,
at 5 C and 10 and 15 C respectively, compared
to the methane potential achieved at 35 C, after
345-days digestion of manure.
The eect of temperature on the achievable
methane potential of manure was investigated by
Hashimoto et al. (1981). It was found that temperatures in the range between 30 and 60 C (at
5 C intervals) did not aect the achievable
methane potential of manure. At higher temperatures (65 C) the experimentally determined
methane potential was lower. The lower potential
was attributed to unstable fermentation rather
than decreased substrate availability at that temperature.
2.3. Hydrolysis
An important step of the anaerobic biodegradation process is the hydrolysis of the complex organic matter. During the anaerobic digestion of
complex organic matter the hydrolysis is the rst
and often the rate-limiting step. The hydrolysis can
be dened as the breakdown of organic substrate
into smaller products that can subsequently be
taken up and degraded by bacteria (Morgenroth
et al. 2002). Substrate for hydrolysis can be directly present in the waste or can be formed by
microbial activity such as internal storage products, or bacterial biomass.
During hydrolysis of macro-pollutants polymers such as lipids, protein, carbohydrates are
depolymerized to glycerol and long chain fatty
acids, to amino acids and to oligo- and monosaccharides for lipids, proteins and carbohydrates
respectively.
2.3.1. Hydrolysis mechanism
Hydrolysis takes place extracellular by enzymes
excreted by the biomass. Three main mechanisms

119
exist for the release of enzymes and the subsequent
hydrolysis of the complex substrate during anaerobic digestion (Batstone 2000).
1. The organism secretes enzymes to the bulk liquid, where they will either adsorb to a particle
or react with a soluble substrate (Jain et al.
1992).
2. The organism attaches to the particle, secretes
enzymes into the vicinity of the particle and next
the organism will benet from the released dissolved substrates (Vavilin et al. 1996).
3. The organism has an attached enzyme that may
also act as a transport receptor to the interior of
the cell. This method requires the organism to
absorb onto the surface of the particle.
Research on aerobic sludge has indicated that
most of the extracellular enzymes are associated to
the biomass (Morgenroth et al. 2002). Only a few
studies have been done on anaerobic sludge but
also in this case the enzymes seem to be cell
associated (Hobson 1987; Philip et al. 1993).
Mechanisms 2 and 3 seem therefore more likely
than mechanism 1. This indicates that good contact between biomass and substrate is a prerequisite to the hydrolysis.
2.3.2. Aspects related to the enzymatic
depolymeriation
Hydrolytic enzymes can be endo-enzymes, which
prefer to cut the bonds towards the middle of the
molecule, or exo-enzymes, which prefer to cut the
bonds near to the edges of the macromolecule. The
enzyme substrate specic activity is thought to
follow MichaelisMenten kinetics.
The overall eect of the digestion temperature
on the hydrolysis rate originates from the combined temperature eect on the enzyme kinetics,
bacterial growth and solubility of the substrate.
For instance, the GibbsHelmoltz equation
gives the relation between temperature and pKa of
the enzymes. The change in charge will have consequences for the structure of the enzyme resulting
in changes of catalytic eciency, amount of active
enzyme and binding of the substrate (Chaplin &
Bucke 1990). In general, the rates of reactions vary
with temperature in accordance with the Arrhenius
equation. Veeken and Hamelers (1999) digested
several biowaste components, such as orange
peels, bark, leaf and grass at 20, 30 and 40 C.
They found a good Arrhenius relation between the

rst order hydrolysis constant and the digestion

temperature (R2 0.9840.999) with an average
standard free energy of activation of 46
14 kJ/
mol.
The eect of the pH on the hydrolysis is complicated. The net eect of pH on the hydrolysis
rate is specied by the pH optima of the dierent
enzymes present in the digester and the eect of
pH on the charge/solubility of the substrate. The
latter especially applies to the digestion of substrates that contain proteins.
The production and activity of enzymes can be
inhibited by hydrolysis products. The production
of proteinases by microbes can be inhibited by
components, such as amino acids, high inorganic
phosphate levels and glucose. With respect to the
production of cellulases similar ndings were made
as for proteinases (Zeeman 1991). The production
of cellulases is inhibited by high glucose levels but is
stimulated by low glucose levels. However, no effects of the concentration of free amino acid on the
production of cellulases were reported (Glenn
1976). Also NH
4 can inhibit the hydrolysis of cellulose (Zeeman 1991). From the results it was
however unclear if the inhibition was directly from
the free ammonia or indirectly from the resulting
high volatile fatty acid levels. El-Mashad (2003)
studied the eect of dierent ammonia concentrations (range 13.7 g NH
4 N/l) on the hydrolysis of
liquid cattle manure at 50 and 60 C in batch
reactors. The inoculum used for the experiments
was digested cow manure adapted to an ammonia
concentration of 1.1 g NH
4 N/l. At both temperatures a negative linear relation between the rst
order hydrolysis constant and both total and free
ammonia concentrations was established.
Accumulation of long chain fatty acids at the
lipid-water interface causes inhibition of the lipase
activity by physicalchemical changes of the
interface, e.g. the surface tension (Verger 1980;
Angelidaki & Ahring 1992).
2.3.3. Aspects related to the physical state
and structure of the substrate
An important factor for the hydrolysis is the
physical state and structure of the substrate and its
accessibility for hydrolytic enzymes. It is therefore
obvious that the hydrolysis rate of particulate
substrates is lower than that of dissolved polymers
as with the former only part of the substrate is
accessible to the enzymes. Macro-pollutants in

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Table 1. Surface related hydrolysis rate assessed for dierent substrates
Substrate

Hydrolysis rate
(mg COD/cm2/day)

Inoculum

Temperature (C)

Reactor

Reference

Starch
Rice
Hay
Cellulose

1.0
1.1
0.01
0.33

Granular sludge from potato factory

Not indicated
Not indicated
MSW leachate

35
35
35
38

Batch
Batch
Batch
CSTR

Sanders et al. (2000)

Palmowski et al. (2001)
Palmowski et al. (2001)
Song (2003)

waste (water) can be found in dierent physical

states, in particles, dissolved or emulsied. Particles are the most commonly found, for example 60
90% of the total organic load in domestic sewage
consists of particles (Elmitwalli 2000).
Chyi and Dague (1994), Hills and Nagano
(1984) and Hobson (1987) indicated that the
hydrolysis of complex substrates is a surface related process and a formation of a biolm on the
particle surfaces is necessary for the complete
anaerobic digestion of organic matter (Song 2003).
The rate of microbial attachment to the substrate
depends on the type of micro-organisms (Song
2003). At full microbial colonization the hydrolysis
rate of particulate substrates is constant on a per
unit surface area basis, although the actual value
seems to depend on the type of inoculum (Song
2003). In Table 1 values for the surface hydrolysis
rate are presented.
The accessibility of a substrate can also be altered by formation of complexes with other compounds. For example, cellulose itself is relatively
easily degradable, but once it is incorporated in a
lignocellulosic complex, the biodegradability of
the cellulose is distinctly lower (Tong et al. 1990).

3. Anaerobic biodegradability assays

Anaerobic biodegradability assays are used to
establish anaerobic biodegradability, for determination of the ultimate methane potential of wastes,
but are also used for determination of the rate of
the biodegradation in general.
3.1. Methods for determination of anaerobic
biodegradation
Biodegradability assays are based on the measurement of either formation of one or more products
involved in the biological reaction under investigation or measurement for substrate depletion.

Methods based on product formation are

monitoring either the end product (biogas) or
intermediates production such as volatile fatty
acids. Most methods are based on monitoring
biogas production. Biogas production is measured
either as volume increase under constant pressure
(volumetric methods), or measurement of pressure
increase in constant volume (manometric methods), or measurement of methane formation by
gas chromatography (Rozzi & Remigi, 2001). Gas
chromatography is used to measure content of
methane and carbon dioxide of the biogas that
ends up in headspace of closed vials (Dolng &
Bloemen, 1985). Soto et al. (1993) compared liquid
displacement systems to gas chromatography
methods and concluded that the latter are more
accurate for low methane productions.
Gas chromatographic methods can be divided
in two groups:
1. Using a GC with thermal conductivity detection
(TCD) where both methane and carbon dioxide
are measured. By using a reference gas e.g.
nitrogen in the headspace and regular sampling
the volume of biogas can be estimated based on
the molar fractions of CH4 , CO2 (Soto et al.
1993).
2. Using a GC with ame ionization detection
(FID), where only methane is measured (Angelidaki and Ahring 1993). The measurement is
compared with methane standard with known
methane content. This method is simple and
fast; one methane measurement takes less than
half a minute. Thus, many samples can be tested with relatively low time consumption.
Methods based on substrate depletion, require
usually more complex analysis. Substrate depletion can be determined either as lumped parameter
(volatile solids (VS), chemical oxygen demand
(COD), dissolved organic carbon (DOC), etc.) or
directly analysis of the compound that is being
used as substrate (Rozzi & Remigi, 2001).

121

Figure 1. Principles for biodegradation assays.

In Figure 1 the principles of biodegradation

assays are summarized. When the biodegradation
assay is used for determination of biodegradation
rates, primary depletion may be only representative of hydrolysis or acidogenesis step, when
methanogenesis is the limiting step, while CH4
production is reecting overall biodegradation
only when methanogenesis is non-limiting.

nal). The latter reactor is actually a system of

several small batch reactors of equal contents that
allows more thorough homogenization than one
large batch reactor. This type of batch reactor is
mainly used for assessment of the biodegradability
and hydrolysis rate of low homogeneity wastes
such as lipid containing wastes.
3.3. Inoculum

3.2. Experimental set-up for biodegradability tests

When evaluating literature it is clear that commonly two dierent experimental set-ups are used
to establish the biodegradability and hydrolysis
rate of particulate substrates, i.e. batch (Veeken &
Hamelers 1999) or continuous (Miron et al. 2000)
experiments. In the batch approach, the selected
substrate (waste) is incubated in closed vials or
asks at a specic temperature with an amount of
methanogenic inoculum. After incubation the degree of degradation of the substrate is assessed at
pre-set time intervals to determine the rate and
ultimate biodegradation or hydrolysis. Controls
with only inoculum added are included in order to
account for the biogas produced from organic
matter contained in the inoculum.
The continuous set-up uses completely stirred
tank reactors (CSTR) operated at a specic temperature and at dierent hydraulic retention times
(HRT). Analyses are made from the euent once
steady state has been established. The continuous
set-up is much more laborious than the batch set-up.
Batch experiments can be performed in a single-ask batch reactor or a multiple-ask batch
reactor (Sanders 2002; Rozzi & Remigi, this jour-

The anaerobic digestion process is a complex

process requiring the presence of several dierent
microorganisms. It is of great importance to nd
appropriate inoculum containing the necessary
microorganisms for the degradation process to
proceed. Digested sludge is often the used inoculum (Owen et al. 1979). However, in some cases,
microorganisms adapted to specic conditions
such as high ammonia concentrations are needed
(Angelidaki & Ahring 1993).
Another important factor is the amount of
inoculum added. Low amount of inoculum is often
wished as inoculum also contributes to product
formation (biogas) and thus can blur the results if
biogas production from inoculum is relatively high
compared to the compound (or waste) under
investigation. On the other hand a relatively small
amount of inoculum can lead to overload of the
process with acidication as a result. If the
ammonia concentration in the medium is high, or
substrate contains high concentration of proteins
(releasing ammonia during degradation), accumulation of VFA will not lead to acidication due
to the buer capacity supplied by ammonia (Angelidaki & Ahring 1993, 1994; Sanders et al.

122
2002a). The degree of primary biodegradation
must of course in this case be assessed via substrate
depletion instead of methane production, or as
sum of product formation (VFA and methane).
Acidogenic conditions can be prevented by
estimation of the maximum VFA production
during the assay, so that sucient inoculum can be
added to provide enough methanogenic activity
to remove the VFA at all times. As the hydrolysis
depends on the concentration of the substrate that
is to be hydrolyzed, the highest production of VFA
is to be expected in the rst day after incubation.
Based on this the minimal amount of inoculum
that has to be added can therefore be calculated as
follows:
Xss Vww kh Vinoculum VSSinoculum Ainoculum

Vreactor
Vreactor
Vinoculum

Xss Vww kh
VSSinoculum Ainoculum

1
2

where Xss is the concentration of hydrolyzable

substrate in waste (water) (g l1 ), Vww , the volume
of waste (water) in batch reactor (l), kh , the rst
order hydrolysis constant (day1 ), Vreactor , the
volume of the batch reactor (l), Vinoculum , the volume of the methanogenic inoculum (l), VSSinoculum
the volatile solids suspended content of the methanogenic inoculum (g VSS l1 ) and Ainoculum is the
methanogenic
activity
of
the
inoculum
(g g1 VSS day1 ).

As the hydrolysis constant is unknown applying Equations 1 and 2 requires an estimated value
for the hydrolysis constant. However, as the minimum amount of inoculum to be used in the
experiment is wanted, it is preferable to use an
overestimated value for the kh than an underestimated value.
3.4. Medium
An important factor associated with the inoculum
is the medium, which can be added to the inoculum
during degradation experiments. Medium consists
of several minerals and does not contain any signicant amount of organic carbon. Synthetic
medium can be added as a source of nutrients of
micronutrients, growth factors vitamins and trace
metals necessary for growth of the microorganisms.
Synthetic media are used in cases that lack of
important for growth components might be limiting microbial growth. Important components needed for growth, are shown in Table 2.
An example of a synthetic basic medium used
in anaerobic tests [modied from previously described basic medium (Angelidaki et al. 1990) is
shown in Table 3.
3.5. pH
pH plays a major part in anaerobic biodegradation. pH inuences the activity of the hydrolytic
enzymes and the microorganisms which are active

Table 2. Components needed in synthetic media (modied from Madigan (2000))

Compound

Function in the cell

Nitrogen

Next most abundant after carbon. Major element in nucleic

acids and amino acids
Phosphorus (P)
For nucleic acids and phospholipids
Sulphur (S)
In amino acids cysteine and methionine, vitamins such as
thiamine, biotin, and lipoic acid, coenzymes A
Potassium (K)
Used by several dierent enzymes
Magnesium (Mg) Stabilizes ribosomes, cell membranes and nucleic acids
Sodium (Na)
Necessary for many enzymes
Calcium (Ca)
Helps stabilize the bacterial cell wall and is important for
stabilizing endospores
Iron (Fe)
Present in cytochromes
Micronutrients
Growth factors

These are usually necessary for specic enzymes

Required in small amounts

Chemical form supplied in synthetic media

NH4Cl, (NH4)2SO4, N2, KNO3
KH2PO4, Na2HPO4
Na2SO4, KH2SO4, Na2S2O3, Na2S, cysteine, etc
KCl, KH2PO4
MgCl2, MgSO4
NaCl
CaCl2
FeCl3, FeSO4, various chelated iron (in complexes with EDTA etc.)
Cr, Co, Cu, Mn, Mo, Ni, Se, V, Zn
Vitamins, amino acids (essential), purines,
pyrimidines

123
within certain, usually narrow pH ranges. The
anaerobic digestion process occurs in the pH
interval of 6.08.3. Most methanogens have a pH
optimum between 7 and 8 while the acid forming
bacteria often have a lower optimum (Angelidaki
& Ahring 1994). If the pH of the waste to be tested
is outside the optimal range, and if enough buer
capacity is not present, the anaerobic process will
be inhibited. This will lead underestimation of the
methane potential.

4. Determination of methane potential

Methane potential (also called biochemical methane potential) of wastes is dened as the ultimate
specic methane production, for indenite degradation time. In practice the degradation time is
denite and the methane potential is estimated by
extrapolation of a methane time degradation
curve. Methane potential can be expressed specifically per amount of waste (l CH4 /kg-waste), volume of waste (l CH4 /l-waste), per mass volatile
solids added (l CH4 /kg-VS) or COD added (l CH4 /
kg-COD). The volume is usually expressed in
standard pressure (1 atm) and temperature (0 C)
conditions (STP conditions). Other variations for
expressing methane potential are also used. By the
same way biogas potential can be dened as the
ultimate biogas production.
Several methods exist for measuring methane
potentials of waste. However, the technical approaches in terms of pretreatment of the sample,
inoculum, gas measurement technique and incuba-

tion vary signicantly among the published methods (Owen et al. 1978; Eleazer et al. 1997; Owens &
Chynoweth 1993; Adani et al. 2001; Harries et al.
2001; Heerenklage et al. 2002; Hansen et al. 2003).
Some of these dierences originate from the purpose of measuring the methane potential and from
the type of waste samples measured.
According to the DTU protocol for determination of the methane potential the sample is
inoculated with 6090% w/w (depending on the
organic load of the waste) digested manure from a
reactor operating at thermophilic temperature
(55 C) (Hansen et al. 2003). Large inoculation
volumes are ensuring high microbial activity, low
risk for overloading and low risk of inhibition.
Thermophilic temperature would ensure fast reaction rates. Furthermore, digested manure has been
shown to be superior to digested sludge as it is rich
to many nutrients, and has a high buer capacity
(Angelidaki & Ahring 1994). Inoculum is degassed
by incubation at 55 C before use for a few days to
ensure that methane production from inoculum is
as low as possible. The sample is diluted with water
at dierent dilutions (from undiluted waste to 90%
w/w dilution) to ensure that possible toxicity of the
sample is not overseen. The dilutions used are
depending on the type of waste and the expected
toxicity or overloading to the anaerobic process.
Serum vials are thoroughly gassed with N2 : CO2
(80 : 20) before addition of inoculum and waste,
sealed with butyl stoppers and closed with aluminium crimps. The vials are incubated at 55 C.
The test is run in triplicates. The accumulated
methane production in the headspace of the vials is

Table 3. Basic anaerobic medium

Description of anaerobic basic medium
The basic medium is prepared from the following stock solutions, (chemicals given below are concentrations in g l)1, in distilled
water)
(A) NH4Cl, 100; NaCl, 10; MgCl26H2O, 10; CaCl22H2O, 5
(B) K2HPO43H2O, 200
(C) Resazurin 0.5
(D) Trace-metal and selenite solution: FeCl24H2O, 2; H3BO3, 0.05; ZnCl2, 0.05; CuCl22H2O, 0.038; MnCl24H2O, 0.05;
(NH4)6Mo7O244H2O, 0.05; AlCl3, 0.05; CoCl26H2O, 0.05; NiCl26H2O, 0.092; ethylenediaminetetraacetate, 0.5; concentrated
HCl, 1 ml; Na2SeO35H2O, 0.1
(E) Vitamin mixture (componets are given in mg/l): Biotin, 2; folic acid, 2; pyridoxine acid, 10; ridoavin, 5; thiamine hydrochloride,
5; cyanocobalamine, 0.1; nicotinic acid, 5; P-aminobenzoic acid, 5; lipoic acid, 5; D L -pantothenic acid.
To 974 ml of distilled water, the following stock solutions were added A, 10 ml; B, 2 ml; C, 1 ml; D, 1 ml and E, 1 ml. The mixture is
gassed with 80% N2 20% CO2. Cysteine hydrochloride, 0.5 g and NaHCO3, 2.6 g, are added and the medium is dispensed to serum
vials and autoclaved if necessary. Before inoculation the vials are reduced with Na2S9H2O to a nal concentration of 0.025%.

124

Figure 2. Experimental set-up for anaerobic biodegradation test used at Denmark Technical University (DTU) for standard biogas
potential tests.

followed by sampling gas from headspace of the

vial with a pressure-lock syringe, and subsequent
analysis of methane content by GC (FID detection). The methane production from the inoculum
(blanks are included where water is added instead
of waste) is subtracted from the methane production of the waste samples. The methane potential is
determined from the vials resulting in the highest
methane potential. At least two dilutions should
result in the highest methane potential, else higher
dilutions of the waste should be tested.
4.1 Biogas/methane potential considerations
Anaerobic degradation of organic matter results in
mainly, methane and carbon dioxide. Methane is
practically non-soluble and ends up for the most in
the gas phase. However, more complex conditions
are valid for carbon dioxide. Carbon dioxide is more
water soluble and at the same time a part of it is
2
mainly ionized to HCO
3 , and a small part to CO3
that might either precipitate or remain as ion. The
distribution of inorganic carbon to gas or liquid
phase is strongly controlled by pH but also by
temperature. Therefore, it is often more reliable to
determine methane potential than biogas potential.
4.2. Theoretical aspects for calculation of the
biogas potential
4.2.1. COD/VS
When considering the biogas process for a specic
application the characteristics of the substrate/

waste is naturally of prime interest. Waste and

wastewater is often of a complex composition,
which is dicult to describe in detail.
The most common single parameters used to
describe the concentration of waste or wastewater
is the chemical oxygen demand (COD) expressed
as g-O2 /l, or the volatile solids content (VS) expressed as g-VS/l or %.
The COD content describes the amount of
oxygen needed to completely oxidize the waste
under aerobic conditions, and is determined
experimentally by measuring the amount of a
chemical oxidizing agent needed to fully oxidize a
sample of the waste. The COD is used as a measure of the oxygen equivalent of the organic matter
content of a sample that is susceptible to oxidation
by a strong chemical oxidant (APHA 1992).
During oxidation 9095% of the organic matter is
oxidized, depending on the method used. The
preferred oxidant used for COD determination is
dichromate, due its superior oxidizing ability,
applicability to a wide variety of samples and ease
of manipulation.
The VS content describes the content of organic material in the waste, and is dened as the
amount of matter in a dried sample lost after 1 h at
a temperature of approximately. 550 C in air
(APHA 1992). The method relies on the fact that
most organic materials ignite and combust at this
temperature, while most inorganic compounds
require higher temperatures.
Both methods are relatively easy to perform,
and give a good rst impression of the strength of

125
a waste. COD is usually used for description of
wastewaters, while VS is used for slurries and solid
wastes.
If the composition of the organic material is
known, the relation between COD and VS content
can be calculated by setting up the stoichiometry
of complete oxidation. As an example the relation
is derived below for glucose (Equations (3) and
(4)):
C6 H12 O6 M 180 6O2 M 32 ! 6CO2 6H2 O

chloride) S, and N will be oxidized to a dierent

degree, thus contributing to the nal COD value.
Thus a compound having the formula
Cn Ha Ob Nc Sd is oxidized with O2 to the following
products (Equation. (7)).
Cn Ha Ob Nc Sd xO2 ! yCO2 zH2 O vNH3 wH2 SO4
7

Alternatively HNO3 can be produced instead of

NH3 . Therefore one should be aware of the denition during such calculations.

and the COD/VS ratio then becomes (Equation 6):

n a4  b2 32
6
COD=VS
12n a 16b

4.2.2. Theoretical biogas potential

When organic material is degraded anaerobically,
the end result is carbon in its most oxidized form
(CO2 ) and its most reduced form (CH4 ), i.e. an
electron transfer between carbon atoms takes
place. The ratio between CH4 and CO2 depends on
the oxidation state of the carbon present in the
organic material, i.e. the more reduced the organic
carbon content is, the more CH4 will be produced.
If the composition of the organic material is
known and all the material is converted to biogas,
the theoretical methane yield potential can be
calculated from the following Buswells equation
(Buswell and Neave 1930):


a b
Cn Ha Ob n   H2 O !
4 2




n a b
n a b
 CH4
 CO2
8
2 8 4
2 8 4

Organic matter consisting of only C, H, and O is

theoretically fully oxidized CO2 and H2 O. When
organic matter contains also S, and N, it is wished
that S, and N stay in the reduced form (H2 S,
NH3 ). However, depending on the method and the
salt content of the sample (e.g. high content of

This equation is derived by balancing the total

conversion of the organic material to CH4 and
CO2 with H2 O as the only external source, i.e.
under anaerobic conditions.
The specic methane yield, usually expressed as
(STP l CH4 )/g-VS might then be calculated as:

COD=VS 6  32=180 1:067 g  COD=g-VS

4
For many types of organic waste the oxidation
state of carbon is close to zero (as for glucose) and
in these cases the COD/VS ratio will be close to
unity.
In a more generalized form, the oxidation
reaction for organic material is given in Equation
(5).


a b
a
Cn Ha Ob n  O2 ! nCO2 H2 O
4 2
2
5

Table 4. Theoretical characteristics of typical substrate components

Substrate

Composition

type
Carbohydrate
Protein*
Lipids
Ethanol
Acetate
Propionate

(C6H10O5)n
C5H7NO2
C57H104O6
C2H6O
C2H4O2
C3H6O2

*Nitrogen is converted to NH3.

COD/VS

CH4 yield

CH4 yield

CH4

g-COD/g-VS

STP l/g-VS

STP l/g-COD

(%)

1.19
1.42
2.90
2.09
1.07
1.51

0.415
0.496
1.014
0.730
0.373
0.530

0.35
0.35
0.35
0.35
0.35
0.35

50
50
70
75
50
58

126
Bo;th

a8  b4 22:4

12n a 16b
n
2



lCH4
STP
g-VS

where 22.4 is the volume of 1 mol of gas at STP

conditions.
If Buswells equation is combined with the
COD/VS relation a similar COD based specic
yield becomes:




n
a
b
lCH4
2 8  4 22:4


Bo;th
STP
10
g-COD
n a4  b2 32
In Table 4 the characteristics of a number of typical organic materials suitable for anaerobic degradation is listed.
4.2.3. Practical biogas potential
Although the theoretical biogas potential gives a
rough idea of the quality of waste and the potential biogas production, the practical yield obtained
in a biogas reactor will always be lower due to a
number of factors:
A fraction of the substrate is utilized to synthesize bacterial mass, typically 510% of the
organic material degraded.
At a nite retention time a fraction of the organic material will be lost in the euent, typically 10%.
Lignin is not degraded anaerobically.
Often a part of the organic material is inaccessible due to binding in particles or structural
organic matter.
Limitation of other nutrient factors.
Under favourable conditions with mainly water-soluble matter, degrees of conversion up to 90
95% can be achieved. If the organic matter is
highly particulate or structural such as manures,
3060% conversion is more normal.
In order to predict the biogas yield to be expected under practical conditions, it is best to nd
experimentally determined biogas yields in the
literature or perform small-scale batch fermentations.
A prediction of the composition of the gas
produced is more complex and depends rst of
all on the amount of CH4 and CO2 produced,
but also on the pH of the reactor content. The
CH4 produced is mainly released to the gas
phase, but CO2 is partly dissolved in the liquid
phase of the reactor or is converted to bicar-

bonate as a function of the pH. Consequently,

the CH4 percentage in the biogas produced will
generally be higher than predicted by the stoichiometric ratio.

5. Assessment of the hydrolysis rate

First order kinetics are most commonly used to
describe the hydrolysis of particulate substrates
during anaerobic digestion (Eastman and Ferguson 1981; Pavlostathis and Giraldo-Gomez 1991).
dXdegr =dt k h  Xdegr

11

with Xdegr is the concentration biodegradable

substrate (kg/ m3 ), t is the time (days) and, kh is
the rst order hydrolysis constant (day1 ).
Despite the fact that the rst order kinetics is
empirical relation it does reect the major aspect
of the hydrolysis of particulate substrates, namely
the fact that the hydrolysis of particles limited by
the amount of surface available. Several
researchers showed that the hydrolysis mechanism
of particulate substrates is surface related (Hills &
Nakano 1984; Sanders et al. 2000). In this case the
amount of enzymes is present in excess relative to
the available surface area (Hobson 1987) and the
hydrolysis rate is determined by the surface area
not by the enzyme activity. Such surface limited
kinetics can be described with a rst order relation
(Vavilin et al. 1996; Valentini et al. 1997; Veeken &
Hamelers 1999; Sanders 2002a). As it is assumed
that the enzyme activity is associated with the
biomass the rst order constant is not aected by
the biomass concentration.
Although the rst order kinetics were only intended to describe the hydrolysis of particles they
can also be used to describe the hydrolysis of dissolved polymers. Sanders et al. (2002b) showed by
statistical calculations that the production of
mono and dimers from a soluble polymeric substrate by a mixture of endo- and exo-enzymes can
be described by rst order kinetics. However, in
contrast to the hydrolysis of particles in this case
the hydrolysis rate is aected by the enzyme
activity, thus biomass concentration.
From Equation (11) the relation between the
hydrolysis constant, digestion time and euent
concentration for a batch system can be derived
(Sanders et al. 2002a).

127
Xss;batch t Xss;added 1  fh
fh Xss;added e

kh t

12

where Xss , batch(t) is the concentration of total

substrate in the batch reactor t days after incubation biodegradable + non-biodegradable part)
(g l1 ), Xss;added the concentration of total substrate
in the added to batch reactor at start of experiment
biodegradable + non-biodegradable part) (g l1 ),
fh the biodegradable fraction of substrate, fh 2
[0;1], kh is the rst order hydrolysis constant
(day1 ), and t is the digestion time of batch (days).
Equation (13) presents the linearized form of
Equation (12). From Equation (13) it can be seen
that the hydrolysis constant is determined via a
linear least square t of the results from the batch
digestion, Xss;batch at several moments in time
during the batch assay. The biodegradability fh
has to be assessed directly from the experimental
results, the maximum methane yield of the substrate (Veeken and Hamelers 1999) or the nal
euent concentration.
ln



Xss;batch t  Xss;added 1  fh
kh  t 13
Xss;added fh

As the concentration of biodegradable substrate is

highest at the start of the experiment so will the
hydrolysis rate. This means that to obtain enough
experimental data for calculation of the hydrolysis
constant it is essential that the reactor is sampled
at smaller intervals in the beginning of the experiment then at the end. Nevertheless, enough data
has to be gathered at the end of the experiment to
establish the biodegradability of the substrate.
A more direct and accurate method for
assessing the hydrolysis constant and biodegradability from batch and continuous experiments
is the non-linear least squares t on the
assessed
euent
concentration
(Sanders
et al. 2002a). This method should be assessed
whenever possible. With these calculations the
gas production or the COD, protein and carbohydrate content of the blank has to be taken into
account.
Additionally, it should be emphasised that the
biodegradability in Equations (12) and (13) refers
to the biodegradability under the applied conditions and may change with the imposed reactor
conditions.

6. Potential problems in estimation of methane

potential of wastes
6.1. Nitrate, sulphate reducers and methanogens
It has been shown that sulphate reducers
and denitriers are able to outgrow the methanogens. This is due to the higher energy gained by
sulphate or nitrate reduction compared to methanogenesis. Therefore, presence of high concentrations of sulphate or nitrate will result in
determination of low methane potentials of the
wastes.
In cases where the waste (water) contains sulphate and the degree of hydrolysis is measured via
methane production the hydrolysis obviously will
also be underestimated due to consumption of
organic matter for the formation of H2 S. Unless
the amount of H2 S produced can be measured
direct measurement of the degradation of the
individual components for this type of waste
(water) is necessary.
6.2. Sorption and bioavailability
Sorption is an important mechanism that inuences the fate and eect of organic compounds. When compounds reside in environments
with high sorption capacity, they may become
unavailable for anaerobic degradation. This can
aect determination of their methane potential.
6.3. Problems during COD determination
Volatile straight-chain aliphatic compounds are
not oxidized to any appreciable degree.
aromatic carbohydrates, and some aromatic
heterocyclic compounds (e.g. pyridine) are not
oxidized.
Halogens (Cl , Br J ) can been oxidized.

NO2
2 exerts a COD of 1.1 mg/mg NO2 N
Reduced inorganic compounds such as ferrous
iron, sulphide, manganous manganese, etc., are
oxidized quantitatively under the species
The various COD methods have been developed
for water and waste water analyses. However,
when samples like manure are analyzed interferences of other additional factors can occur. Furthermore, the samples have to be properly
homogenized and diluted, since agricultural and

128
household wastes contain much higher organic
contents that wastewaters normally contain.
6.4. Inhibition of the process
The experimentally determined methane potential
can be underestimated in cases where waste contains toxicants or the process is overloaded. In
such cases dilution of the waste will result in more
accurate determination of the methane potential.
7. Conclusions
Anaerobic biodegradation is a complex process
and the biological approach to determining
anaerobic biodegradation or methane potentials
leads to substantial uncertainty in the determination. Therefore, the determination procedure
should be carefully considered, anaerobic optimal
growth conditions secured and results carefully
evaluated.
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