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Assessment of the anaerobic biodegradability of macropollutants

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 Reviews in Environmental Science and Bio/Technology 3: 117–129, 2004.
117

2004 Kluwer Academic Publishers. Printed in the Netherlands.

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 final mineralization of organic compounds and the methane potential of com-
pounds. Hydrolysis of complex substrates is also discussed. Furthermore, factors important for anaerobic
biodegradation are shortly discussed.

1. Introduction A substance or a compound is biodegradable

Anaerobic degradation or digestion can be
defined 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 reduc-
tions to its most oxidized state (CO2 ), and its most
reduced state (CH4 ). A wide range of microor-
ganisms catalyze the process in the absence of
oxygen (McInerney et al. 1980). The main prod-
ucts of the process are carbon dioxide and meth-
ane, 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 anaero-
bic digestion process there is an urgent need to
review methods for estimation of the biodegrad-
ability and methane potential of wastes used in for
anaerobic digestion.
if it can be decomposed by the action of micro-
organisms. Microorganisms can use this com-
pound as energy source and as carbon
source. A compound can be mineralized i.e. con-
verted besides to new microbial biomass to the
end carbon products i.e. to carbon dioxide
and methane. In some cases complete mineraliza-
tion is not seen and the compound can be
involved in microbial metabolism with only trans-
formation (also called primary biodegradation)
of the compound to intermediates, but with-
out total conversion to end products. An
organic compound can be processed dur-
ing anaerobic degradation through metabo-
lism (the compound is supplying energy and
carbon source for the micro-organisms) or by co-
metabolism (the compound is converted only at
the presence of another, usually easily degrad-
able 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
/influencing anaerobic biodegradation
There are several physical, chemical and physio-
logical factors in the environment that affect bio-
degradation of organic compounds, such as
availability of the compounds, the availability of
electron donors and acceptors, oxygen concentra-
tion, temperature, pH, moisture, salinity, sorption
of chemicals to particulate material, concentration
of the chemicals. Different factors might have
different influence according the specific charac-
teristics of the compound.
2.1. Redox conditions
One of the major factors governing biodegrada-
tion is the nature and availability of electron ac-
ceptors. 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 ac-
ceptors, such as nitrate, iron, sulphate or carbon
dioxide. In truly anaerobic conditions there is ab-
sence 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 affects survival and growth of
microorganisms and it also influences their meta-
bolic 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
different temperature ranges, i.e. the mesophilic
(25–40 C), the thermophilic (45–60 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 temper-
ature 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 effect of temperature on anaerobic degra-
dation is theoretically only influencing the degra-
dation 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 effect of temperature on the achievable
methane potential of manure was investigated by
Hashimoto et al. (1981). It was found that tem-
peratures in the range between 30 and 60 C (at
5 C intervals) did not affect the achievable
methane potential of manure. At higher tempera-
tures (65 C) the experimentally determined
methane potential was lower. The lower potential
was attributed to unstable fermentation rather
than decreased substrate availability at that tem-
perature.
2.3. Hydrolysis
An important step of the anaerobic biodegrada-
tion process is the hydrolysis of the complex or-
ganic matter. During the anaerobic digestion of
complex organic matter the hydrolysis is the first
and often the rate-limiting step. The hydrolysis can
be defined 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 di-
rectly present in the waste or can be formed by
microbial activity such as internal storage prod-
ucts, or bacterial biomass.
During hydrolysis of macro-pollutants poly-
mers such as lipids, protein, carbohydrates are
depolymerized to glycerol and long chain fatty
acids, to amino acids and to oligo- and monosac-
charides for lipids, proteins and carbohydrates
respectively.
2.3.1. Hydrolysis mechanism
Hydrolysis takes place extracellular by enzymes
excreted by the biomass. Three main mechanisms

exist for the release of enzymes and the subsequent
hydrolysis of the complex substrate during anaer-
obic digestion (Batstone 2000).
1. The organism secretes enzymes to the bulk li-
quid, 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 benefit from the released dis-
solved 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 con-
tact between biomass and substrate is a pre-
requisite 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 specific activity is thought to
follow Michaelis–Menten kinetics.
The overall effect of the digestion temperature
on the hydrolysis rate originates from the com-
bined temperature effect on the enzyme kinetics,
bacterial growth and solubility of the substrate.
For instance, the Gibbs–Helmoltz equation
gives the relation between temperature and pKa of
the enzymes. The change in charge will have con-
sequences for the structure of the enzyme resulting
in changes of catalytic efficiency, 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
119
first order hydrolysis constant and the digestion
temperature (R2 0.984–0.999) with an average
standard free energy of activation of 46
14 kJ/
mol.
The effect of the pH on the hydrolysis is com-
plicated. The net effect of pH on the hydrolysis
rate is specified by the pH optima of the different
enzymes present in the digester and the effect of
pH on the charge/solubility of the substrate. The
latter especially applies to the digestion of sub-
strates 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 findings 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 ef-
fects of the concentration of free amino acid on the
production of cellulases were reported (Glenn
1976). Also NHþ 4 can inhibit the hydrolysis of cel-
lulose (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 effect of different ammonia concentra-
tions (range 1–3.7 g NHþ 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 NHþ 4 –N/l. At both temper-
atures a negative linear relation between the first
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 physical–chemical 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
120
Table 1. Surface related hydrolysis rate assessed for different substrates
Substrate Hydrolysis rate Inoculum
(mg COD/cm2/day)
Starch 1.0 Granular sludge from potato factory
Rice 1.1 Not indicated
Hay 0.01 Not indicated
Cellulose 0.33 MSW leachate
waste (water) can be found in different physical
states, in particles, dissolved or emulsified. Parti-
cles 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 re-
lated process and a formation of a biofilm 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 al-
tered by formation of complexes with other com-
pounds. 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 determi-
nation 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 measure-
ment of either formation of one or more products
involved in the biological reaction under investiga-
tion or measurement for substrate depletion.
Temperature (C) Reactor Reference
35 Batch Sanders et al. (2000)
35 Batch Palmowski et al. (2001)
35 Batch Palmowski et al. (2001)
38 CSTR Song (2003)
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 meth-
ods), 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 (Dolfing &
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 flame ionization detection
(FID), where only methane is measured (An-
gelidaki 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 tes-
ted with relatively low time consumption.
Methods based on substrate depletion, require
usually more complex analysis. Substrate deple-
tion 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).

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 representa-
tive of hydrolysis or acidogenesis step, when
methanogenesis is the limiting step, while CH4
production is reflecting overall biodegradation
only when methanogenesis is non-limiting.
3.2. Experimental set-up for biodegradability tests
When evaluating literature it is clear that com-
monly two different 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
flasks at a specific temperature with an amount of
methanogenic inoculum. After incubation the de-
gree 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 specific tem-
perature and at different hydraulic retention times
(HRT). Analyses are made from the effluent 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 sin-
gle-flask batch reactor or a multiple-flask batch
reactor (Sanders 2002; Rozzi & Remigi, this jour-
121
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
The anaerobic digestion process is a complex
process requiring the presence of several different
microorganisms. It is of great importance to find
appropriate inoculum containing the necessary
microorganisms for the degradation process to
proceed. Digested sludge is often the used inocu-
lum (Owen et al. 1979). However, in some cases,
microorganisms adapted to specific 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 acidification as a result. If the
ammonia concentration in the medium is high, or
substrate contains high concentration of proteins
(releasing ammonia during degradation), accu-
mulation of VFA will not lead to acidification due
to the buffer capacity supplied by ammonia (An-
gelidaki & 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 sufficient 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 first 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
¼ ð1Þ
Vreactor Vreactor
Xss Vww kh
Vinoculum ¼
VSSinoculum Ainoculum
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 first
order hydrolysis constant (day1 ), Vreactor , the
volume of the batch reactor (l), Vinoculum , the vol-
ume of the methanogenic inoculum (l), VSSinoculum
the volatile solids suspended content of the meth-
anogenic inoculum (g VSS l1 ) and Ainoculum is the
methanogenic activity of the inoculum
(g g1 VSS day1 ).
As the hydrolysis constant is unknown apply-
ing Equations 1 and 2 requires an estimated value
for the hydrolysis constant. However, as the min-
imum amount of inoculum to be used in the
experiment is wanted, it is preferable to use an
overestimated value for the kh than an underesti-
mated 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 sig-
nificant 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 limit-
ð2Þ ing microbial growth. Important components nee-
ded for growth, are shown in Table 2.
An example of a synthetic basic medium used
in anaerobic tests [modified from previously de-
scribed basic medium (Angelidaki et al. 1990) is
shown in Table 3.
3.5. pH
pH plays a major part in anaerobic biodegrada-
tion. pH influences the activity of the hydrolytic
enzymes and the microorganisms which are active

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

Compound Function in the cell Chemical form supplied in synthetic media

Nitrogen Next most abundant after carbon. Major element in nucleic NH4Cl, (NH4)2SO4, N2, KNO3
acids and amino acids
Phosphorus (P) For nucleic acids and phospholipids KH2PO4, Na2HPO4
Sulphur (S) In amino acids cysteine and methionine, vitamins such as Na2SO4, KH2SO4, Na2S2O3, Na2S, cysteine, etc
thiamine, biotin, and lipoic acid, coenzymes A
Potassium (K) Used by several different enzymes KCl, KH2PO4
Magnesium (Mg) Stabilizes ribosomes, cell membranes and nucleic acids MgCl2, MgSO4
Sodium (Na) Necessary for many enzymes NaCl
Calcium (Ca) Helps stabilize the bacterial cell wall and is important for CaCl2
stabilizing endospores
Iron (Fe) Present in cytochromes FeCl3, FeSO4, various chelated iron (in com-
plexes with EDTA etc.)
Micronutrients These are usually necessary for specific enzymes Cr, Co, Cu, Mn, Mo, Ni, Se, V, Zn
Growth factors Required in small amounts Vitamins, amino acids (essential), purines,
pyrimidines
 123

within certain, usually narrow pH ranges. The
anaerobic digestion process occurs in the pH
interval of 6.0–8.3. 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 buffer
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 meth-
ane potential) of wastes is defined as the ultimate
specific methane production, for indefinite degra-
dation time. In practice the degradation time is
definite and the methane potential is estimated by
extrapolation of a methane time degradation
curve. Methane potential can be expressed specif-
ically per amount of waste (l CH4 /kg-waste), vol-
ume 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 defined as the
ultimate biogas production.
Several methods exist for measuring methane
potentials of waste. However, the technical ap-
proaches in terms of pretreatment of the sample,
inoculum, gas measurement technique and incuba-
tion vary significantly among the published meth-
ods (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 differences originate from the pur-
pose of measuring the methane potential and from
the type of waste samples measured.
According to the DTU protocol for determi-
nation of the methane potential the sample is
inoculated with 60–90% 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 reac-
tion rates. Furthermore, digested manure has been
shown to be superior to digested sludge as it is rich
to many nutrients, and has a high buffer 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 different 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 alu-
minium 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; MnCl2Æ4H2O, 0.05;
(NH4)6Mo7O244H2O, 0.05; AlCl3, 0.05; CoCl26H2O, 0.05; NiCl26H2O, 0.092; ethylenediaminetetraacetate, 0.5; concentrated
HCl, 1 ml; Na2SeO3Æ5H2O, 0.1
(E) Vitamin mixture (componets are given in mg/l): Biotin, 2; folic acid, 2; pyridoxine acid, 10; ridoflavin, 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 Na2SÆ9H2O to a final 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 detec-
tion). The methane production from the inoculum
(blanks are included where water is added instead
of waste) is subtracted from the methane produc-
tion 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
mainly ionized to HCO 2
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 specific
application the characteristics of the substrate/
waste is naturally of prime interest. Waste and
wastewater is often of a complex composition,
which is difficult 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) ex-
pressed 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 mea-
sure 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 90–95% 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 or-
ganic material in the waste, and is defined 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 first 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 ðM ¼ 180Þ þ 6O2 ðM ¼ 32Þ ! 6CO2 þ 6H2 O
ð3Þ
COD=VS ¼ 6  32=180 ¼ 1:067 g  COD=g-VS
ð4Þ
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 þ n þ  O2 ! þnCO2 þ H2 O
4 2 2
ð5Þ
and the COD/VS ratio then becomes (Equation 6):


n þ a4  b2 32
COD=VS ¼ ð6Þ
12n þ a þ 16b
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
chloride) S, and N will be oxidized to a different
degree, thus contributing to the final 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 þ xO2 ! yCO2 þ zH2 O þ vNH3 þ wH2 SO4
ð7Þ
Alternatively HNO3 can be produced instead of
NH3 . Therefore one should be aware of the defi-
nition during such calculations.
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 Buswell’s equation
(Buswell and Neave 1930):
 
a b
Cn Ha Ob þ n   H2 O !
4 2
  
n a b n a b
þ  CH4 þ  þ CO2 ð8Þ
2 8 4 2 8 4
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 specific methane yield, usually expressed as
(STP l CH4 )/g-VS might then be calculated as:

Table 4. Theoretical characteristics of typical substrate components

Substrate Composition COD/VS CH4 yield CH4 yield CH4

type g-COD/g-VS STP l/g-VS STP l/g-COD (%)

Carbohydrate (C6H10O5)n 1.19 0.415 0.35 50

Protein* C5H7NO2 1.42 0.496 0.35 50
Lipids C57H104O6 2.90 1.014 0.35 70
Ethanol C2H6O 2.09 0.730 0.35 75
Acetate C2H4O2 1.07 0.373 0.35 50
Propionate C3H6O2 1.51 0.530 0.35 58

*Nitrogen is converted to NH3.

126

 
n
2þ a8  b4 22:4 lCH4
Bo;th ¼ STP ð9Þ
12n þ a þ 16b g-VS
where 22.4 is the volume of 1 mol of gas at STP
conditions.
If Buswell’s equation is combined with the
COD/VS relation a similar COD based specific
yield becomes:

 
n a b
2 þ 8  4 22:4 lCH4
Bo;th ¼
STP ð10Þ
n þ a4  b2 32 g-COD
In Table 4 the characteristics of a number of typ-
ical organic materials suitable for anaerobic deg-
radation is listed.
4.2.3. Practical biogas potential
Although the theoretical biogas potential gives a
rough idea of the quality of waste and the poten-
tial 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 syn-
thesize bacterial mass, typically 5–10% of the
organic material degraded.
• At a finite retention time a fraction of the or-
ganic material will be lost in the effluent, typi-
cally 10%.
• Lignin is not degraded anaerobically.
• Often a part of the organic material is inac-
cessible due to binding in particles or structural
organic matter.
• Limitation of other nutrient factors.
Under favourable conditions with mainly wa-
ter-soluble matter, degrees of conversion up to 90–
95% can be achieved. If the organic matter is
highly particulate or structural such as manures,
30–60% conversion is more normal.
In order to predict the biogas yield to be ex-
pected under practical conditions, it is best to find
experimentally determined biogas yields in the
literature or perform small-scale batch fermenta-
tions.
A prediction of the composition of the gas
produced is more complex and depends first 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 stoi-
chiometric 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 Fergu-
son 1981; Pavlostathis and Giraldo-Gomez 1991).
dXdegr =dt ¼ k h  Xdegr ð11Þ
with Xdegr is the concentration biodegradable
substrate (kg/ m3 ), t is the time (days) and, kh is
the first order hydrolysis constant (day1 ).
Despite the fact that the first order kinetics is
empirical relation it does reflect 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 first 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 first order constant is not affected by
the biomass concentration.
Although the first order kinetics were only in-
tended to describe the hydrolysis of particles they
can also be used to describe the hydrolysis of dis-
solved polymers. Sanders et al. (2002b) showed by
statistical calculations that the production of
mono and dimers from a soluble polymeric sub-
strate by a mixture of endo- and exo-enzymes can
be described by first order kinetics. However, in
contrast to the hydrolysis of particles in this case
the hydrolysis rate is affected by the enzyme
activity, thus biomass concentration.
From Equation (11) the relation between the
hydrolysis constant, digestion time and effluent
concentration for a batch system can be derived
(Sanders et al. 2002a).

Xss;batch ðtÞ ¼ Xss;added ð1  fh Þ
kh t
þ fh Xss;added e
where Xss , batch(t) is the concentration of total
substrate in the batch reactor t days after incuba-
tion 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 first 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 fit of the results from the batch
digestion, Xss;batch at several moments in time
during the batch assay. The biodegradability ðfh Þ
has to be assessed directly from the experimental
results, the maximum methane yield of the sub-
strate (Veeken and Hamelers 1999) or the final
effluent concentration.
 
Xss;batch ðtÞ  Xss;added ð1  fh Þ
ln ¼ kh  t ð13Þ
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 exper-
iment 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 biodegrad-
ability from batch and continuous experiments
is the non-linear least squares fit on the
assessed effluent concentration (Sanders
et al. 2002a). This method should be assessed
whenever possible. With these calculations the
gas production or the COD, protein and carbo-
hydrate 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 condi-
tions and may change with the imposed reactor
conditions.
127
6. Potential problems in estimation of methane
potential of wastes
ð12Þ
6.1. Nitrate, sulphate reducers and methanogens
It has been shown that sulphate reducers
and denitrifiers are able to outgrow the methano-
gens. This is due to the higher energy gained by
sulphate or nitrate reduction compared to metha-
nogenesis. Therefore, presence of high concentra-
tions of sulphate or nitrate will result in
determination of low methane potentials of the
wastes.
In cases where the waste (water) contains sul-
phate 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 influ-
ences the fate and effect of organic com-
pounds. When compounds reside in environments
with high sorption capacity, they may become
unavailable for anaerobic degradation. This can
affect 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.
• NO22 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 interfer-
ences of other additional factors can occur. Fur-
thermore, 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 con-
tains 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 determi-
nation. Therefore, the determination procedure
should be carefully considered, anaerobic optimal
growth conditions secured and results carefully
evaluated.
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