EUROPEAN COMMISSION

JOINT RESEARCH CENTRE

Institute for Environment and Sustainability

I-21020 Ispra (VA) Italy

WORKSHOP ON

HARMONISATION OF ANAEROBIC
BIODEGRADATION, ACTIVITY AND
INHIBITION ASSAYS
June 7-8, 2002 Lago dOrta, Italy

Proceedings

Edited by
Jos Ligthart and Hans Nieman

2002

EUR 20535 EN

ANAEROBIC BIODEGRADABILITY, METHANOGENIC

ACTIVITY AND TOXICITY TEST SYSTEMS:
DEFINING THE TEST CONDITIONS

Emer Colleran and Sean Pender

Department of Microbiology, National University of Ireland, Galway, Ireland
Tel: 00353 91 750416, Fax: 00353 91 512510, E-mail: emer.colleran@nuigalway.ie

INTRODUCTION
Until relatively recently, consideration of the microbial degradability of organic pollutants focussed on
aerobic systems and environments, as evidenced by the widespread use of the BOD test (Biochemical
Oxygen Demand) as an index of the organic pollution potential of domestic, industrial and agricultural
wastewaters. It had been widely assumed that many organic contaminants, which were aerobically
biodegradable, accumulated in anaerobic ecosystems. More recent laboratory investigations of the
organic degradability potential of pure cultures or consortia of anaerobic microorganisms have
indicated that anaerobic species are much more metabolically versatile than previously believed
(Battersby and Wilson, 1989; Aeckersberg et al., 1991; Caldwell et al., 1998; Anderson and Lovley,
2000). This greater understanding of the degradative capacities of anaerobic consortia under
methanogenic conditions has led to the increasing adoption, at full-scale, of anaerobic digestion
technology for the treatment of a growing variety of industrial wastewaters (Habets, 1996).

Although procedures for evaluating anaerobic degradability have tended to focus on methanogenic
systems, particularly those prevailing within mesophilic and thermophilic digesters, it is important to
recognise that degradation under anaerobic conditions may be linked to the reduction of a variety of
alternative electron acceptors, such as nitrate, sulphate, manganese and iron (Strevett et al., 2002).
Consequently, in order to fully appreciate the anaerobic degradability of organic contaminants in
natural environments, there is an evident need to develop a battery of biodegradability test systems
which incorporate alternative electron acceptors and which utilise, in so far as possible,
environmentally realistic assay conditions (Lovley et al., 1994; Suflita and Concannon, 1995; Lovley
& Chapelle, 1997). Redox potentials and thermodynamic considerations suggest that methanogenesis
will be favoured in natural environments only after depletion, in the sequence listed, of nitrogen
oxyanions, Fe (III), Mn (IV) and sulphate, if present in the system under study (Lovley, 1997; Lovley
& Chapelle, 1997). The development of such a test battery system is rendered even more complex by
current indications that the various anaerobic processes are not mutually exclusive in natural
environments (Jacobsen et al., 1998) and that the use of H2 determination, together with
thermodynamic calculations, may not be sufficient to provide environmentally meaningful information
on actual anaerobic degradation routes in different ecosystems (Christensen et al., 2000).

The need for test systems utilising different terminal electron acceptors is necessary in order to fully
evaluate in situ anaerobic degradability of contaminants since it cannot be assumed that the metabolic
potential of an anaerobic environment is the same under all terminal electron accepting regimes
(Strevett et al., 2002). Since the inherent physiological capability of the resident anaerobic microflora
of a particular environmental niche is the dominant factor determining biodegradability, individual
organic contaminants may be differentially exploited under different terminal electron accepting
regimes i.e. may be recalcitrant, may be partially transformed to intermediate metabolites that may or
may not accumulate or may be fully mineralised. In some cases, the products of partial mineralisation
may be more toxic than the parent compounds, thus adding further complexity to the interpretation of
anaerobic biodegradability test systems (Brunner et al., 1988; Strevett et al., 2002).

This review will focus on the anaerobic biodegradability of organic contaminants in the controlled
environments provided by psychrophilic, mesophilic and thermophilic anaerobic digesters. Although
anaerobic degradation linked to CO2

reduction

(methanogenesis)

is

the

least

favoured,

thermodynamically, of the anaerobic options, the conditions of digester operation typically favour
methanogenesis. For the majority of influent wastewaters, the involvement of anaerobic species
utilising alternative external electron acceptors is likely to be minimal and is usually discounted. This is
not the case, however, for industrial wastewaters containing high levels of sulphate or nitrate/nitrite as
electron acceptors or ammonium as a potential electron donor (Van de Graaf et al., 1997; OFlaherty et
al., 1998; Fdz.-Polanco et al., 2000; Colleran et al., 2002). In the latter cases, active competition may
take place between methanogens and other anaerobic respiratory species. Consequently, a strictly
methanogenic biodegradability test system may not provide an accurate assessment of the overall
mineralisation capacity of the anaerobic reactor under test. In such cases, it will also be necessary to
determine the biodegradation potential of species utilising alternative external electron acceptors.

In order to compare the suitability of anaerobic sludges to act as seed for the start-up of new reactors
and to evaluate the effect of potential toxicants on the multiple trophic groups involved in the overall
methanogenic biodegradation process, there is also a need to define reproducible assay test systems for
quantification of the specific methanogenic activity (SMA) of digester sludges against direct and
indirect methanogenic substrates and to develop appropriate assay procedures for evaluation of the
potential toxicity of starting or intermediate metabolite organics against the trophic groups involved in
the overall anaerobic digestion process. This review will focus solely on test conditions for the
determination of anaerobic methanogenic biodegradability, anaerobic methanogenic trophic group
activity, and potential toxicity towards trophic groups involved in methanogenesis solely in the
context of application of anaerobic digestion technology for waste/wastewater treatment under
methanogenic conditions.

METHANOGENIC BIODEGRADABILITY, SPECIFIC ACTIVITY

AND TOXICITY TEST SYSTEMS

Methanogenic Biodegradability Tests

Due to the limited substrate range of methanogens, full mineralisation under
methanogenic conditions of the wide variety of organic constituents present in a waste
or wastewater requires the coordinated and sequential involvement of the various
bacterial trophic groups illustrated in Fig. 1. Consequently, in order to determine the
anaerobic biodegradability of a test organic contaminant and its potential complete
mineralisation under methanogenic conditions, the test inoculum must contain
relevant members of all of the trophic groups illustrated in Fig. 1.

ACETATE

4a

COMPLEX
ORGANIC

FERMENTATION

INTERMEDIATES

MOLECULES

CH , CO2

4b
H /CO
2

Fig. 1 Carbon flow in anaerobic digesters: 1 = Hydrolytic/fermentative bacteria; 2 = Obligate

hydrogen producing acetogens; 3 = Homoacetogenic bacteria; 4a = Aceticlastic methanogens; 4b
= Hydrogenotrophic methanogens; 5 = Fatty acid synthesising bacteria.

A variety of test procedures have been developed over the years in order to screen for the susceptibility
of organic chemicals to anaerobic methanogenic biodegradation (Rozzi and Remigi, 2002; Strevett et
al., 2002). These methods rely on (i) measurement of the volume of the produced biogas from the test
organic substrate (respirometric methods either volumetric or manometric); (ii) measurement of the
mass and composition of the produced biogas in terms of its gaseous constituents (CH4 , CO2 etc.), and
(iii) analysis of the depletion rate of the test organic contaminant, either by test compound
determination or by measurement of the depletion of COD (chemical oxygen demand), TOC (total
organic carbon) or DOC (dissolved organic carbon). One of the first screening protocols developed
involved the displacement of the plunger of lubricated glass syringes (manual volumetric method)
inserted into the rubber septum of test vials (Owen et al., 1979). Subsequently, a variety of alternative
manual and automated volumetric respiratory systems were developed (Van den Berg, 1974; Gledhill,
W.E., 1979; Valke and Verstraete, 1983; Shelton and Tiedje, 1984; Pagga and Beimborn, 1993; Rozzi
and Remigi, 2002). Manometric methods initially utilised respirometers (Miller and Wolin, 1974;

James et al., 1990; Campos and Chernicharo, 1991). Biogas production within test systems was also
measured by the use of pressure transducers, which were initially manually operated and subsequently
automated (Gledhill, 1979; Concannon et al., 1988; Colleran et al., 1992). Analysis of biogas
constituents in sealed test vials by gas chromatography of headspace gas samples taken by pressurelock syringes was pioneered by Dolfing and Bloemen (1985). Soto et al. (1993) compared this method
with liquid displacement systems and concluded that the former was more accurate when low biogas
evolution rates were involved.

The necessity to determine the anaerobic biodegradability of organic compounds under methanogenic
conditions has led to the development of standardised international protocols for evaluation of the
anaerobic biodegradability of organic compounds by sewage sludge (ISO 11734, 1995; ISO 62, 1999;
ASTM E 2170, 2001).

Specific Methanogenic Activity Test systems

Analysis of the activity of individual trophic groups involved in the overall process of methanogenesis
(Fig. 1) has, up to now, mainly focussed on determination of the activity of the acetotrophic
methanogen population of digester sludges. This focus on aceticlastic methanogens has been largely
dictated by the crucial role played by this population in methane evolution in digesters.

Rozzi and Remigi (2002) reviewed the range of test systems that have been developed and utilised for
measurement of the activity of hydrolytic, acidogenic, acetogenic and methanogenic (aceticlastic and
hydrogenotrophic) populations in anaerobic digester biomass samples. The techniques utilised have
largely been the same as those developed for methanogenic biodegradability evaluation (i.e.
respirometric; biogas composition measurement or substrate depletion). To date, no internationallyaccepted test protocols have been delineated for determination of the specific activity of individual
trophic populations in anaerobic digester biomass samples.

Toxicity Test systems

Compounds (organic and inorganic) that exhibit toxicity towards individual trophic
groups involved in methanogenesis may be present in the raw waste/wastewater or
may be produced as metabolic intermediates of incomplete organic substrate
degradation. Test methods utilised to date for toxicity determination have been based
on the protocols developed for anaerobic biodegradation and specific activity
measurements and have largely focussed on the sensitivity of the acetotrophic
methanogenic populations of anaerobic digester sludges, biogranules and biofilms.
It is generally accepted that at least 65 % and, in some situations even up to 90 %, of the methane
evolved during digestion derives from heterotrophic methanogenic conversion of acetate (Smith and
Mah, 1966; Cappenberg and Prins, 1974; Mountford and Asher, 1978; Batstone et al., 2002). The
perception also exists that aceticlastic methanogens are more susceptible to toxicity than other trophic

groups involved in the overall methanogenesis process. Consequently, monitoring of the activity of this
trophic group has received priority in terms of development of toxicity evaluation test protocols.

TEST CONDITIONS
Test methods developed to determine the anaerobic biodegradability of wastewater organics have,
typically, been utilised and modified to allow evaluation of the specific activity of individual trophic
groups and to determine the potential toxicity of organic/inorganic compounds towards the populations
involved. It should be pointed out that (i) these test systems are typically batch, rather than continuous,
and consequently may not adequately reflect the conditions prevailing in anaerobic digesters, and (ii)
test conditions designed largely for anaerobic biodegradability evaluation may not be universally
suitable for specific activity and toxicity determination.

Test sludge and time of incubation

Anaerobic biodegradability studies have typically used sludge from full-scale
anaerobic digesters treating either primary sewage sludge or a mixture of primary and
secondary sewage sludges. The standard protocols for evaluation of the anaerobic
biodegradability of organic compounds specify sewage sludge as the test biomass
inoculum (ISO 11734, 1995; ISO 62, 1999; ASTM E 2170, 2001). While sludge
biomass from domestic sewage sludge digesters is typically exposed to a wide range
of organic compounds, it cannot be assumed that competent hydrolytic/fermentative
bacteria capable of metabolising the full range of potential test organic compounds
exist, or are present in relevant numbers in the sewage sludge inoculum utilised in
individual tests. Acclimation of existing competent populations to a test organic
compound may require a significant time period to synthesise the enzymes required
for degradation and may also require time for growth of the population(s) involved.
Consequently, anaerobic biodegradability assays may exhibit long time periods (days,
months) before degradation of the test organic compound is observed.
Battersby and Wilson (1988) reported that degradation of phenol commenced after a lag period of 6
days using a sewage sludge inoculum. By contrast, no degradation of phenol was observed by Colleran
et al. (1992) over a 50 day assay period using a sewage sludge inoculum. When the sewage sludge was
used to seed a laboratory-scale reactor fed a mixture of aromatic chemicals, phenol degradation
commenced after c. 80 days, indicating that species capable of phenol degradation were present in the
test sludge but were not detected in the anaerobic biodegradability assay.
A variety of incubation times have been arbitrarily recommended for anaerobic biodegradability
assays. An incubation time of 8 weeks was recommended by Shelton and Tiedje (1984) and strongly
supported as a minimum test period more recently by Strevett et al. (2002). As suggested by Strevett et
al. (2002), this time period may, for some chemicals, be insufficient. Based on the authors experience,
an incubation time period of 100 days, rather than 8 weeks, may be preferable.

Because of the CSTR (continuously-stirred tank reactor) design of sewage sludge digesters, slowgrowing OHPA and acetotrophic methanogenic bacteria are not typically present at high population
numbers in sewage sludge digester biomass. In the authors laboratory, the inclusion of granular sludge
from an industrial UASB reactor (at a ratio of 3 : 1 : sewage sludge/UASB granular sludge on a VSS
basis) has been found to improve methane production rates in anaerobic biodegradability assays.

For activity and toxicity tests, the test sludge usually derives from high-rate industrial wastewater
digesters. Consequently, the activity of the test populations (typically acetotrophic methanogens) is
higher than in sewage sludge and activity/toxicity tests are generally accomplished within 1 to 2 weeks.

Temperature
Since anaerobic digesters typically operate under mesophilic or thermophilic conditions, there is a need
to define the conditions of sludge handling, storage etc. prior to carrying out biodegradability, activity
or toxicity tests. Thermophilic reactor sludge is particularly susceptible to exposure to lower
temperatures and activity tests may exhibit long lag phases prior to the re-acclimation of the sludge
populations to the test thermophilic temperature.

Little attention has focussed to date on evaluation of the anaerobic degradability potential or the
specific methanogenic activity of anaerobic biomass at psychrophilic temperatures. It is evident from
recent research publications, that anaerobic treatment of domestic and industrial wastewaters is feasible
in psychrophilic high-rate digesters which ensure biomass retention as biogranules or biofilm (Rebac et
al., 1999; Lettinga et al., 2001; Collins et al., 2002). Consequently, protocols for evaluation of
biodegradability, specific methanogenic activity and trophic group toxicity should include a definition
of psychrophilic test temperature.

Test medium
Since anaerobic biodegradability tests may require growth of a population present in the test sludge at
low numbers, the medium used in these tests should provide all of the inorganic nutrients required for
growth. There is a need to define the test medium for anaerobic biodegradability assays, bearing in
mind that methanogenic populations may require organic and inorganic compound supplementation
(i.e. vitamins, amino acids, trace metals) for optimal growth.

By contrast, determination of the specific activity of anaerobic digester sludge populations should
utilise a non-growth anaerobic medium in order to evaluate the "actual" activity of the test populations
within operational reactors. In the authors laboratory, the medium used in activity tests consists only
of the following ingredients: bicarbonate buffer, cysteine hydrochloride, resazurin (Reynolds, 1986;
Coates, 1991; Colleran et al., 1992). The same medium is used in toxicity test systems.

Substrate concentration in biodegradability tests

Degradation of test organic compounds is usually expressed as a percentage of the theoretical gas
production using Buswell's equation (Symons and Buswell, 1933). Problems in biodegradability tests

result from (i) the limited aqueous solubility of some test organics; (ii) the potential toxicity of
individual natural or xenobiotic compounds at low concentrations and (iii) problems in detection of
products of biodegradation at low initial substrate concentrations.

Standard biodegradability test systems define initial test compound concentrations in terms of
BOD/COD. In cases where solubility problems require utilisation of a carrier compound to ensure
adequate concentrations in test vials, it is essential to include appropriate blank/control test vials which
(i) determine the background rate of methanogenesis resulting from conversion of degradable materials
present in the sludge inoculum and (ii) determine the methane production potential of carrier organics
used to solubilise the organic test compound in the test vials.

Substrate concentration in activity and toxicity test vials

For soluble direct and indirect methanogenic intermediate substrates (volatile fatty acids, alcohols,
ketones etc.), there is a need to define initial test substrate concentrations that (i) are present at
sufficiently high concentrations to allow product determination and (ii) are not present at potentially
inhibitory concentrations. The test in vial concentrations utilised in the authors laboratory are 30 mM
for acetate, propionate and ethanol and 15 mM for butyrate. Test vials utilised for determination of
hydrogenotrophic methanogenic activity are pressurised to 1 atmosphere pressure with 80/20 H2 /CO2 .
Since solubility of the H2 substrate is a rate-limiting factor, hydrogenotrophic activity tests are carried
out in 120 ml vials, with a test volume of 10 ml, and incubated horizontally on rotating shakers.

Number of tests/replicates required

In order to adequately analyse the specific methanogenic activity of digester biomass samples, test
analyses should be replicated at least in triplicate. This is essential since the granular nature of the
retained biomass in UASB/IC reactors presents difficulties with respect to uniform sampling. For labscale reactors, the requirement of sludge removal for triplicate analysis may present problems and
necessitates the use of low-volume analytical test systems. In the authors' experience, the use of the
pressure transducer device for measurement of biogas production allows use of small-scale test systems
(60 ml volume vials); facilitates reproducibility (minimum of triplicate assays), and allows frequent
biomass sampling from laboratory-scale reactors without significant impairment of reactor function.

CONCLUSIONS
Although standard methods have been issued for determining the potential anaerobic biodegradability
of organic compounds (ISO 11734, 1995; ISO 62, 1999: ASTM E 2170), significant questions arise as
to the reproducibility of these protocols. In particular, questions arise re the standardisation of the test
inoculum; the test medium; the test conditions and the duration of the biodegradability test.

No standard test conditions have been agreed with respect to determination of the specific
methanogenic activity of digester sludges (acetotrophic and hydrogenophilic methanogens). Evaluation
and quantitative assessment of the role of hydrolytic/fermentative and non-hydrolytic fermentative
bacteria; acetogenic bacteria and homoacetogenic bacteria has not, so far, been adequately addressed.
Without the availability of standardised individual species activity assays, data deriving from currentlyapplied toxicity tests are extremely difficult to interpret and yield little information of direct value to

the operation of full-scale digesters or to knowledge of the in situ of toxicity organic compounds in
environmental ecosystems.

There is, clearly, an evident need to define reproducible and internationally-comparable test systems
for determining (i) anaerobic biodegradability under methanogenic or alternative electron-acceptor test
conditions, (ii) the specific activity of hydrolytic, fermentative, acetogenic and methanogenic bacteria
in digester biomass and environmental samples and (iii) toxicity determination against the diverse
populations involved in anaerobic degradation.

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