Bio Degradation and Persistence

Aerobic and Anaerobic Biodegradation Potentials
of Microorganisms
Walter Reineke
Bergische Universitt Gesamthochschule Wuppertal, Chemische Mikrobiologie,
Fachbereich 9, Gaustrae 20, D-42097 Wuppertal, Germany
E-mail: reineke@uni-wuppertal.de
Microorganisms represent essential components of the global carbon cycle. In addition, it ap-
pears that most xenobiotic industrial chemicals can be degraded by microorganisms, either by
a combination of cometabolic steps, often yielding partial degradation, or by serving as
growth substrate which is accompanied by mineralization of at least part of the molecule.
Using a number of examples, including aromatic, chloroaromatic, aliphatic, and chloroali-
phatic compounds, I have presented some principles on the degradation. The great influence
of some environmental conditions on the degradation, such as the presence or absence of
oxygen, the availability of other electron acceptors such as nitrate or sulfate, has been discussed
with special emphasis on the type of reactions and the rates of degradation that occur.
While aerobic microorganisms use oxidative reactions, the degradation by anaerobic bac-
teria takes place by reductive types of reactions. The oxidative sequences of aromatic and chlo-
roaromatic compounds in aerobic bacteria yield central intermediates with a diphenolic struc-
ture. These compounds are then cleaved by enzymes that use molecular oxygen. In contrast,
the anaerobes degrade aromatic compounds by reductive conversions and the central inter-
mediates ready for hydrolytic ring cleavage bear a 1,3-dioxo structure.
Aerobic bacteria and fungi, especially ligninolytic ones, were shown to use mechanistically
different catabolic pathways and enzymes. The ligninolytic fungi convert oxygen to hydrogen
peroxide which is then used for the formation of an aryl cation radical undergoing spon-
taneous rearrangements and degradation.
The broad variety of mechanisms which brings about dechlorination is another important
part of this work. Although the diversity of the compounds discussed is very large, the strategy
of the organisms used in the degradation includes various analogous reactions.
Another important aspect of discussion is the different degree of degradation. While most
research is done on organisms that are able to use the respective compound as the growth sub-
strate, i. e., carbon dioxide and biomass result, the cometabolic potential of microorganisms
should not be neglected. Cometabolism takes place very much in nature and brings about
some modification of a target compound.
In general, anoxic microbial degradation seems to be of greater relevance in nature than ear-
lier expected. It is remarkable that some chlorinated compounds such as chlorobenzoates, chlo-
rophenols, or tetrachloroethene may function as a physiologically functional electron acceptor
in a type of anaerobic respiration, which leads to non-chlorinated or lower chlorinated products.
Since various compounds will not be degraded totally by one type of organism the com-
plementary potential of anaerobic and aerobic populations in combination is thought to be a
method to bring about complete mineralization.
Finally, the possibility of enhancing the degradative potential of aerobic organisms in the
laboratory, i. e., artificial evolution of enzymes and pathways, by different genetic approaches,
is discussed.
Keywords. Aromatic, chloroaromatic, aliphatic, and chloroaliphatic compounds, Aerobic and
anaerobic bacteria, Ligninolytic fungi, Cometabolism vs productive mineralization, Com-
pounds as carbon and energy sources, Degradative pathways with oxidative sequences, De-
gradative pathways with reductive sequences, Dechlorination mechanisms, Fermentations,
CHAPTER 1
The Handbook of Environmental Chemistry Vol. 2 Part K
Biodegradation and Persistence
(ed. by B. Beek)
Springer-Verlag Berlin Heidelberg 2001
Haloaliphatic and haloaromatic compounds as electron acceptors with dechlorination: deha-
lorespiration, Sequential anaerobic-aerobic processes, Enhancing the degradative potential
by in vivo and in vitro techniques
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1 Redox Processes and Mineralization of Organic Compounds . . . 5
1.2 Energy Yields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3 Distribution of Electron Acceptors in the Environment
and Sequential Redox Conditions . . . . . . . . . . . . . . . . . . . 8
1.4 Organic Compounds as Electron Acceptors . . . . . . . . . . . . . 9
1.5 Limitations to Fermentation . . . . . . . . . . . . . . . . . . . . . . 12
1.6 Various Degrees of Degradation . . . . . . . . . . . . . . . . . . . 13
1.7 Rsum of Introduction . . . . . . . . . . . . . . . . . . . . . . . . 15
2 Degradation of Aromatic Compounds . . . . . . . . . . . . . . . . 15
2.1 Aerobic vs Anaerobic Degradation: Introduction . . . . . . . . . . 15
2.2 Aerobic Degradation of Aromatics: General Differences Between
Prokaryotic and Eukaryotic Organisms in the Initial Reactions . . 17
2.3 Degradation of Aromatic Compounds by Aerobic Bacteria . . . . 18
2.3.1 Reactions Converting Aromatic Compounds into
Ring Cleavage Substrates . . . . . . . . . . . . . . . . . . . . . . . 18
2.3.2 Ring Fission and Carbon Chain Fission . . . . . . . . . . . . . . . 26
2.3.3 Pathways as a Whole for Catechol, Protocatechuate, and Gentisate 29
2.4 Degradation of Aromatics by Fungi . . . . . . . . . . . . . . . . . 32
2.4.1 Degradation by Non-Ligninolytic Fungi . . . . . . . . . . . . . . . 32
2.4.2 Degradation of PAHs by Ligninolytic Fungi . . . . . . . . . . . . . 34
2.5 Rsum: Aerobic Degradation of Aromatic Compounds . . . . . . 39
2.6 Anaerobic Degradation of Aromatic Compounds . . . . . . . . . . 40
2.6.1 Channeling Reactions . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.6.2 Activating Reductive Sequences and Ring Cleavage . . . . . . . . . 48
2.6.3 Anaerobic Degradation of Environmentally Important Aromatics
where Pathway Information is Missing or Minor . . . . . . . . . . 51
2.6.4 Rsum: Anaerobic Degradation of Aromatic Compounds . . . . . 52
2.7 Rsum: Aromatic Compounds . . . . . . . . . . . . . . . . . . . . 53
2.7.1 Degradation in the Presence of Oxygen . . . . . . . . . . . . . . . 53
2.7.2 Degradation in the Absence of Oxygen . . . . . . . . . . . . . . . . 54
3 Degradation of Chloroaromatic Compounds . . . . . . . . . . . . 55
3.1 Chloroaromatic Compounds as Growth Substrate
for Aerobic Bacteria and the Dechlorination Mechanisms . . . . . 55
3.1.1 Elimination of Chlorine Substituents Prior to Ring Cleavage . . . 55
3.1.2 Late Eliminations of Chlorine After or Linked with Ring Cleavage 62
3.1.3 Degradation of Higher Chlorinated Aromatic Compounds
Needs Different Dechlorination Mechanisms . . . . . . . . . . . . 68
3.2 Degradation of Chloroaromatic Compounds
by Ligninolytic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . 71
2 W. Reineke
3.3 Anaerobic Microbial Populations with the Potential
to Dechlorinate Chloroaromatic Compounds . . . . . . . . . . . . 75
3.3.1 Potential of Environmental Materials and Undefined Enrichments 75
3.3.2 Pure Cultures: Chloroaromatic Compounds
as Electron Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.3.3 Pure Cultures: Chloroaromatic Compounds as Growth Substrate . 82
3.3.4 Dechlorinating Organisms, Part of a Food Web . . . . . . . . . . . 82
3.3.5 Phototrophic Bacteria and Chloroaromatic Compounds . . . . . . 83
3.4 Rsum: Chloroaromatic Compounds . . . . . . . . . . . . . . . . 84
4 Degradation of Aliphatic Hydrocarbons . . . . . . . . . . . . . . . 84
4.1 Aerobic Degradation of Aliphatic Hydrocarbons . . . . . . . . . . 84
4.1.1 Alkanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.1.2 Branched Alkanes . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.1.3 Alkenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.2 Anaerobic Degradation of Aliphatic Hydrocarbons . . . . . . . . . 95
4.3 Rsum: Aliphatic Hydrocarbons . . . . . . . . . . . . . . . . . . . 96
5 Degradation of Chloroaliphatic Compounds . . . . . . . . . . . . 96
5.1 Chloroaliphatic Compounds as Growth Substrate
for Aerobic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 96
5.1.1 Hydrolytic Dehalogenation . . . . . . . . . . . . . . . . . . . . . . 99
5.1.2 Glutathione S-Transferase-Dependent Dehalogenation . . . . . . . 103
5.1.3 Lyase-Catalyzed Dehalogenation . . . . . . . . . . . . . . . . . . . 104
5.1.4 Hydratase-Catalyzed Dehalogenation . . . . . . . . . . . . . . . . 104
5.1.5 Dehalogenation by Oxygenases . . . . . . . . . . . . . . . . . . . . 104
5.1.6 Dehalogenation During b-Oxidation . . . . . . . . . . . . . . . . . 105
5.1.7 Dehydrohalogenation . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.1.8 Dehalogenation by Methyltransferase/Dehydrogenase . . . . . . . 106
5.2 Chloroaliphatic Compounds as Growth Substrates
for Anaerobic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.2.1 Fermentative Degradation . . . . . . . . . . . . . . . . . . . . . . . 107
5.2.2 Degradation Under Denitrifying Conditions . . . . . . . . . . . . 109
5.2.3 Degradation Under Methanogenic Conditions . . . . . . . . . . . 109
5.3 Cometabolic Transformations . . . . . . . . . . . . . . . . . . . . . 110
5.3.1 Aerobic Bacteria: Oxidative . . . . . . . . . . . . . . . . . . . . . . 110
5.3.2 Ligninolytic Fungi: Reductive . . . . . . . . . . . . . . . . . . . . . 112
5.3.3 Anaerobic Bacteria: Reductive . . . . . . . . . . . . . . . . . . . . . 112
5.4 Chloroaliphatic Compounds as Electron Acceptors . . . . . . . . . 113
5.5 Rsum: Chloroaliphatic Compounds . . . . . . . . . . . . . . . . 115
6 Sequential Anaerobic-Aerobic Processes for the Degradation
of Problematic Compounds . . . . . . . . . . . . . . . . . . . . . . 116
6.1 Studies with Environmental Materials . . . . . . . . . . . . . . . . 117
6.2 Studies with Undefined Enrichment Cultures . . . . . . . . . . . . 118
Aerobic and Anaerobic Biodegradation Potentials of Microorganisms 3
6.3 Studies with Undefined Enrichment Cultures Supplemented
with Pure Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
6.4 Studies with Pure Cultures . . . . . . . . . . . . . . . . . . . . . . . 120
6.5 Rsum: Sequential Anaerobic-Aerobic Processes . . . . . . . . . . 121
7 Enhancement of the Catabolic Potential of Microbial Strains
in the Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7.1 Uptake of a Target Compound . . . . . . . . . . . . . . . . . . . . 122
7.2 Expansion of the Effector Specificity of Transcriptional Regulators 123
7.3 Alterations in Structural Genes . . . . . . . . . . . . . . . . . . . . 124
7.3.1 Widening of the Substrate Range . . . . . . . . . . . . . . . . . . . 124
7.3.2 Mutations in Structural Genes to Avoid the Formation
of a Toxic Metabolite . . . . . . . . . . . . . . . . . . . . . . . . . . 126
7.4 Use of External Genetic Information to Expand
the Substrate Range . . . . . . . . . . . . . . . . . . . . . . . . . . 127
7.4.1 Chlorobenzoate-Degraders by Conjugal Transfer . . . . . . . . . . 127
7.4.2 Chloronitrophenol-Degraders by Conjugal Transfer . . . . . . . . 128
7.4.3 Chlorobiphenyl-Degraders by Mating Three Strains . . . . . . . . 129
7.4.4 Other Chloroaromatic-Degraders by Conjugal Transfer . . . . . . 129
7.4.5 Chlorobenzoate- and Chlorosalicylate-Degraders
by Genetic Engineering Techniques . . . . . . . . . . . . . . . . . 130
7.4.6 Chlorobiphenyl-Degraders by Genetic Engineering Techniques . . 132
7.4.7 Trihalopropane-Degraders by Genetic Engineering Techniques . . 132
7.5 Construct to Degrade TCE Without Apparent Toxic Effect . . . . . 132
7.6 Creation of a Pathway for the Degradation
of Halogenated Alkanes and Alkenes . . . . . . . . . . . . . . . . . 134
7.7 Creation of a Pathway for the Degradation of Mixtures of Methyl-
and Chloroaromatics by Combination of Pathway Modules . . . . 134
7.8 Rsum: Enhancement of Catabolic Potential . . . . . . . . . . . . 136
8 Concluding Remarks and Outlook . . . . . . . . . . . . . . . . . . 136
9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
List of Abbreviations
BESA bromoethane sulfonic acid
BTEX benzene, toluene, ethylbenzene, xylenes mixture
cDCE or cis-1,2-DCE cis-dichloroethene
DDT 1,1,1-trichloro-bis(p-chlorophenyl)-ethane
DHB dihydrodihydroxybenzoate
EDTA ethylenediaminetetraacetic acid
GSH glutathione (reduced)
IP ionization potential
LiP lignin peroxidase
MnP manganese-dependent peroxidase
4 W. Reineke
NTA nitrilotriacetic acid
PAHs polycyclic aromatic hydrocarbons
PCBs polychlorinated biphenyls
PCE tetrachloroethene
PCP pentachlorophenol
PQQ methoxatin (2,7,9-tricarboxy-1H-pyrrolo(2,3-f )quino-
line-4,5-dione)
TCA cycle tricarboxylic acid cycle = Krebs cycle
TCE trichloroethene
VC vinyl chloride
2,4-D 2,4-dichlorophenoxyacetic acid
1
Introduction
1.1
Redox Processes and Mineralization of Organic Compounds
The biosphere presents a large diversity of different habitats within which
microorganisms can operate provided that an energy source and nutrients are
available and that physical conditions are appropriate. Organic compounds in
nature are distributed throughout aerobic and anaerobic environments. Anoxic
ecosystems are created when oxygen consumption by microorganisms exceeds
its supply, e. g., in soils with impeded drainage, stagnant water, municipal land-
fills, sewage treatment digesters, and sediments of the oceans and other natural
bodies of water. Different groups of microorganisms are found in oxic and an-
oxic situations. Although the biochemical pathways used by microorganisms to
degrade organic compounds are extremely diverse, they are all directed towards
the production of energy and carbon for growth.
Concerning energy-yielding processes, four types of microbial metabolism
are recognized and they are described by the terms photometabolism, fermen-
tation, aerobic respiration, and anaerobic respiration.
Fermentation is a process that does not require oxygen or the presence of
other electron acceptors such as NO
3
, Mn
4+
, Fe
3+
, SO
4
2
, or CO
2
and depends on
the ability of the microorganisms to use part of the organic molecule (often a
metabolite) as an electron acceptor. During fermentation of an organic com-
pound, reduced pyridine nucleotides (NADH) and adenosine triphosphate
(ATP) are produced by the degradative pathway. Such an energy-yielding
sequence of reactions, which is accompanied by substrate level phosphory-
lation, can only continue if the organism has some mechanism to regenerate
oxidized pyridine nucleotides as an acceptor for the further oxidation of the
organic substrate. Oxidized pyridine nucleotides are produced by the transfer
of electrons to intermediates which are formed during metabolism of the
growth substrates. The result of a typical fermentation is a mixture of products
which are more oxidized and others which are less oxidized than the original
substrate (Fig. 1).
1.2
Energy Yields
Metabolism of organic compounds by respiration leads to a much more effi-
cient use of potential chemical energy than fermentative conversion. During re-
spiration electrons in reduced pyridine nucleotides can be transferred to oxy-
gen in the case of aerobic respiration or to various other acceptors such as NO
3
,
Mn
4+
, Fe
3+
, SO
4
2
, or CO
2
in the case of anoxic respiration. The chemical energy
of the redox system is used for the production of a proton gradient when trans-
porting the electrons. The ATP synthetase then converts the energy of the pro-
ton gradient into chemical energy (ATP) (Fig. 2). Together, this constitutes two
6 W. Reineke
Fig. 1. Schematic illustration of the general scheme of fermentation with energy generation
by substrate level phosphorylation and NAD/NADH cycling
Fig. 2. Schematic illustration of the respiratory chain of energy conservation including the
proton translocation steps that establish a proton motive force which is used by the ATP syn-
thetase for ATP formation
to three molecules of ATP generated per electron pair that is channeled through
the electron transport chain.
During oxidative metabolism, the organic substrate is converted to carbon di-
oxide and water and part of it is assimilated into cell material. Molecular oxy-
gen is usually the preferred electron acceptor; it is reduced to water. The oxida-
tion of an organic substrate with oxygen or nitrate as the electron acceptors
leads to a similar high yield of ATP. The reduction of CO
2
to methane and sul-
fate to sulfide is carried out predominantly by strict anaerobes, whereas nitrate
reduction is carried out predominantly by facultative anaerobes if oxygen is not
available. The energetics of these processes are very different. The free energy
change of O
2
and nitrate reduction per two electrons is about the same while the
values are much lower for sulfate and CO
2
reduction (Table 1). This explains the
lower growth yields and rates on an organic substrate with sulfate-reducing
bacteria and methanogens as compared to aerobic bacteria and nitrate-reduc-
ing organisms.
Many complex organic compounds can be oxidized to CO
2
by pure cultures
of bacteria with NO
3
, Mn
4+
, Fe
3+
, or SO
4
2
as electron acceptor, whereas metha-
nogenic metabolism usually requires mixed cultures, since most methanogens
can only ferment simple low molecular weight organic molecules such as ace-
tate or methylamine. At least three physiological types of bacteria operate in
methanogenic systems: fermenters, which convert the initial substrate into or-
ganic acids, such as propionate, butyrate, acetate, formate, succinate, and lactate,
as well as alcohols; acetogenic proton-reducing bacteria, producing acetate; and
acetate and CO
2
plus hydrogen-consuming methanogens.
If light is present in anaerobic environments, photometabolism is also possi-
ble. The phototrophic bacteria use light as the energy source while substrates
such as organic acids (acetate, propionate, butyrate, succinate, glutarate, benzo-
ate) are usually extensively assimilated into cell material. Therefore, the oxida-
tive or fermentative metabolism of a portion of the carbon source is unneces-
sary.
Just recently, bacteria have been isolated and characterized that use either the
oxyanions of arsenate or selenate or both as terminal electron acceptors [1].
Table 1. Free energy changes in aerobic and anaerobic respiration using hydrogen as electron
donor
DG
o
Redox reaction (kJ/mol acceptor) (kJ/mol 2e
)
2 H
2
+ O
2
2 H
2
O 474 237
2.5 H
2
+ NO
3
+ H
+
0.5 N
2
+ 3 H
2
O 560 224
H
2
+ Mn
4+
Mn
2+
+ 2 H
+
187 187
0.5 H
2
+ Fe
3+
Fe
2+
+ H
+
30.9 61.8
4 H
2
+ SO
4
2
+ 1.5 H
+
0.5 H
2
S + 0.5 HS
+ 4 H
2
O 153 38.3
4 H
2
+ CO
2
CH
4
+ 2 H
2
O 135 33.8
1.3
Distribution of Electron Acceptors in the Environment
and Sequential Redox Conditions
The natural abundance of major environmental electron acceptors is summariz-
ed in Table 2, including the concentrations commonly encountered in terrestrial
and aquatic environments. With the exception of sulfate, which is very abundant
in seawater and low but variable in freshwater, the concentrations of the other el-
ectron acceptors are similar in both environments. Both in soil and freshwater, el-
ectron acceptors such as oxidized nitrogen or sulfur compounds or oxidized me-
tal ions such as Fe
3+
or Mn
4+
can serve in anaerobic respiration. In waters of neu-
tral pH, solid forms of Mn and Fe will dominate. The concentration of CO
2
varies
depending on the alkalinity and the organic carbon input into the ecosystem.
Oxygen is the preferred electron acceptor, in agreement with what would be
expected in view of thermodynamics. If the organic carbon influx into an envi-
ronmental compartment such as groundwater is higher than that of oxygen,
anaerobic conditions will occur. Then there is sequential utilization of the al-
ternative electron acceptors NO
3
, MnO
2
(s), Fe(OH)
3
(s), SO
4
2
, and CO
2
in that
order, creating different redox zones (Fig. 3).
The sequential use of electron acceptors is important for biodegradation,
since some compounds can only be converted by certain microorganisms, i. e.,
under certain redox conditions. Consequently, a chemical present as a ground-
water contaminant is likely to be degraded only in that part of the groundwater
plume where favorable redox conditions prevail. Some compounds are degrad-
able under any specific redox conditions, but the degradation rate of such com-
pounds may vary under different redox conditions. Table 3 summarizes data
which roughly show the degradation kinetics occurring at various redox condi-
tions for some compounds. Comparing chlorinated aromatic and chlorinated
aliphatic compounds with a low and high degree of substitution, it appears that
lower chlorinated compounds will generally be degraded rapidly under aerobic
8 W. Reineke
Table 2. Natural abundance and free energy yield of commonly used electron acceptors
Electron acceptor Natural abundance Free energy
(kJ/mol glucose)
Oxygen 300 mmol/l 3190
Nitrate Few mmol/l 3030
Manganese, MnO
2
(birnessite) <mmol/l to >mmol/l 3090
Manganese, MnO
2
(nsutite) <mmol/l to >mmol/l 3050
Manganese, MnO
2
(pyrolusite) <mmol/l to >mmol/l 2920
Nitrate Few mmol/l 2750
Iron, Fe
2
O
3
(hematite) <mmol/l to >mmol/l 1410
Iron, FeOOH (geothite) <mmol/l to >mmol/l 1330
Sulfate <100 mmol/l (freshwater), 380
~28 mmol/l (seawater)
Carbon dioxide Variable 350
Free energies are taken from Froelich et al. [2]. The mineralogical form of the Mn
4+
and Fe
3+
oxides can alter the redox potential significantly.
Fig. 3. Transport of chemicals and microbial reactions in the groundwater with respect to the
presence of electron acceptors and prevailing redox conditions (according to Bouwer and
Zehnder [3] and Schachtschabel et al. [4])
conditions, while the higher chlorinated analogs are preferentially dechlorina-
ted under anaerobic conditions. Table 3 illustrates that a broad variety of com-
pounds are subject to aerobic degradation, while compounds such as hexa-
chlorobenzene are degraded in groundwater only at the most reduced part of
the redox regime. Overall the degradative potential of anaerobic microbial com-
munities is much greater than appreciated until only recently.
The existence of electron acceptor profiles in the above-mentioned order and
their associated microbial processes in the different redox zones of the subsurface
can also be observed in lakes and the marine environment as a function
of water depth, as in the Black Sea, where oxygen reduction only occurs in the top
layer of about 50 m and alternative electron acceptors are used at greater depths.
1.4
Organic Compounds as Electron Acceptors
Besides the inorganic compounds mentioned above, several organic com-
pounds can also serve as electron acceptors in anaerobic respiration [5].
Fumarate respiration is the most widespread type of anaerobic respiration.
Fumarate is formed during the biodegradation of carbohydrates and proteins
and its reduction to succinate is linked to a respiratory chain. Dimethyl-
sulphoxide can be used by many other microorganisms as an electron acceptor
for an anaerobic electron transport with dimethylsulphide as the product. Even
man-made chemicals such as tetrachloroethene and some chloroaromatic com-
pounds such as chlorobenzoates and chlorophenols can function as electron ac-
ceptors in a dehalorespiration (these will be discussed later in detail).
1
0
W
.
R
e
i
n
e
k
e
Table 3. A general picture of microbial transformations of organic compounds at various redox conditions
Oxygen reduction Nitrate reduction Manganese Iron reduction Sulfate reduction Carbon dioxide
reduction reduction
Aromatics:
Benzene (very fast) Benzene Benzene (very slow) Benzene (very slow)
Toluene (very fast) Toluene Toluene Toluene Toluene Toluene
Ethylbenzene (very fast)
Xylenes (very fast) Xylenes
Biphenyl (very fast) Biphenyl (very slow)
Naphthalene (very fast) Naphthalene (slow) Naphthalene (slow) Naphthalene (very slow)
Polycyclic aromatic hydrocarbons Phenanthrene
up to 4 rings (very fast to slow) (very slow)
Benzoate (very fast) Benzoate (fast) Benzoate (fast) Benzoate (fast)
Phenol (very fast) Phenol (slow) Phenol (slow)
Cresols (very fast) Cresols (slow)
Aniline (very fast) Aniline (slow)
Chloroaromatics:
Chlorobenzoates (very fast)
Monochlorotoluenes (very fast)
Mono-, di-, tri-, pentachlorophenol Chlorophenols (slow) Chlorophenols (slow)
(very fast to fast)
Chloroanilines (very fast)
Mono-, dichlorobiphenyls (very fast)
Mono-, di-, tri-, tetrachlorobenzene
(very fast)
Aliphatics:
Propane
C
10
C
22
n-alkanes n-Hexadecane
2,6,10,14-Tetramethylpentadecane (very slow)
Alkenes
A
e
r
o
b
i
c

a
n
d

A
n
a
e
r
o
b
i
c

B
i
o
d
e
g
r
a
d
a
t
i
o
n

P
o
t
e
n
t
i
a
l
s

o
f

M
i
c
r
o
o
r
g
a
n
i
s
m
s
1
1
Table 3 (continued)
Oxygen reduction Nitrate reduction Manganese Iron reduction Sulfate reduction Carbon dioxide
reduction reduction
Haloaliphatics:
Haloalkanes Haloalkanes Haloalkanes
Haloalkenes Haloalkenes Haloalkenes
Haloalkanols
Haloalkenols
Haloalkanoates (very fast)
Haloalkenoates
Classification of growth rate (t
d
) of population: very fast degradation: hours; fast degradation: <1 day; slow degradation: >1 day, <7 days; very slow
degradation: >3 weeks
1.5
Limitations to Fermentation
Most anoxic terrestrial and subsurface environments do not receive sufficient
influx of external electron acceptors for the oxidation of the organic com-
pounds that are present. This leaves fermentation as the only possible physiolo-
gical process for biodegradation. However, many compounds cannot be simply
fermented by pure bacterial cultures for biochemical and energetic reasons.
This is illustrated for some aromatic compounds. Fermentative degradation of
benzoate, phenol, and monohydroxybenzoates to acetate, hydrogen, and carbon
dioxide is not possible in pure culture, since the reactions are endergonic under
standard conditions [68] (Table 4). These fermentations become possible only
if a hydrogen-utilizing methanogenic bacterium keeps the hydrogen partial
pressure low (about 10
4
bar) to render the reactions exergonic (Table 5). Such
so-called syntrophic couplings between different metabolic types of bacteria
are widespread in methanogenic degradative processes [9]. However, the syn-
trophic coupling between fermentative and methanogenic bacteria leads to very
low yields of energy compared to oxidation of benzoate with other electron
acceptors (Table 6). In contrast to the non- and monohydroxylated aro-
12 W. Reineke
Table 4. Fermentation of aromatics
Overall reaction Free energy, DG
o
C
7
H
5
O
2
+ 7H
2
O 3CH
3
COO

+ 3H
+
+ 3H
2
+ HCO
3
+70.6 kJ/mol benzoate

C
7
H
5
O
3
+ 6H
2
O 3CH
3
COO

+ 3H
+
+ 2H
2
+ HCO
3
+5.4 kJ/mol p-hydroxybenzoate

C
6
H
6
O + 5H
2
O 3CH
3
COO

+ 3H
+
+ 2H
2
+6.6 kJ/mol phenol
Table 5. Fermentation of aromatics coupled to methanogenesis
o
C
7
H
6
O
2
+ 4.5H
2
O 3.25CO
2
+ 3.75CH
4
+ H
+
159 kJ/mol benzoate
C
7
H
6
O
3
+ 3.5H
2
O + H
+
3.25CO
2
+ 3.75CH
4
178 kJ/mol p-hydroxybenzoate
C
6
H
6
O + 3.5H
2
O + H
+
2.25CO
2
+ 3.75CH
4
140 kJ/mol phenol
Table 6. Energetics of the oxidation of benzoate coupled to anaerobic respiration (according
to Fuchs et al. [10])
o
Nitrate respiration:
C
7
H
6
O
2
+ 15NO
3
7CO
2
+ 15NO
2

+ 3H
2
O 2116 kJ/mol benzoate
Sulfate respiration:
C
7
H
6
O
2
+ 3.75SO
4
2
+7.5H
+
7CO
2
+ 3.75H
2
S + 3H
2
O 327 kJ/mol benzoate
Carbonate respiration:
C
7
H
6
O
2
+ 4.5H
2
O 3.25CO
2
+ 3.75CH
4
+ H
+
159 kJ/mol benzoate
matics mentioned above, the fermentation of two- or threefold hydroxylated
aromatics is sufficiently exergonic to allow degradation in pure culture [11, 12]
(Table 7).
1.6
Various Degrees of Degradation
Evidence for a microbial role in the transformation of organic chemicals by
samples from the natural environment such as soil, water, and sediments can be
obtained by demonstrating that the compound is transformed in nonsterile but
not in sterilized samples. Chemicals are usually subject to a variety of microbial
reactions, resulting in various types and degrees of degradation. With 4-chloro-
biphenyl as a model compound, the different types of processes which can take
place have been schematically described (Fig. 4).
Biodegradation is often a growth-linked process that brings about total
(complete) degradation or mineralization. As the microorganisms convert the
Table 7. Fermentation of phenolic aromatic compounds
o
C
7
H
5
O
4

+ 5H
2
O 3CH
3
COO

+ 3H
+
+ H
2
+ HCO
3
77.8 kJ/mol protocatechuate

C
7
H
5
O
5

+ 4H
2
O 3CH
3
COO

+ 3H
+
+ HCO
3
160.0 kJ/mol gallate

C
6
H
6
O
2
+ 4H
2
O 3CH
3
COO

+ 3H
+
+ H
2
78.1 kJ/mol catechol
C
6
H
6
O
3
+ 3H
2
O 3CH
3
COO

+ 3H
+
158.3 kJ/mol pyrogallol
Fig. 4. Comparison of the degree of degradation of 4-chlorobiphenyl as a model compound
by cometabolism, by partial degradation, by the use of the compound as an electron accep-
tor, and by total degradation processes. Compound (middle gray), chlorosubstituted metabo-
lite (light gray), biphenyl (dotted line), chloride (dark gray), and cells (pointed line)
organic substrate to fermentation products or carbon dioxide, energy is
released and fixed in the form of ATP, as described in the first sections.
Simultaneously, the organisms convert some of the carbon in the substrate to
cell constituents, using ATP and NAD(P)H produced by oxidation reactions for
these biosynthetic reactions. As a result, the populations increase in numbers
and biomass. Organic chlorine is returned to the mineral state during the min-
eralization process.
With many chemicals a type of microbial conversion quite different from
mineralization takes place. The phenomenon that chemicals are subject to
microbial action and yet do not sustain growth of the organisms has been term-
ed cometabolism, or sometimes cooxidation or fortuitous oxidation [1316].
The responsible organisms are presumably growing on another substrate while
performing cometabolic transformation reactions. The microorganisms in-
volved in cometabolic transformation do not increase in numbers or biomass
as a result of the degradation of the chemical of interest. This lack of growth is
a reflection of the inability of the organisms to use the chemical for energy gen-
eration or biosynthetic purposes, and it is in marked contrast to the increase in
population size or biomass when a mineralizable substrate is introduced into
the same sample. Because populations are usually small, a compound subject to
cometabolism is modified slowly, and the rate of degradation does not increase
with time. The product of cometabolism will often be used by other micro-
organisms.
In some cases the conversion of organic chemicals may support microbial
growth even though no complete mineralization occurs. This was observed,
for example, during the degradation of 4-chlorobiphenyl. Population increase
and chemical disappearance were observed when aerobic biphenyl-degrading
populations were provided with 4-chlorobiphenyl. In contrast to a substrate
that is mineralized, the bacteria could only use part of the molecule (the
nonsubstituted ring) as a carbon and energy source for growth. The bacterial
population size and the rate of decline in concentration of the chemical in-
creased over time. However, elimination of the chlorine substituent, which
would be typical for mineralization, did not take place and the chlorine-con-
taining metabolite 4-chlorobenzoate accumulated. This process is termed par-
tial degradation.
Microbial populations present in anaerobic sediments are often able to deal
with chlorinated chemicals in a quite different way. Compounds such as chloro-
aromatics and chloroaliphatics can function as electron acceptors in a type of
anaerobic respiration [1719]. Evidence for the occurrence of such a process in
natural ecosystems is provided by the observation that reductive dechlorination
does occur in nonsterile but not in sterilized samples. The addition of electron
donors such as pyruvate or hydrogen stimulates the process. This type of re-
ductive dechlorination of 4-chlorobiphenyl has never been studied but in ana-
logy to the observation with chlorobenzoates, chlorophenols, and tetrachloro-
ethene, giving the non- or lower chlorinated products, the formation of bi-
phenyl may occur. Reductive dechlorination proceeds relatively easily with
highly chlorinated compounds and the use of a chlorinated chemical as an
electron acceptor is sometimes called dehalorespiration.
14 W. Reineke
1.7
Rsum of Introduction
Redox processes are of key importance for microbial activity and they usually
require an electron acceptor in addition to the organic substance itself, since the
possibilities of fermentation are restricted. Thus, the availability of an electron
acceptor is essential. Various compounds such as aromatic and aliphatic com-
pounds as well as their chlorinated analogous may be used.
After the description of the more general subjects of microbial metabolism,
the degradative properties of microorganisms will be discussed in more detail
for some selected chemicals. Aromatic and chloroaromatic compounds have
been chosen to document the differences in the degradation that are related to
the presence or absence of special substituents on the aromatic ring. In addi-
tion, the degradation of aliphatic and chloroaliphatic compounds will be dis-
cussed because of the occurrence of these chemicals as pollutants in the en-
vironment. This will be used to illustrate the potential but also the limitations
of some microbial systems in complete degradation. The abundance of reac-
tions involved in the degradative pathways, and also the widespread use of va-
rious analogous reactions, will be discussed. A comparison of pathways used by
aerobic and anaerobic organisms, as well as the differences between bacteria
and fungi with respect to biodegradation, are subjects of the following sections.
2
Degradation of Aromatic Compounds
2.1
Aerobic vs Anaerobic Degradation: Introduction
Aromatic compounds can be degraded under aerobic and anaerobic conditions.
In both cases a key step is the activation of the inert aromatic ring. In the pre-
sence of oxygen this is carried out by oxygen-dependent enzymes. Successive
addition of two oxygen atoms to the ring or direct introduction of molecular
oxygen leads to the formation of diphenolic compounds. These compounds are
then cleaved by enzymes that use molecular oxygen again. While bacteria uses
oxygen for ring activation and cleavage, ligninolytic fungi bring about the ac-
tivation by use of hydrogen peroxide. The formation of an aryl cation radical is
then followed by spontaneous rearrangements and degradation. In contrast,
non-ligninolytic fungi use oxygen to activate the aromatic ring which is follow-
ed by coupling to give O-conjugates. Thus, oxygen plays a vital role both as a ter-
minal electron acceptor and as reagent in the biochemical activation of inert
aromatic compounds under oxic conditions.
Under anoxic conditions various inorganic compounds can function as an
electron acceptor instead of oxygen, but there is apparently no equivalent re-
placement for oxygen with respect to its function in activation reactions.
Therefore, the anaerobes have to degrade aromatic compounds by reductive
conversions. In the anaerobic conversion central intermediates ready for hydro-
lytic ring cleavage bear a 1,3-dioxo structure in the alicyclic ring when resorci-
1
6
W
.
R
e
i
n
e
k
e
Fig. 5. General scheme for the metabolism of aromatic compounds by bacteria under oxic (left) and anoxic (right), and by fungi
under oxic conditions (middle)
nol or phloroglucinol are the central metabolites. When benzoyl-CoA is the cen-
tral metabolite after the channeling reactions, one oxo group is in the alicyclic
ring and the other exocyclic in the ester group.
Thus, different degradation sequences are used under oxic and anoxic con-
ditions, as schematically illustrated in Fig. 5.
2.2
Aerobic Degradation of Aromatics: General Differences Between Prokaryotic
and Eukaryotic Organisms in the Initial Reactions
Higher organisms, including fungi, have evolved a different enzyme system for
the oxidation of aromatic hydrocarbons from that observed in bacteria (Fig. 6).
Most non-ligninolytic fungi are able to oxidize aromatic hydrocarbons by a
cytochrome P-450 monooxygenase. One atom of the oxygen molecule is incor-
porated into the aromatic substrate, while the other oxygen atom is reduced to
water. The arene oxide formed then becomes a substrate for further meta-
bolism. The enzymatic hydration of the arene oxide leads to the formation of a
dihydrodiol with a trans configuration (Fig. 6). Another pathway involves iso-
Fig. 6. Differences in the initial reactions used by prokaryotic and eukaryotic organisms for
the oxidation of aromatic hydrocarbons. The upper reactions 14 are used by fungi: cyto-
chrome P-450 monooxygenase; non-enzymatic rearrangement; coupling reactions;
epoxide hydrolase. The lower reactions are used by bacteria: dioxygenase; cis-dihydro-
diol dehydrogenase; and monooxygenase, ring fission pathways starting with a di-
oxygenase
merization of the arene oxide to form a phenol that can be conjugated with sul-
fate, glucuronic acid, or glucose.
The following two different routes are used by bacteria:
1. An aromatic compound without a hydroxyl group is activated through a di-
oxygenase using molecular oxygen. Subsequently, the resulting cis-dihydro-
diol is converted by a dehydrogenase to give a 1,2-diphenol derivative, re-
gaining the aromatic state. The diphenol is the substrate for ring cleavage, a
reaction which requires another molecule of oxygen.
2. The use of successive monooxygenase reactions has also been described. In
this case, the diphenol is produced in two steps, and the hydroxyl groups
need not to be on adjacent carbon atoms. In contrast to the reaction in the
eukaryotic organisms, no epoxides are formed as intermediates.
Details of the further routes after ring cleavage will be discussed later.
2.3
Degradation of Aromatic Compounds by Aerobic Bacteria
Enzymes occurring in pathways for the aerobic degradation of aromatic com-
pounds have to fulfil various functions. An important point is to overcome the
chemical stability of the aromatic ring. Besides ring activation and cleavage,
further degradative steps have to achieve the formation of common interme-
diates of cell metabolism by cleavage of carbon-carbon bonds and by proces-
sion of functional groups or side-chains of substituted aromatics and multi-
substituted aromatics arising from bi- and polycyclic aromatics. I have focused
on the chemical logic of the overall metabolic pathways to illustrate how a few
types of chemical mechanisms are used by the organisms in linked, sequential
reactions to meet the above requirements.
2.3.1
Reactions Converting Aromatic Compounds into Ring Cleavage Substrates
Aerobic bacteria and especially pseudomonads are able to use various com-
pounds as their source of carbon and energy, whose chemical structures con-
tain one or more benzene rings. Ring cleavage is an important enzymatic step
for the degradation of aromatics. With rare exceptions the introduction of two
hydroxyl groups onto a benzene ring is a prerequisite for ring opening. The sub-
strates for ring cleavage may be 1,2-diphenolic compounds such as catechol,
protocatechuate, and catechol derivatives, which bear either substituents such
as a phenyl, a hydroxy phenyl, and a phenoxy group, or an additional aromatic
ring condensed to the first one (see Fig. 7). Gentisate, a 1,4-diphenolic com-
pound, is a ring cleavage substrate too.
2.3.1.1
Formation of Diphenols from Monocyclic Hydrocarbons
The conversion of a hydrocarbon to a catecholic ring fission substrate involves
two important enzymatic steps: a dioxygenase catalyzes the formation of a cis-
18 W. Reineke
dihydrodiol, which is then oxidized to the corresponding catechol by a dehy-
drogenase. This occurs, for example, with benzene and benzoate degradation
(Fig. 8). In some cases only a dioxygenase is needed to produce a catechol, for
example during aniline degradation, where a 1,2-diphenol is formed by an ani-
line dioxygenase which concomitantly eliminates NH
3
. In phenol and salicylate
degradation a monooxygenase adds one atom of molecular oxygen to the sub-
strate to give catechol, while the other oxygen atom is reduced to water.
The degradation pathways of m-cresol, naphthalene, and phthalate converge
via protocatechuate and gentisate. Monooxygenase as well as dioxygenase reac-
tions are involved in the formation of these diphenols (Fig. 9). The degradation
of salicylate can proceed via catechol (Fig. 8) or gentisate as the ring cleavage
substrate (Fig. 9) depending on the organism.
Aromatic compounds which bear alkyl substituents on the aromatic ring may
undergo oxidation of the side chain before ring activation. Toluene is a good
compound to illustrate the wide variety of reactions which can take place in re-
aching a diphenolic structure (Fig. 10). Five different routes for toluene degra-
dation have been found: (1) side chain oxidation by a monooxygenase and two
dehydrogenases to give the carboxylic acid, which is further degraded by the di-
oxygenase pathway; (2+3) two monooxygenase reactions plus side chain oxida-
tion to form protocatechuate these monooxygenase reactions may start at C-2
or C-3; (4) two subsequent monooxygenase reactions activating the aromatic ring
leading to 3-methylcatechol; (5) the same methylsubstituted catechol is formed
when a dioxygenase and a dehydrogenase are involved in ring activation.
2.3.1.2
Formation of Diphenols from Bi- and Polycyclic Hydrocarbons
The presence of analogous reactions in the degradation pathways of aromatic
compounds has been shown for binuclear compounds like biphenyl, dibenzo-
furan, dibenzo-p-dioxin, and naphthalene (Fig. 11). The general scheme is the
following: ring activation by a dioxygenase, rearomatization by a dehydrogen-
ase to give a 1,2-diphenol, ring cleavage by dioxygenase, fission of the aliphatic
side chain by a hydrolase to give, on the one side, a-oxo acids such as pyruvate
or compounds which are metabolites of the meta pathway (will be discussed la-
ter), such as 4-oxalocrotonate and 2-oxopent-4-enoate and, on the other side, sali-
Fig. 7. Diphenolic compounds that can undergo ring fission
20 W. Reineke
Fig. 8. Degradative steps for monocyclic aromatic compounds that lead to catechol [2028].
benzene; aniline; salicylate; phenol; benzoate. Catechol, the central metabolite,
is further degraded via the meta or ortho pathway
Fig. 9. Sequences for the bacterial degradation of aromatic compounds that converge via pro-
tocatechuate and gentisate: m-cresol via m-hydroxybenzoate and protocatechuate or genti-
sate, p-cresol via protocatechuate; naphthalene via salicylate and gentisate, phthalate via 4,5-
dihydroxyphthalate [2938]. phthalate dioxygenase; 4,5-dihydroxyphthalate decarboxy-
lase; p-hydroxybenzoate hydroxylase; salicylate hydroxylase
cylaldehyde, salicylate, catechol or benzoate. During the degradation of binuclear
aromatics, the formation and cleavage of a 1,2-diphenol takes place twice.
In the degradative pathway of dibenzo-p-dioxin and dibenzofuran the cleav-
age of the stable ether bond is an important step. Interestingly, no special en-
zyme is necessary to bring about this reaction. Angular dioxygenation by the
ring activating enzymes, i. e., oxygenation at a pair of vicinal carbon atoms, one
of which is involved in the bridge between the two aromatic rings, forms cis-
dihydrodiols, which are hemiacetals so that because of their instability the ether
bond is cleaved spontaneously. Therefore, a dehydrogenase reaction, normally
bringing about rearomatization, is unnecessary.
Various polycyclic aromatic hydrocarbons (PAHs) containing up to four
rings, such as pyrene and chrysene, can be mineralized by pure bacterial cul-
Fig. 10. Different routes for the aerobic bacterial mineralization of toluene via catechol, 3-me-
thylcatechol, and protocatechuate. Enzymes: , toluene 2-monooxygenase [39]; , toluene 3-
monooxygenase [40]; , toluene dioxygenase [41]; , toluene cis-dihydrodiol dehydrogen-
ase [42, 43]; , xylene monooxygenase [44]; , benzylalcohol dehydrogenase [45]; , benz-
aldehyde dehydrogenase [46]; , benzoate dioxygenase [47]; , benzoate cis-dihydrodiol
dehydrogenase [48]; , toluene 4-monooxygenase [49, 50]; ; 4-cresol dehydrogenase [51];
, 4-hydroxybenzaldehyde dehydrogenase [51]; , 4-hydroxybenzoate hydroxylase [51]
tures [6374]. Because of the different degrees of solubility (Table 8), the de-
gradation rates vary strongly, with the higher molecular weight PAHs being de-
graded very slowly. These latter PAHs tend to resist degradation and bioaccu-
mulate in biological material due to the hydrophobic character. Complete deg-
radation pathways have been elucidated for anthracene and phenanthrene.
Various metabolites of acenaphthene, acenaphthylene, fluorene, fluoranthene,
pyrene, and benzo[a]anthracene have been identified, allowing some clues on
the catabolic pathways [67, 69, 7882]. The degradation generally follows the
same pathway as described above for the bicyclic aromatic hydrocarbons: ring
activation, rearomatization, ring cleavage, and subsequent fission of the ali-
phatic side chain to give pyruvate and an aromatic ortho-hydroxy acid after oxi-
dation of the aldehyde group. Then decarboxylation yields a diphenolic deriva-
tive of the remaining aromatic ring which is subject to the next cycle for the eli-
mination of an aromatic ring. This cycle of reactions is illustrated for the linear
and angular PAHs, anthracene and phenanthrene, respectively, in Fig. 12.
22 W. Reineke
Fig. 11. Degradation sequences of bicyclic hydrocarbons that converge at catechol. naph-
thalene [52, 53]; dibenzofuran; dibenzo-p-dioxin [5459]; biphenyl [26, 27, 6062].
Catechol, the central metabolite, is further degraded via the meta or ortho pathway. The sites
of cleavage of C-C bonds are marked

Table 8. Polycyclic aromatic hydrocarbons used by pure cultures of bacteria as the sole source
of carbon and energy and important physicochemical properties
Compound Structure Aqueous Octanol/ Degrada- Ionization
solubility water co- tive path- potential
(mg/l) efficient way (eV)
at 25C (log K
ow
)
Naphthalene 31.7 3.37 ++ 8.12
Biphenyl 7.0 3.9 ++
Acenaphthene 3.42 4.33 + 7.61
Acenaphthylene 3.93 4.07 +
Fluorene 1.98 4.18 ++
Anthracene 0.075 4.45 ++ 7.43
Phenanthrene 1.6 4.46 ++ 8.03
Fluoranthene 0.265 5.33 + 7.85
Pyrene 0.148 5.32 + 7.53
Chrysene 0.002 5.61 + 7.81
Benzo[a]anthracene 0.014 5.61 + 7.56
++, well established; +, metabolites identified.
Data compiled from [7577].
24 W. Reineke
Fig. 12. Degradative pathway for anthracene and phenanthrene: path a Pseudomonas [83];
path b Aeromonas, Alcaligenes, Micrococcus, Mycobacterium, Vibrio [8489]
Two modes of initial PAH degradation may occur, as has been shown for
fluorene (Fig. 13). The first pathway, which involves dioxygenation and meta
cleavage of one aromatic ring, with the subsequent release of pyruvate, oxida-
tion of the aldehyde group, followed by decarboxylation [73, 9093], is analo-
gous to the pathways described for phenanthrene and anthracene. An important
reaction is the biological Baeyer-Villiger oxidation of the cyclic ketone, inda-
none, yielding 3,4-dihydrocoumarin, which is then subject of hydrolytic fission
to form 3-(2-hydroxyphenyl) propionate.
The second pathway is initiated by monooxygenation at C-9 to give 9-fluore-
nol, which is then dehydrogenated to 9-fluorenone (Fig. 13). These reactions
Fig. 13. Degradation of fluorene. Pathway (a) with initial dioxygenase; oxidation of cyclic
ketones by Baeyer-Villiger oxidation. Pathway (b) with initial monooxygenase. Dashed ar-
rows indicate two or more successive reactions
precede dioxygenase attack adjacent to the carbonyl group to form the angular
diol 1,1a-hydroxy-1-hydro-9-fluorenone [54, 94]. Cleavage of the five-member-
ed ring then generates a substituted biphenyl, whose further degradation by
reactions similar to those of biphenyl catabolism leads to the formation of
phthalic acid [95, 96]. Phthalate can be further metabolized via 4,5-dihydroxy-
phthalate, protocatechuate, and b-carboxy-cis,cis-muconate [95].
2.3.2
Ring Fission and Carbon Chain Fission
2.3.2.1
Ring Cleavage
There are two distinct modes of oxidative cleavage of the benzene nucleus.
Cleavage of the bond between adjacent carbon atoms that carry hydroxyl
groups is know as ortho or intradiol cleavage (Fig. 14). The pathway by which
the product of such a cleavage is metabolized is termed the ortho or b-ketoadi-
pate pathway. Ortho cleavage of a carbon-carbon bond is catalyzed by a catechol
1,2-dioxygenase or a protocatechuate 3,4-dioxygenase.
In the second mode of fission of the benzene nucleus C-C-bond cleavage oc-
curs between two carbon atoms of which one carries a hydroxyl group and the
other carries another substituent or a hydrogen atom. In this case, the hydroxyl
groups may be either ortho or para to one another, and the enzymes catalyzing
such cleavage reaction are again designated by the position of the carbon bond
which is cleaved. Thus, catechol and protocatechuate are cleaved, respectively,
26 W. Reineke
Fig. 14. Mode of ring fission
by a catechol 2,3-dioxygenase or by a protocatechuate 4,5-dioxygenase or a pro-
tocatechuate 2,3-dioxygenase. Gentisate is cleaved by gentisate 1,2-dioxygenase.
The 1,2-diphenols derived from biphenyl, naphthalene, dibenzofuran, and di-
benzodioxin are all cleaved by meta-cleaving dioxygenases.
2.3.2.2
Carbon Chain Carbon-Carbon Fission
Following ring fission, further reactions yield pyruvate and/or other a-keto
acid intermediates. A key step is carbon-carbon bond cleavage, which may in-
volve stabilization of carbanionic intermediates or transition states. The carbon
chain is cleaved by aldol fission, hydrolytic fission, decarboxylation, or thiolytic
fission after formation of compounds that carry in a b-position to a carbonyl
function a hydroxyl, an oxo, or a carboxyl group (Fig. 15).
2.3.2.3
Aldol Cleavage
Substrates for aldol cleavage have the partial structure shown in Fig. 16.
Oxygen as an electronegative atom stabilizes the incipient negative charge
density during the reaction, to give the enolate as the initial product. Therefore,
an aldehyde and a ketone are the fission products as has been shown with com-
pounds A and B in Fig. 15.
Fig. 15. Carbon chain carbon-carbon bond fission substrates
2.3.2.4
Hydrolytic Fission
The fission of fumarylpyruvate (compound C) is a good example to illustrate
this type of cleavage to occur from the 1,3-dioxo structure (Fig. 17). Inspection
of the fumarylpyruvate skeleton suggests that H
2
O could be added nucleophili-
cally to the keto group of the fumaryl moiety to form a tetrahedral adduct. The
tetrahedral adduct in equilibrium with the dicarbonyl is competent for CC
cleavage, since it could (in a low-energy path) expel the stable enolate anion of
pyruvate and fumarate.
The same type of reaction producing a carboxylic acid and a ketone was
found for compounds D and E in Fig. 15.
2.3.2.5
Decarboxylation
A b-keto group facilitates decarboxylation by stabilizing the carbanionic tran-
sition state as decarboxylation proceeds (Fig. 18). The ready possibility for eno-
lization allows the b-carbonyl group to act as an electron sink, and ketonization
follows. This type of reaction is plausible for compound F in Fig. 15.
2.3.2.6
Vinylogy
Some cleavage substrates have no b-dicarbonyl structure. However, the inser-
tion of a CH=CH group between two functional groups produces a vinylo-
gous compound whose property is similar to the compound without the vinylic
group. The concept is called principle of vinylogy [97]. Therefore compound F,
formed in the meta pathway, is a vinylogous b-keto acid ready for decarboxyla-
tion, while compounds D and E are vinylogous to 1,3-dicarbonyl compounds
which are cleaved by a hydrolytic type of reaction (Fig. 15).
28 W. Reineke
Fig. 16. Aldol cleavage
Fig. 17. Hydrolytic cleavage of fumarylpyruvate
2.3.3
Pathways as a Whole for Catechol, Protocatechuate, and Gentisate
2.3.3.1
Gentisate Pathway
The first mode of preparation for fission between carbon atoms, i. e., forming a
carbonyl group in a b-position to another carbonyl, is seen in the gentisate
pathway (Fig. 19). After the benzene nucleus has been cleaved by a gentisate 1,2-
dioxygenase, one of two reaction pathways is available depending upon the na-
ture of the organism. Some organisms use a sequence in which the ring fission
product, maleylpyruvate, is first isomerized to fumarylpyruvate. Both com-
pounds are in equilibrium with the respective 1,3-diketones which are suitable
substrates for hydrolysis whereas a 1-keto-3-hydroxy grouping permits aldol
fission to occur. The point of ring fission, together with the positioning of the
hydroxyl groups in gentisate, determine that 1,3-diketone substrates will be
furnished for hydrolysis. The function of the isomerase is to ensure that an in-
termediate of the tricarboxylic acid cycle, fumarate, results from the next reac-
tion. In other organisms this isomerase is not present: maleylpyruvate is hydro-
lyzed directly to give maleate which is then hydrated to form D-malate.
2.3.3.2
meta Pathway
In meta fission sequences of catechol the carbon chain is prepared for the fis-
sion into pyruvate and acetaldehyde the following way (Fig. 20). After ring fis-
sion by catechol 2,3-dioxygenase to give 2-hydroxymuconic semialdehyde, cate-
chol is metabolized by two routes. For the particular organisms studied the oxi-
dative route is quantitatively the more important for catechol. The enzyme
catalyzed keto-enol change is known to precede the decarboxylation step since
a vinylogous b-keto acid is formed. Both routes converge to 2-oxopent-4-eno-
ate, which is hydrated to introduce a hydroxyl group at C-4. The carbonyl group
at C-2 of the resulting 4-hydroxy-2-keto acid (4-hydroxy-2-oxovalerate) was
formed by ketonization of the hydroxyl group originally present in the ring fis-
sion substrate and its product. This carbonyl also appears in the pyruvate
molecule formed from the hydroxyketo acid by aldol cleavage.
Fig. 18. Decarboxylation of b-keto acids
2-Hydroxymuconic semialdehyde is also a metabolite in the degradation of
protocatechuate formed by decarboxylation of 2-hydroxy-4-carboxymuconic
semialdehyde.
2.3.3.3
b-Ketoadipate Pathway (3-Oxoadipate)
The b-ketoadipate or ortho fission pathway (Fig. 20) is a multistep convergent
metabolic route used by many microorganisms to convert either of two com-
pounds, protocatechuate and catechol, to succinyl-CoA and acetyl-CoA
[105107]. Reactions of the protocatechuate and the catechol branch both give
rise to the common intermediate, b-ketoadipate enol-lactone. The reactions of
30 W. Reineke
Fig. 19. Gentisate pathway [29, 30, 32, 36, 98104]. The following enzymes are involved:
gentisate 1,2-dioxygenase, GSH dependent or GSH independent isomerase; maleylpy-
ruvate hydrolase; fumarylpyruvate hydrolase, maleate hydratase; fumarase
Fig. 20. Meta and ortho cleavage pathways for degradation of protocatechuate and catechol.
The meta cleavage pathway is illustrated on the left side, while the ortho (or b-ketoadipate)
pathway is given on the right side. Divergence is determined by the nature of the dioxygen-
ases which cleave each diphenolic substrate. The mode of cleavage is shown by a line of stars.
The following enzymes are involved in the meta pathway [108113]: catechol 2,3-dioxyge-
nase; 2-hydroxymuconic semialdehyde dehydrogenase; 4-oxalocrotonate isomerase;
4-oxalocrotonate decarboxylase; 2-hydroxymuconic semialdehyde hydrolase; 2-oxo-
pent-4-enoate hydratase; 4-hydroxy-2-oxovalerate aldolase; acetaldehyde dehydrogen-
ase (acylating). Enzymes of the ortho pathway are as follows [106, 109, 114118]: a, catechol
1,2-dioxygenase; a, protocatechuate 3,4-dioxygenase; b, muconate cycloisomerase (muco-
nate lactonizing enzyme); b, b-carboxymuconate lactonizing enzyme; c, muconolactone iso-
merase; c, g-carboxymuconolactone decarboxylase; d, b-ketoadipate enol-lactone hydrolase;
e, b-ketoadipate succinyl CoA transferase; f, b-ketoadipyl CoA thiolase
the two branches are chemically analogous: dioxygenase-mediated cleavage be-
tween the carbon atoms that carry the hydroxyl groups yields cis,cis-muconate
from catechol and b-carboxy-cis,cis-muconate from protocatechuate. Strictly
analogous enzyme reactions convert the two muconates to muconolactone and
g-carboxymuconolactone, respectively. Decarboxylation of g-carboxymucono-
lactone forces the migration of the double bond within the lactone ring to yield
b-ketoadipate enol-lactone; deprotonation of the g-carbon of muconolactone
gives rise to the same product via an analogous mechanism. Hydrolytic opening
of the b-ketoadipate enol-lactone results in the formation of b-ketoadipate. In
summary, lactonization followed by delactonization is the strategy to form the
3-keto acid. This compound is further oxidized after formation of a coenzyme
A ester analogous to the catabolism of fatty acids. The thiolytic fission of co-
enzyme A esters of 3-keto acids to generate Krebs cycle intermediates repre-
sents the fourth mode of fission of the carbon chain. The coenzyme A ester,
which is formed by the reaction of 3-ketoadipate with succinyl-CoA catalyzed
by a transferase, undergoes thiolysis by reaction with coenzyme A, yielding
acetyl-CoA and generating succinyl-CoA to be used for esterifying another
molecule of 3-ketoadipate.
2.4
Degradation of Aromatics by Fungi
Whereas the mineralization of aromatic compounds including PAHs and the
degradation pathways of bacteria have been well studied, knowledge of similar
activities in fungi is limited to a few species of soil fungi and various white-rot
fungi. In contrast to prokaryotes, fungi rarely utilize aromatics as a sole source
of carbon and energy. An Aspergillus fumigatus was found to be capable of
growth on phenol, p-cresol, and 4-ethylphenol [119121]. Pathways via cate-
chol, protocatechuate, and hydroquinone were proposed. Weber et al. [122] re-
ported the discovery of a toluene-degrading fungus, Cladosporium sphaero-
sperum. In addition to toluene, the organism is able to use styrene, ethylben-
zene, and propylbenzene as the sole source of carbon and energy. There are
indications that the degradation of toluene is initiated by oxidation of the me-
thyl group. Just recently, a hyphomycete Scedosporium apiospermum was isola-
ted which is able to grow on phenol and p-cresol with 3-oxoadipate as the
metabolite [123].
2.4.1
Degradation by Non-Ligninolytic Fungi
A variety of fungi have been found to transform aromatic compounds including
complex polycyclic aromatic hydrocarbons to metabolites that are similar to
those produced by mammalian enzymes. Only a few fungi appear to have the
ability to catabolize PAHs to CO
2
[124].
In Cunninghamella elegans, a non-ligninolytic fungus, and several other
fungi naphthalene is metabolized via a branched pathway to naphthalene trans-
1,2-dihydrodiol, 1-naphthol, 2-naphthol, 4-hydroxy-1-tetralone, 1,4-naphtho-
32 W. Reineke
quinone, and 1,2-naphthoquinone (Fig. 21). Two conjugates, 1-naphthylgluc-
uronide and 1-naphthylsulfate, are also produced by C. elegans. The observa-
tions are consistent with a mechanism for naphthalene trans-1,2-dihydrodiol
formation in which a cytochrome P-450 monooxygenase catalyzes the forma-
tion of naphthalene 1,2-oxide and then an epoxide hydrolase adds water to
form the dihydrodiol. Since naphthalene 1,2-oxide is unstable in solution it
rapidly rearranges to form phenols, principally 1-naphthol.
The same sequence of reactions have been shown for a variety of other poly-
cyclic aromatic hydrocarbons, such as acenaphthene, anthracene, phenan-
threne, benzo[a]pyrene, benzo[a]anthracene, fluoranthene, and pyrene.
Cunninghamella elegans initiates the oxidation of anthracene by incorporating
one atom of molecular oxygen into the aromatic ring to form anthracene 1,2-
oxide, which is hydrated to form anthracene trans-1,2-dihydrodiol (Fig. 22).
Anthracene 1,2-oxide is rearranged rapidly to form 1-anthrol, which is subse-
quently conjugated with sulfate.
Phenanthrene is metabolized by Cunninghamella elegans predominately
at the 1,2-positions to form phenanthrene trans-1,2-dihydrodiol and a glu-
coside conjugate of 1-phenanthrol (Fig. 23). The carbons at the 3,4- and 9,10-
Fig. 21. Proposed pathway for the fungal oxidation of naphthalene. cytochrome P-450
monooxygenase, epoxide hydrolase
positions may also be oxidized to form trans-3,4-dihydrodiol and trans-9,10-
dihydrodiol.
2.4.2
Degradation of PAHs by Ligninolytic Fungi
2.4.2.1
The Ligninolytic System
White rot fungi have the ability to degrade lignin efficiently [125128]. This ca-
pacity results from the activities of a complex system (Fig. 24) composed of ex-
tracellular heme-containing peroxidases, known as lignin peroxidases [129]
and manganese-dependent peroxidases [130], a H
2
O
2
-generating system, other
oxidases, and laccases [126, 131]. The enzymes are produced in response to low
levels of sources of carbon, nitrogen, or sulfur nutrients [132, 133]. Interestingly,
these fungi do not use lignin as a carbon source for growth; instead they de-
grade the lignin to obtain the cellulose that is in the interior of the wood fiber
[134]. Ligninolysis only occurs when other readily degradable substrates are
available.
Extracellular hydrogen peroxide will be produced by oxidases that utilize
compounds such as glucose or glyoxal from molecular oxygen by two-electron
reduction [135].
Lignin peroxidases highly potent oxidizing agents can abstract one elec-
tron from a non-phenolic moiety of the lignin molecule, thus creating a cation
34 W. Reineke
Fig. 22. Proposed pathway for the fungal oxidation of anthracene. cytochrome P-450
Fig. 23. Proposed pathway for the fungal oxidation of phenanthrene. cytochrome P-450
Fig. 24. The extracellular ligninolytic system in ligninolytic fungi. glyoxal oxidase, li-
gnin peroxidase, manganese-dependent peroxidase. The structure represents one aroma-
tic moiety of the complex lignin molecule
radical [136] which in turn initiates a random oxidative chemical reaction that
finally results in the oxygenation and depolymerization of lignin including C-
C-bond cleavage.
Manganese-dependent peroxidases function by oxidizing Mn
2+
to Mn
3+
.
Mn
3+
behaves as a low-molecular-weight mediator that can initiate the oxida-
tion process [137].
Interesting features are the catalytic cycles of lignin and manganese peroxi-
dase (LiP and MnP). The resting lignin peroxidase (ferric state) is oxidized by a
two-electron transfer to H
2
O
2
to form compound I (LiPI, a ferryl (Fe
4+
) p-por-
phyrin cation radical) [138]. LiPI oxidizes a substrate molecule (aryl, Ar) by one
electron, forming compound II (LiPII) and a free radical product (aryl cation
radical, Ar
+
). LiPII reacts with another substrate molecule, forming back the
native enzyme and a free radical. The free radicals then undergo nonenzymatic
reactions to form the final products.
LiP + H
2
O
2
LiPI + H
2
O
LiPI + Ar LiPII + Ar
+
LiPII + Ar LiP + Ar
+
+ H
2
O
As shown in the next equation, the primary reducing substrate in the manga-
nese peroxidase cycle is Mn
2+
, which efficiently reduces both compound I
(MnPI) and compound II (MnPII), generating Mn
3+
, which subsequently oxi-
dizes the organic substrate.
MnP + H
2
O
2
MnPI + H
2
O
MnPI + Mn
2+
MnPII + Mn
3+
MnPII + Mn
2+
MnP + Mn
3+
+ H
2
O
MnPI + Ar MnPII + Ar
+
Mn
3+
+ Ar Mn
2+
+ Ar
+
Compared to most other peroxidases (Table 9), the redox potential of lignin and
manganese peroxidases are more positive. The redox potentials of the ferric/
ferrous couple (Fe
3+
/Fe
2+
) is about 140 mV for lignin peroxidase and 90 mV
for manganese peroxidases, values that are considerably higher than the values
36 W. Reineke
Table 9. Redox potential of peroxidases
Peroxidases: isoenzymes E
m7
(mV) References
Horseradish 278 [139]
Cytochrome c 194 [140]
Lignin peroxidase: H1, H8, H2, H10 142, 137, 135, 127 [141]
Manganese peroxidase: H4, H3 93, 88 [141]
E
m7
, midpoint potential at pH 7.
for horseradish peroxidase (270 mV) or cytochrome c peroxidase (195 mV).
The higher redox potential suggests that the lignin and manganese peroxidase
compound I and II intermediates are more electron-deficient and hence have
higher oxidation-reduction potential. The higher oxidation-reduction potential
of the active intermediates of lignin and manganese peroxidase extent the
number of chemicals that can be oxidized to those of higher redox potential.
In some white-rot fungi, laccases (low-specificity enzymes which act on o-
and p-quinols) are also present. But the role of laccase in ligninolysis by ligni-
nolytic fungi is not clear, since lignin can be rapidly degraded by Phanerochate
chrysosporium, an organism which misses laccase. However, this conclusion
cannot be extended to eliminate the involvement of laccase in ligninolysis in
those fungi that do secrete this enzyme. It is known that laccases oxidize non-
phenolic aromatic compounds as well as Mn
2+
in the presence of other oxidiz-
able substrates [142]. Substrate oxidation by laccase is a one-electron reaction
generating a free radical [143]. The initial product, the carbon-centered cation
radical formed by removing one electron from an aromatic nucleus [144], is
typically unstable and may undergo a second enzyme-catalyzed oxidation (con-
verting phenol to quinone with many substrates). In addition, the radical may
undergo non-enzymic reactions such as hydration or disproportionation.
Although laccase can (directly or indirectly) cleave a significant proportion of
substrates found in lignin, the role of laccase in ligninolysis remains unresol-
ved, but the widespread occurrence of this enzyme in wood-rotting fungi is un-
likely to be coincidental.
2.4.2.2
Ligninolytic Systems and PAHs
The polyaromatic structure of both lignin and polycyclic aromatic hydrocar-
bons (PAHs) prompted the suggestion that the same fungi might be able to de-
grade these ubiquitous pollutants [145].
White-rot fungi are able to degrade PAHs and in some cases to mineralize
them. Most of the work was done with Phanerochaete chrysosporium, demon-
strating its ability to degrade non-specifically a wide range of aromatic pol-
lutants [146161]. P. chrysosporiumcan metabolize a variety of PAHs, including
benzo[a]pyrene, under ligninolytic (N-limited conditions) as well as non-ligni-
nolytic conditions (non-N-limited) [151, 158], i. e., in the presence and absence
of peroxidases. Other white-rot fungi such as Trametes versicolor, Bjerkandera
sp., and Pleurotus ostreatus are thought to be more promising than P. chrysos-
porium in their ability to mineralize PAHs to CO
2
[157, 162, 163]. In addition,
Crinipellis stipitaria has been reported to metabolize pyrene [164, 165]. Sack
and Gnther [157] showed that P. ostreatus is quite efficient in the degradation
of phenanthrene and fluorene, less efficient with fluoranthene, while pyrene
was not degraded to any significant extent. Vyas et al. [163] showed that P.
ostreatus is able to degrade anthracene. In general, notable differences have
been demonstrated among Phanerochate chrysosporium, other Phanerochaete
species, and members of other genera with regard to the extent of PAH miner-
alization and transformation ability.
2.4.2.3
Influence of Ionization Potential on Oxidation of PAHs
Lignin peroxidase from Phanerochaete chrysosporium directly catalyzes one-
electron oxidations of aromatic substrates [166, 167]. The resultant aryl cation
radicals then undergo spontaneous rearrangements and degradation. Hammel
et al. [154] observed transformation by lignin peroxidase of those PAHs with
ionization potential (IP) values of <7.55 eV (see values of ionization potential
of PAHs in Table 8). Isolated lignin peroxidase was unable to degrade com-
pounds with an IP above the threshold value of 7.55 eV. Therefore, the meta-
bolism of PAHs with high IPs (>7.65 eV) such as triphenylene, phenanthrene,
fluoranthene, chrysene, benzo[b]fluoranthene, and benzo[e]pyrene observed
with whole cell cultures could not be explained by the direct action of the lignin
peroxidase. Moen and Hammel [155] reported data that support a role of man-
ganese-dependent peroxidase-mediated lipid peroxidation in phenanthrene
oxidation by P. chrysosporium. Bogan and Lamar [168] gave evidence that the
degradation of three- to six-ring PAHs with IPs between 7.2 eV and 8.1 eV is IP-
dependent during in vivo and in vitro lipid peroxidation. This implies that the
participation of a one-electron oxidant stronger than lignin peroxidase or Mn
3+
is involved. The data presented showed that compounds with up to six rings
were degraded in vitro during manganese peroxidase-dependent lipid peroxi-
dation reactions.
Hammel et al. [152] emphasized that PAHs which are lignin peroxidase sub-
strates are more susceptible to mineralization than PAHs which are not, so that
benzo[a]pyrene, pyrene, and anthracene are all rapidly depleted from N-limit-
ed cultures.
2.4.2.4
Metabolites Formed During Degradation
The degradative pathways for anthracene and phenanthrene by Phanerochate
chrysosporiumare shown in Figs. 25 and 26. The formation of a quinone to pre-
pare the aromatic ring for cleavage is an unusual biodegradative strategy, and it
appears to be of general importance in P. chrysosporium. While the formations
of 9,10-anthraquinone and phthalate were found to be rapid processes, the
further conversion to carbon dioxide is slow. The 2,2-dicarboxylic biphenyl was
found to be the major product in the degradation of phenanthrene.
Yadav and Reddy [169] presented data indicating that P. chrysosporium
mineralizes all BTEX components (benzene, toluene, ethylbenzene, xylenes)
38 W. Reineke
Fig. 25. Proposed pathways for anthracene degradation in ligninolytic P. chrysosporium[153]
either individually or as a mixture to CO
2
under non-ligninolytic conditions,
i. e., when no lignin and manganese peroxidases are produced.
P. ostreatus differs from P. chrysosporium in its lignin degradation mecha-
nism in that it does not involve lignin peroxidase activity [126]. Instead, its
lignin degradation ability is assumed to be due to laccase activity [127, 128, 131,
170]. Laccase was found to be nonspecific as to its reducing substrate as well as
able to oxidize a variety of substrates including polyphenols, methoxy-substi-
tuted phenols, diamines, and a range of other compounds. Bezalel et al. [171,
172] reported that Pleurotus ostreatus is able to mineralize various PAHs to
14
CO
2
such as phenanthrene, pyrene (no degradation in [157]), benzo[a]pyrene,
anthracene, and fluorene, but fails to mineralize fluoranthene.
2.5
Rsum: Aerobic Degradation of Aromatic Compounds
Bacteria and fungi are dealing with aromatic hydrocarbons in a different way.
While bacteria are able to utilize the compounds as the sole source of carbon
and energy fungi cometabolize the aromatics to hydroxylated products, some-
times mineralization takes place by the fungi. In bacteria degradation of an
aromatic compound is initiated by dioxygenase to give a 1,2-diphenol as the
ring cleavage substrate, while cytochrome P-450 catalyzed epoxide formation
is the first step in the degradation by the fungi. The further degradation of the
diphenol in the bacteria leading to intermediates of the metabolism has been
described to occur via meta, ortho or gentisate pathways.
Since the polycyclic aromatic hydrocarbons are ubiquitous pollutants the
biodegradation is currently of increasing interest. As the molecular weight of
PAHs increases, their water solubility decreases. The increasing hydrophobicity
usually correlates with decreasing biodegradability and with increasing poten-
tial for bioaccumulation. Because of the difficulty of isolating bacteria which
can effectively degrade high-molecular-weight PAHs with four or more fused
aromatic rings, the ability of white-rot fungi to degrade the abundant naturally-
occurring polymer lignin has made these fungi appropriate candidates for PAH
degradation. Since white-rot fungi produce extracellular enzymes, the PAH bio-
availability is maximized and toxicity problems for the fungi are avoided.
Considering the fact that the rate of PAH degradation often does not correlate
Fig. 26. Proposed pathway for phenanthrene degradation in ligninolytic P. chrysosporium
[152]. Major reactions and minor ones are shown
with lignin peroxidase activity, several other enzymatic mechanisms are
thought to be used by the white-rot fungi to degrade PAH.
2.6
Anaerobic Degradation of Aromatic Compounds
Contrary to the well-known pathways of the aerobic degradation of aromatics
the mesomeric stabilized aromatic ring will be attacked under anaerobic con-
ditions by reductive rather than oxidative reactions. Since the concept of the
destabilization of the aromatic nucleus in the absence of oxygen has been devel-
oped by Evans [173], several aromatic compounds were examined and found to
be degraded by various types of anaerobic bacteria according to that principle.
As far as is known, the pathways of substrate activation and cleavage are ba-
sically the same, regardless of whether nitrate, ferric ion, sulfate, or carbonate
are used as electron acceptor, or whether light can provide an additional energy
source. In addition, degradation of aromatic compounds is possible under fer-
mentative conditions. Most information on the degradative pathways used for
aromatic compounds has been obtained with denitrifying organisms. Different
aromatics are converted through channeling pathways into a few central reac-
tive intermediates: benzoyl-CoA, resorcinol, phloroglucinol, and possibly
others. After these activation reactions, the central compounds are reduced to
form an intermediate with a 1,3-dioxo structure which is no longer an aroma-
tic compound. This structure allows a nucleophilic attack on a ring carbonyl
group and subsequent ring fission. A b-oxidation pathway that produces three
acetyl moieties follows the ring cleavage. Acetyl-CoA may then further be oxi-
dized to CO
2
.
2.6.1
Channeling Reactions
2.6.1.1
Channeling Reactions to Benzoyl-CoA
Benzoyl-CoA is formed from a large variety of different compounds, such as
phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, p-cresol, phenylacetate, 4-hy-
droxyphenylacetate, mandelate, hydroxymandelate, toluene, 2-aminobenzoate,
4-aminobenzoate, aniline, and many others. The pathways are summarized in
Fig. 27 indicating that some analogous reactions take place with these quite dif-
ferent compounds.
Hydroxybenzoates. The reductive dehydroxylation of aromatic hydroxyl func-
tions, notably with compounds having hydroxyl functions para to a carboxyl
group, is an important reaction in the metabolism of phenol, p-cresol, and 4-hy-
droxyphenylacetate. The case that has been studied in detail is the reductive de-
hydroxylation of 4-hydroxybenzoyl-CoA by 4-hydroxybenzoyl-CoA reductase
(dehydroxylating), indicating the requirement of coenzyme A thioester forma-
tion of the aromatic acids prior to dehydroxylation [177, 182]. The metabolism
40 W. Reineke
Fig. 27. Channeling sequences for various aromatic compounds leading to benzoyl-CoA as
the central metabolite. Analogous reactions such as carboxylation, formation of CoA ester,
and reductive elimination of substituents are marked by different gray boxes. The informa-
tion is compiled from [174194]
of salicylate proceeds in a similar reaction sequence via 2-hydroxybenzoyl-CoA
to give benzoyl-CoA [180].
Phenol. Phenol lacks a carboxylic group and therefore cannot be activated by
coenzyme A, a prerequisite which turned out to be essential for complete ring
reduction in the benzoate pathway. A phenol-degrading nitrate reducer was de-
pendent on CO
2
as cosubstrate for phenol oxidation. Carboxylation of phenol to
4-hydroxybenzoate, analogous to a Kolbe-Schmitt reaction [195], was suggested
to be the initial reaction in phenol degradation, followed by coenzyme A ac-
tivation and reductive elimination of the hydroxy substituent to form benzoyl-
CoA [185]. There are indications that phenylphosphate is the physiological in-
termediate used by the para-specific carboxylase [190]. The use of phenylphos-
phonate instead of phenol renders the carboxylation reaction exergonic under
natural CO
2
and phenol concentrations. In addition, phosphorylation would fa-
cilitate the cellular accumulation of this toxic substrate in a non-toxic reactive
form.
Carboxylation of phenol to a benzoate derivative was also demonstrated in
anaerobic enrichment cultures converting fluorophenols to benzoate [183, 184].
These experiments also proved that phenol is carboxylated at the C-4 carbon,
leading to 4-hydroxybenzoate as an intermediate. Although a net carboxylation
of phenol to 4-hydroxybenzoate has never been demonstrated in a cell-free ex-
tract, this concept acquires more and more support from experiments with sul-
fate-reducing bacteria (Schnell and Schink, unpublished) and defined metha-
nogenic cultures [189].
Catechol and hydroquinone. Studies on the degradation of hydroquinone by a
strictly anaerobic fermenting bacterium indicate that the primary step in hydro-
quinone degradation is a carboxylation to a gentisic acid derivative, followed ap-
parently by subsequent reductive dehydroxylations to benzoyl-CoA [196, 197].
The degradation of catechol by a Desulfobacteriumsp. proceeds via carboxy-
lation to protocatechuate followed by esterification with CoA and reductive de-
hydroxylation to give 3-hydroxybenzoyl-CoA [198].
Aniline and anthranilate. Aniline is degraded in anoxic environments only very
slowly. The degradation pathway has so far only been studied with a pure cul-
ture of a sulfate-reducing bacterium [194]. Aniline degradation by this bacter-
ium depends on CO
2
as cosubstrate, and thus is reminiscent of phenol degra-
dation by nitrate reducers. Aniline is first carboxylated to 4-aminobenzoate, ac-
tivated to the coenzyme A derivative, and then reductively deaminated to
benzoyl-CoA. The conversion of 2-aminobenzoyl-CoA to benzoyl-CoA by 2-
aminobenzoyl-CoA reductase is an additional example of a reductive deamina-
tion [191].
Cresols. p-Cresol is converted by Pseudomonas strains to 4-hydroxybenzylal-
cohol by an oxygen-independent reaction [199]. The alcohol oxygen is derived
from water which is added to a quinone methide intermediate formed by a de-
hydrogenation reaction [186]. The hydroxybenzyl alcohol can easily be oxidized
42 W. Reineke
to 4-hydroxybenzoate and further degraded as mentioned above. The same
pathway appears to underlie sulfate-dependent p-cresol degradation [200, 201].
It should be emphasized that the methyl hydroxylation requires an electron-
attracting group in the 4- or 2-position to stabilize the quinone methide inter-
mediate. Therefore, this reaction is also applicable to o-cresol, but not to m-cre-
sol degradation. In methanogenic and sulfidogenic consortia m-cresol appears
to be activated by carboxylation to 2-methyl-4-hydroxybenzoate, analogous to
phenol activation [202, 203]. The further degradation of 4-hydroxy-2-methyl-
benzoate proceeds via 4-hydroxybenzoate and benzoate demonstrated with
another m-cresol-degrading methanogenic consortium [204]. The carboxyla-
tion strategy seems to be used also for o-cresol by a consortium under metha-
nogenic conditions [205].
The anaerobic degradation of m-cresol was later studied with a denitrifying
bacterium [181]. The transient accumulation of 3-hydroxybenzoate and benzo-
ate when using inhibitory compounds supports conversion of m-cresol via ini-
tial anaerobic methyl oxidation to 3-hydroxybenzoate, followed by reductive
dehydroxylation to benzoate or benzoyl-CoA. The same oxidative pathway se-
quence was observed with a sulfate-reducing organism, Desulfotomaculum sp.
strain Groll [201, 206].
ADesulfobacterium cetonicumwas only recently reported, which oxidized m-
cresol completely with sulfate as electron acceptor [207]. 3-Hydroxybenzyl-
succinate was detected as a metabolite indicating that the methyl group is ac-
tivated by the addition to fumarate as in the case of anaerobic toluene metabo-
lism. A further metabolite observed was 3-hydroxybenzoyl-CoA formed by CoA
thioesterification and oxidation of 3-hydroxybenzylsuccinate.
Phenylacetate, phenylpropionate, mandelate. Phenylacetate is first converted
to phenylacetyl-CoA by a specific CoA ligase [208]. 4-Hydroxyphenylacetate
seems to be activated by a different ligase. The thioester apparently activates the
a-methylene carbon enough to allow its dehydrogenation and hydroxylation
with water as the oxygen source, i. e., anaerobic a-oxidation. Phenylglyoxylate
or 4-hydroxyphenylglyoxylate are the products formed by the nitrate-reducing
Pseudomonas strains [209] and Thauera aromatica [210]. The oxidative decar-
boxylation of phenylglyoxylate to benzoyl-CoA by phenylglyoxylate: NAD
+
oxi-
doreductase (CoA benzoylating) [211] is not an unusual reaction, analogous to
the oxidative decarboxylation of pyruvate. Mandelate can be oxidized via
phenylglyoxylate to benzoyl-CoA [212, 213]. Phenylpropionate is easily b-
oxidized to benzoate [214] and further degraded as such.
Phthalates. Degradation of phthalates requires elimination of the additional
carboxyl group, and further degradation analogous to benzoate. However, di-
rect decarboxylation is chemically quite difficult. Phthalates appear to be first
converted to the CoA mono thioester by CoA ligases, followed by decarboxyla-
tion of o-, m-, or p-phthaloyl-CoA to benzoyl-CoA [215217].
Toluene and xylenes. Monoaromatic hydrocarbons such as toluene and xylenes
are known to be degraded in the absence of molecular oxygen, i. e., under
nitrate-reducing [174, 218227], Fe
3+
-reducing [228230], Mn
4+
-reducing [231,
232], sulfate-reducing [222, 233239], and/or methanogenic conditions
[240243]. Just recently, an anoxygenic phototrophic bacterium, Blastochloris
sulfoviridis strain ToP1, was isolated, which is able to use toluene for growth
[244].
Well-characterized bacteria capable of degrading toluene under anaerobic
conditions include several denitrifying species such as Thauera aromatica and
Azoarcus tolulyticus [174, 219, 220, 223, 226, 238], the ferric-ion-reducing
Geobacter metallireducens [230], and the sulfate-reducing species Desulfobacula
toluolica and strain PRTOL1 [234, 237].
While toluene was found to be the most readily degradable aromatic com-
pound under anaerobic conditions [245], a plausible pathway has been obtained
only recently. Diverse compounds have been detected accumulating in different
organisms during the toluene metabolism which hardly fit into one pathway
scheme.
There was strong evidence that toluene oxidation in denitrifying bacteria oc-
curs via benzoate as a common metabolite [174, 178, 226, 230, 233, 246249].
Seyfried et al. [249] reported the transient accumulation of benzaldehyde and
benzoate after toluene was added, while 3-methylbenzaldehyde and 3-methyl-
benzoate were the products formed after the addition of m-xylene. Benzyl al-
cohol and benzaldehyde have been detected in the anaerobic metabolism of
toluene in the denitrifying Thauera aromatica strain K172 (Pseudomonas) [174,
250]. In contrast, p-cresol accumulated in a mixed methanogenic culture grown
with toluene [241, 242]. Carboxylation of the methyl carbon of toluene as sug-
gested by Altenschmidt and Fuchs [174] might explain the accumulation of
phenylacetate.
Benzylsuccinate and benzylfumarate have been reported to accumulate dur-
ing anaerobic degradation of toluene under denitrifying conditions by strain
T1 [246], Pseudomonas sp. strain T [249], Thauera aromatica K172 [249, 251],
and Azoarcus tolulyticus Tol-4 [252], as well as under sulfate-reducing condi-
tions by strain PRTOL1 [234, 253]. Phenylitaconate was identified by Migaud et
al. [254] during growth of Azoarcus tolulyticus Tol-4 with toluene. Other strains
of Azoarcus tolulyticus with the ability to degrade toluene anaerobically [223,
255] also synthesized similar amounts of phenylitaconate during toluene meta-
bolism. These data support the assumption that an alternative pathway may
function in these strains. In analogy to the attack of the methyl group by suc-
cinyl-CoA forming benzylsuccinate and benzylfumarate, activation of toluene
by an oxidative condensation of toluene with acetyl-CoA to yield phenylpro-
pionyl-CoA is proposed by Evans et al. [246], followed by conversion to benzoyl-
CoA via b-oxidation.
The pathway for the initial attack on toluene has recently been elucidated in
cell-extracts of Thauera aromatica strain K172 [179, 256] and Azoarcus sp.
strain T [257, 258]. Toluene is condensed with fumarate by benzylsuccinate
synthase to give benzylsuccinate as the first intermediate. There is strong evi-
dence that benzylsuccinate formation is accomplished in nitrate-reducing bac-
teria via formation of an enzyme-bound radical [256259]. CoA-dependent
conversion of benzylsuccinate to phenylitaconate or the CoA thioester [257]
44 W. Reineke
and subsequently to benzoyl-CoA follows [179, 257]. Present knowledge, shown
in Fig. 28, includes activation and b-oxidation.
While anaerobic growth of pure cultures on m-xylene has often been shown
with denitrifying bacteria [219, 223, 238, 249, 261], strains that grows on o- or
p-xylene have been isolated only recently [262].
Other monoaromatic hydrocarbons. Anaerobic degradation of ethylbenzene,
propylbenzene, and p-cymene has been studied. Anaerobic mineralization of
ethylbenzene has been reported in three denitrifying bacteria [176, 238]. All
three isolates are closely related to each other, and are affiliated with the genus
Azoarcus. A pathway for anaerobic oxidation of ethylbenzene to benzoyl-CoA
has been proposed. Benzoate was detected as a transient intermediate [176].
Formation of 1-phenylethanol and acetophenone from ethylbenzene was de-
monstrated [176, 193]. The proposed initial reaction of the pathway is the oxida-
tion to 1-phenylethanol. The oxygen atom of the hydroxyl group is derived from
water. 1-Phenylethanol is further oxidized to acetophenone. Both enzymes re-
sponsible for the formation of these intermediates have been demonstrated [263].
Only minor evidence is available for the further reaction involved in aceto-
phenone conversion to benzoyl-CoA. It is proposed that acetophenone is car-
boxylated to benzoylacetate in a reaction analogous to reactions found in ae-
robic and anaerobic degradation of aliphatic ketones [264, 265]. Benzoylacetate
is proposed to be activated to the CoA thioester and to be cleaved thiolytically
to acetyl-CoA and benzoyl-CoA.
Information on the pathway used for the degradation of p-cymene is rare.
Harms et al. [266] observed the accumulation of p-isopropylbenzoate during
growth on p-cymene, indicating an initial attack on the methyl group.
Fig. 28. Anaerobic toluene pathway in denitrifying organisms according to Leutwein and
Heider [260]
2.6.1.2
Channeling Reactions to Resorcinol (1,3-Dihydroxybenzene)
and Phloroglucinol (1,3,5-Trihydroxybenzene)
Dihydroxy- and trihydroxybenzoates. Aromatic acids with two or more hy-
droxyl functions may become decarboxylated if the resulting product contains
a diol system in the 1,3-position. These compounds have little aromaticity and
can easily be reduced. Examples are the decarboxylation of resorcylates (2,4-
dihydroxy-, and 2,6-dihydroxybenzoates) to resorcinol [267] (Fig. 29).
Similarly, gallate is decarboxylated to pyrogallol (1,2,3-trihydroxybenzene)
followed by a transhydroxylation to give phloroglucinol (Fig. 30). Phloro-
glucinol results directly from phloroglucinate by decarboxylation. In general,
the decarboxylation of aromatic acids with a hydroxyl function para to the car-
boxyl group is a chemically favored reaction.
Trihydroxybenzenes. Anaerobically fermenting bacteria such as Eubacterium
oxidoreducens, Pelobacter acidigallici, Pelobacter massiliensis, and the homo-
acetogenic Holophaga foetida degrade trihydroxybenzenes via phloroglucinol
[268, 270273].
Pyrogallol is converted to phloroglucinol [269] by an unusual reaction which
includes 1,2,3,5-tetrahydroxybenzene as cosubstrate [268]. The hydroxyl group
is transferred from the tetrahydroxybenzene to pyrogallol, thus yielding phlo-
roglucinol and a new tetrahydroxybenzene molecule [274, 275]. Since the co-
substrate is cyclically regenerated in the course of the reaction, it has to be con-
sidered as a cocatalyst. Its function is to donate one hydroxyl group to C-5 of
pyrogallol.
Hydroxyhydroquinone (1,2,4-trihydroxybenzene) is degraded by the fer-
menting organisms through phloroglucinol using a different hydroxyl transfer
reaction [276]. Three hydroxyl transfers seem to be involved (Fig. 31). First, the
substrate is disproportionated to 1,3-dihydroxy- and 1,2,4,5-tetrahydroxyben-
zene. Then the tetrahydroxybenzene is isomerized to the 1,2,3,5-tetrahydroxy
46 W. Reineke
Fig. 29. Decarboxylation of dihydroxybenzoates leading to resorcinol [11, 267]
Fig. 30. Degradation of trihydroxybenzoates to phloroglucinol involving decarboxylation
and transhydroxylation [268, 269]. The hydroxyl group moving within the sequence is mark-
ed. Gallate: top sequence; phloroglucinate: bottom
Fig. 31. Sequential transhydroxylation of hydroxyhydroquinone to phloroglucinol in Pelo-
bacter massiliensis according to Brune et al. [276]. The hydroxyl group moving within the se-
quence is marked
isomer by an unknown reaction. Finally, 1,2,3,5-tetrahydroxybenzene forms
phloroglucinol by transferring its 2-hydroxyl group to either hydroxyhydroqui-
none or resorcinol, thus also regenerating the cosubstrate involved in earlier re-
actions of the sequence [276].
Besides the strategy of isomerization of 1,2,4-trihydroxybenzene to phloro-
glucinol and further degradation through the phloroglucinol pathway (see la-
ter), which is used by all fermenting bacteria, recently alternative pathways for
1,2,4-trihydroxybenzene have been observed with nitrate- and sulfate-reducing
bacteria [277280].
2.6.2
Activating Reductive Sequences and Ring Cleavage
2.6.2.1
Benzoate Pathway
Utilization of benzoate by some species of nonsulfur purple bacteria has been
known for many years [281285], but is restricted to a few strains of Rhodo-
pseudomonas palustris [282, 286, 287], Rhodocyclus sp. [288], Rhodospirillumsp.
[289], and Rhodomicrobium sp. [290]. A characteristic feature of the phototro-
phic metabolism of these bacteria is that the growth substrate is usually exten-
sively assimilated into cell material. This follows from their use of light as
energy source, so eliminating the need for oxidative or fermentative manipula-
tion of a portion of the carbon source.
In addition, various denitrifying Pseudomonas, Alcaligenes, and Moraxella
species are able to use benzoate in the absence of oxygen [175, 291294].
It is clear that the reduction of the aromatic ring is preceded by coenzyme A
thioesterification of benzoate, first observed by Hutber and Ribbons [187] with
crude extract of Rhodopseudomonas palustris, enabling the cells to accumulate
an otherwise permeant molecule [286]. Benzoate is activated by a benzoyl-CoA
synthetase reaction. ATP is cleaved into AMP and pyrophosphate which sug-
gests the occurrence of an intermediate acyl-AMP. Pyrophosphate is thought to
become subsequently hydrolyzed. This renders the overall reaction strongly
exergonic.
The further pathway has become clearer by studies with enzymes of
Rhodopseudomonas palustris [295] and of Thauera aromatica strain K172
[296298] (Fig. 32). Buckel and Keese [299] proposed a possible mechanism for
the benzoyl-CoA reductase (for a further discussion see Buckel and Golding
[300]). The reaction proceeds in two successive one-electron reactions to give
cyclohex-1,5-diene-1-carboxyl-CoA, analogous to a chemical Birch reduction
[301]. During the first circle of this biological Birch reduction a highly reactive
ketyl radical may be generated, having its origin in the transfer of a superre-
ductive activated electron to the thioester carbonyl group of benzoyl-CoA.
Uptake of a proton in para-position would then neutralize the charge making
the radical able to accept the second electron followed by the addition of the se-
cond proton. The CoA thioester may play an important role in this catalytic pro-
cess. Facilitation of binding of the substrate and correct positioning in the ac-
48 W. Reineke
tive center of the enzyme might be one function. In addition, the thioesterified
carboxyl group is an important substituent mechanistically as the entrance
point of the electrons and thermodynamically by lowering the midpoint poten-
tial of the electron transfer step and of the whole process. However, although
the reduction of benzoyl-CoA is facilitated by the CoA thioester, the reductase
requires input energy of two ATP molecules to overcome the considerable
activation energy, one ATP for each electron introduced [296, 297]. The nitrate-
reducing bacteria can recover the high energy input for substrate-activation
and for dearomatization through the further breakdown of the cleavage prod-
uct, which is totally oxidized to CO
2
. Since fermenting bacteria can recover
only little energy in the further breakdown they apply a different reaction for
benzoyl-CoA dearomatization to have the reaction exergonic so that no ATP is
necessary. They introduce four electrons and protons from NAD(P)H into the
ring structure which directly leads to a cyclohexene derivative [302].
Fig. 32. Reaction sequences of benzoyl-CoA degradation according to [295298, 302, 303,
305, 306]. Path (a) Rhodopseudomonas palustris and Syntrophus gentianae. Path (b) Thauera
aromatica
Therefore, different pathway branches were found in the denitrifying orga-
nism Thauera aromatica, the phototrophic organism Rhodopseudomonas pa-
lustris, and a fermenting organism. In Thauera aromatica the next intermediate
from the diene is 6-hydroxycyclohex-1-ene-1-carboxyl-CoA which is derived by
addition of water. Then there is a gap between 6-hydroxycyclohex-1-ene-1-car-
boxyl-CoA and 3-hydroxypimelyl-CoA, the first non-cyclic intermediate isolat-
ed. The easiest explanation is the addition of water to the double bond in 6-hy-
droxycyclohex-1-ene-1-carboxyl-CoA, the oxidation of the resulting alcohol to
give an oxo group, and the hydrolytic cleavage of the ring. In Rhodo-
pseudomonas palustris the cyclic diene is further reduced to cyclohex-1-ene-1-
carboxyl-CoA. Subsequent b-oxidation results in the formation of a cyclic b-
oxo compound, followed by hydrolytic carbon ring opening yielding pimelyl-
CoA, which is subsequently oxidized via 3-hydroxypimelyl-CoA as in Thauera
aromatica [303]. Recently, the pathway branch via cyclohex-1-ene-1-carboxyl-
CoA used by the phototrophic organism has also been demonstrated in
Syntrophus gentianae when fermenting benzoate [302].
The further b-oxidation of 3-hydroxypimelyl-CoA yields glutaryl-CoA plus
acetyl-CoA. The oxidation of glutaryl-CoA to 2 acetyl-CoA plus CO
2
proceeds
via glutaconyl-CoA and crotonyl-CoA and is catalyzed by a glutaryl-CoA dehy-
drogenase, which is present at a significant level [304].
2.6.2.2
Resorcinol Pathway
In the resorcinol molecule, the two meta-oriented hydroxy substituents polarize
the p-electron cloud in such a way that selective reduction by two electrons to
dihydroresorcinol becomes possible, thus abolishing the molecules aromatic
character. Resorcinol-degrading fermenting bacteria (Clostridium sp.) follow
this degradation strategy and 1,3-dioxocyclohexane is formed as the ultimate
alicyclic compound (Fig. 33). The Clostridium also channels the carboxylated
derivatives 2,4-dihydroxybenzoate and 2,6-dihydroxybenzoate (resorcylic
acids) through initial decarboxylations into this pathway [11, 267]. Obviously,
the C-3 atom of 1,3-dioxocyclohexane carries sufficient positive charge to allow
hydrolytic cleavage to 5-oxocaproic acid.
An obligate nitrate-reducing resorcinol degrader employs a different path of
resorcinol degradation which does not involve an initial ring reduction.
50 W. Reineke
Fig. 33. Proposed degradative sequences for resorcinol (top) and phloroglucinol (bottom)
Resorcinol is hydrolytically transformed in a one-step reaction to a non-cyclic
product, 5-oxo-hex-2-enecarboxylic acid [307].
2.6.2.3
Phloroglucinol Pathway
The three hydroxy substituents of phloroglucinol polarize the p-electron cloud
even more than the two hydroxy substituents of resorcinol. As a consequence,
the trioxo tautomer prevails in aqueous solution. Consequently, phloroglucinol
has no aromatic character and can easily become reduced chemically by mild
reducing agents.
Anaerobic phloroglucinol-degrading bacteria, such as Rhodopseudomonas
gelatinosa [285], Coprococcus sp. [308], Pelobacter acidigallici [309], or
Eubacterium oxidoreducens [270], first reduce phloroglucinol to dihydrophlo-
roglucinol (1,3-dioxo-5-hydroxycyclohexane) in an NADPH-dependent reac-
tion. The corresponding enzyme of E. oxidoreducens has been purified and cha-
racterized [310].
Nucleophilic attack on one of the carbonyl groups of dihydrophloroglucinol
opens the ring to form a 3-hydroxy-5-oxocaproic acid. The further degradation
of the partially oxidized caproic acid residue no longer poses basic biochemical
problems. Details have been studied with E. oxidoreducens and P. acidigallici
[270, 311, 312].
2.6.3
Anaerobic Degradation of Environmentally Important Aromatics where
Pathway Information is Missing or Minor
Benzene. Benzene persists in most anoxic environments [313, 314]. However,
partial mineralization of benzene to carbon dioxide and methane in the ab-
sence of molecular oxygen has been observed in enrichment cultures [241] and
methanogenic river sediments [315].
Benzene was completely mineralized to carbon dioxide in enrichment cul-
tures in which sulfate was provided as a potential electron acceptor [235].
Lovley et al. [316] observed that after an adaptation period benzene was rapidly
oxidized to carbon dioxide with the reduction of sulfate in petroleum-contami-
nated sediments from San Diego Bay, California. Recently, enrichment cultures
from marine sediments were found to be able to mineralize benzene while
using sulfate as the terminal electron acceptor [317]. However, cultures from
river marsh failed to show the activity. Weiner and Lovley [318] showed that
supplementing aquifer sediments with benzene-oxidizing sulfate reducers can
greatly accelerate anaerobic benzene degradation.
Major et al. [319] reported that the degradation of benzene occurred under
denitrification conditions with material from an aquifer. Benzene degradation
linked to nitrate reduction has been found recently with enrichment cultures
developed from soil and groundwater microcosms [320].
Anaerobic oxidation of benzene coupled to Fe
3+
as an electron acceptor has
been documented by Lovley et al. [321]. Stoichiometric studies demonstrated
that the electrons derived from benzene oxidation to carbon dioxide were
transferred to Fe
3+
, when the availability of Fe
3+
had been artificially modified
with a chelator such as nitrilotriacetic acid (NTA). Fe
3+
chelated to compounds
such as ethylenediaminetetraacetic acid (EDTA), N-methyliminodiacetic acid,
ethanol diglycine, humic acids and phosphates stimulated benzene oxidation
coupled to Fe
3+
reduction in anaerobic sediments from a petroleum-contami-
nated aquifer as effectively as or more effectively than when being chelated to
NTA [322].
A study by Kazumi et al. [323] showed that benzene degradation takes place
under methanogenic conditions.
Information on the degradative pathway for benzene is not available. Studies
performed with H
2
18
O revealed that, in enrichment cultures, the oxygen from
water was incorporated into benzene as a hydroxyl group with the formation of
phenol [242], but these results could not be confirmed later. No pure cultures of
benzene-degrading bacteria have been isolated to date, which, however, are im-
portant for gaining a detailed understanding of the biochemistry of anaerobic
benzene degradation.
Polycyclic aromatic hydrocarbons (PAHs). Various studies [324332] have in-
dicated that PAHs are not degraded in the absence of oxygen [64]. Other studies
have suggested that some PAHs can be degraded in the absence of oxygen if
nitrate is available as an electron acceptor, but that PAHs persist under sulfate-
reducing or methanogenic conditions [333336]. Recently, Coates et al. [337]
reported that (
14
C) naphthalene and phenanthrene were oxidized to
14
CO
2
with-
out a detectable lag period under strictly anaerobic conditions in sediments
from San Diego Bay, California, which were heavily contaminated with PAHs,
but not in less contaminated sediments. When molybdate, a specific inhibitor of
sulfate reduction [338], was added to the sediments the production of
14
CO
2
from naphthalene and phenanthrene was immediately inhibited. First informa-
tion on the metabolites of PAH degradation under anaerobic conditions were
obtained by Bedessem et al. [339] and Zhang and Young [340]. Naphthalenol
was tentatively identified as a potential metabolic intermediate of naphthalene
degradation from aquifer enrichments under sulfate-reducing conditions [339].
Zhang and Young [340] gave evidence using sulfidogenic consortia that car-
boxylation is an initial key reaction for the anaerobic metabolism of naphtha-
lene and phenanthrene forming 2-naphthoate and phenanthrenoate, respec-
tively. A pure culture of a naphthalene-degrading sulfate-reducing bacterium
has recently been described which now allow biochemical investigations in an-
aerobic degradation of PAHs [341].
2.6.4
Rsum: Anaerobic Degradation of Aromatic Compounds
It appears as a general pattern that different aromatic compounds are conver-
ted to give one of three central aromatic intermediates: benzoyl-CoA, resorci-
nol, and phloroglucinol. Subsequently, the aromatic nucleus is destabilized by
reduction in all three pathways. Cleavage of the ring is possible by a nucleophi-
52 W. Reineke
lic, probably hydrolytic, attack when the 1,3-dioxo structure is reached. The 1,3-
dioxo structure exists in the cyclic, non-aromatic ring when resorcinol or phlo-
roglucinol are the metabolites. In contrast, when benzoyl-CoA is the central me-
tabolite after the channeling reactions, one oxo group is in the cyclic ring and
the other exocyclic as part of the CoA-ester group. In all cases the ring is broken
down to three acetyl residues which are either excreted as acetate or further oxi-
dized to CO
2
.
Whereas those compounds which enter directly one of the basic pathways
mentioned (benzoate, resorcinol and resorcylic acids, trihydroxybenzenes) are
degraded relatively quickly, other compounds which depend on endergonic
activation reactions (e. g., phenol, m-cresol, catechol, hydroquinone, aniline,
phthalates) or dehydrogenations at a comparably high redox potential (p-cre-
sol, toluene) are decomposed more slowly, indicating that the modification re-
actions limit the transformation rates. Enrichment for anaerobic utilizers of
these substrates takes, in general, much longer than with the previously men-
tioned substrates (benzoate, resorcinol and resorcylic acids, trihydroxyben-
zenes). It is obvious that in the case of p-cresol and toluene the type of alterna-
tive electron acceptor available will also influence the degradation kinetics: a
nitrate reducer can derive more energy from the oxidation of an aromatic com-
pound than a sulfate reducer or a methanogenic association.
Summarizing, it can be concluded that all mononuclear aromatics can be de-
graded anaerobically if they carry at least one carboxy, hydroxy, amino, or me-
thyl substituent. Nonetheless, the degradation kinetics differ considerably:
whereas fermenting bacteria degrading resorcinol have doubling times of 68 h,
catechol or hydroquinone degrading anaerobes have doubling times of several
days. These differences in degradation efficiency of isomeric substrates can to
some extent be explained by the basically different pathways outlined above.
It should be emphasized that non-substituted aromatic compounds such as
benzene and naphthalene are also subjects of anaerobic degradation, although
no or minor information on the pathway used is presently available.
2.7
Rsum: Aromatic Compounds
The section has attempted to summarize what is known about the metabolism
of aromatic compounds by bacteria and fungi. The results presented clearly
show that some general features have emerged which suggest that it may be
possible to predict the types of reactions that will occur with different sub-
strates and different microorganisms. The availability of molecular oxygen is
the important factor which clearly determines which strategy is in use by the
microorganisms to degrade an inert aromatic compound.
2.7.1
Degradation in the Presence of Oxygen
In all cases that have been examined, bacteria initiate the oxidation of unsub-
stituted aromatic compounds by incorporation of molecular oxygen into the
aromatic nucleus to form cis-dihydrodiols. These are further oxidized to di-
phenolic intermediates such as catechols. The dihydroxylated intermediates
undergo intra- or extradiol ring cleavage by dioxygenases to form aliphatic car-
boxylic acids which are further degraded to intermediates of central metabolic
pathways.
In contrast, fungi oxidize unsubstituted aromatic hydrocarbons by the inser-
tion of one atom of molecular oxygen into the aromatic ring by P-450-catalyzed
monooxygenase reaction. The reactive arene oxides can isomerize to phenols or
can undergo enzymatic hydration to yield trans-dihydrodiols. The phenols are
coupled to sulfate, glucose, or glucuronic acid.
The ligninolytic fungi such as Phanerochate chrysosporiumcan use a battery
of extracellular enzymes, cosubstrates, and molecular oxygen to degrade aro-
matics. Lignin peroxidases, manganese-dependent peroxidases, and laccases,
which are one-electron oxidants, produce aromatic cation radical intermediates
that undergo spontaneous fission reactions and formation of highly reactive
quinones. H
2
O
2
needed for the peroxidase reaction is produced by an extracel-
lular oxidase which oxidizes glyoxal or glucose and reduces O
2
.
It is important to underline the different function of the degradation of aro-
matic compounds by bacteria and fungi. Bacteria appear to have evolved suites
of enzymes for the degradation of aromatic hydrocarbons to smaller molecules
that can support growth. In contrast, fungi appear to have evolved a detoxifica-
tion system for the cellular elimination of aromatic hydrocarbons.
2.7.2
Degradation in the Absence of Oxygen
Because of the absence of the molecular oxygen for ring activation and cleav-
age, the anaerobic bacteria used a completely different strategy to break down
aromatic compounds. The general feature is that the aromatic ring is reduced
and the alicyclic ring formed is cleaved hydrolytically. The anaerobic pathways
may be divided into the following general steps:
1. Reactions channeling the variety of substrates into a few central interme-
diates such as benzoyl-CoA, phloroglucinol, or resorcinol and thereby pre-
paring the substrates for ring reduction.
2. Ring reduction, formation of 1,3-dioxo structure, and hydrolytic cleavage.
3. A type of b-oxidation to central metabolites (acetyl-CoA).
The easy laboratory handling of aerobic bacteria allows their isolation in pure
culture much more readily than anaerobic bacteria. However, pure cultures are
prerequisites for elucidation of degradative pathways. This clearly explains the
higher number of studies on the aerobic degradation in former times. In the last
few years, because of the interest in application of organisms to the clean-up of
contaminated aquifers, mostly lacking oxygen, the degradative potential of an-
aerobic bacteria attracted much interest. In addition, the ability of the an-
aerobes to carry out unusual biochemical reactions made them highly inter-
esting candidates for research activities.
54 W. Reineke
3
Degradation of Chloroaromatic Compounds
Microorganisms can degrade chloroaromatic compounds under aerobic and
anaerobic conditions. First the degradation of chloroaromatic compounds,
which are used by aerobic bacteria as the sole carbon and energy source, will be
discussed. Then we will deal with the degradation potential of some fungi,
especially some ligninolytic species such as Phanerochaete chrysosporium to-
wards chloroaromatics. The role of chloroaromatics as electron acceptors for
growth of anaerobic populations is the final topic.
3.1
Chloroaromatic Compounds as Growth Substrate for Aerobic Bacteria
and the Dechlorination Mechanisms
The biodegradation of a chlorosubstituted arene can be considered complete
only when its carbon skeleton is converted into intermediary metabolites and
its organic chlorine is returned to the mineral state. The crucial point is the re-
moval of chlorine substituents from the organic compound. This may occur at
an early stage of the degradative pathway prior to cleavage of the aromatic ring.
Alternatively, degradation proceeds through chlorinated diphenols as central
metabolites, and HCl is eliminated from aliphatic structures, which are generat-
ed after ring cleavage, or is linked with ring cleavage.
Both dechlorination mechanisms, i. e., early and late eliminations, may take
place with multiple chloroaromatics. If the chloroaromatic bears only one chlo-
rine substituent, initial dechlorination reactions lead to the formation of di-
phenolic ring cleavage substrates, which are further degraded in a way similar
to normal aromatics. In contrast, the early dechlorination of higher chlorinated
aromatics leads to chlorocatechols, chloroprotocatechuates, or chlorohydroqui-
nones. The degradation of chlorohydroquinones proceeds through the so-cal-
led hydroquinone pathway, which includes elimination of chlorine substituents
from the aromatic structure. However, chlorocatechols are subject to degrada-
tion through the so-called modified ortho pathway, where two late dechlorina-
tions take place from non-aromatic structures. The degradation pathways of
chlorocatechols or chlorohydroquinones converge at the stage of (chloro)-ma-
leylacetates. A step later 3-oxoadipate is the common metabolite formed in the
degradation pathways used for aromatics and chloroaromatics, if all chlorine
substituents have been eliminated (see simplified overview in Fig. 34). Chloro-
3-oxoadipates occur from the higher chlorinated compounds. Besides this
funneling of pathways, some divergence is seen for chlorocatechols and chloro-
protocatechuates leading into the meta pathway.
3.1.1
Elimination of Chlorine Substituents Prior to Ring Cleavage
The mechanisms of the dechlorination prior to ring cleavage with hydrolytic,
oxygenolytic or reductive elimination of chlorine from the aromatic ring are
schematically summarized in Fig. 35.
3.1.1.1
Replacement of Chlorine by a Hydroxyl
Hydrolytic dechlorinations have been observed in the degradation of 4-chloro-
benzoate and some chlorophenols. The mechanism of a hydrolytic dechlorina-
tion process was clarified for 4-chlorobenzoate initially by labeling experiments
using
18
O
2
and H
2
18
O [342, 343]. The data indicated that the dechlorination
reaction utilizes water as the hydroxyl donor and not molecular oxygen. This
mechanism has been shown for the degradation of 4-chlorobenzoate by
Micrococcus spp., Pseudomonas spp., Nocardia sp., Alcaligenes sp., and Arthro-
bacter spp. to give 4-hydroxybenzoate [344355].
56 W. Reineke
Fig. 34. Scheme of the convergence of degradative pathways for aromatic and chloroaromatic
compounds with (chloro)-maleylacetate and (chloro)-oxoadipate as the common intermedia-
tes. The aromatic ring is a representative for any aromatic structure (e. g., benzene, phenol,
benzoate) and the chlorinated ring for a chloroaromatic structure. Aromatic compound de-
graded via catechol and the 3-oxoadipate pathway. Monochloroaromatic compound con-
verted via catechol: chlorine elimination as part of the peripheral pathway. Chloroaromatic
compound (such as pentachlorophenol) converted via (chloro)-hydroquinone. Trichloro-
and tetrachloroaromatic compounds converted via chlorocatechol. One chlorine elimination
step as part of the peripheral pathway. Chloroaromatic compound converted via chloro-
catechol. Chlorocatechols dechlorinated as part of the modified ortho pathway.
Chlorocatechol or chloroprotocatechuate dechlorinated as part of a meta pathway
The 4-chlorobenzoate dehalogenase system from Pseudomonas sp. strain
CBS3 has been shown to be a three component enzyme complex [356, 357]. The
role of each component of the system dehalogenating 4-chlorobenzoate has
been clarified by cloning of the respective genes [358360] and by detailed
studies with purified enzymes (Fig. 36).
Activation of the substrate to its coenzyme A derivative needs ATP and is car-
ried out by a ligase [361363]. The 4-chlorobenzoate:coenzyme A ligase shares
significant sequence similarity with proteins, which catalyze similar chemistry
in the b-oxidation pathway [364].
The activation reaction precedes dehalogenation, which is catalyzed by a
dehalogenase that has sequence similarity to crotonyl-CoA hydratase [364]. The
data support a proposal that the ligase and dehalogenase evolved from a b-oxi-
dation pathway.
The studies of the Pseudomonas sp. strain CBS3 4-chlorobenzoyl-CoA deha-
logenase have shown that it utilizes a unique form of catalysis in which an ac-
Fig. 35. Schematic presentation of the early dechlorinations prior to ring cleavage. hydro-
lytic; oxygenolytic; reductive dechlorination
Fig. 36. Activation and hydrolytic dechlorination of 4-chlorobenzoate. The following en-
zymes are involved: 4-chlorobenzoate-CoA ligase, 4-chlorobenzoyl-CoA dehalogenase,
4-hydroxybenzoyl-CoA thioesterase
tive site carboxylate (aspartate functions as the active site nucleophile) bonds to
C-4 of the benzoyl ring of the bound substrate to form a Meisenheimer-like
complex. Expulsion of the chloride from the Meisenheimer complex with con-
comitant rearomatization of the benzoyl ring generates an arylated enzyme as
the second reaction intermediate. Hydrolysis of the arylated enzyme occurs by
addition of a water molecule to the acyl carbonyl carbon to form a tetrahedral
intermediate which expels the hydroxylbenzoyl group to generate the catalytic
carboxylate residue and form 4-hydroxybenzoyl-CoA (Fig. 37).
The last reaction step in the reaction to form 4-hydroxybenzoate is carried
out by the 4-hydroxybenzoate:coenzyme A thioesterase leading into the proto-
58 W. Reineke
Fig. 37. Proposed chemical pathway for the hydrolysis of 4-chlorobenzoyl-CoA by dehaloge-
nase (according to [365369]). The role of the amino acids functioning in the catalysis is
shown. substrate; Meisenheimer intermediate; arylated enzyme; arylated enzyme;
tetrahedral intermediate; product
catechuate pathway [357, 361, 370373]. No sequence homology of the thio-
esterase with other proteins was found [364].
Dehalogenation is restricted to halobenzoates substituted in the para-posi-
tion [349, 374].
Hydrolytic eliminations from other chloroaromatics such as various chloro-
phenols and the chlorohydroquinones have been postulated to occur in the so-
called hydroquinone pathway (Fig. 38). A hydrolytic elimination brings about
the formation of 6-chlorohydroxyhydroquinone from 2,6-dichlorohydroqui-
none in the degradation pathway of 2,4,6-trichlorophenol of Azotobacter sp.
GP1 and Streptomyces rochei 303 [375377] as well as 5-chlorohydroxyhydro-
quinone from 2,5-dichlorohydroquinone in the degradation pathway of 2,4,5-
trichlorophenol by Burkholderia cepacia AC1100 [378].
Hydrolytic elimination of chlorine was found to initiate the degradation of te-
trachloro-p-hydroquinone the metabolite in the pentachlorophenol mineraliza-
tion yielding trichloro-1,2,4-trihydroxybenzene in Rhodococcus chlorophenoli-
cus PCP-1 [379] as well as Mycobacterium fortuitumCG2 [380] and Sphingomonas
chlorophenolica (formerly Flavobacteriumsp.) ATCC 39723 [381].
The following conclusion can be reached: hydrolytic dechlorination of an
aromatic ring is difficult, since substituents are difficult to remove by nucleo-
philic displacement from a p-electron rich system and, therefore, the ring must
be activated by CoA or by the presence of hydroxyl or halogen substituents.
3.1.1.2
Chlorine-Carbon Bond Cleavage by Use of Mono- and Dioxygenase Reaction
Ring activating dioxygenase. Dechlorination by ring activating dioxygenases is
another mechanism to remove chlorine from chloroaromatic compounds.
Catechols are produced. The oxygen of the hydroxyl groups originates from
Fig. 38. Hydrolytic eliminations as part of the hydroquinone pathway
molecular oxygen. The oxygenolytic elimination has been shown for 2-chloro-,
3-chloro-, 2,4-dichloro-, 2,5-dichloro-, and 3,4-dichlorobenzoate, 1,2,4,5-te-
trachlorobenzene, and 4-chlorophenylacetate [382396]. Other haloaromatics
such as 2-fluorobenzoate, 2-bromobenzoate, and 3-fluorotoluene were also de-
graded by use of this elimination mechanism [397402].
Initial dioxygenases, which are responsible for ring activation, produce cis-
dihydrodiols, which are further transformed by dehydrogenases to give cate-
chols. The molecular oxygen is placed to the aromatic ring by a benzoate 1,2-di-
oxygenase, a benzene 1,2-dioxygenase, a benzoate 3,4-dioxygenase, or a phenyl-
acetate 3,4-dioxygenase in such a way that one of the vic-hydroxyl groups in the
cis-dihydrodiol is bound to the same carbon as the chlorine substituent (see
Fig. 39 with 2-chlorobenzoate). From such an unstable vic-dihydrodiol, the
chlorine substituent will be eliminated to give an ortho-diphenolic compound.
For instance, in the case of the dehalogenation of 2-fluorobenzoate the na-
ture of the dehalogenation seems to be a spontaneous reaction. While the mu-
tant B9 of Alcaligenes eutrophus, which is defective in the dihydrodihydroxy-
benzoate dehydrogenase, whose function is to form catechol in the benzoate
pathway from dihydrodihydroxybenzoate, fails to grow with benzoate, 2-fluoro-
benzoate can function as growth substrate although the dehydrogenase is miss-
ing [397]. 2-Chlorobenzoate cannot be tested since the benzoate 1,2-dioxygen-
ase is highly specific and does not tolerate the bulky chlorine substituent in
ortho-position. In Alcaligenes eutrophus especially, the benzoate 1,2-dioxygen-
ase has its function in the degradation of benzoate and the activity with benzo-
ate is higher than with the halogenated one. Therefore, the dehalogenation pro-
cess seems to be a fortuitous one. In contrast, phenylacetate 3,4-dioxygenase
prefers the halogenated compound in comparison to the non-halogenated sub-
strate, so that the physiological function seems to be that of a dehalogenase.
Because of the site specificity of the introduction of oxygen in positions 1
and 2, a benzoate 1,2-dioxygenase can only bring about elimination of a chlo-
rine substituent present at the position 2 in benzoates. The same narrow elimi-
nation potential has been observed with the phenylacetate 3,4-dioxygenase.
While 4-chlorophenylacetate is a substrate, the enzyme fails to use other chlori-
nated phenylacetates.
Monohalosubstituted substrates such as 2-chloro- and 2-fluorobenzoate, 3-
fluorotoluene, and 4-chlorophenylacetate, respectively, are oxygenolytically de-
halogenated to yield catechol, methylcatechol, protocatechuate, and dihydroxy-
60 W. Reineke
Fig. 39. Oxygenolytic dechlorination of 2-chlorobenzoate by benzoate 1,2-dioxygenase.
benzoate 1,2-dioxygenase, spontaneous, ring cleavage
phenylacetate. The products are subject to further degradation by enzymes
used for the mineralization of non-halogenated aromatic compounds. In con-
trast, higher chlorinated substrates will be oxidized and partially dechlorinated
to chlorocatechols, which will be further degraded via the modified ortho
pathway (see later). 2,4-Dichloro- and 2,5-dichlorobenzoate are converted to
4-chlorocatechol, whereas 3,5-dichlorocatechol is the product formed from
2,3,5-trichlorobenzoate. 1,2,4,5-Tetrachlorobenzene is dechlorinated to yield
3,4,6-trichlorocatechol. 3,4-Dichlorobenzoate is dechlorinated to 5-chloropro-
tocatechuate, which is subject of meta cleavage (see later).
Monooxygenase. A dechlorination of pentachlorophenol (PCP) by a hydrolytic
displacement of chlorine in Rhodococcus chlorophenolicus PCP-1 was discussed
for quite a while. However, the results were questionable because of the contra-
dicting results published by Apajalahti and Salkinoja-Salonen [403] and Schenk
et al. [404]. Uotila et al. [405] presented evidence that dechlorination proceeds
by cytochrome P-450-mediated hydroxylase.
It is now well established that the initial dechlorination of pentachlorophe-
nol occurs by a monooxygenase reaction [406, 407] producing tetrachloro-p-
hydroquinone. The enzymes catalyzing chlorohydroquinone formation from
pentachlorophenol have been studied in an Arthrobacter, Flavobacterium, and
Mycobacterium fortuitum [380, 408411].
In analogy, an oxygenase-reaction is proposed to bring about the elimination
of a chlorine substituent from 2,4,6-trichlorophenol, yielding 2,6-dichlorohy-
droquinone by Azotobacter sp. strain GP1 [412], Pseudomonas pickettii [413],
and Streptomyces rochei 303 [375]. Similarly, the degradation of 2,4,5-trichloro-
phenol by Burkholderia cepacia AC1100 proceeds through 2,5-dichlorohydro-
quinone [378]. The chlorophenol 4-monooxygenase from strain AC1100 has
recently been purified [414]. Hydroquinone was detected as the transient inter-
mediate in the degradation of 4-chlorophenol by Arthrobacter ureafaciens
[415].
3.1.1.3
Reductive Displacement of Chlorine
A reductive dechlorination mechanism has been shown as part of the degrada-
tion of chloroaromatic compounds like chlorobenzoate or pentachlorophenol
by aerobic pure cultures. A corynebacterium strain NTB-1 and Corynebac-
terium sepedonicum strain KZ-4 were found to degrade 2,4-dichlorobenzoate
via 4-chlorobenzoate [416, 417]. Recently, the degradation was shown to start
with the formation of 2,4-dichlorobenzoyl-CoA followed by a NADPH-depen-
dent ortho dehalogenation yielding 4-chlorobenzoyl-CoA, hydrolytic removal
of chlorine from the para-position to generate 4-hydroxybenzoyl-CoA, and
hydrolysis to form 4-hydroxybenzoate [417].
Reductive dechlorination steps are also involved in the degradation of the
metabolites of pentachlorophenol through the so-called hydroquinone path-
way. In a Flavobacterium sp. and a coryneform-like strain [418, 419] reductive
dechlorinations of tetrachloro-p-hydroquinone followed the initial oxidative
dechlorination of pentachlorophenol. The reductive dehalogenase has been
purified by Xun et al. [420]. A chlorine is removed from tetrachloro-p-hy-
droquinone through substitution with glutathione. Glutathione is then removed
by displacement of the aromatic moiety by a second glutathione molecule,
producing oxidized glutathione. This reaction sequence occurs a second time,
leading to the formation of 2,6-dichlorohydroquinone (Fig. 40). The net re-
action is equivalent to reductive dechlorination occurring in anaerobic or-
ganisms.
In contrast, the pathway of pentachlorophenol degradation in Myco-
bacterium fortuitum CG-2 is different [380] (Fig. 41). Tetrachloro-p-hydro-
quinone, which is formed from pentachlorophenol by an oxygenase-reaction,
seems to be initially ortho-hydroxylated to produce trichloro-1,2,4-trihydroxy-
benzene followed by three reductive dechlorinations to give 1,2,4-trihydroxy-
benzene.
62 W. Reineke
Fig. 40. Glutathione transferase catalyzed dechlorination of tetrachloro-p-hydroquinone to
2,6-dichlorohydroquinone
Fig. 41. Hydrolytic and reductive dechlorinations in Mycobacterium fortuitum CG-2
3.1.2
Late Eliminations of Chlorine After or Linked with Ring Cleavage
Chlorocatechols are key intermediates in the degradation of several chloro-
aromatics (Fig. 42). The enzymatic reactions bringing about the formation of
chlorocatechols are similar to the peripheral sequences used for the degrada-
tion of non-chlorinated aromatic compounds. This means that a number of re-
actions dealing with chlorinated intermediates take place before dechlorination
steps are reached. The late dechlorinations occur after ring cleavage from non-
aromatic structures or are linked with the ring cleavage.
3.1.2.1
Dechlorination as Part of the Modified Ortho Pathway
A common feature of a chlorocatechol degradative pathway (Fig. 43), used by
many organisms able to grow with chloroaromatics such as chlorinated anili-
nes, benzenes, biphenyls, benzoates, naphthalenes, phenols, phenoxyacetates,
salicylates, and toluenes, is the ortho cleavage of chlorocatechols by chlorocate-
chol 1,2-dioxygenases with consumption of molecular oxygen to produce the
corresponding chloro-cis,cis-muconates [421429].
The elimination of the first chlorine substituent was assumed to occur spon-
taneously after 2-chloro- and 3-chloro-cis,cis-muconate have been converted by
chloromuconate cycloisomerases to 5-chloro- and 4-chloromuconolactone, re-
spectively [430]. Dienelactones are formed due to the anti-elimination of hy-
drogen chloride and the formation of an exocyclic double bond [430, 431].
While cis-dienelactone is formed from 3-chloro-cis,cis-muconate, trans-diene-
lactone is the product from 2-chloro-cis,cis-muconate. 2,4-Dichloro-cis,cis-
Fig. 42. Schematic presentation of the mineralization of chloroaromatics with chlorocate-
chols as key metabolites
64 W. Reineke
Fig. 43. Modified ortho pathway for the degradation of 3-
chloro-, 4-chloro-, and 3,5-dichlorocatechol. chlorocate-
chol 1,2-dioxygenase, chloromuconate cycloisomerase,
dienelactone hydrolase, maleylacetate reductase. The bro-
ken arrows indicate a spontaneous elimination of chlorine
substituents from intermediates in parentheses
muconate, the metabolite in the 3,5-dichlorocatechol degradation, is converted
to 2-chlorodienelactone probably in the cis-configuration in analogy to the
conversion of 3-chloro-cis,cis-muconate.
With respect to spontaneous elimination of chlorine during 2-chloro-cis,cis-
muconate cycloisomerization, this view was challenged by the observation that
(+)-5-chloromuconolactone is a stable compound when formed by muconate
cycloisomerase, an enzyme of the normal 3-oxoadipate pathway [432]. The data
of Vollmer and Schlmann [433] corroborate the assumption that the chloro-
muconate cycloisomerase of the 2,4-D-degrading Alcaligenes eutrophus JMP134
purified by Kuhm et al. [434] or the 3-chlorobenzoate-degrading Pseudomonas
putida AC866 have the ability to catalyze chlorine elimination from (+)-5-chlo-
romuconolactone, the primary product of 2-chloro-cis,cis-muconate cycloiso-
merization and are therefore dehalogenases. Overall, a chloromuconate cycloi-
somerase brings about the conversion of 2-chloro-cis,cis-muconate, the product
of the ortho cleavage of 3-chlorocatechol, to trans-dienelactone.
The dienelactones are converted into the respective maleylacetates by diene-
lactone hydrolases.
The following enzyme, maleylacetate reductase, plays a major role in the de-
gradation of chloroaromatic compounds either in the modified ortho pathway
or as part of the hydroquinone pathway. The original function is the reduction
of the double bond by using NADH to channel maleylacetate into the 3-oxoadi-
pate pathway.
In the case of maleylacetates with chlorine substituents in the 2-posi-
tion such as 2-chloromaleylacetate, the intermediate in the degradation of 3,5-
dichlorocatechol, the substrate is reduced to the respective chlorinated 3-oxo-
adipate. This product is converted back into maleylacetate without a substi-
tuent in position 2. This step, probably a spontaneous one, is accompanied by
the elimination of chloride. Therefore, two moles of NADH per mole substrate
are consumed for the conversion of maleylacetates which contain a chlorine
substituent in the 2-position [435439]. In contrast, only 1 mol of NADH was
necessary to convert 1 mol of those substrates without a chlorine substituent in
the 2-position as it is in maleylacetate or 3-chloro- and 5-chloromaleylacetate.
The modified ortho cleavage pathway described tolerates substitution at the
aromatic ring of up to three chlorine atoms (see pathway for the degradation of
1,2,4,5-tetrachlorobenzene later). Two dechlorination steps have been describ-
ed up to now. Whether tetrachlorocatechol can serve as substrate for the known
chlorocatechol sequence is at present unknown.
3.1.2.2
Dechlorination Linked with Ring Cleavage
For a long time the degradation of chloroaromatics has not been shown to oc-
cur via the meta pathway. One reason has been found in the formation of a sui-
cide product, a reactive acyl chloride, from 3-chlorocatechol by the catechol 2,3-
dioxygenase of Pseudomonas putida PaW1 [440], which leads to inactivation of
the ring cleavage enzyme (Fig. 44). In addition, 3-chlorocatechol is able to inac-
tivate reversibly a catechol 2,3-dioxygenase because of its potential to chelate
the ferrous ion [441]. Some publications postulated that compounds degraded
via catechols chlorinated in the 4-position might be mineralized via the meta
pathway [442448]. However, information about the way in which the products
are dechlorinated is not available.
Recently, Pseudomonas putida GJ31 was found to degrade chlorobenzene
rapidly via 3-chlorocatechol and uses a meta cleavage pathway [449] (Fig. 44).
In contrast to other catechol 2,3-dioxygenases, which are subject of inactiva-
tion, the chlorocatechol 2,3-dioxygenase of strain GJ31 productively converts
3-chlorocatechol [450, 451]. Stoichiometric displacement of chloride occurs,
leading to the production of 2-hydroxymuconate, which is further converted
through the meta pathway.
A productive meta cleavage without suicide effect has been known for more
than 15 years. Kersten et al. [452, 453] reported that a distal extradiol cleaving
66 W. Reineke
Fig. 44. Degradation of 3-chlorocatechol through the meta pathway. Conversion leading to
the suicide inactivation of the 2,3-dioxygenase; productive conversion in strain GJ31 grow-
ing with chlorobenzene using a chlorocatechol 2,3-dioxygenase
protocatechuate 4,5-dioxygenase catalyzes 2-pyrone-4,6-dicarboxylic acid for-
mation by nucleophilic displacement of a halide ion from protocatechuates sub-
stituted with a halogen at the C-5 of the nucleus (Fig. 45). This indicates that
cyclization entailing nucleophilic displacement of halogen provides an effective
alternative to the enzyme suicide inactivation that occurs when a nucleophilic
group of the dioxygenase undergoes acylation. An important aspect of this me-
chanism is that the ring fission product remains bound to the enzyme during a
complete configuration change that precedes nucleophilic displacement.
Hydrolysis of the pyrone is followed by degradation through a meta pathway.
In contrast, in the case of 3-chlorocatechol cleavage by the chlorocatechol 2,3-
dioxygenase pyrone formation does not take place. Instead, the reaction of the
acyl chloride with water directly leads to an intermediate of the meta pathway.
A similar type of oxygen-dependent, acyl chloride forming ring cleavage is
assumed to occur in the degradation of g-hexachlorocyclohexane (lindane) and
pentachlorophenol [454456] (Fig. 46). There is evidence that 2-chlorohydro-
quinone, the intermediate in the lindane degradation, is directly subject to ring
cleavage by a new dioxgenase. 2,6-Dichlorohydroquinone, the metabolite in the
pentachlorophenol degradation, is cleaved by an oxygen-dependent reaction
Fig. 45. Degradation of 5-chloroprotocatechuate through a meta pathway
Fig. 46. Dechlorination in the degradation of lindane and pentachlorophenol linked with
oxygen-dependent ring cleavage
rather than hydrolyzed as published in former times. The ring cleavage pro-
ducts of both reactions, acyl chlorides, seems to react with water to give maley-
lacetate and 2-chloromaleylacetate plus HCl.
3.1.3
Degradation of Higher Chlorinated Aromatic Compounds
Needs Different Dechlorination Mechanisms
Early and late dechlorinations allow the degradation of tetrachlorobenzene
by a Pseudomonas sp. [394]. While the steps involved in the elimination of the
first three chlorine substituents are well documented (Fig. 47), i. e., oxygenoly-
tic dechlorination by the benzene dioxygenase, dehydrochlorination by the
chloromuconate cycloisomerase, and reductive elimination by the maleylace-
tate reductase, the fourth elimination step remains unclear as yet.
68 W. Reineke
Fig. 47. Degradation pathway for 1,2,4,5-tetrachlorobenzene by Pseudomonas sp. strain PS14
involving early and late chlorine elimination steps according to Sander et al. [394]. The fol-
lowing enzymes are involved: benzene dioxygenase; spontaneous; chlorocatechol 1,2-
dioxygenase; chloromuconate cycloisomerase; dienelactone hydrolase; maleylacetate
reductase; probably 3-oxoadipate: succinylCoA transferase and 3-oxoadipylCoA thiolase;
unknown sequence
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The degradative pathway for 3,4-dichlorobenzoate (Fig. 48) is another exam-
ple where elimination of chlorine substituents takes place due to the ring ac-
tivation by benzoate 3,4-dioxygenase followed by ring cleavage by protocate-
chuate 4,5-dioxygenase, i. e., oxygenolytic and nucleophilic displacements of
chloride following dioxygenase reactions.
The occurrence of a sequence of different types of chlorine eliminations, i. e.,
oxygenolytic, hydrolytic, and reductive dechlorinations, can also be illustrated
with the hydroquinone pathway used for the degradation of pentachlorophenol
and trichlorophenols, the different steps of which were discussed above (Fig. 49).
Ring cleavage of 6-chlorohydroxyhydroquinone or the nonchlorinated ana-
logue by 6-chlorohydroxyquinol 1,2-dioxygenase brings about the formation of
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Fig. 49. Proposed hydroquinone pathway for chlorophenols involving different types of dechlorination mechanisms [375377, 381, 410413, 419, 420,
456, 457]. The following enzymes are involved: monooxygenase; tetrachloro-p-hydroquinone reductive dehalogenase; 2,6-dichloro-p-hydro-
quinone chlorohydrolase; oxygen-dependent ring cleavage followed by reaction with water; 6-chlorohydroxyquinol 1,2-dioxygenase; maleyl-
acetate reductase
chloromaleylacetates or maleylacetate, which are also metabolites formed in the
modified ortho pathway. In contrast, recent results indicate that the dichlorinat-
ed hydroquinone rather than chlorohydroxyhydroquinone is the subject of di-
rect ring cleavage in Sphingomonas chlorophenolica ATCC39723.
3.2
Degradation of Chloroaromatic Compounds by Ligninolytic Fungi
Unlike bacteria, fungi generally do not utilize chloroaromatic compounds as a
source of carbon and energy. Degradation of chloroaromatics and of many
other xenobiotic compounds is not the consequence of enzyme systems target-
ed to this function. Fungal enzyme systems generally exist to serve other pur-
poses such as degradation of wood components like ligninocellulose. Enzymes
isolated and identified as having chloroaromatic degradative potential are the
phenol oxidases, lignin peroxidases, manganese peroxidases, and laccase in
ligninolytic fungi. Phanerochaete chrysosporium and other white-rot fungi are
such organisms bearing the biodegradative capabilities that encompass a broad
range of organopollutants like chlorinated anilines, benzenes, phenols, phe-
noxyacetates, biphenyls, and dibenzo-p-dioxins.
Arjmand and Sandermann [458] found that chlorinated anilines are minera-
lized by P. chrysosporium.
P. chrysosporium can substantially degrade and mineralize monochloro-
benzene and dichlorobenzenes under nutrient-rich culture conditions, in which
the lignin peroxidases and manganese peroxidases are not produced [459]. This
indicates that the lignin peroxidases and manganese peroxidases are not requir-
ed for degradation of the chlorobenzenes.
Identical results concerning the non-necessity of ligninolytic enzymes were
obtained with 2,4-D as the substrate. Yadav and Reddy [460] presented evidence
for mineralization of 2,4-D in nutrient-rich media by P. chrysosporium and by a
peroxidase-negative mutant of this organism with about 40% of initial radioac-
tivity found as
14
CO
2
, indicating that the ligninolytic enzymes are not necessary.
The fungal degradation of PCBs has been studied in various laboratories.
Eaton [461] reported that P. chrysosporiummineralized a significant fraction of
a
14
C-labeled Aroclor 1254. Bumpus et al. [148] found significant rates of
14
CO
2
evolution from radiolabeled DDT and lindane, but did not observe significant
rates from two different polychlorinated biphenyls. The previous and recent
studies by Bumpus et al. [148] and Thomas et al. [462] indicated only low levels
of mineralization of 0.91.1% for individual PCB congeners such as 3,3,4,4-te-
trachlorobiphenyl, 2,2,4,4-tetrachlorobiphenyl, and 2,2,4,4,5,5-hexachloro-
biphenyl by P. chrysosporium. Results of Zeddel et al. [463] showed that degra-
dation of a nonspecified PCB mixture by white-rot fungi Pleurotus ostreatus and
Trametes versicolor was limited to mono- and dichlorinated congeners. Yadav
et al. [464] presented evidence for substantial degradation of PCB mixtures by
P. chrysosporium based on congener-specific gas chromatographic analysis.
Degradation of Aroclor 1242, 1254, and 1260 (60%, 30%, and 18% by weight, re-
spectively) was observed in both ligninolytic as well as non-ligninolytic media.
Elimination of chlorine substituents was shown to be nonspecific, involving
ortho-, meta-, and para-substitution. Data from Dietrich et al. [465] provided
further insight into the degradative activities of P. chrysosporium with the fol-
lowing model PCB congeners: 4,4-dichlorobiphenyl, 3,3,4,4-tetrachlorobi-
phenyl, and 2,2,4,4,5,5-hexachlorobiphenyl. Extensive degradation of 4,4-
dichlorobiphenyl was found while negligible mineralization and metabolism of
3,3,4,4-tetrachlorobiphenyl, and 2,2,4,4,5,5-hexachlorobiphenyl was observ-
ed. 4-Chlorobenzoate and 4-chlorobenzyl alcohol were identified as metabolites
produced from 4,4-[
14
C]-dichlorobiphenyl.
Information on the degradative sequence was obtained for 2,4-dichloro- and
2,4,5-trichlorophenol [466, 467]. Extensive mineralization of 2,4-dichlorophenol
occurred only under nutrient nitrogen-limiting conditions [467], i. e., the
ligninolytic enzymes are essential for degradation. Valli and Gold [467] elucidat-
ed a pathway for the degradation of 2,4-dichlorophenol with purified lignin pero-
xidase and manganese peroxidase as well as cultures of P. chrysosporiumbased on
isolation and characterization of metabolites formed and transformed. The
pathway involves several cycles. Oxidative dechlorination by either peroxidase
produces a p-quinone. The p-quinone intermediate is then converted by intra-
cellular enzymes and methylated to generate a peroxidase substrate. Such a cycle
of oxidative dechlorination, quinone reduction, and hydroquinone methylation
leads to the removal of the second chlorine atom in the second turn (Fig. 50).
P. chrysosporium rapidly mineralizes 2,4,5-trichlorophenol in nitrogen-limi-
ted culture. Overall, the multistep pathway for 2,4,5-trichlorophenol resembles
the 2,4-dichlorophenol pathway. It involves cycles of peroxidase-catalyzed oxi-
dative dechlorination reactions followed by quinone reduction reactions to
yield the key intermediate 1,2,4,5-tetrahydroxybenzene, which is presumably
ring cleaved. The removal of all three chlorine atoms occurs before the ring
cleavage followed by degradation to CO
2
[466].
Mileski et al. [468] reported that P. chrysosporiumalso oxidizes pentachloro-
phenol. In general, a negligible amount of PCP is mineralized by most fungi
studied. Most of the PCP was transformed, often by O-methylation, to interme-
diates such as pentachloroanisole. Phanerochaete spp. including P. sordida have
also been shown to degrade pentachlorophenol [469]. In addition, high-mole-
72 W. Reineke
Fig. 50. Proposed pathway for the degradation of 2,4-dichlorophenol by Phanerochaete
chrysosporium. The compounds are converted by lignin peroxidase (LiP), manganese peroxi-
dase (MnP), or whole cells
cular-weight polymers can be produced by enzymes of P. chrysosporium from
pentachlorophenol [470].
Like P. chrysosporium the white-rot fungi Pleurotus ostreatus, Phellinus wei-
rii, and Polyporus versicolor also mineralized DDT [471]. In general, the extends
of mineralization varied very much with significant amounts of water-soluble
degradation products also observed in some cases.
Bumpus et al. [148] also reported that 2,3,7,8-tetrachlorobenzo-p-dioxin was
mineralized, but only low
14
CO
2
evolution was observed (2% of total), and the
formation of metabolites was not elucidated. However, recently a mixture of
polychlorinated dibenzo-p-dioxin was degraded at high yield by Phanerochaete
sordida YK-624 in low-nitrogen medium [472]. 4,5-Dichlorocatechol was de-
tected as metabolite from 2,3,7,8-tetrachlorodibenzo-p-dioxin, while tetrachlo-
rocatechol resulted from the degradation of octachlorodibenzo-p-dioxin. Since
the strain does not excrete lignin peroxidase, and breakdown was not mediated
by manganese peroxidase, enzymes other than these ligninolytic enzymes are
responsible for the degradation of polychlorinated dibenzo-p-dioxins.
A pathway for the model dioxin, 2,7-dichlorodibenzo-p-dioxin, was elucidat-
ed by characterization of fungal metabolites generated by lignin peroxidase,
manganese peroxidase, and crude intracellular cell-free extracts [473]. The mul-
tistep pathway shown in Fig. 51 involves the degradation of 2,7-dichlorodibenzo-
p-dioxin and subsequent intermediates by oxidation, reduction, and methylation
reactions to yield the key intermediate 1,2,4-trihydroxybenzene. P. chrysospo-
rium extensively degrades 2,7-dichlorodibenzo-p-dioxin only under nutrient-
limiting conditions, suggesting that a lignin-degradative system is involved.
The dechlorination and cleavage of the dioxin was thought to function as fol-
lows (Fig. 52). The first step is the one-electron oxidation by the oxidized en-
Fig. 51. Proposed pathway for the degradation of 2,7-dichlorodibenzo-p-dioxin by Phan-
erochaete chrysosporium (LiP, lignin peroxidase, MnP, manganese peroxidase)
7
4
W
.
R
e
i
n
e
k
e
Fig. 52. Proposed mechanism for the dechlorination and ring cleavage of 2,7-dichlorodibenzo-p-dioxin by lignin peroxidases of Phanerochaete
chrysosporium
zyme intermediate LiPI resulting in the formation of the aryl cation radical A,
which is probably short-lived. Attack of H
2
O at the cation would result in the
loss of chloride and the formation of the carbon-centered radical intermediate
B. One-electron oxidation by LiP or MnP would result in the formation of the
cation intermediate C. Attack of H
2
O on this intermediate would lead to the first
C-O-C bond cleavage and the formation of the quinone intermediate D.
Subsequent oxidation of the phenolic function would generate the phenoxy ra-
dical E which is in resonance with the carbon-centered radical E. Oxidation by
either LiP or MnP would yield the cation F. Finally, attack of H
2
O on the cation
would result in the cleavage of the second C-O-C bond and generation of 4-
chloro-1,2-benzoquinone and 2-hydroxy-1,4-benzoquinone.
In general, the data obtained with ligninolytic fungi concerning mineraliza-
tion show an indistinct direction. Some degradation rates are very low.
3.3
Anaerobic Microbial Populations with the Potential
to Dechlorinate Chloroaromatic Compounds
The anaerobic biodegradation of a chloroaromatic compound was first demon-
strated for pentachlorophenol in 1972 by Ide et al. [474], and later by several
other groups with both flooded soil and sewage sludge incubation systems
[474478]. However, the significance of reductive dechlorination of chlorinated
aromatic compounds by anaerobic bacteria has gained recognition only in the
last few years, beginning with the report by Suflita et al. [479].
In most cases, the microbial activities have only been shown in situ or with
environmental material, i. e., sewage sludge, sediment, aquifer material, and
with undefined enrichments. While, in the main, the responsible microbes have
not been identified, a few studies are available now with defined consortia and
with isolated anaerobic bacteria.
In addition, phototrophic bacteria can metabolize chloroaromatics.
3.3.1
Potential of Environmental Materials and Undefined Enrichments
By analyzing concentration changes, anaerobic dechlorination has been shown to
occur in anoxic materials with a large variety of chloroaromatic compounds
(Table 10) under denitrification, sulfate reduction and methanogenic conditions.
Clear evidence for the role of microbial processes in dechlorination reactions
with environmental materials came from the following observations:
1. No dechlorination occurs in autoclaved samples.
2. Dechlorination is very specific and different in different systems: the reactions
are specific only certain congeners were used as the substrate such as meta-
substituted benzoates or meta- and para-substituted PCBs [479, 492, 527]. Other
systems ortho-dechlorinate PCB congeners [519]. Such a pronounced specificity
for a definite substitution pattern would not be expected to occur from abiotic
reactions. Enrichments from different sediments clearly indicate different pat-
terns of dechlorination when adding the same congener mixture [527, 531].
Enrichment cultures from sewage sludge adapted to a certain monochloro-
phenol show different dechlorinating activities with various chlorophenols
[500]. Complete dechlorination of pentachlorophenol was obtained when a
mixed culture was produced from single cultures which are able to degrade
different single chlorophenol isomers. In contrast, each single culture alone
failed to show this property towards pentachlorophenol [506]. The regio-
selectivity of reductive dehalogenation in methanogenic sediments depends
on the source of the microbial community and adaptation conditions
[539541].
3. The more highly chlorinated PCB congeners generally appear to be more
readily dechlorinated than the lower chlorinated congeners, which causes ac-
cumulation of mono- and dichlorobiphenyls [524, 528].
4. The dechlorination of the lower substituted congeners started when the
higher chlorinated aromatics were totally converted into the lower chlorinat-
ed ones [479].
5. The reductive dechlorination can be stimulated by the addition of organic
electron donors, such as lactate, acetate, pyruvate, ethanol, and glucose. H
2
can also function as electron donor. In some cultures the addition of an or-
ganic electron donor is essential for the dechlorination.
A different degree of degradation by anoxic microbial populations has been ob-
served. While some chloroaromatic compounds will be mineralized to give
CO
2
, others such as PCBs and chlorobenzenes are only partial dechlorinated
and lower chlorinated congeners are formed.
Dehalogenation and degradation of halogenated aromatic compounds by
anaerobic bacteria populations have mostly been demonstrated under me-
thanogenic conditions [542, 543]. Reductive dechlorination is followed by cleav-
age of the aromatic ring, so that the chloroaromatic compound is ultimately
mineralized to CH
4
and CO
2
. In addition, reductive dechlorination has been de-
monstrated in other than methanogenic conditions. Inhibitors, such as bromo-
ethanesulfonic acid (BESA) (for methanogens) or molybdate (for sulfate-reduc-
ing bacteria), are used to determine which certain group of organisms is re-
sponsible for the dechlorination. Often inhibition of the methanogenic activity
by BESA did not show an effect on the dechlorinating activities [484], indicating
that methanogenic organisms do not always take part in the dechlorination.
76 W. Reineke
Table 10. Chloroaromatic compounds dechlorinated by anoxic microbial populations
Compound(s) References
Chlorobenzenes [480488]
Chloroanilines [489491]
Chlorobenzoates [479, 492498]
Chlorophenols [494, 499508]
Chlorophenoxyacetates [505, 507, 509, 510]
Chlorocatechols [511513]
Chlorobiphenyls (PCBs) [514536]
Chlorodibenzo-p-dioxins [537, 538]
Another interesting observation is that the presence of other electron accep-
tors such as nitrate, sulfate, and other sulfur oxyanions may lead to a partial or
total inhibition of the reductive dechlorination of aromatics by consortia and
populations from sediments [490, 502, 515, 544549]. However, the effect of sul-
fate, for instance, might be in most instances a question of competition for elec-
trons, since DeWeerd et al. [544] observed with resting-cells of Desulfomonile
tiedjei, a model organism with aryl reductive dechlorination potential, that H
2
uptake was much faster when sulfate was available as the electron acceptor in-
stead of 3-chlorobenzoate, the substrate for dechlorination. In addition, thiosul-
fate and sulfite, but not sulfate, were found to be potent inhibitors and to repress
induction of the aryl dechlorination activity of Desulfomonile tiedjei [549].
However, the inhibition of the dechlorination in a consortium by electron ac-
ceptors other than CO
2
is not always the case, as these alternative electron ac-
ceptors may indeed support anaerobic degradation of halogenated aromatic
compounds [503, 509, 550]. It has been demonstrated that chlorinated phenols
and benzoates can be degraded under sulfidogenic conditions in both freshwa-
ter (Hudson and Nile Rivers) and estuarine sediments [551553]. The depen-
dency on sulfate reduction and inhibition by molybdate [552] suggests that sul-
fate-reducing bacteria may be directly responsible for chlorophenol degrada-
tion [554]. The reductive dechlorination as the initial step in chlorophenol
degradation by the sulfate-reducing consortium was confirmed by using
chlorofluorophenols as analogous compounds and the detection of the stoi-
chiometric accumulation of fluorophenols [555].
Kazumi et al. [556] presented data indicating that Fe
3+
can serve as a termi-
nal electron acceptor in the microbial degradation of monochlorinated aromat-
ic compounds such as phenols and benzoates in anoxic sediment enrichments.
A systematic evaluation of the utilization of monochlorobenzoates under deni-
trifying, Fe
3+
-reducing, sulfidogenic and methanogenic conditions showed that
anaerobic microbial consortia from the River Nile have the capacity to degrade
all three chlorobenzoate isomers in the absence of oxygen and in the presence
of the alternative electron acceptors nitrate, ferric ion, sulfate, or carbon dioxide
[553]. The degradation of chlorobenzoates was coupled stoichiometrically to NO
3
loss, Fe
2+
production, SO
4
2
loss, or CH
4
production, indicating that the chloro-
benzoates were oxidized to CO
2
. The loss of chlorobenzoate isomers was fastest
under denitrifying conditions when compared to the other reducing condi-
tions. There was little difference in the rate of initial substrate loss among Fe-
reducing, sulfidogenic, and methanogenic conditions. Degradation of mono-
chlorobenzoates with the population from the River Nile was dependent not
only on the electron acceptor present but also on the position of the chlorine sub-
stituent with the pattern meta>para>ortho. This relative degradability has pre-
viously also been observed with cultures from the Hudson and East Rivers [551].
3.3.2
Pure Cultures: Chloroaromatic Compounds as Electron Acceptors
Although a great number of dechlorinations of aromatics have been reported to
occur under anoxic conditions, only a few pure bacterial cultures have been iso-
lated until now which are able to dechlorinate an aromatic compound reduc-
tively. While the physiological function of the dechlorination of 1,2,4-trichloro-
benzene by the intestinal bacterium Staphyloccoccus epidermis [487, 488] is un-
known, probably being a cometabolic step, the other strains use the chlorinated
aromatic compound as an electron acceptor in an anaerobic respiration
(Table 11).
The first detailed data on the dechlorination mechanism were obtained from
Desulfomonile tiedjei DCB-1, which was isolated from a methanogenic 3-chlo-
robenzoate-degrading consortium. The strain dechlorinates 3-chlorobenzoate
to benzoate but cannot utilize the benzoate. The strain, a gram-negative, stric-
tly anaerobic rod, was found to be a sulfate-reducing organism. The same was
true for other isolates able to use 3-chlorobenzoate. However, the idea that sul-
fate-reducing organisms in general have dechlorinating activities was found to
be wrong. The oxidation of formate is coupled to the reductive dechlorination
of 3-chlorobenzoate leading to energy for growth [557, 558]. Although the
strain is also able to dechlorinate chlorophenols and tetrachloroethene, these
dechlorinations seem not to be coupled to growth [559, 560]. The dechlorinat-
ing activity towards 3-chlorobenzoate is inducible and co-induced with a te-
trachloroethene dechlorinating activity [561]. The dehalogenating activity of
Desulfomonile tiedjei, located in the membrane, was found to be active in cell-
free extracts [562]. The measurement of proton release in a cell suspension due
to the addition of 3-chlorobenzoate clearly indicates that the reductive dechlo-
rination of 3-chlorobenzoate results in the formation of a proton gradient
across the cytoplasma membrane [563]. The 3-chlorobenzoate reductase has
been purified from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1
and characterized [564]. The dechlorination was found to represent a novel
type of anaerobic respiration [479, 496, 565]. Knowledge of the components in-
volved in electron transfer from a donor molecule to the electron-accepting 3-
chlorobenzoate is limiting. Louie et al. [566] found a unique membrane-bound
cytochrome c induced in Desulfomonile tiedjei DCB-1 which is co-induced with
the 3-chlorobenzoate-dechlorinating activity. Louie and Mohn [567] demon-
strated that the reductive dehalogenase is oriented towards the cytoplasm in the
membrane of Desulfomonile tiedjei DCB-1, and the active site seems to be loca-
ted on the cytoplasmic side of the membrane. Protons are produced in the pe-
riplasm generating a proton motive force by a scalar mechanism, i.e., no pro-
tons are translocated.
A model for the process is given in Fig. 53.
Other pure cultures dechlorinate ortho-substituted phenols. Desulfito-
bacterium dehalogenans strain JW/IU DC1 is a gram-positive strictly anaerobic
bacterium, which is able to use besides the chloroaromatic compounds
other electron acceptors such as nitrate, fumarate, sulfite, thiosulfate, sulfur, and
3-chloro-4-hydroxyphenylacetate [568, 569]. The dechlorinated products from
2-chlorophenol or 3-chloro-4-hydroxyphenylacetate, phenol, and 4-hydroxy-
phenylacetate, respectively, will not be used further by the strain. Desulfito-
bacterium dehalogenans strain JW/IU DC1 has now been shown to grow via
dehalorespiration [570]. The ortho-chlorophenol reductive dehalogenase of
strain JW/IU DC1 has now been purified and characterized [571]. In addition,
78 W. Reineke
A
e
r
o
b
i
c

a
n
d

A
n
a
e
r
o
b
i
c

B
i
o
d
e
g
r
a
d
a
t
i
o
n

P
o
t
e
n
t
i
a
l
s

o
f

M
i
c
r
o
o
r
g
a
n
i
s
m
s
7
9
Table 11. Properties of strains using halogenated aromatic compounds as electron acceptors
Strains E-donor E-acceptor C-source t
d
(h) with the chloro-
aromatic compound
as electron acceptor
Desulfomonile tiedjei DCB-1 H
2
, formate, lactate, meta-Substituted chlorobenzoates, sulfate, CO
2
and organic 26 (3-chlorobenzoate)
pyruvate, benzoate, sulfite, thiosulfate compounds
methoxybenzoates
Desulfitobacterium H
2
, formate, lactate, 3-Chloro-4-hydroxyphenylacetate, ortho- Organic compounds 3.5 (3-chloro-4-
dehalogenans JW/IU-DC1 pyruvate, substituted chlorophenols, nitrate, fumarate, (yeast extract) hydroxyphenylacetate)
sulfite, thiosulfate, sulfur
Strain 2CP-1 Formate, acetate ortho-Substituted chlorophenols, oxygen n. d. 89 (2-chlorophenol)
(<6%), fumarate
Desulfitobacterium sp. Lactate, pyruvate, Tetrachloroethene, 2-chlorophenol, 2,4,6-tri- Lactate 23 (3-chloro-4-
strain PCE1 butyrate, formate, chlorophenol, 3-chloro-4-hydroxyphenyl- hydroxyphenylacetate)
succinate, ethanol acetate, sulfite, thiosulfate, fumarate
Desulfitobacterium Pyruvate Pentachlorophenol sulfite, thiosulfate nitrate n. d. n. d.
frappieri PCP-1T
Desulfitobacterium chloro- Formate, butyrate, 2,3-Dichlorophenol, 2,6-dichlorophenol, n. d. 23 (3-chloro-4-
respirans Co23 crotonate, lactate, 2,4,6-trichlorophenol, 3-chloro-4-hydroxy- hydroxyphenylacetate)
pyruvate, H
2
benzoate, 3-chloro-4-hydroxyphenylacetate,
sulfite, thiosulfate, sulfur
Desulfovibrio sp. strain TBP-1 Lactate, pyruvate, H
2
, 2-Bromo-, 4-bromo-, 2,4-dibromo-, n. d. n. d.
fumarate 2,6-dibromo-, 2,4,6-tribromophenol,
sulfate, sulfite, thiosulfate, sulfur
n.d.: no data.
the cloning of the gene coding the dehalogenase showed structural resemblance
with haloalkane reductive dehalogenases.
The strain 2CP-1 is a facultative anaerobic gram-negative rod, more closely
related to the myxobacteria than to D. tiedjei or other sulfidogens. It is able to
grow under semi-aerobically conditions (oxygen concentration<6%) [572]. 2-
Chloro- and 2,6-dichlorophenol were substrates for dechlorination. Phenol, the
product of the dechlorination, is excreted into the medium and supports cell
growth. The strain was enriched from a culture supplied with 2-chlorophenol as
an electron acceptor and formate and acetate as potential electron donors. Cole
et al. [572] added BESA to the enrichment culture to block methanogenesis as
well as the syntrophic fermentation of the expected phenol. It is probable that
strain 2CP-1 gains energy by using the chlorinated substrate as a respiratory
electron acceptor. The range of preferred substrates for dehalogenation by
2CP-1 appears extremely limited. Dehalogenation activity is not constitutive in
this isolate but is induced by the presence of 2-chlorophenol, implying specific
recognition of the substrate. These data indicate that the dechlorination is not
a cometabolic reaction.
It is unknown if dechlorination is coupled to growth via dehalorespiration
in strain DCB-2 [573]. Strain DCB-2 is capable of rapidly catalyzing ortho de-
chlorination of pentachloro-, 2,4,5- and 2,4,6-trichloro-, and 2,4-dichlorophe-
80 W. Reineke
Fig. 53. Scheme of the chemiosmotic coupling between reductive dechlorination of 3-chloro-
benzoate and energy generation in Desulfomonile tiedjei (according to Louie and Mohn [567])
nol. A unique property is the meta dechlorination of 3,5-dichlorophenol. Re-
cently the strain was identified as Desulfitobacterium hafniense [574]. It was
reported that DCB-2 is able to dechlorinate reductively 3-chloro-4-hydroxy-
phenylacetate to 4-hydroxyphenylacetate.
More recently, a tetrachloroethene-dechlorinating strain PCE1 was isolated
from a tetrachloroethene-dechlorinating enrichment culture [575, 576]. The
strain was placed within the genus Desulfitobacteriumand was found to use se-
veral ortho-substituted phenolic compounds as electron acceptors in addition
to tetrachloroethene when lactate or pyruvate were added as electron donors.
2,4,6-Trichlorophenol was reductively dechlorinated via 2,4-dichloro- to 4-
chlorophenol, whereas ortho dechlorination of 2-chlorophenol resulted in the
formation of phenol. Reductive dechlorination of 3-chloro-4-hydroxyphenyla-
cetate to 4-hydroxyphenylacetate supported growth of strain PCE1 on lactate.
Other chlorinated aromatics, such as hexachlorobenzene, 2,5-dichloro-, 3,4-
dichloro-, and 2,3,6-trichlorobenzoate, were not dechlorinated.
An anaerobic bacterium, PCP-1T, characterized as the new species Desulfito-
bacterium frappieri, was isolated from a methanogenic consortium with penta-
chlorophenol as the electron acceptor [577]. The organism dechlorinates several
different chlorophenols in ortho-, meta-, and para-position to the hydroxyl group.
Pentachlorophenol is dechlorinated via 2,3,4,5-tetra-, 3,4,5-trichloro-, and 3,5-
dichloro- to 3-chlorophenol. The strain fails to dechlorinate 2,3-dichloro-, 2,5-
dichloro-, 3,4-dichloro-, and monochlorophenols. There are indications that two
dechlorination systems are involved in the dechlorination of pentachlorophenol,
one for the ortho and the other for the meta and para dechlorination. Ortho- and
para-dechlorinating activities were found to be induced by different chlorophe-
nols [578]. Sulfite, thiosulfate, and nitrate, but not sulfate, function as electron ac-
ceptors when pyruvate and yeast extract were used for growth.
An anaerobic spore-forming microorganism, Desulfitobacterium chlorore-
spirans strain Co23, was enriched on the basis of the ability to grow with 2,3-
dichlorophenol as its electron acceptor [579]. Chlorines in ortho-positions were
removed from di- and trichlorophenols such as 2,3-dichloro-, 2,6-dichloro-, and
2,4,6-trichlorophenol, but not from monochlorophenols as well as 2,4-dichloro-,
2,5-dichloro-, 2,3,5-trichloro-, and pentachlorophenol, when lactate was added
as electron donor. In addition to the chlorophenols, Desulfitobacterium chloro-
respirans strain Co23 could dechlorinate 3-chloro-4-hydroxy-, 3,5-dichloro-4-
hydroxybenzoate, and 3-chloro-4-hydroxyphenylacetate, but fails to used
chlorobenzoates as electron acceptors. Additional electron donors were pyru-
vate, formate, butyrate, crotonate, and H
2
. Strain Co23 also used sulfite, thiosul-
fate, and sulfur as electron acceptors for growth, but did not use sulfate, nitrate,
or fumarate.
In contrast to most other anaerobic dechlorinating organisms, strain Co23
grows rapidly and attains high cell densities on pyruvate in the presence of 3-
chloro-4-hydroxybenzoate as the electron acceptor. 3-Chloro-4-hydroxybenzo-
ate was shown to be the optimum electron acceptor for growing the cells, since
the product of dechlorination, 4-hydroxybenzoate, was not inhibitory at high
concentrations, while 3-chlorophenol, the product from the dechlorination of
2,3-dichlorophenol, was inhibitory to growth and dechlorination activity. The
dechlorination activity is inducible and was found to be membrane-bound and
oxygen-insensitive. It exclusively dechlorinates chlorophenols in ortho-posi-
tions [580]. Interestingly, cell-free membrane preparations dechlorinated some
chlorophenols, such as 2,3,5-trichloro- and pentachlorophenol, that could not
serve as electron acceptors supporting growth of strain Co23. Overall the de-
chlorination takes place with a wide range of chlorophenols, i. e., from dichloro-
to pentachlorophenol, but chlorobenzoates or tetrachloroethene were not de-
chlorinated.
Boyle et al. [581] described the isolation and characterization of a Desul-
fovibrio sp., designated strain TBP-1, capable of reductive dehalogenation from
estuarine sediments. The obligately anaerobic bacterium removes bromine sub-
stituents in the ortho- and para-positions of brominated phenols (2-bromo-, 4-
bromo-, 2,4-dibromo-, 2,6-dibromo-, and 2,4,6-tribromophenol) but not 3-bromo-
or 2,3-dibromophenol or monobrominated benzoates. It does not dehalogenate
chlorinated phenols. Strain TBP-1 possesses the ability to grow by coupling the
oxidation of lactate (electron donor) to the reductive dehalogenation of 2,4,6-tri-
bromophenol, yielding stoichiometric amounts of phenol. Pyruvate, hydrogen as
well as fumarate can function as electron donor besides lactate. Alternative elec-
tron acceptors are sulfate, sulfite, sulfur, thiosulfate, but not nitrate.
A 16S rRNA sequence analysis showed a highly phylogenetic relationship be-
tween chlorophenol-dechlorinating organisms. Desulfitobacterium chlorore-
spirans strain Co23 is related to Desulfitobacterium dehalogenans JW/IU DC1,
Desulfitobacterium sp. PCE1, and Desulfitobacterium hafniense DCB-2 with se-
quence similarities of 97.2%, 96.8%, and 98.5%, respectively [579]. Desulfito-
bacterium frappieri PCP-1T exhibits 95% similarity with Desulfitobacterium de-
halogenans JW/IU DC1 [577].
Overall, little is known at present of the biochemical mechanism of the re-
ductive dechlorination.
3.3.3
Pure Cultures: Chloroaromatic Compounds as Growth Substrate
Hggblom and Young [582] recently isolated a denitrifying bacterium, Thauera
aromatica strain 3CB-1, from Hudson River sediment after enrichment on 3-
chlorobenzoate under anoxic, denitrifying conditions. Other halobenzoates
with substituents in the 3-position are also degraded with stoichiometric re-
lease of halide under conditions supporting anaerobic growth by denitrifica-
tion. Complete oxidation of the substrates to CO
2
was observed. Oxygen could
not replace nitrate as the electron acceptor. The degradation was specific to the
position of the halogen substituent: the strain did not utilize 2- and 4-chloro-
benzoate as sole carbon source.
3.3.4
Dechlorinating Organisms, Part of a Food Web
Studies with Desulfomonile tiedjei DCB-1 suggest possible reasons why an-
aerobes capable of reductive dechlorination of chloroaromatics are difficult to
82 W. Reineke
isolate; they are found most readily in complex communities (Fig. 54). D. tied-
jei DCB-1 was isolated from a 3-chlorobenzoate-degrading methanogenic con-
sortium consisting of strain DCB-1, a fermentative benzoate-degrading bacter-
ium and a Methanospirillum sp., which consumes the hydrogen and CO
2
, pro-
duced by the fermenting bacterium, to form methane. D. tiedjei DCB-1 feeds on
hydrogen and acetate as sources for redox equivalents and carbon, which are
produced by the fermenting organism. 3-Chlorobenzoate is used as the electron
acceptor by D. tiedjei DCB-1 and is reduced to benzoate and chloride. Possible
electron donors for this process include hydrogen and formate.
3.3.5
Phototrophic Bacteria and Chloroaromatic Compounds
Although the degradation of aromatic compounds by phototrophic bacteria has
been known of for a long time, it is only recently that phototrophic mineraliza-
tion of chloroaromatics has gained some attention. A phototrophic enrichment
culture using acetate as carbon source partially dechlorinated 2,3,5,6-tetra-
chlorobiphenyl in the presence of light [584]. Ortho chlorines were removed
preferentially. Two Rhodopseudomonas palustris strains, WS17 and DCP3, as
well as the non-classified phototrophic bacterium H45-2, are able to photo-
metabolize 3-chlorobenzoate when grown with benzoate and forming stoichio-
metric amounts of chloride [585, 586]. In contrast to strains WS17 and H45-2,
Fig. 54. Syntrophic relationships in a 3-chlorobenzoate-degrading consortium (based on
[479, 493, 496, 544, 583])
strain DCP3 is the only photoheterotroph capable of using 3-chlorobenzoate for
growth independently of the presence of benzoate [586]. In addition, Rhodo-
pseudomonas palustris DCP3 3-chlorobenzoate-grown cells can also use 2-
chloro-, 4-chloro-, and 3,5-dichlorobenzoate.
3.4
Rsum: Chloroaromatic Compounds
This section has attempted to summarize data on the microbial conversion of
chloroaromatic compounds, some of which are presently known to be natural.
Many chloroaromatics serve as carbon and energy source for aerobic bac-
teria. Diverse dechlorination mechanisms exist either pre or post ring cleavage,
including hydrolytic, reductive, and oxygenolytic mechanisms. Various of these
steps were found to be spontaneous in nature, i. e., the enzymes convert a chlo-
rinated substrate to an unstable product, eliminating the chlorine substituent.
Other dechlorination reactions were catalyzed by dehalogenases. Overall much
biochemical data concerning the aerobic degradation of chloroaromatic com-
pounds are available.
Anoxic microbial degradation of chloroaromatics was shown to take place
with electron acceptors such as nitrate, ferric ion, sulfate, or carbon dioxide. Most
of the respective populations showed their dechlorinating potential only in mixed
culture or as black box material from the environment. In addition, the chloro-
aromatics can also function as an alternative electron acceptor in a novel type of
anaerobic respiration, termed dehalorespiration, as has been shown with pure
cultures dealing with chlorobenzoates and chlorophenols. The biochemical me-
chanisms involved in the anoxic dechlorination of chloroaromatic compounds
are presently unknown. However, recent years have show a rapid increase in in-
formation concerning the anaerobic degradation of chloroaromatics.
Besides these activities of bacteria, fungi, especially the ligninolytic ones, can
oxidatively dechlorinate chloroaromatic compounds in a cometabolic type of
process by using exoenzymes such as lignin and manganese peroxidases nor-
mally used for cleaving lignin.
4
Degradation of Aliphatic Hydrocarbons
4.1
Aerobic Degradation of Aliphatic Hydrocarbons
4.1.1
Alkanes
4.1.1.1
Gaseous Alkanes (Short-Chain Alkanes)
Organisms termed methanotrophs, such as Methylomonas, Methylosinus,
Methylococcus, etc., use methane as the sole source of carbon and energy. The
bacteria comprise a distinct group of obligatory methylotrophic organisms re-
84 W. Reineke
viewed by Anthony [587]. They grow well on methane but often rather poorly
on methanol. Methanotrophs do not grow on higher alkanes, which clearly dis-
tinguishes them from other alkane-utilizing bacteria.
For the methane molecule to be assimilated for cell growth, this most highly
reduced form of carbon must be oxidized. The initial oxidation of methane is
catalyzed by methane monooxygenase (Fig. 55). Only one atom of the dioxygen
molecule is inserted into the methane molecule to produce methanol; the other
oxygen atom is reduced to water as has been shown by Higgins and Quayle
[588] by using oxygen-18.
The methanol can be further oxidized to formaldehyde. This molecule can
either be used in an assimilatory way to make cell material or continue to be
oxidized to CO
2
in a dissimilatory fashion to produce reduced pyridine nucleo-
tides for energy generation. The formaldehyde will be assimilated either via the
ribulose monophosphate cycle (type I methanotrophs such as Methylomonas,
Methylococcus, Methylobacter) or the serine pathway (type II methanotrophs
such as Methylosinus, Methylocystis).
A remarkable feature of the methane monooxygenases is the wide variety of
substrates whose oxygenation they catalyze [589591]:
1. Alkanes, yielding primary and/or secondary alcohols.
2. Monosubstituted aromatic compounds, yielding hydroxylated derivatives;
for example, the saturated side chain of ethylbenzene is oxidized to the pri-
mary alcohol, while the aromatic ring is oxidized to give a hydroxyl group in
the para-position.
3. Alkenes, yielding epoxides, which are sometimes chemically stable.
This cometabolic potential of the methane monooxygenase will be discussed
latter in the section on the degradation of chloroaliphatic compounds.
The ability of microorganisms to utilize propane as sole carbon source is well
documented [592]. The organisms belong mainly to the Corynebacterium-
Mycobacterium-Nocardia complex, a loosely defined group of gram-positive
Fig. 55. Degradation of methane. methane monooxygenase; methanol dehydrogenase;
formaldehyde dehydrogenase; formate dehydrogenase. PQQ, methoxatin, (2,7,9-tricar-
boxy-1H-pyrrolo(2,3-f)quinoline-4,5-dione)
bacteria containing other genera such as Rhodococcus, Brevibacterium, and
Arthrobacter [592, 593]. As with methane, the initial metabolic attack on pro-
pane is by a monooxygenase, which produces propanol or isopropanol, i. e., by
terminal or sub-terminal oxidation. The metabolism of 1-propanol will run
through propanoate, and further the methyl malonate pathway [594, 595].
86 W. Reineke
Fig. 56. Pathways of propane metabolism [596599]. terminal oxidation via propanoate;
, sub-terminal oxidation via acetol and hydroxymethylacetate; sub-terminal oxidation
via pyruvate; sub-terminal oxidation via methyl acetate
Isopropanol degradation proceeds via acetone, which is further metabolized via
acetol (1-hydroxy-2-propanone) and hydroxymethylacetate, via pyruvate or via
methyl acetate to reach the central metabolism (Fig. 56). For an alternative de-
gradative acetone pathway see Sect. 4.1.3.
4.1.1.2
Long-Chain n-Alkanes
The utilization of C
1
C
4
alkanes is restricted to specialized species, which can
easily be enriched. n-Alkanes in the C
5
C
9
range are toxic to many microor-
ganisms but can be biodegraded by some specific strains that have the correct
set of catabolic enzymes. n-Alkanes of the C
10
C
22
range have been found to be
readily degradable in the environment and support growth of laboratory cul-
tures [600603]. Higher-molecular weight alkanes tend to be solid waxes and
are not readily biodegraded; however, slow biodegradation of n-alkanes up to
C
44
has been shown [604].
In most cases microorganisms such as Pseudomonas spp., Nocardia spp.,
Mycobacterium spp., and certain yeasts such as Candida spp. and molds the
initial metabolic attack on medium chain-length n-alkanes is by a monooxyge-
nase to produce the corresponding alkane-1-ol. Attack by dioxygenase enzymes
has also been reported but is less common. In such cases the n-alkanes are con-
verted to give the corresponding hydroperoxides which are reduced to yield an
alkane-1-ol (Fig. 57).
The sub-terminal oxidation of alkanes to yield secondary alcohols is rarer.
Certain Aspergillus, Fusarium, and Bacillus strains were found to carry out sub-
terminal oxidation of medium chain-length alkanes, producing alcohols with a
hydroxyl group in the 4-, 5-, or 6-position. Lower quantities of 2- and 3-substi-
tuted compounds were produced [605].
Subsequent metabolism of the alcohols may follow a number of pathways as
illustrated in Fig. 58. The alcohol is normally oxidized to the corresponding
aldehyde and fatty acid. Less commonly, w-oxidation may result in the produc-
tion of a,w-dioic acids and/or w-hydroxy fatty acids [605]. The fatty acids pro-
duced by all the pathways are then further metabolized by b-oxidation.
Secondary alcohols produced by sub-terminal oxidation are further oxidized by
a Baeyer-Villiger type of reaction to the corresponding ester and hydrolytically
cleaved to produce an acid and alcohol. The alcohol is then oxidized and the
fatty acid is also subjected to b-oxidation.
The b-oxidation of fatty acids metabolism has been reviewed by Finnerty
and Makula [606]. Fatty acyl-CoA synthetase activates the fatty acid. Although
Fig. 57. Dioxygenase catalyzed activation of alkanes
88 W. Reineke
Fig. 58. Pathways involved in the metabolism of n-alkanes. The three metabolic routes are:
terminal oxidation; sub-terminal oxidation; w-oxidation. They have been demon-
strated to occur in various microorganisms, with terminal oxidation being the most common
TCA cycle
b-oxidation
this reaction is theoretically reversible, the equilibrium is shifted since AMP
and pyrophosphate are generated rather than ADP and ortho-phosphate:
RCOOH + HS-CoA + ATP RCO-SCoA + AMP + PPi + H
2
O
The pyrophosphate is readily hydrolyzed by a pyrophosphatase which ensures
irreversibility of the first reaction.
The b-oxidation cycle involves four separate reactions fatty acyl-CoA de-
hydrogenase, 2,3-enoyl-CoA hydratase (crotonase), 3-hydroxylacyl-CoA dehy-
drogenase, and 3-oxoacyl-CoA thiolase (thiolase) yielding one acetyl-CoA per
cycle (Fig. 59).
Fig. 59. b-Oxidation cycle. Enzyme reactions involved: fatty acyl-CoA dehydrogenase;
2,3-enoyl-CoA hydratase; 3-hydroxyacyl-CoA dehydrogenase; 3-oxoacyl-CoA thio-
lase
For unsaturated acids, such as oleic acid, 18: 1 (c9), the b-oxidation cycle can
proceed only for three complete sequences before a metabolic block occurs
[607] (Fig. 60). The product is 3-cis-dodecanoyl-CoA which has the double
bond in the wrong position and in the wrong configuration. The double bond
should be at the 2-position and the intermediate should have trans- not cis-con-
figuration. Accordingly, an isomerase then converts 3-cis-dodecanoyl-CoA to
the corresponding 2-trans isomer. This then continues in the b-oxidation se-
quence as before hydratase, dehydrogenase, and thiolase to give decanoyl-
CoA which is then handled without further deviations.
4.1.2
Branched Alkanes
In general branched chain alkanes are more slowly degraded than their
straight-chain counterparts. However, it has become apparent that many of
these compounds are more degradable than had previously been thought. It is
generally true that highly branched compounds are more recalcitrant than
simpler compounds, and particularly recalcitrant are b-branched and quater-
nary compounds due to steric hindrance of oxidation enzymes [608, 609].
The ability of diverse microorganisms to grow at the expense of branched-
chain hydrocarbons is variable with the indication that 2-methyl-branched al-
kanes are usually good growth substrates, whereas 3-methyl-branched alkanes
are attacked by very few microorganisms.
Degradation of 2,6,10,14-tetramethylpentadecane (pristane) has been parti-
cularly well studied. Griffin and Cooney [610] found that 5 of 21 bacterial and
11 fungal isolates obtained from freshwater environments could degrade this
compound. The metabolic pathways responsible for pristane (Fig. 61) have been
studied in detail in Brevibacterium sp., Corynebacterium sp., and Rhodococcus
sp. [611613] and may involve b- or w-oxidation [614]. One turn, involving b-
oxidation, yielding one propionyl-CoA, is followed by another one with acetyl-
CoA as the product. These two turns follow each other in sequence.
Nakajima et al. [615] isolated a Rhodococcus sp. capable of degrading other
complex branched chain alkanes such as 2,6,10,14-tetramethylhexadecane,
2,6,10-trimethylpentadecane, and 2,6,10-trimethyldocedane, which were used
90 W. Reineke
Fig. 60. b-Oxidation sequence of unsaturated fatty acids: oleic acid; cis-D3-trans-D-2-enoyl-
CoA isomerase
Fig. 61. Degradative pathways of 2,6,10,14-tetramethylpentadecane (pristane)
as sole sources of carbon and energy. Metabolism proceeded by oxidation of
isopropyl units to yield terminal alcohols and then fatty acids.
The problem of alkyl branching at the b-methyl position can be circumven-
ted by b-alkyl removal as demonstrated in the citronellol pathway [616621].
The main features of this pathway are a cis-geranyl-CoA carboxylase and a 3-
hydroxy-3-isohexenylglutaryl-CoA lyase which catalyzes the removal of the car-
boxylated b-methyl group. The overall result is the conversion of the 3-methyl-
acyl-CoA to a 3-ketoacyl-CoA intermediate, which can be subsequently cleaved
via b-oxidation. The citronellol pathway could provide the mechanism for oxi-
dation of b-methylsubstituted alkanes or alkenes as has been shown by Fall et
al. [622]. In combination with the initial monooxygenation as part of the alkane
pathway the use of the citronellol pathway allowed the growth of a strain with
2,6-dimethyl-2-octene for example (Fig. 62).
There is only one report on the microbial utilization of quaternary car-
bon compounds because these hydrocarbons are extremely resistant to
biodegradation. An Achromobacter sp. was found to use 2,2-dimethylheptane as
sole carbon and energy source [623]. The organism simply attacked the unhin-
dered terminus of the branched alkane and accumulated 2,2-dimethylpropionic
acid.
4.1.3
Alkenes
Olefins tend to be more toxic to microorganisms and, at least under aerobic
conditions, are less readily utilized than the corresponding alkanes [600].
Conversion of unsaturated aliphatic hydrocarbons may be initiated either via
attack on the double bond or by the same mechanisms employed in n-alkane
metabolism. It should be noted that some organisms capable of growth on
short-chain alkenes cannot grow on the corresponding alkanes since they can
only initiate metabolic attack at the double bond.
Four main patterns of initial attack can be recognized (Fig. 63): oxygenase
attack upon a terminal methyl group to produce the corresponding alkene-1-ol;
sub-terminal oxygenase attack to produce an alkenol with the hydroxyl group
at a non-terminal carbon; oxidation across the double bond to give an epoxide;
oxidation across the double bond to produce a diol.
The metabolism of short-chain alkenes (C
6
and below) is thoroughly review-
ed by Hartmans et al. [624] and Watkinson and Morgan [625].
Recently, an additional aspect of metabolism of alkenes has been obtained.
The metabolism of propylene is initiated by a monooxygenase to the corre-
sponding epoxide followed by a carboxylation reaction that forms acetoacetate
as a product [627629]. Further degradation in the Xanthobacter strain Py2,
isolated by van Ginkel and de Bont [630], is proposed to proceed through ace-
toacetyl-CoA and thiolysis to give two acetyl-CoA (Fig. 64). Carboxylation to
give acetoacetate is also a significant strategy for acetone metabolism by aero-
bic bacteria [631, 632].
92 W. Reineke
A
e
r
o
b
i
c

a
n
d

A
n
a
e
r
o
b
i
c

B
i
o
d
e
g
r
a
d
a
t
i
o
n

P
o
t
e
n
t
i
a
l
s

o
f

M
i
c
r
o
o
r
g
a
n
i
s
m
s
9
3
Fig. 62. Combined action of alkane monooxygenase and citronellol pathway for the degradation of 2,6-dimethyl-2-octene, a branched alipha-
tic compound substituted in the b-position. 2,6-dimethyl-2-octene; citronellol; citronellic acid; citronellyl-CoA; cis-geranyl-
CoA; isohexenylglutaconyl-CoA; 3-hydroxy-3-isohexenylglutaryl-CoA; 2-methyl-6-oxo-2-octenyl-CoA
94 W. Reineke
Fig. 64. Proposed pathway for propylene in Xanthobacter strain Py2 and aerobic bacteria for
acetone
Fig. 63. Metabolic pathways involved in the microbial degradation of alkenes [626]
4.2
Anaerobic Degradation of Aliphatic Hydrocarbons
In contrast to the detailed studies on the aerobic degradation of aliphatic hy-
drocarbons, little is known about the degradation of alkanes under anoxic con-
ditions, where oxygen-initiated reactions cannot occur. Although the degrada-
tion by pure cultures of Desulfovibrio under sulfate-reducing conditions has
been reported in the early literature [633635], the potential degradability of
aliphatic hydrocarbons under anoxic conditions has remained a matter for de-
bate. Over the past decades some studies on the alkane degradation with en-
richment cultures or in microcosms were reported [636642].
The first reliable proof of an anoxic degradation was provided for a culture
of a sulfate-reducing bacterium, strain Hxd3 [643, 644]. Growth of this culture
with hexadecane is very slow, with doubling times of more than one week un-
der optimal conditions. The following stoichiometric relationship between n-
hexadecane degradation and sulfate-reduction was observed:
C
16
H
34
+ 12.25SO
4
2
+ 8.5H
+
16HCO
3
+ 12.25H
2
S + H
2
O
No intermediates were detected and the metabolic pathway involved remains
unknown. Since that key publication by Aeckersberg et al. in 1991 [643] three
novel alkane-degrading, sulfate-reducing bacteria, strain TD3 [245, 645], strain
Pnd3 [646], and strain AK-01 [647] have been isolated from anoxic and hydro-
carbon polluted environments, which now allow studies on the mechanism of
the anaerobic alkane metabolism.
So and Young [648] provided evidence that alkanes are oxidized to fatty acids
by strain AK-01 under strictly anaerobic conditions. Subterminal addition of a
carbon to the hydrocarbon chain as an initial reaction is demonstrated to give
a carboxylic acid. This represents a novel mechanism by which alkanes can be
activated without oxygen in contrast to the well-known hydroxylation reaction
mediated by monooxygenases in aerobic organisms.
Recently, three denitrifying bacteria, strains HxN1, OcN1, and HdN1, were
isolated in pure culture, which are able to utilize alkanes such as n-hexane, n-
octane, or n-hexadecane anaerobically [649].
In many deep sediments, oxygen, nitrate, ferric ion, and sulfate are depleted,
leaving methanogenesis as the only terminal degradation process. Zengler et al.
[650] showed that under strict anoxic conditions long-chain alkanes (hexade-
cane) can be transformed by enrichment cultures to methane with the follow-
ing stoichiometry:
4C
16
H
34
+ 3H
2
O 49CH
4
+ 15CO
2
There has been one report of the anaerobic degradation of unsaturated long-
chain hydrocarbons such as hexadecene and squalene by methanogenic en-
richments [651]. However, the degradation was very slow. A pathway with hy-
dration of the terminal double bonds to give the corresponding primary al-
cohols and complete degradation via b-oxidation was proposed.
Acetylene as a highly unsaturated hydrocarbon is fermented fast compared
to acetate and ethanol by disproportionation through acetaldehyde, which is
formed by a hydratase [652, 653].
Harder and Probian [654] reported that Pseudomonas citronellolis can grow
anaerobically on 3,7-dimethyl-1-octanol and citronellol with nitrate as an elec-
tron acceptor.
4.3
Rsum: Aliphatic Hydrocarbons
Oxygen as the substrate for oxidation of aliphatic hydrocarbons is necessary.
The following statement about the relationship between structure and biode-
gradability for alkanes and alkenes can be made: Long-chain n-alkanes are as-
similated more readily than short chains. Saturated aliphatic hydrocarbons are
degraded more readily than unsaturated ones. Branched-chain compounds are
degraded less readily than straight-chain ones. In general, the degradation pro-
ceeds to form fatty acids which are subject to b-oxidation.
Aliphatic hydrocarbons were thought to be more or less recalcitrant in the
absence of oxygen. However, according to recent literature the anaerobic alkane
degradation by sulfate reducers may be a more widespread phenomenon than
was previously thought.
5
Degradation of Chloroaliphatic Compounds
Chlorinated aliphatic compounds form one of the most important group of in-
dustrially produced chemicals. Several of these compounds are poorly degrad-
ed in the environment. This lack of biodegradation is mainly related to bio-
chemical factors rather than thermodynamics. Both oxidative conversion of
chlorinated compounds with oxygen as the electron acceptor and reductive de-
gradation to methane or alkanes should yield sufficient energy to support
growth [655].
Five types of action of microorganisms on chloroaliphatic compounds are
known:
1. The compound serves as a sole carbon and energy source for the growth of a
pure culture of aerobic bacteria (see Table 12).
2. The compound serves as growth substrate for organisms that use an electron
acceptor other than oxygen (nitrate respiration).
3. The compound serves as a growth substrate in an acetogenic fermentation.
4. The compound serves as a substrate for some enzymes in aerobic and an-
aerobic bacteria, while the microorganism grows with another compound,
i. e., cometabolism (see Table 13).
5. The compound serves as an electron acceptor under anaerobic conditions
(see Table 14).
5.1
Chloroaliphatic Compounds as Growth Substrate for Aerobic Bacteria
The degree of recalcitrance of chlorinated aliphatic compounds to aerobic
degradation generally increases with an increasing degree of chlorine substi-
96 W. Reineke
tution. Attempts to isolate organisms that grow on compounds such as tri-
and tetrachloroethene, chloroform, and 1,1,1-trichloroethane have been unsuc-
cessful.
A significant amount of work on the degradation of chloroaliphatics has
been carried out with cultures that grow on hydroxylated or carboxylated chlo-
roaliphatics. This includes organisms growing on 2-chlorocarboxylic acids
[666, 669, 670, 682, 747752], 2-chloroethanol [677, 678], chloroallyl alcohols
[675, 686], epichlorohydrin [753], 3-chloroacrylic acid [686, 687], and various
chloropropanols [675, 679, 753].
Organisms capable of degrading 2-chlorocarboxylic acids are easily isolated
from soil [754] and use hydrolytic dehalogenases. Oxidative conversion may oc-
cur in organisms that grow on chlorinated alkanes [670], but has been poorly
studied. Figure 65 schematically shows the enzymatic reactions responsible for
the dehalogenation.
Table 12. Halogenated aliphatic as growth substrates for aerobic bacteria
Substrate Reference
Haloalkanes
Methyl chloride CH
3
Cl [656658]
Dichloromethane CH
2
Cl
2
[659664]
Ethyl chloride CH
3
CH
2
Cl [665]
1,2-Dichloroethane CH
2
ClCH
2
Cl [666669]
1-Chloropropane CH
3
CH
2
CH
2
Cl [667]
1-Bromooctane CH
3
(CH
2
)
6
CH
2
Br [670, 671]
1-Chlorooctane CH
3
(CH
2
)
6
CH
2
Cl [665]
1,3-Dichloropropane CH
2
ClCH
2
CH
2
Cl [667]
1,9-Dichlorononane CH
2
Cl(CH
2
)
7
CH
2
Cl [672]
Haloalkenes
Vinylchloride CH
2
=CHCl [673, 674]
1,3-Dichloropropene CH
2
ClCH=CHCl [675, 676]
Haloalkanols
2-Chloroethanol CH
2
CH
2
OH [677, 678]
2,3-Dichloro-1-propanol CH
2
ClCHClCH
2
OH [679]
1,3-Dichloro-2-propanol CH
2
ClCHOHCH
2
Cl [680]
Haloalkenols
2-Chloroallyl alcohol CH
2
=CClCH
2
OH [681]
3-Chloroallyl alcohol CHCl=CHCH
2
OH [675]
Haloalkanoates
Chloroacetate CH
2
ClCOOH [682]
Dichloroacetate CHCl
2
COOH [683]
Trichloroacetate CCl
3
COOH [684]
2,2,-Dichloropropionate CH
3
CCl
2
COOH [685]
Haloalkenoates
3-Chloroacrylate CHCl=CHCOOH [686, 687]
3-Chlorocrotonate CH
3
CCl=CHCOOH [688]
98 W. Reineke
Table 13. Examples of halogenated aliphatic compounds as substrates for cometabolic processes
Substrate Growth substrate Organism(s) Reference
a) Transformation aerobic/oxidative
Trichloroethene Methane Mixed culture [689691]
Methanotroph strain 461 [692]
Methylosinus trichosporium [693]
Methylosinus trichosporium OB3b [694696]
Methylocystis sp. [697]
Methylomonas methanica 681 [698]
Methane and propane mixed culture [699]
Propane Mycobacterium vaccae JOB5 [700]
Rhodococcus ssp. [701]
Phenol Burkholderia cepacia G4 [702]
Alcaligenes eutrophus JMP134 [703]
Toluene Pseudomonas putida F1; [704, 705]
toluene 2,3-dioxygenase pathway
Burkholderia cepacia G4; [704,
toluene 2-monooxygenase pathway 706709]
Burkholderia pickettii PKO1;
toluene 3-monooxygenase pathway [704]
Pseudomonas mendocina KR1; [50, 51,
toluene 4-monooxygenase pathway 704]
p-Cymene Rhodococcus erythropolis [710]
Propene Xanthobacter sp. [711, 712]
Isoprene Alcaligenes denitrificans JE75 [713]
Ammonia Nitrosomonas europaea [714, 715]
Chlorinated Methane Mixed culture [690]
ethenes
Chloroform Methane Mixed culture [689]
Toluene Pseudomonas sp. ENVBF1; [716]
toluene 2-monooxygenase pathway
Pseudomonas mendocina KR1; [716]
toluene 4-monooxygenase pathway
Vinylchloride Propane Actinomycetales [717]
Rhodococcus ssp. [701]
Halomethanes, Methane Methylococcus capsulatus (Bath) [718, 719]
ethanes, ethenes
b) Transformation by pure cultures anaerobic/reductive
Tetrachloro- Trichloromethane, Methanobacterium [720, 721]
methane dichloromethane, CO
2
thermoautotrophicum
Trichloromethane, Methanosarcina barkeri [722]
dichloromethane
Trichloromethane, Desulfobacterium autotrophicum [720, 721,
dichloromethane, CO
2
723]
Trichloromethane, Acetobacterium woodii [720, 723]
dichloromethane,
chloromethane, CO
2
Table 13 (continued)
Substrate Product(s) detected Organism(s) Reference
Tetrachloro- Trichloromethane, Clostridium thermoaceticum [723]
methane dichloromethane,
chloromethane, CO
2
Trichloromethane, Clostridium sp. [724]
dichloromethane,
unidentified
Trichloromethane Escherichia coli [725]
Formate, CO
2
Pseudomonas sp. strain KC [726, 727]
Trichloromethane Shewanella putrefaciens 200 [728]
Trichloromethane Shewanella putrefaciens MR-1 [729]
Trichloromethane Dichloromethane, Methanosarcina spp. [730]
chloromethane
1,2-Dichloro- Chloroethane, ethene Methanogens [721, 731,
ethane 732]
1,1,1-Trichloro- 1,1-Dichloroethane Methanobacterium [721]
ethane thermoautotrophicum
1,1-Dichloroethane Desulfobacterium autotrophicum [721]
1,1-Dichloroethane Acetobacterium woodii [720]
1,1-Dichloroethane, Clostridium sp. [724]
acetate, unidentified
Bromoethane Ethane Methanogens [731]
1,2-Dibromo- Ethene Methanogens [731]
ethane
Tetrachloro- Trichloroethene Methanogens [561, 721,
ethane 733]
Trichloroethene Desulfomonile tiedjei [561]
Trichloroethene Methanosarcina sp. strain DCM [734]
Trichloroethene Acetobacterium woodii [723]
1,2-Dibromo- Acetylene Methanogens [731]
ethene
Trichlorofluoro- CHFCl
2
, CH
2
FCl, CO, Methanosarcina barkeri [735]
methane fluoride
(Freon 11)
5.1.1
Hydrolytic Dehalogenation
Hydrolytic dehalogenases catalyze a nucleophilic displacement reaction with
water as the sole cosubstrate. The hydrolytic cleavage has been found in micro-
organisms that grow with chlorocarboxylic acids and haloalkanes. The removal
of the halogens from chlorocarboxylic acids results in the formation of hy-
droxyalkanoic acids from monosubstituted compounds and oxoalkanoic acids
from disubstituted compounds.
1
0
0
W
.
R
e
i
n
e
k
e
Table 14. Properties of bacteria reductively dechlorinating tetrachloroethene
Strain E-donor E-acceptor Product t
d
(h) References
of PCE
dechlorination
Dehalospirillum multivorans H
2
, formate, lactate, PCE, TCE, fumarate, nitrate cis-1,2-DCE 2.5 [736]
pyruvate, ethanol, glycerol
Dehalobacter restrictus strain H
2
PCE, TCE cis-1,2-DCE 19 [737]
PER-K23
Desulfitobacterium sp. strain Formate, lactate, pyruvate, PCE, ortho-chlorinated phenolics, TCE 58 [576]
PCE-1 ethanol, butyrate, succinate fumarate, sulfite, thiosulfate
Desulfitobacterium frappieri H
2
, acetate, pyruvate PCE, TCE, fumarate cis-1,2-DCE n. d. [738, 739]
strain PCE-S
Dehalococcoides ethenogenes H
2
PCE, TCE Ethene 19 [740, 741]
strain 195
Isolate TEA H
2
PCE, TCE cis-1,2-DCE n.d. [742]
Desulfuromonas chloroethenica Acetate, pyruvate PCE, TCE, fumarate, Fe(III)NTA cis-1,2-DCE 4896 [743, 744]
strain TT4B
Desulfitobacterium frappieri H
2
, formate, lactate, ethanol, PCE, TCE, fumarate, nitrate, sulfite, cis-1,2-DCE 9 [745]
strain TCE1 butyrate, crotonate thiosulfate
Isolate MS-1 Polymers, carbohydrates, O
2
, nitrate cis-1,2-DCE n. d. [746]
esters, carboxylic acids,
amides, aromatics, alcohols
n. d., no data.
Fig. 65. Mechanisms for the cleavage of carbon-chlorine bonds in microorganisms that grow
on chlorinated aliphatic compounds. hydrolytic cleavage as occurring with chlorocar-
boxylic acids and chloroalkanes; haloalcohol dehalogenases catalyze a lyase reaction pro-
ducing an epoxide from vic-haloalcohols; hydratase-like mechanism as occurring with
chloroacrylic acids; dehalogenation catalyzed by a glutathione transferase; monooxyge-
nation of a chloro-substituted carbon atom produces a gem-chloroalcohol that decomposes
to an aldehyde; dehydrohalogenation from highly substituted chlorinated compounds;
dehalogenation by methyltransferase (X=unknown)
At least four different classes of hydrolytic 2-halo acid dehalogenases have

been grouped according to their substrate specificity and stereospecific action
on 2-chloropropionic acid. Two classes of enzymes are active with either the
L- or D-substrate, yielding products with inversion of configuration at the chiral
C-2 carbon atom. The two other classes act on both stereo-isomers, one with
inversion of configuration, the other with retention (Fig. 66). In general, high
amino acid sequence identities are observed among the dehalogenases within
any of the separate classes. Mostly no homology is evident between the 2-halo-
acid dehalogenases from different classes.
1. About ten members of the class of hydrolytic dehalogenases, which is speci-
fic for the L-isomer of 2-chloropropionate and dehalogenates the substrate
with inversion of configuration at the chiral carbon atom to produce the cor-
responding D-2-hydroxy acid, are known and a catalytic mechanism has been
proposed [755763].
2. The dehalogenase of Pseudomonas putida strain AJ1 belongs to the class,
which is highly specific for the D-isomer of 2-chloropropionate and brings
about inversion to form L-hydroxy acids [764].
3. DL-2-haloacid dehalogenases such as from Pseudomonas sp. strain 113 cat-
alyze the hydrolytic dehalogenation of both enantiomers of 2-haloalkanoic
acids, producing the corresponding 2-hydroxyalkanoic acids with inversion
of the C-2 configuration [765].
The enzyme of Pseudomonas sp. strain 113 showed significant sequence sim-
ilarity with D-2-haloacid dehalogenases, but little with L-2-haloacid dehalo-
genases [766].
4. DL-2-haloacid dehalogenases (retention type) dehalogenate both enantio-
mers of 2-haloalkanoic acids to the corresponding D- and L-2-hydroxyalka-
noic acids respectively [767].
Another type of dehalogenase is the enzyme from Moraxella sp. B that converts
fluoroacetate, and is mechanistically and structurally similar to haloalkane de-
halogenase of Xanthobacter autotrophicus GJ10 [768].
A strain growing on trichloroacetate [769] dehalogenates chloroacetate or
dichloroacetate to glycolate and glyoxylate. No oxalate was formed from tri-
102 W. Reineke
Fig. 66. Inversion and retention during hydrolytic elimination of chloride from enantiomers
of 2-chloropropionate to the corresponding 2-hydroxypropionate (broken arrow: retention;
full arrow: inversion)
Fig. 67. Catabolic pathway of 1,2-dichloroethane in strains of Xanthobacter autotrophicus and
Ancylobacter aquaticus [667, 669]
chloroacetate; instead carbon-carbon bond cleavage was observed, indicating a
different mechanism of dehalogenation.
Hydrolysis of 2-bromobutane was accompanied by inversion of configura-
tion [770].
Two hydrolytic dehalogenating enzymes have been shown in Xanthobac-
ter autotrophicus and Ancylobacter aquaticus functioning in the degradation
of 1,2-dichloroethane. The compound is degraded via 2-chloroethanol, chloro-
acetaldehyde, and chloroacetate to glycolate in four successive reactions before
it enters the central metabolic routes (Fig. 67). The dehalogenase (DhlA) con-
verting 1,2-dichloroethane belongs to the a/b-hydrolase fold group [771].
A modified aldehyde dehydrogenase is then essential for the conversion of the
toxic aldehyde [772]. The conversion of chloroacetate proceeds by a chloro-
acetate dehalogenase (DhlB) that belongs to the L-specific haloacid dehaloge-
nases.
A haloalkane dehalogenase hydrolyzing 1,3-dichloropropene to 3-chloro-
allyl alcohol has recently been characterized fromPseudomonas cichorii 170 [676].
The whole pathway, including the second dechlorination step, is given in Fig. 70.
5.1.2
Glutathione S-Transferase-Dependent Dehalogenation
The role of glutathione transferases in degradation of halogenated compounds
is the nucleophilic displacement of a halogen substituent and further spon-
taneous decomposition of the resulting glutathione adduct (Fig. 68). The first
evidence for the involvement of a glutathione transferase in bacterial dehaloge-
nation was obtained with facultative and obligate methylotrophs of the genera
Methylobacterium and Hyphomicrobium that can utilize dichloromethane as a
carbon source for growth [661].
The role of these enzymes and their mechanistic aspects were recently re-
viewed [773, 774]. Dichloromethane dehalogenases are atypical enzymes
among glutathione S-transferases. In contrast to most enzymes of the gluta-
thione S-transferase superfamily, their substrate range is very narrow and
restricted to dihalomethanes. Halomethanes and dihaloethanes are not con-
verted.
5.1.3
Lyase-Catalyzed Dehalogenation
Haloalcohol lyases catalyze the intramolecular nucleophilic displacement of
vic-haloalcohols to their respective epoxides. Both the enzymes from
Corynebacterium sp. strain N-1074 [776] and from Arthrobacter sp. strain AD2
[680] are more active with bromoalcohols than with chloroalcohols. The en-
zyme reaction (Fig. 69) is reversible, allowing formation of epoxides from vic-
haloalcohols, conversion of the epoxides to haloalcohols, and trans-halogena-
tion reactions of haloalcohols. The enzymes use 2-propanols with halogen sub-
stituents in the 1- or 3-position.
A lyase reaction has recently been detected to be involved in the degradation
of 1,2-dibromoethane by Mycobacterium sp. strain GP1 [777]. The first step in
the degradation is catalyzed by a hydrolytic haloalkane dehalogenase. The re-
sulting vic-haloalcohol, 2-bromoethanol, is then rapidly converted by the lyase
to ethene oxide. The further metabolism is unclear.
5.1.4
Hydratase-Catalyzed Dehalogenation
A hydration type of dehalogenation reaction has been proposed for the conver-
sion of aliphatic acrylates (Fig. 70) [686, 687]. The degradation of cis- and trans-
3-chloroacrylate by coryneform bacteria is catalyzed by dehalogenases that are
specific for the cis and trans isomers. Two 3-chloroacrylate dehalogenases have
also been found as part of the degradation pathway of 1,3-dichloropropene of
Pseudomonas cichorii 170 [676].
5.1.5
Dehalogenation by Oxygenases
Little biochemical information is available on the role of oxygenases in dehalo-
genation of halogenated aliphatic compounds. Oxygenation can lead to dehalo-
104 W. Reineke
Fig. 69. Microbial dehalogenation of chloropropanols via chloroepoxides. (a) haloalcohol
dehalogenase, (b) epoxide hydrolase
Fig. 68. Degradation of dichloromethane [775]
genation if a saturated carbon atom to which a chlorine is bound is hydroxyla-
ted to yield a gem-chloroalcohol. These compounds are chemically unstable and
decompose to produce aldehydes. A monooxygenase may play a role in the uti-
lization of methyl chloride by Hyphomicrobium sp. strain MCl [657] and in
growth of Pseudomonas sp. strain DE2 on 1,2-dichloroethane [668].
Just recently, a monooxygenase reaction was shown to initiate the degradation
of 1,2-dichloroethane by Pseudomonas sp. strain DCA1, an organism exhibiting
high affinity to the substrate (Fig. 71) [778]. Oxidation results in the formation of
an unstable intermediate, 1,2-dichloroethanol, which spontaneously releases
chloride yielding chloroacetaldehyde. The further degradation proceeds through
the pathway described for X. autotrophicus andAncylobacter aquaticus [667, 669].
5.1.6
Dehalogenation During b-Oxidation
There is evidence that dehalogenation of chlorinated carboxylic acids may oc-
cur after formation of CoA-derivatives (Fig. 72). Kohler-Staub and Kohler [688]
Fig. 70. Pathway of cis-1,3-dichloropropene bringing about the formation of malonic semial-
dehyde. haloalkane dehalogenase; alcohol dehydrogenase; aldehyde dehydrogenase;
3-chloroacrylate dehalogenase. The same pathway functions for the trans isomer [676]
Fig. 71. Catabolic pathway of 1,2-dichloroethane in Pseudomonas sp. strain DCA1 [778]
showed that dechlorination of trans-3-chlorocrotonate and 3-chlorobutyrate,
used as a sole carbon and energy source by Alcaligenes sp. strain CC1, occurs af-
ter activation of the acids to their coenzyme A derivatives. The dechlorination
reaction is not yet understood. The same could be the case in the organism iso-
lated with chloroallyl alcohols as the growth substrates [681].
5.1.7
Dehydrohalogenation
The first step in the metabolism of lindane (g-hexachlorocyclohexane) is an eli-
mination by a dehydrochlorinase (Fig. 73). The initial product is g-pentachlo-
rocyclohexene, which is further converted by the same enzyme to 1,3,4,6-te-
trachloro-1,4-cyclohexadiene. Two hydrolytic dechlorinating steps follow yield-
ing 2,5-dichloro-2,5-cyclohexadiene-1,4-diol which is then metabolized by a
dehydrogenase to 2,5-dichlorohydroquinone [779782].
106 W. Reineke
Fig. 72. Proposed degradation sequence used for trans-3-chlorocrotonate [688]
Fig. 73. Pathway for lindane (g-hexachlorocyclohexane) including dehydrohalogenation and
hydrolytic dechlorination
5.1.8
Dehalogenation by Methyltransferase/Dehydrogenase
Aerobic methylotrophic bacteria of the genera Hyphomicrobium and Methylo-
bacterium have been characterized, which are able to utilize chloromethane
as a growth substrate [656, 657]. Physiological and genetic studies suggest
that Methylobacteriumsp. strain CM4 metabolizes chloromethane by initial de-
halogenation via a methyl transfer reaction (Fig. 74), followed by a series of
dehydrogenase-based steps which are different from those involved in the
downstream steps of methanol metabolism in the same organism [658].
Dichloro- and trichloromethane are neither growth substrates nor are they de-
chlorinated by strain CM4 cells, indicating a narrow specificity of the dechlori-
nation step.
5.2
Chloroaliphatic Compounds as Growth Substrates for Anaerobic Bacteria
5.2.1
Fermentative Degradation
It is conceivable that chlorinated C
1
hydrocarbons are utilized as carbon and
energy sources by anaerobic bacteria, a process that has been observed for chlo-
romethane [783, 784] and dichloromethane [785788].
Traunecker et al. [784] reported the isolation of a strictly anaerobic, homo-
acetogenic bacterium, fermenting methyl chloride as the sole energy source
with a doubling time of near 30 h. Strain MC does not grow with dichlorome-
thane as an energy source. Cells formed about 3 mol of acetate per 4 mol of
CH
3
Cl consumed:
4CH
3
Cl
(g)
+ 2CO
2(g)
+ 2H
2
O 3CH
3
COO

+ 7H
+
+ 4Cl
DG
o
= 419.9 kJ/reaction
More recently, Memer et al. [789] reported the development of an enzyme as-
say to determine the activity of the methyl chloride dehalogenase of the homo-
acetogen strain MC. They showed that the dehalogenase activity was dependent
on the presence of substoichiometric amounts of ATP. The pathway for chloro-
methane degradation is presented in Fig. 75.
A strictly anaerobic two component culture able to grow with a doubling
time of 20 h on a medium containing dichloromethane as the carbon and
energy source was established by Mgli et al. [790]. The strain DMC was able to
grow acetogenically with dichloromethane when it was associated with Aceto-
bacterium woodii, Methanospirillum hungatei, or Desulfovibrio sp. strain DMB.
Strain DMC contained CO dehydrogenase activity and is responsible for both
the dehalogenation of dichloromethane and the acetogenesis. The obligatory
dependence on a partner for growth might be due to the need for a growth fac-
tor provided by the associated organism. The partial mass balance for growth
with dichloromethane is compatible with the following fermentation balance:
2CH
2
Cl
2
+ 2H
2
O CH
3
COO
+ 5H
+
+ 4Cl
DG
o
= 492.7 kJ/reaction
and is thus thermodynamically favorable. A major portion of this free energy,
however, is associated with the dehalogenation step:
2CH
2
Cl
2
+ 2H
2
O 2HCHO + 4H
+
+ 4Cl
DG
o
= 344 kJ/reaction
Later strain DMC was isolated from the dichloromethane-fermenting, two com-
ponent mixed culture and characterized as Dehalobacterium formicoaceticum
Fig. 74. Chloromethane metabolism involving methyltransferase (X=unknown acceptor, not
glutathione)
[791]. The organism converted in a mineral medium with vitamins 5 mmol/l
dichloromethane within 7 days to formate plus acetate in a molar ratio of 2: 1
and to biomass. Only dichloromethane supported growth, while other com-
pounds including chloromethane (50 potential substrates were tested) were not
used by the organism.
Dichloromethane is converted to methylene tetrahydrofolate (Fig. 76), of
which two-thirds are oxidized to formate while one-third gives rise to acetate by
incorporation of CO
2
in the acetyl coenzyme A synthetase reaction. The reduc-
ing equivalents generated by the oxidation through the acetyl CoA pathway are
used by the methylene tetrahydrofolate reductase in the formation of acetate
through the CO dehydrogenase pathway. The dehalogenating activity was found
to be dependent on the presence of ATP, methyl viologen, and hydrogen [787].
Co(I) corrinoid seems to be involved in this anoxic dehalogenation of dichlo-
romethane. The presence of a sodium-independent ATP synthetase suggests a
chemiosmotic mechanism of energy conservation.
108 W. Reineke
Fig. 75. Metabolism of chloromethane by the homoacetogenic bacterium MC according to
Memer et al. [789]. The enzymes are the following: methyl chloride dehalogenase;
methylene tetrahydrofolate reductase; methylene tetrahydrofolate dehydrogenase;
CHFH
4
cyclohydrolase; formyl tetrahydrofolate synthetase; formate dehydro-
genase; CO dehydrogenase; phosphotransacetylase; acetate kinase; methyl trans-
ferase. CH
3
-Co-E=corrinoid enzyme
5.2.2
Degradation Under Denitrifying Conditions
The facultative methylotroph Hyphomicrobium sp. DM2 has been shown to be
capable of growth with dichloromethane in the absence of oxygen using nitrate
as a terminal electron acceptor [792].
5.2.3
Degradation Under Methanogenic Conditions
The anaerobic degradation of chloroacetates has only been reported by Egli et
al. [793]. A methanogenic stable mixed culture degraded chloroacetate accord-
ing the following overall stoichiometry:
4ClCH
2
COO
+ 7H
2
O 5HCO
3
+ 3CH
4
+ 4Cl
+ 5H
+
The data suggest that the anaerobic chloroacetate degradation proceeds via gly-
colate formed by hydrolytic dehalogenation. Glycolate is then cleaved to carbon
dioxide and hydrogen, the substrates of the carboxidotrophic methanogens.
Fig. 76. Proposed pathway for the metabolism of dichloromethane by D. formicoaceticum
strain DMC [787]. The enzymes are the following: dichloromethane dehalogenase; me-
thylene tetrahydrofolate dehydrogenase; CHFH
4
cyclohydrolase; formyl tetrahydro-
folate synthetase; methylene tetrahydrofolate reductase; methyl transferase; CO de-
hydrogenase; phosphotransacetylase; acetate kinase. CH
3
-Co-E=corrinoid enzyme
5.3
Cometabolic Transformations
Cometabolism is the process whereby enzymes or cofactors that have evolved to
degrade other substrates fortuitously convert another compound.
5.3.1
Aerobic Bacteria: Oxidative
The potential of bacteria for oxidative cometabolic degradation has been stud-
ied with chloroethenes and chloromethanes. Only the monochlorinated conge-
ner of the chloroethenes, vinyl chloride, is known to serve as growth substrate
for aerobic microorganisms. The di- and trichlorinated congeners are meta-
bolized exclusively co-metabolically, and tetrachloroethene is not degraded at
all by microorganisms under aerobic conditions.
The presence of C=C bonds renders vinylic halogens rather resistant to
nucleophilic substitution reactions. With these compounds, dehalogenation by
oxidative mechanisms involving the addition of oxygen atoms to the double
bond is possible. The enzymes responsible for the fortuitous dehalogenation are
mono- or dioxygenases: toluene 2-monooxygenase, toluene 3-monooxygenase,
toluene 4-monooxygenase, toluene 2,3-dioxygenase, methane monooxygenase,
isoprene monooxygenase, propane monooxygenase, ammonia monooxygenase
(Table 13). They are present in bacteria growing on e. g. methane [693, 695], tol-
uene [704, 705, 707, 708, 794, 795], phenol [702, 703], isoprene [713], p-cymene
[710], propene [711], propane [700], or ammonia [714].
Since the chlorinated ethenes such as trichloroethene (TCE) are converted
only cometabolically, the natural substrate (e. g., toluene or phenol, etc.) is
normally required for induction of the degradative enzyme. Recently, orga-
nisms have been found in which the TCE-degrading activity (toluene 2,3-di-
oxygenase) can be induced by TCE [796, 797].
Cometabolic oxidative degradation results in the formation of products,
some of which can be very toxic to the organisms that produce them and inac-
tivate the enzyme responsible for the conversion. The toxic intermediate in the
case of trichloroethene is thought to be trichloroethene epoxide. Thus, the con-
version of the epoxides is a critical step in the degradation route of chlorinated
ethenes. The selection of organisms, that are able to convert the potentially
toxic intermediates fast enough to preclude any harm being done by these
intermediates, seems likely to lead to better degradation of trichloroethene.
Isoprene-degrading organisms may contain epoxide-converting enzymes that
also efficiently convert chlorooxiranes. Toxic effects of trichloroethene con-
version by the isoprene degraders were indeed not observed [713].
The results presented by Fox et al. [694] obtained with purified me-
thane monooxygenase from the type II methanotroph Methylosinus tricho-
sporium OB3b strongly imply that neither TCE nor the immediate enzyme-
catalyzed oxidation products, TCE epoxide and chloral, are responsible for the
inactivation reaction. A diffusible intermediate derived from the non-enzyme-
catalyzed hydrolysis or isomerization of TCE epoxide, such as glyoxyl chloride,
110 W. Reineke
formyl chloride, dichloroacetyl chloride, or dichlorocarbene, is thought to be
the modifying agent. The methane monooxygenase oxidizes TCE predo-
minantly to TCE epoxide, while 2,2,2-trichloroacetaldehyde (chloral) is pro-
duced at 6% of the total product yield [694]. The various products arising dur-
ing conversion of TCE by the methane monooxygenase are summarized in
Fig. 77.
The toluene 2,3-dioxygenase oxidizes TCE to formate and glyoxylate in a 2: 1
ratio [798].
In contrast to the methane monooxygenase and the toluene 2,3-dioxygenase,
the toluene 2-monooxygenase shows that Burkholderia cepacia G4 oxidizes TCE
with little apparent inactivation [702, 799]. The stable products of TCE oxida-
tion by toluene 2-monooxygenase were shown to be carbon monoxide, formate,
and glyoxylate [800].
Besides the dechlorination of TCE chloroform was tested with different
toluene-oxidizing bacterial strains harboring mono- and dioxygenases towards
elimination of chlorine substituents [716]. Pseudomonas mendocina KR1, which
induces a toluene 4-monooxygenase, as well as Pseudomonas sp. ENVBF1, which
appears to produce toluene 2-monooxygenase, oxidize chloroform at respect-
able rate. The same is true for Escherichia coli harboring the respective cloned
genes of the monooxygenases. Degradation of
14
C-chloroform and ion analysis
revealed that a great part was mineralized to carbon dioxide (approximately
3057% of the total products). Chloride was approximately 75 % of the ex-
pected yield.
However, Burkholderia cepacia G4 producing toluene 2-monooxygenase,
Pseudomonas putida producing toluene 2,3-dioxygenase, and Burkholderia
picketti PKO1 producing toluene 3-monooxygenase fail to show oxidation of
chloroform. So overall the picture of the potential of the oxidizing enzymes to-
ward chloroform is not totally clear.
Fig. 77. Oxidation of trichloroethene by methane monooxygenase of Methylosinus trichospo-
riumOB3b and following reactions according to Fox et al. [694]. main reaction of methane
monooxygenase; side reaction of methane monooxygenase; spontaneous; reduction;
oxidation
5.3.2
Ligninolytic Fungi: Reductive
The enzyme system of the ligninolytic fungi is known to oxidize a number of che-
micals including polycyclic hydrocarbons. Chloroaliphatics are highly electron
deficient and therefore cannot be oxidized by the lignin peroxidase. However, a li-
gnin peroxidase-dependent reductive pathway seems to be possible (Fig. 78)
[801]. The veratryl alcohol cation formed by the lignin peroxidase is an oxidizing
species which can effectively oxidize certain chemicals by one electron. EDTA was
shown to be a good reductant for the veratryl alcohol cation radical and will be
oxidized to the organic acid radical. The EDTA anion radical reduces other chem-
icals such as tetrachloromethane, resulting in the dechlorination and formation
of trichloromethyl radical and the decarboxylation of EDTA. In summary, CCl
4
,
which is neither a substrate for lignin peroxidase nor a good reductant, is degrad-
ed via free radicals generated by lignin peroxidase under reducing conditions.
112 W. Reineke
Fig. 78. Proposed scheme for the reduction of CCl
4
by lignin peroxidase (LiP)
5.3.3
Anaerobic Bacteria: Reductive
Considerable evidence has accrued in studies of anaerobic microcosms and cul-
tures for reductive dechlorination of tetra- to trichloroethene (PCE, TCE) [560],
to dichloroethene isomers [802804], or to vinyl chloride (VC) [805, 806]. More
important, complete dechlorination to ethene [805, 807] or ethane [808] has
been reported.
Freedman and Gossett [805] demonstrated the reductive dechlorination of
PCE and TCE to ethene by anoxic aquifer material when methanol, glucose, H
2
,
and other electron donors were added. No conversion to CO
2
and CH
4
was ob-
served. The authors propose a pathway for the reductive dechlorination that fol-
lows the course PCE, TCE, cis-1,2-dichloroethene (cDCE), vinyl chloride, ethene
(Fig. 79). The dechlorination process was inhibited by 2-bromoethane sulfonic
acid, a methanogen inhibitor, indicating that these organisms may play a key
role in the anaerobic biotransformation of PCE and TCE.
It is unclear why some anaerobic systems only partially dechlorinate PCE
while others effect complete dechlorination. Reductive dechlorination of TCE
and PCE under methanogenic conditions can proceed to VC [805807], where-
as cDCE has tended to accumulate under sulfate-reducing conditions [802, 809].
A clearer picture of the microorganisms and enzymes involved in reductive
dechlorination emerges with studies using pure cultures. Reductive dehaloge-
nation of molecules such as dibromo- and dichloroethane has been demon-
strated with pure cultures of methanogens [731]. The anaerobic dechlorination
of chlorinated ethenes has also been demonstrated with corrinoids (Co(II)-
containing) and nickel porphyrins, which play a role in the normal anaerobic
metabolism. Thus, transition metal-containing cofactors or enzymes are likely
to be responsible for the reductive dechlorination in the anaerobes.
5.4
Chloroaliphatic Compounds as Electron Acceptors
The above-mentioned dechlorinating anaerobic strains cometabolically trans-
form the chlorinated compounds and thus do not benefit from the exergonic re-
action which they catalyze. Halogenated aliphatic compounds are quite strong
oxidants: hexachloroethane is a stronger electron acceptor than is oxygen, and
several halogenated compounds, such as tetrachloromethane, tetrachloro-
ethene, and trichloromethane, are stronger acceptors than nitrate [810]. There-
fore, they can in principle serve as terminal electron acceptors in an anaerobic
respiration.
Although there have been many indications that reductive dechlorination re-
actions are catalyzed by bacteria that couple this reduction to growth, the isola-
tion of these bacteria appears to be very difficult. To date, eight pure cultures
have been obtained that dechlorinate tetrachloroethene at high rates and utilize
the chlorinated compound in an anaerobic respiration as an electron acceptor
(Table 14). In another pure strain, isolate MS-1, which is able to dechlorinate
PCE, the respiratory growth with PCE has not yet been shown. With respect to
the origin of the organisms, both contaminated material as well as material
without known history of contamination with chloroethenes has been the
Fig. 79. Proposed degradative pathway for PCE
source. Dehalobacter restrictus strain PER-K23 was isolated from an anaerobic
fixed-bed column fed with lactate and PCE [737, 803]. However, while PCE had
been completely dechlorinated to ethane at high rate by the sediment from
Rhine river, which has functioned as the source for strain PER-K23 [808], strain
PER-K23 brings about only partial dechlorination to cis-dichloroethene.
Dehalospirillum multivorans was isolated from activated sludge on a medium
containing pyruvate plus PCE [811], while Desulfitobacterium frappieri strain
PCE-S originating from PCE contaminated soil was enriched with hydrogen,
acetate, yeast extract, and PCE [738, 739]. Desulfitobacterium frappieri strain
TCE1 was isolated from a PCE-dechlorinating chemostat mixed culture that
had been enriched by using soil obtained from a chloroethene-polluted loca-
tion. Formate plus glucose were used as the electron donor [745].
The tetrachloroethene-dechlorinating bacteria isolated so far belong to five
phylogenetically different groups of bacteria. Physiologically they range from
facultative anaerobes, nitrate reducers, and sulfoxy anion reducers to strict
tetrachloroethene reducers. Five isolates (Desulfitobacterium sp. strain PCE1,
Desulfitobacterium frappieri strains PCE-S and TCE1, isolate TEA, and PER-
K23) relate phylogenetically to the gram-positive bacteria with a low DNA G+C
content. Strain MS-1, a facultative anaerobe, belongs to the Enterobacteriaceae.
Dehalospirillum multivorans is a member of the e-subdivision of the Proteo-
bacteria and Desulfuromonas chloroethenica strain TT4B of the d-subdivision,
while the classification of Dehalococcoides ethenogenes strain 195 is unclear.
Strain MS-1 is able to oxidize a broad spectrum of substrates. Dehalobacter re-
strictus strain PER-K23, isolate TEA, and Dehalococcoides ethenogenes strain 195,
on the other hand, can only utilize hydrogen as the electron donor. The other te-
trachloroethene dechlorinators can use two to six different electron donors.
Desulfitobacterium sp. strain PCE1 dechlorinates tetrachloroethene mainly
to trichloroethene and is physiologically much more versatile than isolate TEA
and Dehalobacter restrictus. On the basis of electron donors and electron ac-
ceptors utilized, strain PCE1 has much more in common with Dehalospirillum
multivorans.
Dehalospirillum multivorans and Dehalobacter restrictus show similarities re-
garding the tetrachloroethene dechlorination. Both organisms dechlorinate te-
trachloroethene to cis-1,2-dichloroethene and can couple this reaction to hy-
drogen oxidation, and both contain b-type cytochromes and menaquinones that
are possibly involved in electron transfer [812]. The purified tetrachloroethene
reductive dehalogenase of Dehalospirillum multivorans and the tetrachloro-
ethene reductase of Dehalobacter restrictus are corrinoid-containing enzymes
[813, 814]. A major difference between the two organisms has been found in the
localization of the tetrachloroethene-reducing enzyme. While the tetrachloro-
ethene reductive dehalogenase of Dehalospirillum multivorans is localized in the
cytoplasmic fraction [811, 815], the tetrachloroethene reductase of Dehalobacter
restrictus is membrane-bound [812]. The reductase of D. multivorans has been
purified and characterized and its gene has been cloned [816, 817].
The ability of both strains to grow on mineral medium with H
2
and PCE as
sole energy source gave evidence that the reductive dechlorination of PCE in
these organisms is coupled to the synthesis of ATP. Since neither the oxidation
114 W. Reineke
of H
2
nor the reductive dechlorination of PCE is mechanistically coupled to
substrate level phosphorylation, ATP synthesis must proceed via chemiosmotic
mechanism. The two enzymes, hydrogenase and dehalogenase, catalyze the
following overall reaction:
PCE + 2H
2
cis-1,2-DCE + 2H
+
+ 2Cl
DG
o
= 376 kJ/mol
During the oxidation of H
2
, protons are produced at the outside of the cyto-
plasmic membrane. PCE-reduction in the cytoplasm uses protons. Only due to
the consumption of protons inside and the production outside an electro-
chemical proton potential will be created, which will be used for the conserva-
tion of energy by the proton-translocating ATP synthetase. The participation of
a proton pump from the inside to the outside seems to be non-essential in this
type of respiration. A simplified scheme of the process is given in Fig. 80.
5.5
Rsum: Chloroaliphatic Compounds
Chloroaliphatic compounds are the subject of degradation by microorganisms
in different ways. Some of them can serve as growth substrates for aerobic bac-
teria. A set of different dechlorination mechanisms is known to be involved.
Since for highly chlorinated compounds such as tetrachloro- and trichloro-
ethene, both are well known pollutants, no aerobic populations are available, the
cometabolic potential of various aerobic systems has been studied especially for
the application in the cleanup processes. Besides degradation in the presence of
oxygen, under anaerobic conditions mineralization has been shown.
Fermentation as well as degradation under methanogenic conditions is known.
Fig. 80. Simplified scheme of the energy conservation during the reductive dechlorination of
tetrachloroethene with H
2
as the electron donor (modified with information from [17, 818])
In a couple of recently isolated bacteria a chloroaliphatic compound such as
tetrachloroethene can serve as an electron acceptor in a respiration leading to
reductive elimination of chlorine substituents.
6
Sequential Anaerobic-Aerobic Processes for the Degradation
of Problematic Compounds
Aerobic biological processes are widely employed as cost-effective, reliable sys-
tems for the removal of organic material from municipal wastewater. In addi-
tion, the use of sequential anaerobic and aerobic processes allows the removal
of nitrogen and phosphorus from the wastewater. The question arises whether
sequential environments might also be a possibility for the mineralization of
some man-made organic compounds mentioned in the former paragraphs and
found to be recalcitrant. Considerable interest exists to develop systems to over-
come the recalcitrance.
Separate aerobic and anaerobic environments each have limitations in their
biodegrading abilities, but they often complement each other when they are
combined. One limitation of aerobic processes involves the recalcitrance of highly
chlorinated chemicals, such as hexachlorobenzene, tetrachloroethene, and carbon
tetrachloride. Quite the opposite is true for the reductive dechlorination reactions
catalyzed by some anaerobes. While under aerobic conditions the persistence of
chlorinated compounds generally increases with increasing chlorine substitution
and this may enhance anaerobic degradation. However, in many cases the reduc-
tive dechlorination by anaerobic bacteria is incomplete but yields less-halogenat-
ed compounds, which may be efficiently degraded by aerobic bacteria.
Theoretically, sequential anaerobic-aerobic systems can be developed for both
highly chlorinated aliphatic and aromatic compounds. The first candidates for
these systems are tetrachloroethene and carbon tetrachloride. Initial anaerobic
stages may accomplish reductive dechlorination, producing trichloroethene and
chloroform. Subsequent aerobic, methanotrophic stages may convert trichloro-
ethene and chloroform to carbon dioxide and water. Alternatively, anaerobic re-
ductive dechlorination may produce vinyl chloride and chloromethane, which
may degrade in conventional aerobic processes if volatilization losses are mini-
mized. Another example of sequential anaerobic-aerobic processes is the minera-
lization of chlorinated aromatic compounds such as hexachlorobenzene and
PCBs. Reductive dechlorination may occur in anaerobic stages, producing less
chlorinated congeners, which may be degraded under aerobic conditions.
Indeed, the studies of anaerobic-aerobic systems performed thus far have
focused on the degradation of chlorinated compounds, and anaerobic reductive
dechlorination followed by aerobic degradation of the less chlorinated products
has been realized. Studies with non-classified indigenous microflora of environ-
mental materials were performed. Besides biostimulation, i. e., the addition of
substrates to help the native organisms, bioaugmentation has been studied,
which involves the supplementation of known bacterial populations to the indi-
genous populations. In addition, an artificial system consisting of two bacterial
strains has been investigated towards sequential anaerobic-aerobic degradation.
116 W. Reineke
6.1
Studies with Environmental Materials
Tetrachloroethene, chloroform, and hexachlorobenzene have been degraded in
a two-stage biofilm reactor consisting of an anaerobic column followed by a
conventional aerobic one [819]. Reductive dechlorination occurred in the an-
aerobic column, and trichlorinated and dichlorinated products were formed. In
the aerobic column the lesser chlorinated intermediates were substantially
transformed into carbon dioxide and nonvolatile products. The two-stage pro-
cess resulted in 61%, 49%, and 23% mineralization of chloroform, tetrachlo-
roethene, and hexachlorobenzene, respectively. Dechlorination was most exten-
sive when acetate served as the primary substrate, but it occurred to a lesser ex-
tent also when glucose and methanol served as primary substrates.
Gerritse et al. [576] investigated the degradation of tetrachloroethene by com-
bining the abilities of anaerobic bacteria, capable of reductive dechlorination of
tetrachloroethene, with those of aerobic methanotrophic bacteria, capable of co-
metabolic degradation of the less-chlorinated ethenes formed by the reductive
dechlorination of tetrachloroethene. It was demonstrated that complete degra-
dation of tetrachloroethene was possible by combining two columns in series.
An anaerobic community reductively dechlorinating tetra-, trichloro-, and di-
chloroethenes was used for inoculating an anoxic fixed-bed upflow column that
subsequently converted tetrachloroethene mainly to cis-dichloroethene. The
oxic fixed-bed downflow column contained aerobic methanotrophic bacteria
that grew with methane and cometabolized the less-chlorinated ethenes formed
in the anoxic column. The sensitivity of the methanotrophic bacteria to chlori-
nated intermediates represented the bottleneck in the sequential anoxic-oxic de-
gradation process of tetrachloroethene. In a similar approach, the second oxic
step, bringing about the dechlorination of the products of the partially anoxic
dechlorination of tetrachloroethene, is substituted by the cometabolic reactions
of aerobic phenol-grown bacteria [820]. The maximum capacity for chloroethe-
nes degradation was significantly higher than reported thus far.
In a simpler approach the simultaneous anaerobic and aerobic degradation
of tri- and tetrachloroethene has been demonstrated by Enzien et al. [821].
Under aerobic conditions, a column containing three sediments from different
horizons from the Savannah River site was run with groundwater of an uncon-
taminated well supplemented with methane, oxygen, methanol, and the chloro-
aliphatic compounds. About 90% removal of tri- and tetrachloroethene was ob-
served when comparing the feed and the exit water. Enumerations of the micro-
bial populations in the column indicated the presence of both aerobic and
anaerobic populations throughout the experiment of more than one year.
Methanotrophs were detected at low numbers. The presence of methanogens
suggested that anaerobic zones or microsites existed, allowing the simultaneous
presence of both aerobic and even strict anaerobic microorganisms. These re-
sults may have important implications for in situ and on-site tri- and tetrachlo-
roethene bioremediation projects.
Other compounds have been successfully mineralized under sequential an-
aerobic-aerobic conditions. C
14
-labeled 2,2,2-trichloro-1,1-bis(p-methoxy-
phenyl) ethane (methoxychlor) was degraded by bacteria that were initially in-
cubated for three months under anaerobic conditions and subsequently incu-
bated under aerobic conditions [822]. Cultures exposed to the sequence of en-
vironments showed a 10- to 70-fold increase in labeled carbon dioxide produc-
tion compared with cultures maintained under aerobic conditions only. At all
concentrations of methoxychlor studied, the anaerobic-aerobic system pro-
duced significantly more labeled carbon dioxide than did the purely aerobic
system.
Results have been obtained from investigations describing sequential an-
aerobic-aerobic processes for the destruction of PCBs in river sediment. The
biotransformations that occurred when a mixture of PCB congeners (Aroclor
1242) in sediment is incubated under anaerobic conditions and then under
aerobic conditions have been described [823]. Methanol was added as a primary
substrate. During the anaerobic period, reductive dechlorination occurred, and
the mass of tri-, tetra-, penta-, and hexachlorobiphenyl decreased, whereas
mono- and dichlorobiphenyl concentrations increased. Under subsequent
aerobic conditions, significant degradation of all mono- and dichlorobiphenyl
congeners occurred. Still, 43% of the 300 mg of PCBs/kg of soil initially added
remained after treatment. The authors also describe a conceptual model in
which the in situ mineralization of PCBs may be accomplished by injecting
methanol or other primary substrates into river sediment, monitoring the ex-
tent of dechlorination, and finally injecting hydrogen peroxide and methanol to
stimulate aerobic mineralization of less chlorinated homologues.
6.2
Studies with Undefined Enrichment Cultures
A single microbial population enriched from anaerobic sludge allowed the de-
gradation of 2,4,6-trichlorophenol by cycling between anaerobic and aerobic
conditions [824, 825]. In the first step, 4-chlorophenol was the most significant
product formed from the target compound during incubation with diluted di-
gester fluid. The anaerobic population was subsequently transferred to aerobic
conditions, resulting in a very slow mineralization. The results indicate that
there is no need for a second microbial population to achieve the goal of min-
eralization of the target compound. Instead the addition of oxygen to the an-
aerobic population to shift facultative organisms to aerobic degradation path-
ways is sufficient to bring about mineralization. An increasing rate of dehaloge-
nation was observed at high pH under anaerobic conditions, while neutral pH
was essential for the aerobic step. Therefore the sequential anaerobic-aerobic
process has to involve a pH adjustment. The population was found to be robust
and resistant to fluctuations in pH.
6.3
Studies with Undefined Enrichment Cultures Supplemented with Pure Cultures
The sequential degradation of 2,3,6-trichlorobenzoate using anaerobic and
aerobic organisms was reported by Gerritse and Gottschal [826] (Fig. 81). A
118 W. Reineke
2,3,6-trichlorobenzoate dechlorinating methanogenic enrichment culture
growing with medium containing yeast extract, peptone, benzoate, and a fatty
acid mixture (acetate, propionate, butyrate, 2-methylbutyrate, isobutyrate, vale-
rate, and isovalerate) was cultivated anaerobically in a chemostat in which a
nylon bag filled with particles of the clay mineral vermiculite was placed to pre-
vent wash-out of bacteria. 2,5-Dichlorobenzoate was produced under these con-
ditions. Aeration of the reactor neither brought about further degradation nor
killed the methanogenic culture in the bag. The addition of a culture of
Pseudomonas aeruginosa JB2, which is able to grow aerobically with 2,5-dichlo-
robenzoate [387], resulted in total degradation of 2,3,6-trichlorobenzoate. The
fact that reductive dechlorination of 2,3,6-trichlorobenzoate and oxidative min-
eralization of 2,5-dichlorobenzoate occurred simultaneously in the aerated re-
actor and resulted in almost complete mineralization of 2,3,6-trichlorobenzoate
shows that the strictly anaerobic and aerobic bacteria can successfully be com-
bined at low oxygen tensions. O
2
concentrations in the nylon bag with vermi-
culite were much lower than in the liquid phase, thus ensuring suitable growth
Fig. 81. Scheme of chlorobenzoate degradation in the mixed culture reactor under aerated
conditions. 2,3,6-Trichlorobenzoate enters the vermiculite where reductive dechlorination
takes place. The anaerobic dechlorination product 2,5-dichlorobenzoate is degraded further
by Pseudomonas aeruginosa once it has reached the microaerobic liquid phase
conditions for strict anaerobes. A biological purification system harboring ha-
bitats for both anaerobic and aerobic bacteria is thought to have advantages
over systems which are only (or sequentially) aerobic or anaerobic: inactivation
or death of aerobes or anaerobes due to the periodic absence or presence of O
2
,
respectively, is prevented.
In a soil slurry microcosm study, Evans et al. [827] investigated the degrada-
tion of weathered Aroclor 1248, i. e., decreased levels of trichlorophenyls com-
pared to the original congener mixture, in historically contaminated soil with a
low organic carbon content. The PCB concentration was approximately 100 mg/
kg dry soil. Three systems were studied. The sandy soil was inoculated with
PCB-dechlorinated microorganisms from Hudson River sediment. In a second
incubation strain LB400 (Burkholderia cepacia LB400), an organism with a high
potential for the aerobic transformation of complex Aroclor mixtures, was ad-
ded plus the supplementation of biphenyl as the growth substrate. The effi-
ciency of a sequential anaerobic-aerobic scheme was tested in a third incuba-
tion. The aerobic treatment alone proved quite effective in reducing the total
PCB concentration by 67% after 19 weeks, leaving mainly tetra- and pentachlo-
robiphenyls. The sequential anaerobic-aerobic incubation after 19 weeks show-
ed a reduction of about 70% of the total PCB concentration. A further dechlori-
nation was not shown for the next 60 weeks. The higher efficiency in compari-
son to the study by Anid et al. [823] was thought to be due to the greater ability
of strain LB400 to degrade trichlorobiphenyls.
Shannon et al. [828] reported that more than 80% of the PCBs from a 1240-
ppm Aroclor 1248-contaminated sediment was biodegraded using a two-stage
anaerobic/aerobic microbial system. However, few details of the procedure were
presented. Comparing the differences in the Shannon and Evans studies, the
greater degradation noted by Shannon et al. may be contributed to the presence
of easily degraded trichlorobiphenyls which are absent in the weathered soil in-
vestigated by Evans et al. [827].
6.4
Studies with Pure Cultures
Beunink and Rehm [829] developed an anaerobic-aerobic process by immobi-
lizing two strains in calcium-alginate beads to degrade 4-chloro-2-nitrophenol
(Fig. 82). The conversion of 4-chloro-2-nitrophenol by Enterobacter cloacae,
growing with glucose as the substrate, led to the formation of 4-chloro-2-ami-
nophenol plus minor amounts of 4-chloro-2-acetaminophenol. The main reac-
tion product was further mineralized under aerobic conditions by an
Alcaligenes sp. strain TK-2. Whereas both degradative steps excluded one an-
other in homogenous systems with free cells, a coupled reductive and oxidative
degradation took place in an aerated reactor system with alginate beads. The
outer bead region was an aerobic environment, while the inner bead regions
were anaerobic because of the slow diffusion of oxygen in the beads and the
consumption in the outer bead region.
120 W. Reineke
6.5
Rsum: Sequential Anaerobic-Aerobic Processes
The examples presented clearly show the potential of sequential anaerobic-
aerobic microbial processes in the degradation of xenobiotic compounds. These
may be carried out in two-stage reactor systems or synchronously in a single re-
actor where both anaerobic and aerobic sites occur. The idea of sequential an-
aerobic-aerobic microbial degradation can also be applied to groundwater
cleanup by manipulating the environmental conditions, additions of substrates
to stimulate special bacteria of the indigenous populations, or the injection of
effective populations.
However, much work remains to be done before sequential conversions are
employed to their fullest potential.
7
Enhancement of the Catabolic Potential of Microbial Strains
in the Laboratory
In a number of cases, a great deal is known about the molecular changes involv-
ed in the alteration of existing metabolic capabilities, resulting in the selection
of mutant strains with the ability to grow on novel growth substrates.
I will describe with a few examples why a particular pollutant may not
support growth of a single microbial species and the methods used to eliminate
the respective bottleneck. Mutagenesis, transfer of genetic information, and
genetic engineering techniques will be discussed. The compounds of interest,
Fig. 82. Model of the reductive and oxidative degradation steps in a Ca-alginate bead for im-
mobilization of a mixed culture to mineralize 4-chloro-2-nitrophenol
where data are available, include aromatic, haloaromatic, and chloroaliphatic
compounds and their intermediates.
Figure 83 schematically illustrates different steps which might form a bottle-
neck for a target compound.
7.1
Uptake of a Target Compound
In principle, the cell membrane, consisting of a bimolecular phospholipid layer,
is impermeable to hydrophilic substances, and therefore bacteria have a variety
of specific systems by which hydrophilic compounds of biological importance,
such as carbohydrates or amino acids, are transported into the cell. Lipophilic
compounds, however, can pass the membrane by simple diffusion. Halogenated
aromatic compounds are generally more apolar, depending on the number of
halogen substituents, than the unsubstituted analogues. Therefore, if the halo-
gen-free compound is acceptable as a growth substrate entering the cell by dif-
fusion, one can assume that the halogen compound will permeate as well.
Besides permeation by a simple diffusion process, specific transport systems
have been implicated for aromatic compounds and metabolites. Evidence for an
inducible active transport of mandelate and benzoate has been reported [830,
831]. The aromatic compounds enter the cells by a facilitated diffusion process
[832]. Later the transport of benzoate, 4-hydroxybenzoate, and protocatechuate
via an inducible permease was reported [833837]. Recently, the transport of
low concentrations of 2,4-D was shown to be dependent on the presence of a
transport protein [838].
122 W. Reineke
Fig. 83. Scheme summarizing the steps which might be responsible for recalcitrance present-
ed for a chloroaromatic compound. Uptake into the cell, induction (effector specificity),
conversion by enzymes (enzyme specificity), formation of toxic products
The necessity of permeability mutants and transport systems for the use of
the polar metabolites 3-oxoadipate, cis,cis-muconate, and g-carboxy-cis,cis-mu-
conate as the growth substrate has been documented [839843].
In general, information concerning the mechanisms for the production of
transport mutants is rare. Overall, the transport into the cell seems to be a
bottleneck only for ionized compounds.
7.2
Expansion of the Effector Specificity of Transcriptional Regulators
Biological activities that mineralize pollutants generally consist of multistep
pathways. Degradation of simple compounds such as toluene can involve ten or
more enzymatic reactions. The expression of the enzymes is carefully control-
led by multicomponent regulatory networks.
One mechanism for enhancing the range of inducers is the alteration of tran-
scription circuits of catabolic operons. The genes of catabolic pathways are
typically organized in operons that assure coordinated synthesis of the com-
ponent enzymes of the pathways. Transcription of such operons from the
operon promoters is generally controlled by positively acting regulatory pro-
teins that are activated by pathway substrates or metabolites, i. e., the effectors.
Among the genes for degradative pathways the xyl genes, laying on the TOL
plasmid pWW0, are the best characterized ones. These genes, which are essen-
tial for the total degradation of toluene and xylenes, form two functional clus-
ters, the so-called upper and the meta operon. A simplified model is given in
Fig. 84.
The upper operon xylCMABN encodes three enzymes which oxidize tol-
uene and xylenes to benzoate and toluates, respectively. The promoter of the
upper pathway genes, P
u
, is positively regulated by the regulatory gene xylR.
This gene encodes a protein which enhances transcription after the binding of
an inducing molecule (toluene, m-xylene, and the respective benzoates).
Subsequently, benzoate and toluate are transformed to the respective catechols
and mineralized by the enzymes of the meta pathway, encoded by the genes of
the meta operon. The meta operon contains the genes xylXYZ and xylL, which
Fig. 84. Simplified model of the regulation of xyl gene operons (xyl, xylene). The upper
operon (xylCMABN) codes xylene monooxygenase, benzylalcohol and benzaldehyde de-
hydrogenase, while 13 enzymes are encoded by the meta operon (xylXYZLTEGFJQKIH),
including toluate 1,2-dioxygenase, toluate dihydrodiol dehydrogenase and catechol 2,3-di-
oxygenase. The regulator genes xylS and xylR are shown in gray. The promoter regions are
marked by small boxes. The arrows indicate induction by XylR and XylS regulatory protein in
concert with the respective aromatic effectors. Data from [844850]
encode the enzymes toluate 1,2-dioxygenase and toluate dihydrodiol dehydro-
genase. Not less than nine enzymes are co-ordinately expressed in the polycis-
tronic meta operon from the P
m
promoter. For efficient expression of the meta
operon by the P
m
promoter, the product of the xylS gene is needed. Inducing
compounds like benzoate and toluate bind to XylS, bringing it to an activated
form.
Studies on mutants with expanded substrate range and induction patterns
have shown that the xylS gene is the target for adaptive mutations. Some ben-
zoate analogues that can be metabolized by the enzymes of the pathway, such as
toluate dioxygenase and toluate dihydrodiol dehydrogenase, fail to induce syn-
thesis of these enzymes. Instead, these non-effector benzoate analogues compe-
titively inhibited effector-mediated activation of the regulator protein. These
compounds clearly interacted with the effector-binding site of the regulator but
failed to establish the productive contacts needed to induce the conformational
change leading to activation of the protein. It was relatively easy to isolate mu-
tant bacteria producing regulators that were activated by such benzoate analo-
gues [849]. A mutant of the XylS regulatory protein can mediate three- to eight
times higher levels of transcription than the wild type regulator [851].
Subsequent studies showed that selective elevation of XylS expression and an
increase in the intracellular level of XylS can be obtained either by replacing the
relatively weak native promoter with a stronger one or by increasing xylS gene
dosage. This results in a substantial increase in P
m
activation [852, 853].
Selective changes in the 10 region of the P
m
promoter can also increase
transcription of the meta pathway enzymes in a fully regulated manner [854].
Other mechanisms which have been shown to expand the substrate range of
catabolic pathways include activation by insertion elements [855, 856]. van der
Ploeg et al. [855] showed that the substrate range of a chloroacetate-utilizing
strain of Xanthobacter autotrophicus can be expanded to include bromoacetate
by spontaneous insertion of an insertion element, which copies itself from an-
other position on the chromosome to a site closely in front of the dehalogenase
gene. This leads to a five- to tenfold increase in expression.
By judicious manipulation of regulatory proteins and their levels of expres-
sion and of the structure of the cognate promoters of the regulators, one can
achieve very high levels of expression of catabolic operons and create effective
degradative organisms. It has been shown that the substrate range can be ex-
panded. However, from a practical point of view it should be noted that most of
the new substrates such as 4-ethylbenzoate can also be degraded by naturally
occurring bacteria that can be enriched from polluted environmental samples.
7.3
Alterations in Structural Genes
7.3.1
Widening of the Substrate Range
An excellent example of mutations in a structural gene that alter enzyme speci-
ficity to allow a strain to grow with a novel substrate is given by Pries et al. [857].
124 W. Reineke
They expressed the haloalkane dehalogenase (DhlA) of Xanthobacter autotro-
phicus that hydrolyses short-chain (C
2
C
4
) 1-chloro-n-alkanes to the corre-
sponding alcohols in a strain of Pseudomonas that grows on long-chain alco-
hols. Different spontaneous mutants were selected able to use 1-chlorohexane
as the growth substrate. All the mutants showed alterations, deletions, point
mutations, or tandem repeats, in a distinct region of the dehalogenase, the so-
called cap domain. The kinetic constants indicate that the mutants are better
adapted to use the novel substrate than the wild type but lost some efficiency
against the original substrate 1,2-dibromoethane. Pries et al. [857] found that
the generation of duplication was a common event during adaptation to 1-
chlorohexane. Since duplications were also present in the cap domain of wild
type dehalogenase they suggest that the wild type enzyme has undergone re-
cent adaptive mutations leading to utilization of 1,2-dichloroethane.
Besides these natural processes, site-directed mutagenesis allows the con-
struction of enzyme variants that exhibit broader substrate specificities.
Biphenyl dioxygenases catalyze the first step in the aerobic degradation of chlo-
rinated biphenyls. The nucleotide and amino acid sequences of the biphenyl di-
oxygenases from Pseudomonas sp. strain LB400 and Pseudomonas pseudoalca-
ligenes KF707 were found to be nearly identical, yet these enzymes exhibited
dramatically different substrate specificities for chlorinated biphenyls (LB400
broad substrate spectrum, KF707 narrow substrate spectrum). Site-directed
mutagenesis of the LB400 bphA gene, encoding the large subunit of the termi-
nal dioxygenase, resulted in an enzyme combining the broad congener specifi-
city of LB400 with an increased activity against several congeners, including
double para-substituted ones, which is characteristic for KF707 dioxygenase
[858]. The mutagenesis procedure altered a region of the LB400 bphA gene en-
coding a block of four amino acids, which differ from those of strain KF707. The
region of amino acids 335 to 341 in LB400 BphA (TFNNIRI) was converted to
the corresponding KF707 sequence (AINTIRT) at the underlined positions.
This multi-amino acid modification brought about the greatest improvement
in activity. The novel dioxygenase combined the broad congener specificity of
LB400 with the increased activity against several congeners characteristic of
KF707.
Later, Mondello et al. [859] examined the BphA sequences from a variety of
bacteria whose PCB substrate specificities differ from that of strains LB400 and
KF707 but which contain related bphA genes. With that database four regions
were identified in which specific sequences were associated with either broad or
narrow PCB substrate specificity. The most important one was region III which
has been modified in the Erickson and Mondello study [858]. Based on these as-
sociations, site-directed mutagenesis was used to alter specific regions of the
LB400 bphA nucleotide sequence and the effects of these mutations on PCB
substrate specificity were determined. The most important region again was re-
gion III, but also a modification of one amino acid in region IV can contribute
to the broader substrate specificity.
Similarly, Kimura et al. [860] produced chimeric enzymes from the LB400
and KF707 dioxygenases combining the substrate range of both parental en-
zymes by exchanging restriction fragments.
Besides the rational, site-directed approaches, which allow the exploration of
only very limited sequence space at a time,irrational approaches such as DNA
shuffling, a technique using PCR [861], can be alternatives for the production of
enzymes with altered substrate specificity. In two independent studies [862,
863] the substrate range of biphenyl dioxygenases towards PCB congeners have
been successfully extended using the random cross-breeding of the genes from
strain LB400 and KF707 by DNA shuffling. One major advantage of the in vitro
DNA shuffling of enzymes over the structural remodeling by site-directed mu-
tagenesis is that only a minimum of prior information is required.
7.3.2
Mutations in Structural Genes to Avoid the Formation of a Toxic Metabolite
Pseudomonas sp. strain B13, which is able to use 3-chlorobenzoate as the growth
substrate but fails to use 2-fluorobenzoate, was adapted to growth with the lat-
ter compound over a period of six months in a chemostat in which 3-chloro-
benzoate was stepwise replaced by 2-fluorobenzoate [397]. The resulting strain
B13-1 was then cultivated in sequential batch cultures for 250 generations with
2-fluorobenzoate as the sole carbon and energy source. The adapted strain
B13-2, growing at high rate with the new substrate, degrades 2-fluorobenzoate
for 95% via pathway a, with oxygenolytic elimination of fluoride and catechol
(Fig. 85), while pathway b is not much used. Instead, strain B13 degrades 2-fluo-
robenzoate for 22% via pathway b, which yields production of high amounts of
3-fluorocatechol, which accumulates and negatively affects the cells so that no
growth occurs with 2-fluorobenzoate. The improved strain B13-2 is able to grow
126 W. Reineke
Fig. 85. Alternative pathways for the degradation of 2-fluorobenzoate due to the attachment
of the substrate to the dioxygenase with substituents in the 2- or 6-position
in the presence of 2-fluorobenzoate due to a change in selectivity of the initial
benzoate 1,2-dioxygenase towards degradation via 2-fluorodihydrodihydroxy-
benzoate. Therefore, only about 5% of the 2-fluorobenzoate will be converted in
strain B13-2 to give the toxic metabolite 3-fluorocatechol.
A quite different strategy in avoiding the formation of a toxic metabolite was
shown to allow a mutant of Alcaligenes eutrophus to grow with 2-fluorobenzo-
ate. The wild type organism uses benzoate as the sole source of carbon and
energy. 2-Fluorobenzoate will be converted via pathway a (20%), so that great
amounts of the toxic metabolite 3-fluorocatechol accumulate. The accumula-
tion is avoided in the mutant B9 by a defect of the dihydrodihydroxybenzoate
dehydrogenase. 6-Fluorodihydrodihydroxybenzoate will not be converted
further to the toxic 3-fluorocatechol. Growth resulted because of the minera-
lization of catechol formed after spontaneous elimination of fluoride.
These examples show how modification of enzyme selectivity prevents mis-
routing to give toxic metabolites.
7.4
Use of External Genetic Information to Expand the Substrate Range
7.4.1
Chlorobenzoate-Degraders by Conjugal Transfer
The first report of the development of a catabolic pathway for the mineraliza-
tion of chlorinated aromatics using external genetic information for the acqui-
sition of a novel phenotype described work with Pseudomonas sp. strain B13
and Pseudomonas putida PaW1 and the novel growth substrates 4-chloro- and
3,5-dichlorobenzoate. Strain B13 was isolated by enrichment culture with 3-
chlorobenzoate. It oxidizes 3-chlorobenzoate to 3- and 4-chlorocatechol and
uses the modified ortho pathway for further breakdown. Strain B13 is unable to
utilize 4-chloro- and 3,5-dichlorobenzoate, since the benzoate 1,2-dioxygenase
has a very narrow specificity and will not accept 4-chloro- and 3,5-dichloro-
benzoate as substrates. However, strain B13 can oxidize 4-chloro- and 3,5-di-
chlorocatechol, the expected metabolites in the degradation of 4-chloro- and
3,5-dichlorobenzoate. The toluate 1,2-dioxygenase in Pseudomonas putida
PaW1, which is isofunctional to the benzoate 1,2-dioxygenase, encoded by the
TOL plasmid, has a broader range than the B13 enzyme and can accept 4-
chloro- and 3,5-dichlorobenzoate as a substrate.
In one enrichment experiment the two organisms were grown in a chemostat
initially with 3-chlorobenzoate (substrate for strain B13) and 4-methylbenzoate
(substrate for strain PaW1). After four weeks of operation, 4-chlorobenzoate
was added as an additional carbon source since it could not support the growth
of either organism. After another four-week period, the culture was switched to
only 4-chlorobenzoate and during the next twelve weeks 3,5-dichlorobenzoate
was added and colonies able to grow on 3,5-dichlorobenzoate were isolated.
Eventually, Pseudomonas sp. strain WR912 was isolated and shown to be cap-
able of growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate [864]. The
complexity of this experiment, with the prolonged selection period, made it dif-
ficult to interpret, but one of the predictions was that strain B13 would acquire
the toluate 1,2-dioxygenase coded by the TOL plasmid of Pseudomonas putida
PaW1. That a novel catabolic pathway could be developed in this way was later
confirmed by direct transfer experiments, but the results were unexpected
[865867]. Transconjugants from a mating between Pseudomonas sp. strain B13
and Pseudomonas putida PaW1 were obtained on plates containing 4-methyl-
benzoate to select for TOL plasmid transfer and streptomycin to contraselect
against the Pseudomonas putida parent. Strain WR211 had the phenotype of
strain B13 with the additional ability to grow on 3- and 4-methylbenzoate, but
was unable to grow on 4-chlorobenzoate. Strain WR211 was plated on 4-chloro-
benzoate and gave rise to strains, such as WR216, which had gained the ability
to utilize 4-chlorobenzoate but had lost the ability to utilize the methylbenzoa-
tes. 4-Chlorobenzoate utilizers were derived directly from other plate matings.
Plasmid transfer by itself was clearly inadequate for the development of the
novel pathway.
The TOL plasmid of Pseudomonas putida PaW1 is known to undergo various
rearrangements of its DNA. In the development of the 4-chlorobenzoate utilizer
it was suggested that the events are as follows [868]:
1. Transfer of TOL plasmid into strain B13.
2. Integration of a 56-kb segment of TOL DNA into the chromosome.
3. Deletion of a 39-kb segment from TOL.
4. Insertion of a DNA segment of about 3 kb into the xylE gene, the gene encod-
ing the catechol 2,3-dioxygenase.
For the selection of the 4-chlorobenzoate derivatives of WR211 it was essential
that the meta pathway is inactivated. The catechol 2,3-dioxygenase has a high
affinity for 4-chlorocatechol, which is channeled into the meta cleavage path-
way, resulting in the production of dead-end metabolites. Strain WR216 has
no catechol 2,3-dioxygenase activity and 4-chlorocatechol is catabolized via the
ortho cleavage pathway.
In addition to the bottlenecks concerning the turnover of substrates, some-
times the induction of a pathway by the novel compound does not take place.
One example for this fact is 3,5-dichlorobenzoate. While 3,5-dichlorobenzoate
fails to induce the toluate 1,2-dioxygenase in the strains WR211 and WR216, the
compound is an effector in the strains which have occurred on solid media
containing 3,5-dichlorobenzoate from the respective origins.
7.4.2
Chloronitrophenol-Degraders by Conjugal Transfer
The chlorocatechol degradative genes of strain B13 and JMP134 have also been
used to obtain strains able to grow with 4-chloro-2-nitrophenol (Fig. 86).
Pseudomonas sp. N31, isolated with 3-nitrophenol and succinate as sole source
of nitrogen and carbon, respectively, expresses a nitrophenol oxygenase elimi-
nating nitrite from 4-chloro-2-nitrophenol to produce 4-chlorocatechol.
Conjugal transfer of the genes coding the modified ortho pathway from B13 or
JMP134 into strain N31 allowed the isolation of hybrid strains such as N31-1
128 W. Reineke
able to use 4-chloro-2-nitrophenol as sole source of carbon, nitrogen, and
energy [869].
7.4.3
Chlorobiphenyl-Degraders by Mating Three Strains
Some chlorobiphenyls fail to be growth substrates because the peripheral en-
zymes do not convert them to the chlorocatechols. In these cases a novel path-
way has to be constructed in one organism by segments from at least three or-
ganisms. Such a mating is summarized schematically in Fig. 87 for various chlo-
robiphenyls. However, the nature of some events which have to take place
during the development is presently unknown.
7.4.4
Other Chloroaromatic-Degraders by Conjugal Transfer
The development of hybrid pathways by DNA transfer with whole degradative
plasmids has been demonstrated for various other mono- and dichlorosub-
stituted aromatics (Fig. 88). The procedure is superior due to its technical sim-
plicity and effective positive selection. Hybrid strains can be developed by the
well-aimed mating within weeks, since the organisms with the suitable pathway
segments are inoculated together on solid media, making a gene transfer easy.
However, the selection steps have to be done in the right order. It is important
to establish the chlorocatechol degradative sequence at the beginning of the de-
velopment of the hybrid strains, since otherwise the accumulated chlorocate-
chols will harm the cells.
Various chloroaromatic-degrading strains have been isolated by enrichment
techniques, such as chlorobenzenes, chlorophenols, and chlorophenoxyacetates
degraders. An inspection of the pathway in some strains indicated that they are
also a product of the patchwork assembly described above.
In principle, similar hybrid strains can also be constructed using genetic
engineering techniques. The major disadvantage is the huge amount of research
necessary prior to the in vitro construction experiment leading to the hybrid
strains. Cloning of structural as well as regulatory genes has to be done follow-
ed by establishment in a suitable host.
Fig. 86. Pathway for 4-chloro-2-nitrophenol in the hybrid strain N311
7.4.5
Chlorobenzoate- and Chlorosalicylate-Degraders by Genetic Engineering Techniques
Using genetic engineering techniques, chlorobenzoate-degrading strains were
prepared. A DNA module carrying the genes of toluate dioxygenase (xylXYZ)
and of the subsequent enzyme of the pathway, toluate dihydrodiol dehydro-
130 W. Reineke
Fig. 87 ac. Hybrid pathways for the mineralization of chlorobiphenyls in hybrid strains:
a BN210, 3-chlorobiphenyl
+
; b KE210, 3-chlorobiphenyl
+
, 4-chlorobiphenyl
+
; c JHR22, 2-chlo-
robiphenyl
+
, 3-chlorobiphenyl
+
, 4-chlorobiphenyl
+
, 2,4-dichlorobiphenyl
+
, 3,5-dichlorobi-
phenyl
+
. The color or the hatch on the right side of the pathway characterizes the origin of the
respective pathway segment in the hybrid strains: , biphenyl degrading strains JHR (c) or
BN10 (a, b); , p-toluate degrading strain PaW1; , o-toluate degrading strain WR401,
, modified ortho pathway of strain B13, and , late 3-oxoadipate pathway segment of
Pseudomonas putida strain BN10 (a, b) or Burkholderia cepacia strain WR401 (c).
Information compiled from [870, 871, K. Engelberts and W. Reineke, unpublished results]
genase (xylL), plus the P
m
promoter and the xylS regulatory gene, was cloned
into a broad-host-range plasmid vector and introduced into Pseudomonas sp.
strain B13 [886]. The B13 derivative could grow on 3-chloro- and 4-chloro-
benzoate, and synthesis of all catabolic enzymes involved in their metabolism
was fully regulated. The B13 derivative did not grow on 3,5-dichlorobenzoate,
even though the catabolic enzymes present in 4-chlorobenzoate-grown bacte-
ria can degrade this compound, because of the inability of the XylS regulator
to be activated by 3,5-dichlorobenzoate. However, 3,5-dichlorobenzoate be-
came a substrate for the hybrid pathway when a XylS mutant regulator that is
activated by this compound was also recruited by Pseudomonas sp. strain B13
[886].
Central pathways, such as the chlorocatechol ortho cleavage pathway, can be
used as a base upon which to assemble additional enzymatic steps to permit
the catabolism of more complex compounds. Construction of a derivative cap-
able of degrading chlorosalicylates represents a simple example of vertical
expansion of the chlorocatechol pathway of Pseudomonas sp. B13. Strain B13
cannot degrade either salicylate or chlorosalicylates, and such bacteria are not
readily isolated from soil. Plasmid NAH7 specifies a pathway for the catabolism
of naphthalene via salicylate and catechol. Salicylate hydroxylase encoded by
NAH7 exhibits a relaxed substrate specificity and oxidizes salicylate and
methyl- and chlorosalicylates to the corresponding catechols. A DNA fragment
of the NAH7 plasmid containing the gene encoding salicylate hydroxylase, its
promoter, and the regulator gene was introduced into Pseudomonas sp. B13,
which thereby acquired the ability to grow on 3-, 4-, and 5-chlorosalicylates
[886].
Fig. 88. Hybrid pathways for the mineralization of chloroaromatics developed by in vivo con-
struction using peripheral pathway segments plus the modified ortho pathway (data com-
piled from [865, 867, 870885])
7.4.6
Chlorobiphenyl-Degraders by Genetic Engineering Techniques
Hrywna et al. [887] reported that the use of sequenced and well-characterized
chlorobenzoate dehalogenase genes is an effective strategy for the construction
of hybrid organisms able to grow on ortho- and para-substituted chlorobi-
phenyls. Two plasmids were introduced separately by transformation into the
biphenyl-growing, chlorinated biphenyls-cometabolizing Comamonas testo-
steroni VP44. Plasmid pE43 carries the cloned gene coding for the terminal oxy-
genase of the ortho-halobenzoate 1,2-dioxygenase [888], which performed oxy-
genolytic ortho dehalogenation of 2-chlorobenzoate, a dead-end product in the
2-chlorobiphenyl transformation by strain VP44. Hydrolytic dechlorination of
4-chlorobenzoate is catalyzed by 4-chlorobenzoate dehalogenase coded by
cloned genes on plasmid pPC3 [889]. The resulting hybrid strains harboring
one of these plasmids expressed a dehalogenating enzyme, grew rapidly on, and
completely dechlorinate high concentrations of 2-chloro- or 4-chlorobiphenyl,
respectively (Fig. 89).
This is an example where the addition of one enzymatic reaction, missing in
the host organism, directly brings about the formation of a normalmetabolite
of the aromatic degradation so that growth with the new compound can occur.
7.4.7
Trihalopropane-Degraders by Genetic Engineering Techniques
The rational combination of catabolic segments from different organisms in
one recipient strain creating a complete metabolic route has been shown for tri-
halopropanes, for which mineralization has not yet been described [890]. Broad
host-range plasmids were constructed, which contained the gene coding for
haloalkane dehalogenase from Rhodococcus sp. strain M15-3, an enzyme cap-
able of efficient transformation of trihalopropanes to dihalopropanols, under
the control of different heterologous promoters. Recombinant organisms were
obtained, which are able to grow on trihalopropanes, by introduction of these
plasmids into Agrobacterium radiobacter AD1, capable of utilizing dihalogenat-
ed propanols for growth.
7.5
Construct to Degrade TCE Without Apparent Toxic Effect
Cometabolic conversion of trichloroethene by mono- and dioxygenases of wild
type cells resulted in toxic effects which drastically reduced the conversion rate.
To overcome this problem, Winter et al. [891] have introduced the gene of a to-
luene monooxygenase in Escherichia coli under the control of the Trp-Lac(tac)
promoter. This construct, in contrast to the situation in the wild type, was then
able to degrade TCE without apparent toxic effects.
132 W. Reineke
A
e
r
o
b
i
c

a
n
d

A
n
a
e
r
o
b
i
c

B
i
o
d
e
g
r
a
d
a
t
i
o
n

P
o
t
e
n
t
i
a
l
s

o
f

M
i
c
r
o
o
r
g
a
n
i
s
m
s
1
3
3
Fig. 89. Hybrid pathways for growth with chlorobiphenyls by introduction of dehalogenase genes (for details of dehalogenating reac-
tions see Figs. 36 and 39)
7.6
Creation of a Pathway for the Degradation of Halogenated Alkanes and Alkenes
The design of a new pathway to carry out sequential reductive and oxidative re-
actions in an organism is another example demonstrating the value of using the
knowledge of catabolic enzymes and recombinant DNA technology. Hur et al.
[892] and Wackett et al. [893] reported the construction of a useful metabolic
pathway for the degradation of fluoro, chloro, bromo, and chlorofluoro alkanes
and alkenes. Three genes coding cytochrome P-450
CAM
monooxygenase from
Pseudomonas putida, which is able to dehalogenate reductively polyhalo-
genated compounds [894], were combined with four genes coding the toluene
dioxygenase of Pseudomonas putida F1 to construct the cometabolic dehalo-
genation sequence of consecutive reductive and oxidative reactions. The con-
version of pentachloroethane by the recombinant bacterium is given in Fig. 90.
7.7
Creation of a Pathway for the Degradation of Mixtures
of Methyl- and Chloroaromatics by Combination of Pathway Modules
The existence of both ortho and meta cleavage enzymes produces problems for
the conversion of a mixture of methyl and chloroaromatic compounds.
Catechol and chlorocatechols are generally subjected to ortho fission, whereas
methylcatechols suffer meta fission. Although both pathways may exist in indi-
vidual microorganisms, only one is usually functional at any given moment de-
pending on the available substrate. However, when both chloro- and methyl-
catechols are formed from mixtures of chloro- and methylaromatics, both path-
way types are functional, and the catechols are subjected to both types of
cleavage. Although ortho cleavage of chlorocatechols leads to their productive
metabolism, ortho cleavage of methylcatechols leads to the formation of dead-
end products. Similarly, whereas the meta cleavage of methylcatechols leads to
their productive metabolism, the meta cleavage of chlorocatechols leads to the
formation of either dead-end products or products that inactivate catechol 2,3-
dioxygenase, the ring cleavage enzyme. The misrouting of catechol cleavage
products during simultaneous metabolism of chloro- and methylsubstituted
aromatics creates a sort of biochemical anarchy and eventually perturbs the
productive metabolism of aromatics [895].
Before designing a catabolic route in Pseudomonas sp. B13 for chloro- and
methylaromatics (Fig. 91) that employs only one type of catechol-ring fission
mode, the peripheral pathway was expanded [895]. Pseudomonas sp. B13 pos-
134 W. Reineke
Fig. 90. Hybrid pathway for the degradation of pentachloroethane
Fig. 91 AC. Construction of 4-methylbenzoate degraders using the ortho cleavage pathway for the mineralization of mixtures of chloro- and methyl-
substituted aromatics by combining pathway segments: A pathway segments involved in the construction: white: from strain B13, gray: segments co-
ded by xylXYZL, i. e., toluate 1,2-dioxygenase and toluate dihydrodiol dehydrogenase of strain PaW1; dark-gray: 4-methyl-2-enelactone isomerase from
A. eutrophus. Full arrows indicate pathways used for growth, broken arrows those pathways able to convert compounds; B hybrid pathway for the de-
gradation of 4-methylbenzoate in strain FR1(pFRC20P)-1; C genealogy of the strains. Abbreviations: 3CB, 3-chlorobenzoate; 4CB, 4-chlorobenzoate;
4MB, p-toluate; 4CP, 4-chlorophenol; 4MP, 4-methylphenol
(A)
(B)
(C)
sesses only an ortho cleavage route for catechols; it can grow on 3-chloroben-
zoate and acquires the ability to grow on 4-chlorobenzoate when it recruits a re-
laxed substrate-specificity toluate dioxygenase through transfer of the xylXYZ
genes of the TOL plasmid. To create a stable B13 derivative that can degrade 4-
chlorobenzoate and partially metabolize 4-methylbenzoate, the cloned TOL
gene module (xylXYZL including their promoter P
m
, and xylS) was inserted into
transposon Tn5; the hybrid transposon was then transposed into the B13 chro-
mosome. The derivative strain, FR1, grew on 3- and 4-chlorobenzoate and co-
metabolized 3- and 4-methylbenzoate via ortho cleavage to the dead-end pro-
ducts 2- and 4-methyl-2-enelactone, respectively.
Strain FR1 like B13 grew on 3-methyl-2-enelactone as a sole source of
carbon and energy. Therefore, the degradation of 4-methylbenzoate by strain
FR1 in principle requires recruitment only of an isomerase that converts 4-me-
thyl-2-enelactone to 3-methyl-2-enelactone. The recruitment of 4-methyl-2-
enelactone isomerase from Alcaligenes eutrophus which is able to transform
4-methyl- into 3-methyl-2-enelactone [896] resulted in the transformation of 4-
methylbenzoate to 3-methyl-2-enelactone, which was then mineralized by B13
enzymes.
This pathway was further expanded through mutational activation of the
previously cryptic phenol hydroxylase of strain B13 to produce catechols which
allowed metabolism of 4-methylphenol exclusively via ortho cleavage. 3-Me-
thylbenzoate and 3-methylphenol are no substrates, since they are mainly co-
metabolized to 2-methyl-2-enelactone [897, 898], which is not further metabo-
lized by B13 enzymes nor by the 4-methyl-2-enelactone isomerase cloned from
A. eutrophus.
Thus, a novel ortho cleavage pathway for the degradation of mixtures of 3-
and 4-chloro- and 4-methylbenzoates and 4-chloro- and 4-methylphenol has
been constructed by the rational assembly of pathway modules from three dif-
ferent bacteria: P. putida PaW1, Pseudomonas sp. B13, and A. eutrophus JMP134.
7.8
Rsum: Enhancement of Catabolic Potential
The identification of pathways and genes have allowed an understanding of the
biochemical causes of recalcitrance and degradability of chlorinated aromatic
and aliphatic compounds. Blocks have been identified and enzyme sequences
with broader specificity have been obtained. Based on this knowledge, new
pathways have been constructed. Some of them seem to be copies of those
found in strains enriched from environmental samples. A drawback for the ra-
tional design of novel microorganisms is that a lot of biochemical knowledge is
needed, which is not available for many compounds.
8
Concluding Remarks and Outlook
In this chapter I have been concerned with drawing together information from
a variety of sources to illustrate the current state of knowledge of microbial de-
136 W. Reineke
gradation of some natural and man-made molecules, and aliphatic and aromat-
ic compounds and their chlorinated counterparts were chosen. In former times
the chlorinated hydrocarbons were classified as being solely anthropogenic and
therefore should be attributed to be xenobiotics. However, today it is known
that more than 2400 organohalogen chemicals are produced by living orga-
nisms and over half of these contain chlorine as part of their innate molecular
structure [899]. The difference between the anthropogenic and natural organo-
halogen compounds is mostly the difference in the amounts produced. While
the chemical production is in the range of 10
6
tonnes per year world-wide the
majority of the natural organohalogens can be termed to be niche products.
However, in contrast, some natural halogenated compounds exceed the anthro-
pogenic production drastically, e. g. natural generation of chloromethane was
estimated to be 28 10
6
tonnes per year [900] compared to an estimated annual
industrial world production of about 0.7 10
6
tonnes [901].
There was and is one compelling reason among several why attention should
be focused upon the special area of microbial metabolism of haloorganics. A
large number of these compounds either deliberately or unintentionally have
found their way into our environment as a consequence of the activity of
modern industry and agriculture, and by persisting there for various periods of
time have caused concern to society. We need to understand why some chemi-
cals persist while others disappear [902].
While in those days simpler answers were given, such as it is the structural
element chlorine substituent on an aromatic ring which is responsible for the
recalcitrance of these compounds, todays answers show a more complex pic-
ture.
Biochemical reactions have to follow chemical principles: the negative in-
ductive effect of halogen substituents decreased the nucleophilic character of
an aromatic ring and thus impedes the electrophilic attack of dioxygenases.
However, this simple view of a chemist, when trying to explain recalcitrance of
chloroaromatic compounds, is misleading. Steric effects more drastically con-
tribute to the slow down of dioxygenase reactions. In one organism a dioxyge-
nase reaction might be a bottleneck with chlorosubstituted substrate analo-
gues, but not a bottleneck in another one. It is hard to give predictions on the
stereospecificity of an enzyme. The only simple argument which can be given is
that if an enzyme tolerates substrates with a methylsubstituent, it will also be
able to deal with a chlorosubstituted substrate because of the identical size of
both substituents. In contrast, the substitution of a hydrogen in the natural sub-
strate by a chlorine substituent results in dramatic effects because of the great
difference in size, while a fluorine substituent will be tolerated from this point
of view.
Another observation along the same lines was that a high number of chlorine
substituents on an aromatic ring strongly reduced the electron density and hin-
dered an electrophilic reaction. This implies that an aerobic degradation of
compounds such as tetrachlorobenzene or pentachlorophenol cannot take
place. However, this simplifying view of a chemist does not fit in with reality.
Tetrachlorobenzene and pentachlorophenol are the subjects of degradation by
aerobic pure cultures.
In reference to the argument that anaerobic degradation has little impact in
comparison to aerobic oxidation, it should be noted that there are numerous
natural anaerobic environments. The great influence of some environmental
conditions on the degradation, such as the presence or absence of oxygen, or the
availability of electron acceptors such as nitrate or sulfate, is evident. Different
types of reactions occur and the rates of degradation are different under oxic
and anoxic conditions. In general, anoxic microbial degradation of aromatics
seems to be of greater relevance in nature than earlier expected.
A very important explanation for the persistence of a chemical is the fact that
the physico-chemical behavior might bring about low bioavailability as shown
for PAHs, with the result that microbial degradation cannot take place or pro-
ceeds slowly. A possible way to enhance the bioavailability and the biodegrada-
tion is the application of (bio)surfactants. However, recent observations show
that single surfactants can have contrasting effects on the degradation of or-
ganic pollutants by different bacteria [903]. Surfactants were found to enhance
the oxidation of phenanthrene by a Pseudomonas but inhibited its oxidation
and growth on various aromatic compounds by a Sphingomonas strain.
Therefore there is a need for design of optimal (bio)surfactants.
Reaction sequences must make chemical sense, a view which has been ad-
dressed by Dagley [902]: Catabolic pathways are usually economical and direct
to the point of elegance, if that is not the case it will probably be incorrect.
Attempts to discuss common aspects of the degradative pathways kept this
statement in mind.
The book of Gibson from 1984 [904] has elegantly summarized the state of
the art on the subject of microbial degradation available at the beginning of the
1980s. Since then considerable progress has been made in understanding the
mineralization of chlorinated compounds. In contrast, only smaller pieces of
fundamental knowledge in the field of non-substituted aliphatic and aromatic
compounds were collected such as ether cleavage of dibenzofuran and dibenzo-
p-dioxin. While most of the information on the aerobic degradation was avail-
able in 1984, astonishing unexpected results have been obtained in recent
years concerning anoxic degradation.
Degradation of aliphatic and aromatic compounds is possible without molec-
ular oxygen contrary to opinion in former times. The degradation of toluene is
an example of the substantial increase in knowledge in the field of the anoxic
degradation of aromatic compounds. However, also for the alkanes, which were
considered to be non-degradable without molecular oxygen, pure cultures have
recently been enriched which now allow biochemical investigations to elucidate
the strategy used by the organisms to activate alkanes without oxygen.
Dechlorination reactions involved in the aerobic degradation of chloroaro-
matic compounds via the modified ortho pathway are now known in detail. The
hydroquinone pathway as an alternative to the modified ortho pathway has
been elucidated more recently.
In addition, some results which formerly brought about some generalization
have now to be assessed more carefully: It was assumed for a long time to be im-
possible to metabolize 3-chlorocatechols via the meta pathway, because the re-
action product would inactivate the extradiol dioxygenase. However, a novel
138 W. Reineke
chlorocatechol 2,3-dioxygenase that can effectively cleave 3-chlorocatechol,
leading to simultaneous ring cleavage and dechlorination, allows a pseudomo-
nad to degrade chlorobenzene rapidly via a meta cleavage pathway.
The problems with generalizing rules and statements can be illustrated by
another example. Anaerobic bacteria have to degrade aromatic compounds by
reductive conversions. Gallus and Schink [905] and Philipp and Schink [277]
presented evidence that the reductive strategy for ring destabilization is not the
only one used in anaerobic degradation of aromatic compounds. Instead, an-
aerobic bacteria were isolated which use oxidation rather than reduction to
overcome stability of an aromatic ring.
Optimistic microbiologists of the early 1980s believed that some man-made
compounds which were considered to be persistent at that time will turn out to
be biodegradable in the future when a few microorganisms acquire the neces-
sary catabolic expertise and then transmit it to others through the agency of
plasmids. I think they were right. Some new properties for instance were found
to be the result of patchwork assembly of preexisting pathway segments which
in combination function as hybrid pathways [906, 907]. Besides the natural
development and the construction in the laboratory by use of conjugation, ra-
tional design of pathways for novel compounds has been shown by use of
genetic engineering techniques.
In recent years a big change in emphasis of interest in the field of degrada-
tion has taken place. So information pertaining to the genetic basis of the de-
gradative pathway is widely available today, but mostly omitted in this chapter.
Instead, emphasis has been placed here on the biochemical activities of micro-
organisms with respect to the degradation or transformation. So most of the
chapter is written with the view of a biochemical microbiologist working with
pure cultures and single compounds rather than of a molecular biologist or an
environmentalist.
I believe that traditional microbiological approaches such as the enrichment
of new organisms give the chance to recruit presently unexpected, useful reac-
tion sequences, for instance in the field of anaerobic degradation. The natural
pool harbors a great diversity of reactions formed during natures evolution,
which should be used and not neglected.
Some authors have felt that laboratory studies with pure cultures and single
compounds have nothing to contribute to the solution of environmental prob-
lems. Our understanding of microbial action in the environment is still in its
infancy; there can be no doubt that these fundamental contributions from the
laboratory, including the genetic tools, are respectable and necessary starting
points to understand the fate, survival, and activities of microorganisms in the
environment and during bioremediation processes. Syntrophic microbial popu-
lations, whose partners cannot be isolated in pure culture but which seem to be
responsible for degradation in various anoxic environments, can be
characterized with these genetic tools.
So there is much work remaining for microbiologists, biochemists, molecular
biologists, ecologists, environmental chemists, and chemical engineers to
answer the above-mentioned questions and to help in solving problems with
current and future pollutants.
Acknowledgements. I am grateful to Dick B. Janssen for critical reading of the first draft ver-
sion of the manuscript and highly productive comments. In addition, I thank Bernd Beek for
patience and fruitful comments.
I thank all my collaborators for their enthusiastic participation in the research in my la-
boratory. I wish to acknowledge support provided by the European Community and the
Deutsche Forschungsgemeinschaft.
9
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Biodegradation of Xenobiotics in Environment
and Technosphere
Tom N. P. Bosma, Hauke Harms, Alexander J. B. Zehnder
Swiss Federal Institute of Environmental Science & Technology, Ueberlandstrasse 133,
CH-8600 Dbendorf, Switzerland, E-mail bosma@mep.tno.nl
Microorganisms play an important role in the removal of synthetic organic compounds from
the environment. This chapter gives an overview of the evolution of biodegradation pathways
and describes the strategies that microorganisms have evolved to transform important molec-
ular structures. The actual effectiveness of biodegradation in the environment is determined
by the bioavailability of the compounds. As a general rule, one could state that the release rates
of synthetic compounds should not exceed the environments ability to degrade them.
Keywords. Biodegradation, Xenobiotics, Pathways, Bioavailability, Technosphere, Remedia-
tion, Evolution, Recalcitrance
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
1.1 Behavior of Chemicals in the Biosphere . . . . . . . . . . . . . . . 164
1.2 Behavior of Chemicals in the Physical Environment . . . . . . . . 166
1.3 Decontamination by Microorganisms . . . . . . . . . . . . . . . . 166
2 Biodegradation Pathways . . . . . . . . . . . . . . . . . . . . . . . 168
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2.1.1 Evolution of Metabolic Pathways . . . . . . . . . . . . . . . . . . . 169
2.1.1.1 Events in Vertical Evolution . . . . . . . . . . . . . . . . . . . . . . 169
2.1.1.2 Events in Horizontal Evolution . . . . . . . . . . . . . . . . . . . . 172
2.1.1.3 Generation of Novel Degradative Pathways . . . . . . . . . . . . . 172
2.2 Fate of Substituents . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
2.2.1 Removal of Halogen . . . . . . . . . . . . . . . . . . . . . . . . . . 174
2.2.1.1 Nucleophilic Substitution of Halogen . . . . . . . . . . . . . . . . 177
2.2.1.2 Dehydrodehalogenation . . . . . . . . . . . . . . . . . . . . . . . . 178
2.2.1.3 Oxidative Dehalogenation . . . . . . . . . . . . . . . . . . . . . . . 178
2.2.1.4 Reductive Dehalogenation . . . . . . . . . . . . . . . . . . . . . . . 179
2.2.2 Nitro Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
2.2.2.1 Nitroaromatic Compounds . . . . . . . . . . . . . . . . . . . . . . 181
2.2.2.2 Nitrate Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.2.3 Sulfonic Acid Groups . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.2.4 Organophosphonates . . . . . . . . . . . . . . . . . . . . . . . . . . 184
2.3 Degradation of the Carbon Backbone . . . . . . . . . . . . . . . . 184
2.3.1 Aliphatic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . 184
2.3.1.1 Aerobic Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
2.3.1.2 Anaerobic Hydrocarbon Degradation . . . . . . . . . . . . . . . . 185
CHAPTER 2
(ed. by B. Beek)
2.3.2 Aromatic Rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.3.2.1 Aerobic Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.3.2.2 Anaerobic Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . 191
2.3.2.3 Fungal Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
2.3.3 Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
2.3.4 Chiral Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
2.3.5 Complexing Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
2.4 Recalcitrance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
3 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . 196
4 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
1
Introduction
Due to extensive production and use in almost all human activities, synthetic
organic compounds are widely distributed in the environment. They enter the
environment via waste disposal, accidental spills, application as pesticides, and
via losses during transport, storage, and use. It has been recognized since the
early 1960s that environmental pollution with low concentrations of organic
chemicals is world-wide. In the last two decades, the danger of heavily polluted
sites to nature and mankind has received increased attention.
The objective of the current chapter is to present the state-of-the-art knowl-
edge on the biodegradation of xenobiotics in the environment and the techno-
sphere on the basis of biodegradation mechanisms that have evolved in the past
decades. The introduction (1) will discuss the behavior of man-made chemicals
in the biosphere and the response of ecosystems on their presence. Then the
evolution of new biodegradation pathways and the transformation mechanisms
themselves will be discussed (2). It appears that microorganisms use convergent
strategies to transform chemicals so that they can be channeled into existing me-
tabolic pathways. The mechanisms of enzymatic conversions are discussed from
the point of view of molecule constituents (for example, halogen or nitro groups)
instead of discussing the biodegradation of certain groups of compounds (for
example, halogenated vs non-halogenated substances). The impact of environ-
mental conditions on the possible biodegradation and the implications for the
engineering of bioremediation are discussed, together with the factors govern-
ing exposure of microorganisms to contaminants in soil and groundwater.
1.1
Behavior of Chemicals in the Biosphere
The response of the biosphere to the appearance of man-made compounds can
be understood by using the concepts developed in systems ecology. The bio-
sphere is characterized by the cycling of matter through the expenditure of
energy flowing through the system and is thus able to maintain a status of high
entropy. Upon their release in the environment, man-made chemicals enter the
164 T. N. P. Bosma et al.
biosphere, and may be taken up by organisms or become part of the pool of
non-living matter (the contaminant pool, Fig. 1). Biota take up contaminants di-
rectly from the contaminant pool, e.g., via leaves or the skin, or they ingest them
by feeding on a lower trophic level. Organisms have systems at their disposal to
excrete or detoxify contaminants. Excretion brings contaminants back to the
contaminant pool, while detoxification results in a decontamination as indicat-
ed in Fig. 1.
Plants and animals are not always able to detoxify or excrete contaminants
after uptake. The inability of organisms to deal with xenobiotic compounds
may have several causes. One example is the absence of appropriate enzymes to
transform the compounds, another the accumulation in (animal) fat tissue be-
fore excretion or enzymatic transformation has taken place. Contaminants will
then accumulate in the food chain. Accumulation is indicated by the use of dif-
ferent gray shades in Fig. 1.
The population of decomposers (Fig. 1) is specialized in the uptake and
conversion of all kinds of dead organic material, like for instance dead animals
and plant debris. Decomposers are crucial for the functioning of ecosystems be-
cause they recycle nutrients back to the nutrient pool. Contaminants which are
accumulated in the tissue of organisms are recycled back to the contaminant
Biodegradation of Xenobiotics in Environment and Technosphere 165
Fig. 1. Cycling of hydrophobic organic contaminants in ecosystems. Input to the system oc-
curs as a result of human activities. Output occurs via chemical or biological pathways result-
ing in the formation of harmless products or in complete mineralization. Microorganisms
that are able to transform and mineralize hazardous organic compounds may be viewed as
decontaminators and belong to the ecological group of the decomposers
pool together with the recycling of nutrients back to the nutrient pool. Some
bacteria and fungi are able to detoxify and mineralize man-made organic com-
pounds like chlorinated benzenes and polyaromatic hydrocarbons. Thus, they
prevent the accumulation of such chemicals in the environment. These micro-
organisms may therefore be viewed as the decontaminators of ecosystems and
the biosphere (Fig. 1).
1.2
Behavior of Chemicals in the Physical Environment
Global exchange of organic chemicals mainly occurs via trade, the atmosphere,
the oceans, and biota [16]. The world-wide character of pollution is illus-
trated by the presence of man-made organics in Arctic snow and in the air of
the Northern and Southern hemispheres [1, 7]. The spread of chemicals is caus-
ed by high production and release rates combined with their stability against
biotic and abiotic transformation and their relative mobilities in air, water, soil,
and biota [8].
PCBs, dioxins, and PAH are released directly to the atmosphere in combus-
tion processes and can exist there both unbound and bound to particles [2, 4,
6]. Wet and dry deposition then lead to soil and water pollution [2, 4, 6].
Subsurface contamination results from infiltration of contaminated surface
water into river borders, from deposition with settling particles onto the sedi-
ment in sedimentation areas of rivers and large water bodies, and from seepage
from the top-soil to deeper layers [912]. Thus, the atmosphere and subsurface
are two major sinks where hydrophobic organic contaminants accumulate
(Fig. 2). Volatile compounds accumulate more in the atmosphere while less
volatile compounds accumulate more in the subsurface. Mineralization of these
otherwise recalcitrant compounds in one of these sinks is the only pathway
removing these compounds from our environment.
Transformation reactions in the atmosphere are almost exclusively
(photo)chemical and may interfere with other atmospheric compounds.
Volatile CFCs (chlorofluorocarbons), for instance, can survive unchanged for
more than 100 years in the atmosphere due to their physical and chemical inert-
ness. They diffuse eventually upward into the stratosphere where chlorine radi-
cals are released by short-wavelength UV-radiation [13]. This reaction initiates
the well-known breakdown of the stratospheric ozone-layer and is the only re-
moval mechanism of CFCs known to occur in the atmosphere [13].
1.3
Decontamination by Microorganisms
Photochemical reactions are not possible in the subsurface, where microbially
mediated transformations constitute the dominant removal mechanism of per-
sistent organic compounds. Many microorganisms live in soil and groundwater
where hazardous compounds may accumulate. It was recognized early in this
century that bacteria are able to oxidize rapidly complex, chemically stable or-
ganic structures originating from petroleum, paraffin, and benzine [14]. Their
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p
h
e
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6
7
Fig. 2. Global exchange of hydrophobic contaminants. They tend to volatilize or to bind to soil due to their physical-chemical properties. As a conse-
quence, the subsurface and the atmosphere are major sinks where these contaminants accumulate. Exchange between the northern and southern he-
mispheres is minor and occurs via human intervention (transport by air and sea) and biota (animals traveling over the equator)
capacity to detoxify anthropogenic chemicals under similar environmental
conditions is variable among various habitats. This may be related to previous
exposure of the microorganisms to the compound under consideration. An ad-
apted microflora capable of converting and mineralizing new compounds may
evolve after a long exposure time. The microflora in a not pre-exposed en-
vironment may not be able to detoxify the same compound. Dichloropropene
and 2,4-D (dichlorophenoxy acetic acid) are examples of pesticides that micro-
organisms have learned to transform. Degradation of these compounds in the
field can be so rapid nowadays that their effectiveness as pesticides is strongly
reduced. As a result, farmers have to apply considerably larger amounts of these
pesticides than was necessary when they were first being used.
2
Biodegradation Pathways
2.1
Introduction
For organisms to grow, electron donors and acceptors, a carbon source, and
nutrients need to be present. In addition to the naturally occurring organic
substrates, many anthropogenic compounds can fulfill the growth requirements
of microorganisms [15]. Many aliphatic and aromatic contaminants serve as
electron donors, thereby undergoing substantial transformation or even min-
eralization to inorganic end products such as carbon dioxide, water, and in-
organic ions. Intermediates of the degradation may be assimilated as the car-
bon source for the formation of biomass, and functional groups of the con-
taminants such as amino, nitro, and sulfonate groups may be used as nutrients.
The electron acceptor for contaminant degradation may be molecular oxygen
or, under anoxic conditions, the oxidized inorganic compounds nitrate, metal
ions, sulfate, and carbon dioxide. It appears that not all of these electron ac-
ceptors are functional with any organic contaminant. The fate of many organic
contaminants therefore relies on the presence of the appropriate electron ac-
ceptors, or even the absence of other electron acceptors. Oxygen, for instance,
cannot be replaced by other compounds for its function as a direct reactant
[16], whereas the absence of oxygen, i. e., a reducing environment, may be
required to allow reductions which are involved in the catabolism of certain
contaminants.
Several important pollutants, such as polychlorinated biphenyls (PCBs) [17],
polychlorinated dibenzofurans, and dibenzo-p-dioxins (PCDF/D) [18], and
dichlorodiphenyltrichloroethane (DDT) [19] do not support growth of the
microorganisms, which achieve their primary degradation. Such fortuitous or
co-metabolism of pollutants may be catalyzed by enzymes which are needed for
the metabolism of the growth-supporting substrate, but also exert activity on
these co-substrates due to relaxed substrate specificities. The strategy of many
fungi is to non-specifically oxidize the organic matter in their neighbourhood,
such as lignin. The fungi seem to benefit not only directly from the oxidation
products but also indirectly from the fertilization of their habitat by growing on
other organisms or on excretion products of those organisms. The effectiveness
and the low substrate specificity of the fungal exo-enzymes leads to the co-oxi-
dation of many pollutants.
2.1.1
Evolution of Metabolic Pathways
Microorganisms have evolved a broad range of biochemical pathways in order
to be able to utilize all naturally occurring organic compounds [2023].
Microbial communities which are exposed to xenobiotic compounds, have of-
ten been found to adapt to the utilization of these chemicals [2426]. Such ad-
aptation events may be the result of induction of specific enzymes in members
of the community or the growth of subpopulations capable of metabolizing the
xenobiotic. However, there is a wealth of information suggesting that micro-
organisms use genetic mechanisms to evolve metabolic pathways for the degra-
dation of xenobiotic compounds. The possibility was considered that microor-
ganisms may eventually be able to degrade any kind of molecule [21, 27]. The
fact that evolution of metabolic activities has occurred becomes evident from
the similarities between individual genes and enzymes involved in the degra-
dation of different xenobiotics and natural substrates and the similarities be-
tween the arrangements of genes encoding for different metabolic pathways.
Direct evidence comes from studies on experimental evolution. Figure 3 shows
schematically the two principle groups of mechanisms involved in the genetic
adaptation to xenobiotic compounds. Genetic changes can be subdivided in
those acting on the level of the cell, like point mutations and DNA rearrange-
ments which are transmitted to the descendants (vertical evolution), and those
involving the transfer of genes between related or non-related organisms (hori-
zontal evolution) [22].
2.1.1.1
Events in Vertical Evolution
Point mutations as well as changes affecting larger DNA sequences occur at low
frequencies as a result of errors in DNA replication or repair. It has been shown
that point mutations, i. e., changes only affecting a single base pair of the DNA,
can alter the specificities of enzymes and regulatory proteins or affect the re-
cognition of promoter sequences. An example is the extension of the specificity
of the catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 to 4-ethylcate-
chol by an exchange of a single amino acid [28]. There are indications that en-
vironmental stresses may lead to increases in the frequency of point mutations,
thereby accelerating vertical DNA evolution [29]. Increased mutation rates were
observed in non-growing bacterial cultures in the presence of substrates, which
potentially could provide energy or carbon for growth [22]. However, it is still
unclear whether external factors may also control the direction of changes, for
instance, by specifically promoting useful point mutations. Divergence of DNA
sequences may also result from erroneous repair of defective DNA or during
DNA replication. Such mutations are particularly frequent in DNA regions
where sequence repetitions or palindromic sequences facilitate false hybridiza-
tion of DNA strands or the shift of the template DNA. A number of recent re-
views and articles summarize the current knowledge of such mutational events
[3035]. Mobile DNA sequences, so-called insertion elements (IS-elements)
which are present in high number in bacterial genomes [21, 36] are often sus-
pected to be involved in DNA rearrangements. The replication of such elements
and integration into other regions of the DNA may, however, disturb the func-
tionality of genes. On the other hand IS-elements may contain transcription
signals, such as promoter sequences which, after insertion, activate formerly
unrecognized genetic information (silent genes) in their neighborhood. Mobile
DNA elements may also co-mobilize genes for degradation of pollutants. A well-
documented example is the tcbAB element encoding for the chlorobenzene di-
oxygenase of Pseudomonas sp. P51 which is flanked by two IS-elements of the
same type. The composite transposable element, referred to as a transposon,
was shown to be capable of inserting at random into the bacterial genome. It is
obvious that transposons may translocate DNA sequences containing large
amounts of genetic information, thereby substantially rearranging bacterial ge-
nomes [37].
Fig. 3. Scheme of the two principle mechanisms in molecular evolution. Vertical evolution
comprises genetic events such as replication errors leading to single base-pair substitutions
(1) or deletions (2). Horizontal evolution refers to the mobilization of DNA fragments or
molecules from one organism to another
Most gene mutations will result in deactivation of the enzyme they encode
and in case of the destruction of an essential function will not propagate.
However, when mutation occurs in combination with a preceding gene duplica-
tion, one of the gene copies may not be subject to selective pressure and may
therefore act as a playground for mutational changes [38]. The two isoforms of
the 6-aminohexanoate dimer hydrolase in Flavobacterium sp. strain K172, for
example, differed by two orders of magnitude in their activity [39]. A detailed
summary of the mechanisms known to alter the DNA and examples for the ef-
fects of these alterations in degradation of aromatic compounds is given by van
der Meer et al. [21].
Comparison of the genetic information for degradation of xenobiotic com-
pounds revealed that the operational DNA regions (operons) and gene clusters
encoding different pathways have genes in common. The abundance of similar-
ities of gene functions between pathways suggests that the assembly and re-
combination of existing genetic material is the most important mechanism for
evolving or expanding metabolic pathways [21, 22]. The TOL plasmid pWW0
contains two separate operons, one encoding the enzymes oxidizing toluenes to
benzoates, the other one encoding the meta cleavage pathway enzymes de-
grading methylbenzoates to pyruvic acid and acetaldehyde (Fig. 4) [40, 41]. The
meta cleavage pathway encoded by the NAH7 plasmid is homologous to that of
the TOL plasmid but contains the gene encoding for the salicylate 1-hydro-
xylase required to channel salicylic acid into the catechol meta cleavage path-
way instead of the multicomponent aromatic ring dioxygenase necessary for
benzoate dioxygenation [42].
Fig. 4. Organization of the TOL plasmid-encoded pathway for the degradation of alkylben-
zenes. The genetic information for the catabolic enzymes is organized in two regulated
operons. The XylR protein stimulates transcription of the upper operon when activated by,
e. g., toluene. The XylS regulator protein stimulates transcription of the meta operon when
activated by, e. g., benzoate (redrawn from [45])
2.1.1.2
Events in Horizontal Evolution
Horizontal evolution comprises those genetic events which involve the transfer
of genes between organisms, and are believed to have resulted in the obvious
distribution of common genetic information in the microbial community. The
observation that slightly different homologous operons encoding for degrada-
tion pathways have been frequently found in phylogenetically distant orga-
nisms suggests the occurrence of extensive horizontal gene transfer. Examples
for highly mobile DNA are self-transmissible plasmids such as the homologous
TOL, NAH, and SAL plasmids [43]. These plasmids not only encode for the de-
gradation pathways of toluene, naphthalene, and salicylate, respectively, but also
contain the instructions for their transfer into other organisms. As a conse-
quence, these plasmids are found in a wide range of bacterial species. In micro-
cosm experiments, such catabolic plasmids could be transferred from labora-
tory-derived organisms to indigenous recipient bacteria [44]. Alternative me-
chanisms of gene transfer known to occur in the environment are transduction,
i. e., gene transfer mediated by bacteriophages, and transformation, i. e., the
uptake of free DNA. Novel metabolic activities of bacteria have been created in
many studies on experimental evolution. There are examples for both the suc-
cessful expansion of existing pathways by addition of peripheral enzymes and
the broadening of substrate ranges by exchange of narrow substrate range en-
zymes with enzymes having low substrate specificity.
2.1.1.3
Generation of Novel Degradative Pathways
Most of the information on the evolution of metabolic pathways reviewed so far
has been deduced from comparative biochemical and molecular analysis of
currently existing microorganisms. The direct investigation of the underlying
natural evolutionary events is nearly impossible because of their low frequency
and randomness [21]. Therefore, many studies on experimental evolution in the
laboratory were conducted which aimed at the change, extension, or de novo
construction of metabolic pathways. Different approaches to the laboratory
evolution of metabolic pathways were used [45]:
1. Long-term selection may involve the progressive replacement of a utilizable
substrate by the recalcitrant target substrate and the use of mutagens. An ex-
ample for such an approach is the successful adaptation of naphthalene de-
grading microorganisms to the degradation of naphthalenesulfonic acid [46].
2. In vivo construction of pathways, i.e., the recruiting of genes of one organism
into another, is achieved by a stimulation of the natural genetic transfer pro-
cesses, such as transduction, transformation, and conjugation. By means of a
stimulation of conjugation, the degradation range of 3-chlorobenzoate de-
grading Pseudomonas sp. strain B13 could be expanded to chlorosalicylates,
chlorobiphenyls, chloroanilines, and chloronitrophenols [4750].
3. In vitro construction of pathways involves the directed and selective combina-
tion of well-characterized genes by molecular techniques [45]. The construc-
tion of a degradative pathway can be very straightforward when the recal-
citrant target compound exhibits substantial structural analogy to a compound
which is readily degradable by a known pathway [51]. It involves the identifica-
tion of the steps in the known pathway which are not permissive for the target
compound and their modification into permissive ones. Such a modification
can be the broadening of the substrate or effector specificity of a certain en-
zyme or regulator protein, respectively, or the upward extension of the known
pathway. Timmis achieved the modification of the toluene degradation path-
way of Pseudomonas putida encoded by the TOL plasmid pWW0 [45] (Fig. 5).
Fig. 5. Schematic representation of the successful modification of the TOL plasmid-encoded
pathway for the degradation of alkylbenzenes leading to the broadening of its substrate range
to 4-ethylbenzoate. Unlike 4-methylbenzoate (1), 4-ethylbenzoate (3) is neither an effector
molecule for the XylS regulator protein (see Fig. 2) nor a substrate for the meta pathway.
Mutation of the xyl S gene led to a XylS* regulator protein that accepted 4-ethylbenzoate and
activated the meta cleavage pathway. This resulted in the conversion of 4-ethylbenzoate to 4-
ethylcatechol (4), which inactivated the ring cleavage protein. Mutation of the xyl E gene led
to an inactivation resistant ring cleavage enzyme, permitting complete 4-ethylbenzoate de-
gradation (redrawn from [45])
A sequence of changes was required to restructure the toluene pathway in such
a way that eventually the formerly recalcitrant analogue 4-ethyltoluene was
utilized [28, 52, 53]. The effector specificity of the XylS regulatory protein con-
trolling the expression of the meta cleavage pathway turned out to be too nar-
row to accept 4-ethylbenzoate as an inductor. XylS mutants with a relaxed
effector specificity could be obtained by chemical mutagenesis. One of the
mutants degraded 4-ethylbenzoate to 4-ethylcatechol. This product, however,
was not further metabolized since it appeared to be a suicide substrate for the
catechol 2,3-dioxygenase. Clones with a catechol 2,3-dioxygenase being resis-
tant to 4-ethylcatechol were also obtained by chemical mutagenesis. These or-
ganisms mineralized 4-ethylbenzoate, but not 4-ethyltoluene. An analysis of the
substrate specificity of the three enzymes leading from toluene to benzoate and
the effector specificity of the XylR protein controlling the expression of this up-
per pathway revealed that only the toluene oxidase was not permissive for 4-
ethyltoluene. Chemical mutagenesis resulted in mutants with relaxed substrate
specificity of the toluene oxidase. These mutants completely mineralized 4-
ethyltoluene via the meta cleavage pathway.
An alternative approach to new degradation pathways is the patchwork assem-
bly of enzymes from different organisms [54]. One of the problems associated
with the degradation of mixtures of aromatic pollutants arises when these che-
micals simultaneously activate both the meta and the ortho cleavage pathways
of catechols. The ortho route productively degrades chlorinated catechols,
whereas alkylated catechols are converted to dead-end metabolites (Fig. 6). The
meta cleaving catechol 2,3-dioxygenase is appropriate for alkylated catechols,
but converts 3-chlorocatechol and 4-chlorocatechol to metabolites which ra-
pidly inactivate the enzyme [55]. Pseudomonas sp. B13, a strain degrading 3-
chlorobenzoate via the ortho pathway, served as the basis for the construction
of a derivative which simultaneously metabolized chlorinated and methylated
phenols and benzoates via the ortho cleavage pathway (Fig. 7). The transforma-
tion of 4-chlorobenzoate, 3-, and 4-methylbenzoate to the corresponding cate-
chols was achieved by adding a broad substrate range toluate dioxygenase
encoded by the TOL plasmid pWW0 [49]. The production of dead-end meta-
bolites from 3- and 4-methylbenzoate was overcome by adding a 4-methyl-2-
enelactone isomerase from Ralstonia eutropha (i. e., the former Alcaligenes eu-
trophus) [54].
2.2
Fate of Substituents
2.2.1
Removal of Halogen
Although many halogenated organic molecules occur in nature [56], industri-
ally produced halo-organics constitute a large group of environmental pol-
lutants and are often considered the quantitatively most important compounds
of xenobiotic character. Examples for the widespread industrial and agricul-
B
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a
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Fig. 6. Differential degradation routes for halogenated and alkylated catechols. Productive further degradation of 3-chlorocatechol A and 4-
chlorocatechol B requires ortho-cleavage, whereas degradation of methylcatechol degradation requires initiation by meta-cleavage C
tural application of synthetic halogenated compounds are their use as solvents,
intermediates, hydraulic and heat transfer fluids, plastics, pesticides, and inter-
mediates for chemical syntheses. In contrast to natural halogenated com-
pounds, which readily degrade [56], many man-made haloorganics were found
to persist in the environment [57].
Fig. 7. Patchwork assembly of a catabolic pathway for the simultaneous degradation of
chloro- and methylaromatics. The modified ortho pathway for the degradation of chlorocate-
chol from Pseudomonas sp. B13 was extended by gene functions for the conversion of alkyl-
benzoates from the TOL plasmid pWW0 (see also Fig. 2). This expanded the degrada-
tion range to include 4-chlorobenzoate and methylbenzoates. Extension by gene functions
from Ralstonia eutropha (the former Alcaligenes eutrophus) for the conversion of 4-methyl-
2-enelactone to 3-methyl-2-enelactone allowed the complete metabolization of alkylben-
zoates. Methylphenols and chlorophenols became subject to degradation upon mutational
activation of the expression of the phenol hydroxylase of Pseudomonas sp. B13 (redrawn
from [45])
The crucial reaction in the microbial degradation of halogenated compounds
is the removal of the halogen substituent. This is reflected by the large number
of review articles dealing with bacterial dehalogenation [5881]. Carbon-halo-
gen bonds are either cleaved enzymatically or by chemical dehalogenation.
Four widespread enzymatic dehalogenation reaction types will be discussed
here:
1. In nucleophilic substitution reactions, the halogen is displaced by an other
nucleophile, such as the hydroxyl ion OH
in the course of hydrolytic deha-

logenation.
2. During dehydrodehalogenation a halogen is removed together with an adja-
cent hydrogen resulting in the formation of a double-bond.
3. Oxidative dehalogenation is often achieved by monooxygenase or dioxygen-
ase activity replacing the halogen by a hydroxyl group, the oxygen of which
originates from molecular oxygen.
4. Reductive dehalogenation comprises hydrogenolytic dehalogenation, dihalo-
elimination, coupling of halogenated molecules, and hydrolytic reduction
[8284].
2.2.1.1
Nucleophilic Substitution of Halogen
In hydrolytic dehalogenation reactions, the halogen substituent is displaced
through a hydroxyl group which is derived from water. The oxidation state of
the reacting molecule remains unchanged (Fig. 8A). Hydrolytic dehalogenation
of halo-benzoates seems to be very common, and has been shown to act on flu-
orinated, chlorinated, brominated, and iodinated compounds. Under anaerobic
conditions halobenzoates are often activated with coenzyme A (CoA) prior to
hydrolytic dehalogenation [62]. Dechlorination of dichloroethane by Xantho-
bacter autotrophicus GJ10 was achieved by two hydrolytic reactions, the first
Fig. 8A, B. Nucleophilic substitution of halogen from: A 1,1-dichloroethane by hydroxyl [85];
B dichloromethane by reduced glutathion and hydroxyl [86, 87]
forming 2-chloroethanol and the second dechlorinating chloroacetic acid to
glyoxylate [85]. Dechlorination of dichloromethane by Hyphomicrobium sp.
strain DM2 involved the replacement of the first chlorine atom through reduc-
ed glutathion (thiolytic dehalogenation) followed by hydrolytic removal of the
second chlorine atom (Fig. 8B) [86, 87]. Subsequently, the reduced glutathion
was recovered and formaldehyde was formed. An example of a hydrolytic de-
chlorination not involving cofactors is the enzymatic removal of chlorine from
S-triazine deethylsimazine by Rhodococcus corallinus [88]. Chloride is non-en-
zymatically eliminated from intermediates of the ortho cleavage pathway of
chlorocatechol degradation (see also Fig. 6). Muconate cycloisomerase II con-
verts 2-chloro-cis,cis-muconate and 3-chloro-cis,cis-muconate, respectively, to
the corresponding 4-carboxychloromethylbut-2-en-4-olides which spontane-
ously release HCl to give the trans and cis isomers of 4-carboxymethylbut-2-en-
4-olide, respectively (for overview see [78]). The overall reaction is an intra-
molecular substitution of halogen by hydroxyl.
2.2.1.2
Dehydrodehalogenation
In dehydrodehalogenation halogen is removed together with a hydrogen atom
from an adjacent carbon resulting in the formation of a double bond (Fig. 9).
Pseudomonas paucimobilis UT26 dehydrodechlorinated g-HCH via penta-
chlorocyclohexene and, possibly, via the unstable metabolite 1,3,4,6-tetra-
chloro-1,4-cyclohexadiene to 1,2,4-trichlorobenzene [89, 90]. Eukaryotic orga-
nisms were shown to dehydrodehalogenate DDT to DDE [91].
2.2.1.3
Oxidative Dehalogenation
None of the previously described reaction mechanisms involved molecular oxy-
gen. Hydroxylation of pentachlorophenol to tetrachloro-p-hydroquinone by
Arthrobacter sp. and Flavobacterium sp. depended on NADPH and oxygen
(Fig. 10A). Labeling experiments revealed that the oxygen of the hydroxyl ori-
ginated from molecular oxygen [92]. The broad substrate range of the pentach-
lorophenol 4-monooxygenase allowed the removal of halogen, nitro, amino,
and cyano groups. Oxygenolytic dehalogenation was also found in the course of
the dioxygenation of halogenated aromatic compounds. In the degradation of
1,2,4,5-tetrachlorobenzene by Pseudomonas sp. strain PS14, initial dioxygena-
Fig. 9. Stepwise dehydrodehalogenation of g-hexachlorocyclohexane to 1,2,4-trichloroben-
zene (redrawn from [89, 90])
tion at C5 and C6 is followed by rearomatization due to the release of hydro-
chloric acid yielding 3,4,6-trichlorocatechol (Fig. 10B) [93]. The oxidation of
trans-1,2-dichloroethylene by a culture of methanotrophic bacteria involved
the spontaneous release of chloride from the intermediately formed epoxide
[94] (Fig. 10C).
2.2.1.4
Reductive Dehalogenation
Four different mechanisms of reductive dehalogenation have been distinguish-
ed (Fig. 11) [82]. First, in hydrogenolytic dehalogenation, the halogen substi-
tuent is replaced by hydrogen at the expense of reduction equivalents [74]. This
reaction is therefore mainly found under anaerobic conditions, although hy-
drogenolytic dehalogenation by facultatively anaerobic microorganisms has
been reported [95]. Aromatic and aliphatic compounds can be subject to hy-
drogenolytic dehalogenation. Hydrogenolytic dechlorination of aromatic com-
pounds has been proposed for various chlorophenols including pentachloro-
phenol, 2,4,5-trichlorophenoxyacetic acid, polychlorinated benzenes, polychlo-
rinated biphenyls, and polychlorinated dibenzo-p-dioxins [17, 18, 26, 9698].
The redox potentials of chlorinated aliphatic or aromatic hydrocarbon couples
range between E0 = 0.3 V and 0.5 V. Thus, from the energetic point of view, the
Fig. 10AC. Different types of oxidative dechlorination: A monooxygenation of pentachloro-
phenol yielding tetrachloro-p-hydrochinone [93]); B dioxygenation of 1,2,4,5-tetrachloro-
benzene yielding 3,4,6-trichlorocatechol [93]; C epoxide formation by monooxygenation of
trans-1,2-dichloroethylene followed by spontaneous dechlorination [94]
utilization of chlorinated hydrocarbons as electron acceptors is possible [99,
100]. Accordingly, hydrogenolytic dechlorination of perchloroethylene by the
strictly anaerobic bacterial isolates Dehalobacter restrictus and Desulfitobac-
terium sp. strain PCE1 and of 3-chlorobenzoate by the sulfidogenic bacterium
Desulfomonile tiedjei DCB-1 was found to be coupled to chemiosmotic energy
conservation [65, 99101]. The halogenated compounds are used as the termi-
nal electron acceptor for the growth of these organisms. In the course of the me-
tabolism of 2,4-dichlorophenoxyacetic acid by the aerobic Alcaligenes eutro-
phus JMP134, the NADH dependent maleylacetate reductase was shown to hy-
drogenolytically dehalogenate 2-chloromaleylacetate. Although dehalogenation
generally decreases the xenobiotic character of a chemical, in some cases it may
form more hazardous chemicals than the educts [102]. The hydrogenolytic de-
Fig. 11. Different types of reductive dehalogenation (redrawn from [82])
chlorination of tetrachloroethylene to vinylchloride is an example of such an
undesired reaction [103]. Second, in dihalo-elimination, halide is removed and
an additional carbon to carbon bond is formed. Dichloro-elimination from
dichloroethane by methanogenic bacteria yielded ethene. g-Hexachlorocyclo-
hexane (lindane) was dihalodechlorinated to g-tetrachlorocyclohexene [104,
105]. Third, coupling of two chloromethane molecules resulted in reductive de-
halogenation and the concomitant formation of ethane [82]. Fourth, the oxida-
tions of tetrachloromethane and 1,1,1-trichoroethane to CO
2
and acetate, re-
spectively, were found to be initiated by two electron reductions, followed by
hydrolysis [106].
2.2.2
Nitro Groups
2.2.2.1
Nitroaromatic Compounds
The use of nitroaromatic compounds as explosives, pesticides, and dyes, and as
intermediates in many chemical syntheses led to their entrance in all major
compartments of the environment. Since organic nitro compounds of natural
origin are very rare, virtually all nitroaromatic compounds nowadays found in
aqueous systems [107, 108], terrestrial systems [109, 110], and the atmosphere
[111, 112] were formed by human activities. Nitroaromatic compounds may be
toxic and/or mutagenic to microorganisms, plants, animals, and humans. The
explosive 2,4,6,trinitrotoluene (TNT), one of the most abundantly produced
nitro compounds, has been shown to cause anemia in humans [113]. The muta-
genicity of airborne 3-nitrodibenzofuran was shown to be three times stronger
than that of benzo(a)pyrene [114].
In spite of their xenobiotic character, microorganisms have developed en-
zymatic mechanisms to degrade nitro compounds, i. e., to convert or release the
nitro group prior to further metabolism. A number of recent reviews deal with
the degradation of nitroorganic compounds [115121]. In the following, the
fate of the nitro group will be used for a classification of degradation mecha-
nisms.
Oxidative Release of the Nitro Group. The degradation of a number of nitro-
benzenes, nitrotoluenes, and nitrophenols is initiated by an NADPH- or FAD-
dependent monooxygenase attack of the aromatic ring that replaces the nitro
group by hydroxyl and results in the release of nitrite (Fig. 12A) [122, 123].
Dioxygenolytic attack liberated nitrite from compounds such as 2,4,5-trichlo-
ronitrobenzene, 3-nitrobenzoic acid, and nitrobenzene, and resulted in the for-
mation of the corresponding catechols [93, 124, 125].
Reductive Removal of the Nitro Group. Nitrite can also be reductively removed
from the aromatic ring (Fig. 12B). The nucleophilic attack of picric acid result-
ed in the transient formation of a hydride-Meisenheimer complex, which re-
aromatized by releasing nitrite [126].
Reduction of the Nitro Group. The nitro groups are reduced prior to the clea-
vage of the carbon-nitrogen bonds of nitrobenzoic acids, nitrotoluenes, and
nitrophenols (for an overview see [117, 119]). The reduction is catalyzed by
nitroreductases and proceeds via a nitroso and a hydroxylamino to an amino
group (Fig. 12C). The oxygenolytical removal of the amino group yields the cor-
responding catechol as the substrate for further metabolism. Recently, in the
course of the degradation of 4-nitrobenzoate, nitrobenzene, and nitrotoluene,
the hydrolytic removal of the hydroxylamino group from the aromatic nucleus
yielding the corresponding catechol has been described [127129]. The general
observation that the ability to reduce the nitro group of nitroaromatic compo-
unds is much more common than the utilization of the products has been at-
tributed to the broad substrate ranges of many nitroreductases. The reduction
of various substituted nitrobenzenes to the corresponding amino compounds
in anaerobic aquifer material occurred by a reaction with surface bound iron
species, which served as mediators for the transfer of electrons originating from
the oxidation of organic material by iron-reducing bacteria [130].
Aromatic amino compounds tend to form complexes with the humus frac-
tion of soils. There is an ongoing discussion as to whether such complexes may
be regarded as stable and, as a consequence, desirable deposits of amino aro-
matic compounds or represent reservoirs of remobilizable hazardous metabo-
lites [131].
Fig. 12AC. Mechanisms of nitrogen removal from nitroaromatic compounds: Aoxidative re-
lease of the nitro group from 4-nitrophenol yielding p-hydroquinone [122]; B reductive re-
moval of the nitro group from picric acid involves the formation of a hydride-Meisenheimer
complex [126]; C reduction of the nitro group prior to oxidative deamination (redrawn from
[119])
2.2.2.2
Nitrate Esters
Nitrate esters are abundantly used in ammunition and for the therapy of car-
diovascular diseases. Up to now, no naturally occurring nitrate esters are
known. Despite the strict xenobiotic character, the hydrolytic microbial cleav-
age of the nitrate ester bonds of the explosives glycerol trinitrate, ethylene gly-
col dinitrate, pentaerythritol tetranitrate, cellulose nitrate, and the pharmaceu-
tical isosorbide 2,5-dinitrate, and the concomitant liberation of nitrite, have
been shown [118, 121].
2.2.3
Sulfonic Acid Groups
Sulfonated organic contaminants are very rare in nature. Large amounts of
these compounds arise as lignosulfonates in the course of the industrial pro-
duction of paper from wood, as surface active alkylarylsulfonates, and as
amino- and hydroxynaphthalenesulfonic acids which serve as building blocks
in the synthesis of azo dyes. It appears that the removal of the bulky sulfonate
group is the first step in the degradation of many sulfonated compounds.
Methanesulfonic acid degradation by marine microorganisms was initiated by
NADH-dependent monooxygenase reaction yielding formaldehyde and sulfite
[132]. The degradation of naphthalene-2-sulfonic acid and naphthalene-2,6-di-
sulfonic acid was found to be initiated by the dioxygenolytic removal of a sul-
fonate group as sulfite (Fig. 13) [46]. In the latter case, the resulting sulfonated
dihydroxynaphthalene is channeled into the naphthalene metabolism. The re-
maining substituent is removed later in the metabolism by hydroxylation of 5-
sulfosalicylic acid yielding gentisic acid [133]. Several bacteria are able to use
arylsulfonates as the sole sources of sulfur. Pseudomonas putida S-313 [134]
converted a number of structurally diverse sulfonated aromates, including sur-
factants and azo dyes, to the corresponding phenols. The resulting molar yield
of biomass was as high as with sulfate.
Fig. 13. Oxygenolytic desulfonation of naphthalene-2-sulfonic acid [46]
2.2.4
Organophosphonates
Organophosphonates are a class of compounds possessing one or more carbon-
phosphorus (C-P) bonds. C-P bonds are chemically stable and withstand hy-
drolysis, thermal decomposition, and photolysis [135]. Organophosphonates
are chemicals of environmental concern. Although their toxicity has long been
known, they are widely used as herbicides, insecticides, lubricant and polymer
additives, corrosion inhibitors, and antibiotics [136]. Organophosphonates are
readily catabolized and utilized as source of carbon, phosphorus, or nitrogen by
a large number of bacterial and fungal isolates [138140]. There are a number
of reports on the partial degradation of the organophosphonate molecule prior
to the cleavage of the C-P bond [141, 142]. In a recent report, Bujacz et al. [140]
distinguish three enzymatic mechanisms of C-P bond cleavage (Fig. 14): (i) the
hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate
by means of a phosphonatase [141] and (ii) the metal cation-assisted hydroly-
sis of phosphonoacetate to acetate and inorganic phosphate by means of the
phosphonoacetate hydrolase [137]; (iii) the hydrolysis of a broad spectrum of
organophosphonates by the so-called C-P lyases in fact comprises reductive
and oxidative pathways [143145].
2.3
Degradation of the Carbon Backbone
2.3.1
Aliphatic Compounds
Aliphatic compounds comprise a great number of straight-chain and branched
molecules including gases, liquids, and long-chain molecules which are solid at
physiological temperatures [146]. Many of these molecules serve as growth sub-
Fig. 14. Enzymatic reactions involved in the cleavage of carbon-phosphorus bonds (redrawn
from [140])
strates for a very wide variety of microorganisms [146152]. The low water so-
lubility of most aliphatic hydrocarbons is the major obstacle for the uptake of
these compounds. Microorganisms have therefore developed various mecha-
nisms to overcome substrate limitation. Some yeasts are known to take up hy-
drocarbon droplets [153], whereas many bacteria excrete emulsifying agents or
possess highly hydrophobic cell surfaces, mediating their permanent associa-
tion with hydrocarbon droplets [154158].
2.3.1.1
Aerobic Pathways
Aliphatic compounds from C
1
up to C
44
are subject to microbial degradation
[159]. The most readily degraded hydrocarbons are the straight chain alkanes
from C
10
to C
18
[151]. The principle strategy of the metabolism of aliphatic com-
pounds is to convert the alkane chains into fatty acids (Fig. 15). Under aerobic
conditions, the alkane is attacked by an NADH
2
-dependent monooxygenase (al-
kane-hydroxylase) system consisting of the terminal oxidase and two or three
electron transfer components. The monooxygenase forms a fatty alcohol by
transferring one oxygen atom of molecular oxygen onto the terminal C-atom of
the molecule. Another effective multicomponent enzyme known to catalyze this
reaction is the methane monooxygenase of methylotrophic bacteria [151].
Alcohol dehydrogenase further oxidizes the fatty alcohol to the corresponding
aldehyde, which is then oxidized to a carboxylic acid by means of an aldehyde
dehydrogenase. The carboxylic acid is channeled into the central metabolism
for further oxidation by b-oxidation. b-Oxidation is initiated by the formation
of a CoA-thioester of the fatty acid by means of the activity of ATP-dependent
acyl-CoA synthetase. The products of each b-oxidation cycle are reduction
equivalents and acetyl-CoA. b-Oxidation of C-odd fatty acids yields propionyl-
CoA in the last oxidation cycle which is carboxylated to methylmalonyl-CoA
and can enter the central metabolism after being isomerized to succinyl-CoA.
The aerobic degradation of alkenes can be initiated in various ways. Besides in-
itial terminal or subterminal oxygenation, the reactive double bond can be sub-
ject to epoxidation or hydroxylation [147]. Straight-chain aliphatic compounds
are generally more rapidly degraded than branched compounds, although com-
plete metabolism of a number of branched compounds has been reported.
Examples are the utilization of the isoprenoid pristane (2,6,10,14-tetramethyl-
pentadecane) and of 2,2,4,4,6,8,8-heptamethylnonane by various bacteria [160,
161]. The metabolism of alkynes, such as acetylene, by both aerobic and anaer-
obic bacteria is initiated by hydration of the triple bond [148]. For information
on the microbial degradation of cyclic aliphates, the reader is referred to re-
views by Trudgill [152] and Perry [162].
2.3.1.2
Anaerobic Hydrocarbon Degradation
Under anoxic conditions, oxygen is unavailable for the initial oxidation of al-
kanes and for a long time it was believed that alkanes (with the exception of
Fig. 15. Aerobic and anaerobic mechanisms of aliphatic hydrocarbon degradation (redrawn
from [84])
methane) are anaerobically recalcitrant [146, 163]. Recently, nitrate-reducing
and sulfate-reducing bacteria which were isolated from an oil production plant
were shown to grow anaerobically with straight chain alkanes as the sole car-
bon sources. Strains exhibited different specificities with respect to the range of
alkanes that could be utilized. A sulfate-reducing bacterium was found to grow
with short-chain alkanes (C
6
to C
13
), whereas nitrate-reducing strains were en-
riched under anoxic conditions with hexadecane as the substrate [164, 165].
One of the sulfate reducing isolates did not utilize alcohols, indicating that there
are no intermediates in alkane degradation. The pattern of total cell fatty acids
suggested that the alkane oxidation occurred via an elongation of the carbon
chain by a C
1
-unit [166]. The double bonds of alkenes can be hydrated anaerobi-
cally to form an alcohol (Fig. 15) [167]. The further conversion to a fatty acid is
then performed by oxygen-independent enzymes as in the aerobic pathway.
2.3.2
Aromatic Rings
Next to the glucosyl residues, the benzene ring, as one of the main constituent
of wood, is the most widely distributed structural unit in nature [168].
Understandably, many microorganisms have evolved enzymatic pathways to
make use of the gigantic source of carbon and energy provided by aromatic
compounds. The recycling of carbon bound in aromatic rings actually relies al-
most entirely on microbial activities. Nowadays, there is much concern about
anthropogenic aromatic bulk chemicals such as benzene, toluene, and xylene,
which are widely used as solvents [169] and polyaromatic hydrocarbons (PAH),
which were formed and unintentionally entered soils and groundwater during
manufacturing of combustible gases [170]. The thermodynamic stability of
benzene, substituted benzenes, and condensed aromatic rings caused by their
possession of a negative resonance energy, required the development of special
degradation mechanisms.
2.3.2.1
Aerobic Pathways
The main strategy of microorganisms to degrade aromatic pollutants aerobi-
cally is to use a range of peripheral enzymes which convert the substances to a
key intermediate, which is most often a (substituted) catechol (Fig. 16). This
strategy appears to be very economic, since it allows one to channel a large va-
riety of substrates into the same central catabolic pathway. Different mecha-
nisms of catechol formation exist in eukaryotes and prokaryotes (Fig. 17).
Eukaryotic organisms produce catechols from aromatic compounds by insert-
ing one atom of molecular oxygen by means of a monooxygenase yielding epox-
ides. Subsequent addition of water leading to trans-dihydrodiols is followed by
dehydrogenation. Prokaryotes introduce an entire oxygen molecule by a dioxy-
genase reaction forming a cis-dihydrodiol which is then subject to dehydroge-
nation. Aromatic hydrocarbons, such as benzene, naphthalene, higher condens-
ed polyaromatic compounds, and biphenyl are attacked in this way. Aliphatic
substituent groups on the benzene ring offer the possibility of alternative
modes of biodegradation; either side-chain attack or ring attack. It seems that
alkylbenzenes with chains of up to 7 C-atoms are preferably attacked at the
aromatic rings, whereas the initial oxidation of the alkyl chain is the preferred
attack when chain lengths exceed 7 carbon atoms [169]. However, the degrada-
tion of toluene is a well-examined example for the realization of both degrada-
tion pathways, the initial attack being either ring dioxygenation (Fig. 18A) or
the stepwise oxidation of the methyl group to benzoic acid as the substrate for
ring dioxygenation (Fig. 18B). In eukaryotes and in prokaryotes, catechol and
various substituted catechols are opened by dioxygenase reactions by either
ortho- (intradiol-) or meta- (extradiol-) cleavage.
The ortho-cleavage of catechol by catechol 1,2-dioxygenase activity yields
cis,cis-muconic acid (Fig. 19). Further breakdown via muconolactone and 3-
oxoadipate enol-lactone leads to 3-oxoadipate which enters the central metabo-
lism as succinate and acetyl-CoA. Halogenated aromatic compounds are most
often degraded in this manner, although recently a meta cleavage pathway for
4-chlorobenzoate has been reported [171]. The chlorines are eliminated after
ring cleavage depending on their position in the molecule during the cycloiso-
merization or even later in the breakdown (see also Fig. 6). The enzymes involv-
ed in the breakdown of chlorocatechols have broader substrate specificities
than the ordinary ortho cleavage pathway enzymes. An example is the modified
Fig. 16. Converging pathways of aromatic hydrocarbon degradation (redrawn from [84])
ortho pathway involved in the degradation of 2,4-dichlorophenoxy acid by
Ralstonia eutropha JMP134 (formerly Alcaligenes eutrophus) [172].
The meta-cleavage of catechol (Fig. 19) by catechol 2,3-dioxygenase brings
about 2-hydroxymuconic semialdehyde, which after decarboxylation to 2-oxo-
penta-4-enoate is cleaved into pyruvate and acetaldehyde. Alkylated and phenyl
substituted catechols are most often subject to meta cleavage [68, 169].
Alternative substrates for ring cleavage are the para-dihydroxylated aromat-
ic acids gentisic acid (Fig. 20) and homogentisic acid, which are cleaved be-
tween the carboxy group and the adjacent hydroxy group [173]. Homogentisic
acid is known as key intermediate in the catabolism of the aromatic amino
acids phenylalanin and tyrosin. The ring cleavage products of homogentisate
and gentisate enter the central metabolism as fumarate and acetoacetate, and
fumarate and pyruvate, respectively. Interestingly, none of the reactions follow-
ing ortho or meta cleavage of catechol or the cleavage of homogentisic and gen-
tisic acid requires oxygen. Further information on the aerobic biodegradation
Fig. 17. Different ways of catechol formation from mononuclear aromatic compounds by eu-
karyotes and prokaryotes (redrawn from [84])
Fig. 18A, B. Toluene biodegradation initiated by: A dioxygenation; B successive oxidation of
the methyl group (redrawn from [169])
1
9
0
T
.
N
.
P
.
B
o
s
m
a

e
t

a
l
.
Fig. 19. The ortho-(intradiol-) pathway and the meta-(extradiol-) pathway of catechol degradation
Fig. 20. The gentisate pathway of aromatic ring-cleavage
of aromatic compounds can be obtained from several of reviews and mono-
graphs [57, 168, 169, 173181].
2.3.2.2
Anaerobic Pathways
Anaerobic degradation of aromatic compounds is carried out by phototrophic
bacteria, by fermenting, manganese-reducing, iron-reducing, nitrate-reducing,
and sulfate-reducing bacteria, and by methanogenic consortia. The destabiliza-
tion of the aromatic ring prior to cleavage is achieved by reduction (Fig. 21).
The key intermediate in this pathway is cyclohexanone or one of its derivatives.
Ring opening proceeds through hydration of the cyclohexanone. As early as
1968 the intermediates of the anaerobic biodegradation of benzoic acid by
Rhodopseudomonas palustris were identified and a degradative pathway was
proposed [182]. Benzoic acid was found to be activated by with co-enzyme A
followed by complete reduction of the aromatic ring. The consecutive sequence
of dehydrogenation forming a double-bond in the a, b-position, hydration, de-
hydrogenation, and thiolysis resulting in ring cleavage follows the scheme of the
b-oxidation of fatty acids. The first linear product was pimelyldi-CoA. Phenol
reduction by nitrate-reducing enrichment cultures was observed by Bakker
[183]. Here caproate was the product of ring-cleavage. Nitrate-reducing
Pseudomonas strains exhibited phenol carboxylase activity forming 4-hydroxy-
benzoate, which after dehydroxylation to benzoate was channeled into the re-
Fig. 21. Reductive pathways of anaerobic aromatic hydrocarbon degradation (redrawn from
[84])
ductive pathway [184]. Catechol degradation by methanogenic consortia pro-
ceeded via dehydroxylation to phenol followed by reduction and ring cleavage
to give caproate or, alternatively, adipate. An oxidative initiation of the an-
aerobic degradation of toluene, phenol, and p-cresol was proposed by Lovley
and Lonergan [185]. They could show that the oxidation of the methyl group of
(substituted) toluene to the carboxy group and the further degradation of ben-
zoate was coupled to the reduction of Fe (III). The anaerobic biodegradation of
aromatic compounds has been dealt with in more detail in several review
articles [186191].
2.3.2.3
Fungal Metabolism
White-rot basidiomycetes exhibit the ability to transform a wide range of ali-
phatic and aromatic compounds including many environmental pollutants.
Most of the degradative mechanisms involve the activity of lignin peroxidase
and manganese peroxidase, the major components of the lignin-degradative
system [192, 193]. The non-specific nature of the peroxidases, which use hydro-
gen peroxide to achieve a one-electron oxidation of chemicals to free radicals,
allows them to attack complex mixtures of pollutants. However, primary growth
substrates such as cellulose and glucose are required for the fungal co-metabo-
lism of pollutants. The fact that peroxidases are generally excreted, allows the
transformation of insoluble and polymeric compounds, such as polycyclic aro-
matic hydrocarbons or lignin. Since the products of co-oxidation are often not
metabolized further by the fungi, mixed populations of fungi and bacteria are
usually required to achieve the complete mineralization of organic contami-
nants. Fungi might prove valuable should molecules which are not easily trans-
ported into bacterial cells or metabolized by bacteria have to be oxidized.
Detailed information on fungal metabolism is available in several articles, re-
views, and monographs [192201].
2.3.3
Ethers
The ether bond is a structural feature present in many natural compounds, such
as lignin and its degradation products. Synthetic ether compounds are used as
agrochemicals, such as the phenoxyalkanoate herbicide 2,4 dichlorophenoxy-
acetic acid (2,4-D), as additives, such as the antifreeze agent polyethylene glycol
(PEG), and as detergents. Ether compounds of considerable concern are the
highly toxic polychlorinated dibenzofurans (PCDF) and dibenzo-p-dioxins
(PCDD) which are unintentionally formed in various incineration processes
[202]. Due to its chemical stability, the ether linkage represents a major chal-
lenge to enzymatic attack. Microorganisms have, nevertheless, evolved a num-
ber of mechanisms resulting in ether cleavage. A common mechanism of
aerobic ether cleavage involves the insertion of oxygen into one of the carbon
atoms next to the ether bond by either monooxygenase [203, 204] or dioxygen-
ase activity [205]. This yields unstable hemiacetal structures which sponta-
neously dismutate into alcohol and aldehyde (Fig. 22A). Hydroxylation of the
adjacent C-atom can also be achieved by the addition of water to a C=C double
bond next to the ether linkage (Fig. 22B). Such a mechanism is involved in the
aerobic and the anaerobic metabolism of polyethylene glycol [16, 206]. A furth-
er common mechanism of ether cleavage is the oxidation of one of the ether
carbons to a keto group yielding an ester, which is subject to enzymatic hydro-
lysis (for an overview see [207]). Whereas all mechanisms discussed thus far
achieve ether cleavage by an initial destabilizing reaction, followed by a sponta-
neous C-O fission or ester hydrolysis, carbon-oxygen lyases represent a means
of direct ether cleavage by a b-elimination reaction as shown in Fig. 22C [208].
Fig. 22AC. Different mechanisms of ether cleavage: A destabilization of 4-chlorodiphenyl
ether by angular dioxygenation is followed by spontaneous cleavage [205]; B destabilization
of the ether bond by addition of water to an adjacent double bond [206]; C direct ether cleav-
age by a carbon-oxygen lyase [208]
2.3.4
Chiral Compounds
Many of the chemical compounds in use are chiral. Chirality refers to the sym-
metry properties of the molecules. A chiral molecule is not superimposable
(i. e., not identical) with its mirror image. In the most simple case of a molecule
containing one asymmetric carbon atom (a carbon atom connected to four dif-
ferent residues) there are two stereo-isomers, called enantiomers. Many chem-
icals in agricultural and medical use are marketed as racemates, i. e., mixtures
of equal amounts of both enantiomers. A review by Arins gives a good ac-
count of the manifold occurrence of racemates among pesticides and drugs
[209]. Other chiral compounds of environmental relevance are the linear alkyl-
benzene sulfonates (LAS) which are constituents of commercial detergents,
with an annual production of 12 million tons [210]. Enantiomers have iden-
tical chemical and physical properties only in a symmetrical environment.
Enzymes, however, are generally asymmetrical, due to their composition of
chiral amino acids. Compared with the quantitative importance of racemates,
relatively few studies considered the environmental degradation of chiral com-
pounds [211]. There are many examples found in the literature of the enantio-
selective degradation of racemates, i.e., the preferred usage of one of the enan-
tiomers [212216]. A recent investigation of the complete microbial degrada-
tion of the racemate of the herbicide mecoprop [(RS)-2-(4-chloro-2-
methylphenoxy) propionic acid] revealed, that both enantiomers were degrad-
ed by different mechanisms [217]. These reports clearly indicate the necessity to
treat enantiomers separately with respect to environmental degradation studies
and environmental regulations.
2.3.5
Complexing Agents
Complexing aminopolycarboxylates such as nitrilotriacetic acid (NTA) and
ethylenediaminetetraacetic acid (EDTA) form water-soluble metal complexes.
They are used in household detergents to inhibit the formation of insoluble
Ca
2+
and Mg
2+
salts from tensides or carbonate. Nowadays, these compounds
substitute phosphate-based complexing agents which had been found to cause
the world-wide eutrophication of rivers and lakes [218]. NTA is readily degrad-
ed in laboratory cultures by several taxonomically different bacteria as well as
during waste water treatment [219]. Aerobic primary biodegradation involves
the cleavage of NTA by the oxygen and NADH-dependent monooxygenase giv-
ing iminodiacetic acid and glyoxylate (Fig. 23). Interestingly, the same products
are formed under denitrifying conditions by the NTA dehydrogenase [219].
EDTA, in contrast, has been found to persist in wastewater treatment plants, ri-
vers, and in aerobic groundwater infiltration zones [220222]. The recal-
citrance of EDTA in natural environments is of considerable concern since the
effective metal-binding properties are suspected to have undesirable environ-
mental consequences such as the remobilization of heavy metals from river se-
diments [223].
2.4
Recalcitrance
Chemicals that resist biodegradation are known as recalcitrant molecules.
Alexander listed several examples of anthropogenic compounds which per-
sisted in soils for between 3 and more than 20 years [57]. Recalcitrance may be
the result of adverse environmental factors preventing the degradation of a che-
mical. This implies that changes of the environment may turn formerly recal-
citrant chemicals readily biodegradable. Polychlorinated biphenyls (PCBs) and
polychlorinated benzenes carrying more than seven and four chlorine atoms,
respectively, are recalcitrant in the presence of oxygen. However, under an-
aerobic conditions they are readily dechlorinated to biphenyl and benzene
which, in turn, persist under anaerobic conditions, but readily degrade under
oxic conditions. Recalcitrance of highly chlorinated aromates may therefore be
overcome either by environmental conditions changing between anoxic and
oxic or by the transport of intermediates from anoxic to oxic zones.
Recalcitrance in the strict sense refers to inherently non-biodegradable che-
mical structures. Several reasons for the intrinsic recalcitrance of molecules
have been suggested [57]: (i) Enzymes able to attack chemicals of strong xeno-
biotic character may not exist. This may for instance explain the considerable
resistance of highly branched molecules and molecules containing bulky sub-
stituents, which may mask potential sites for enzymatic cleavage. (ii) Uptake
systems allowing the intracellular degradation of the chemicals may not exist.
Fig. 23. Reaction scheme of the degradation of nitrilotriacetate (NTA) (adapted from [230])
The considerable recalcitrance of most polymers can be at least partly attribut-
ed to the impracticability of the uptake in combination with the non-existence
of extracellular enzymes. The observed recalcitrance of polyesters and nylon in
contrast to the ready degradability of the respective oligomers is a strong indi-
cation for such a mechanism of recalcitrance [39]. (iii) Regulatory proteins able
to recognize the chemicals and induce the enzyme synthesis may not exist. This
is understandable in terms of the necessity to reconcile the evolution of degra-
dative enzymes with the parallel evolution of appropriate regulator proteins
[21]. An example of the lack of such a parallel evolution is the aerobic metabo-
lism of PCBs which requires the presence of biphenyl or monochlorobiphenyls
as an inductor of the degradation pathway [68, 224]. (iv) The intrinsic toxicity
of the molecule may, in case of an unspecific mechanism of action, affect all
microorganisms with degradative potential. The recalcitrance of tetralin may at
least partly be attributed to its accumulation in cell membranes, causing mem-
brane expansion and adversely affecting membrane function [57, 225]. Bacteria
were found to overcome such mechanisms of recalcitrance by specific adapta-
tion. For instance, the solvent tolerances of Pseudomonas putida strains were
mediated by changed membrane compositions and modified lipopolysacchari-
des [226]. In some cases not the anthropogenic pollutants but metabolites resist
biodegradation. The poor effectiveness of the cometabolic degradation of PCBs
in the environment has recently be attributed to the fortuitous formation of the
broad spectrum antibiotic protoanemonin from 4-chlorocatechol by the natur-
al microflora via the widespread ortho cleavage pathway of catechol [227].
3
Concluding Remarks
Microbial transformation is required to achieve detoxification of organic con-
taminants that accumulate in soil and groundwater. Microorganisms can there-
fore be viewed as a sub-population of the decomposers with a special function,
namely detoxification of the environment. Microbial processes have been used
in the fields of waste water technology, the clean-up of polluted air streams in
biofilters and in soil and groundwater remediation. The effectiveness of micro-
bial processes in soil and groundwater can be reduced by the relative immobi-
lity of organic compounds in the soil matrix where microorganisms live.
Pollutants which are immobilized in the soil due to sorption are considered to
be unavailable for biodegradation. Other reasons for a limited bioavailability in
soil can be the presence of the compounds as pure solids or liquids as is often
the case at former gas manufacturing plants [170] or the formation of bound re-
sidues due to chemical binding to organic matter [228].
The relative availability of a compound can be expressed in a bioavailability
number Bn which simply is the ratio of the rate of mass transfer to the degrad-
ing organisms to the rate of biodegradation [229]. Bn expresses control of bio-
degradation by mass transfer at values less than unity and control by microbial
activity at values greater than unity [229]. A lot of effort in the past has been put
into resolving the limitations to biodegradation caused by mass transfer from
sorbed phases or pure solids or liquids. There has been a trend recently to ad-
apt the strategy in bioremediation technologies to the limited availability of
pollutants rather than try to speed up the remediation to faster than the mass
transfer rates. This approach has been facilitated by the acceptance that classi-
cal clean-up goals (for instance, the Dutch list) can only be reached through
costly in terms of money and energy ex situ treatment technologies of pol-
luted soil. Thus, it became possible to design approaches that are adapted to the
limitations set by mass transfer. An excellent example of such a remediation
strategy is intrinsic bioremediation or natural attenuation. This strategy makes
use of intrinsic degradation processes for the remediation of polluted soil and
groundwater. The attenuation of the pollutants is the overall result of physical,
chemical, and biological removal processes. An important clue to natural at-
tenuation is that the technology is adapted to the limited availability of pol-
lutants for biodegradation. Thus, the realm of biodegradation pathways as de-
scribed in the previous pages becomes available for the solution of the prob-
lems arising from environmental pollution with man-made chemicals, especially
in soil and groundwater.
From an ecological stand-point, it can be argued that production and release
rates of toxicants have to be smaller than in situ biotransformation rates to keep
environmental pollution within acceptable limits. Treatment as close to the
source as possible during the manufacturing and use of chemicals will be an
important strategy to reach such a goal. The use of pesticides should be regula-
ted such that the amount applied in a growth season is completely transformed
in situ in the same season.
4
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Dordrecht
Protozoa in Wastewater Treatment:
Function and Importance
Wilfried Pauli
1
, Kurt Jax
2
, Sandra Berger
1
1
Institut fr Biochemie und kotoxikologie, Freie Universitt Berlin, Ehrenbergstr. 2628,
D-14195 Berlin, Germany, E-mail: wpauli@zedat.fu-berlin.de
2
Zentrum fr Ethik in den Wissenschaften, Universitt Tbingen, Keplerstrasse 17,
D-72074 Tbingen, Germany
Protozoa constitute a major link between the highly productive and nutrient retaining micro-
bial loop and the metazoans of the classical food web. Protozoa are efficient at gathering
microbes as food, and they are sufficiently small to have generation times that are similar to
those of the food particles on which they feed. They are, in quantitative terms, the most im-
portant grazers of microbes in aquatic environments, balancing bacterio-plankton produc-
tion. Protozoa not only play an important ecological role in the self-purification and matter
cycling of natural ecosystems, but also in the artificial system of sewage treatment plants. In
conventional plants ciliates usually dominate over other protozoa, not only in number of spe-
cies but also in total count and biomass. It is generally accepted that their feeding on bacteria
improve the treatment, resulting in a lower organic load in the output water of the treated
wastes. Due to their biodegradation potential some attempts have been made to use ciliates
specifically in environmental biotechnology. As biosensors they could provide valuable infor-
mation regarding adverse effects of environmental chemicals on this part of the biocoenosis
essential for the effective operation of biological waste-water treatment processes.
Keywords. Protozoa, Ciliates, Ecology, Sewage treatment, Environmental biotechnology
1 Ecological Role of Aquatic Protozoa with Special Regard
to Ciliates Within the Microbial Food Web . . . . . . . . . . . . . 205
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
1.2 Traditional Food Webs and Microbial Food Webs . . . . . . . . . . 205
1.3 The Role of Protozoa in Aquatic Food Webs . . . . . . . . . . . . . 208
1.4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
2 Protozoa in Wastewater Treatment . . . . . . . . . . . . . . . . . . 212
2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
2.1.1 Wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
2.1.2 Biological Treatment Processes . . . . . . . . . . . . . . . . . . . . 214
2.1.3 Bacterial Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.1.4 Activated Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.2 Protozoa in Biological Wastewater Treatment Plants . . . . . . . . 217
2.2.1 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
2.2.2 Species Composition . . . . . . . . . . . . . . . . . . . . . . . . . . 218
2.2.3 Plant Specific Basic Communities . . . . . . . . . . . . . . . . . . . 220
2.2.4 Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
2.2.5 Ecological Framework . . . . . . . . . . . . . . . . . . . . . . . . . 221
2.2.5.1 Sludge Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
CHAPTER 3
(ed. by B. Beek)
2.2.5.2 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.2.5.3 pH-Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.2.5.4 O
2
-Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2.3 Significance of Protozoa for Wastewater Treatment . . . . . . . . . 225
2.3.1 Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.3.2 Reduction and Elimination of Suspended Particles and Bacteria . 227
2.3.2.1 Clearing Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
2.3.2.2 Experimental Findings . . . . . . . . . . . . . . . . . . . . . . . . . 228
2.3.2.3 Field-Observations . . . . . . . . . . . . . . . . . . . . . . . . . . 231
2.3.3 Elimination of Dissolved Substances . . . . . . . . . . . . . . . . . 232
2.3.4 Flocculation and Composition of the Bacterial Community . . . . 232
2.3.5 Reduction of the Total Biomass . . . . . . . . . . . . . . . . . . . . 235
2.3.6 Influence of Protozoa on Bacterial Metabolism . . . . . . . . . . . 237
2.3.7 Filamentous Bacteria and Protozoa . . . . . . . . . . . . . . . . . . 239
3 Impairments of Protozoa: Consequences for Water Purification . 241
4 Environmental Biotechnological Aspects . . . . . . . . . . . . . . 243
4.1 Biodegradation Potentials of Ciliates . . . . . . . . . . . . . . . . . 243
4.2 Ciliates as Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . 245
5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
List of Abbreviations
BOD
(5)
biological oxygen demand (index: within 5 days)
COD chemical oxygen demand
dw dry weight
f
m
sludge loading [g BOD (g MLSS day)
1
or g BOD (g MLVSS day)
1
],
also known as food to micro-organism (F/M) ratio
F/M-ratio see f
m
MLSS Mixed-liquor suspended solids, sludge solids (g m
3
; concentration
of the suspended solids in an aeration tank including inorganic mat-
ter)
MLVSS Mixed-liquor volatile suspended solids (g m
3
; corresponds to the or-
ganic, i. e., combustible content of the sludge, which amounts to ca.
70% of the sludge solids: 0.7 MLSSMLVSS; this parameter is often
used as indicator of microbial concentration, although it does not
distinguish between biochemically active material and inert or dead
material in the sludge)
EC/LC
50
50% effective and lethal concentration, respectively
204 W. Pauli et al.
1
Ecological Role of Aquatic Protozoa with Special Regard
to Ciliates Within the Microbial Food Web
1.1
Introduction
There is hardly any place on earth in which protozoa cannot be found. They are
abundant in terrestrial as well as in aquatic systems. In the latter they are pre-
sent in high numbers of species and individuals both in the oceans and in fresh-
water habitats. Some taxa live attached to solid substrates or within the sedi-
ment, some as part of the plankton. An overview of the data about the abun-
dance of protozoa in aquatic habitats gives a first indication that these organisms
are not negligible in aquatic environments although in fact they are still often
neglected. In the plankton of highly productive lakes, densities of small flagel-
lates (<20 mm body size) of more than 10
6
cells per ml were reported [1] and in
studies on the periphyton of small bodies of waters maximum values of more
than 1350 cells per cm
2
of the much larger testate amoebae specimens were en-
countered [2]. However, these numbers do not make any statements about the
ecological interactions in which the species are involved and the role they play
within those processes which mostly are seen as the essence of ecosystem dy-
namics, namely the fluxes of energy and material. It is the objective of this pa-
per to provide a short introduction to the current knowledge of these roles as
regards aquatic environments.
1.2
Traditional Food Webs and Microbial Food Webs
Traditionally, food webs in aquatic systems were illustrated as in Fig. 1. Going
back to the limnologist August Thienemann, the different species within a body
of water were characterized by the categories of producers, consumers of differ-
ent order (primary consumers, secondary consumers and so on) and decom-
posers [3]. The latter live on the dead organic matter and mineralize the organ-
ic compounds to inorganic nutrients, e. g., phosphorus, nitrogen, etc. These ca-
tegories were also the basis on which Raymond Lindeman [4] built his famous
trophic dynamic concept of ecology which was the first implementation of
Arthur Tansleys ecosystem concept [5]. Energy enters the system as light and is
processed as organic matter along the food chain or food web until most of the
energy is dissipated by respiration.
In aquatic habitats these functional categories trophic levels in Lindemans
parlance were commonly attributed to phytoplankton (producers), zooplank-
ton (primary consumers), and different kinds of vertebrates on the higher
trophic levels. Protozoa and particularly bacteria were seen as decomposers,
mainly restricted to sediments and other surfaces, but of minor importance in
the pelagic food web.
This association of bacteria and protozoa with decaying matter was recog-
nized and used for applied purposes rather early. Protozoa were used as bioin-
Protozoa in Wastewater Treatment: Function and Importance 205
dicators for the saprobic states of natural and manmade freshwaters as early as
1908 (e. g., [7, 8]). Their dynamics in the process of decomposition of organic
substances were clarified by the middle of the century. Meanwhile, classical stu-
dies on this topic were made by Bick and co-workers (e. g., [9, 10]) who investi-
gated the succession of micro-organisms, in particular ciliated protozoa, in the
course of the self-purification of water enriched with sewage and other organ-
ic substances.
However, during the last two decades there have been some new insights
which have broadened and fundamentally changed our way of looking at the
water of lakes and oceans and which affect the role protozoa and other micro-
organisms are supposed to play within aquatic systems. These insights were in-
itiated by the appearance of some new actors on the stage of the ecological thea-
ter which also radically changed the roles in which protozoa were perceived. In
1974 Pomeroy [11] presented a paper in which he developed new ideas about
the interactions of the pelagic organisms. Although these ideas were first devel-
oped in connection with marine systems they were soon transferred to fresh-
water habitats. The main point made is that, besides and connected with the
classical macroscopic food web, there exists a microbial food web. The reason
why these microbial food webs were discovered so late can, to a high degree, be
attributed to the development of new methods in aquatic ecology.
206 W. Pauli et al.
Fig. 1. Diagram of the classical food web in lakes. Modified, according to [6]
By the early 1970s it was recognized that an important part of the pelagic or-
ganisms had been neglected as a result both of the methods used and of the
theories regarding interactions in the water. Using direct counts of bacteria with
epifluorescence methods instead of plate counts, it turned out that the abun-
dance of bacteria in the open water had been underestimated by orders of mag-
nitude. Only 0.11% of the actual abundance had been counted [12]. Fur-
thermore, most investigations of marine and freshwater plankton used plank-
ton nets with a mesh size of 20 mm or even 60 mm, while all smaller organisms
were thought to be of minor importance. Finally, the methods of conserving
planktonic protozoa were inadequate and even larger protozoa were neglected
or underestimated as components of the pelagic species assemblages [13].
What was collected and counted were those fractions of the plankton which we
now call the micro- and macroplankton, i.e., organisms bigger than 20 mm
(Table 1).
Thus, not only all smaller organisms, the pico- and nanoplankton consist-
ing of bacteria, Cyanobacteria, small protozoa, and small eukaryotic algae [14]
but also many larger protozoans were to a large extent excluded from the
quantitative sampling. However, it turned out that especially this small sized
fraction of the plankton is of extreme importance in terms of energy- and ma-
terial fluxes. New measurements revealed that the major part of the metabolic
activity in plankton was displayed by the size fraction below 10 mm [15]. The
most productive component of the pelagic food webs was not, as thought ear-
lier, the planktonic eukaryotic algae of the microplankton, but the tiny
Cyanobacteria, mostly of the genus Synechococcus, and some small eukaryotic
algae. The percentage of primary production in terms of carbon varies between
1% and 90% in marine waters with higher ratios in more oligotrophic condi-
tions and 1670% in fresh waters [16]. For oligotrophic lakes 5070% are do-
cumented, while the autotrophic picoplankton amounts to 1045% of the total
phytoplankton biomass (standing stock, measured as chlorophyll) [17]. Data for
marine habitats give estimates of 2080% [18]. Similarly, the abundance of he-
terotrophic picoplankton, i. e., heterotrophic bacteria, is much higher than pre-
viously thought and can approach 10
9
cells in highly eutrophic fresh waters [1].
However, the new theory incorporates some new links rather than just adding
picoplankton to the classical food web. Figure 2 presents a very simple diagram
of what a microbial food web might look like, given the current status of knowl-
edge.
Table 1. The size classes of planktonic organisms
Picoplankton Nanoplankton Microplankton Macroplankton
0.22 mm 220 mm 20200 mm >200 mm
Bacteria Algae Algae Ciliates
Cyanobacteria Flagellates Rhizopods Rotatoria
Algae Rhizopods Ciliates Crustaceans
Flagellates Ciliates Rotatoria Fish larvae
Ciliates Nauplii
The earlier food chain from algae via macrozooplankton to fish still exists
but is supplemented by a new section which is commonly called the microbial
loop. This consists of the picoplankton (algae, i. e., Cyanobacteria and he-
terotrophic bacteria), protozoa, and a compartment of non-living material, i. e.,
dissolved organic matter (DOM). DOM is lost and excreted in substantial
amounts by both algae and Cyanobacteria and constitutes the energy source for
the heterotrophic bacteria. The rate of fixed carbon lost by phytoplankton cells
may vary between 10% and 40% depending on the physiological status of the
cells [13]. The picoplankton is grazed by protozoa which themselves are preyed
upon by the metazoan zooplankton, thus coupling the microbial loop to the tra-
ditional parts of the food web. As cells with a size of up to 2 mm hardly get lost
through sedimentation, the microbial loop not only adds some new links to the
classical food web but keeps the nutrients (DOM and inorganic nutrients) with-
in the water body and minimizes losses to the deeper, non-productive regions
of the waters or even the sediment. This seems to be particularly important dur-
ing the summer stratification of oligotrophic lakes, in which the epilimnion, the
upper and photosynthetically active region of the lake the euphotic zone is
temporarily cut off from the richer nutrient supply of the deeper waters [17].
1.3
The Role of Protozoa in Aquatic Food Webs
From this scheme the new role of protozoa within the food webs of aquatic sys-
tems seems obvious. They are not only in the same way as bacteria decom-
posers associated with the decay of organic material, but they are a link between
208 W. Pauli et al.
Fig. 2. The food web of the lake plankton. The classical food chain (open circles) is supple-
mented by the elements of the microbial loop (filled ovals and square). DOM: dissolved or-
ganic matter
the highly productive and nutrient retaining microbial loop and the metazoans
of the classical food web. Most microplankton organisms are unable to utilize
particles smaller than 5 mm directly [18]. Protozoa repack the organic mate-
rial into edible portions and thus make it available to crustaceans, rotatoria, and
other metazoans. There is empirical evidence that planktonic protozoa graze ef-
fectively on picoplankton and also that protozoa constitute a valuable diet for
crustaceans [19]. Thus both necessary links between picoplankton and metazoa
have been established.
The details of the microbial webs, however, are still the subject of research
and discussion. The specific pathways and the number of steps over which
energy and nutrients are transferred are subject to much variation. There is
temporal variation, e. g., seasonally, [20] and there is spatial variation both with-
in lakes and even more if different lakes are compared.
The compartment of protozoa can be divided in several ecological relevant
ways. Not only is there a taxonomic division between flagellates and ciliates, but
also a physiological one, relating to the nutritional mode (autotroph, he-
terotroph, mixotroph, etc.) which does not correspond with the classic taxonom-
ic or trophic level boundaries [21]. Furthermore the body sizes of the differ-
ent taxa are important features for their position within the food webs.
In many cases bacteria are grazed upon mainly by small heterotrophic fla-
gellates, the heterotrophic nanoplankton (HNAN), which in most cases turned
out to be the most efficient predators of bacteria that were able to control the
bacterial populations even during their highest productivity (e. g., [1, 22]).
Berninger et al. [1] found a clear correlation between the abundance of bacteria
and HNAN in comparing samples from more than hundred freshwater sites of
different trophic states. The numbers of the two groups of organisms differed
by two or three orders of magnitude, with maxima of more than 10
6
specimen
of HNAN and 10
9
specimens of bacteria per ml. They inferred predator-prey re-
lationships between these groups.
HNAN are sometimes grazed upon directly by metazoa, while in other bo-
dies of water ciliates constitute the main predators [17, 23]. Heterotrophic fla-
gellates, possessing high turnover rates, inhabit a central position in the trans-
fer of organic carbon in most microbial food webs.
But what about the ecological roles of ciliates? In some cases, especially in pro-
ductive waters, ciliates can also graze effectively on picoplankton and can even be
the most important bacterivores, taking a key position for the transfer of matter
to the metazoan links [23]. However, smaller bacterivorous ciliates with high
grazing efficiencies need a threshold abundance of bacteria to persist on this diet.
Beaver and Crisman [24] gave an estimate that small ciliates (2030 mm) were
largely excluded from lakes having <5 10
6
5 10
8
bacteria ml
1
a concen-
tration normally found only in more productive systems. Large ciliates
(>50 mm), being mainly phytophagous and grazing on nanoplanktic algae, do-
minate the ciliate assemblages in oligotrophic lakes, with low bacterial abun-
dance. Mixotrophic ciliates with endosymbiotic algae can even contribute sub-
stantially to pelagic autotrophic biomass in some lakes (15% of annual total [25]).
The overall number of planktonic ciliates in lakes is correlated with the tro-
phic state of the water bodies. While under oligotrophic conditions abundancies
of 310 cells ml
1
were recorded, 90215 cells ml
1
were recorded in hypereu-
trophic waters [25].
The length of the food chain originating from bacteria and Cyanobacteria
and the identity of links involved is important to the still unresolved question
as to whether the microbial loop is acting as a link or a sink for organic mate-
rial. Adherents of the latter position argue that a microbial food chain with four
steps will be unlikely to transfer any substantial amount of organic carbon to
the metazoan part of the web [15, 26, 27]. The answer to this question is depen-
dent on several variables. Besides the trophic states of the waterbodies, other
abiotic variables such as temperature and acidity are relevant for the specific
patterns of the microbial web [25] and also the species composition of the
whole food web [28].
In some cases organic material is transferred from picoplankton via he-
terotrophic flagellates to larger ciliates and then to crustaceans or other meta-
zoans. In other cases crustaceans may directly feed on nanoplankton, while ci-
liates are of minor importance [29]. Even though most metazoans cannot feed
effectively on small particles of the order of few mm, some freshwater species, in
particular cladocera of the genus Daphnia, can effectively control bacterial
abundance (although they may not persist on bacteria alone), thus shortcutting
the microbial loop [17, 28]. The presence or absence of a single species can thus
change the pathways completely, deciding the coupling or decoupling of the
microbial loop from the metazoan web. The proportion to which different
groups of organisms contribute to different nutritional types in a lake is also
seasonally variable [17, 20, 28].
In this regard, the scheme displayed in Fig. 3 comes closer to the perceived
processes than many other representations, in that a multitude of pathways is
possible which may be more or less important at different times.
210 W. Pauli et al.
Fig. 3. Diagram of the food web in lake plankton. In contrast to the scheme in Fig. 2, the com-
partment of protozoa has been differentiated. Note that not all pathways are realized at any
one time. See also text. DOM: dissolved organic matter
As mentioned above, the microbial loop is not only important for the trans-
fer of energy in the form of organic carbon, but also for the cycling and reten-
tion of nutrients. This is especially important in oligotrophic situations, where
nutrients like phosphorus and nitrogen are scarce at least during certain
times of the year. The phosphorus dynamics of the pelagic zone seem to be
strongly determined by the interactions of algae, bacteria, and protozoan gra-
zers. Algae and bacteria compete for P, with bacteria being more efficient in the
uptake of P. Bacterial grazing by protozoa was demonstrated to enhance phos-
phorus turnover and mineralization [30]. As grazed bacteria populations grow
faster their excretion of P also becomes stronger. Furthermore, protozoan gra-
zers increase the amount of organic P by excretion, which seems to be of spe-
cial importance for phytoplankton [31]. Although this compound is also excret-
ed by micro- and macrozooplankton, the high metabolic rate of protozoa leads
to higher excretion rate of this group of organisms. Buechler and Dillon [32]
estimated that if ciliates only contribute 1% to the biomass of a zooplankton as-
semblage, they should be able to contribute 50% to the release of dissolved P.
A similar situation exists with regard to nitrogen in cases where nitrogen is
a limiting factor for the growth of algae and bacteria. Bacteria can also out-
compete phytoplankton for N and thus serve as a sink for nitrogen within the
food web. However, as has been demonstrated experimentally, the presence of
bacterivorous protozoan grazers leads to a partial remineralization of N and al-
lows an increase in algal biomass [33]. The degree to which this process is of im-
portance depends on the carbon available for the bacteria. As Caron et al. [33]
concluded: the role of bacterivorous protozoa as mineralizers of a growth-
limiting nutrient is maximal in situations where the carbon:nutrient ratio of the
bacterial substrate is high.
1.4
Outlook
Most of the interactions described above were investigated in the pelagic part
of aquatic habitats. However, as mentioned above, many protozoa are closely re-
lated to surfaces within the water bodies, be they sediments, plants, and stones,
or even microscopic aggregates within the pelagic zone. In lakes or oceans the
main metabolic activity is certainly associated with the pelagic zone. Regarding
streams or small water bodies, the surface-related biota gain in importance for
the fluxes of energy and materials. In streams, a true plankton only exists in the
slow flowing lower reaches of large rivers. Thus, most organismic activities are
found in and on the benthic parts. Many of the aspects discussed above will also
be valid in these environments. However, there will surely be differences.
Although some data is available on the numbers and production of protozoa in
these microhabitats [3436], our understanding of the complex web of interre-
lations is much less than for the open water. To a considerable degree this seems
to be a consequence of the methodical difficulties. Benthic assemblages are
highly heterogeneous in space and time and this heterogeneity, i. e., the small
scale spatial arrangement of the different components, is by itself of importance
for the nature of the interactions between protozoa and the other parts of these
assemblages. Thus we are only just beginning to delve deeper into the compli-
cated patterns and dynamics of those biofilms. There is now important evi-
dence that these biofilms are also highly productive but also very retentive in
regard to nutrients [37]. Nutrient pulses are retained much longer within the
periphyton assemblages of streams than would be expected on the basis of a
continuous water flow.
There are certainly many other important ways in which protozoa are in-
volved in the ecology of aquatic systems. For example, little is known about
informational relations between protozoa and other members of the species
assemblages, although there may be indications in this direction (e. g., [38]).
Also, our view of microbial food webs may change during the next years with
the new awareness that even the pelagic zone of lakes is not as homogenous as
it seems at first sight. In addition to rather macroscopic stratifications of abio-
tic factors and the related stratifications of organisms, the role of tiny and in
the realm of human time-scales fleeting aggregates of small detritus particles,
bacteria, protozoa and algae come into prominence, the so called lake snow.
These aggregates may turn out to be hot spots of microbial activity, and especi-
ally for the grazing activities of protozoa. There are data that indicate that ciliate
bacterivory is especially high in lakes with high amounts of suspended organic
matter [39]. Similar to biofilms on solid substrates, the microenvironment on,
in, and around these aggregates can be chemically strangely different from the
average water column data. It remains to be seen, what these new insights will
bring about for the understanding of the ecological processes in freshwater
habitats.
2
Protozoa in Wastewater Treatment
2.1
Background
2.1.1
Wastewater
Wastewater includes municipal, industrial, and agricultural wastewater as well
as rainwater. The relative proportions of wastewater for West Germany (1980)
were 32% municipal, 47% industrial, and 1% agricultural wastewater, plus 20%
rainwater run-off in areas with main drainage. All wastewater produced in
towns and communities is termed municipal sewage. This expression covers
domestic wastewater (50%), extraneous water (leachates 14%), and wastewater
from industry and commerce (36%) [40].
Municipal sewage is treated as follows:
Initial mechanical purification or sedimentation
Biological purification or clarification
Further purification, e. g., elimination or reduction of the nitrogen, sulfur, or
phosphate content, polishing, filtration
The treated wastewater is then discharged into the receiving stream (Fig. 4)
212 W. Pauli et al.
P
r
o
t
o
z
o
a

i
n

W
a
s
t
e
w
a
t
e
r

T
r
e
a
t
m
e
n
t
:
F
u
n
c
t
i
o
n

a
n
d

I
m
p
o
r
t
a
n
c
e
2
1
3
Fig. 4 ac. Types of common sewage treatment plants flow
diagram of: a activated sludge plants; b, c biofilm processes
(trickling filter and Rotating Biological Contactor, RBC, re-
spectively). In the activated sludge process (a) the wastewater
is exposed to a mixed microbial population in the form of a
flocculent suspension. In fixed medium systems the waste-
water is brought into contact with a film of microbial slime (b)
on the surfaces of the packing medium, (the wastewater
trickles through the bed, most commonly consisting of
stacked stones), or (c) on a partly submerged support medium
which rotates slowly on a horizontal axis in a tank through
which the wastewater flows
All substances present in sewage are classified according to their significance
for wastewater treatment plants. Organic content is of particular importance for
degradation processes. It is quoted in terms of the chemical or biochemical oxy-
gen demand (COD, BOD) of the organic substances. Furthermore, a differentia-
tion is made between suspended and dissolved wastewater components.
Approximately two-thirds of the total load (organic and inorganic) of muni-
cipal sewage is in solution. With regard to the organic load almost half is in so-
lution, the rest consists of colloidal material (25%) or is bound to particles
which sediment (75%). Similarly, about half of the oxygen demand of biochem-
ically degradable organic compounds is attributed to the dissolved fraction, of
the other half one third to floating and two thirds to particulate matter. After
a 2 h sedimentation period, two-thirds of the total organic load remains in the
supernatant (also two-thirds of the total BOD). About 25% of the dissolved or-
ganic load is bound to colloids and particles which do not sediment (Table 2).
Carbohydrates are not usually present in municipal wastewater plants. They are
metabolized on route in the sewage. Proteins are also hydrolyzed in the sewers.
The main task of the wastewater treatment plant is then to eliminate fatty acids
and the amino acids formed by protein hydrolysis.
Municipal sewage averages an organic load of 300 mg BOD
5
l
1
(ca. 450 mg l
1
organic content). Activated sludge plants aim for effluent values <20 mg
BOD
5
l
1
, i. e., a reduction in the organic content of more than 90% [41]. For in-
dustrial as opposed to municipal wastewater, no generalizations can be
made regarding type and amount of load. Diverse organic and inorganic loads
are produced by different industrial sectors. Even within a sector values vary
according to the production methods and environmental requirements.
Wastewater from the chemical industry often exhibits toxic or inhibitory ef-
fects.
2.1.2
Biological Treatment Processes
It is well known that a microbial degradation of organic substances takes place
in natural flowing waters. This natural, self-purifying capacity of water became
overtaxed by the increase in population and industrialization. Attempts were
then made to pre-treat partially or fully sewage by mechano-biological pro-
cesses, before discharging it into the surface water.
214 W. Pauli et al.
Table 2. Average contribution of settleable (sedimentation within 2 h) and non-settleable
matter and their respective biochemical oxygen demand (BOD5) to the total organic load of
municipal sewage, according to [157]
Organic load (in Settleable: 33% (w/v) or
total ca. 450 mg/l) 150 mg/l, 33% (BOD)
Non-settleable: 67% (w/v) Dissolved: 83% (w/v)
or 300 mg/l, 67% (BOD) or 250 mg/l, 75% (BOD)
Suspended: 17% (w/v)
or 50 mg/l, 25% (BOD)
A conscious use of biological degradation began after bacteria were discov-
ered in the nineteenth century. Two principles were implemented: activated and
fixed-bed processes. The latter have been in use since 1882 and utilize the slime
growth of organisms in the receiving stream. The activated sludge process,
which takes advantage of the self-purification properties of the suspended or-
ganisms in the receiving water body, was developed in 1913, and the first
German plant was operational in 1926 [42]. Both methods are still in use today.
In Germany the activated sludge technique has taken precedence, due to its
higher performance capacity, particularly for extended wastewater treatment
including nutrient elimination. However fixed-bed reactors in combination
with activated sludge techniques are finding increased application today. As
submerged aerators they increase the active biomass and the age of the sludge
in activated sludge plants, making a positive contribution to the purification ef-
ficiency [43].
The underlying principle of biological wastewater treatment is to transform
the majority of dissolved and suspended substances into biomass which can
then be removed either by sedimentation (activated sludge) or by fixing (sub-
merged aerator contactors). In this way, a nutrient concentration exceeding the
degradation capacity of local surface waters, resulting in disruption or even de-
struction of natural biological systems, can be avoided: Direct discharge of sub-
stances would result in anaerobic or aerobic burdening of the sediment of sur-
face waters; high oxygen consuming, organic content (BOD
5
) in the effluent can
overtax the oxygen household of the water, through its rapid conversion by he-
terotrophic organisms; direct discharge of plant nutrients, particularly nitrogen
compounds and phosphates, encourages algal growth, with negative effects on
the water (larger pH- and O
2
-fluctuations, sludge formation). At the same time,
however, the discharge of bacteria used for the fixation of wastewater sub-
stances should be kept to a minimum.
All biological processes have in common that they involve sectors of natural
metabolic cycles. In wastewater treatment plants, the only difference from na-
tural processes is that part of the reaction chain is technically controlled. The
performance is dependent not on one specific species with a high degradation
capacity, but on the interaction of a wide range of different organisms. Over the
last 20 years the traditional model of a vertical material and energy flow, start-
ing from nutrients through to decomposers and primary producers and both
primary and secondary consumers, has been replaced by a more complex eco-
logical web, which takes into account the network of microbial systems and
their significance for turnover of matter (see Sect. 1.2).
In treatment plants, due to the high organic content of the wastewater, a bio-
coenosis of organisms forms, primarily made up of members of the group of
decomposers, i. e., saprophytic bacteria. The majority of the bacteria degrade
dead organic matter, in the presence of oxygen, to carbon dioxide and water.
Nitrogen is released in the form of ammonia. Bacteria are significant in waste-
water treatment due to their large surface area in relation to their body volume
and their associated high metabolic and reproductive rates. Apart from these
prokaryotic forms of life, protozoa (unicellular, animal organisms) are the next
most important group of organisms in the wastewater biocoenosis. Together
with bacteria they form a closely related microbial system which forms the ba-
sis of the so-called natural self-purification process.
2.1.3
Bacterial Biofilms
In both fixed-bed and activated sludge processes, microbial biofilms either as
slime growth or flocs are fundamental for the turnover of organic waste. The
colonization of surfaces by bacteria is a widespread process in the environment.
In natural biotopes, bacteria favor the colonization of suspended particles and
sediment. By far the majority (99%) of all bacteria in the environment adhere
to surfaces such as stones, sediment, and soil. Important physico-chemical pro-
cesses, forming the basis for the biomass layer, precede the attachment of a bio-
film. Dissolved organic molecules (polysaccharides, proteins, humic acids) ac-
cumulate spontaneously on the surface of very different materials forming a
conditioning film, on which bacteria colonization follows. The cells are im-
mobilized and produce extra-cellular polymeric substances which anchor the
organisms to the surface and to each other. Embedded in this matrix, microbial
communities of complex composition are built up, usually in several layers.
Biofilms are not static systems, rather a dynamic equilibrium exists between
freely suspended bacteria and those adhering to particles. From the moment a
bacterial biofilm forms, a detachment of cells or cell-aggregates takes place [44],
dependent on the prevailing conditions. Several bacteria species, dependent on
their nutrient supply, can exist either freely suspended or mainly aggregated in
both pure and mixed cultures [45].
2.1.4
Activated Sludge
Existing literature regarding protozoa and wastewater treatment deals mainly
with aerobic processes, with the focus on activated sludge technology. This
is due to the significance of this technology for wastewater treatment on the
one hand and that suspended activated sludge is more easily accessible for bio-
logical investigations than slime-growth areas of fixed-bed reactors on the
other.
Activated sludge processes operate with typical sludge concentrations be-
tween 23 g l
1
[46]. About 70% of the activated sludge is organic content and
30% inorganic (clay: Si; Al; Fe; ferric oxide; calcium phosphate) [47]. Non- or
not easily oxidizable organic matter makes up 2025% of the sludge [41].
In a conventional activated sludge tank flocculate suspended material con-
tains about 6 10
9
bacteria ml
1
, i. e., 13 10
12
bacteria g
1
dry weight [48].
They represent about 90% of the total biomass of the activated sludge. The pro-
portion of living or metabolically active bacteria found in the flocs varies con-
siderably, depending on the method of analysis. Estimates based on glucose,
stearate and acetate uptake rates imply active proportions of 813%, 1428%,
and 510% of the total biomass, respectively [48]. More recently, direct mea-
surements by fluorescence-microscopy indicate a proportion of 3540% (de-
216 W. Pauli et al.
hydrogenase activity [49]) and 70% (rRNA directed oligonucleotide probes,
[50]), whereby a similar level of activity was assumed for all zones of the floc
[51].
2.2
Protozoa in Biological Wastewater Treatment Plants
2.2.1
Occurrence
Systematic investigations at a large number of wastewater treatment plants re-
veal protozoa as typical components of the biocoenosis (Table 3). Thus, for ex-
ample, in all ten South African activated sludge plants studied by Bux and Kasan
[52] basic communities of protozoa, typical for sewage plants were found.
Similarly, Curds and Cockburn [53] found protozoa biocoenoses in 53 of 56
British activated sludge plants and all 52 biological percolation filter plants
studied. In New Jersey, Chung and Strom [54] found protozoa in all the rotating
disc contactors and according to Madoni and Ghetti [55], typical ciliate com-
munities were detected in 38 of 39 activated sludge plants and 47 of 49 rotating
disc contactors in the Emilia region of Italy. The presence of protozoa is closely
associated with biofilms and restricted mainly to aerobic processes and there-
fore to certain areas of the wastewater treatment plant; only a few specialists
among the protozoa take part in anaerobic processes. Thus protozoan commu-
nities can be typically encountered in activated sludge tanks as well as in the se-
dimentation tanks, whereas no protozoa are found in sludge digestion or in the
supernatant of the sedimentation tank (effluent), with the exception of malfunc-
tions [56].
Table 3. A survey of the protozoan fauna in sewage treatment plants (only microfaunistic in-
vestigations based on ten and more plants are taken into consideration), according to [5255]
Type of plant No. of plants Occurrence of Typical Protozoa
investigated typical protozoan protozoan absent
(country) communities communities
absent
Activated sludge 56 (Great Britain) Within 53 plants 2 plants
a
1 plant
39 (Italy) Within 38 plants
b
1 plant
b
?
10 (South Africa) Within all 10 plants
Trickling filter 52 (Great Britain) Within all 52 plants
Rotating biological 49 (Italy) Within 47 plants
b
2 plants
b
?
contactor
10 (USA) Within all 10 plants
a
No ciliates, but flagellates present.
b
Only ciliates investigated, no comments on other protozoan groups such as flagellates and
amoebae.
2.2.2
Species Composition
The majority of microfaunal investigations confirms that all of the three main
groups of protozoa flagellates, ciliates, and amoebae (naked and shell) can
be found in wastewater treatment plants, whereby ciliates form the largest pro-
portion with regard to biomass and number of species, both in activated sludge
[53, 5762] and in fixed-bed processes (percolation filters: [53, 59]; rotating disc
contactors: [6365]), compare Table 4.
It should be noted, however, that the composition of the protozoan biocoe-
nosis, as well as that of the total biomass involved in the purification process,
is mainly dependent on the composition of the wastewater, together with phy-
sical conditions and factors arising from the process technology used. In the
case of malfunctions, or in the initial stage of a plant, very different composi-
tions can be encountered. Sydenham [57] observed 2 municipal activated sludge
plants over a period of 12 months and identified amoebae as the dominant
group with regard to biomass. In sludge with a high organic load, Curds and
Cockburn [66] and Mudrack and Kunst [67] report high population densities of
flagellates. The age of the sludge also has an effect on the composition of the
protozoan community. Kinner and Curds [63] quote 612 months as the length
of time required to establish a steady-state community of protozoa in a pilot
rotating disc contactor plant supplied with domestic effluent. Bacteria were vi-
sible on the disc surfaces within one day of startup followed within a few days
by flagellates and small amoebae. Free-swimming bacterivorous ciliates appear-
ed within 810 days. Subsequently, sessile peritrichous forms accompanied by
carnivorous ciliates, rotatoria, and large amoebae make up the stable commu-
nity. Parallel to sludge aging, a typical chronological succession of dominant
protozoa populations can also be observed in activated sludge plants. After the
initial phase of 12 weeks where flagellates, naked amoebae, and free-swim-
218 W. Pauli et al.
Table 4. Structure of the protozoan community in three urban activated-sludge plants, oper-
ating at different organic loading rates and dissolved oxygen concentrations (observation
over a one year period), according to [62]. Biomass calculation is based on data, given by [61]
Plant 1 Plant 2 Plant 3
Organic load
a
0.230.38 0.210.35 0.50.8
O
2
-conc. (mg O
2
/l) 3.65.2 1.83.0 1.01.3
Densities and biomass ind./ml mg/l ind./ml mg/l ind./ml mg/l
Ciliates 3000 1843 8600 5099 4500 2693
7400 17000 16000
Flagellates (<20 mm) 43000 2.25.2 89000 4.651 38000 2083
600000 980000 1600000
Naked Amoebae (<50 mm) 4000 0.215.3 800 0.04 77000 4.15.4
100000 130000 6.9 101000
a
kg BOD5/(kg MLVSS) day.
ming ciliates predominate, more and more crawling and sessile forms appear,
which remain dominant throughout the stabilization phase and can be regard-
ed as typical representatives of mature sludge [62, 65, 6870]; see also Fig. 5.
Unlike the free-swimming forms, which arrive at the plant with the sewage and
are flushed out at the end of the process, the existence of sessile and crawling
forms is closely associated with the development of slime growth or sludge
Fig. 5. Composition of the bacterivorous ciliate community during the establishment of a
mature sludge. Stabilization, i. e., steady-state occurs after about 50 (activated sludge, above fi-
gure) and ca. 80 days (RBC, below figure), respectively. Bars lower than 100% indicate the ad-
ditional presence of carnivorous and omnivorous ciliates, after [65]
flocs. Bound to biofilms as fixed slime growths (fixed-bed) or as sedimentable
sludge, they are retained in the treatment plant and can thus build up a stable
community with the bacterial flora. Whereas characteristic population succes-
sion takes place in both plant types, in percolation filters, due to the unequal
distribution of the organic load, a physical separation of the organisms is ob-
served, dependent on the filter depth [68].
Figure 5a, b shows results from studies on the colonization behavior of cili-
ates in a pilot rotating disc contactor plant as well as in an operational activated
sludge plant [65]. Both plants were fed with domestic wastewater. Whereas in
the initial stage of the activated sludge plant ciliates make up between 0.17%
and 0.44% of the total biomass, in the stabilizing phase they account for more
than 9% of the sludge biomass. In the initial phase free-swimming forms from
the wastewater dominate. After 1015 days their numbers drop markedly and
crawling (Aspidisca cicada, A. lynceus, Euplotes affinis, Chilodonella uncinata)
as well as sessile (Vorticella convallaria, V. microstoma, Epistylis plicatilis,
Opercularia coarctata) ciliates characterize the protozoan fauna. Similarly, in
the rotating disc contactor plant, ciliates makes up only 45% of the slime bio-
mass in the colonization phase, as opposed to 1219% under steady-state con-
ditions. Here, too, essential changes take place during the colonization of the
submerged contact aerator and the typical ciliate biocoenosis develops in the
plant itself. In the initial phase, free-swimming ciliates such as Paramecium
putrinum and Uronema nigricans are present; in the stable phase sessile forms
such as Opercularia coarctata and Vorticella convallaria dominate. Investiga-
tions by Madoni [64, 65] make it clear that in both types of plants (submerged
contact aerator and activated sludge) a significant positive correlation exists
between the increase of the sludge, biofilm and ciliate biomass (r
2
= 0.927 and
r
2
= 0.853). This implies a close relationship between the size of the ciliate
population and the bacterial biomass.
2.2.3
Plant Specific Basic Communities
The relative abundance of an organism in a particular habitat can be consider-
ed as a measure for its significance within the ecological structure of the bio-
logical system concerned. Alongside amoebae and flagellates, Curds and
Cockburn [53] identified 67 and 53 ciliate species in 56 activated sludge plants
and 52 percolation filter plants in Britain, respectively. Madoni and Ghetti [55]
detected 45 and 47 ciliate species in 39 activated sludge plants and 49 percola-
tion filter plants in Northern Italy. Of note is that the British and Italian activat-
ed sludge plants revealed very similar ciliate fauna [55]. Nevertheless, not all
species in the individual samples can be regarded as typical, as to their presence
and population density, for the respective wastewater treatment process. The
majority of the species are found only sporadically in a few samples and usually
with a low population density. The overall picture of the ciliate population is de-
termined by a few, primarily sessile (peritrichous) and crawling (hypotrichous),
species most of which are bacterivorous (compare with Fig. 6). With cell counts
of, on average, more than 10
4
ml
1
, ciliate densities are 1001000 times higher
220 W. Pauli et al.
here, than in the plankton of oligotrophic (10 ml
1
) and eutrophic (100 ml
1
)
waters [24]. Table 5 summarizes the dominant ciliate species in the basis com-
munity of each plant type identified by Curds and Cockburn [53] and Madoni
and Ghetti [55]. The specific biocoenosis differs according to plant type and to
the current operating conditions [55]: in areas with a high organic load an in-
crease in free swimming species is observed [61, 66] along with a decrease in the
diversity of species [71, 72]; with these limitations, the community forms given
in Table 5 can be considered average for municipal plants.
2.2.4
Biomass
In activated sludge plants a high proportion of the eukaryotic biomass is com-
prised of protozoa. Investigations carried out by Sydenham [57] revealed that
protozoa made up over 90% of the total eukaryotic biomass of two municipal
wastewater treatment plants. According to Aescht and Foissner [61], protozoa
made up 99100% of the eukaryotes in a pharmaceutical plant with a bacterial
nutrient load. The average proportion of protozoa in relation to total solids
(dw) is 5% [59, 73]. Ciliates alone make up 10% of the total biomass (pro- and
eukaryotic dry weight). Even higher numbers of ciliates are encountered in mu-
nicipal rotating disc contactors where proportions of about 20% of the total
biomass of the slime-growth can be observed [64, 65].
2.2.5
Ecological Framework
The biocoenosis in wastewater treatment plants should not be regarded as a
community with a rigid composition and constant characteristics but rather as
Fig. 6. Examples of free swimming (holotrichous), crawling (hypotrichous), and sessile (pe-
ritrichous) ciliates in waste water treatment plants
222 W. Pauli et al.
Table 5. Ciliate species dominating and occurring with a high frequency in sludge samples of
British (GB) and North Italian (I) sewage treatment plants, respectively, after [53, 55].
Dominating refers to the relative cell density, whereas present indicates the number of
samples, in which the respective species independent of its individual numbers could be
observed
Dominant Present Life form Nutrition
a
(%) (%) Ecological type
GB I GB I
Activated sludge (GB and I)
Aspidisca costata
a
35 85 69 90 Crawling Bacterivorous
Vorticella convallaria
a
19 77 58 84 Sessile Bacterivorous
Trachelophyllum pusillum
a
15 30 64 58 Free swimming Carnivorous
Opercularia coarctata
a
Carchesium polypinum
a
Vorticella alba (GB) 11 38 Sessile Bacterivorous
Vorticella microstoma (GB) 10 10 75 10 Sessile Bacterivorous
Euplotes moebiusi (GB) 5 5 35 7 Crawling Bacterivorous
Vorticella fromenteli (GB) 4 31 Sessile Bacterivorous
Euplotes affinis (I) 59 11 69 Crawling Bacterivorous
Zoothamnium pygmaeum (I) 33 33 Sessile Bacterivorous
Trochilia minuta (I) 2 23 12 25 Crawling Filamentous
b
Trickling filter (GB)
Opercularia micodiscum 44 81 Sessile Bacterivorous
Carchesium polypinum 15 62 Sessile Bacterivorous
Vorticella convallaria 10 83 Sessile Bacterivorous
Chilodonella uncinata 4 90 Crawling Filamentous
b
Opercularia phryganeae 4 90 Sessile Bacterivorous
Opercularia coarctata 2 56 Sessile Bacterivorous
Vorticella striata 2 52 Sessile Bacterivorous
Aspidisca costata 56 Crawling Bacterivorous
Cinetochilum margaritaceum 54 Crawling Bacterivorous
Rotating biological contactor (I)
Euplotes moebiusi 53 79 Crawling Bacterivorous
Paramecium caudatum 46 79 Free swimming Bacterivorous
Trachelophyllum pusillum 41 59 Free swimming Carnivorous
Vorticella convallaria 53 57 Sessile Bacterivorous
Opercularia microdiscum 41 45 Sessile Bacterivorous
Opercularia coarctata 33 37 Sessile Bacterivorous
Paramecium trichium 27 43 Free swimming Bacterivorous
Cinetochilum margaritaceum 23 37 Crawling Bacterivorous
Chilodonella cucullulus 18 41 Crawling Filamentous
b
a
Dominant both in British and Italian plants.
b
Filamentous: ciliates with a specialized oral apparatus, enabling the ingestion of rod-shap-
ed, filamentous bacteria.
not present.
an artificial but biological segment of natural self-purification processes, the
composition of which is influenced by ecological conditions and physico-chem-
ical factors, thus differing from plant to plant and even within a plant over
time.
2.2.5.1
Sludge Loading
Sludge loads with f
m
-values between 0.2 and 0.6 [g BOD (g MLSS day)
1
] are
considered optimal for the purification sequence at conventional municipal ac-
tivated sludge plants (e. g., [47, 67]). Ciliate densities of 600030,000 ml
1
are
found in sludge with these loads [71, 74]. However, similar concentrations of
ciliates are also encountered in sludge with both higher and lower loads:
Salvado and Gracia [71] observed a constant ciliate population density in a mu-
nicipal plant with f
m
-values varying from 0.03 to 0.4. Experiments by Lee et al.
[74] confirm only slight changes in ciliate counts at sludge loadings between
0.11.4 [g BOD (g MLVSS day)
1
]. Only under very heavy loads [1.82.4 g BOD
(g MLVSS day)
1
], was a reduction in cell density observed.
Although the population density remains constant over a wide range, the
organic load influences the number of species and the composition of domi-
nant ciliates in the basis community. The number of species present sinks with
increasing organic content of the wastewater [66, 71, 72]. According to Curds
and Cockburn [66], activated sludge with a relatively low organic load
[f
m
=0.10.3 g BOD (g MLSS day)
1
] shows the greatest species diversification,
whereby all three groups of ciliates peritrichs (sessile), hypotrichs (crawling),
and holotrichs (free swimming) are represented with approximately the same
number of species. In the medium load range of f
m
=0.30.6, peritrichous spe-
cies dominate and by high organic loads of f
m
=0.60.9 equal portions of peri-
trichs and holotrichs are present (Fig. 7).
2.2.5.2
Temperature
Temperatures in municipal plants are generally slightly above the outside tem-
perature in winter and slightly below in summer. Performance is optimal be-
tween 10C and 25C [41]. No negative effects on ciliate fauna are found up to
30 C; experimental activated sludge investigations reveal a decline in ciliates at
temperatures above 30C and their disappearance above 40C [74]. The authors
discuss the concomitant deterioration of the settling properties of the sludge as
possibly resulting from the collapse of the ciliate population.
2.2.5.3
pH-Value
Activated sludge has a relatively high buffer capacity. If no strongly acidic or al-
kaline effluents are introduced, mainly from industrial processes, pH-values
generally fluctuate between 6.5 and 8 [41, 67, 75]. Therefore not only the tem-
perature, but also the pH-values of municipal plants are in a favorable range for
protozoan growth [75].
2.2.5.4
O
2
-Content
Conventional processes of biological wastewater treatment utilize the meta-
bolism of the organic load, which is faster, more thorough, and easier to control
under aerobic conditions. Aerobic conditions are also a prerequisite for a high
incidence of protozoa. Few specialists can survive strictly anaerobic conditions
and little knowledge is available regarding their distribution or function in an-
aerobic degradation processes. The number of facultative anaerobic protozoa is
slightly higher, but almost all species seem to be able to survive low oxygen con-
centrations or even the absence of oxygen, at least for a short period [75]. Apart
from plant malfunctions (e. g., breakdown of the aeration), this ability is also
important in the normal cycle of activated sludge processes, where the organ-
isms are constantly alternating between the aerobic activated sludge tanks and
the sedimentation tanks, in which anaerobic conditions arise for short periods
224 W. Pauli et al.
Fig. 7. Composition and species number of ciliates in activated sludge plants operated at dif-
ferent sludge loadings [food to micro-organism (F/M) ratio]. Results from an investigation of
52 British plants made by Curds and Cockburn [53]. Peritrichous, hypotrichous, and holo-
trichous ciliates represent sessile, crawling, and free swimming ciliates, respectively. In con-
ventional municipal plants, treating domestic wastewater, a sludge loading between 0.2 and
0.6 is regarded to be optimum for the functioning of the sewage treatment process
in the deeper layers of the settling sludge (less than 4 h [41]). Only longer and
repeated oxygen deprivation over several hours (continual alternation between
6 h aerated and 24 h without aeration) leads to a marked decline of the ses-
sile ciliates Vorticella convallaria and Opercularia coarctata, typically found in
wastewater treatment plants [76].
2.3
Significance of Protozoa for Wastewater Treatment
As already described (Sect. 2.2.2), the majority of protozoa in aerobic biological
purification systems are sessile or crawling ciliates. Whereas free-swimming ci-
liates are flushed out with the clarified water, crawling and especially sessile
forms are bound to bacterial biofilms (flocs and slime growths) [59]. In the case
of fixed-bed plants they remain bound to the biofilms in the plant; in activated
sludge processes they sediment with the sludge and are retained in the plant
due to continual sludge recycling.
To understand the role of protozoa and classify their position in the artificial
system of biological wastewater treatment, the following characteristics have to
be considered: type of motion (free swimming, crawling, or sessile); form of
nutrition (e. g., filter-feeders, browsers); sources of nutrition (abiotic colloids
and particles, bacteria, algae, other protozoa). From their form of nutrition and
their trophic level, functional aspects important for wastewater treatment be-
come apparent. New understanding of natural systems as well as experimental
results on the physiology, energy budget, and nutrient cycling of both aquatic
and terrestrial protozoa provide extensive information regarding the ecological
role of this group of organisms, which, although quantitatively less significant
than bacteria, make a considerable contribution to wastewater treatment.
2.3.1
Nutrition
Several possibilities are open to ciliates for nutrient-uptake. On the one hand,
similar to bacteria, substances can be transferred directly through the plasma
membrane into the interior of the cell. Active and passive, carrier-mediated
uptake mechanisms through the plasma membrane have been described for
Tetrahymena for amino-acids [7779], di-peptides [80], acetate, glucose [81,
82], and even for such complex nutrient solutions as proteose-peptone-yeast ex-
tract (PPY) medium [83]. Another method of nutrient uptake is pinocytosis
[84, 85]. It describes the active transport of dissolved substances in sub-micro-
scopic, particle-free vacuoles or vesicles from the plasma membrane to the cell
interior, where they undergo normal lysosomal digestion processes. Finally,
ciliates have a highly specialized oral apparatus for taking up particulate mat-
ter by phagocytosis. The particles are not simply ingested with the surrounding
solution but rather undergo a highly efficient filtration process, facilitating the
concentration of particulate matter from a large volume of liquid, prior to their
intake in food vacuoles [85]. This process involves the production of a water
current by cilia (Fig. 8) and the extraction of particles from the flowing water
with the aid of a ciliary sieve, which retains in the case of bacterivorous spe-
cies particles sized between 0.3 mm and 5 mm [8587]. The particles, thus con-
centrated, are subsequently ingested. Apart from food, abiotic and even indige-
stible matter of the size of bacteria are efficiently ingested [8689]. Paramecia
concentrate food particles in this manner in their oral cavity up to 1000-fold
[90]. A similarly high concentration capacity can be assumed for Tetrahymena:
Whereas a volume of 5080 nl is cleared of particles per hour and cell [87, 91],
a more than 1000 times lower water volume of 36 pl h
1
and cell is actually in-
gested by the food vacuoles [92]. The efficiency of this form of nutrition is un-
derlined by investigations comparing the growth kinetics of Tetrahymena pyri-
formis with particulate and dissolved substances as nutrient source, respec-
tively [93]. While under monoxenic conditions with particulate bacterial
substrate the half maximum growth rate is already attained with a bacteria con-
tent of 12 mg l
1
Klebsiella aerogenes (5.5 mg carbon l
1
), 200 times that con-
centration of organic matter is required in case of dissolved nutrients (2.4 g l
1
proteose-peptone-yeast medium=1.3 g carbon l
1
).
226 W. Pauli et al.
Fig. 8. Mechanisms of filter-feeding (ambiguously often referred to as grazing) used by pro-
tozoa. Water currents are created by flagella or the coordinated activity of cilia, that bring sus-
pended food to the mouth region of the cell
2.3.2
Reduction and Elimination of Suspended Particles and Bacteria
2.3.2.1
Clearing Rate
The volume of water cleared per individual and hour depends on cell size. Small
protozoa with cell diameters of less than 5 mm, such as flagellates, filter less than
1 nl h
1
at temperatures between 9C and 17C [20]. Higher filtration rates are
observed for larger ciliates. Sanders et al. [20] quote a yearly fluctuation range
of 12156 nl h
1
for the filtration performance of planktonic ciliates. In labora-
tory experiments with the ciliates Halteria grandinella (diameter: 25 mm) and
Strombidium sp. (size: 15 21 mm) filtration rates of 8090 nl h
1
at 9C and
120140 nl h
1
at 17C were determined. In the case of Vorticella microstoma
(average cell dimensions: 60 30 mm), a ciliate frequently present in wastewa-
ter treatment plants, filtration rates as high as 156 nl h
1
at bacteria densities of
10
6
ml
1
are reported. Tetrahymena (cell dimensions: 40 20 mm), a species
present but not dominant in wastewater treatment plants, has a filtration per-
formance of 80 nl h
1
[91]. Fenchel [87] observed filtration rates of 50 nl h
1
and
cell at 2022C for Tetrahymena pyriformis and 2001000 nl h
1
for larger
(100200 mm) representatives of crawling and free-swimming ciliates such
as Euplotes, Paramecium, or Blepharisma. Assuming average filtration rates of
100 nl h
1
and cell and ciliate densities of 10,000 ml
1
and above [61, 9497], this
implies that the entire liquid of an activated sludge plant can be filtered in less
than 1 h. The enormous predator and selection pressure exerted on the bacteria
is illustrated by the following examples.
Many heterotrophic bacteria in activated sludge have the ability to divide
every 2040 min under optimal laboratory conditions [41, 48]. Under field
conditions, such as those prevailing in wastewater treatment plants, their
growth is generally much slower due to sub-optimal physical (temperature)
and physiological (nutrients, pH-values) parameters. The actual bacterial divi-
sion rates under constant operating conditions and good nutrient availability
can be estimated from the ratio of the surplus (drawn off) sludge to the total
sludge in the activated sludge plant [98]. For low to high organic loads
(f
m
=0.050.6 g BOD per g MLSS and day), growth rates can vary from 450%
per day [41] or, expressed in other terms, the bacteria population in the sludge
doubles every 48 h at most, i. e., in a time span by no means adequate to com-
pensate for potential protozoan feeding.
Highly loaded wastewater contains ca. 10
6
bacteria ml
1
. The majority are
medically harmless but others are pathogenic and bear health risks. Con-
ventional wastewater purification involves an initial pre-clarification step of
2030 min, after which the wastewater is fed into the activated sludge tank and
aerated for 4 h. In the aerated and agitated system of the activated sludge tank
the wastewater is brought into contact with a mixed microbial population in the
form of a flocculent suspension. When the desired degree of treatment has been
achieved, the flocculent microbial mass, known as the sludge, is separated for
24 h from the treated wastewater in a separate, specifically designed sedimen-
tation tank. The supernatant from the separation stage is the treated waste-
water, and should be virtually free of sludge. Most of the settled sludge from the
separation stage is returned to the aeration stage to maintain the sludge con-
centration in the aeration tank at the level needed for effective treatment and to
act as a microbial inoculum. Some of the sludge is removed for disposal, and is
known as waste or surplus sludge. In both the activated sludge and the sedi-
mentation tanks, the resident ciliate community has sufficient time to filter the
entire wastewater several times, thus removing bacteria and abiotic particles of
similar size (see Sects. 2.3.1 and 2.3.2).
2.3.2.2
Experimental Findings
It has long been known that protozoa are present in wastewater treatment
plants and that their species composition reflects the prevailing conditions in
the plant. However, scientific opinion was less unanimous with regard to the ac-
tual contribution of protozoa to the purification process. Although Ardern and
Lockett [99], Pillay and Subrahmanyan [100], Pillay et al. [101] and McKinney
and Gram [102] referred to a connection between protozoa and the quality of
the water discharged from the plant, proof of a causal relationship was lacking
or inconclusive.
Curds et al. [103] succeeded in selectively removing protozoa from activated
sludge and further cultivating this protozoan-free sludge in bench-scale treat-
ment plants over a long period. Through the subsequent re-introduction of ty-
pical sludge ciliates they observed, under various starting conditions, positive
effects on a series of parameters describing the success of the purification pro-
cess (Table 6). The principal observation of their experiments was that in the
absence of protozoa the effluent of the plant was turbid, due to its high content
of suspended bacteria; this turbidity almost disappears after re-introduction of
the protozoa (Fig. 9).
Similar findings are published by Sridhar and Pillai [104] and Macek [105] in
protozoan-free, pasteurized sludge and in bacteria cultures isolated from ac-
tivated sludge. The addition of sessile, crawling, and free-swimming ciliates
228 W. Pauli et al.
Table 6. Effects of ciliated protozoa on the effluent quality of bench-scale activated-sludge
plants. Results are given in mg l
1
unless otherwise noted; after [103]
Effluent analysis Without ciliates With ciliates Mean reduction
BOD 5370 724 75%
COD 198250 134142 38%
Permanganate value (4 h) 83106 6270 30%
BOD after filtration 3035 39 81%
COD after filtration 3150 1425 39%
Organic nitrogen 1421 710 51%
Suspended solids 86118 2634 71%
Optical density at 620 nm 0.951.42 0.230.34 76%
Viable bacteria counts (10
6
ml
1
) 160 19 97%
(Epistylis articulata, Vorticella microstoma, Aspidisca cicada, Chilodonella unci-
nata, Stylonychia putrina, Colpidium camylum) reduces high COD values and
suspended matter content. Farrah et al. [106] confirm the causal relationship
between the presence of ciliates and a clear, almost bacteria-free effluent with a
low organic content. Departing from a typical pro- and eukaryotic sludge bio-
coenosis, the authors show that a largely selective reduction of protozoa, by the
addition of sodium fluoride (0.2 mol l
1
) or sodium azide (620 mmol l
1
) re-
sults in a notably higher content of freely suspended bacteria including strep-
tococci. After application of the eukaryotic cell toxins, the total count of fecal
streptococci increases about threefold and the proportion of suspended bac-
teria, as compared to those bound to flocs, increases from 0.3% to 64%.
Kakiichi et al. [107] made essentially the same observations. The effects of two
amphoteric detergents (orthodichlorobenzene and polyhexamethylene bigua-
nide hydrochloride) with known effects on bacteria and protozoa were studied
and a causal relationship between poor quality of the outflow (increased turbi-
dity and COD values) from batch cultures of activated sludge and the inhibitory
effect (reduction in population density) on the protozoa was observed. A corre-
lation between the effluent quality and the population density of protozoa is
also implied by Lee et al. [74]. Studies with a bench-scale activated sludge plant
(organic load: 0.10.4 g BOD per g MLSS and day) show that the selective de-
cline of the ciliate population density, due to running temperatures of 36C and
over (see Sect. 2.2.5.2), corresponds to a more than twofold increase in suspen-
ded matter in the effluent.
Experiments with bacteria-free synthetic wastewater (e. g., [103]) exhibit that
freely suspended bacteria, originating from the autochthonous microflora of
the activated sludge itself, are substantially reduced in the presence of protozoa.
Fig. 9. Influence of ciliates on the bacteria content in the effluent of a bench-scale activated
sludge plant, after [103]
bacterial density
Furthermore Curds and Fey [108] observed that bacteria originating from the
influent wastewater are also effectively removed in the presence of protozoa.
After mechanical destruction of the protozoan population, by means of a ball
mill, the authors determined concentrations of 6.5 10
5
culturable E. coli ml
1
in the effluent of a continuously operating bench-scale activated sludge plant;
after re-inoculation of the activated sludge with ciliates (Opercularia coarctata,
Vorticella microstoma, Hypotrichidium conicum, Tetrahymena pyriformis) and
the establishment of a stable protozoan community, this count was reduced ten-
fold to 6.3 10
4
ml
1
. The half life of E. coli in the activated sludge was reduced
from 16 h to 1.8 h.
Filter-feeding ciliates in wastewater treatment plants are, in principal, not
selective consumers. Along with harmless bacteria, a series of pathogenic
strains causing, for example, diphtheria, cholera, typhoid, and streptococcal in-
fections are also phagocytosed (for reviews [75, 109]. Investigations by Farrah
et al. [106], with activated sludge in batch cultures, illustrate the significance of
this elimination of pathogenic bacteria from wastewater treatment plants. After
selective reduction of the protozoan fauna by sodium fluoride (200 mmol l
1
) or
sodium azide (20 mmol l
1
), cultures of Salmonella typhimuriumand E. coli, ad-
ded in densities of 10
5
ml
1
almost treble within 24 h (S. typhimurium and so-
dium fluoride) or only decrease by ca. 50% (E. coli and sodium azide), whereas
in untreated controls with protozoa, both bacteria are reduced to less than 5%
of their initial density. Moreover, under conditions of aerobic sludge stabiliza-
tion, the authors show that even low densities of protozoa (660 ml
1
) lead to a
substantial elimination of bacteria. Figure 10 shows results with Streptococcus
faecalis.
230 W. Pauli et al.
Fig. 10. Effect of sodium azide (6 mmol l
1
, selectively reducing protozoan activity) on
Streptococcus fecalis in activated sludge (laboratory scale), after [106]. CFU: colony forming
units
2.3.2.3
Field-Observations
Field observations leave no doubt that the results found in the laboratory
microcosms are transferable to pilot and full-scale plants and that the presence
of a typical protozoan community is reflected by the improved quality of the
plant effluent.
First, a close negative correlation is observed between the population density
of, mainly crawling and sessile, ciliate populations and the proportion of sus-
pended matter in the effluent of wastewater treatment plants (domestic and
municipal wastewater [56, 110114] and brewery wastewater [115]). Results
from a three-year investigation of three activated sludge plants with different
organic loads in Spain [114] reveal on average for all plants a highly signifi-
cant correlation coefficient between total ciliate population density and biolog-
ical oxygen demand of r = 0.868. In the presence of protozoa the effluent BOD
ranges from 4 mg l
1
to 18 mg l
1
, rising to values of up to 67 mg l
1
in their ab-
sence. An almost identical correlation between effluent quality (COD) and the
population density of typical activated sludge ciliates was observed by Sudo and
Aiba [111] for six municipal wastewater treatment plants in Tokyo. Mean COD
values of 10 mg l
1
were found with ciliate densities of ca. 10
4
ml
1
; these in-
crease to 40 mg l
1
when ciliate densities drop to 10
2
ml
1
(Fig. 11).
Second, according to Curds and Cockburn [53], plants without ciliates can be
recognized by the low quality of their effluent: 3 out of 53 activated sludge
plants were selected due to the high content of suspended material in their
effluent. In one of these plants no protozoa could be found at all, in the other
two no ciliates, only small flagellates, could be detected. The highest BOD values
measured in the three plants occurred in the plant with no protozoa.
Fig. 11. Relationship between protozoan densities and effluent COD, observed in municipal
activated sludge plants of Tokyo, after [111]. Symbols represent different plants
Finally, as compared to normal activated sludge processes, the clarifying ef-
fect of protozoa in activated sludge processes with submerged fixed-bed filters
a technology which creates additional surfaces for slime growth and primar-
ily sessile ciliates [116118] improves, which is basically due to low bacteria
and suspended matter content in the fixed-bed plant effluent [117, 119].
(Evidently, protozoa find optimum living conditions on the filter installed in the
activated sludge tank, an adequate oxygen supply and plenty of food, so that the
dense population of mainly ciliates even crowds out attached bacterial growths.
In contrast to the common activated sludge process, where ciliates contribute to
about 10% of the total, bacteria dominated biomass, an almost inverse relation
of 68% protozoan and 32% bacterial biomass (dw) is found for the biofilms of
submerged fixed-bed filters [116].)
2.3.3
Elimination of Dissolved Substances
The bulk of dissolved substances entering the wastewater treatment plant are
amino-acids, products of protein hydrolysis in the sewage system, and fatty
acids. Carbohydrates are usually completely degraded in the sewage before
reaching the plant.
Although many protozoa can take up organic substances [85, 89, 120, 121],
their contribution to the degradation of these substances in wastewater treat-
ment plants is negligible: For these substances the essential activity comes from
the bacteria population. They dominate the biomass and possess a higher me-
tabolic efficiency as a result of their high surface to volume ratio [41, 4648, 67].
An impression of the different degradation efficiencies can be gathered from
measurements of amino-acid uptake by Escherichia coli and T. pyriformis [122].
Even under the assumption that all ciliates present in wastewater treatment
plants can metabolize not only bacteria but also dissolved substances similar to
T. pyriformis, the experiments reveal an 80-times higher uptake of amino-acids
by bacteria. Results from Hrudey [123] can also be well interpreted in the light
of the significantly higher degradation rate of dissolved substances by bacteria.
After addition of peptone, a protein hydrolysate rich in amino-acids, an imme-
diate rise in the bacterial biomass was observed, whereas ciliates were scarcely
able to convert the available peptone into their own biomass and could only re-
produce substantially after the bacterial content increased considerably.
2.3.4
Flocculation and Composition of the Bacterial Community
Apart from the feeding activity of protozoa, another factor is discussed as con-
tributing to the reduction of the content of suspended matter and bacteria in
bench and full-scale plants. In the presence of protozoa, freely suspended, single
bacteria form compact flocs, which then settle [59, 105, 106, 111, 124128].
This is attributed, on the one hand, to polymer, particle-aggregating excre-
tion products (polysaccharides) from protozoa [59, 125], which are possibly re-
leased into the media to facilitate a more effective uptake of particles [24, 129].
232 W. Pauli et al.
On the other hand, this flocculation is believed to be associated with the
exocytosis of indigestible, originally finely dispersed material as a digested
bundle [130, 131], which in turn could serve as a settlement surface for solitary
bacteria [132134]. However, wastewater itself contains a high proportion of
chemically complex particles of differing sizes, and bacteria themselves, domi-
nant with regard to their biomass in wastewater treatment plants, produce ex-
tracellular polymeric substances (polysaccharides), to which they can effec-
tively adsorb [135, 136]. For these reasons, protozoa, by excretion of digested re-
mains and polymers, probably play only a minor role in floc formation in
wastewater treatment plants.
Bacteria feeding itself seems, not only quantitatively but also qualitatively, a
significant stimulus for complex bacterial growth forms. As a result of the pre-
dator-prey relationship between protozoa and bacteria, a collapse of the bacte-
ria population in the activated sludge and a reduced elimination efficiency of
the system as a whole would be expected (see Sects. 2.1.2 and 2.3.3). Such
collapses or phase-shifted oscillations between predator and prey can be obser-
ved in model systems [111, 137141] and in natural ecosystems [20, 142146]
and led originally to the view that protozoa are harmful for the clarification
process [147].
Only a few protozoa, e. g., amoebae, mostly present at low densities in waste-
water treatment plants, are principally capable of taking up larger particles, due
to their ability to entrap their prey. Ciliates, typical representatives of protozoa
found in wastewater treatment processes, possess a highly specialized oral ap-
paratus for highly efficient filtration, which at the same time exclude particles
of several micrometers in diameter [86]. The ability of bacteria to develop larg-
er forms, to grow collectively, or to merge as micro-colonies protects them
against the predator pressure from the protozoa [148153]. The development of
growth forms resistant to filter-feeding can thus be seen as an essential process
in the evolution of bacterial flocs and biofilms [45, 111, 127, 148].
To what extent a qualitative selection of floc and biofilm forming bacteria is
possible [148], and what could be gained from a quantitative shift within a spe-
cies to larger or aggregating phenotypes [45, 149], cannot be decided in the light
of the present literature. Gde [148] observed selection of bacteria populations
which aggregate in pilot wastewater treatment plants. On the other hand
Shikano et al. [149] find that, in the presence of the ciliate Cyclidium sp., phe-
notypes of considerably larger dimensions appear within a bacteria species.
Gurijala and Alexander [154] provide evidence of lower feeding pressure by the
ciliate Tetrahymena thermophila on bacteria with hydrophobic surfaces in
other words on phenotypes with water-repellent properties which enhance
their adhesive, i. e., aggregation, ability [155].
Many bacteria are also capable of organizing themselves spontaneously into
biofilms in the absence of protozoa, thus forming flocs [102, 135, 156].
Nevertheless, the extent and persistence of the flocs seem to be influenced by
the presence of protozoa. Farrah et al. [106] show that in the absence of proto-
zoa autochthonous aerobic bacteria and cultures of Salmonella typhimurium
and E. coli introduced into the sewage sludge are predominantly freely suspend-
ed (4368%). In the presence of protozoa, the proportion of freely suspended
bacteria drops significantly to 115%. The majority can now be found in or
adhering to flocs (8599%); compare also Fig. 10.
Experiments in model wastewater treatment plants [105] show that different
ciliate species induce flocculation to different degrees. With the exception of the
crawling Aspidisca costata, which, even at low population densities, induces
good flocculation when added to protozoan free (pasteurized: 50C, 5 min) se-
wage sludge, the tendency of bacteria to aggregate in the laboratory fermenter
varies considerably for free-swimming (Colpidium campylum), crawling
(Chilodonella uncinata, Stylonichiaputrina), and sessile (Vorticella microstoma)
forms, essentially independent of their population density.
Ciliates feed selectively, not only as shown for Tetrahymena with regard
to the physico-chemical surface structure of their prey [154], but also regarding
the size of the phagocytosed particles: This was shown by Fenchel [86] with fil-
ter-feeding ciliates, characteristic for the ciliate fauna in wastewater treatment
plants. Each ciliate species can only filter specific size ranges of food particles,
i. e., different ciliate species feed in their respective sometimes distinct ni-
ches (Fig. 12). Dependent on the selection mechanism, different effects on the
composition of the bacterial populations and the development of more or less
aggregated growth forms become apparent.
234 W. Pauli et al.
Fig. 12. Clearing rate (volume of water the organisms can clear of particles per unit time
at low particle concentrations, here in multiples of the ciliates own volume per h) for three
ciliate species as function of particle size, from [86]
2.3.5
Reduction of the Total Biomass
In order not to exceed a sludge concentration favorable for the purification per-
formance of the wastewater treatment plant, an amount equal to the daily pro-
duction must continuously be drawn off. This excess sludge is subsequently
concentrated, digested, and drained and must finally be disposed of as a poten-
tially pathogenic and frequently toxic waste product. Excess sludge is therefore
an economic factor, even within the wastewater treatment plant itself. A reduc-
tion in sludge production corresponds to savings in personnel, energy, and run-
ning costs.
Since the function of the sedimentation tank is merely to separate the bio-
mass from the purified water, the effluent concentration must already be attain-
ed in the well-mixed activated sludge tank. The organisms therefore live in an
environment with low nutrient concentrations, resulting in slow growth [46,
67]. The average age of activated sludge (sludge residence time) for organically
burdened municipal sewage, where the main emphasis is on the elimination of
the carbon compounds, is 4 days [41, 46]. If nitrification is an objective, the
sludge residence time increases to 810 days [157]. This means that the sludge
biomass doubles after 4 days, at the earliest. Generation times in this range im-
ply not only stationary growth for the majority of heterotrophic bacteria but
also sub-optimal, reduced growth rates for the ciliate fauna having generation
times of 515 h; see Table 7.
In principal, a lengthening of the food chain results in a reduction of the orig-
inally available energy. In every heterotrophic link, part of the assimilated food
is converted into biomass. The remaining carbon compounds are used as
energy source for metabolic processes. When the chain becomes longer, less
energy will remain locked into biomass. This means more carbon-mineraliza-
tion and less biomass production.
Table 7. Doubling times of activated sludge and ciliates isolated from activated sludge plants
Doubling time (h) Temperature ( C)
Activated sludge
a
3.310 20
Aspidisca costata
b
13.6 20
Aspidisca lynceus
b
12.4 20
Vorticella microstoma
b
5.0 20
Vorticella convallaria
b
7.6 20
Carchesium polypinum
b
9.3 20
Opercularia spec
b
5.0 20
Epistylis plicatilis
b
10.2 20
Colpidium campylum
b
4.7 20
Tetrahymena pyriformis
b
4.5 20
Paramecium caudatum
b
12.0 20
a
[158].
b
[111].
Protozoa assimilate about 85% of readily exploitable nutrients after uptake.
They are converted into individual biomass or respired for energy purposes. The
remaining 15% are eliminated as compact digestion bundles (exocytosis) or dis-
solved substances (excretion) [159]. Under optimal growth conditions, ca. 50% of
the nutrients taken up by protozoa are converted into individual biomass, which
corresponds to the metabolic efficiency of prokaryotes [160]. Different circum-
stances are encountered under inhibited or stationary growth conditions. Here
the emphasis is on basal metabolism, not growth: The metabolic performance is
reduced and energy consumption, as mineralized carbon in the form of CO
2
, in-
creases [128, 161]. This diminished ability to utilize available nutrients for bio-
mass production as a result of reduced growth rates is demonstrated by Ratsak et
al. [128] with Tetrahymena pyriformis. At a high growth rate (generation time of
5.5 h near the optimum of 3.4 h), 51% of phagocytosed bacterial biomass
(Pseudomonas fluorescens) are converted into ciliate biomass, whereas at a low
growth rate with a generation time of 17 h only 39% of the prey is converted into
predator biomass. At the same time the ratio of respired mineralized carbon to
that converted into cell biomass increases from 0.65 to 1.2.
In municipal activated sludge plants ciliates are present in densities of
10
4
ml
1
and over [61, 75, 9497]. The number of bacteria required to maintain
this ciliate population can be estimated based on data from Macek [162]. Under
steady-state conditions (20C) and generation times of 5 days, free-swimming
ciliates such as Colpidium campylumand sessile forms such as Vorticella micro-
stoma at densities of 1.3 10
4
ml
1
and 0.59 10
4
ml
1
consume, over the 5-day
period, 2.5 10
9
and 2.1 10
9
bacteria ml
1
(450 and 420 mg COD l
1
), respec-
tively. The bacterial content of sewage arriving at the plant is on average
10
6
ml
1
. With flow-through times of 2 h or more in municipal activated sludge
plants [41], no more than 0.5 10
6
bacteria are available per ml and hour for
the ciliates. Based on the findings of Macek [162], however, a typical ciliate den-
sity of 10
4
ml
1
would require more than 17 10
6
bacteria ml
1
and hour (23
10
5
ml
1
in 5 days). Therefore, the suspended bacterial content in the influent
sewage cannot essentially contribute to the production of protozoan biomass.
To supply adequately the protozoan population a 30-times higher bacterial con-
tent in the influent would be required.
It is known that bacterivorous species are capable of effective filtration and
ingestion of abiotic particles with diameters of 0.35 mm [8587; see also
Sect. 2.3.1] and exploiting them, if possible, for cell reproduction or to increase
individual biomass. Thus in bench-scale plants, the addition of emulsified li-
pids, which form suspended particulate fat droplets, leads to a rapid increase in
sessile ciliates, which can accumulate these lipids in their cytoplasm [123].
Similarly, Tetrahymena is able to convert particulate suspended skimmed-milk
for reproduction, thereby attaining high population densities [163, 164]. It is un-
clear however to what extent particulate abiotic organic materials (e. g., protein
rich colloids from feces) in municipal sewage are suitable, in terms of chemical
composition, size, and content, to be utilized in the biomass production of ty-
pical sewage plant protozoan fauna.
The composition of the ciliate community in wastewater treatment plants is
primarily made up of bacterivorous filter-feeding organisms which efficiently
236 W. Pauli et al.
concentrate and ingest particulate matter the size of bacteria from the sur-
rounding liquid (see Sects. 2.2.3 and 2.3.1). Bacteria occur both in activated
sludge and fixed-bed processes as complex, aggregated cell formations (flocs
and slime growth). Firmly embedded in these structures, they are protected
against their protozoan predators. However, there is a dynamic equilibrium be-
tween flocculation and de-flocculation (see Sects. 2.1.3 and 2.3.4) which, in the
presence of protozoa, shifts towards more complex micro-colonies and, in their
absence, leads to high concentrations of single suspended bacteria (see
Sects. 2.3.2 and 2.3.4). That ciliates indeed can exploit the micro-flora of the
sludge itself as their primary source of nutrition is confirmed by experiments
with sterile synthetic wastewaters, e.g., [103, 123]. Activated sludge with an al-
most exclusively bacterial biomass was supplied with sterile synthetic waste-
water as nutrient source (see Sect. 2.3.3) and nonetheless, a typical protozoan
biocoenosis is developing.
The average sludge concentration at municipal plants is quoted as 23 g (dw)
l
1
[46], which corresponds to ca. 6 10
9
bacteria ml
1
[48]. In conventional
plants this bacterial mass is reproduced in 4 or more days (sludge residence
time). Referring to data from Macek [162], typical ciliate populations in activat-
ed sludge consume 1.52.9 10
9
bacteria ml
1
. In other words, even at shorter
retention times in a plant aimed primarily at the elimination of carbon com-
pounds, a considerable proportion (2548%) of the bacteria can be phago-
cytosed by ciliates: This corresponds to a 1019% reduction of the accumula-
ted sludge, based on a mineralization of around 40% of the bacterial food [128].
Observations with submerged fixed-bed filters in activated sludge plants reveal
a similar picture with regard to the reduction of the accumulated sludge by pro-
tozoa. In the activated sludge tank (volume 756 m
3
) a contact aerator, whose
slime-growth makes up almost 18% of the biomass (dw) of the tank, leads to a
reduction of the BOD sludge accumulation of about 25% [117]. Such sub-
merged fixed-beds are primarily colonized by protozoa whereby ciliates domi-
nate [116, 117, 165, 166], comprising around 68% of the total biomass [116].
Based on these data, an additional biomass of 12% consisting exclusively of
ciliates (18% additional biomass, 68% of it ciliates) effects a sludge reduction
of 25%. A transfer of these results to conventional activated sludge plants
would mean that the autochthonous ciliate fauna, as the second link in the food
chain and representing 9% (dw) of the total biomass [64, 65], is in a position
to reduce sludge accumulation by 19%.
2.3.6
Influence of Protozoa on Bacterial Metabolism
A series of studies on degradation efficiency in bench-scale wastewater treatment
plants show that in the presence of protozoa in spite of their antagonistic effects
as bacteria predators the physiological performance of the bacteria is maintain-
ed or even increased: In bench-scale plants, ciliates show no effects on the nitrifi-
cation bound to flocs [103, 126, 127]. The degradation of nitrilotriacetic acid by
bacteria is equally unaffected by the presence of ciliates; however, here a shift
from single suspended to complex aggregate growth forms is observed [126, 127].
Clear indications of an increase in bacterial metabolic activity were found by
Curds et al. [103]: Under experimental conditions they observed, in the pre-
sence of protozoa, an increased degradation (BOD, COD) of the dissolved, non-
filterable portion of organic materials, attributed almost exclusively to the ac-
tivated sludge flora (see Sect. 2.3.3 and Table 6). Findings by Wiggins and
Alexander [167] also imply a positive influence of protozoa on bacterial degra-
dation processes with regard to the organic pollutants 2,4-dichlorophenol (2,4-
DCP) and 2,4-dichlorophenoxyacetic acid (2,4-D). Although protozoan feeding
reduced the mixed culture of freely suspended bacteria by more than one order
of magnitude leading to delayed degradation compared to protozoa-free cul-
tures after 15 days the environmental chemicals 2,4-DCP and 2,4-D were min-
eralized in the presence of protozoa to 70% and 90%, respectively: Whereas in
cultures where the protozoa were inhibited by nystatin and cycloheximide, de-
gradation of only 40% (2,4-DCP) and 10% (2,4-D) were observed over the same
period (Fig. 13).
In biocoenoses other than wastewater, i. e., in microcosms with pure and
mixed cultures of typical aquatic and terrestrial bacteria, an increased bacterial
metabolism in the presence of protozoa is observed. Various explanatory at-
tempts emphasize the direct influence of the protozoan metabolic activity;
others attach more importance to bacteria feeding and its indirect consequen-
ces on the size and composition of bacteria populations and some correlate the
micro-currents, generated by the ciliates, with an improved food and oxygen
supply of the bacterial flocs or multi-layer biofilms. Protozoa are capable of me-
tabolizing bacterial metabolic products such as acetic-acid, butyric acid, and
ethanol [77, 168] and could thus avert end-product inhibition [169]. On the
238 W. Pauli et al.
Fig. 13. Effects of protozoa on the mineralization of 0.1 mg l
1
2,4-dichlorophenol and 2,4-
dichlorophenoxyacetic acid (2,4-D) in sewage. Cycloheximide (250 mg l
1
) and nystatin
(30 mg l
1
) were used to suppress protozoa; from [167]
other hand, protozoa release a series of organic substances such as amino-acids
[170] and growth factors, having chemical structures not characterized in de-
tail [22, 109, 171175], into the surrounding medium, leading to activation of
bacterial metabolism and growth. Furthermore, protozoa have the highest
excretion rate of inorganic phosphate and nitrogen, relative to biomass, within
the zooplankton [176]. In addition, in the presence of protozoa, an accelerated
bacterial phosphorus mineralization is observed [177]. This mutually advantag-
eous interaction by nitrogen and phosphorus re-mineralization is emphasized
by many authors [22, 145, 177183]. To what extent these additional organic and
inorganic substances introduced into the wastewater cycle play a part in waste-
water treatment processes is controversial, but due to the composition of the
wastewater, rather unlikely [75, 184]. On the one hand, municipal sewage itself
is a complex nutrient solution with a heavy organic load; on the other hand,
nitrogen and phosphorus are present in excess in wastewater treatment plants,
in contrast to most limnic, marine, and terrestrial ecosystems (a BOD: N: P ra-
tio of 100: 5: 1 is considered to be the optimal substrate composition of sewage
compared to this nutrient balance, municipal sewage with average BOD: N: P
ratios of 100: 17: 5 [41] contains an excess of nitrogen and phosphorus).
However, in the case of commercial and industrial wastewaters with high car-
bon loading and comparatively low concentrations of nitrogen and phosphorus
(e. g., vegetable processing businesses, fiberboard works, paper and cardboard
factories, coking plants, as well as chemical and pharmaceutical industries [41,
185]) catalytic effects on bacterial metabolism by interactions with N and P set
free by protozoa are quite conceivable.
It is not self-evident that bacteria feeding and their subsequent reduction of
bacterial populations should have positive effects on bacterial metabolic turn-
over. A possible cause could be the qualitative shifting of the selection condi-
tions for the bacteria and therefore the composition of mixed bacteria popula-
tions and their organizational forms. The success of a bacteria population is not
only dependent on its adaptation to the nutrients on offer but also on whether
it is edible for protozoa [148]. As discussed in Sect. 2.3.4, the selection of feed-
ing-resistant bacterial growth forms can be viewed mainly as a result of pha-
gocytic activity of protozoa: Freely suspended bacteria are succeeded by aggre-
gated, sessile growth forms [136, 148, 186]. That this shift can be accompanied
by a simultaneous intensification of the microbial metabolic processes is shown
by studies on marine bacteria, which as adherent cells in biofilms (marine
snow) display faster growth (incorporation of thymidin into DNA [187]), an
increase in electron transport (reduction of tetrazolium salts to formazan
[188]), and higher hydrolytic activity [189], than as freely suspended single
cells.
2.3.7
Filamentous Bacteria and Protozoa
Filamentous bacteria are present in the bacterial flora of almost all activated
sludge. Due to their large surface area, they are well-equipped for the adsorp-
tion and metabolism of organic compounds. At low densities, they contribute to
the stabilization of activated sludge flocs. However processing problems arise if
mass reproduction of filamentous bacteria occurs in the activated sludge tank.
The enlarged surface area of the flocs hinders the settling and thickening pro-
cesses in the sedimentation tank which can, in extreme cases, due to the forma-
tion of light, fluffy, poorly settling flocs, result in the discharge of sludge into na-
tural waters. This phenomenon, known as bulking sludge, used to be caused
by high load bacteria such as Sphaerotilus sp. or filamentous types 1863 and
0961. Today, however, many of the filamentous bacteria found in sewage treat-
ment are adjusted to low carbon concentrations [low F/M ( food: micro-orga-
nism) bulking], e. g., types 0041, 0675, 0092, 1851, or Microthrix parvicella.
Experience shows that putrid wastewater, rich in H
2
S or with high carbohydrate
or short-chain organic acid content (i. e., wastewater from food processing, pa-
per and textile industries), as well as low nitrogen, phosphorus, or oxygen con-
240 W. Pauli et al.
Fig. 14. Degeneration of bulking sludge (decrease of sludge volume index: SVI) in the aera-
tion tank of an activated sludge plant and in laboratory experiment by the filamentous pre-
dacious protozoan Trochilioides recta; from [194]
tent, stimulates the development of bulking sludge. Various chemical (e. g., lim-
ing, chlorination, addition of H
2
O
2
, iron salts, and nitrogen and phosphorus
compounds) and physical (e. g., increased oxygen supply) methods are imple-
mented to combat bulking. Sometimes even operational conditions of plants
are altered (e.g., increasing the return-flow rate, by-passing the pre-clarifica-
tion, aerobic, and anaerobic selectors) [67, 185].
In principal, autochthonous ciliates appear suited to counteract abundant
development of filamentous bacteria. However, only a few species capable of
taking up filamentous bacteria are present in activated sludge plants, e. g.,
Trithigmostoma cucullus (Chilodonella cucullus), Trochilioides recta, Trochilia
minuta, and Chilodonella uncinata. If these ciliates attain a high population
density, a pronounced decline in filamentous bacteria and degeneration of
bulking sludge is observed within a few days, both in bench-scale and operatio-
nal plants [190193]; see Fig. 14. Effective cell densities for Trochilioides sp. are
quoted as 1000 ml
1
[190] and for Trithigmostoma cucullus and Trochilioides
recta as 2000 ml
1
[193].
3
Impairments of Protozoa: Consequences for Water Purification
Ciliated protozoa are very numerous in all types of aerated biological treatment
systems (compare Sects. 2.2.3 and 2.2.4). They play an important role in the
purification process removing, through predation, the major part of dispersed
bacteria that cause highly turbid, i.e., low quality effluent. It has been generally
recognized that changes in the population density and community structure of
ciliates affect the food web of this artificial ecosystem, thus influencing the per-
formance of plants. Excess influx of toxic wastes with detrimental effects on ci-
liates would prevent clarification, thereby severely threatening the degradation
process. A variety of chemicals can limit growth of ciliates. As with organisms
from other taxonomic, functional, and trophic levels, the toxicological effects
induced by organic and inorganic chemicals on ciliates vary widely, i. e., EC
50
-
values ranking from some mg l
1
to some g l
1
(reviewed by [194, 195]). Sub-
stances having toxic effects which diminish or even paralyze the purification
performance frequently find their way into wastewater treatment plants with
commercial and industrial wastewater. Risks are particularly great from metal-
finishing works with electrochemical processes and wastewater from iron and
steel pickling plants, accumulator-charging stations, stereotype, photocopy,
photographic and printing works, dry-cleaning premises, industries producing
pesticides, herbicides, and disinfectants, as well as tanneries, leather goods
manufacturers and coking plants. In order to estimate the hazard potential and
to lay down maximal concentrations, in addition to bacterial tests, biological
tests with ciliates are indispensable to reflect potential risks of hazardous sub-
stances on the biological system of wastewater treatment as a whole.
Tests with typical wastewater protozoa have been carried out for a number of
toxic substances. Gracia et al. [196] observed effects of copper (sulfate) in con-
centrations of 1 mg Cu
2+
l
1
on species diversity and population density espe-
cially of the ciliates of natural sludge samples. Madoni et al. [197] determined
the 50% lethal effect concentrations of Cu<Hg<Cd<Pb<Cr<Zn (1 mg l
1
50 mg l
1
) on various ciliates isolated from activated sludge, whereby the au-
thors report differences in species sensitivities of up to two orders of magni-
tude, dependent on the heavy metal tested. Kakiichi et al. [107, 198200] report
inhibitory effects of disinfectants and surfactants on typical activated sludge
ciliates. A comparison of the effect potential of 4 disinfectants towards the
wastewater bacteria Alcaligenes faecalis and the wastewater ciliate Colpoda as-
pera reveals an almost 10-fold higher sensitivity of the ciliates [200]. Higher
sensitivities of ciliates in comparison to bacteria were also found by Yoshioka et
al. [201] for 32 wastewater relevant environmental chemicals. Results from the
OECD activated sludge respiration test (RI Test, [202]) considered as an indi-
cator for acute effects of chemicals on heterotrophic bacterial flora and
growth tests with Tetrahymena, a ciliate typical in polysaprobic surface waters,
but also found in activated sludge and submerged contactor plants [53, 55, 111,
112, 115, 203209] were compared: 50% effect concentrations were, on average,
10 times lower with the ciliate test. Furthermore, certain substances proved
highly toxic in the Tetrahymena test, and showed only weak effects in the respi-
ration test; out of a total of 32 substances, just 6 cases had a (toxic) effect po-
tential of less than 100 mg l
1
. The weak correlation of r
2
=0.17 confirms the dis-
crepancy between the two tests (Fig. 15). Similar observations of a low correla-
tion were made by Pauli and Berger [210]. Figure 16 illustrates toxic responses
of 4 ciliate species and standard tests with activated sludge towards industrial
chemicals (data taken from the International Uniform ChemicaL Information
Database, IUCLID, including toxic data of a wide variety of industrial chemi-
242 W. Pauli et al.
Fig. 15. Acute effects of chemicals on the bacterial flora of activated sludge (OECD
Respiration Inhibition Test) in comparison to those on the ciliate Tetrahymena pyriformis
(growth inhibition) and on fish (OECD lethality test with Oryzias latipes); after [202]
cals). Although a generally higher sensitivity of ciliates cannot be observed for
this data set, the random distribution of points around the bisector confirms
the dissimilarity of ciliate and activated sludge toxicities (r
2
<0.01, n=35).
Evidently ciliates are not only sensitive to pollutant induced stress, but test
results reflect a series of additional toxic interactions, not represented by tests
with bacteria in activated sludge. That this different toxic profile is probably due
to the more complex cell-physiological eukaryotic organizational structure
of the ciliates is implied by QSAR studies for heterogeneous chemical classes
[211], which revealed a high correlation between the LC
50
values found in the
widely accepted fish lethality test (r
2
=0.78) with Tetrahymena growth, but not
with bacteria test.
4
Environmental Biotechnological Aspects
4.1
Biodegradation Potentials of Ciliates
Although it is well known that ciliate grazing on bacteria fulfills important tasks
in the biological purification of sewage (compare Sects. 2.3.2.2 and 2.3.2.3) and
that a number of technical methods and plant operation parameters obviously
improve the purification efficiency by favoring ciliate growth (see Sects. 2.2.5,
2.3.2.2, and 2.3.2.3); only recently some pioneering attempts have been made to
specifically use ciliates in biodegradation processes.
Generally, large amounts of biosludge are formed in biological wastewater
treatment processes and the separation, dewatering, treatment and disposal of
this sludge represents major investment and operating costs. One of the poten-
Fig. 16. Comparison of results from standard activated sludge respiration tests and bioassays
with ciliates (data from IUCLID); from [210]
tially useful assemblies for reducing the sludge yield is the two-stage cascade
used in many experiments for the study of ciliate-bacterial interactions, e. g.,
[140, 212215]. The technique of a two-stage system enables one to manipulate
the artificial ecosystem of conventional treatment processes so that dispersed
bacteria are growing in the first part of the process and being consumed by pro-
tozoa in the last. Whereas in conventional treatment due to the growth of floc
or film forming bacteria most of the bacterial biomass is protected against pre-
dation (see Sect. 2.3.4), dispersed bacteria can be readily taken up and meta-
bolized by protozoa (see Sect. 2.3.2), resulting in a lower sludge yield (see
Sect. 2.3.5). Operating the first part of the treatment process as an aerated tank
reactor without biomass retention and at an hydraulic retention time short
enough to prevent a significant growth of protozoa is a simple way to stimulate
this growth of dispersed bacteria. Cultivations using synthetic wastewater and
defined cultures of bacteria and ciliates in a two-stage chemostat cascade have
shown that protozoan grazing can result in a considerable biomass reduction
[128]. By introducing a predation trap (second stage) it was possible to obtain
a decrease of 1243% in biomass yield in comparison with a system without ci-
liate grazing. Studies of Lee and Welander [216, 217] confirm this potential of a
two-stage system to reduce the sludge yield. Employing synthetic wastewater
and mixed cultures of bacteria, protozoa and metazoa from activated sludge
they observed a sludge yield around 3050% of the yields typically obtained in
conventional aerobic processes [216]. If authentic instead of synthetic waste-
water was used as bacterial food supply the sludge production was also con-
siderably lower than in conventional treatment [217].
Cox and Deshusses [218] developed a strategy to control biomass growth in
biotrickling filters for waste air treatment by engineering predation of bacteria
by protozoa. It was shown that clogging of bench-scale biotrickling filters could
be slowed down with the use of protozoa. Interestingly, it was found that the re-
actor with protozoa had a shorter start-up time, possibly because of bacterial
growth factors secreted by the protozoa.
For the biodegradation of whey, the ciliate Tetrahymena had been chosen by
Bonnet at al. [219] as a micro-organism capable of degrading and modifying
the whey biologically in order to diminish its pollutant effect (whey is the
aqueous phase that separates from the curds during cheese making or casein
production). Disposal of crude whey completely arrested operation of lagoon
pilots serving as example of receptor media, whereas the effects of biodegraded
whey were only temporary, and normal operation was recovered within a few
days. The authors stress that this method could be a valuable tool for small
dairy farms, being unable to use complex industrial treatment technologies to
forestall pollution by waste whey.
Clearly, optimal conditions for protozoan activity need to be further evaluat-
ed and pilot scale experiments have to be performed to prove the influence of
biomass predators in real treatment systems. Nonetheless these findings are
auspicious, suggesting that specific use of ciliates can be made to improve bio-
degradation processes.
244 W. Pauli et al.
4.2
Ciliates as Biosensors
As a constitutive group within the microbial food web, ciliates not only play an
important ecological role in the self-purification and matter cycling of natural
aquatic ecosystems, but also in the artificial system of sewage treatment plants.
Their feeding on bacteria improve the treatment, resulting in higher trans-
parency, i. e. lower organic loads in the output water of the treated wastes (see
Sects. 1 and 2). This status of ciliates as an important functional group, improv-
ing the process in municipal sewage treatment, and furthermore that values
from ciliate growth inhibition tests are relevant for the risk assessment for
sewage treatment plants has been recently acknowledged by a Technical
Recommendation of the EEC [220].
There is a broad consensus in ecotoxicology that taxonomic similarity (i. e.,
close relationship, in terms of phylogeny) generally implies similar toxicologi-
cal responses, e. g., [221, 222]. This is reflected in aquatic toxicology by selecting
certain fish, crustacean, and algae species to represent trophic and taxonomic
levels as a whole. A transferability of toxicological data for ciliates is also indi-
cated. Although there exists an extraordinary amount of evolutionary distance
between different genera and even between species of the same genus [223,
224], comparisons between the ciliates Colpidium, Colpoda, Paramecium,
Tetrahymena, Uronema, and Vorticella reveal an almost comparable toxicologi-
cal susceptibility [210]. Despite the lack of standardized ciliate test protocols,
only 2 substances out of 13 exert a toxic effect differing by a factor of more than
100, whereas for the rest of the chemicals the deviations lie within about one or-
der of magnitude (Fig. 17).
The early use of ciliates in toxicity testing was reviewed by Persoone and Dive
[225]. Among the ciliates, the organism of choice in aquatic toxicity testing has
become the common freshwater hymenostome Tetrahymena [195, 226, 227].
Many features have contributed to making Tetrahymena particularly the species
T. pyriformis and T. thermophila favorite models in cell biology and facilitated
their modern day use as aquatic toxicity test organisms. It is worth mentioning,
not only that these unicellular organisms can be grown under axenic, i. e., bac-
teria-free conditions, but also that they combine important advantages from two
groups of organisms. Indeed, they belong to the higher cells, the eukaryotes, but
they can be cultured both easily and economically like the prokaryotic bacteria.
An innovative tool with the potential of a wide application has recently been
offered by the introduction of a commercialized microtoxicity test kit with
Tetrahymena (Protoxkit F, Creasel Ltd., Belgium). The test is specially designed
for the use of environmental samples, thereby providing a helpful means to as-
sess risks of sewage contaminants and their possible detrimental effects on the
performance of waste water treatment plants. Following the concept of ready-
to-use microbiotests, with the test kit a ciliate multi-generation (growth) assay
can be conducted by non-experts without sophisticated sample preparation and
expensive equipment.
Growth impairment tests with Tetrahymena have generally reached the high-
est degree of acceptance and standardization [195, 227, 228]: Based on an inter-
national pilot ring test, a growth test with the ciliate Tetrahymena is recom-
mended by the German Federal Environmental Agency for ecotoxicological risk
assessment [229]. A final ring test to establish an internationally recognized Test
Guideline has been initiated an important step to include a traditionally un-
tested, but ecologically important group of organisms in comprehensive eco-
toxicity test batteries.
5
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226. Schultz TW (1996) Tetrahymena in aquatic toxicology: QSARs and ecological hazard as-
sessment. In: Pauli W, Berger S (eds) Proceedings of the International Workshop on a
Protozoan Test Protocol with Tetrahymena in Aquatic Toxicity Testing. Umweltbundes-
amt-Texte 34/96, Berlin, Germany, p 31
227. Gilron GL, Lynn DH (1997) Ciliated protozoa as test organisms in toxicity assessment.
In: Wells PG, Lee K, Blaise C (eds) Microscale testing in aquatic toxicology. CRC Press,
Boca Raton
228. Pauli W, Berger S (1996) Proceedings of the International Workshop on a Protozoan Test
Protocol with Tetrahymena in Aquatic Toxicity testing. Umweltbundesamt-Texte 34/96,
Berlin, Germany
229. Heger W, Jung S, Martin S, Rnnefahrt I, Schiecke U, Schmitz S, Teichmann H, Peter H
(1998) Chemikaliengesetz Heft 11, kotoxikologische Testverfahren mit aquatischen
Organismen. Texte 58/98, Umweltbundesamt, Berlin, Germany
252 W. Pauli et al.
Predictability of Biodegradation on the Environment:
Limits of Prediction from Experimental Data
Johanna B. Wesnigk
1
, Maike Keskin
2
, Wilfrid Jonas
3
, Klaus Figge
3
,
Gerhard Rheinheimer
4
1
BLG Consult Bremen GmbH, Hafenstr. 55, 28217 Bremen, Germany,
E-mail: Wesnigk.BLG-Consult@t-online.de
2
proDERM, Institut fr Angewandte Dermatologische Forschung, Industriestr. 1,
22869 Schenefeld, E-mail: mkeskin@proderm.de
3
NATEC, Institut fr naturwissenschaftlich-technische Dienste GmbH, Behringstr. 154,
22763 Hamburg, Germany
4
Institut fr Meereskunde an der Christian-Albrechts-Universitt Kiel, Dsternbrooker
Weg 20, 24105 Kiel, Germany
This chapter deals with the description and interpretation of different biodegradation tests
dealing with the degradation of chemicals by mixed microbial communities derived from dif-
ferent natural and semi-natural habitats. Prescribed standardized laboratory tests and their
limitations are listed and compared to more complicated tests systems such as different
simulation tests, micro- and mesocosm tests, and field studies. Where possible examples are
given to illustrate possible outcomes of some tests, using the substances 4-nitrophenol, te-
trachlorobenzene, and NTA. Tests with and without soil or sediments are described. The im-
portance of environmental factors like the concentration of the chemical, size of the degrad-
ing population, grazing, temperature, sorption, and others are illustrated. A focus lies on the
importance of adaptation phenomena. Interactions of the factors and their impacts on the
predictability of biodegradation are discussed and recommendations for further research as
well as management advice are given.
Keywords. Biodegradation, Predictability, Adaptation, Test systems, Environmental factors
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
2 Typical Environmentally Relevant Substances . . . . . . . . . . . 256
2.1 4-Nitrophenol (4-Np) . . . . . . . . . . . . . . . . . . . . . . . . . 256
2.2 Chlorobenzenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
2.3 Nitrilotriacetate or Acetic Acid (NTA) . . . . . . . . . . . . . . . . 256
3 Analytical Methods and Their Implications . . . . . . . . . . . . . 257
3.1 Analytical Methods Used in Screening Tests . . . . . . . . . . . . . 257
3.1.1 Dissolved Organic Carbon (DOC Die-Away Test and Modified
OECD Screening Test, MOST) . . . . . . . . . . . . . . . . . . . . . 257
3.1.2 Biochemical Oxygen Demand (BOD Closed Bottle Test) . . . . . . 259
3.1.3 Carbon Dioxide (CO
2
Evolution Test, Previously Called
Modified Sturm Test) . . . . . . . . . . . . . . . . . . . . . . . . . . 259
3.2 Common Features for All Screening Tests
for Biodegradability . . . . . . . . . . . . . . . . . . . . . . . . . . 260
3.3 Radioanalytical Methods . . . . . . . . . . . . . . . . . . . . . . . 260
3.4 Analytical Separation and Quantitation Methods . . . . . . . . . . 261
3.5 Methods for Identification of Compounds . . . . . . . . . . . . . . 262
CHAPTER 4
(ed. by B. Beek)
4 Which Test Systems are Used for Which Purpose? . . . . . . . . . 262
4.1 Screening Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
4.2 Simulation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
4.2.1 Degradation Studies in Water/Sediment Systems . . . . . . . . . . 266
4.2.1.1 Limitations of Simulation Studies Using Soil or Sediments . . . . 268
4.2.2 Possibilities to Predict from Simulation Test Results . . . . . . . . 268
4.3 Mesocosms and Field Studies . . . . . . . . . . . . . . . . . . . . . 269
4.3.1 Possibilities to Predict from Mesocosm and Field Test Results
and Comparison of Test Systems . . . . . . . . . . . . . . . . . . . 271
5 Potentials and Limitations for Prediction
from Laboratory Results . . . . . . . . . . . . . . . . . . . . . . . . 272
5.1 Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
5.2 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
5.3 Sorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
5.4 Oxygen Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
5.5 Sediments and Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
5.6 Grazing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
5.7 Interactions Between Concentration, Growth, Grazing,
and Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
6 What is Persistence, and When is a Substance Biodegradable? . . 283
6.1 A Practitioners View . . . . . . . . . . . . . . . . . . . . . . . . . . 283
6.2 An Ecologists View . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
7 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
7.1 Some Research Suggestions for Better Predictability . . . . . . . . 286
7.2 Some Steps Towards Sustainable Development . . . . . . . . . . . 287
8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
1
Introduction
Of major importance for environmental management are questions relating to
the timely discovery and estimation of the potential dangers stemming from
chemical substances. To do this it is necessary to study two fundamental aspects
of pollutants in detail: their fate and effect in the ecosystem. The fate can be de-
scribed by the paths of transport, the distribution, and the degradation pat-
terns. The effect refers to unwanted, deleterious, toxic or other chronic or acute
effects and their mechanisms.
The prediction of expected paths and distribution patterns of the chemicals
present in the environment, involuntarily or intentionally, is important for
management and therefore often attempted. A synopsis of work related to this
topic to date shows that this question has been looked at almost exclusively
254 J. B. Wesnigk et al.
from a theoretical point of view. The focus of research activities so far has been
on developing models and related calculations. They predict the behavior of
chemicals in the environment according to the underlying theory of the model.
These models are based on assumptions, several of which are specific for
each theoretical model on the nature of the different processes under natural
conditions. As examples may serve:
1. The assumed relevance of a defined balance (steady state) for the distribu-
tion due to the chemical and physical properties of the substance in question.
2. The importance of several chosen transfer constants for the passage through
boundaries between the compartments.
3. The kinetic model used for the degradation of the chemicals in the separate
compartments.
In addition, the properties of the environment itself necessitate assumptions
specific for each model, i. e., whether it can be represented by homogenous com-
partments with related, amenable volumina. This chapter will try to give an an-
swer to the important question as to whether degradation patterns calculated
from models or laboratory test results are conforming to the results of measur-
ing the reaction more directly in a natural environment. Furthermore, com-
monly used test procedures will be described and evaluated in this regard.
In addition to the degradation characteristics, it is necessary to evaluate the
behavior of chemicals in the environment to gain reliable data on their ecotoxi-
cological effects. This evaluation is complicated by possible synergisms and an-
tagonisms. A pollutant can have different effects when entering an organism in
different ways. Certain pollutants can be present in varying physical and chem-
ical states, which might change their toxicity, while others may be very specific
or quite broad ranging in their effects. A recent example for a new pollutant ef-
fect are the hormone mimicking substances or endocrine disruptors. They are
chemically unrelated but have a structural element in common which in several
cases locks onto estrogen receptors, thus amplifying and confusing hormonal
signals in very low doses.
It has been surprisingly hard to identify and causally relate unwanted effects
in nature to a single substance. However, todays development of modern eco-
toxicological methods makes it possible to determine changes in the environ-
ment and relate them to pollutants. In order to analyze effects it is not enough
to look only at segments of the ecosystem in question. To get the full picture it
is necessary to study all ecosystem levels including all compartments, i. e., wa-
ter, soil, air, and biota.
This leads to the following conclusions: practical experiments regarding
transport, distribution, degradation, and effect of chemical substances in ecosys-
tems will continue to play an important role in preventative environmental re-
search. These kinds of studies are difficult in open, natural systems. Thus stan-
dardized laboratory tests or small scale ecosystem models later named micro-
or mesocosms have to be used. If one of these systems is used, the following
questions regarding the transfer of laboratory results to the field will arise:
Can the results obtained in simplified microcosms reflect the reality in com-
plex natural ecosystems?
Predictability of Biodegradation on the Environment: Limits of Prediction from Experimental Data 255
Is it acceptable to use laboratory data for the calculations in theoretical eco-
system models?
To what extent can a cost-effective simplified microcosm be used as a prac-
tical model to obtain results relevant for the environment?
This chapter will mainly focus on the question of how far experimental labora-
tory data can predict the degradation of chemicals in nature.
2
Typical Environmentally Relevant Substances
The following substances were chosen as examples. Their degradation pathways
are presented in chapters 1 of this volume.
2.1
4-Nitrophenol (4-Np)
This substance was studied extensively by several authors. It shows a somewhat
erratic behavior in standard tests, being sometimes mineralized and sometimes
not [1, 2], and is therefore classified as a typical gray list substance. In the
United States 2- and 4-nitrophenol are classified as priority pollutants [3]. 4-Np
is supposedly a major degradation product of the widely used insecticide para-
thion and can be generated in the atmosphere from car exhausts like benzene
and NO
x
[4]. The degradation pathway has been identified [5] but there have
been indications that 4-Np can be metabolized to the more persistent 4-amino-
phenol instead of being mineralized [6].
2.2
Chlorobenzenes
Chlorobenzenes with different chlorination grades can be found worldwide in
all compartments of the environment [7, 8] and even in human blood and fat
tissue [9] since they are used as solvents or heat conductors and as basics for the
production of insecticides, other biocides, and colors [10]. They have been clas-
sified as priority pollutants by the US Environment Protection Agency [3].
Their high persistence is mainly due to chloro-substituents which lower the
electronic density of the aromatic ring usually cleaved by electrophilic oxy-
genases [11]. Therefore, highly chlorinated aromatics cannot be degraded by
most living organisms. Only for the last fifteen years several bacterial cultures
capable of using chlorobenzenes as sole source of carbon and energy have been
isolated [12, 13]. This implies the evolution of new strains which have adapted
themselves to the environmental contamination.
2.3
Nitrilotriacetate or Acetic Acid (NTA)
This detergent builder and chelating agent has been intensively studied before
and during its large scale introduction into surface waters as a phosphate sub-
stitute in Canada [1416]. It can be degraded after an adaptation phase and it
can interact with metals [1719]. There is still some doubt about the concen-
trations and temperatures necessary for adaptation and about its degradability
in seawater [2022].
3
Analytical Methods and Their Implications
Chemical analytical work in biodegradation studies has the following aims:
The qualitative and quantitative determination of the test substance and of
its known metabolites at different time points and the spatial distribution in
the test system, including a balance of substance distribution and metabo-
lization.
The identification of unknown metabolites occurring during the study in or-
der to develop a degradation pathway.
The determination of the proportion of total mineralization.
These objectives can best be achieved by using radiolabeled compounds in
connection with suitable radiochemical, chemical, and physical analytical me-
thods. However, before carrying out sophisticated studies using radiolabeled
compounds, it is reasonable to perform screening tests in water in order to line
out the readily biodegradable compounds and to focus resources for expensive
studies on problematic compounds and in more heterogenous media such as
soils. This stepwise procedure (i. e., starting the assessment of biochemical de-
gradability with a screening test) is generally required for registration of a chem-
ical in Europe.
3.1
Analytical Methods Used in Screening Tests
The most commonly used screening tests for testing biodegradability are listed
in Table 1 [23]. One common feature in these tests is the analysis of sum para-
meters as measures for the biodegradability instead of analyzing the test sub-
stance directly. The most obvious advantage of analyzing sum parameters is the
low cost. Therefore these tests are the most effective way of testing a large num-
ber of chemicals. In many cases, these test give satisfying answers to the ques-
tion if a chemical is readily degraded. The limits of the screening tests in gen-
eral will be given below. In this section, the principles and the limits of the ana-
lytical methods are described.
3.1.1
Dissolved Organic Carbon (DOC Die-Away Test and Modified
OECD Screening Test, MOST)
The principle of this method is the oxidation of the organic matter in a sample
by combustion or by using a strong oxidant and measurement of the infrared
(IR) absorption of CO
2
by an IR-detector. If the sample is used directly, the to-
Table 1. Test methods for screening tests for ready and inherent biodegradability [23]
Test name Parameter Concen- Inoculum [cfu
a
/ml] Suitable Suitable
for analysis tration for poorly for volatile
soluble soluble
substances substances
Ready:
DOC Die- DOC 1040 mg 10
4
10
5
; active sludge,
Away DOC/l sewage effluent, surface
water, soil or mixture
CO
2
evolu- CO
2
1020 mg 10
4
10
5
; see above +
tion DOC/l
Modified DOC 100 mg/l 10
4
10
5
; mixture of 10 dif- +/
MITI
b
(I) ferent sites, incl. Industrial
sewage effluent, acclimation
in lab. 13 months
Closed bottle BOD 210 mg/l 1010
3
; secondary sewage +/ +
effluent (domestic), and/or
surface water
Modified DOC 1040 mg 10
2
; secondary sewage
OECD DOC/l effluent (domestic)
screening
Manometric BOD 100 mg/l 10
4
10
5
; like DOC + +/
respirometry Die-away test
BODIS
c
BOD 100 10
4
10
5
; supernatant of + +
ThOD/l settled activated sludge
(domestic)
Special test:
Biodegrada- DOC or 540 mg No cfu-conc. prescribed, +/ +/
tion in BOD DOC/l seawater
seawater
Inherent:
Modified DOC 20 mg/l >10
5
; activated sludge from
SCAS
d
a suitable treatment plant,
adaptation permitted
Modified DOC 10100 DOC (inoc.) /DOC
Zahn- mg/DOC/l (test subst.) = 2.5/1 to 4/1;
Wellens activated sludge; adapta-
tion permitted
Modified BOD 30 mg/l >10
5
; like MITI (I) +
MITI
b
(II)
a
cfu cell forming units.
b
MITI Ministry of International Trade and Industry, Japan.
c
BODIS BOD-Test for Insoluble Substancesa modification of the Closed Bottle Test, issued
by the German Umweltbundesamt.
d
SCAS Semi-continuous activated sludge test.
tal carbon (TC) is determined. If the sample is filtered or centrifuged before
measurement, the total dissolved carbon is analyzed. For separation of organic
and inorganic carbon (carbonates), the sample is acidified and the CO
2
of inor-
ganic origin is stripped before oxidation. This means air or inert gas is blown
through the sample. After this pretreatment, the DOC can be analyzed. Limits of
this method are:
Volatile organic test substances or metabolites are not detected.
The limit of detection is approximately 2 mg DOC/l.
This means that the starting concentration needs to be a minimum of
7 mg DOC/l in order to allow measurement of at least 70% degradation.
3.1.2
Biochemical Oxygen Demand (BOD Closed Bottle Test)
The determination of biodegradation by means of BOD measurements is an in-
direct method. The theoretical oxygen demand (ThOD) from the complete oxi-
dation of a test substance is calculated on the basis of the formula of the test
substance or by measurement of the chemical oxygen demand (COD). In
screening tests using this parameter the oxygen depletion is determined during
the tests using oxygen sensitive electrodes or manometers. The measured oxy-
gen depletion is compared to the theoretical oxygen demand. Limits of this
method are:
It is only applicable for aerobic degradation.
The limit of detection is 45 mg ThOD/l.
This means that the starting concentration needs to be a minimum of 10 mg
ThOD/l in order to allow measurement of at least 60% degradation.
3.1.3
Carbon Dioxide (CO
2
Evolution Test, Previously Called Modified Sturm Test)
The CO
2
evolution test is also an indirect method. The theoretical CO
2
evolution
(ThCO
2
) after complete oxidation of a test substance is calculated on the basis
of the formula of the test substance. The CO
2
produced during the tests is trap-
ped in barium or sodium hydroxide and is measured by titration of the residual
hydroxide, or as inorganic carbon. The CO
2
evolution is compared to the
ThCO
2
. Limits of the test are:
It is not applicable to volatile test substances or metabolites.
It is relatively laborious compared to other screening methods.
The limit of detection depends on the concentration and amount of barium hy-
droxide in the traps (it corresponds to the limits given in the DOC Die-Away
test).
3.2
Common Features for All Screening Tests for Biodegradability
The following features are those all screening tests mentioned so far have in
common:
The test media are mineral media containing prescribed concentrations of
potassium and sodium phosphates plus ammonium chloride, magnesium
sulphate, and iron (III) chloride. For the modified OECD screening test, trace
elements (Mn, B, Zn, Mo) and yeast extract are added.
A reference compound is run in parallel to check the operation of the proce-
dures.
Pass levels for ready degradability are 70% removal of DOC and 60% of
ThOD or ThCO
2
production.
For ready biodegradability tests, the inoculum may be pre-conditioned to the
experimental conditions but not pre-adapted to the test substance. For inhe-
rent biodegradability tests, this pre-adaptation is allowed.
Test duration is 28 days for ready biodegradability tests. It can be shortened
if the substance is degraded before the end of this period. It can be extended
if the plateau phase has not been reached. The test duration of the inherent
biodegradability tests depend on the individual adaption periods.
The incubation temperature is 20C or room temperature.
3.3
Radioanalytical Methods
The most common isotope used for environmental studies is
14
C, simply be-
cause carbon is the most common atom in organic compounds and because of
the relatively easy detection methods available for this carbon isotope. If the
synthesis of a [
14
C]-labeled compound is too complicated, it may be necessary
to use
3
H. The use of this isotope, however, may cause problems in the interpre-
tation of the data since
3
H is known to be replaced intramolecularly. If unknown
degradation products are expected, additional labeling with
13
C,
32
P,
15
N,
35
S,
36
Cl,
19
F, and others may be reasonable in order to facilitate the interpretation of
NMR- and mass spectrometer data.
The position of the label within the test substance is very important for the
study design. Generally, a
14
C-label will be positioned in the part of the mole-
cule which is the least susceptible for biodegradation in order to be able to trace
as much of the metabolites as possible. If a molecule carries a short ester group,
for example, this group will normally be quickly separated by enzymatic or
non-enzymatic ester cleavage. If this group carries the radioactive isotope one
will find a quick degradation but will not know what happened to the major
part of the compound. The most clear-cut results can be expected if the test
substance is labeled with one atom in a hardly susceptible position such as the
ring of an aromatic system. Two labels in one molecule may increase the sensi-
tivity but may complicate the interpretation of the results, especially if metabo-
lites have to be identified.
The following equipment and radioanalytical methods are commonly used:
Liquid scintillation counter (LSC) suitable for counting b-decay events in
samples mixed with scintillation cocktails. The number of decay events per
minute is a measure for the amount of test substance present in the sample.
This apparatus is absolutely necessary.
Combustion apparatus: this apparatus is necessary for determination of
samples bound to or present as insoluble solid compounds such as soil or
sediment, plant or animal tissues. A defined amount of the sample is weighed
into a cellulose cone, this cone is transferred into the combustion coil (Pt-Rh-
alloy) and combusted at 800C in an oxygen flux. The combustion gas con-
tains besides other non-labeled compounds the residues of the radiola-
beled test substance as
14
CO
2
. This radiolabeled carbon dioxide is collected in
an automatic trapping system containing a special absorber scintillation cock-
tail. The radioactivity in this cocktail is determined in an LSC (see above).
Linear analyzer for radio thin layer chromatography (radio-TLC-scanner):
the TLC-scanner enables the detection and quantitative determination of
compounds separated by thin-layer chromatography.
Digital autoradiograph (DAR): a detection system for samples separated by
two-dimensional TLC or for sliced tissue samples containing a two-dimen-
sional pattern of radioactive spots.
Radio-high performance liquid chromatography (radio-HPLC): a normal
HPLC-apparatus with a detector for radiolabeled compounds. There are two
general principles:
a) The detector contains a solid scintillator.
b) A scintillator liquid is mixed with the eluate of the HPLC-column.
In both cases, an integrator processes the signals of the counter in the
detector and calculates the peak area resulting in a measure for quantifica-
tion of each compound separated in an HPLC-run.
Radio-gas chromatography (radio-GC): this method is only used in few cases
when substances with high volatility are investigated.
3.4
Analytical Separation and Quantitation Methods
The analytical procedures for separation and quantitation of non-radiolabeled
compounds are similar to the methods listed for radioanalytical methods: TLC,
HPLC, and GC. The main difference is the detection method and the fact that
more effort is necessary for separation of the test substance and its metabolites
from compounds occurring naturally in the respective matrix. All results there-
fore have to be compared to blank values of the particular matrix. In addition,
the samples more often need more extensive pretreatment (clean-up proce-
dures) before injecting them into the chromatographic systems.
For the preparation of samples for chromatographic analysis one or more of
the following steps may be necessary:
Homogenization of the matrix
Liquid extraction from the matrix (e. g., in a Soxhlett apparatus)
Separation of polar and nonpolar components by:
Liquid-liquid separation
Solid-liquid separation
Derivatization (e. g., esterification)
Commonly used GC-detection methods are:
Universaldetectors such as the flame ionization detector (FID): suitable for
substances which are well combustible.
Selectivedetectors such as the electron capture detector (ECD): suitable for
substance with high affinity for electrons such as molecules containing halo-
gens or aromatic compounds with nitro- or cyano substituents.
Specific detectors such as the nitrogen/phosphorus selective detector: suit-
able for substances containing several atoms of nitrogen and/or phosphorus.
Mass spectrometer (MS): GC-MS is normally used for identification pur-
poses but can also be used for the quantitation of compounds not detectable
with other GC-detectors.
Commonly used HPLC-detection methods are:
UV/VIS-detector: suitable for substances absorbing UV- or visible light.
Polarization detector.
Mass spectrometer (MS): HPLC-MS is normally used for identification pur-
poses but can also be used for the quantitation of compounds not detectable
with other HPLC-detectors.
Besides the methods mentioned above, the eluates from the chromatography
columns can be collected and analyzed using any suitable method not mentio-
ned before (nucleo-magnetic resonance NMR, atom-absorption spectrometer
AAS, or infrared spectrometer IR).
3.5
Methods for Identification of Compounds
Before attempting the identification of a certain compound in a mixture, this
compound has to be separated from the other compounds present in this
mixture. The separation methods are the same as mentioned above. For identi-
fication, the separation method is combined with a mass spectrometer system
(GC-MS or HPLC-MS) or with NMR-analysis. The function of these detectors is
described in the common analytical literature.
4
Which Test Systems are Used for Which Purpose?
Some biodegradation tests have been used for quantitative risk assessment
which is mostly focused on toxicity data. For example, in the GESAMP hazard
evaluation procedures (with 6 overall criteria (AF) organized in 19 sub-co-
lumns), biodegradation features only in subcolumn A2, whereas toxicity-related
parameters are present in all other criteria (BF) [24]. This overall process re-
views chemical, biological, and physical properties to evaluate the fate of a chem-
ical in the environment. In the past it has been used mostly for new chemi-
cals, for which test data have to be submitted for registration in advance, e. g.,
under the Toxic Substances Control Act in the USA or the EC Directives 67/548,
79/831, 91/414 and 92/32, and related national legislation in the European
Union.
4.1
Screening Tests
Two extremes in biodegradability testing are screening tests for ready and in-
herent biodegradation and field tests. If these different test systems are com-
pared, the analytical methods have to be considered, as they and the test setup
itself have a major influence on the validity of predicting biodegradation in na-
tural environments. For screening tests indirect methods are mostly used, de-
termining parameters like oxygen demand (BOD or COD) or the changing con-
centrations of dissolved organic carbon (DOC). This is done for the sake of easy
determination and to get an indication of the extent of degradation, but it has
some drawbacks. When the different screening methods were tested against
each other they sometimes gave the same relative order of biodegradation of
different chemicals but the time frame for each of them was different [25].
Other authors found widely differing results, especially for those compounds
used as examples in this chapter [1, 26].
Only the biodegradation of chemicals studied with the same test can be eas-
ily compared, and therefore the name of the test should always be mentioned
[27]. These authors compared the degradation of several chemicals in the Zahn-
Wellens-, the MOST-, the Closed-Bottle-, and in their self-devised GSF-Test (for
details of the tests see Table 1). For 4-nitrophenol (4-Np) the results ranged
from 55% degradation to only 1% mineralization and 35% metabolites formed.
For hexachlorobenzene only one test was appropriate, the GSF-test using 50 mg/l
of radiolabeled substrate, as the solubility of this chemical was too low. It resul-
ted in 1% CO
2
and metabolites each. The different drawbacks of screening tests
like problems with water insoluble compounds and the high initial concentra-
tions needed are discussed in more detail above and in Howard et al. [25] and
Struijs and van den Berg [28].
Most of the screening tests only yield qualitative results to classify substances
in groups:
1. With the potential to be (readily) biodegraded, so-called soft or white list
compounds.
2. With the potential for degradation under specific circumstances (e. g., in the
Zahn-Wellens Test with a very high population density), leading to gray list
compounds.
3. With no biodegradation in the test period, leading to hard or black list
compounds.
The results cannot be used to determine degradation rate constants, and there-
fore they cannot be used for a quantitative prediction of the fate of a chemical
in a natural environment. For a prediction it needs to be known how fast and to
what extent a substance will be mineralized under natural conditions.
This should be obvious if the inocula are considered (see Table 1). For almost
all screening tests sewage or sludge is or can be used, sometimes even elaborate
concoctions of the two with soil added (MITI-test). To determine whether bac-
teria with the ability to degrade a certain chemical exist, this approach a clas-
sical bacteriological technique called enrichment culture seems feasible. But
it cannot under any circumstances be used to extrapolate to degradation rates
in nature, i. e., in a surface water body, where several environmental biotic and
abiotic factors are at work simultaneously. This holds true especially when little
or no waste water input is received by the surface water.
Some tests may show the potential for degradation of a chronically polluted
river (MOST, Closed Bottle), but their results cannot be regarded as more than
a potential. In reality the key controlling feature is the environment, which in-
cludes the substrate [29]. Very often laboratory tests are based on environ-
mental irrelevancies, using unnaturally high concentrations of chemicals and
ignoring important microbial interactions [30].
Even the applicability of some screening tests using sewage or sludge, e. g.,
the Zahn-Wellens Test, to what is happening in real sewage treatment plants re-
mains questionable, as the residence time of waste water in such a plant may
vary but is mostly short.
To sum up, screening tests can be used to rank chemicals in a specific rela-
tive order of potential biodegradability if the same test and standardized refe-
rence compounds are used. The results can give management advice insofar as
new substances can be grouped into white, gray, and black lists accordingly. The
black list substances should not be registered or should be phased out as soon
as possible. The gray ones should be submitted to additional testing, e. g., simu-
lation or field tests using natural conditions, i. e., low concentrations, low tem-
peratures, or low oxygen concentrations. This procedure may lead to differen-
tiated results, advising phase outs or restrictions of use/emission in certain en-
vironmental compartments. During this evaluation period the precautionary
principle should be enacted, i. e., no registration for new substances.
The white compounds which are degraded easily in all screening tests for
ready biodegradation may not be a problem. But depending on their mode of
application they may not end up in adequate sewage treatment plants. If they
can get into the environment or monitoring studies already prove they are
there, simulation tests are necessary to determine degradation rates in the rele-
vant compartments and to identify problem areas as described for gray list sub-
stances above.
4.2
Simulation Tests
The next steps in testing are the much more specific simulation tests. They are
an opportunity and a risk at the same time as much needs to be known about
the simulated environment. Very important are the source of the inoculum and
its preconditioning, like acclimation to laboratory conditions, adaptation to the
chemical under study, or pre-adaptation to it in the environment where the
sample was taken from.
Instead of using mineral salts media, natural water, sediment, or soil samples
are mostly used. These are brought to the laboratory and treated or stabilized,
e. g., by filtering, acclimation in an aquarium, or other methods. Most simula-
tion tests are static tests, but in some systems a flow-through mechanism is
maintained. Another important feature is the means of agitation, as the oxygen
concentration in samples with sediment or soil needs to be maintained in an
enclosed system. This can be achieved by stirring, shaking, or by continuous
flow of aerated water or air through the system.
Since 1955 the River Die-Away Test has been used. It has been recommended
by the US-American EPA since 1978 for biocide registration, requiring radio-
labeled test compounds [25]. A major advantage of this technique is the mea-
surement of mineralization of the test compound with a sensitive and specific
method allowing low concentrations to be used. A drawback is the use of water
without sediment.
Therefore the core-chamber method with intact sediment cores came into
use, as described, together with other systems, by Bourquin et al. [31]. These
eco-cores are artificial laboratory microcosms that closely mimic natural con-
ditions for studying microbial interactions. They belong to the group of micro-
cosms, of which an array of different systems exist. They are considered simpli-
fied small-scale models for ecosystems and can consist of more than just water
and sediment components. Often they are used to quantify transport, bioaccu-
mulation, and toxicity mechanisms, sometimes using flow-through systems,
which limit the degradation. Every method has to be studied closely to deter-
mine the advantages and drawbacks of the system, and of the environment sim-
ulated.
A good example, how the size and setup of microcosms can influence results,
is shown by Spain et al. [32]. In their experiments with flasks, eco-cores, and two
other kinds of larger microcosms, the rate of biodegradation of 4-Np varied
with the type of test and the adaptation status of the inoculum from initial ra-
tes of 0.020.48 mg l
1
h
1
to rates from 2.5617.82 mg l
1
h
1
(after 88460 h of
adaptation).
Very often mass balancing is not done in microcosm studies, and therefore
the disappearance of the substrate has to be interpreted with care, especially
when the compound under study is not used in its radiolabeled form. The use
of proper abiotic controls is absolutely necessary. This was shown by van Veld
and Spain [33] in their experiments comparing the degradation in shake flasks
with and without sediment with that in intact sediment/water cores, in which
they could follow the mineralization. Only in the eco-cores and in one sediment
shake flask a significant decrease of 4-Np was observed. But only 613% turn-
ed out to be carbon dioxide, 3965% was tightly bound to the sediment (in the
abiotic control 23%), pointing to degradation products with stronger sorption
capacities.
Shaking a sediment fully changes its properties, mainly enhancing the ac-
tivity and available number of the microflora and also facilitating adsorption by
exposing more surfaces. This phenomenon may be naturally occurring, e. g., in
tidal waters, but as a rule sediment and soils are more stable. Therefore un-
disturbed sediment samples should be used to simulate biodegradation in most
natural environments.
For a good simulation test the objectives and environmental conditions sim-
ulated have to be clearly stated as there is no fixed definition for a simulation or
a microcosm test. As the design has a large influence on the kinetics and the ex-
tent of degradation, the size, the sediment/soil to water ratio, and the supply
with water and oxygen has to be appropriate for the simulated environment. It
should be the same when the mineralization of different chemicals is compared.
4.2.1
Degradation Studies in Water/Sediment Systems
As an example for a typical simulation test for the investigation of biodegration
in soil or sediments, one exemplary procedure used for water/sediment studies
is described in detail below. Other studies using soil as a matrix have different
intentions but they are similar with respect to the methodology and the limita-
tions posed by the analytical methods.
Water/sediment studies are required especially for the registration of new ac-
tive ingredients used in biocides (plant protectives) if the standard laboratory
tests have not been positive. Since there are no international guidelines, some
countries have developed national ones. The German guideline was issued by
the Biologische Bundesanstalt (BBA) in Braunschweig [34].
Two water sediment systems are collected from an uncontaminated freshwa-
ter, differing in the organic carbon content. The water is characterized by deter-
mination of oxygen concentration, redox potential, pH, total N, total P, DOC,
and water hardness. The sediment is characterized by determination of particle
size distribution, organic carbon, pH, total N, total P, microbial biomass, dry
matter, redox potential, and cation exchange capacity.
Prior to the test, the water is separated from the sediment. Weighed amounts
of the wet sediment and of the water are transferred, resulting in a sediment
layer of 2.5 cm and a supernatant water layer of 6 cm. The flasks are placed on
an orbital shaker in the dark at 20C and are allowed to equilibrate for approxi-
mately 4 weeks respectively, before the
14
C-labeled substance is applied. The
amount to be applied is calculated on the basis of the maximum field applica-
tion rate and, assuming a homogenous distribution, to a depth of 30 cm in the
natural system (water of creek/lake close to fields).
Through stirring, aerobic conditions are achieved in the water, whereas a
gradient in the redox potential is maintained in parts of the sediment (see
Fig. 1). As an alternative, anaerobic conditions can be achieved if nitrogen is in-
troduced into the aqueous phase in the flasks, for example by introducing a
small Teflon tube through the septum.
The experimental conditions analyzed are dissolved oxygen, redox potential,
temperature and pH in the water, and redox potential in the sediment. The test
substance concentration is measured by means of radioactivity measurements
and chromatographic methods (TLC and/or HPLC) in order to separate parent
compound from the metabolites. The radioactivity is measured separately for
water and for sediments. The compounds in the sediment can only be charac-
terized after extraction of the radioactivity using organic solvents and/or basic
or acidic conditions. Chromatographic separation of parent compound from
metabolites can be carried out in liquid systems only, whereas determination of
the total radioactivity is possible by submission of sediment samples to com-
bustion. Additionally the radioactivity in the traps is analyzed to determine vo-
latile metabolites and CO
2
.
Water/sediment studies as described above simulate conditions occurring in
the environment very well. The test systems are selected sections of real eco-
systems, the environmental conditions corresponding well to the conditions oc-
curring in summer months in the real environment, only light effects not being
considered. Since the test method allows differentiation between parent com-
pound and degradation products, and since several samples are collected at dif-
ferent time points, the generation and further degradation of the metabolites
can be recorded in the aqueous phase as well as in the sediment. Since a clos-
ed system is used, the study is carried out with radiolabeled test substances and
the volatile compounds are also collected, a total balance of all degradation pro-
ducts can be achieved.
Fig. 1. Water/sediment system as prescribed by the German BBA for the testing of plant pro-
tectives. The incubation flask (volume ca. 1.0 l) is equipped with a device for slow stirring of
the water only, with a trap for volatile components and a septum allowing the introduction of
gas. The trap contains quartz wool impregnated with paraffin oil and soda lime and is per-
meable to air. Sampling is done after 0 h, 6 h, 24 h, 2 d, 7 d, 14 d, 30 d, 60 d, and more than
100 d after application of the test substance. One flask is used for each sampling date
4.2.1.1
Limitations of Simulation Studies Using Soil or Sediments
Studies using complex systems such as soil and sediment have their limitations.
Often there are residues bound to the soil which cannot be extracted using
normal chemical extraction methods. Little is known about these compo-
nents. Depending on the type of test substance, the reasons for the amount
and the occurrence of bound residues can be very different. One mechanism is
the irreversible adsorption of parent compound and/or metabolites to differ-
ent kinds of soil particles. Other mechanisms which are discussed are the in-
tegration of degradation products into humic and fulvic acids or other com-
ponents of the soil. It is also possible that some parts of the test substance are
used for microbial cell compounds such as structural proteins, cell wall com-
ponents, or extracellular polymers which are adsorbed to surfaces of soil par-
ticles. Most studies end up with a certain amount of bound residues and it is
not known how relevant they are for a hazard assessment of the chemical sub-
stance.
Another problem is very polar degradation products which cannot be sepa-
rated in normal phase TLC-systems or in reversed phase systems. In long-term
studies such as lysimeter studies, these metabolites often comprise the main
fraction of radioactivity found in leachates or in soil extracts. These substances
are regarded as a complex mixture of polar components in the soil metabo-
lites of the normal physiology of soil microorganisms and hydrolysis or oxida-
tion products of these metabolites. Since chemical characterization would be
very expensive and would probably give no relevant information, leachates con-
taining high concentrations of these substances (more than 0.5 mg/l test sub-
stance equivalents) are used for ecotoxicity tests. If these leachates have no ef-
fects, e. g., on crustaceans, they are regarded as harmless.
4.2.2
Possibilities to Predict from Simulation Test Results
Simulation tests can lead to habitat specific predictions, especially when dif-
ferent habitats like rivers or lakes are compared with the same test setup.
Alexander and co-workers found differences in rates and extent of mineraliza-
tion in lakes of different trophic status. But additionally the substrate concen-
tration influenced the results, leading to a threshold for degradation or to long
lag times before degradation started in some cases. Under these conditions they
strongly advise against extrapolation from laboratory conditions to natural
waters [30, 35, 36].
Again the results do not represent the absolute truth on the biodegradability
of a substance but they indicate in which environment and under which condi-
tions major problems with a chemical might be encountered. A chemical may
be mineralized in samples from a polluted river or coastal environment but not
in ocean water, or the same may happen in samples with and without sediment
[37, 38].
4.3
Mesocosms and Field Studies
The next steps of sophistication in measuring degradation rates close to natural
ones are mesocosms and finally field studies. The exact line between micro- and
mesocosms is hard to draw: mesocosms are larger and sometimes they are used
outdoors or at least with some outdoor features like a natural light regime. They
can be artificial ponds or canals, large plastic enclosures mounted in a lake or
the sea, open or enclosed aquatic-terrestrial systems, or wholly terrestrial me-
socosms with single or different compartments. Up to six compartments have
been analyzed for the fate of the test compound and its metabolites and the size
varied from a half to hundreds or thousands of cubic meters in volume [39].
Table 2 explains two types of lysimeter, one of which has to be used in
Germany (and Europe) for the registration of biocides if there is reason to be-
lieve that the chemical itself or its degradation products can reach the ground-
water in concentrations of 0.1 mg/l or 0.5 mg/l respectively. Modeling of the dis-
tribution after application, e. g., with the PELMO pesticide leaching model [40]
is used to estimate the potential risk. The test has a duration of 3 years.
A natural soil core is utilized in both cases. The larger closed lysimeter is
more complex as more factors can be controlled. But it has turned out to be too
much of an effort for routine practice. Nevertheless some interesting results
have been obtained and one example will be given below.
Both lysimeters can be used:
1. To obtain substance specific data on distribution, degradation and residues
required for registration.
2. To provide information on the effects of various factors, e. g., application rate
or soil type on distribution and degradation.
3. To enable quantitative analysis of partitioning, accumulation, and the degra-
dation process of chemicals, including the atmosphere to obtain a mass ba-
lance.
Table 2. Comparison of two environmental test systems modelling terrestrial ecosystems [41]
Type of Function Number Size Surface Weather Analysis Analytical Mass-
system and use of (diam. area regime method compart- balance
replicas depth) ments
Large- Legal 2 113 cm 1.0 m
2
natural
14
C P, L, S, Pl No,
scale require- 110120 open
ments cm system
Large- Fate/me- 1 71 cm 0.4 m
2
chosen
14
C P, L, S, Pl, Yes
scale tabolism 50 cm Air
P = Percolation water; L = Leachate; S = Soil; Pl = Plants.
The system uses legally prescribed soil, consisting of loamy sand with low humus content, ta-
ken as an undisturbed core and acclimated to the study site conditions for 2 months: pH =
6.16.3; water capacity = 13.833.5; organic carbon = 0.021.31 (depending on depth).
In all systems the temperature of the lower half of the soil and the amount of rainfall is con-
trolled to stay at 8C and no less than 800 mm of rain.
To give an example of the use of the closed lysimeter, the degradation of te-
trachlorobenzene in two different setups with soil is described and compared.
Figure 2 demonstrates the differences in mineralization of 1,2,4,5-tetrachloro-
benzene in soil in a laboratory study and this mesocosm inoculated with a pure
culture of bacterial degraders. For both studies the same soil and the same
number of cells per gram of contaminated soil was used. The closed mesocosm
or lysimeter used (see Table 2) was incubated under the climatic conditions of
an average day in June in Northern Germany regarding temperature, humidity,
light conditions, and wind during a 24 h day. Only the top 5 cm (33 kg) were
removed, homogeneously contaminated with the chemical and later on the bac-
teria were added and then the contaminated material was reapplied onto the
lysimeter surface; for the flasks the whole soil sample (50 g) was contaminated.
The glass flasks in the laboratory were incubated at 15C, the mean temperature
of the 24 h period of the June day [42].
The degradation process within the controlled mesocosm was much slower
than under similar conditions obtained in a laboratory (see Fig. 2). This result
might have been caused by a multitude of different factors and/or their combi-
nation, possibly the periodical variation of the climate (temperature, water con-
tent, etc.). One can only speculate about the exact reasons, and this example
shows again that there is no simple correlation between data obtained in the la-
boratory and mesocosm data, not to mention natural ecosystems.
Fig. 2. Comparison of mineralization of 1,2,4,5-tetrachlorobenzene by the bacterial strain
Acidovorax sp. PS14 under different conditions. Unsterile soil (BBA standard soil) was used
in glass vessels containing 50 g soil () and in a mesocosm (soil core: 71 cm diameter and
40 cm depth) (b). The tetrachlorobenzene concentration was 10 ppm in the laboratory study,
the inoculum 10
7
cells PS14/g soil, the incubation temperature 15C, the water content 40%
of the maximal water holding capacity. The tetrachlorobenzene concentration of the meso-
cosm was 19 ppm, which was only applied to the top 5 cm (=33 kg), the inoculum 10
7
cells
PS14/g soil with chemical. The mesocosm was incubated under the climatic conditions of a
mean day in June in Northern Germany
Field studies are mostly done in soil systems, and sometimes with ponds or
small sections of rivers [32, 43]. The mesocosms used by Kuiper and co-workers
[44, 45] are the closest approach to a field study in a marine ecosystem.
A disadvantage of field studies compared to mesocosms is the necessity to
use specific analytical methods to measure the disappearance of the substrate,
leading to indirect evidence of biodegradation only. Besides, there is no possi-
bility for abiotic controls. A mass balance in a field test and the distinction of
abiotic and biotic processes remain a formidable task and may not be possible
[46].
Accompanying lab tests for mineralization are often done to obtain more in-
sight into the biotic process, but they may not reflect what happens outdoors, as
could be shown by Kuiper and Hanstveit [45]. 4-Np was mineralized in bottles
incubated under almost precisely the same conditions in the laboratory, but not
degraded in the outdoor mesocosms in up to 50 days in their tests.
It seems that the best but most specific system for information on biodegrada-
tion close to in situ conditions is a well modeled mesocosm in which factors at the
ecosystem level, which are thought to be relevant to biodegradation, are at work
but can be controlled. The size has to be chosen according to the system under
study, e. g., for an oligotrophic system the mesocosm needs to be larger [44].
Big differences remain between systems with and without sediment. The im-
portance of integrating the sediment compartment is specific for the chemical
in question, e. g., it is more important for hydrophobic substances, but it should
be done more often, even if analyzing sediment samples is more of an effort.
4.3.1
Possibilities to Predict from Mesocosm and Field Test Results and Comparison
of Test Systems
It has to be kept in mind that it cannot be assumed that the physiological status
of microorganisms is unaltered after a sample of water, soil, or sediment is re-
moved from the field [46]. In his review Madsen states that, despite decades of
debate, the problems of extrapolating from laboratory results to the field have
never been solved, one reason being the impasse due to the methodological
limitations mentioned above. It has been hard to prove degradation in situ un-
equivocally. His recommendation is an elaborate stepwise strategy for deter-
mining in situ biodegradation, explaining the complexities and the necessary
array of parameters to be measured. Only if proof of biodegradation in the field
can be obtained, e. g., by enhanced numbers of protozoan predators or unique
metabolites appearing, the classical microbiological methods of isolation and
enumeration of biodegrading organisms and flask assays can be used as con-
firmatory evidence. Adaptation alone is not conclusive evidence for in situ bio-
degradation [46]. Results can be taken directly for predictions for specific habi-
tats if no or slow change is documented, i. e., by monitoring data. If a fast dis-
appearance is determined, additional lab tests should verify the mechanism,
focusing if possible on mineralization and excluding sorption, leaching, sedi-
mentation, volatilization, or bioconcentration. A promising field seems to be the
study of combined photo- and biodegradation.
Up to this time it remains a challenge to determine in situ biodegradation ra-
tes. As several competing biotransformation processes may act on the sub-
strate(s) under study, the extent of mineralization and the fate of transforma-
tion products need to be studied under field conditions.
A promising approach was shown by Ulrich and co-workers [47]. They com-
pared measurements of the NTA concentration in the Swiss Greifensee with re-
sults of a mathematical model, simulating transport and transformation pro-
cesses. In the epilimnion NTA disappeared with a rate of 0.020.05 per day
without any seasonal trend. They attribute this process to biodegradation.
Several habitats need to be studied, and their selection should proceed ac-
cording to the application and fate of the chemical in question. An extrapola-
tion from fresh water test results to what will happen in salt water should not be
done. Similarly, tests with soil or sewage cannot be used for prediction of the
fate in aquatic systems or vice versa. The most sensitive and exposed habitat
should be chosen through a series of tests and calculations and the fate of the
compound tested in depth in the resulting compartment(s) to derive advice for
management purposes.
5
Potentials and Limitations for Prediction from Laboratory Results
A lot of factors have to be taken into account, which influence the predictability
of biodegradation in nature. A main focus in this chapter will be on the inter-
actions between some of these factors. These interactions can be additive, syn-
ergistic, or antagonistic. A lot of studies have tried to identify and quantify
single factors, but the identification and even more the quantification of the in-
teractions is just beginning.
5.1
Concentration
Starting with the limitations, it should be clear that one of the main influencing
factors is the concentration of the substance in question. It has to be seen in cor-
relation with the number of bacteria capable of mineralizing the chemical
[4850]. Dilution can either bring the number of degrading bacteria down so
that no effect is measurable and/or dilute the chemical so that no growth is pos-
sible anymore. Additionally it is very unlikely that concentrations below 1 mg/l
can induce enzymes, as Hanne et al. [51] determined at much higher con-
centrations for an Arthrobacter and a Nocardia strain. Even if an experiment
with long incubation periods can force the enrichment/activation of degrading
bacteria there still is no guarantee that this will happen in nature where the
organisms are not starved to biodegrade.
An especially interesting phenomenon, which impedes the prediction from
laboratory results, is the threshold for growth of the microorganisms capable of
degrading chemicals [52]. It should be assumed that the interaction between the
substrate concentration and growth is not linear at low concentrations. Van Veld
and Spain [33] noticed the following effect in fresh water sediment cores. When
a first dose of 4-Np was below 10 mg/l, they found a slow degradation rate which
was not increased after a second addition of 10 mg/l. When the first dose was
60 mg/l or higher, a second addition was degraded faster. It seemed that 10 mg/l
was a threshold for adaptation to faster degradation and was very likely related
to the missing growth of the degrader population.
Typical graphs of mineralization after an adaptation phase cannot be model-
ed with models regularly used to describe degradation kinetics. The subtraction
of the lag is arbitrary, and first order kinetics do not fit. Therefore data from this
kind of degradation cannot be used as an input for most models. The multitude
of possible reasons causing an adaptation phase or, as it is often called acclima-
tion phase, singly or in co-operation was intensively studied by Wiggins [53].
Even if a small population is present which can degrade a low concentration
of the chemical in question, the kinetics often differ from that at higher con-
centrations, i. e., with a less steep incline (i. e., a lower rate) and lower final ex-
tent of mineralization as can be seen in Fig. 3.
All samples with 4-Np concentrations ranging from 0.08 mg/l to 250 mg/l
showed no mineralization during the first 10 days of incubation at 20C (Fig. 3).
Obviously an adaptation was necessary before degradation could start. Then
different concentrations of 4-Np were degraded in two different patterns. The
higher concentrations showed a steep increase after 13 days. All concentrations
below 10 mg/l were degraded at a much slower rate and to a lower final extent.
Maximum degradation rates were 0.21%/h for 0.08 and 0.8 mg/l (for 8 no rate
was determined) whereas they were 0.62%/h and 0.77%/h respectively for
80 mg/l and 250 mg/l. Therefore the increase was not proportional, the two high-
Fig. 3. Mineralization of 4-nitrophenol in different concentrations in natural seawater sam-
ples from the Kiel Fjord. The incubation temperature was 20C. Natural mixed communities
and radiolabeled 4-Np were used. The sampling months were December for 8 mg l
l
and May
for all other concentrations
er concentrations showing a much higher degradation rate than could be ex-
pected from extrapolation alone or vice versa.
MPN counts showed that the number of bacteria capable of degrading 4-Np
had increased substantially after 16 days in both incubations with high concen-
trations. At the low concentrations and in a control without 4-Np no degraders
could be measured with the MPN method till the end of the experiment after
35 days (limit of detection >3/ml).
The adaptation phenomenon is complicated as some chemicals need more or
less rare enzymes to be mineralized. These enzymes will not be constitutional
in most cases but will have to be induced as is the case for 4-Np [5]. Therefore
some authors postulate a threshold for adaptation, which may be related to the
formation of these non-constituent enzymes. It should be noted in this context
that a lot of the genetic information for the degradation of xenobiotics is plas-
mid coded, i. e., the ability for degradation of toluene, 2,4-D, or p-chlorobi-
phenyl [54]. All enzymes involved in a plasmid coded pathway are inducible by
the primary substrate [54]. Whether enzymatic adaptation can be the only
reason for the often observed slow or postponed degradation, or if slow or no
growth is an additional factor, has not been resolved for degradation under
close to natural conditions. Probably both factors act together.
An interesting fact is that slow degradation remains possible at low concentra-
tions. But the kinetics are totally different from those at higher concentrations. To
give an example the mineralization of 4-Np in a water sample from the Baltic Sea
is illustrated in Fig. 4. After 80 days of incubation 1 g/l 4-Np was mineralized and
a second addition did not show enhanced degradation (Fig. 4). There probably
Fig. 4. Mineralization of 1 mg l
1
4-nitrophenol in a water sample from the Central Baltic Sea.
After 110 days a second dose of 5 g l
1
was added (A). Or the bacteria were harvested and in-
cubated in freshly prepared sterile media with a second dose of 1 mg l
1
4-Np (B freshly ta-
ken Baltic Sea water, C aged brackish water). The incubation temperature was 20C. The
standard deviation is only shown when it is higher than 1.5%
was no growth and only minimal degradative capacity present in the biota of the
original sample. It was not enhanced by the low dose but it was still present after
a prolonged incubation. Other experiments incubating samples without 4-Np de-
monstrated a loss of the degradative capability in a few weeks [55].
Examples for substances where adaptation is often observed are 4-Np, NTA,
and methyl-parathion [17, 26, 5658]. The frequent practice of just subtracting
these lag periods, sometimes without even mentioning them in the publication,
does not lead to better predictability of degradation in nature. After adaptation
the test system is changed either irrevocably or at least for a prolonged period.
There are obviously different kinds of substrates, those enabling growth,
those only degradable through cometabolism, and probably some in between,
for which other carbon sources may enhance the degradation as they are no suf-
ficient growth substrates themselves. The resulting differences in character-
istics and behavior of the two types of biodegradation were summarized by
Tiedje [59] using NTA as a case study. Important for this chapter are the differ-
ences in kinetics: exponential kinetics with growth vs first order kinetics with-
out growth.
In biodegradation studies there will be a major effect if mineralization with
growth or only cometabolism happens. If growth can happen and is necessary
for the chemical to disappear in an adequate time, anomalous behavior due to
thresholds (for induction and/or growth) is possible as low ambient concentra-
tions will influence this time. If this is compared to cometabolic processes with-
out induction they will also start with a very slow rate, so we may be fooled into
thinking that we see acclimation. The main difference should be that a cometa-
bolic rate does not increase over time, just a certain percentage of the chemical
will be slowly transformed. Robertson and Alexander [60] found that pesticides
not supporting growth at all, like simazine and carbofuran, were not subject to
accelerated biodegradation in soil samples, whereas 2,4-D and propham (IPC)
initially showed a lag-phase, then an increase in degrader numbers and in min-
eralization rate. A second addition was only mineralized faster in the last two
cases. In extrapolation and decision making it is of paramount importance to
know if a substance can be mineralized with growth, because then quantitative
degradation in nature can be expected. Still a prediction of how long this will
take can be difficult. A strong influence of environmental factors on degrada-
tion has to be taken into account, requiring precise rate measurements under
relevant conditions. If a substance is only cometabolized, extremely slow rates
have to be expected in any case and possibly recalcitrant metabolites.
However, the problem with low ambient concentrations not allowing any
growth under natural conditions remains. Can there be adaptation in nature
without growth? This still remains to be proven, thus necessitating a graded ap-
proach towards prediction of degradation in different habitats.
5.2
Temperature
The next factor often overlooked is temperature or to be more specific the in-
fluence of naturally low ones. There is no proof that bacteria react simply as ex-
pected in a chemical reaction for which the Arrhenius equation holds true. The
microbial ecologists have separated natural populations of bacteria into psy-
chrophilic, mesophilic, and thermophilic groups because the incubation tempe-
rature (or that occurring in their natural habitat) has a major influence on
selecting one of these groups. Room temperature may be adequate for a simu-
lation of sewage treatment, even if it is more likely to be an artifact of time pres-
sure to get results fast and of restricted access to temperature controlled rooms
on the site of the research institution. But for natural aquatic systems in north-
ern Europe a standard incubation temperature of 10C would make much more
sense as there is no general way to extrapolate downward from higher tempe-
ratures. There are very few studies on the effect of temperature on adaptation of
bacteria and on the kinetics and extent of mineralization. Some authors could
show a much bigger effect than expected by simple extrapolation [21, 55, 61].
This effect is more pronounced if low concentration and low temperature act
together. For example when uninduced microbial communities were incubated
with low concentrations of 4-Np at low temperature, nothing happened in
months whereas adapted communities took only a few days longer, degrading
the chemical at 10C rather than at 20C, at higher concentration [55]. For the
effect of a low concentration see Fig. 7.
Hales and Ernst [21] could measure NTA mineralization at 5C in a river
estuary. The rate depended on the salinity and the sediment content, and filter-
ing the sample halved the rate. The low temperature had a stronger influence
when concentrations in the lower microgram range were used compared with
when 1000 mg/l were used. At 12C they measured half-lives of 429 days
whereas the mean retention time of the water body in the estuary is 17
days. Palmisano et al. [20] noted than NTA was degraded not only faster but
also more completely at higher temperatures in samples from different river
compartments in a polluted river.
NTA is an interesting example for a substance which is measured in the
environment in spite of being biodegradable in several standard tests. Perry et
al. [14] stated in a review article that NTA often leaves sewage treatment plants
untreated in substantial amounts. It can mobilize heavy metals and pollute
ground and drinking water and it is therefore now routinely measured in some
European monitoring programs, together with the even more recalcitrant
EDTA. He notes that NTA seems to be degraded very slowly at temperatures un-
der 7C as concentrations up to 100 mg/l could be measured in winter whereas
only 10 mg/l or less were determined in summer.
5.3
Sorption
Sorption can be caused by different mechanisms like van der Waals forces,
charge transfer complexation, hydrogen bonding, and hydrophobic interactions
[62]. All adsorption processes except for covalent bonds are reversible.
Chemical substances covalently bound to the humus matrix are called bound
residues. Sorption and degradation processes are dependent on each other
[63]. The limitation of degradation processes by sorption was found to differ
with different bacterial strains. This implies that the bioavailability of soil-sorb-
ed substances (e. g., atrazine [64], naphthalene [65]) also depends upon the de-
sorption efficiency accomplished by a bacterial strain.
Several authors noted a strong adsorption of metabolites of the pesticide
methyl parathion in sediments up to 48%. In sterile controls there was only
little adsorption, and therefore a biologically mediated sorption was postulated
[33, 66].
5.4
Oxygen Content
Oxygen is a prerequisite for most mineralization processes. It is usually not a
problem if only water samples are tested and the concentration of substrate is
not extremely high. But in sediments and soils, oxygen can be a very important
factor.
The water content in a soil is inversely proportional to its gas content.
Therefore, the oxygen content of a soil decreases with increasing water content.
Hardly any aerobic degradation of chemical substances can be found in a water
saturated soil if no oxygen is brought in by mixing (e. g., parathion [67]). The
smaller the grains of a soil and therefore the finer its porosity, the slower will be
the oxygen exchange within the soil. No optimal water content can be defined,
but in different soils an optimal degradation is achieved with different water
contents.
This is demonstrated in Fig. 5. In all soils (A, B, C) the bacterial degradation
of tetrachlorobenzene was lowest at 100% water saturation. In the sandy soil A
with the largest grains and pores, no difference was found for the degradation
with 40% and 70% water saturation. Here, a fast oxygen and water exchange
could take place. Soil B with medium grain and pore size showed a better de-
gradation with the lowest water content of 40%. Apparently, the oxygen content
was not sufficient for the strictly aerobic microorganisms at the higher water
contents. In soil C with a high clay content, equaling a very small grain size,
much water was bound by the clay particles and therefore inaccessible for the
microorganisms. In this case, the degradation was better with the higher water
content of 70% [42].
5.5
Sediments and Soil
There are not many examples of a transfer of experiments performed in the la-
boratory to the pilot scale. On a larger scale the degradation process is often re-
stricted by suboptimal water content, e. g., dryness or insufficient oxygen sup-
ply accompanying a high water content, as well as deficient bioavailability of the
degradable substance or other nutrients [68]; conditions of the natural envi-
ronment often do not lead to such positive results as those obtained under con-
trolled laboratory conditions.
Many single factors have been investigated by the research group of
Alexander and coworkers [69, 70], but the complex interactions between these
Fig. 5AC. Soil types (see text). Influence of different water contents in sterile soils upon the
mineralization rates of 1,2,4,5-tetrachlorobenzene by the bacterial strain Acidovorax sp. PS14.
Water contents: 40% (), 70% (), and 100% () respectively, of the maximal water holding
capacity of the soil; tetrachlorobenzene concentration: 10 ppm; inoculum: 10
7
cells/g soil; in-
cubation temperature: 25C
factors can hardly be covered by laboratory experiments. In a concrete reme-
diation case described by Steilen et al. [71], none of four remediation techniques
applied led to the successful degradation of a PAH-contaminated soil, but the
authors had not been able to forecast this.
The toxicity of a xenobiotic substance to the autochthonous soil microflora
was shown to be higher in smaller samples than in large-scale experiments [72].
This can be explained by the greater variance of the microflora found in larger
samples, so different effects can be balanced. Larger samples also have a higher
probability of containing microorganisms capable of degradation. Additionally,
abiotic procedures like the draining reaction of chemical substances were shown
to be different in natural soil compared with disturbed soil columns [73].
A special problem arises with soil and sediments exposed to xenobiotic sub-
stances for a longer period of time prior to remediation (e. g., old neglected de-
posits) so that complex sorption processes have taken place. These time-depen-
dent reactions can hardly be simulated in the laboratory [74]. It is often tried to
forecast the behavior and degradation of chemical substances in soil eco-
systems with the help of mathematical models [75, 76], but these models are
only applicable for defined chemical classes, defined soils with defined water
contents etc., and have no general validity.
An interesting experiment was performed by the research group of Short et
al. [77, 78]. They created a genetically engineered microorganism (GEM) of
Pseudomonas putida with a plasmid which enabled the microorganism to
transform 2,4-D to 2-chloromaleyl acetate in soil. Subsequently this metabolite
was mineralized by the endogenous microorganisms of the soil [77]. After these
positive results the GEM was introduced into another soil type. Here they found
that 2,4-D was only metabolized to 2,4-dichlorophenol, and not any further.
This metabolite accumulated in the soil. Because of the toxicity of this com-
pound, which is higher than that of 2,4-D, the number of fungi decreased more
than 400-fold resulting in a reduced soil respiration rate in comparison to a soil
containing 2,4-D. This means that the degradation pathway of the GEM led to
different metabolites in different soils and it was not possible to transfer the re-
sults from one soil type to another [78].
5.6
Grazing
Another phenomenon is the influence of grazers i. e., heterotrophic nanoflagel-
lates, ciliates, or other protozoa living on bacteria. Galvao [79] observed that
only at flagellate numbers of more than 68 10
3
/ml significant grazing can be
observed. Additionally it is important to note that flagellates thrive at a temper-
ature of around 10C. Andersen and Fenchel [80] determined that a minimum
of 10
6
bacteria/ml are necessary for grazing to start. Therefore the concentra-
tion of total bacteria and of grazers is decisive for grazing to start. If part of the
population grows only slowly they may not survive the grazing pressure and
can be severely decimated or perhaps even eliminated [69, 81]. If this part in a
mixed microbial community happens to be the one degrading the substance in
question, the mineralization may stop or never start. The slow growth rate may
of course be caused by low substrate concentrations and/or low temperatures.
Furthermore the influence of grazing on the adaptation process has been
shown to prolong the lag phase [82].
An example for the influence of grazing on an adapted population of degrad-
ing bacteria will be given below.
5.7
Interactions Between Concentration, Growth, Grazing, and Temperature
Many authors found some kind of correlation between the numbers of bacteria
able to degrade 4-Np and the onset and kinetics of 4-Np mineralization [55,
8385]. If the number is small, growth is necessary for the population to thrive
and bring about measurable change in the substrate concentration. This growth
and the consequential degradation is influenced by several factors, i. e., low tem-
perature can severely restrict degradation and high grazing pressure can do the
same, especially as 4-Np is not such a good substrate for growth. Several authors
found only small numbers of bacteria able to degrade a specific chemical, even
in adapted ecosystems (20/l to 80/ml in unadapted water, up to 780/ml in
pre-adapted natural water, compared to a total bacterial number (TBN) of
10
5
10
7
/ml). Therefore it seems logical that so far a correlation between TBN
and degradation has never been found.
To give an example, the degradation of 4-Np will be presented under differ-
ent conditions typical for ecological stress factors. With this substance it is
already known from laboratory experiments that 4-Np is principally bio-
degradable. But an adaptation period is usually necessary to provide a large
enough number of bacteria with induced enzymes to attack 4-Np.
A simulation experiment was designed to determine the influence of biotic
factors like growth, the presence of other bacteria, and grazing by protozoa, as
well as abiotic factors like other carbon sources and temperature on degrada-
tion. The experimental setup simulated the introduction and dilution of 4-Np
and adapted degrading bacteria into seawater as might happen around sewage
outfalls, or when coastal waters are swept out into the open sea by currents.
A coastal seawater sample was incubated with 250 mg/l 4-Np at 20C till all 4-
Np was degraded to get an adapted inoculum. Then 5 vol % of this culture were
added to differently treated sea water subsamples, so that around 10
5
degrading
bacteria were present per litre. The amount of bacteria introduced was derived
from 125 ng 4-Np absolutely or equivalent to 12.5 mg/l 4-Np after dilution. They
could be expected to multiply roughly up to ten times on the added 8 mg/l ra-
dioactive 4-Np, assuming that 1 pg of organic substrate supports the growth of
a single bacterial cell [60]. The uptake, mineralization, and percentage remain-
ing in solution were monitored using three replicas and a control.
Three treatments (BW, FF, and CY) were incubated at 20C to simulate opti-
mum summer conditions, two treatments (abbreviated as10C and 3D) at 10C,
which is the optimum temperature for most psychrophilic bacteria and close to
the annual mean temperature of Baltic Sea surface water [86].
The treatment with sterile aged brackish water a typical laboratory test set-
up promoted the fastest mineralization (BW, Fig. 6). Most of the 4-Np was de-
graded in the first 4 days before any multiplication of the total population could
be measured. Therefore the fastest degradation possibly took place with little or
no growth. An assumption, which is further proven by the shape of the curve,
which looks like a first order reaction and could be fit with a first-order model
(MARQFIT curve fitting program by Schmidt and Simkins as described in [49,
50]).
In the treatment with natural Baltic Sea water plus cycloheximide, which was
added to stop protozoan growth, the degradation rate was lower. The final per-
centage of CO
2
formed was about the same (around 70%). The best curve fit was
obtained with a model for logistic growth which is indicative of growth (CY,
Fig. 6). Some growth of the degrading population can be assumed, as this treat-
ment showed the highest uptake of 4-Np into the cells (7% uptake after 7 days)
through the whole incubation time and a sevenfold increase of the total bacteria
count.
Under conditions coming closest to nature a small amount of degraders
introduced into a natural water sample with protozoa, other bacteria and nu-
trients already present the mineralization of 4-Np was the slowest (FF, Fig. 6).
For the first 4 days the degradation in the fresh seawater treatment (FF) follow-
ed the CY treatment, which used the same water. No significant increase of the
CO
2
formed could then be observed at all for 24 h, and the mean value at day 5
was even lower than at day 4. This curve could not be fitted adequately with any
1
4-nitrophenol after adaptation. Influence of differently treat-
ed water. A mixed inoculum adapted to the degradation of 250 mg l
1
was diluted with new
media containing only inorganic nutrients (BW), freshly taken seawater without protozoa
(CY), or freshly taken seawater with competing bacteria, protozoa, organic, and inorganic nu-
trients (FF). The incubation temperature was 20C. The standard deviation is only shown,
when it is higher than 1.5%
model. When the experiment was ended, only 56% was mineralized to CO
2
(Fig. 6). There were always flagellates present.
To enhance the possible influence of lower temperature and grazing by pro-
tozoa on 4-Np degradation, a second set of treatments was incubated at 10C
(10C and 3D, Fig. 7). Half of the samples were incubated for 3 days without 4-
Np and without additional degrading bacteria so that a bloom of heterotrophic
nanoflagellates (3.8 10
3
/ml) had already developed (3D (3 days), Fig. 7). Then
the adapted degrading population was added.
After a one-day lag phase, the degradation started in the 10C treatment un-
til 25% CO
2
were produced in 7 days (Fig. 7). The mean value of mineralization
decreased thereafter from day 7 to day 9. Then the mineralization increased
again, probably leveling of at 43% CO
2
after 16 days. The 3D (three-day) treat-
ment showed a linear increase of CO
2
-production to almost 30% in 11 days and
a very long stagnation period till day 20 (Fig. 7). After 25 days the CO
2
-produc-
tion stopped again and did not reach more than 48% at the end of the experi-
ment (Fig. 7).
The slow growth rate, due to low temperature, grazing, and especially to pre-
incubation resulting in higher initial flagellate numbers, led to lag-periods, dur-
ing which the CO
2
-production did not increase. In all mineralization graphs
only the treatments with active flagellates showed high standard deviations
(Fig. 6, FF and 7). The described factors combined can result in slow and
incomplete mineralization as can be seen in Fig. 7, even when adapted bacteria
1
4-nitrophenol after adaptation. Influence of temperature and
grazing. Sample preparation was as described for Fig. 6 (FF), only the incubation temperature
was 10C. One set of samples was pre-incubated without 4-Np until a bloom of heterotrophic
nanoflagellates could be microscopically observed after 3 days (3D). Then the adapted ino-
culum and the 4-Np was added. The standard deviation is only shown when it is higher than
1.5%
are utilized. Competition with other bacteria for nutrients or additional carbon
sources can change the kinetics as well (Fig. 6).
It is possible that similar effects happen during the first days of 4-Np degra-
dation in unadapted samples. The very small population tries to grow, but is
slowed down at a much lower level of CO
2
-production than could be shown
in these experiments, thus leading to the often perceived adaptation phenome-
non (e. g., Fig. 3).
6
What is Persistence, and When is a Substance Biodegradable?
6.1
A Practitioners View
Halogenated dibenzofurans and dioxins are commonly regarded as typical ex-
amples for highly toxic and highly persistent chemicals. The electronegativity of
the halogen atoms make it very difficult to oxidize the aromatic rings present in
these molecules. Therefore these substances are regarded as not susceptible to
biodegradation. Also the non-halogenated cores of these molecules dibenzo-
furan and dioxin were regarded as non-biodegradable until special strains
were isolated by Rolf Wittich in 1989 at the University of Hamburg. The NATEC
laboratory, however, has found that up to 7080% of
14
C-radiolabeled dibenzo-
furan (concentration in soil of 1 mg/kg) is turned over to
14
CO
2
within 1 year by
the autochthonic microflora in soil without supplementation of specialized
microbial strains [87]. Half of this amount was degraded to
14
CO
2
within
1 month. After adding a specialized strain to the soil, the degradation was accel-
erated the residues were degraded within 1 week. At a higher concentration
(1 mg/kg), the degradation rates were not as high: the maximum was 50% after
1 year (10% after 1 month).
These results show that some degradation processes are only a matter of
time. They also show that the mechanisms resulting in degradability or non-de-
gradability can run in several directions: normally higher degradation rates
would be expected at higher start concentrations. If 1 mg/kg can be degraded,
normally, 1 mg/kg (if it is not toxic at that concentration) should also be degrad-
able lower concentrations tend to cause more problems since the substance
could be adsorbed into to the soil and would therefore not be available for bio-
degradation. The experiments showed that obviously other things happened.
The mechanisms are open for speculations: perhaps the bioavailablity of di-
benzofuran depended on the water solubility or maybe the soil microflora
grows a biofilm on surfaces contaminated with dibenzofuran later on the in-
ner bacteria of this biofilm may not be able to graze any more hindering other
bacteria from reaching the substrate.
Persistence is not a general feature which can be assigned unequivocally to
all chemical substances. There is a number of chemical substances which can be
regarded as persistent to biodegradation under regularly occurring environ-
mental conditions without supplementation of specialized strains (e. g., many
highly halogenated aromatic compounds). There is also a number of chemical
substances susceptible to ready biodegradation. But there is a large group in be-
tween. They may be biodegradable within a certain range of environmental
conditions (temperature, light regime, water activity, available nutrients) and
within a certain range of substance concentrations in a specific matrix. The bio-
degradability may also depend on the history of the contaminated spot, e. g.,
have there been contaminations with the same substance or with antagonistic
or synergistic substances?
6.2
An Ecologists View
For an ecologist the environment determines almost everything. The individual
organism and one specific substance are only one combination of interacting
factors in a large array of abiotic and biotic factors, some of which have been
described before. One half-life or one rate constant as the all encompassing
number to characterize the behavior of a chemical in nature (as happens more
and more frequently in hazard assessment) is not an acceptable option for an
ecologist.
First, it depends largely on the type of test system used what kind of a rate
constant is determined. What bacteria are in it, what concentration of chemical
is used? Are natural conditions simulated (if yes, which?) or are the conditions
optimized to enhance biodegradation (i. e., 20C, plus suspended sediment, plus
nutrients and so forth)?
Second, what about the rate constant, when the rate increases only after a
certain time during the degradation process? When ecologists take samples into
the laboratory they are painfully aware that a lot of changes will happen in the
microbial community, making the reaction differ from their natural one in
unpredictable ways. This so-called bottle effect starts immediately after taking
a sample and is the more pronounced the longer a sample is incubated.
For these two reason alone it can be deduced that it cannot be enough to use
standard tests to determine half-lives of the chemical in question.
In addition to these system-specific and microbiological factors, the en-
vironmental conditions are at least as important. They are at work at the same
time as the microbe-chemical interactions, which are normally studied isolated
in the laboratory. To name but a few abiotic factors like currents and temper-
ature regime, salinity, water quality and sediment content and characteristics
have to be taken into account. On the biotic side, competition, predation, and
biodiversity can play a major role. On top of these basic ecological descriptors,
some interactions of the microbe-chemical system happen mainly on an eco-
system level, i. e., the pollution history, the dilution factor, photo-oxidation, se-
dimentation, and adsorption phenomena. Therefore, a substance can be de-
gradable in a (polluted) river estuary, but not in the open sea or in an oligotro-
phic lake. This may happen not because the right bacteria are not there, but
because the environmental conditions are not the right ones for substantial
mineralization to happen.
Important is the time scale for rivers the residence time of a chemical is
short, for estuaries and oceans longer. There should not be a persistence prob-
lem if the major part of the yearly load can be mineralized in several tide cycles
up to one season, a period corresponding to half-lives less than 80 days [88]. But
if the chemical or its primary degradation products are toxic and/or bioaccu-
mulate, such a long residence time may cause other problems. Additionally the
longer a substance stays in the system without biodegradation, the higher is the
chance in an open system for dilution to levels too low for degradation to start,
for evaporation/volatilization and further transport, or for sedimentation and
transfer to a different environment.
The environmental factors correlated with the protection of chemicals from
microbial attack in waters have not been adequately defined as the well known
microbial ecologist M. Alexander said in 1980 [89]. Therefore the ecologist has
to stress the importance of a clear differentiation between potential for degra-
dation estimated in lab tests and proof of mineralization in situ. The examples
mentioned before may have already shown that recent research has led to some
answers and to even more questions.
Keeping all this in mind, the following tentative definitions of terms relevant
to biodegradation are given from an ecological point of view:
Persistance: no mineralization in one growing season or in an appropriate
time span for the ecosystem under study, therefore potential for accumula-
tion and toxicity if input is permanent or recurring.
Biodegradable: mineralization of a concentration encountered in nature un-
der natural temperature and other conditions, in appropriate time span for
ecosystem under study. If a substance or its metabolites significantly adsorb
to sediment, the sediment should be integrated into the study.
In rivers, half-life should be days.
In lakes, half-life should be no more than half of the time between the two
mixing periods.
In estuaries, half-life should be no more than half of the residence time.
In oceans, half-life may not be a useful parameter (if first-order model).
Adaptation should be possible in situ with concentrations measured in
situ, and a sufficient number of bacteria able to degrade the chemical in
question should be present.
In sediments/soils, mineralization should be possible in undisturbed sedi-
ments/soils and under naturally occurring conditions (oxygen, temper-
ature, etc.). The time spans should be as described for the different eco-
systems before.
Detoxification: no mineralization but transformation, i. e., transfer into
humic substance, leading to products which are tightly bound and will not
be released in their original form anymore. Proof has to be obtained that no
toxic intermediates are generated.
7
Outlook
To sum up the text of this chapter and its main conclusions, some suggestions
for further research and some management advice will be given. A leading role
has been taken by some recently updated international conventions for the pro-
tection of the northern oceans (North-East Atlantic and the Baltic Sea). An at-
tempt has been made to incorporate the precautionary and the polluter pays
principles. It is stated that:
hazardous substances are those which are persistent, liable to accumulate or toxic The
target [is] their cessation within one generation (25 years) with the ultimate aim of concen-
trations in the environment close to zero concentrations for man-made synthetic sub-
stances (OSPAR Convention, Convention for the Protection of the Marine Environment of
the North-East Atlantic).
This statement has recently been updated with the enlightened ecological in-
terpretation that other substances may require a similar approach, even if they
do not meet all the criteria mentioned above, but give rise to an equivalent le-
vel of concern. Transformation products, which generate concern, even if the
original chemical does not, are explicitly mentioned. Persistence is defined in
the draft as:
if the conversion of a substance or of its degradation products in the marine environment
and in particular in the water column is slow enough to permit long-term occurrence and wi-
despread distribution from its point of release (OSPAR Draft 1997 [90]).
This shows clearly that persistence should be seen in the context of residence
times and typical environmental factors in the ecosystem under study as elabo-
rated in this chapter. Acclimation and adaptation phenomena have to be con-
sidered when studies are designed to determine the fate of a specific chemical
in the environment. After lab tests established that a chemical can (only) be de-
graded after adaptation the guiding questions should be: are adapted bacteria
present in the receiving environment and, if not, can the natural microbial com-
munity adapt to the chemical under study under close to natural conditions?
Interactions between several factors are the main problem for predictability,
i. e., low concentration and temperature, or low temperature and low oxygen
content or the additional necessity of adaptation (enzymatically and/or because
the population is too small). Natural ecosystems are highly dynamic and not
made to maintain stable or optimum conditions for fast degradation.
Therefore one step of a strategy to ease the chemical burden on the environ-
ment should be an optimized understanding and functioning of sewage treat-
ment facilities where these factors can be controlled much better. For those
chemicals which have to be used in open systems and which will not reach
sewage treatment plants a more detailed array of tests is recommended taking
the described factors and influences into account.
7.1
Some Research Suggestions for Better Predictability
It seems that at least for the protection of the oceans the first steps have been ta-
ken to prevent further pollution. But the work of prioritizing which chemicals
should be monitored, controlled, reduced, or eliminated still needs to be done. The
following text tries to give some advice how microbiological research can help:
Instead of or additionally to simulation tests, an estimation of the initial
number of potentially degrading bacteria in the habitat under study, perhaps
with two different concentrations, might help to find problem areas where
regulation of input should be a priority.
Better kinetic models taking growth or no growth into account are needed.
They should not be independent of concentration of the chemical (and
perhaps even take second substrates into account).
Ecosystem modeling should include other members of the food chain (com-
petition, grazing), other transformation pathways (photodegradation,
(bio)sorption), and all relevant compartments (sediment, surface layer).
Ecological knowledge should be better integrated into the design of sewage
treatment facilities. A better understanding of processes in sewage treatment
plants may lead to new forms of controlled small scale treatment, tailored to
problem chemicals.
7.2
Some Steps Towards Sustainable Development
As has been said before, when the knowledge about a chemicals behavior in na-
ture is still generally unknown or the results, e. g., of degradation tests are con-
tradictory, the precautionary principle should be applied and the use of the sub-
stance restricted and controlled until the results allow a final decision. Final is
meant in a relative sense as new toxic effects may be found, e. g., the ozone dam-
aging substances some years ago or the hormone mimicking substances re-
cently. On the other hand, new treatment methods, e. g., with adapted inocula,
may allow controlled uses of formerly restricted chemicals.
Encouraging the development of principally degradable chemicals and the
concurrent methods for their disposal after use might lead to a thorough green-
ing of the chemical industry and make its products more socially acceptable.
Responsible care or other environmental management initiatives can be en-
couraged by clear framework regulations and research incentives by the rele-
vant government or international authorities.
It should be legitimate to assume that a new substance or substance group,
which contain features proven to slow down degradation like branched side
chains, chloro-or nitro-groups, isomeric mixtures, belong to the persistent cate-
gory until the opposite has been proven at the cost of the party interested in its
introduction. If this rule was applied, new problem chemicals like toxaphene or
nonylphenol (polyethoxylates) would never have been introduced after the ex-
periences with the first generation of detergents and PCB.
8
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The Assessment of Biodegradation and Persistence
Bernd Beek, Stella Bhling, Christian Franke, Ulrich Jhncke,
Gabriele Studinger, Elisabeth Thumm
Federal Environmental Agency, Seecktstrasse 6-10, D-13581 Berlin, Germany,
E-mail: bernd.beek@uba.de
Testing and assessment strategies for biodegradation and persistence of chemicals and their
impact on microbial activity within the framework of environmental legislations are out-
lined. An integrated assessment concept for biodegradation and persistence of chemicals in
the environment is presented, taking into account primary degradation, mineralization and
bound residues. Results of simulation tests from soil and water/sediment systems are assign-
ed to four classes of these three criteria and aggregated to an overall assessment resulting in
four persistence categories allowing for a more comprehensive estimation of fate and behav-
ior of a chemical in soils, surface waters, and sediments. Examples are given for an applica-
tion of this assessment concept comparing data sets from simulation studies with plant pro-
tection agents in soils and water/sediment systems. The proposed assessment scheme may
also be applied for risk assessment in context with registration and notification procedures
of any kind of chemical substances by environmental authorities. An assessment scheme is
also proposed for the biodegradation and elimination in sewage treatment plants as well as
the toxic impact of substances on microbial activity with regard to impaired biodegradation
potentials in sewage treatment plants, soils, surface waters, and sediments. Deficiencies and
future needs are addressed for achievement of a more realistic risk assessment of fate and be-
havior of chemicals in the environment.
Keywords. Biodegradation, Persistence, Microbial toxicity, Risk Assessment, Deterioration
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
2 Some Basic Conditions for the Determination of Biodegradation 294
2.1 Aerobic vs Anaerobic Biodegradation . . . . . . . . . . . . . . . . 294
2.2 Primary Degradation vs Mineralization . . . . . . . . . . . . . . . 294
3 Test Systems for Biodegradation and Elimination . . . . . . . . . 296
3.1 Screening Tests for Ready Biodegradation . . . . . . . . . . . . . . 298
3.2 Screening Tests for Inherent Biodegradation . . . . . . . . . . . . 300
3.3 Simulation Tests and Persistence Categories . . . . . . . . . . . . . 301
3.3.1 Simulation Tests for Surface Waters and Soils . . . . . . . . . . . . 303
3.3.2 Simulation Tests for Sewage Treatment Plants . . . . . . . . . . . . 307
4 Biodegradation Rate Constants . . . . . . . . . . . . . . . . . . . . 308
4.1 Determination of Biodegradation Rate Constants
from Screening Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 308
CHAPTER 5
(ed. by B. Beek)
4.2 Prerequisites for the Derivation of Biodegradation
Rate Constants from Simulation Tests . . . . . . . . . . . . . . . . 309
4.3 Field Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
5 Microbial Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . 311
5.1 Assessment Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 311
5.2 Intrinsic Properties of Chemicals and Consequences for
Choice and Performance of Tests . . . . . . . . . . . . . . . . . . . 314
5.3 Risk Assessment and Safety Factors . . . . . . . . . . . . . . . . . 316
6 Deficits and Perspectives . . . . . . . . . . . . . . . . . . . . . . . 317
7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
1
Introduction
In addition to natural substances, a large number of anthropogenic chemicals
circulate in the environment today, the disposal of which frequently poses great
problems for humans and the environment. The most important process mini-
mizing this hazard potential in water and soil is biodegradation. In the en-
vironmental compartment air degradation is mediated by physico-chemical
processes.
The demand for products of high persistence is opposed to the demand for
their almost complete degradation after use, apart from further utilization
(e. g., recycling). Although nearly every substance is liable to transformation,
i. e., degradation, continuous discharge can result in the achievement of a
steady-state concentration in the environment or, if the decrease is slow, in a
steady accumulation (geoaccumulation). Furthermore, chemicals can accumu-
late in environmental compartments to persist there for very long periods of
time and also in organisms (bioaccumulation; see [1]) even after long distance
transport as far as into arctic and antarctic regions where conditions are par-
ticularly impeded as to biodegradation (Persistent Organic Pollutants, POPs).
Spectacular examples encountered today in many environmental samples are
dioxines, polychlorinated biphenyls (PCBs), and DDT.
Biodegradation has always been one of the main fields of microbiological re-
search, focussing on the identification of degradation pathways and metabolic
processes. Consequently, the conditions of these tests aimed rather at the opti-
mization of test conditions and selection of degradation-potent micro-organ-
isms rather than simulation of environmentally relevant parameters.
Yet it was not until the 1950s and 1960s that the issue of biodegradation in
the environment became a matter of public interest when, resulting from the
widespread use of detergents, poorly degradable surfactants reached surface
waters and foam formation made the problem visible. As a result more degrad-
able detergents were produced and test methods were developed in order to
quantitatively pursue their biodegradability.
292 B. Beek et al.
In the time that followed, all industrial nations developed degradation tests
which, aiming at mutual acceptance of test results, were internationally har-
monized by the Organisation for Economic Co-operation and Development
(OECD) and later in the European Union (EU).
Since the implementation of the German Chemicals Act [2] in 1982, new no-
tified substances are subjected to biodegradation tests according to Annex V [3,
4, 12] of the Council Directive 67/548/EEC [5].
For existing substances the Government of the Federal Republic of Germany
has in its Existing Chemicals Programme chosen the means of cooperation be-
tween Industry, Research, and Governmental Institutions, and this was followed
by all EU member states. After priority setting, all available information on exis-
ting environmentally relevant chemicals, including biodegradation, is collected,
assessed, and summarized in dossiers containing proposals for appropriate
measures.
Since 1986 the German Federal Environmental Agency has been included as
an authority of consent into the implementation of the German Plant
Protection Act [6], decisive information on biodegradation of many hazardous
substances being obtained from sophisticated testing.
In contrast to the Chemicals Act, which requires the determination of biode-
gradability only in the aquatic milieu, the Plant Protection Act stipulates man-
datory degradation testing of pesticides in soil and sediments, thus making data
available to provide a deeper insight into the complex proceedings of biodegra-
dation processes.
Data on degradation of detergents gathered from the implementation of the
German Detergents and Cleansing Agents Act [7] increasingly broaden knowl-
edge of degradation processes. This also applies to data concerning the biode-
gradation of non-agricultural biocides.
From the environmental point of view, however, nearly all tests applied with-
in the different fields of environmental legislation have in common that they ex-
amine biodegradation in too high test concentrations and under non-represen-
tative test conditions. Some standardized tests simulating environmental con-
ditions in water and soil and in sewage treatment plants (STP) are available but
laborious and relatively costly.
Numerous chemicals classified according to laboratory tests as readily
biodegradable are, however, detected in the environment. For a realistic assess-
ment of biodegradation in the environment this uncertainty forces us to re-
consider present strategies and also requires in part more sophisticated test
methods.
The objective of this contribution to investigation and assessment of bio-
degradation within the scope of the implementation of environmental acts is
to impart the present state of knowledge and to show up current activi-
ties regarding the progress of further developments in testing and assessment
strategies.
The Assessment of Biodegradation and Persistence 293
2
Some Basic Conditions for the Determination of Biodegradation
2.1
Aerobic vs Anaerobic Biodegradation
Biochemical-enzymatic transformations performed in the presence of oxygen es-
pecially by micro-organisms such as fungi and bacteria leading to the oxidation
of the substance characterize the aerobic biological degradation of organic sub-
stances. In the various transformation steps generally more polar, hydrophilic
metabolites are produced, equally subject to further decomposition. As a rule end
products of aerobic degradation are carbon dioxide, water, and inorganic salts.
In the absence of oxygen, i.e., under anaerobic conditions, ultimate degrada-
tion leads to the formation of methane, carbon dioxide, and inorganic salts as
end products (cf. Table 5).
Where oxidative degradation processes, e. g., catalyzed by oxygenases or
peroxidases, are hindered due to lack of oxygen, reductive degradation proces-
ses, e. g., the reduction of nitro-groups, will prevail. Halogenated hydrocarbons
can to a great extent be transformed anaerobically.
Anoxic conditions occur not only in the digesters of sewage treatment plants,
but also in many surface water sediments, deeper soil layers, parts of ground
water, and in dumping sites. Especially poorly water-soluble and highly adsorb-
ing substances are predominantly transferred to anaerobic sites. The impor-
tance of anaerobic biodegradation processes seems to have been underestima-
ted until now (see contribution by Reineke, this volume).
2.2
Primary Degradation vs Mineralization
For the determination of primary degradation or mineralization, i. e., ultimate
biodegradation of a substance, different analytical parameters are used. Table 1
lists the analytical parameters of tests most commonly applied within the prac-
tice of chemicals legislation.
Test methods measuring ultimate biological degradation are mostly based on
the determination of summary parameters such as oxygen consumption (bio-
chemical oxygen demand, BOD), carbon dioxide evolution (CO
2
), and dissolved
organic carbon (DOC) or chemical oxygen demand (COD) removal.
The use of radiolabeled substances allows for both the analysis of primary-
and ultimate degradation, especially of substrates in low concentrations. By
means of appropriate labeling an unspecific parameter such as CO
2
may be-
come a substance specific parameter (
14
CO
2
).
By measuring the oxygen consumption or the carbon dioxide or methane
production, no discrimination is possible between energy metabolism and bio-
mass production. DOC and total organic carbon (TOC) both represent a mea-
sure for the organic substances present; the degree of oxidation of the substance
or its metabolites has no influence on these parameters, as opposed to the COD-
determination.
294 B. Beek et al.
If abiotic elimination processes (adsorption, hydrolysis, photolysis, volati-
lization) can be excluded, in the case of single substance determinations the
extent of the DOC- or TOC-removal is a measure of the degree of biological
degradation. However, possible preceding oxidative degradation steps not lead-
ing to an elimination of C-atoms from the parent molecule remain uncon-
sidered.
In the case of adsorbing, poorly water-soluble, or volatile substances, a clear
distinction between biological degradation processes and elimination (e. g., via
adsorption, precipitation, stripping effects) is not possible. These latter proces-
ses can only be measured and quantified by means of a sterile adsorption con-
trol. For substances with the properties mentioned above, it must generally be
assumed that, if such controls are missing, measurements based on DOC-ana-
lysis cannot be regarded as degradation but only as elimination tests, and the
results assessed accordingly.
Using summary parameters for the degradation of a mixture of substances,
all organic compounds present in the test are measured jointly; a decrease of
the amount of the different compounds cannot be differentiated. Furthermore,
if metabolites are formed their amounts cannot be quantified. Thus it cannot be
differentiated whether the observed partial degradation results from the com-
plete degradation of one constituent as opposed to others being not degraded
at all or all substances undergoing only partial degradation.
Tests based on summary parameters are therefore only applicable to single
substances.
Table 1. Analytical parameters of biodegradation tests
Criterion Parameter
Primary degradation 1. Specific analysis for groups of substances, e. g.,
Anionic tensides: decline of MBAS
a
Non ionic tensides: decline of BiAS
b
Cationic tensides: decline of DSBAS
c
Hydrocarbons: decline of IR
d
absorption
2. Analysis of single substances, e. g., active ingredients
of plant protection products
Ultimate degradation 1. Summary parameter
(mineralization) Consumption of O
2
(BOD
e
in % ThOD
f
)
Evolution of CO
2
(CO
2
in % ThCO
2
g
)
Removal of DOC
h
2. Analysis of single substances (in case of radioactive labeling),
e. g., active ingredients of plant protection products
a
MBAS = Methylene Blue Active Substance.
b
BiAS = Bismuth Active Substance.
c
DSBAS = Disulfin Blue Active Substance.
d
IR = Infra Red.
e
BOD = Biochemical Oxygen Demand.
f
ThOD = Theoretical Oxygen Demand.
g
ThCO
2
= Theoretical Carbon Dioxide evolution.
h
DOC = Dissolved Organic Carbon.
3
Test Systems for Biodegradation and Elimination
In the beginning of the 1960s the first biodegradability tests for detergents were
developed, aiming at the prediction of the fate of the substances in the environ-
ment from the results. It is therefore not surprising that the oldest legislative re-
gulations to examine biological degradation originate from the field of deter-
gents and cleansing agents.
Of these tests, some are still, although in a modified version, used today.
Generally, substance or group specific methods were applied to analyze primary
degradation. In the meantime test methods were expanded to include all or-
ganic compounds, at the same time both broadening the test program and in-
creasing the stringency of some tests with respect to biodegradation potential.
In general, testing for ultimate biological degradation, complete mineralization
is prescribed; however exceptions to this rule still exist for several substance
groups, e. g., tensides.
Today in chemicals legislation, a great number of tests exist, predominantly
to be conducted according to standardized test procedures and thus possessing
mutual international acceptance:
For the testing of chemicals including the determination of their biodegrad-
ability the Organisation for Economic Co-operation and Development
(OECD) recommends a three-tier testing hierarchy naming appropriate test
methods for each of these tiers [8].
The EU has adopted this tiered program in its guidelines for New Substances
[9]; depending on the amount placed on the market or imported quantities,
the stipulated test methods provided are to a large extent identical with those
of the OECD.
The German Chemicals Act [2] based on the EU-Directives and in Article 2,
paragraph 4 of the Ordinance on Test Certifications and other Registration
and Notification Documents under the Chemicals Act (ChemGPrfV) [10]
explicitly refers to the test methods cited there.
Furthermore, a great number of so far internationally not standardized test
methods to examine biological degradation exists, especially for the testing of
biodegradation under environmentally more realistic conditions. In the light of
a testing strategy tailored to fit every single substance, greater access to these
tests should gain in importance.
Screening tests such as the tests for ready and inherent biodegradability de-
scribed below cannot consider the various circumstances of natural conditions.
The investigation of the degradation behavior of a substance in an appropriate
simulation model is extremely difficult, taking adequately into account the real
local environment into which the substance is discharged or released in the
event of an accident. Nevertheless, to assess ultimately a substance with respect
to its biodegradability or persistence it is necessary to investigate biodegrad-
ability in a suitable simulation model.
Principally two different types of degradation tests can be distinguished:
screening tests and simulation tests.
296 B. Beek et al.
First, in screening tests with relatively simple test conceptions, substances are
tested in high concentrations compared to those normally found in the envi-
ronment (but not inhibiting bacteria) in an aqueous solution or suspension in
test vessels together with a small amount of a polyvalent inoculum. This means
that no specialized or adapted micro-organisms are added. Instead the inocu-
lum is generally taken from municipal sewage treatment plants (STP), river wa-
ter, and/or soil suspension in order to represent a realistic spectrum of degrad-
ing organisms present in the environment. The test substance as sole source of
carbon is incubated in the dark for 28 days under conditions favoring bio-
degradation with respect to pH-value, O
2
-content, and temperature. Biodegra-
dation is generally followed by means of summary parameters.
Depending on the test substance loading, two test types are distinguished:
1. Tests with low inoculum concentration and high initial substrate concentra-
tion (tests for ready biodegradation). Low micro-organism concentrations
are encountered, e. g., in surface waters.
2. Tests with high inoculum concentration and, compared with the inoculum
amount, low initial substrate concentration (tests for inherent biodegrada-
tion/elimination). Such conditions prevail in municipal STP.
Screening tests should allow for a general statement concerning the biode-
gradation potential of a substance and do not simulate any specific environ-
mental situation. Hence the degradation rates obtained cannot be transferred to
environmental conditions.
Tests for ready biodegradation were designed to be stringent. It can be assum-
ed that substances reaching the degradation pass levels in tests for ready bio-
degradation within a defined period of time (10 days-window) will be ultima-
tely degraded in the environment within surveyable periods of time (see Fig. 1).
Fig. 1. The concept of the 10 days-window. Idealized curve of a DOC-Die-Away-Test for ready
biodegradability
Second, simulation tests should simulate the degradation behavior in a spe-
cific environmental compartment as far as possible.
The results from simulation tests enable the classification of a substance into
persistence categories (see below). Furthermore, field-studies may be per-
formed, the evaluation of which permit an assessment of the biodegradability
of the substance under concern on an ecosystematic level.
3.1
Screening Tests for Ready Biodegradation
If the amount of a new chemical placed on the market exceeds 100 kg/year or
500 kg cumulative (base-set level), it is first subjected to testing for ready bio-
degradation in a screening test.
Table 2 gives an overview of the internationally standardized tests of the
base-set level [11, 12].
A further development of the CO
2
Evolution-Test is the CO
2
Headspace-Test
(ISO DIS 14593) [13]. Apart from the CO
2
in the gaseous phase, this modifica-
tion of the CO
2
Evolution-Test also measures the solubilized CO
2
in the aqueous
phase. This test is suited for water-soluble, poorly water-soluble, and volatile
substances.
Decisive for the evaluation of the tests for ready biodegradation are both the
degradation percentage after 28 days and the fulfillment of the 10 days-window
criterion.
The biodegradation assessments are as follows.
If:
60% of ThOD
1
OR
60% of ThCO
2
2
OR
70% DOC removal AND
the criterion of the 10 days-window
3
is fulfilled
the assessment will be readily biodegradable
4
Reaching the pass-levels mentioned above but failing the 10 days-window re-
sults in the assessment readily biodegradable, but failing 10 days-window.
In both cases further testing for inherent biodegradability on tier 2 is not ne-
cessary.
If the pass levels are not met, the substance is classified as not readily biode-
gradable. In this case further testing for inherent biodegradability on tier 2 is
necessary.
Classifications other than the above stated from the tests for ready biodegrad-
ability within the scope of chemicals legislation for New Substances are not
feasible.
298 B. Beek et al.
1
ThOD = Theoretical Oxygen Demand
2
ThCO
2
= Theoretical Carbon Dioxide evolution
3
exceptions: (1). The 10 days-window criterion does not apply to the Mod. MITI I-Test [11,
12] (2). According to the instructions of the Closed Bottle-Test [11, 12] weekly measure-
ments are permitted. Consequently only a 14 days-window applies.
4
According to Technical Guidance Document (TGD) of the EU [14].
Given only a BOD
5
/COD ratio (BOD
5
=BOD after 5 days of incubation), a ra-
tio >0.5 is classified as indication of ready biodegradability
5
.
Due to less stringent test conditions this quotient is from our point of view
not comparable with results from screening tests for biodegradation and can
therefore only be assessed as an indication. Consequently, a ratio BOD
5
/COD
<0.5 is classified as no indication of ready biodegradability.
Table 2. Screening tests for ready biodegradability comparison of present and former na-
mes of tests
Present name Guideline Former name Guideline Comment
of test of test
OECD EU OECD EU
[11] [12] [8] [3]
DOC Die 301 A C.4-A Modified 301 A C.4
a
Away-Test AFNOR-Test
CO
2
Evolution- 301 B C.4-C Modified Sturm- 301 B C.5
a
Test Test
Modified MITI 301 C C.4-F Modified MITI 301 C C.7
b
I-Test I-Test
Closed Bottle- 301 D C.4-E Closed Bottle- 301 D C.6
c
Test Test
Modified OECD 301 E C.4-B Modified OECD 301 E C.3
d
Screening-Test Screening-Test
(MOST)
Manometric 301 F C.4-D
a, e
Respirometry-
Test
a
By standardization of inoculum concentration resulting in a maximum of 30 mg substance
(dry weight) per liter test medium or 100 ml effluent of sewage treatment plant per liter test
medium.
b
By specification of inoculum concentration, i. e., measurement of cell density per liter test
medium; restriction of valid range of temperature and approximation of this range with the
range in other tests.
c
By enhancement of inoculum concentration to a maximum of 5 ml effluent of sewage tre-
atment plant per liter test medium.
d
By enhancement of inoculum concentration to a maximum of 0.5 ml effluent of sewage tre-
atment plant per liter test medium.
e
Newly included; coincides with the European version of the Japanese Modified MITI I-Test
with only one inoculum and decreased range of temperature.
5
This classification deviates from the TGD. Some EU member countries accept the BOD
5
,
others do not. In our opinion the BOD
5
/COD-ratio cannot replace a complete and valid test
for ready biodegradability.
3.2
Screening Tests for Inherent Biodegradation
On level 1, i. e., at 100 tonnes/year or already at 10 tonnes/year triggered by cer-
tain hazard criteria, substances classified as not readily biodegradable are sub-
jected to further investigation of their biodegradation potential in screening
tests for inherent biodegradation.
In these tests the amount of inoculum is increased as compared to the base-
set tests, thus leading to better conditions for biodegradation.
The EU-guidelines [12] or OECD-guidelines [11] comprise 2 or 3 test me-
thods: Mod. S. C. A. S.-Test [4, 8], Zahn-Wellens-Test [4, 8, 11], and Mod. MITI
II-Test [8].
The German Federal Environmental Agency accepts the performance of the
Mod. MITI II-Test with only one inoculum from municipal STP as opposed to
the requirement of using ten different inocula in the original Japanese version.
In both cases a direct parameter for mineralization is measured (O
2
-consump-
tion). Of equal scientific value and therefore also accepted are CO
2
Evolution-
[11, 12] or Respirometer-Tests [11, 12], in which, however, the substrate/inocu-
lum relationship is in inverse proportion to the respective test for ready biode-
gradability (C.4-C or C.4-D). This is in accordance with the differences between
the Mod. MITI I-Test [4, 8] and Mod. MITI II-Test [8].
In general, both the Zahn-Wellens-Test and the S. C. A. S.-Test do not distin-
guish between biological degradation and other elimination mechanisms (mea-
sured parameter DOC, open system). In the assessment this is taken into ac-
count by the supplement biodegradable/eliminable. This differentiation is not
found in the Technical Guidance Document (TGD) of the EU [14]. In both tests
the only assessment provided for is in terms of degradability. Also no assess-
ment of results from the tests on inherent biodegradability failing the pass le-
vel, i. e., 2070%, is adequately considered in the TGD. Since in this range the
occurrence of stable metabolites cannot be excluded, the term partial degra-
dation is used here. To derive degradation rate constants for biodegradation
(see below) the TGD clearly states that results from the Zahn-Wellens-Test or
from the Mod. MITI II-Test are only to be considered if biological degradation
is clearly identified. To enable such a conclusion, various requirements which
are to be met in the tests are listed below.
The S. C. A. S.-Test is not comparable with other tests for inherent biodegra-
dability due to its test design (i.e., discontinuous operation modus, high inocu-
lum concentration, nutrients addition, long adaptation phase). A positive result
in the S.C.A.S.-Test can therefore only be regarded as an indication for inhe-
rent biodegradability.
Biodegradation < 20% Non-biodegradable/eliminable
Biodegradation 2070% Indication of partial biodegradability/ elimination
Biodegradation > 70% Indication of inherent biodegradability/elimination
Even if positive results are achieved, the TGD does not put the S. C. A. S.-Test on
an equal level with the other tests for inherent biodegradability and assigns a
degradation rate constant of 0.
300 B. Beek et al.
For the Zahn-Wellens-Test the following terminology of assessment is used:
Biodegradation < 20% Non-biodegradable/eliminable
Biodegradation 2070% Partially biodegradable/eliminable (with indication
of formation of stable metabolites)
Biodegradation > 70% Inherently biodegradable/eliminable
According to TGD the assessment inherent biodegradable can only be applied
if the following criteria are fulfilled: the biodegradation pass-level of 70% must
be reached within seven days, log-phase may be no longer than three days, and
elimination (e. g., by adsorption) prior to begin of biodegradation must be less
than 15%.
If by appropriate test controls abiotic elimination processes, e. g., adsorption,
volatilization, precipitation can be excluded, the term eliminablemay be omit-
ted.
For the Mod. MITI II-Test the following terminology of assessment is used:
Biodegradation < 20% Non-biodegradable
Biodegradation 2070% Partially biodegradable (with indication of forma-
tion of stable metabolites)
Biodegradation > 70% Inherently biodegradable (with indication of mine-
ralization)
According to TGD, the assessment inherent biodegradablecan only be applied
if the following criteria are fulfilled: the biodegradation pass level of 70% must
be reached within 14 days and the log-phase may be no longer than 3 days.
A tabular compilation of the classification of biodegradation potentials of a
substance on the basis of screening tests for ready and inherent biodegradabi-
lity is given in Table 3.
3.3
Simulation Tests and Persistence Categories
Substances are subjected to simulation tests to verify their degradation poten-
tials and to investigate their degradation behavior in specified, exposure rele-
vant compartments by means of test designs as close to environment as possi-
ble. From the results of such investigations a classification into persistence clas-
ses may be derived.
Depending on which environmental compartment degradation and dissipa-
tion processes are to be simulated, various test systems are currently available,
essentially belonging to three groups:
Simulation tests for surface waters
Simulation tests for soils
Simulation tests for sewage treatment plants (STP)
The simulation of sewage treatment plants represents a special case, since not
an environmental milieu but a technical plant is to be simulated. From this dif-
ference a separate assessment concept results for sewage treatment plant simu-
lation tests.
Considering physico-chemical characteristics of the substance, exposure
scenarios, and the results from screening tests, the appropriate tests are selected
in dialogue with the notifier.
Currently the following standardized simulation tests are available in
Germany:
Degradation and fate of plant protection agents in a water/sediment system.
BBA-guideline, part IV, 51 [17]
Fate of plant protection agents in soil degradation, transformation, and
metabolism BBA-guideline, part IV, 41 [18]
302 B. Beek et al.
Table 3. Classification of biodegradation potential
Readily Not readily biodegradable
biodegradable
Indication of Mineralizable Inherently bio- Partially bio- Non bio-
persistence degradable and degradable and degradable
category I indication of indication of
(see Table 4) mineralization
a
formation of
stable metabolites
Persistence of a substance
Screening tests for ready Screening tests for inherent biodegradability
biodegradability
Biodegradation: Biodegradation:
60% of ThOD 70% > 20% to < 70% 20%
60% of ThCO
2
70% DOC removal
and fulfilling and not ful-
10 days-window filling 10 days-
criterion window criterion
Closed Bottle-Test Modified MITI II-Test [8]
Modified MITI I-Test
Manometric Respirometry-Test BODIS-Test [15]
CO
2
Evolution-Test
Modified OECD Screening-Test Zahn-Wellens-Test [4, 8]
DOC Die-Away-Test [11, 12] Modified S.C.A.S-Test [4, 8]
(relation of BOD
5
/COD > 0.5)
ISO 11734 (Test on anaerobic biodegradability, ECETOC-Test) [16]
Stringency of tests
a
Equivalent to inherently biodegradable fulfilling specific criteria according to Technical
Guidance Document.
For deriving biodegradation rate constants for low concentrations of chemicals
in surface waters without sediment, an ISO guideline is currently in the process
of adoption: ISO/CD 14592 Water Quality Evaluation of the aerobic biodegrad-
ability of organic compounds at low concentrations, Part 1: shake flask batch
test with surface water or surface water/sediment suspensions [19].
3.3.1
Simulation Tests for Surface Waters and Soils
Simulation tests for surface waters (water/sediment systems) and soils are as-
sessed according to identical criteria. In addition to the quantitative base para-
meters primary degradation (dt
50
, disappearance time of 50% of the substance),
mineralization, and bound residues, further qualitative parameters, especially
the degradation kinetics and the metabolism scheme, are included in the
assessment and formation of persistence classes.
A proposal for a comprehensive assessment concept is presented in Table 4.
From the use of different sediments, soils, or temperature in different tests,
different ranges of results may arise. This has to be kept in mind. By inclusion
of these parameters the persistence class may change.
In accordance with the assessment practice of the TGD the results of degra-
dation tests are taken for the calculation of the predicted environmental con-
centration (PEC).
An example of the application of this assessment concept is presented in the
following.
In context with the registration of plant protection products, fate studies on
the degradation and distribution of active substances in soil and water/sedi-
ment simulation test systems were evaluated and assessed based on the para-
meters primary degradation, mineralization, and bound residues.
The studies were conducted based on the above-mentioned guidelines [17,
18].
The evaluation is based on 294 comparable data sets for soil systems and 253
data sets for water/sediment systems, respectively, and have been classified ac-
cording to the assessment scheme outlined above (Table 4).
As can be seen in Fig. 2, primary degradation rates in soils and water/sedi-
ment systems reveal that the disappearance from the water phase is much faster
than in soils (class I, rapid primary degradation, i. e., dt
50
<10 days).
Whereas in water/sediment systems the rapid disappearance from the water
phase is mostly due to transfer and adsorption of the parent compound to se-
diment, the elimination of the parent compound in soil systems is caused by
primary degradation, i. e., the transformation of the molecule. Primary degra-
dation is a slower process than physico-chemical reactions, leading to the ob-
served rapid disappearance from the water phase.
As shown in Fig. 3, mineralization in water/sediment systems is less effective
than in soil systems.
A comparison of especially class I in Figs. 2 and 3 shows that the mineraliza-
tion in water/sediment systems is remarkably lower than the primary degrada-
tion (dissipation) from the water phase.
As can be seen in Fig. 4, higher amounts of bound residues have been found
in soil systems (classes III and IV) as compared to sediment systems (classes I
and II).
In spite of the differences found in primary degradation, mineralization, and
the amount of bound residues between soil and water/sediment systems, the
overall assessments reveal comparable persistence categories, i. e., comparable
304 B. Beek et al.
Table 4. Persistence classes and persistence categories
1st criterion: primary degradation
dt
50
Class Assessment
< 10 days I Rapid primary degradation
1030 days II Delayed primary degradation
30100 days III Slow primary degradation
> 100 days IV Negligible primary degradation
2nd criterion: mineralization (after 100 d)
CO
2
Class Assessment
> 50% I Extensive mineralization
2550% II Moderate mineralization
1025% III Limited mineralization
< 10% IV Negligible mineralization
3rd criterion: bound residues (after 100 d)
Amount Class Assessment
< 10% I Low plateau
1025% II Moderate plateau
2550% III High plateau
> 50% IV Very high plateau
Calculation of Persistence Category
The three criteria mentioned above, respectively the resulting classes are equally taken for
calculation of the overall persistence category (average by rounding): sum of single classes:
number of parameters = persistence category
I Low persistence
II Moderate persistence
III High persistence
IV Not biodegradable
On a case-by-case basis the degradation curve as well as the metabolism scheme is consid-
ered for obtaining the overall persistence category
Example:
A substance shows the following properties:
dt
50
3 days I Rapid primary degradation
CO
2
12% III Limited mineralization
Bound residues 60% IV Very high plateau
8: 3 =2.7 (rounded: 3)
Consequently the substance has to be considered as highly persistent (persistence category
III). This example clearly demonstrates that a classification on the basis of primary degrada-
tion alone (class I) would have resulted in a wrong assessment of the real persistence
Fig. 2. Primary degradation (class IIV; see Table 4) of plant protection substances in water/
sediment (water phase only) and soil systems
Fig. 3. Mineralization (class IIV; see Table 4) of plant protection substances in water/sedi-
ment and soil systems
biodegradation/elimination behavior in both systems as presented in Figs. 5
and 6.
Conclusions and consequences from the results of the test systems used are:
Primary degradation and mineralization must be assessed separately.
Primary degradation/disappearance in water in the presence of sediment is
faster than in soil.
Mineralization in soil is more effective than in sediment.
Amount of bound residues in soils is higher than in sediments; this is rele-
vant because bound residues may be bioavailable for soil/sediment orga-
nisms.
The overall degradation/disappearance rates in soils and water/sediment sy-
stems (persistence categories) are almost equal.
306 B. Beek et al.
Fig. 4. Bound Residues (class IIV; see Table 4) of plant protection substances in water/sedi-
ment and soil systems
Fig. 5. Overall assessment of persistence categories (IIV; see Table 4) of plant protection
substances in water/sediment systems
3.3.2
Simulation Tests for Sewage Treatment Plants
Simulation tests for sewage treatment plants must permit an assessment of the
degradation and dissipation behavior of a substance in an STP. An approach
orientated solely at measuring the elimination capacity is insufficient as, e. g.,
the transfer of a problematic compound from the aqueous compartment to the
soil compartment via sludge or into air by stripping (volatilization) is not con-
sidered.
For an assessment information on the following should be available:
Primary degradation and formation of metabolites
Mineralization
Adsorption onto sewage sludge
Volatilization
An inclusion of these requirements into the internationally harmonized test
protocols is still lacking.
Representing a simulation test on a laboratory scale, in chemicals legislation
only the Coupled Units-Test [8] is currently available. Due to its test design
(open system) and its analytics (DOC) it is applicable only to sufficiently water-
soluble, non-adsorbing, and non-volatile substances. The test does not enable a
differentiation between biodegradation and abiotic elimination mechanisms,
like adsorption and volatilization, and hence cannot be considered to be a true
simulation test. Since alternative test methods are lacking at present, the results
from a Coupled Units-Test are taken into consideration on a case-by-case basis
for a quantitative estimation of the elimination capacity of a mechanical-bio-
logical sewage treatment plant.
The OECD 303A is currently under revision and will comprise two parts, in-
cluding far-reaching improvements.
Fig. 6. Overall assessment of persistence categories (IIV; see Table 4) of plant protection
substances in soil systems
Aspects such as the percentage connection to STP, variable loading rates,
operational disturbances due to intoxication or fluctuating substance concen-
trations (e. g., campaign operation, other temporal fluctuations), as well as dis-
charges resulting from rainwater overflows must be considered in a final ex-
posure analysis. The qualitative assessment of the removal efficiency in
Germany is based on the legal requirements of the Federal Water Act (WHG)
[20], Appendix 22, in compliance with generally accepted rules of the state of
the art.
The elimination capacities measured in the Coupled Units-Test are not suffi-
cient as a basis for a quantitative exposure estimation. Kinetic rate constants
could be derived if the appropriate parameters like sludge age and hydraulic re-
tention time are taken into consideration as provided by the updated draft of
the OECD 303A.
For a tentative estimation of the elimination capacity of STP, the elimination
classification from results of STP simulation tests given in Table 5 may be used.
4
Biodegradation Rate Constants
Biodegradation rate constants, i.e., the biodegradation within defined time in-
tervals, are essential for the estimation of the fate of a chemical in the different
environmental compartments. For this estimation first order biodegradation
kinetics are assumed. Due to mostly low concentrations of chemicals and rela-
tively low microbial density, biodegradation processes in the environment fre-
quently follow such kinetics.
4.1
Determination of Biodegradation Rate Constants from Screening Tests
The determination of biodegradation rate constants from screening tests is
generally not possible.
For exposure estimation, based on results from biodegradation tests on
ready and inherent biodegradability, kinetic rate constants for the biodegrada-
tion of a substance in various environmental compartments (STP, surface wa-
ters, sediments, soils) have nevertheless been derived as default values, present-
ed in Tables 6 and 7.
These parameters were agreed upon internationally within the context of ad-
opting the TGD and are used for exposure estimation as long as no substance
specific data are available from higher quality degradation tests (simulation
308 B. Beek et al.
Table 5. Classification of elimination performance of sewage treatment plants
Degree of elimination Class Assessment
> 95% I Substantial elimination
7595% II Medium elimination
< 75% III Poor elimination
tests), which may substitute the tentative rate constants derived from screening
degradation tests.
4.2
Prerequisites for the Derivation of Biodegradation Rate Constants
from Simulation Tests
To derive biodegradation rate constants from results of simulation tests, at least
the following prerequisites should be fulfilled:
The elimination portion resulting from adsorption in an aquatic test system
has to be determined. If no direct experimental data are available this por-
tion is estimated by an additional adsorption control in a test for inherent
biodegradability, e. g., Zahn-Wellens-Test [4, 8, 11], by an experimentally de-
termined Koc [21], or by a calculated Koc (from the Kow or from water solub-
ility).
The abiotic part of elimination either by adsorption, precipitation, or volatil-
ization is subtracted from the total elimination. This gives the amount of de-
gradation, from which the biodegradation rate constant will be calculated.
As a general rule, the test conditions should simulate real conditions as close as
possible.
Table 6. First order degradation rate constants in sewage treatment plants derived from re-
sults of screening tests for ready or inherent biodegradability
Tests for Ready Biodegradability (28 days)
Tests 92/69/EEC C.4 A F respectively OECD 301 AF or tests which
are considered scientifically equal (expert judgement)
Pass-level 60/70% with 60/70% without < 60/70%
10 d-window 10 d-window
Assessment Readily bio- Readily biodegrad- Not readily
degradable able, but failing biodegradable
10 d-window
Degradation rate in k
a
1st order
= 1 h
1
k
1st order
= 0.3 h
1
k
1st order
= 0 h
1
models for sewage
treatment plants
Tests for Inherent Biodegradability (28 days)
Tests 88/302/EEC respectively OECD 302 B C or tests which are con-
sidered scientifically equal (expert judgement)
Pass-level 70% 20 to <70% <20%
Assessment Inherently bio- Partially biodegrad- Not bio-
degradable, fulfilling able/eliminable degradable
specific criteria
Degradation rate in k
1st order
= 0.1 h
1
k
1st order
= 0 h
1
k
1st order
= 0 h
1
models for sewage
treatment plants
a
Equivalent to dt
50
= 0.7 h.
In water/sediment studies and/or simulation tests for biodegradation in soil,
a fractionation into extractable and non-extractable residues (bound residues)
has to be conducted in order to differentiate between easily bioavailable and
less available fractions. Hence the extraction method applied has to be consid-
ered. For the characterization of bound residues soft extraction methods should
be used in order to simulate environmental conditions in sediments and soils.
4.3
Field Studies
Field studies should give information on the biological degradation and dissi-
pation processes on an ecosystematic level thus allowing for a complex assess-
ment of the environmental behavior of a substance; since in field studies some
processes like volatilization, leaching, and metabolism can hardly be investigat-
ed in an appropriate manner, a combination of both laboratory and field studies
may lead to a comprehensive assessment of fate and behavior of a chemical in
the environment. Such studies are to be designed and performed in close co-
operation with the assessing authority. Due to the high complexity of a field
ecosystem and the resulting methodological problems in evaluating and inter-
310 B. Beek et al.
Table 7. First order degradation rate constants for different environmental compartments de-
rived from results of screening tests for ready or inherent biodegradability (according to
TGD)
Compartment Biodegradation 1st Order
Potential Degradation Rate
Constant
Fresh water Water Readily bio- k
1st order
= 0.047 day
1
(river, lake) Phase degradable
Readily biode- k
1st order
= 0.014 day
1
gradable, but failing
10 d-window
Inherently bio- k
1st order
= 0.0047 day
1
degradable, fulfilling
specific criteria
Inherently bio- k
1st order
= 0.00047 day
1
degradable, not ful-
filling specific criteria
Sediment Water All biodegradation Same values as for
Phase Phase potentials water phase
Solid Compared to water k
1st order
= 0 day
1
Phase phase biodegradation (default)
potential is lower
Sea and brackish Compared to water k
1st order
= 0 day
1
water phase biodegradation (default)
potential is lower
preting results from field studies, currently no standardized test system exists,
on the one hand fulfilling the requirements of legislation and on the other hand
those of the above-mentioned scientific minimum requirements.
5
Microbial Inhibition
Testing for toxic or inhibiting effects of a substance to micro-organisms is con-
ducted together with biodegradation testing under the following aspects:
For interpreting test results from biodegradation tests it is essential to have
knowledge of the impact of potentially toxic or inhibiting effects of the sub-
stance of concern to the degrading micro-organisms.
By estimating the risk potential for sewage treatment plants the risk of a
technical malfunction/breakdown due to intoxication can be identified.
Prospective disturbances of biogenic cycles in the primarily exposed envi-
ronmental compartments are to be detected.
A microbial inhibition below 10% or 20% with respect to the control depend-
ing on the test system used is considered as not significant due to methodolo-
gical variability. For the calculation of a PNEC (predicted no effect concentra-
tion) such results (EC
10
/EC
20
) are treated as NOEC (no observed effect concen-
tration) values.
For a risk assessment concerning microbial toxicity it is necessary to com-
pare exposure concentrations (PEC, predicted environmental concentration)
with effect concentrations (PNEC) as is usually applied in ecotoxicology.
The PNEC
micro-organisms
is generally derived from a NOEC supplemented by
an appropriate safety/uncertainty factor depending on the test system used
(endpoint tested, sensitivity) and environmental compartment under consider-
ation (water, soil, STP). Safety factors are applied to minimize the risk of dam-
age to more sensitive micro-organisms as used in the respective test systems.
In Tables 8 and 9 standardized test methods on inhibition of microbial ac-
tivity are compiled, including the most important parameters of test perfor-
mance.
Additionally, a test guideline has recently become available from ISO [22]
based upon the inhibition of growth of sewage sludge bacteria.
5.1
Assessment Criteria
When assessing the results from microbial inhibition tests in the aquatic milieu
a qualitative and quantitative assessment of results is performed. In addition
the test results EC
50
(inhibition concentration of 50%) or NOEC are given,
stating the test method applied, the measured endpoint, and the test duration.
In soils the inhibition of microbial activity of soil micro-organisms can be
determined according to the guideline IV 11 Auswirkung auf die Aktivitt der
Bodenmikroflora/Side-effects on soil microflora [32] of the German Federal
Research Center for Agriculture and Forestry.
The endpoints respiration rates, dehydrogenase-activity, and nitrogen turn-
over are measured within the registration procedure in fulfillment of the
German Plant Protection Act.
Accordingly, the test duration is generally 28 days and can provided effects
>15% are encountered be prolonged up to 56 days or 100 days. The respective
effects, i. e., both increase and decrease of activities, are expressed as percent de-
viation from an untreated control and assessed according to a model described
by Malkomes [33] as shown in Fig. 7. The results from these tests, carried out
with fivefold and tenfold maximum rate of application in order to simulate the
accumulation of the substance in deeper soil layers, represent a realistic worst-
312 B. Beek et al.
Table 8. Inhibition of microbial activity comparison of test methods part 1
Method Inhibition of Inhibition con- Activatet sludge Inhibition of
oxygen control of the respiration dehydrogenase-
sumption with closed bottle inhibition test activity
Pseudomonas test
a
putida
References DIN 38 412, OECD 301 D EU 88/302/EEC [26]
part 27 [23] [8, 11] L 133 [4]
EU C.4 E [12] OECD 209 [24]
ISO 8192 [25]
Inoculum
Origin
Bacilliculture P. putida
Mixed culture Effluent of sewage Activated sludge Activated sludge
treatment plant or
surface water
Density 5 ml/l 0.81.6 g dw/l 0.42.4 g/l
Approx. cells/l No information 10
4
10
6
10
9
10
10
No information
Temperature 211 C 222 C 202 C 21 C
pH-Value 7.50.5 7.40.2 7.258.0 No information
(optimum)
Test duration 30 min 14 d 30 min and/ 15 or 30 min
or 3 h
Measured Inhibition of Inhibition of O
2
- Inhibition of O
2
- Inhibition of
parameters O
2
-consumption consumption of consumption reduction of redox
a readily degrad- dye-stuffs by
able reference microbial dehy-
substance drogenase
Evaluation Determination Graphical com- Graphical dose- Graphical dose-
of dilution grade parison of BOD: effect relation, effect relationship;
causing an inhi- reference sub- ship; EC
20
, EC
50
EC
50
bition of < 20% stance vs.
reference sub-
stance + test
substance
a
This applies to all tests for ready biodegradation. In all these tests the performance of a
toxicity control is optional.
case. Such an accumulation can be observed with strongly-adsorbing sub-
stances as well as under extremely dry weather conditions.
In the context of implementing the Council Directive 91/414/EEC of 15th
of July 1991 [34] concerning the placing of plant protection products on the mar-
ket, the following classification based on the same parameters is currently used:
Table 9. Inhibition of microbial activity comparison of test methods part 2
Method Inhibition of Growth inhibi- Inhibition of Side-effect on the
nitrification tion test with light emission activity of soil
Pseudomonas microflora
putida
a) Dehydrogenase
activity
b) Short time
respiration
c) Metabolic active
biomass
d) Nitrogen turn-
over
References ISO 9509 [27] DIN EN ISO DIN EN ISO BBA Guideline,
10712 [28] 11348 Part 1 to 3 Part VI, 11, [32]
[29, 30, 31]
Inoculum
Origin
Bacilliculture P. putida Vibrio fisheri
Mixed culture Activated Two agricultural
sludge soils with micro-
flora of differing
activity
Density 1.5 g/l
Approx. cells/l No information 10
9
2 10
9
No information
Temperature 2025C 21 1C 15 2C 20 2C
pH-Value 7.6 7.4 7.0 0.2 soil 1: 5.57.0
soil 2: 6.07.5
Test duration 4 h 16 1 h 15 and 30 min Different; depend-
ing on parameter
to be tested
Measured Inhibition of Inhibition of Decline of a) Reduction of
parameters nitrification cell multipli- luminescence redox dye-stuffs
(oxidation of cation intensity by microbial
ammonium) (turbidity) dehydrogenase
b) O
2
-consumption
c) CO
2
-evolution
d) Determination of
NH
4
+
, NO
3
, (NO
2
),
N-total
Evaluation Graphical dose- Graphical dose- Graphical dose- Graphical dose-
effect relation- effect relation- effect relation- effect relationship;
ship; EC
50
ship; EC
10
; EC
50
EC
20
; EC
50
EC
50
Category Toxicity Deviation of activity After x days
from control (in %)
I very weak < 2.5 28
II weak < 25 28
III moderate < 25 56
IV high < 25 100
V very high > 25 100
The first category with a deviation of <2.5% from control, however, is in view
of methodological aspects, not applicable because effects will only be signifi-
cant above 1015%. Therefore we propose to omit this category completely.
5.2
Intrinsic Properties of Chemicals and Consequences for Choice
and Performance of Tests
The issue of which test design is suited to clarify the microbial toxicity of a test
substance depends on the selection of an appropriate endpoint, the respective
314 B. Beek et al.
Fig. 7. The decrease of microbial toxicity with time according to Malkomes [33]: 1 negligible,
2 tolerable, 3 critical, 4 non-tolerable toxicity
environmental compartment, and the intrinsic properties of the substance un-
der concern.
As shown in Tables 8 and 9 (see above), the summarized tests exhibit differ-
ent toxicological endpoints, sensitivities, and test durations. Generally, short-
term measurements in terms of hours (e. g. 10 h) are preferred, in accordance
with the retention time in an STP.
For soils only one test system is available in Germany (BBA, 1990) [17]. Two
guidelines for microbial toxicity in soils are currently under development by the
OECD (based upon the inhibition of transformation of carbon and nitrogen in
soils). The other test systems refer to aquatic compartments and STP.
Concerning the intrinsic properties of substances, poor water solubility and
adsorbance are especially problematic.
For such difficult substances guidance documents are currently under de-
velopment by ISO, OECD, and EU. From a pragmatic point of view, substances
may be defined as being poorly water soluble if their solubility is below
100 mg/l. The use of solubilizers for increasing the solubility of poorly water
soluble substances should be avoided because the results may be misleading.
Instead, stirring a nominal of, e. g., 100 mg/l for 24 h ensuring the maximum sol-
ubility and subsequent testing is recommended as a limit test.
In such a test performance the real substance concentrations are often below
detection limits, i. e., unknown. For this the term Water Accommodated
Fraction (WAF) may be used as has originally been proposed for mixtures of
poorly water soluble substances [35].
If testing microbial toxicity in serial dilutions of a stock solution of a poorly
water soluble substance, as is usually done with well soluble substances, mis-
leading results will be obtained as exemplified in the following.
Given a poorly water soluble substance with a solubility of 1 mg/l which,
however, was not analytically determined, a stock solution with a nominal con-
centration of 100 mg/l is prepared exerting a slight but significant effect. Hence
the LOEC (lowest observed effect concentration) would be noted as 100 mg/l
nominal. Given further that a 1: 1 dilution would stop the effect, then the NOEC
(no observed effect concentration) would be noted as 50 mg/l.
However, preparation of a stock solution of a nominal 50 mg/l would also
contain 1 mg/l dissolved substance exerting the same effect as 25 mg/l and so
on, until 1 mg/l is dissolved and further diluted 1: 1, leading now to the true
NOEC of 0.5 mg/l instead of 50 mg/l as above.
Testing should consequently be performed with the supernatant of WAFs
without filtration or centrifugation procedures.
For the OECD 209 Activated sludge respiration inhibition test [24] the test
duration of 3 h is recommended for poorly water soluble substances to en-
counter possible delayed effects. This is true also for the other test systems lis-
ted in Tables 8 and 9. The rationale behind this and an actual example is given
in the following.
A poorly water soluble substance (identity confidential) was tested in an
OECD 209 test within a notification procedure. After 30 min an EC
50
>1000 mg/l
was measured, whereas after 3 h an EC
50
of 400 mg/l and a NOEC of 100 mg/l
was obtained.
If after 3 h testing no effects are found, no further testing is required and the
substance is classified as being not inhibiting the microbial activity up to, e. g.,
100 mg/l nominal.
For STP and surface waters, no risk is assumed from microbial toxicity, and
no PNECs are derived.
If significant effects occur, WAFs of single concentrations within a concen-
tration range should be prepared to establish a dose-response relationship.
With water soluble substances, i. e., water solubility>100 mg/l, testing of
microbial toxicity may start with a limit test as well, preferably with 1000 mg/l,
and then in case of significant effects (>10% or 20% depending on the test sys-
tem) regular testing with serial dilutions has to be performed as described
above to establish a dose-response relationship to derive EC
x
-values and the
NOEC for the calculation of the PNEC
micro-organisms
.
If the exposure concentration exceeds the test concentration of, e. g.,
100 mg/l, a test with higher concentration either as a limit test or with serial di-
lutions has to be performed.
A further problematic property is the adsorption tendency of a substance,
since in this case an adsorption onto suspended solids, soils, sediments, vessel
walls, and onto the inoculum must be expected. This may lead to a reduction of
the true test concentration in a test system. We assume an essential adsorption
if the substance has a log K
OW
>3 (octanol/water partition coefficient) or if an
adsorption potential can be proven in an adsorption/desorption screening test
(OECD 106, ISO draft).
Moreover, delayed toxicity may occur due to depot effects. Quarternary am-
monia compounds for example are known to cause such effects.
Apart from regular tests on microbial toxicity mentioned above, inhibi-
tion controls from tests on ready biodegradability may be used for a preli-
minary screening for toxic effects. In such assays the influence of a test sub-
stance on the biodegradation of a well-degradable reference substance is deter-
mined. However, results are only obtained for one single concentration (see
Table 8).
5.3
Risk Assessment and Safety Factors
For risk assessment of a sewage treatment plant a PEC
STP
/PNEC
micro-organisms
ratio
is calculated. As already mentioned, for the calculation of a PNEC
micro-organisms
the NOEC is taken as a basic value supplemented with an appropriate safety fac-
tor according to the TGD [14] listed in Table 10.
The rationale behind the different safety factors is given by differences in
sensitivities, end points, and test durations of the respective test system. Thus,
test systems with higher safety factors are the less sensitive ones.
For the calculation of the predicted environmental concentration for sewage
treatment plants (PEC
STP
) the influent concentration (c
inf
), i. e., in the sewer
should be taken rather than, as suggested by the TGD, the effluent concentration
(c
eff
), because toxicity already occurs in the sewer, possibly giving rise to inhi-
bition effects of microbial activity in the sewage sludge.
316 B. Beek et al.
Given a PEC
STP
/PNEC
micro-organisms
of <1, no indication of microbial toxicity in
STP is assumed and no further testing is required.
In case of a PEC
STP
/PNEC
micro-organisms
of >1, an indication of microbial tox-
icity for STP is given and further testing of microbial toxicity is required using
different toxicological endpoints, preferably with regard to the intrinsic pro-
perty of the test substance.
The same procedure is applied for the compartments soil and surface water.
Protozoa (ciliates) are an integral and important part of a functioning bio-
cenosis of an STP, mainly due to the elimination of germs and improvement of
carbon and nitrogen metabolism in an STP (see contribution by Pauli et al., this
volume). Besides microbial toxicity the role of protozoa in STP has recently be
considered in a Technical Recommendation of the European Chemicals Bureau
[36] as follows:
All valid ciliate growth impairment data should be taken into account for the
derivation of a PNEC
STP
. Protozoa have to be regarded as additional species, not
as an additional trophic layer. The PNEC
STP
should be derived on the basis of
the most sensitive species regardless of whether this is from a test with activat-
ed sludge, relevant bacteria, or ciliated protozoa.
6
Deficits and Perspectives
The modified MITI II-Test (guideline OECD 302 C) [8] should be included in
the test repertoire of the EEC as a test on inherent biodegradability, since the
true biodegradation is measured separate from adsorption and/or volatiliza-
tion. As to the practical performance, the use of one inoculum from a muni-
cipal STP instead of ten as in the original Japanese prescription may be ac-
cepted for reasons of simplification.
The Two Phase Closed Bottle-Test (BODIS-Test, ISO 10708) [15] for poorly
water-soluble substances should be included in the inventory of tests for ulti-
mate biodegradability. However, the test conditions regarding the agitation
procedure should be standardized.
Table 10. Safety factors in accordance with the TGD
Test Safety factor applied Safety factor applied
to NOEC or EC
10
/EC
20
to EC
50
Growth Inhibition Test with 1 10
Pseudomonas putida
Inhibition of nitrification 1 10
Inhibition of luminescent bacteria 1 10
Other tests with single species 1 10
Activated Sludge Respiration Inhibition 10 100
Test (OECD 209)
Other tests with mixed inoculum 1 or 10 (depending 10 or 100
on sensitivity of test)
There is a need for standardized test guidelines and assessment schemes for
screening and simulation tests on the anaerobic biodegradation of sub-
stances in hydrosphere, soil, and sludge for risk assessment.
A test system has been developed sponsored by the German Federal
Environmental Agency for testing fate and behavior of chemicals in surface
waters (rivers, lakes) including sediment. A draft has recently been submit-
ted to ISO.
There is a need for tests simulating biodegradation and microbial toxicity in
estuaries, coastal areas, and the open sea.
Besides the existing tests on nitrification in water and soil there is a need for
further development of denitrification test systems.
At present, standardized tests covering the inhibition of microbial activity
under anaerobic conditions are missing (e.g., inhibition of methane produc-
tion). This is also relevant for inhibition controls in anaerobic biodegrada-
tion tests mentioned above.
For testing difficult substances (volatile, poorly water soluble, adsorbing)
there is still no appropriate guidance available. However, ISO, OECD, and EU
have respective guidance documents currently in progress.
Likewise, still no guidance exists for situations where conflicting results on
biodegradability have been obtained from tests of the same level, e. g., base
set screening tests on ready biodegradability as experienced within the noti-
fication of New Chemicals according to the German Chemicals Act. Some
member states of the EU have suggested the best test results be taken if va-
lidity of the performance had been ensured, while others rely on expert
judgement (weight of evidence). In cases where a sufficient number of
independent and valid test results exists, the calculation of a median value
(geometric mean value) is proposed for discussion.
Biodegradation and persistence have become of increasing importance for en-
vironmental risk assessment of chemicals during the last decade. This is also re-
flected by ongoing international activities of the United Nations Environmental
Program (UNEP) concerning Persistent Organic Pollutants (POPs).
Thus, the above-mentioned deficiencies urgently need to be removed.
7
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320 B. Beek et al.

Bio Degradation and Persistence

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Bio Degradation and Persistence

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Aerobic and Anaerobic Biodegradation Potentials

Redox reaction (kJ/mol acceptor) (kJ/mol 2e

+70.6 kJ/mol benzoate

+5.4 kJ/mol p-hydroxybenzoate

77.8 kJ/mol protocatechuate

160.0 kJ/mol gallate

At least four different classes of hydrolytic 2-halo acid dehalogenases have

in the course of hydrolytic deha-

kopov I (1993) Int Revue ges Hydrobiol 78: 557

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