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Transcript
6 Aerobic Degradation by
Microorganisms
WOLFGANG FRITSCHE
MARTIN HOFRICHTER
Jena, Germany
1 Introduction: Characteristics of Aerobic Microorganisms Capable of Degrading Organic
Pollutants 146
2 Principles of Bacterial Degradation 147
2.1 Typical Aerobic Degrading Bacteria 147
2.2 Growth-Associated Degradation of Aliphatics 148
2.3 Diversity of Aromatic Compounds – Unity of Catabolic Processes 149
2.4 Extension of Degradative Capacities 152
2.4.1 Cometabolic Degradation of Organopollutants 152
2.4.2 Overcoming the Persistence by Cooperation of Anaerobic and Aerobic
Bacteria 154
3 Degradative Capacities of Fungi 156
3.1 Metabolism of Organopollutants by Microfungi 157
3.1.1 Aliphatic Hydrocarbons 157
3.1.2 Aromatic Compounds 158
3.2 Degradative Capabilities of Basidiomycetous Fungi 160
3.2.1 The Ligninolytic Enzyme System 161
3.2.2 Degradation of Organopollutants 163
4 Conclusions 164
5 References 164
146
6 Aerobic Degradation by Microorganisms
List of Abbreviations
AsO
BTX
DBDs
DBFs
DCA
DCP
DDT
DNT
KCN
LiP
MnP
PAHs
PCBs
PCP
TCC
TCE
TNT
arsenic-containing organic compounds
benzene, toluene, xylenes
dibenzo-p-dioxines
dibenzofurans
3,4-dichloroaniline
2,4-dichlorophenol
1,1,1-trichloro-2,2b-bis(4-chlorophenyl)ethane
2,4-dinitrotoluene
potassium cyanide
lignin peroxidase
manganese peroxidase
polycyclic aromatic hydrocarbons
polychlorinated biphenyls
pentachlorophenol
tricarboxylic acid cycle
trichloroethene
2,4,6-trinitrotoluene
1 Introduction:
Characteristics of Aerobic
Microorganisms Capable of
Degrading Organic
Pollutants
The most important classes of organic pollutants in the environment are mineral oil constituents and halogenated products of petrochemicals. Therefore, the capacities of aerobic
microorganisms are of particular relevance for
the biodegradation of such compounds and
are exemplarily described with reference to
the degradation of aliphatic and aromatic hydrocarbons as well as their chlorinated derivatives. The most rapid and complete degradation of the majority of pollutants is brought
about under aerobic conditions (RISER-ROBERTS, 1998).
The essential characteristics of aerobic microorganisms degrading organic pollutants are
(Fig. 1):
Fig. 1. Main principle of aerobic degradation of hydrocarbons: growth associated processes.
2 Principles of Bacterial Degradation
(1) Metabolic processes for optimizing the
contact between the microbial cells and
the organic pollutants. The chemicals
must be accessible to the organisms
having biodegrading activities. For example, hydrocarbons are water-insoluble and their degradation requires the
production of biosurfactants.
(2) The initial intracellular attack of organic pollutants is an oxidative process, the
activation and incorporation of oxygen
is the enzymatic key reaction catalyzed
by oxygenases and peroxidases.
(3) Peripheral degradation pathways convert organic pollutants step by step into
intermediates of the central intermediary metabolism, e.g., the tricarboxylic
acid cycle.
(4) Biosynthesis of cell biomass from the
central precursor metabolites, e.g., acetyl-CoA, succinate, pyruvate. Sugars required for various biosyntheses and
growth must be synthesized by gluconeogenesis.
A huge number of bacterial and fungal genera
possess the capability to degrade organic pollutants. Biodegradation is defined as the biologically catalyzed reduction in complexity of
chemical compounds (ALEXANDER, 1994). It is
based on two processes: growth and cometabolism. In the case of growth, organic pollutants
are used as sole source of carbon and energy.
This process results in a complete degradation
(mineralization) of organic pollutants as demonstrated in Sect. 2.2. Cometabolism is defined
as the metabolism of an organic compound in
the presence of a growth substrate which is
used as the primary carbon and energy source.
The principle is explained in Sect. 2.4.
Enzymatic key reactions of aerobic biodegradation are oxidations catalyzed by oxygenases and peroxidases. Oxygenases are oxidoreductases that use O2 to incorporate oxygen
into the substrate. Degradative organisms
need oxygen at two metabolic sites, at the initial attack of the substrate and at the end of
the respiratory chain (Fig. 1). Distinct higher
fungi have developed a unique oxidative
system for the degradation of lignin based on
extracellular ligninolytic peroxidases and laccases. This enzymatic system possesses increas-
147
ing significance for the cometabolic degradation of persistent organopollutants.Thus it is in
particular the domain of basidiomycetous fungi, which requires a deeper insight and an extensive consideration. Therefore, this chapter
has been divided in two sections: bacterial and
fungal degradation capacities.
2 Principles of Bacterial
Degradation
2.1 Typical Aerobic Degrading
Bacteria
The predominant degraders of organopollutants in the oxic zone of contaminated areas
are chemo-organotrophic species able to use a
huge number of natural and xenobiotic compounds as carbon sources and electron donors
for the generation of energy. Although many
bacteria are able to metabolize organic pollutants, a single bacterium does not possess the
enzymatic capability to degrade all or even
most of the organic compounds in a polluted
soil. Mixed microbial communities have the
most powerful biodegradative potential because the genetic information of more than
one organism is necessary to degrade the complex mixtures of organic compounds present in
contaminated areas. The genetic potential and
certain environmental factors such as temperature, pH, and available nitrogen and phosphorus sources, therefore, seem to determine
the rate and the extent of degradation.
The predominant bacteria of polluted soils
belong to a spectrum of genera and species
listed in Tab. 1. The composition of this list of
bacteria is determined by the fact whether
they can be cultured on nutrient-rich media.
We have to consider that the majority of bacteria present in soils cannot be cultured in the
laboratory yet.
Pseudomonads, aerobic gram-negative rods
that never show fermentative activities, seem
to have the highest degradative potential, e.g.,
Pseudomonas putida and P. fluorescens. Further important degraders of organic pollutants
can be found within the genera Comamonas,
148
6 Aerobic Degradation by Microorganisms
Tab. 1. Predominant Bacteria in Soil Samples Polluted with Aliphatic and Aromatic Hydrocarbons, Polycyclic Aromatic Hydrocarbons, and Chlorinated
Compoundsa
Gram-Negative
Bacteria
Gram-Positive
Bacteria
Pseudomonas spp.
Acinetobacter spp.
Alcaligenes sp.
Flavobacterium/
Cytophaga group
Xanthomonas spp.
Nocardia spp.
Mycobacterium spp.
Corynebacterium spp.
Arthrobacter spp.
Bacillus spp.
a
The reclassification of bacteria has been based on
phylogenetic markers resulting in changes of some
genera and species. This is why the names of species
are not mentioned.
Burkholderia, and Xanthomonas. Some species utilize `100 different organic compounds
as carbon sources. The immense potential of
the pseudomonas does not solely depend on
the catabolic enzymes, but also on their capability of metabolic regulation (HOUGHTON and
SHANLEY, 1994). A second important group of
degrading bacteria are the gram-positive rhodococci and coryneform bacteria. Many species,
now classified as Rhodococcus spp. had originally been described as Nocardia spp., Mycobacterium spp., and Corynebacterium spp.
Rhodococci are aerobic actinomycetes showing considerable morphological diversity. A
certain group of these bacteria possess mycolic acids at the external surface of the cell.
These compounds are unusual long-chain alcohols and fatty acids, esterified to the peptidoglycan of the cell wall. Probably, these lipophilic cell structures have a significance for the
affinity of rhodococci to lipophilic pollutants.
In general, rhodococci have high and diverse
metabolic activities and are able to synthesize
biosurfactants.
2.2 Growth-Associated
Degradation of Aliphatics
The aerobic initial attack of aliphatic and cycloaliphatic hydrocarbons requires molecular
oxygen. Fig. 2 shows both types of enzymatic
reactions involved in these processes. It depends on the nature of the substrate and the
enzymatic equipment of the involved microorganisms, what kind of enzymatic reaction is
realized. n-Alkanes are the main constituents
of mineral oil contaminations (HINCHEE et al.,
1994). Long-chain n-alkanes (C10UC24) are
degraded most rapidly by mechanisms demonstrated in Fig. 3. Short-chain alkanes (less than
C9) are toxic to many microorganisms, but
they evaporate rapidly from petroleum contaminated sites. Oxidation of alkanes is classified as being terminal or diterminal. The
monoterminal oxidation is the main pathway.
It proceeds via the formation of the corresponding alcohol, aldehyde, and fatty acid.
b-Oxidation of the fatty acids results in the
formation of acetyl-CoA. n-Alkanes with an
uneven number of carbon atoms are degraded
to propionyl-CoA, which is in turn carboxylated to methylmalonyl-CoA and further converted to succinyl-CoA. Fatty acids of a physiological chain length may be directly incorporated into membrane lipids, but the majority of
degradation products is introduced into the
tricarboxylic acid cycle. The subterminal oxidation occurs with lower (C3UC6) and longer
alkanes with the formation of a secondary alcohol and subsequent ketone. Unsaturated 1alkenes are oxidized at the saturated end of
the chains. A minor pathway has been shown
to proceed via an epoxide, which is converted
to a fatty acid. Branching, in general, reduces
the rate of biodegradation. Methyl side groups
do not drastically decrease the biodegradability, whereas complex branching chains, e.g., the
tertiary butyl group, hinder the action of the
degradative enzymes.
Cyclic alkanes representing minor components of mineral oil are relatively resistant to
microbial attack. The absence of an exposed
terminal methyl group complicates the primary attack. A few species are able to use cyclohexane as sole carbon source; more common is its cometabolism by mixed cultures.
The mechanism of cyclohexane degradation is
shown in Fig. 4. In general, alkyl side chains of
cycloalkanes facilitate the degradation.
Aliphatic hydrocarbons become less water
soluble with increasing chain length. Hydrocarbons with a chain length of C12 and above
are virtually water insoluble. Two mechanisms
2 Principles of Bacterial Degradation
149
Fig. 2. Initial attack on xenobiotics by oxygenases.
Monooxygenases incorporate one atom of oxygen of
O2 into the substrate, the
second atom is reduced to
H2O. Dioxygenases incorporate both atoms into the
substrate.
are involved in the uptake of these lipophilic
substrates: the attachment of microbial cells at
oil droplets and the production of biosurfactants (HOMMEL, 1990). The uptake mechanism
linked to attachment of the cells is still unknown, whereas the effect of biosurfactants
has been studied well (Fig. 5). Biosurfactants
are molecules consisting of a hydrophilic and a
lipophilic moiety. They act as emulsifying
agents, by decreasing the surface tension and
by forming micelles. The microdroplets may be
encapsulated in the hydrophobic microbial
cell surface. The products of hydrocarbon degradation, introduced to the central tricarboxylic acid cycle, have a dual function. They are
substrates of the energy metabolism and building blocks for biosynthesis of cell biomass
and growth (Fig. 1). The synthesis of amino
acids and proteins needs a nitrogen and sulfur
source, that of nucleotides and nucleic acids a
phosphorus source. The biosynthesis of the
bacterial cell wall requires activated sugars
synthesized by gluconeogenesis.
Products of growth-associated degradation
are CO2, H2O, and cell biomass. The cells act as
the complex biocatalysts of degradation. In addition, cell biomass may be mineralized after
exhaustion of the degradable pollutants in a
contaminated site.
2.3 Diversity of Aromatic
Compounds – Unity of Catabolic
Processes
Aromatic hydrocarbons, e.g., benzene, toluene, ethylbenzene and xylenes (BTEX compounds), and naphthalene belong to the large
volume petrochemicals, widely used as fuels
and industrial solvents. Phenols and chlorophenols are released into the environment as
150
6 Aerobic Degradation by Microorganisms
Fig. 3. Peripheral pathways
of alkane degradation. The
main pathway is the terminal oxidation to fatty acids
catalyzed by a n-alkane
monoxygenase, b alcohol
dehydrogenase and c aldehyde dehydrogenase.
products and waste materials from industry.
Aromatic compounds are formed in large
amounts by all organisms, e.g., as aromatic
amino acids, phenols, or quinones. Thus, it is
not surprising that many microorganisms have
evolved catabolic pathways to degrade aromatic compounds. In general, man-made organic
chemicals (xenobiotics) can be degraded by
microorganisms, when the respective molecules are similar to natural compounds. The diversity of man-made aromatics shown in Fig. 6
can be converted enzymatically to natural intermediates of the degradation: catechol and
protecatechuate. In general, benzene and related compounds are characterized by a higher
thermodynamic stability than aliphatics are.
Only few reports on bacteria capable of
attacking benzene have been published
(SMITH, 1990). The first step of benzene oxidation is a hydroxylation catalyzed by a dioxygenase (Fig. 2).The product, a diol, is then converted to catechol by a dehydrogenase. These initial reactions, hydroxylation and dehydrogenation, are also common to pathways of degradation of other aromatic hydrocarbons.
The introduction of a substituent group onto the benzene ring renders alternative mechanisms possible to attack side chains or to oxidize the aromatic ring. The versatility and
adaptability of bacteria is based on the existence of catabolic plasmids. Catabolic plasmids
have been found to encode enzymes degrading
2 Principles of Bacterial Degradation
Fig. 4. Peripheric metabolic pathway of cycloaliphatic compounds (cycloparaffins).
naturally occurring aromatics such as camphor, naphthalene, and salicylate. Most of the
catabolic plasmids are self-transmissible and
have a broad host range. The majority of gramnegative soil bacteria isolated from polluted
areas possess degradative plasmids, mainly the
so called TOL plasmids. These pseudomonads
are able to grow on toluene, m- and p-xylene,
and m-ethyltoluene. The main reaction involved in the oxidation of toluene and related
arenes is the methyl group hydroxylation. The
methyl group of toluene is oxidized stepwise
to the corresponding alcohol, aldehyde, and
carboxylic group. Benzoate formed or its alky-
151
lated derivatives are then oxidized by toluate
dioxygenase and decarboxylated to catechol
(SMITH, 1990).
The oxygenolytic cleavage of the aromatic
ring occurs via o- or m-cleavage. The significance of the diversity of degradative pathways
and of the few key intermediates is still under
discussion. Both pathways may be present in
one bacterial species. “Whenever an alternative
mechanism for the dissimilation of any compound becomes available (ortho- versus metacleavage of ring structures, for example) control
of each outcome must be imposed” (HOUGHTON and SHANLEY, 1994). The metabolism of a
wide spectrum of aromatic compounds by one
species requires the metabolic isolation of
intermediates into distinct pathways. This kind
of metabolic compartmentation seems to be
realized by metabolic regulation. The key enzymes of the degradation of aromatic substrates are induced and synthesized in appreciable amounts only when the substrate or
structurally related compounds are present.
Enzyme induction depends on the concentration of the inducing molecules. The substrate
specific concentrations represent the threshold of utilization and growth and are in the
magnitude of µM. A recent report on the regulation of TOL catabolic pathways has been
published by RAMOS et al. (1997).
Fig. 7 shows the pathways of the oxygenolytic ring cleavage to intermediates of the central
metabolism. At the branchpoint catechol either is oxidized by the intradiol o-cleavage, or
the extradiol m-cleavage. Both ring cleavage
reactions are catalyzed by specific dioxygenases. The product of the o-cleavage – cis,cismuconate – is transferred to the instable enollactone, which is in turn hydrolyzed to oxoadipate. This dicarboxylic acid is activated by
transfer to CoA, followed by the thiolytic
cleavage to acetyl-CoA and succinate. Protocatechuate is metabolized by a homologous set
of enzymes. The additional carboxylic group is
decarboxylated and, simultaneously, the double bond is shifted to form oxoadipate enollactone. The oxygenolytic m-cleavage yields 2hydroxymuconic semialdehyde, which is metabolized by the hydrolytic enzymes to formate, acetaldehyde, and pyruvate. These are
then utilized in the central metabolism. In general, a wealth of aromatic substrates is degrad-
152
6 Aerobic Degradation by Microorganisms
Fig. 5. Involvement of
biosurfactants in the
uptake of hydrocarbons. The figure demonstrates the emulsifying
effect of a rhamnolipid
produced by Pseudomonas spp. within the
oil–water interphase
and the formation of
micelles. Lipid phases
are printed in bold.
ed by a limited number of reactions: hydroxylation, oxygenolytic ring cleavage, isomerization, hydrolysis.The inducible nature of the enzymes and their substrate specificity enable
bacteria with a high degradation potential, e.g.,
pseudomonads and rhodococci, to adapt their
metabolism to the effective utilization of substrate mixtures in polluted soils and to grow at
a high rate.
2.4 Extension of Degradative
Capacities
2.4.1 Cometabolic Degradation of
Organopollutants
Cometabolism, the transformation of a substance without nutritional benefit in the presence of a growth substrate, is a common phenomenon of microbial activities. It is the basis
of biotransformations (bioconversions) used
in biotechnology to convert a substance to a
chemically modified form. Microorganisms
growing on a particular substrate gratuitously
oxidize a second substrate (cosubstrate). The
cosubstrate is not assimilated, but the product
may be available as substrate for other organisms of a mixed culture.
The prerequisites of cometabolic transformations are the enzymes of the growing cells
and the synthesis of cofactors necessary for enzymatic reactions, e.g., of hydrogen donors (reducing equivalents, NADH) for oxygenases.
The principle is shown in Fig. 8. The example
demonstrated in Fig. 8 has been used in field
experiments for the elimination of trichloroethylene (THOMAS and WARD, 1989). Methanotrophic bacteria used in this experiment can
utilize methane and other C1 compounds as
sole sources of carbon and energy. They oxidize methane to CO2 via methanol, formaldehyde, and formate. The assimilation requires
special pathways, and formaldehyde is the
intermediate assimilated. The first step of
2 Principles of Bacterial Degradation
153
Fig. 6. Degradation of a broad
spectrum of aromatic natural
and xenobiotic compounds into
two central intermediates: catechol and protocatechuate.
methane oxidation is catalyzed by methane
monooxygenase, which attacks the inert CH4.
It is an unspecific enzyme that also oxidizes
various other compounds, e.g., alkanes, aromatic compounds, and trichloroethylene
(TCE). The proposed mechanism of TCE
transformation according to HENRY and
GRBIĆ -GALLIĆ (1994) is shown in Fig. 8.TCE is
oxidized to an epoxide excreted from the cell.
The unstable oxidation product breaks down
to compounds, which may be used by other microorganisms. Methanotrophic bacteria are
aerobic indigenous bacteria, in soil and aqui-
fers, but methane has to be added as growth
substrate and inducer for the development of
methanotrophic biomass. The addition of
methane as substrate limits the application for
bioremediation.
Cometabolism of chloroaromatics is a widespread activity of bacteria in mixtures of industrial pollutants. KNACKMUSS (1997) demonstrated that the cometabolic transformation of
2-chlorophenol gives rise to dead end metabolites, e.g., 3-chlorocatechol. This reaction
product may be auto-oxidized or polymerized
in soil to humic-like structures. Irreversible
154
6 Aerobic Degradation by Microorganisms
Fig. 7. The two alternative pathways
of aerobic degradation of aromatic
compounds: o- and m-cleavage, a
phenol monoxygenase, b catechol
1,2-dioxygenase, c muconate lactonizing enzyme, d muconolactone isomerase, e oxoadipate enol-lactone
hydrolase, f oxoadipate succinylCoA transferase, g catechol 2,3-dioxygenase, h hydroxymuconic semialdehyde hydrolase, i 2-oxopent-4enoic acid hydrolase, j 4-hydroxy-2oxovalerate aldolase.
binding of dead end metabolites may fulfill the
function of detoxification. The accumulation
of dead end products within microbial communities under selection pressure is the basis for
the evolution of new catabolic traits (REINECKE, 1994).
2.4.2 Overcoming the Persistence
by Cooperation of Anaerobic and
Aerobic Bacteria
As a rule, recalcitrance of organic pollutants
increases with increasing halogenation. Substi-
tution of halogen as well as nitro and sulfo
groups at the aromatic ring is accomplished by
an increasing electrophilicity of the molecule.
These compounds resist the electrophilic attack by oxygenases of aerobic bacteria. Compounds that persist under oxic conditions are,
e.g., PCBs (polychlorinated biphenyls), chlorinated dioxins, some pesticides, e.g., DDT.
To overcome the relatively high persistence
of halogenated xenobiotics, reductive attack of
anaerobic bacteria is of significance. The degradation of environmental pollutants by anaerobic bacteria is the subject of Chapter 7,
this volume. Because of the significance of reductive dehalogenation for the first step in the
2 Principles of Bacterial Degradation
Fig. 8. Cometabolic degradation of trichloromethane by the methane monoxygenase system of
methanotrophic bacteria.
degradation of higher halogenated compounds, this process has been announced. Reductive dehalogenation effected by anaerobic
bacteria is either a gratuitous reaction or a new
type of anaerobic respiration (ZEHNDER, 1988).
The process reduces the degree of chlorination
and, therefore, makes the product more accessible to mineralization by aerobic bacteria.
The potential of a sequence of anaerobic
and aerobic bacterial activities for the mineralization of chlorinated xenobiotics is described
in Fig. 9. PCBs, which are selected as an example for degradation of halogenated compounds, are well-studied objects (TIEDJE et al.
1993; SYLVESTRE and SANDOSSI, 1994; BEDARD
and QUENSEN, 1995). The scheme demonstrates the principle of enzymatic dehalogenation mechanisms. The realization of the reactions depends on the structure of the chemical
compounds as well as on the microorganisms
and conditions in a polluted ecosystem. We
have to distinguish between the general degradation potential and the actual conditions necessary for its realization. Reductive dehalogenation, the first step of PCB degradation, requires anaerobic conditions and organic substrates acting as electron donors. The PCBs
155
have the function of an electron acceptor to allow the anaerobic bacteria to transfer electrons to these compounds. Anaerobic bacteria
capable of catalyzing reductive dehalogenation seem to be relatively ubiquitous in nature.
Most dechlorinating cultures are mixed cultures (consortia). Aanaerobic dechlorination
is always incomplete, products are di- and
monochlorinated biphenyls. These products
can be metabolized further by aerobic microorganisms. The substantial reduction of PCBs
by sequential anaerobic and aerobic treatment
has been demonstrated in the laboratory (ABRAMOWICZ, 1990).
The principle of aerobic microbial dehalogenation reactions of chloroaromatics are described in Fig. 9. Hydrolytic dechlorination has
been elucidated using 4-chlorobenzoate as
substrate for Pseudomonas and Nocardia spp.
A halidohydrolase is capable of replacing the
halogen substituent by a hydroxy group originating from water. This type of reaction seems
to be restricted to halobenzoates substituted in
the p-position. Dechlorination after ring cleavage is a common reaction of the o-pathway of
chlorocatechols catalyzed by catechol 1,2dioxygenases to produce chloromuconates.
The oxygenolytic dechlorination is a rare fortuitous reaction catalyzed by mono- and dioxygenases. During this reaction, the halogen
substituent is replaced by oxygen of O2.
Higher chlorinated phenols, e.g., pentachlorophenol, have been widely used as biocides.
Several aerobic bacteria that degrade chlorophenols have been isolated (Flavobacterium,
Rhodococcus). The degradation mechanism
has been elucidated in some cases (MCALLISTER et al., 1996). Thus, Rhodococcus chlorophenolicus
degrades
pentachlorophenol
through a hydrolytic dechlorination and three
reductive dechlorinations, producing trihydroxybenzene (APAJALAKTI and SALKINOJASALONEN, 1987). The potential of these bacteria is limited to some specialists and specific
conditions. Therefore, the use of polychlorinated phenols has been banned in many countries.