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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.