6 Aerobic Degradation by Microorganisms - marnotanahfpubmarno.lecture.ub.ac.id/files/2012/05/BIODEGRADASI-AEROBIK-OLEH-MIKRO... · 6 Aerobic Degradation by Microorganisms WOLFGANG

6 Aerobic Degradation byMicroorganisms

WOLFGANG FRITSCHEMARTIN HOFRICHTER

Jena, Germany

1 Introduction: Characteristics of Aerobic Microorganisms Capable of Degrading Organic Pollutants 146

2 Principles of Bacterial Degradation 1472.1 Typical Aerobic Degrading Bacteria 1472.2 Growth-Associated Degradation of Aliphatics 1482.3 Diversity of Aromatic Compounds – Unity of Catabolic Processes 1492.4 Extension of Degradative Capacities 152

2.4.1 Cometabolic Degradation of Organopollutants 1522.4.2 Overcoming the Persistence by Cooperation of Anaerobic and Aerobic

Bacteria 1543 Degradative Capacities of Fungi 156

3.1 Metabolism of Organopollutants by Microfungi 1573.1.1 Aliphatic Hydrocarbons 1573.1.2 Aromatic Compounds 158

3.2 Degradative Capabilities of Basidiomycetous Fungi 1603.2.1 The Ligninolytic Enzyme System 1613.2.2 Degradation of Organopollutants 163

4 Conclusions 1645 References 164

1 Introduction:Characteristics of AerobicMicroorganisms Capable ofDegrading OrganicPollutants

The most important classes of organic pollu-tants in the environment are mineral oil con-stituents and halogenated products of petro-chemicals. Therefore, the capacities of aerobicmicroorganisms are of particular relevance forthe biodegradation of such compounds andare exemplarily described with reference tothe degradation of aliphatic and aromatic hy-drocarbons as well as their chlorinated deriva-tives. The most rapid and complete degrada-tion of the majority of pollutants is broughtabout under aerobic conditions (RISER-RO-BERTS, 1998).

The essential characteristics of aerobic mi-croorganisms degrading organic pollutants are(Fig. 1):

146 6 Aerobic Degradation by Microorganisms

List of AbbreviationsAsO arsenic-containing organic compoundsBTX benzene, toluene, xylenesDBDs dibenzo-p-dioxinesDBFs dibenzofuransDCA 3,4-dichloroanilineDCP 2,4-dichlorophenolDDT 1,1,1-trichloro-2,2b-bis(4-chlorophenyl)ethaneDNT 2,4-dinitrotolueneKCN potassium cyanideLiP lignin peroxidaseMnP manganese peroxidasePAHs polycyclic aromatic hydrocarbonsPCBs polychlorinated biphenylsPCP pentachlorophenolTCC tricarboxylic acid cycleTCE trichloroetheneTNT 2,4,6-trinitrotoluene

Fig. 1. Main principle of aerobic degradation of hy-drocarbons: growth associated processes.

2 Principles of Bacterial Degradation 147

ing significance for the cometabolic degrada-tion of persistent organopollutants.Thus it is inparticular the domain of basidiomycetous fun-gi, which requires a deeper insight and an ex-tensive consideration. Therefore, this chapterhas been divided in two sections: bacterial andfungal degradation capacities.

2 Principles of BacterialDegradation

2.1 Typical Aerobic DegradingBacteria

The predominant degraders of organopollu-tants in the oxic zone of contaminated areasare chemo-organotrophic species able to use ahuge number of natural and xenobiotic com-pounds as carbon sources and electron donorsfor the generation of energy. Although manybacteria are able to metabolize organic pollu-tants, a single bacterium does not possess theenzymatic capability to degrade all or evenmost of the organic compounds in a pollutedsoil. Mixed microbial communities have themost powerful biodegradative potential be-cause the genetic information of more thanone organism is necessary to degrade the com-plex mixtures of organic compounds present incontaminated areas. The genetic potential andcertain environmental factors such as temper-ature, pH, and available nitrogen and phos-phorus sources, therefore, seem to determinethe rate and the extent of degradation.

The predominant bacteria of polluted soilsbelong to a spectrum of genera and specieslisted in Tab. 1. The composition of this list ofbacteria is determined by the fact whetherthey can be cultured on nutrient-rich media.We have to consider that the majority of bac-teria present in soils cannot be cultured in thelaboratory yet.

Pseudomonads, aerobic gram-negative rodsthat never show fermentative activities, seemto have the highest degradative potential, e.g.,Pseudomonas putida and P. fluorescens. Fur-ther important degraders of organic pollutantscan be found within the genera Comamonas,

(1) Metabolic processes for optimizing thecontact between the microbial cells andthe organic pollutants. The chemicalsmust be accessible to the organismshaving biodegrading activities. For ex-ample, hydrocarbons are water-insolu-ble and their degradation requires theproduction of biosurfactants.

(2) The initial intracellular attack of organ-ic pollutants is an oxidative process, theactivation and incorporation of oxygenis the enzymatic key reaction catalyzedby oxygenases and peroxidases.

(3) Peripheral degradation pathways con-vert organic pollutants step by step intointermediates of the central intermedi-ary metabolism, e.g., the tricarboxylicacid cycle.

(4) Biosynthesis of cell biomass from thecentral precursor metabolites, e.g., ace-tyl-CoA, succinate, pyruvate. Sugars re-quired for various biosyntheses andgrowth must be synthesized by gluco-neogenesis.

A huge number of bacterial and fungal generapossess the capability to degrade organic pol-lutants. Biodegradation is defined as the bio-logically catalyzed reduction in complexity ofchemical compounds (ALEXANDER, 1994). It isbased on two processes: growth and cometab-olism. In the case of growth, organic pollutantsare used as sole source of carbon and energy.This process results in a complete degradation(mineralization) of organic pollutants as dem-onstrated in Sect. 2.2. Cometabolism is definedas the metabolism of an organic compound inthe presence of a growth substrate which isused as the primary carbon and energy source.The principle is explained in Sect. 2.4.

Enzymatic key reactions of aerobic biodeg-radation are oxidations catalyzed by oxygen-ases and peroxidases. Oxygenases are oxido-reductases that use O2 to incorporate oxygeninto the substrate. Degradative organismsneed oxygen at two metabolic sites, at the in-itial attack of the substrate and at the end ofthe respiratory chain (Fig. 1). Distinct higherfungi have developed a unique oxidativesystem for the degradation of lignin based onextracellular ligninolytic peroxidases and lac-cases.This enzymatic system possesses increas-

Burkholderia, and Xanthomonas. Some spe-cies utilize `100 different organic compoundsas carbon sources. The immense potential ofthe pseudomonas does not solely depend onthe catabolic enzymes, but also on their capa-bility of metabolic regulation (HOUGHTON andSHANLEY, 1994). A second important group ofdegrading bacteria are the gram-positive rhod-ococci and coryneform bacteria. Many species,now classified as Rhodococcus spp. had origi-nally been described as Nocardia spp., Myco-bacterium spp., and Corynebacterium spp.Rhodococci are aerobic actinomycetes show-ing considerable morphological diversity. Acertain group of these bacteria possess mycol-ic acids at the external surface of the cell.These compounds are unusual long-chain al-cohols and fatty acids, esterified to the pepti-doglycan of the cell wall. Probably, these lipo-philic cell structures have a significance for theaffinity of rhodococci to lipophilic pollutants.In general, rhodococci have high and diversemetabolic activities and are able to synthesizebiosurfactants.

2.2 Growth-AssociatedDegradation of Aliphatics

The aerobic initial attack of aliphatic and cy-cloaliphatic hydrocarbons requires molecularoxygen. Fig. 2 shows both types of enzymatic

reactions involved in these processes. It de-pends on the nature of the substrate and theenzymatic equipment of the involved microor-ganisms, what kind of enzymatic reaction is realized. n-Alkanes are the main constituentsof mineral oil contaminations (HINCHEE et al.,1994). Long-chain n-alkanes (C10UC24) aredegraded most rapidly by mechanisms demon-strated in Fig. 3. Short-chain alkanes (less thanC9) are toxic to many microorganisms, butthey evaporate rapidly from petroleum con-taminated sites. Oxidation of alkanes is classi-fied as being terminal or diterminal. Themonoterminal oxidation is the main pathway.It proceeds via the formation of the corre-sponding alcohol, aldehyde, and fatty acid.b-Oxidation of the fatty acids results in theformation of acetyl-CoA. n-Alkanes with anuneven number of carbon atoms are degradedto propionyl-CoA, which is in turn carboxylat-ed to methylmalonyl-CoA and further con-verted to succinyl-CoA. Fatty acids of a phys-iological chain length may be directly incorpo-rated into membrane lipids, but the majority ofdegradation products is introduced into thetricarboxylic acid cycle. The subterminal oxi-dation occurs with lower (C3UC6) and longeralkanes with the formation of a secondary al-cohol and subsequent ketone. Unsaturated 1-alkenes are oxidized at the saturated end ofthe chains. A minor pathway has been shownto proceed via an epoxide, which is convertedto a fatty acid. Branching, in general, reducesthe rate of biodegradation. Methyl side groupsdo not drastically decrease the biodegradabili-ty, whereas complex branching chains, e.g., thetertiary butyl group, hinder the action of thedegradative enzymes.

Cyclic alkanes representing minor compo-nents of mineral oil are relatively resistant tomicrobial attack. The absence of an exposedterminal methyl group complicates the pri-mary attack. A few species are able to use cy-clohexane as sole carbon source; more com-mon is its cometabolism by mixed cultures.The mechanism of cyclohexane degradation isshown in Fig. 4. In general, alkyl side chains ofcycloalkanes facilitate the degradation.

Aliphatic hydrocarbons become less watersoluble with increasing chain length. Hydro-carbons with a chain length of C12 and aboveare virtually water insoluble. Two mechanisms


Tab. 1. Predominant Bacteria in Soil Samples Pollut-ed with Aliphatic and Aromatic Hydrocarbons, Po-lycyclic Aromatic Hydrocarbons, and ChlorinatedCompoundsa

Gram-Negative Gram-Positive Bacteria Bacteria

Pseudomonas spp. Nocardia spp.Acinetobacter spp. Mycobacterium spp.Alcaligenes sp. Corynebacterium spp.Flavobacterium/ Arthrobacter spp.

Cytophaga groupXanthomonas spp. Bacillus spp.

a The reclassification of bacteria has been based onphylogenetic markers resulting in changes of somegenera and species. This is why the names of speciesare not mentioned.


are involved in the uptake of these lipophilicsubstrates: the attachment of microbial cells atoil droplets and the production of biosurfac-tants (HOMMEL, 1990). The uptake mechanismlinked to attachment of the cells is still un-known, whereas the effect of biosurfactantshas been studied well (Fig. 5). Biosurfactantsare molecules consisting of a hydrophilic and alipophilic moiety. They act as emulsifyingagents, by decreasing the surface tension andby forming micelles.The microdroplets may beencapsulated in the hydrophobic microbialcell surface. The products of hydrocarbon deg-radation, introduced to the central tricarboxyl-ic acid cycle, have a dual function. They aresubstrates of the energy metabolism and build-ing blocks for biosynthesis of cell biomass and growth (Fig. 1). The synthesis of aminoacids and proteins needs a nitrogen and sulfursource, that of nucleotides and nucleic acids aphosphorus source. The biosynthesis of the

bacterial cell wall requires activated sugarssynthesized by gluconeogenesis.

Products of growth-associated degradationare CO2, H2O, and cell biomass.The cells act asthe complex biocatalysts of degradation. In ad-dition, cell biomass may be mineralized afterexhaustion of the degradable pollutants in acontaminated site.

2.3 Diversity of AromaticCompounds – Unity of CatabolicProcesses

Aromatic hydrocarbons, e.g., benzene, tolu-ene, ethylbenzene and xylenes (BTEX com-pounds), and naphthalene belong to the largevolume petrochemicals, widely used as fuelsand industrial solvents. Phenols and chloro-phenols are released into the environment as

Fig. 2. Initial attack on xe-nobiotics by oxygenases.Monooxygenases incorpo-rate one atom of oxygen ofO2 into the substrate, thesecond atom is reduced toH2O. Dioxygenases incor-porate both atoms into thesubstrate.

products and waste materials from industry.Aromatic compounds are formed in largeamounts by all organisms, e.g., as aromaticamino acids, phenols, or quinones. Thus, it isnot surprising that many microorganisms haveevolved catabolic pathways to degrade aromat-ic compounds. In general, man-made organicchemicals (xenobiotics) can be degraded bymicroorganisms, when the respective molecu-les are similar to natural compounds. The di-versity of man-made aromatics shown in Fig. 6can be converted enzymatically to natural in-termediates of the degradation: catechol andprotecatechuate. In general, benzene and re-lated compounds are characterized by a higherthermodynamic stability than aliphatics are.

Only few reports on bacteria capable ofattacking benzene have been published(SMITH, 1990). The first step of benzene oxida-tion is a hydroxylation catalyzed by a dioxygen-ase (Fig. 2).The product, a diol, is then convert-ed to catechol by a dehydrogenase. These ini-tial reactions, hydroxylation and dehydrogena-tion, are also common to pathways of degrada-tion of other aromatic hydrocarbons.

The introduction of a substituent group on-to the benzene ring renders alternative mecha-nisms possible to attack side chains or to oxid-ize the aromatic ring. The versatility andadaptability of bacteria is based on the exis-tence of catabolic plasmids. Catabolic plasmidshave been found to encode enzymes degrading


Fig. 3. Peripheral pathwaysof alkane degradation. Themain pathway is the termi-nal oxidation to fatty acidscatalyzed by a n-alkanemonoxygenase, b alcoholdehydrogenase and c al-dehyde dehydrogenase.


naturally occurring aromatics such as cam-phor, naphthalene, and salicylate. Most of thecatabolic plasmids are self-transmissible andhave a broad host range.The majority of gram-negative soil bacteria isolated from pollutedareas possess degradative plasmids, mainly theso called TOL plasmids. These pseudomonadsare able to grow on toluene, m- and p-xylene,and m-ethyltoluene. The main reaction in-volved in the oxidation of toluene and relatedarenes is the methyl group hydroxylation. Themethyl group of toluene is oxidized stepwiseto the corresponding alcohol, aldehyde, andcarboxylic group. Benzoate formed or its alky-

lated derivatives are then oxidized by toluatedioxygenase and decarboxylated to catechol(SMITH, 1990).

The oxygenolytic cleavage of the aromaticring occurs via o- or m-cleavage. The signifi-cance of the diversity of degradative pathwaysand of the few key intermediates is still underdiscussion. Both pathways may be present inone bacterial species. “Whenever an alternativemechanism for the dissimilation of any com-pound becomes available (ortho- versus meta-cleavage of ring structures, for example) controlof each outcome must be imposed” (HOUGH-TON and SHANLEY, 1994). The metabolism of awide spectrum of aromatic compounds by onespecies requires the metabolic isolation ofintermediates into distinct pathways. This kindof metabolic compartmentation seems to berealized by metabolic regulation. The key en-zymes of the degradation of aromatic sub-strates are induced and synthesized in appre-ciable amounts only when the substrate orstructurally related compounds are present.Enzyme induction depends on the concentra-tion of the inducing molecules. The substratespecific concentrations represent the thresh-old of utilization and growth and are in themagnitude of µM.A recent report on the regu-lation of TOL catabolic pathways has beenpublished by RAMOS et al. (1997).

Fig. 7 shows the pathways of the oxygenolyt-ic ring cleavage to intermediates of the centralmetabolism. At the branchpoint catechol ei-ther is oxidized by the intradiol o-cleavage, orthe extradiol m-cleavage. Both ring cleavagereactions are catalyzed by specific dioxygen-ases. The product of the o-cleavage – cis,cis-muconate – is transferred to the instable enol-lactone, which is in turn hydrolyzed to oxoad-ipate. This dicarboxylic acid is activated bytransfer to CoA, followed by the thiolyticcleavage to acetyl-CoA and succinate. Proto-catechuate is metabolized by a homologous setof enzymes. The additional carboxylic group isdecarboxylated and, simultaneously, the dou-ble bond is shifted to form oxoadipate enol-lactone. The oxygenolytic m-cleavage yields 2-hydroxymuconic semialdehyde, which is me-tabolized by the hydrolytic enzymes to for-mate, acetaldehyde, and pyruvate. These arethen utilized in the central metabolism. In gen-eral, a wealth of aromatic substrates is degrad-

Fig. 4. Peripheric metabolic pathway of cycloaliphat-ic compounds (cycloparaffins).

ed by a limited number of reactions: hydroxy-lation, oxygenolytic ring cleavage, isomeriza-tion, hydrolysis.The inducible nature of the en-zymes and their substrate specificity enablebacteria with a high degradation potential, e.g.,pseudomonads and rhodococci, to adapt theirmetabolism to the effective utilization of sub-strate mixtures in polluted soils and to grow ata high rate.

2.4 Extension of DegradativeCapacities

2.4.1 Cometabolic Degradation ofOrganopollutants

Cometabolism, the transformation of a sub-stance without nutritional benefit in the pres-ence of a growth substrate, is a common phe-nomenon of microbial activities. It is the basisof biotransformations (bioconversions) used

in biotechnology to convert a substance to achemically modified form. Microorganismsgrowing on a particular substrate gratuitouslyoxidize a second substrate (cosubstrate). Thecosubstrate is not assimilated, but the productmay be available as substrate for other organ-isms of a mixed culture.

The prerequisites of cometabolic transfor-mations are the enzymes of the growing cellsand the synthesis of cofactors necessary for en-zymatic reactions, e.g., of hydrogen donors (re-ducing equivalents, NADH) for oxygenases.The principle is shown in Fig. 8. The exampledemonstrated in Fig. 8 has been used in fieldexperiments for the elimination of trichloro-ethylene (THOMAS and WARD, 1989). Methan-otrophic bacteria used in this experiment canutilize methane and other C1 compounds assole sources of carbon and energy. They oxi-dize methane to CO2 via methanol, formalde-hyde, and formate. The assimilation requiresspecial pathways, and formaldehyde is theintermediate assimilated. The first step of


Fig. 5. Involvement ofbiosurfactants in theuptake of hydrocar-bons. The figure demon-strates the emulsifyingeffect of a rhamnolipidproduced by Pseudo-monas spp. within theoil–water interphaseand the formation ofmicelles. Lipid phasesare printed in bold.


methane oxidation is catalyzed by methanemonooxygenase, which attacks the inert CH4.It is an unspecific enzyme that also oxidizesvarious other compounds, e.g., alkanes, aro-matic compounds, and trichloroethylene(TCE). The proposed mechanism of TCEtransformation according to HENRY andGRBIC-GALLIC (1994) is shown in Fig. 8.TCE isoxidized to an epoxide excreted from the cell.The unstable oxidation product breaks downto compounds, which may be used by other mi-croorganisms. Methanotrophic bacteria areaerobic indigenous bacteria, in soil and aqui-

fers, but methane has to be added as growthsubstrate and inducer for the development ofmethanotrophic biomass. The addition ofmethane as substrate limits the application forbioremediation.

Cometabolism of chloroaromatics is a wide-spread activity of bacteria in mixtures of in-dustrial pollutants. KNACKMUSS (1997) demon-strated that the cometabolic transformation of2-chlorophenol gives rise to dead end metab-olites, e.g., 3-chlorocatechol. This reactionproduct may be auto-oxidized or polymerizedin soil to humic-like structures. Irreversible

Fig. 6. Degradation of a broadspectrum of aromatic naturaland xenobiotic compounds intotwo central intermediates: cate-chol and protocatechuate.

binding of dead end metabolites may fulfill thefunction of detoxification. The accumulationof dead end products within microbial commu-nities under selection pressure is the basis forthe evolution of new catabolic traits (REIN-ECKE, 1994).

2.4.2 Overcoming the Persistenceby Cooperation of Anaerobic andAerobic Bacteria

As a rule, recalcitrance of organic pollutantsincreases with increasing halogenation. Substi-

tution of halogen as well as nitro and sulfogroups at the aromatic ring is accomplished byan increasing electrophilicity of the molecule.These compounds resist the electrophilic at-tack by oxygenases of aerobic bacteria. Com-pounds that persist under oxic conditions are,e.g., PCBs (polychlorinated biphenyls), chlori-nated dioxins, some pesticides, e.g., DDT.

To overcome the relatively high persistenceof halogenated xenobiotics, reductive attack ofanaerobic bacteria is of significance. The deg-radation of environmental pollutants by an-aerobic bacteria is the subject of Chapter 7,this volume. Because of the significance of re-ductive dehalogenation for the first step in the


Fig. 7. The two alternative pathwaysof aerobic degradation of aromaticcompounds: o- and m-cleavage, aphenol monoxygenase, b catechol1,2-dioxygenase, c muconate lacton-izing enzyme, d muconolactone iso-merase, e oxoadipate enol-lactonehydrolase, f oxoadipate succinyl-CoA transferase, g catechol 2,3-di-oxygenase, h hydroxymuconic semi-aldehyde hydrolase, i 2-oxopent-4-enoic acid hydrolase, j 4-hydroxy-2-oxovalerate aldolase.


degradation of higher halogenated com-pounds, this process has been announced. Re-ductive dehalogenation effected by anaerobicbacteria is either a gratuitous reaction or a newtype of anaerobic respiration (ZEHNDER, 1988).The process reduces the degree of chlorinationand, therefore, makes the product more ac-cessible to mineralization by aerobic bacteria.

The potential of a sequence of anaerobicand aerobic bacterial activities for the mineral-ization of chlorinated xenobiotics is describedin Fig. 9. PCBs, which are selected as an exam-ple for degradation of halogenated com-pounds, are well-studied objects (TIEDJE et al.1993; SYLVESTRE and SANDOSSI, 1994; BEDARD

and QUENSEN, 1995). The scheme demon-strates the principle of enzymatic dehalogena-tion mechanisms. The realization of the reac-tions depends on the structure of the chemicalcompounds as well as on the microorganismsand conditions in a polluted ecosystem. Wehave to distinguish between the general degra-dation potential and the actual conditions nec-essary for its realization. Reductive dehalog-enation, the first step of PCB degradation, re-quires anaerobic conditions and organic sub-strates acting as electron donors. The PCBs

have the function of an electron acceptor to al-low the anaerobic bacteria to transfer elec-trons to these compounds. Anaerobic bacteriacapable of catalyzing reductive dehalogena-tion seem to be relatively ubiquitous in nature.Most dechlorinating cultures are mixed cul-tures (consortia). Aanaerobic dechlorinationis always incomplete, products are di- andmonochlorinated biphenyls. These productscan be metabolized further by aerobic micro-organisms. The substantial reduction of PCBsby sequential anaerobic and aerobic treatmenthas been demonstrated in the laboratory (AB-RAMOWICZ, 1990).

The principle of aerobic microbial dehalog-enation reactions of chloroaromatics are de-scribed in Fig. 9. Hydrolytic dechlorination hasbeen elucidated using 4-chlorobenzoate assubstrate for Pseudomonas and Nocardia spp.A halidohydrolase is capable of replacing thehalogen substituent by a hydroxy group origi-nating from water. This type of reaction seemsto be restricted to halobenzoates substituted inthe p-position. Dechlorination after ring cleav-age is a common reaction of the o-pathway ofchlorocatechols catalyzed by catechol 1,2-dioxygenases to produce chloromuconates.The oxygenolytic dechlorination is a rare for-tuitous reaction catalyzed by mono- and di-oxygenases. During this reaction, the halogensubstituent is replaced by oxygen of O2.

Higher chlorinated phenols, e.g., pentachlo-rophenol, have been widely used as biocides.Several aerobic bacteria that degrade chloro-phenols have been isolated (Flavobacterium,Rhodococcus). The degradation mechanismhas been elucidated in some cases (MCALLIS-TER et al., 1996). Thus, Rhodococcus chloro-phenolicus degrades pentachlorophenolthrough a hydrolytic dechlorination and threereductive dechlorinations, producing trihy-droxybenzene (APAJALAKTI and SALKINOJA-SALONEN, 1987). The potential of these bacte-ria is limited to some specialists and specificconditions.Therefore, the use of polychlorinat-ed phenols has been banned in many coun-tries.

Fig. 8. Cometabolic degradation of trichlorometh-ane by the methane monoxygenase system ofmethanotrophic bacteria.

6 Aerobic Degradation by Microorganisms - marnotanahfpubmarno.lecture.ub.ac.id/files/2012/05/BIODEGRADASI-AEROBIK-OLEH-MIKRO... · 6 Aerobic Degradation by Microorganisms WOLFGANG

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