General Microbiology - Schlegel H. 1987

Decomposition of natural substances
Hydrocarbons

Even chemically stable substances such as paraffins, petroleum, and rubber are subject to microbial decomposition. Noticeable breakdown does not occur only in the absence of O₂ (for example, in oil deposits or, under special conditions, in hard coal seams). Several practical questions are of great importance: does petroleum entering soil or Water undergo Biological Oxidation? Are there microorganisms that specifically utilize hydrocarbons? And finally, can the likely presence of oil or natural gas be judged by the Abundance of hydrocarbon-oxidizing microorganisms?

Until recently, the growth of microorganisms on petroleum was considered a very rare phenomenon. It was assumed that Bacteria capable of utilizing petroleum occur exclusively where petroleum itself is found (oil fields, oil storage facilities). Based on this assumption, a new method for oil exploration was proposed: researchers hoped to discover oil deposits by counting the number of bacteria capable of utilizing petroleum in soil samples. However, contemporary data indicate that such bacteria are widespread and can be isolated from any field, forest, or meadow soil. Furthermore, The ability to use petroleum as an energy source is not restricted to isolated specialized forms, but is characteristic of many Fungi and bacteria. These findings are consistent with recent analytical data on The chemical composition of bacterial, plant, and animal Cells. Hydrocarbons are present in numerous organisms and are continuously synthesized by bacteria and plants. These apparently include the waxy substances that coat plant leaves. Thus, hydrocarbons are not merely remnants of ancient plant life that have survived to the present day; they are also secondary metabolites currently synthesized by green plants in significant quantities.

14.11.1 Methane

Among hydrocarbons, methane occupies a unique position. It is utilized and oxidized by bacteria that are incapable of cleaving long-chain hydrocarbons. Such bacteria should be regarded not as hydrocarbon-oxidizing microorganisms, but rather as a group specialized in utilizing C₁ compounds. Therefore, methane-oxidizing bacteria, together with all bacteria (and Yeasts) capable of utilizing methanol, methylated amines, dimethyl ether, formaldehyde, and formate, are classified as methylotrophic organisms. In enrichment cultures with methane as the sole carbon and energy source, bacteria belonging to various genera—such as Methylomonas, Methylococcus, and Methylosinus—develop. Some of these grow only on media containing methane, methanol, or dimethyl ether, and cannot utilize sugars, organic acids, or other alcohols (except methanol).

Energy generation. Reducing equivalents for energy production can be generated through The oxidation of methane via The formation of methanol, formaldehyde, and formate up to CO₂. The oxidation of methane to methanol is accompanied by the incorporation of one oxygen atom into the molecule; this reaction is catalyzed by methane monooxygenase.

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Cellular material synthesis. Cell material is synthesized, as a rule, from formaldehyde—an intermediate product of methane oxidation. This synthesis can proceed via different pathways, of which the ribulose monophosphate cycle of formaldehyde fixation and the Serine pathway are the most thoroughly studied.

In the ribulose monophosphate cycle, formaldehyde (the product of methane oxidation) is converted via aldol Condensation with ribulose-5-phosphate into arabino-3-hexulose-6-phosphate; the latter undergoes an isomerization reaction to form fructose-6-phosphate (Fig. 14.6). From fructose-6-phosphate, pentose phosphate is regenerated again (much like the process occurring during CO₂ fixation in the ribulose bisphosphate cycle). The cycle does not involve the reaction catalyzed by sedoheptulose-1,7-bisphosphatase and thus proceeds According to the scheme shown on the left in Fig. 11.2.

Fig. 14.6. Ribulose monophosphate cycle of formaldehyde fixation. Key Enzymes (circled numbers): 1 — hexulose-phosphate synthase; 2 — hexulose-phosphate isomerase. (Ström et al., Biochem. J., 144 [1974], 465–476.)

The serine pathway (Fig. 14.7) has been studied in Pseudomonas MA and Hyphomicrobium X. Other microorganisms likely possess modifications of this pathway. Glycine serves as the formaldehyde acceptor. Organisms assimilating C₁ compounds via this cycle typically contain detectable levels of hydroxypyruvate reductase, malate thiokinase, malyl-CoA lyase, and isocitrate lyase.

Fig. 14.7. Assimilation of C₁ compounds via the serine pathway. The C₁ unit is incorporated into serine with the participation of tetrahydrofolic acid. Subsequent reactions lead to the synthesis of intermediates (phosphoenolpyruvate and Pyruvate) and, ultimately, cellular material, as well as the regeneration of glycine. Key enzymes (circled numbers): 1 — serine hydroxymethyltransferase; 2 — hydroxypyruvate reductase; 3 — malate thiokinase; 4 — malyl-CoA lyase; 5 — isocitrate lyase. (Bollion, Hersh, Arch. Biochem. Biophys., 153 [1972], 368; Harder et al., J. Gen. Microbiol., 78 [1973], 155.)

Yeasts are capable of utilizing only methanol, not methane. In A number of yeasts (Candida boidinii, Hansenula polymorpha), the incorporation of methanol into cellular material also proceeds via formaldehyde, but through a different pentose phosphate, namely xylulose-5-phosphate. In this xylulose monophosphate cycle of formaldehyde fixation, formaldehyde reacts with xylulose-5-phosphate via a specific transketolase to yield glyceraldehyde-3-phosphate and dihydroxyacetone. Both products (dihydroxyacetone after phosphorylation by triokinase) enter biosynthetic pathways.

The utilization of methanol by bacteria is initiated with the participation of methanol dehydrogenase. A previously unknown prosthetic group—methoxatin, or pyrroloquinoline quinone (PQQ)—was discovered in this enzyme. It is now known that methoxatin is a component of many bacterial membrane-bound Alcohol dehydrogenases.

14.11.2 Ethane, propane, and butane

While enrichment cultures containing only pure methane along with CO₂ and O₂ develop predominantly Methylomonas methanica, cultures grown on natural gas, which contains ethane alongside methane, support the growth of ethane oxidizers exclusively. A significantly greater number of species are capable of oxidizing ethane than methane. Most ethane-oxidizing microorganisms belong to the genera Mycobacterium, Flavobacterium, and Nocardia. Some ethane-utilizing bacteria can also oxidize molecular hydrogen. An even larger number of bacterial species develop in enrichment cultures at the expense of propane. Furthermore, bacteria capable of oxidizing butane (Mycobacterium and Pseudomonas) have been isolated.

14.11.3 Long-chain alkanes (aliphatic hydrocarbons)

Long-chain alkanes are utilized by a vast number of bacteria. Chain length is of critical importance here: as the paraffin chain lengthens, both the number of species capable of utilizing these compounds and their utilization activity increase. Mycobacteria, nocardiae, and corynebacteria participate in their degradation. For a long time, the ability of bacteria to grow on media containing hydrocarbons was regarded as a biological curiosity. Interest in hydrocarbon-oxidizing microorganisms arose in connection with two observations. In 1950, at the Institute for Fermentation Industries in Berlin, two Yeast species—Candida lipolytica and Candida tropicalis—were isolated from enrichment cultures containing various hydrocarbon fractions as an energy source. It turned out that C. lipolytica utilizes all higher hydrocarbon homologues starting from C₁₅ compounds. Subsequently, it was established that the majority of Candida species oxidize hydrocarbons. Screening of strains from yeast collections revealed that the ability to utilize hydrocarbons is inherent to a great many of them. Hydrocarbon utilization proceeds with an unusually high yield of biomass ($Y$). While the yield coefficient $Y$ is approximately 0.5 when utilizing CARBOHYDRATES, it ranges from 0.7 to 1.0 for hydrocarbons.

Mechanism. Many pseudomonads oxidize hydrocarbons so completely that no intermediate products accumulate. Only in Acinetobacter calcoaceticus are oxidation products released into the medium, whereas in Nocardia they accumulate intracellularly. The Nature of the products depends on the Nature of the substrate. When A. calcoaceticus grows on a medium with hexadecane, cetyl palmitate can be isolated from the culture liquid. Cetyl palmitate is an ester of palmitic acid and cetyl alcohol (hexadecanol)—two oxidation products of hexadecane. The accompanying diagram (Fig. 14.8) shows that paraffin Cleavage begins with the oxidation of the terminal carbon.

Oxygen is involved in the initial attack on the hydrocarbon chain. In the absence of molecular oxygen, paraffins are not oxidized. The oxidation is catalyzed by a monooxygenase (alkane monooxygenase):

Paraffin + O₂ + NADH₂ → Aliphatic alcohol + NAD + H₂O

Fig. 14.8. Formation of cetyl palmitate from hexadecane by Acinetobacter calcoaceticus. Growth on a medium with ordinary water (H₂¹⁶O) in an atmosphere enriched with heavy oxygen (¹⁸O₂) yields cetyl palmitate in which 75% of the oxygen consists of ¹⁸O. This is consistent with the concept that The conversion of hexadecane begins with the oxidation of the terminal carbon mediated by alkane monooxygenase.

Fig. 14.9. Degradation of alkanes (paraffins) via terminal oxidation involving a monooxygenase and subsequent ß-Oxidation to acetyl-CoA. Participating enzymes: 1 - monooxygenase (alkane 1-hydroxylase); 2 - Alcohol dehydrogenase; 3 - aldehyde dehydrogenase; 4 - acyl-CoA synthetase; 5 - acyl-CoA dehydrogenase; 6 - 3-hydroxyacyl-CoA hydro-lyase; 7 - 3-hydroxyacyl-CoA dehydrogenase; 8 - ß-ketothiolase.

Further oxidation of paraffin proceeds via the pathway known as the ß-oxidation of long-chain Fatty acids (Fig. 14.9). When heptane-oxidizing pseudomonads are subjected to a reduced partial pressure of oxygen, C₃, C₅, and C₇ fatty acids accumulate in the culture medium. Growing bacterial cells in the presence of hexane and subsequently incubating them with heptane reveals the accumulation of propionic acid. This is presumably because cells grown in the presence of hexane lack the enzymes required to oxidize propionate via methylmalonyl-CoA.

14.11.4 Aromatic Hydrocarbons

Plants synthesize a vast array of compounds containing aromatic rings, of which Lignin is the most quantitatively abundant, accounting for 20% of wood by dry weight. Many bacteria and fungi possess the ability to cleave such compounds by breaking the aromatic ring. Some pseudomonads grow faster on media containing benzoate than on media with sugars. Molecular oxygen is essential for the rapid degradation of aromatic substances. The pathways of such cleavage are discussed in this section. According to recent findings, Aromatic Compounds can also undergo anaerobic degradation, though its metabolic mechanisms will not be covered here.

Preparation for ring cleavage. Most naturally occurring aromatic compounds are initially degraded by bacteria to pyrocatechol (catechol) or protocatechuic acid. Compounds with either a single substituent or two substituents at positions 1 and 2—such as mandelic acid, phenylalanine, toluene, benzene, phenol, benzoic acid, and salicylic acid—are typically cleaved via pyrocatechol.

Aromatic substances with two substituents at positions 1,3 or 1,4, as well as those with a higher number of substituent groups (e.g., 4-hydroxybenzoic, quinic, vanillic, and shikimic acids), are degraded with the formation of protocatechuic acid. In all cases, hydroxyl groups are introduced into the ring. Molecular oxygen provides the oxygen atoms for these hydroxyl groups. For non-phenolic aromatic compounds, the 1,2-dihydroxybenzene Structure required for ring fission is generated through double hydroxylation.

For instance, the unsubstituted benzene ring is hydroxylated by a dioxygenase (dual hydroxylase) to yield cis-1,2-dihydro-1,2-dihydroxybenzene, which is subsequently dehydrogenated (rearomatized) to form pyrocatechol.

Phenolic aromatic compounds are hydroxylated by Monooxygenases. One oxygen atom from molecular oxygen is incorporated into the substrate, while the other is reduced to water. Pyridine NUCLEOTIDES can serve as hydrogen Donors.

Substituents on the aromatic ring are frequently (though not always) removed prior to ring cleavage. For example, chlorine atoms, nitro groups, and sulfo groups can be replaced by hydroxyl groups. Aliphatic side chains may undergo diverse modifications and truncations or remain entirely unchanged.

Ring cleavage. The Cleavage of the aromatic ring is carried out by Dioxygenases, which incorporate molecular oxygen directly into the substrate. Fission occurs either between two adjacent hydroxyl groups or between a hydroxylated carbon and an adjacent non-hydroxylated carbon. Enzymes isolated from various Pseudomonas species are the best characterized among those involved in this process. Figure 14.10 illustrates the most important types of aromatic ring cleavage.

Ortho-cleavage. Fission of the ring between two adjacent hydroxylated carbon atoms (ortho-cleavage, or intradiol cleavage) yields a dicarboxylic acid. The process presumably begins with The addition of an O₂ molecule to the hydroxyl groups of adjacent carbon atoms to form a cyclic peroxide, followed by an intramolecular rearrangement that breaks the carbon-carbon bond, yielding cis,cis-muconic acid:

Pyrocatechol is cleaved by ortho-pyrocatechase (catechol 1,2-dioxygenase), whereas protocatechuic acid is degraded by protocatechuate 3,4-dioxygenase. The resulting intermediates—cis,cis-muconic acid and 3-carboxy-cis,cis-muconic acid—converge during further Catabolism through a shared intermediate, 3-oxoadipic acid. The latter is activated by a CoA transferase and cleaved into succinyl-CoA and acetyl-CoA, which then enter central Intermediary METABOLISM (Fig. 14.10).

Meta-cleavage. The cleavage of the aromatic ring between a hydroxylated and a non-hydroxylated carbon atom (meta-cleavage, or extradiol cleavage) is likewise catalyzed by dioxygenases. The resulting products are 2-hydroxymuconic semialdehydes (Fig. 14.11), which are subsequently converted (depending on the substituents) into pyruvate, acetaldehyde, oxaloacetate, fumarate, acetoacetate, succinate, or other intermediates channeled into intermediary metabolism.

Research has demonstrated that the pathways of aromatic compound degradation are remarkably diverse. Both the preparatory stages preceding ring cleavage and the nature of the cleavage itself can vary. In some bacteria, the synthesis of ortho- or meta-cleavage enzymes depends on the growth phase and environmental conditions. Certain pseudomonads degrade aromatics channeled through pyrocatechol via the ortho-pathway, while those routed through protocatechuic acid are degraded via the meta-pathway.

Convergent degradation pathways. A considerable variety of reactions is involved in The breakdown of aromatic compounds. Ultimately, diverse degradation pathways converge, leading to the formation of either pyrocatechol or protocatechuic acid (Figs. 14.12 and 14.13). Consequently, the CATABOLISM OF AROMATIC compounds serves as an excellent model for studying The regulation of convergent Catabolic pathways (Section 16.1.1).

Fig. 14.10. Ortho-cleavage of the aromatic ring and the 3-oxoadipate pathway. Participating enzymes: 1 - pyrocatechase (catechol 1,2-dioxygenase); 2 - muconate cycloisomerase; 3 - muconolactone isomerase; 4 - protocatechuate 3,4-dioxygenase; 5 - 3-carboxymuconate cycloisomerase; 6 - 4-carboxymuconolactone decarboxylase; 7 - 4-oxoadipate enol-lactonase; 8 - 3-oxoadipate succinyl-CoA transferase; 9 - 3-oxoadipyl-CoA thiolase.

Naphthalene, anthracene, and other polyaromatic compounds. Certain bacteria are capable of degrading polycyclic hydrocarbons, among which naphthalene, anthracene, and phenanthrene are the most notable. When bacteria are cultivated on a medium containing one of these compounds, salicylic acid can often be detected in the culture broth; this presumably proceeds via transformations analogous to those described above for monocyclic compounds (Fig. 14.13).

In summary, naturally occurring hydrocarbons are partially or completely oxidized by microorganisms. Under favorable conditions, even asphalt undergoes biodegradation, albeit very slowly. In microbially active soils, even graphite is subject to oxidation.

Fig. 14.11. Meta-cleavage of the aromatic ring. Participating enzymes: 1 - metapyrocatechase (catechol 2,3-dioxygenase); 2 - protocatechuate 4,5-dioxygenase.

Fig. 14.12. Pathways of aromatic compound degradation leading to the formation of pyrocatechol.

Oil pollution. In cases of soil contamination by petroleum, it should be kept in mind that hydrocarbons undergo rapid and complete degradation in non-sterile, aerated soils. Only under conditions of heavy contamination and restricted air access, or when oil penetrates deep into the soil layers, does it pose a risk of persisting for a long time and eventually reaching drinking water sources. Oil spilled on the sea surface initially presents a severe hazard to flora and fauna, but it is subsequently degraded by bacteria as well. However, long-chain alkanes, polyaromatic hydrocarbons, and asphalt-like substance mixtures remain; all of these compounds resist biodegradation for extended periods.

Fig. 14.13. Pathways of aromatic compound degradation leading to the formation of protocatechuic acid.

Xenobiotics. Foreign compounds (xenobiotics), particularly various pesticides (fungicides, herbicides, insecticides, and nematicides), can accumulate in the soil following Treatment because microorganisms capable of degrading and detoxifying these substances fail to proliferate.

Aromatic compounds bearing ring substituents such as halogens, sulfo groups, and nitro groups degrade extremely slowly and can resist microbial attack for many years. One of the most persistent substances is dichlorodiphenyltrichloroethane (DDT). Plastics such as polyethylene, polypropylene, and the like are harmless, but apparently completely resistant to microbial breakdown. The plasticizers they contain undergo gradual oxidation, whereas the polymer backbone remains intact.

Cometabolism. Apparently, certain compounds are degraded by microorganisms only in conjunction with readily utilizable substrates. The Transformation of a substance that cannot serve as a nutrient on its own in the presence of a so-called cosubstrate—a compound utilized by cells for growth—is termed cometabolism or co-oxidation. The phenomenon of cometabolism can be useful, for instance, in the treatment of industrial wastewater containing poorly degradable synthetic products by co-treating it with municipal wastewater in the same wastewater treatment facilities.