Hydrocarbonoclastic bacteria

See also

Bioremediation Oil spill Microbial ecology

Hydrocarbonoclastic bacteria (also known as hydrocarbon degrading bacteria, oil degrading bacteria or HCB) are a heterogeneous group of prokaryotes which can degrade and utilize hydrocarbon compounds as source of carbon and energy. Despite being present in most of environments around the world, several of these specialized bacteria live in the sea and have been isolated from polluted seawater.

Taxonomy and distribution

The taxonomic diversity of hydrocarbon-degrading bacteria has not changed dramatically if we consider the higher taxa, many studies have provided information on 25 kinds of hydrocarbon-degrading bacteria and 25 kinds of fungi isolated from marine environments. Bacterial genera such as Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia and Mycobacterium isolated from oil have been shown to be potential organisms for hydrocarbon degradation. Cerniglia et al. observed that nine cyanobacteria, five green algae, one red alga, one brown alga and two diatoms could oxidise naphthalene. Temperature is crucial because it influences microbial physiology and diversity; the rate of biodegradation generally decreases as the temperature decreases.

Hydrocarbonoclastic bacteria are diazophilic, i.e. they can live in environments extremely poor in nitrogen compounds, which allows them to distribute themselves throughout the environment. They are extremely useful for environmentally friendly biosanitation; the fastest and most complete degradation occurs under aerobic conditions.

Hydrocarbons occur in marine environments where there are oil spills, which makes us understand that they are nutritionally independent of nitrogen sources, a characteristic due to their ability to fix atmospheric nitrogen. In Lagos in a city in Nigeria, nine bacterial strains Pseudomonas fluorescens, P. aeruginosa, Bacillus subtilis, Bacillus sp., Alcaligenes sp., Acinetobacter lwoffi, Flavobacterium sp., Micrococcus roseus and Corynebacterium sp, isolated from the polluted flow that could degrade crude oil, were detected ; in north-east India they were also detected. In the Louisiana incident in the Gulf of Mexico, about 100 strains were detected and studied, revealing that the isolates all belong to the phylum Proteobacteria and three classes (Alteromonadales, Rhodospirillales and Enterobacteriales).

These organisms are normally present in very small numbers, which gives them an advantage over hydrocarbons such as carbon and energy, as they grow and multiply rapidly. Alcanivorax-like bacteria have been detected in oil-affected environments around the world, including the US, Germany, the UK, Spain, Italy, Singapore, China, the western Philippines, Japan, the mid-Atlantic ridge near Antarctica, and from deepwater sediments in the eastern Pacific Ocean.

Physiology and metabolism Hydrocarbonoclastic bacteria have two fundamental characteristics: (1) specific membrane-bound dioxygenases and (2) mechanisms for optimizing contact with water-insoluble hydrocarbons. Microbial biodegradation occurs wherever oil contamination occurs. However, biodegradation rates are slow and as a result there are severe toxic effects on marine life in the water and on the coast.

The hydrocarbons contained in petroleum have a different behavior in water depending on their chemical nature. This process is called weathering, those with low molecular weight volatilize when they reach the surface.

The rest is attacked by bacteria that are able to do this. These bacteria do not adhere to the oil and do not have a high hydrophobicity of the cell surface. The next stage of degradation involves microorganisms with high cell surface hydrophobicity, which can adhere to residual high molecular weight hydrocarbons. Adhesion is due to hydrophobic fimbriae, fibrils, lipids and proteins of the outer membrane and some small molecules of the cell surface, such as gramicidin S and prodigiosin.

All petroleum products are derived from crude oil whose major constituents are hydrocarbons, that can be separated into four fractions: saturated, aromatic, resin and asphaltene fractions. The susceptibility of hydrocarbons to microbial degradation can be generally ranked as follows: linear alkanes branched alkanes > small aromatics > cyclic alkanes.J. J. Perry, "Microbial metabolism of cyclic alkanes," in Petroleum Microbiology, R. M. Atlas, Ed., pp. 61–98, Macmil- lan, New York, NY, USA, 1984.W. Ulrici, "Contaminant soil areas, different countries and contaminant monitoring of contaminants," in Environmental Process II. Soil Decontamination Biotechnology, H. J. Rehm and G. Reed, Eds., vol. 11, pp. 5–42, 2000. Some compounds, such as the high molecular weight polycyclic aromatic hydrocarbons (PAHs), may not be degraded at all, asphaltenes and resins are considered to be recalcitrant to biodegradation.

Alkane degradation pathways Alkanes are readily biodegraded aerobically in the sea by several different pathways.

Terminal oxidation The degradation of medium-length ones by Pseudomonas putida starts from the alkane hydroxylase, this enzyme is made up of three components: the membrane-bound oxygenase component and two soluble components called rubredoxin and rubredoxin reductase.

From the oxidation of the methyl group of n-alkanes by the alkane hydroxylase, n-alkanols are released which are further oxidized by a membrane-bound alcohol dehydrogenase in n-alkanals. The n-alkanals are subsequently transformed into fatty acids and then into acyl CoA, respectively by the aldehyde dehydrogenase and by the acyl-CoA synthetase.

CH3-R-CH3 -> CH3-R-CH2OH -> CH3-R-CHO -> CH3-R-COOH -> (CH2OH)-R-COOH

CH3-R-CH2OH -> (CH2OH)-R-CH2OH -> (CH2OH)-R-CHO -> (CH2OH)-R-COOH

(CH2OH)-R-COOH -> CHO-R-COOH -> HOOC-R-COOH

Subterminal oxidation This path leads to the release of secondary alcohols. The n-alkanes are oxidized by monooxygenase to secondary alcohols, then to ketones and finally to fatty acids.

R1-(CH2)(CH2)-R2 -> R1-(CH2)(CHOH)-R2 -> R1-(CH2)(CO)-R2 -> R1-(CH2)O(CO)-R2 -> R1-COOH + R2-COOH

Cycloalkane degradation pathways Cycloalkanes are degraded by a co-oxidation mechanism, the process leading to the formation of a cyclic alcohol and a ketone. A monooxygenase introduces an oxygen into the cyclic ketone and the cyclic ring is cleaved.

Aromatic hydrocarbon degradation pathways For aromatic compounds there are different pathways, considering toluene at least five are known, each of these is present in specific bacterial species, Burkholderia sp. strain JS150 is unique in using multiple pathways for toluene metabolism:

TOL pathway: This path takes the name of the homonymous plasmid that codes for it. Toluene is degraded to alcohol to benzyl, to benzaldehyde and then to benzoate, which is further transformed into the intermediates of the TCA cycle. F1 pathway: P.putida is able to undertake this pathway, which consists in the introduction of two hydroxyl groups into toluene, forming cis-toluene dihydrodiol. This intermediate is then converted to 3-methylcatechol. KR1 pathway: Pseudomonas mendocina KR1, is able to convert toluene into p-cresol, by the enzyme toluene 4-monooxygenase. Subsequently, p-hydroxybenzoate is formed through oxidation of the methyl side chain. PK01: Pseudomonas pickettii PKO1 oxidizes toluene with the enzyme toluene 3-monooxygease to m-cresol, which is further oxidized to 3-methylcatechol by another monooxygenase. G4: The G4 pathway was observed in Bukholderia cepacia G4, where toluene is converted into o-cresol by toluene 2-monooxygenase and subsequently another monooxygenase converts it to 3-methylcatechol.

Anaerobic degradation pathways

Oil components that are trapped in marine sediments tend to persist in anaerobic conditions. Some hydrocarbons can be oxidized under anaerobic conditions when nitrate reduction, sulfate reduction, methane production, Fe3+ reduction or photosynthesis are coupled to hydrocarbon oxidation.

Anaerobic bacterium strain HD-1 grows on in the presence of or tetradecane. In the absence of , tetradecane is degraded, and the major metabolic intermediate is 1-dodecene

Factors influencing the biodegradation The biodegradation of hydrocarbons is limited by a number of chemical, physical and biological factors.

Biosurfactants: The contact between bacteria and hydrocarbons is fundamental because the first degradative step involves the use of oxygenase. Contact is favored by adhesion and emulsifying mechanisms Bacteria that break down hydrocarbons often produce bioemulsifiers as secondary metabolites. These can be divided into low molecular weight molecules which effectively lower surface and interfacial tensions, and high molecular weight polymers which bond firmly to surfaces. Some bioemulsifiers promote the growth of bacteria on hydrophobic substrates insoluble in water by increasing their bioavailability, increasing their surface, desorbing them from surfaces and increasing their solubility. Biosurfactants have an amphiphilic nature, which allows the microorganisms that produce them to exploit hydrophobic substrates, allowing motility, avoiding competitors. The hydrophobic part usually comprises saturated or unsaturated fatty acids, fatty hydroxy acids or fatty alcohols with a chain length between 8 and 18 carbon atoms. The hydrophilic components consist either of small hydroxyl, phosphate or carboxylic groups, or of portions of carbohydrates or peptides. Biosurfactants are predominantly anionic and non-ionic compounds. Nutritional requirements for growth: The HCB also need large amounts of nitrogen and phosphorus. It has been estimated that 150 g of nitrogen and 30 g of phosphorus are required for 1 kg of oxidized hydrocarbon. Temperature: The biodegradation of petroleum hydrocarbons occurs in an optimum temperature between 20 °C and 50 °C, while at lower temperatures the deterioration rate is slower.Bartha, R. and Bossert, I. (1984). The treatment and disposal of petroleum wastes. In: Petroleum Microbiology, R. M. Atlas, Macmillan, New York, NY, USA. 553–578. pH and oxygen: Bacteria require a neutral pH, and in this the same oil can help neutralize environments that are too acidic for microbial growth. Oxygen is critical for aerobic degradation.

Ecology At the moment, most studies of the ecology of hydrocarbonoclastic bacteria refer to a wide group of genera found principally in marine environments. Since each of them is characterized by a different metabolism, these organisms work together in order to degrade all types of hydrocarbon compounds in a very efficient way. They also play a fundamental role in the carbon biogeochemical cycle and several studies show that some species can create intricate relationships with different marine organisms.

Oil degrading microbial communities and ecological successions When a release of oil (or whichever kind of hydrocarbon compound) happens in a specific marine area, a lot of bacterial species begin to colonize it, changing the microbial community already present there. Analyzing the dynamics of those communities has led to the discovery of common patterns that are associated with biodegradation, and that information can be useful for the improving of bioremediation methods. Microbial community in situ shuffles since the quantities of nutrients change as the presence of hydrocarbons increases: this ecological situation is able to select only those organisms which can use hydrocarbons as an energy source and possess all the enzymes to do so. In addition, most oil biodegrading species require specific quantities of phosphorus and nitrogen to carry out their catabolic processes. It is possible to state therefore, that hydrocarbonoclastic bacteria rate is limited by the availability of nitrogen and phosphorus mainly.