marine microorganisms make a meal of oil

© 2006 Nature Publishing Group

Hydrocarbon-degrading bacteria were first isolated almost a century ago1, and a recent review lists 79 bacterial gen-era that can use hydrocarbons as a sole source of carbon and energy, as well as 9 cyanobacterial genera, 103 fungal genera and 14 algal genera that are known to degrade or transform hydrocarbons2. Several hydrocarbon-degrading bacteria have been used as model organisms to elucidate the biochemistry, genetic basis and regula-tion of hydrocarbon-degrading pathways3, and it has been known for some time that hydrocarbon degradation in the environment is typically limited by the bioavailabil-ity of nutrients such as nitrogen and phosphorus4. In organic-compound-rich environments associated with long-term hydrocarbon contamination, the bioavailabil-ity of the hydrocarbons themselves is also an important limiting factor in biodegradation, and even in situations in which biodegradation of surface oil contamination occurs, buried oil can persist on beaches as a result of limitations on mass transport of hydrocarbons, nutrients or oxygen.

Despite there being a considerable amount of lit-erature on microbial hydrocarbon degradation, until recently we had limited knowledge about which organ-isms are the most important hydrocarbon degraders in the environment and what the biological fate of hydro-carbons is under anoxic conditions. Furthermore, we have lacked a theoretical basis to underpin and predict the behaviour and interactions of hydrocarbon -degrading bacteria in situ. There have been many recent, excit-ing advances in our understanding of the degradation of hydrocarbons in the absence of oxygen. These have recently been reviewed5–7, so this article focuses on our growing understanding of the bacteria that are responsi-ble for hydrocarbon removal in oxic environments, with an emphasis on marine systems.

Composition of crude oil Crude oil is perhaps the most complex mixture of organic compounds that occurs on Earth. Recent advances in ultra-high-resolution mass spectrometry have allowed the identification of more than 17,000 distinct chemi-cal components, and the term petroleomics has been coined to express this newly uncovered complexity 8. Furthermore, crude oil is not a homogeneous mat erial, and different crude oils have a range of chemical and physical properties that affect their susceptibility to biodegradation and their environmental fate. Within this complexity, however, crude oil can be classified into four main operationally defined groups of chemicals: the saturated hydrocarbons and the aromatic hydrocarbons, and the more polar, non-hydrocarbon components the resins and the asphaltenes.

Light oils are typically high in saturated and aromatic hydrocarbons, with a smaller proportion of resins and asphaltenes. Heavy oils, which result from the bio-degradation of crude oil under anoxic conditions in situ in petroleum reservoirs, have a much lower content of saturated and aromatic hydrocarbons and a higher proportion of the more polar chemicals, the resins and asphaltenes9 (FIG. 1). Biodegradation of crude oil in sur-face environments results in similar changes in crude-oil composition, and the loss of saturated and aromatic hydrocarbons, together with an increase in the relative abundance of the polar fractions (which are more resist-ant to biodegradation), is a characteristic signature of crude-oil biodegradation. Because saturated hydro-carbons constitute the largest fraction of crude oil by mass, the biodegradation of saturated hydrocarbons is quantitatively the most important process in the removal of crude oil from the environment. Nevertheless, the aromatic hydrocarbons and polar fractions, which are

*School of Civil Engineering and Geosciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK.‡Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.Correspondence to I.M.H.e-mail: [email protected]:10.1038/nrmicro1348

Hydrocarbons Organic compounds that contain only carbon and hydrogen.

Marine microorganisms make a meal of oilIan M. Head*, D. Martin Jones* and Wilfred F. M. Röling‡

Abstract | Hundreds of millions of litres of petroleum enter the environment from both natural and anthropogenic sources every year. The input from natural marine oil seeps alone would be enough to cover all of the world’s oceans in a layer of oil 20 molecules thick. That the globe is not swamped with oil is testament to the efficiency and versatility of the networks of microorganisms that degrade hydrocarbons, some of which have recently begun to reveal the secrets of when and how they exploit hydrocarbons as a source of carbon and energy.

NATURE REVIEWS | MICROBIOLOGY VOLUME 4 | MARCH 2006 | 173

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Bioremediation The use of biological organisms such as plants or microorganisms to aid in the removal of hazardous substances from a polluted area.

Laboratory microcosm A laboratory incubation system that is designed to simulate environmental conditions. This can be as simple as a flask or serum bottle, or it can be more complex (for example, a system that incorporates diurnal and tidal cycles).

more toxic and persistent, could be of greater long-term environmental significance.

Global players in hydrocarbon degradationOf the diverse hydrocarbon-degrading bacterial types that are known, are there any that seem to have particular significance for the removal of hydrocarbons from the environment? To answer this question, it is important to garner evidence from culture-based studies, which define the catabolic capabilities of candidate organisms, and to couple this evidence with data from environmental studies, which indicate the qualitative and quantitative importance of particular organisms in situ.

The clearest evidence of particular bacteria assuming greatest importance in hydrocarbon degradation comes from marine environments. In the past 10 years, several interesting marine bacteria that are specialists adapted to hydrocarbon degradation have been isolated (FIG. 2). These bacteria use hydrocarbons almost exclusively as a carbon source, and they include Alcanivorax spp.10, Cycloclasticus spp.11, Oleiphilus spp.12, Oleispira spp.13,

Thalassolituus spp.14 and some members of the genus Planomicrobium (pre viously known as Planococcus)15. Alcanivorax spp., Oleiphilus spp., Oleispira spp. and Thalassolituus spp. use a variety of branched- and/or straight-chain saturated hydrocarbons, as does Planomicrobium alkanoclasticum MAE2 (previously known as Planococcus alkanoclasticus MAE2). By con-trast, Cycloclasticus spp. have evolved to use a range of polycyclic aromatic hydrocarbons.

Several culture-independent studies of oil-impacted marine environments have shown that some of these bacteria are rapidly and strongly selected when hydro-carbon degradation is stimulated by the addition of nutrients. Alcanivorax spp., for example, were shown to increase from being undetectable in oil-treated sea water to constituting 70–90% of prokaryotic cells in oil-treated sea water within 1–2 weeks of nutrient amendment16 (FIG. 3). Similarly, in oil-spill bioremediation experiments carried out in laboratory microcosms and in the field, 16S ribosomal RNA (rRNA)-gene sequences from Alcanivorax spp. were undetectable in control experi-ments in which samples were not treated with oil, but within 2 weeks of oil treatment, they constituted more than 30% of the sequences in libraries of 16S-rRNA-gene clones constructed from oil-treated sediments and more than 70% of the sequences recovered from sediment treated with oil and inorganic nutrients17,18. These results were mirrored by the detection of alkB genes, which encode the catalytic component of alkane hydroxylase, only in samples in which Alcanivorax spp. 16S-rRNA genes were abundant (W.F.M.R. and I.M.H., unpublished observations). It is generally thought that these org anisms are normally present in very small num-bers, and by providing conditions that allow them to take advantage of the hydrocarbons as a carbon and energy source, they grow and multiply rapidly.

Alcanivorax-like bacteria have now been detected in oil-impacted environments across the globe. They have been isolated or detected in culture-independent bacterial-community surveys from the United States19,20, Germany10, the United Kingdom17,18, Spain21, Italy22, Singapore (GenBank accession number AF062642), China23, the West Philippines (GenBank accession number AB166953), Japan24,25, the Mid-Atlantic Ridge near Antarctica26, and from deep-sea sediments from the eastern Pacific Ocean23. It has been suggested that the success of Alcanivorax spp. could relate to their ability to use branched-chain alkanes more effectively than do other hydrocarbon-degrading bacteria, giving these species a selective advantage27. Branched-chain alkanes such as pristane not only enter the sea from oil spills but also are naturally produced by some marine plankton, possibly explaining the widespread occurrence of Alcanivorax spp.

In most cases in which Alcanivorax spp. have been found to be dominant in oil-impacted environments, samples have been analysed within days of the oiling or bioremediation event. If the first samples to be analysed are taken weeks or more after the initial oiling event, Alcanivorax spp. do not seem to be detected. In the case of an oil-spill bioremediation field trial carried out in Delaware (United States)28, as well as after the Nakhodka

Figure 1 | The effects of biodegradation on oil composition. a | Composition of a light North Sea crude oil (top panel) and a slightly biodegraded (heavy) oil (bottom panel). The resins and asphaltenes are complex mixtures of polar compounds. The degraded oil is characterized as being slightly biodegraded on the basis of its detailed molecular composition. Most resolvable saturated hydrocarbons have been biodegraded, as have the non-cyclic terpenoids pristane and phytane. The cyclic terpenoids, however, are intact, and only the two- and three-ring aromatic hydrocarbons have been extensively degraded. b | Gas-chromatogram traces showing separation of the components of whole oil that is increasingly biodegraded (from top to bottom). The main peaks that are lost are the resolvable saturated hydrocarbons. The large peaks on the right that do not decrease with biodegradation are internal standards that are added to the oil before analysis for quantification of individual components of the oil.

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Listeria monocytogenes

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tanker accident, on the Sakondani Coast (Japan)29, and during experimental oil-spill bio remediation in Spitsbergen, samples were collected more than 8 weeks after the start of the experiments30,31 and therefore might have been collected after the bloom of Alcanivorax spp., which seem to be early colonizers after nutrient amend-ment of an oil spill. This is consistent with the observa-tion that, after an initial rapid increase in population size, Alcanivorax spp. decline to much lower numbers within a few weeks16–18,32, and this correlates with the removal of the bulk of the saturated hydrocarbons. Interestingly, in experiments using seawater samples that were collected within 2 weeks of the Nakhodka running aground and sinking, Alcanivorax spp., although detected, were not predominant24. This could reflect the fact that heavy oil was spilled from the Nakhodka, because heavy oils con-tain a lower proportion of saturated hydrocarbons than is found in light crude oils.

There is an analogous situation for Cycloclasticus spp., which seem to have a global role in the removal of aromatic hydrocarbons from oil spilled in marine environments. Cycloclasticus spp. were found to be particularly enriched

in oil-contaminated gravel that was treated with inorganic nutrients: numbers were shown to increase by ∼5 orders of magnitude relative to uncontaminated sediment and by 2 orders of magnitude compared with gravel that was contaminated with oil but not amended with nutrients33. In a study of seawater and oil-paste samples collected following the Nakhodka oil spill, bands corresponding to Cycloclasticus spp. 16S-rRNA-gene fragments were only detected in denaturing-gradient-gel electrophoresis profiles from oil paste towards the end of an 11-week sampling period, at which time most alkanes had been degraded25. Short-term studies of oil degradation in beach microcosms are consistent with this observation, and only relatively small numbers of Cycloclasticus-like 16S-rRNA sequences were recovered from oil-contaminated-sed iment microcosms where appreciable degradation of the polycyclic aromatic hydrocarbons had not occurred during the short time course of the experiment17. Culture-based studies have also shown that Cycloclasticus spp. are abundant polycyclic-aromatic-hydrocarbon-degrading bacteria in the Gulf of Mexico and Puget Sound (United States), especially in contaminated sites34,35.

Figure 2 | A phylogenetic tree illustrating the diversity of aerobic hydrocarbon-degrading bacteria. Aerobic bacteria that degrade hydrocarbons are increasingly being thought of as key players in the removal of hydrocarbons from oil-polluted marine environments. The organisms shown in blue can degrade saturated hydrocarbons, whereas those shown in red can degrade polycyclic aromatic hydrocarbons. The organisms shown in black do not degrade hydrocarbons.

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Community dynamics Changes in community size and composition that result from various forces (such as climate changes, nutrient concentrations, habitat destruction, predation and so on) that control and regulate communities over time.

Chao1 A non-parametric statistic that allows the species richness of an environment to be estimated by analysis of data from a small sample.

Community dynamics and nutrientsMicrobial community dynamics are mainly followed using cultivation-independent, 16S-rRNA-gene-based meth-ods or functional-gene-based methods. Monitoring the dynamics of microbial communities can lead to the discovery of common patterns that are associ-ated with biodegradation, which in turn will help to develop new tools to evaluate ongoing bioremediation pro cesses rapidly. Important issues include the following: whether different treatments have a systematic effect on biodegradation and the associated microbial communi-ties, and the extent to which the community response varies between bio remediation treatments; whether the results of small-scale laboratory experiments can be extrapolated to the field; and how community dynam-ics compare in different environmental settings. These issues have been examined in the context of nutrient-enhanced bioremediation of beach oil spills, allowing some tentative conclusions to be drawn.

Do different treatments have a systematic effect on bio-degradation and the associated microbial communi-ties? Owing to the high carbon content of hydrocarbons and the low concentrations of other nutrients that are essential for microbial growth, the rate and the extent of degradation of freshly spilled oil are, in general, lim-ited by the availability of nitrogen and phos phorous4. A profitable way of increasing biodegradation of petroleum hydrocarbons, therefore, is fertilization with inorganic nitrogen and phosphorus. The systematic effects of nutrient amendment on biodegradative micro-bial populations and the progress of bioremediation have been studied in laboratory microcosms17,36. Hydrocarbon biodegradation in oiled, control microcosms that were

not treated with nutrients was negligible, but amend-ment with liquid fertilizer, spanning a wide range of concentrations of nitrogen and phosphorus, significantly improved oil degradation within a few days. However, the final extent of oil degradation was similar for all microcosms, indicating that the lowest concentrations of nutrients used in this experiment were sufficient to pro-duce a similar bioremediation end-point, even though the initial biodegradation rate was increased at higher nutrient concentrations.

Interestingly, bacterial and archaeal communities on beaches seem to have relatively low diversity17,18,28,36. One method that has been used to estimate the diversity of microbial communities is use of the estimator Chao1 (REF. 37). On the basis of Chao1, bacterial communities, before the addition of oil to beach sediments, were esti-mated to comprise 117.1±26.8 operational taxonomic units (OTUs); and archaeal communities, 9.4±3.4 OTUs36. This is towards the low end of prokaryotic-community diversity that has been estimated using Chao1 in other environments; it is comparable to esti-mates for fresh and marine water but much lower than the several hundreds of OTUs typically estimated for soils37,38. In addition to their relatively low diversity, the prokaryotic communities on beaches also seem to be relatively stable over time in the absence of any perturbation17,18,36.

Oil contamination and bioremediation treatments do, however, have a significant effect on community composi-tion. In beach microcosms, Archaea could no longer be detected following exposure to oil, and marked changes in bacterial-community composition occur within days17,36. Clearly, rapid and extensive changes in bacterial - and archaeal-community structure are engendered by

Figure 3 | Changes in the composition of spilled oil and corresponding changes in the abundance of key organisms. This schematic diagram represents general changes that have been observed in several studies. Slight variations are likely, both in the specific organisms that are involved and in the extent of biodegradation of different crude oils, which have a range of physical and chemical properties that affect their fate in the environment.

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Interstitial waters The water that surrounds sediment particles in aquatic environments. The composition of interstitial waters is controlled by the physical, chemical and biological activity of the sediment.

Phenanthrenes Readily biodegradable, relatively water-soluble polycyclic aromatic compounds that are found in crude oil. Phenanthrene, the parent molecule, is a tri-aromatic hydrocarbon, and substituted phenanthrenes have alkyl substitutions of varying number and position on the aromatic rings.

Dibenzothiophenes Sulphur-containing, non-hydrocarbon compounds that are found in crude oils. Dibenzothiophene, the parent molecule, comprises two benzene rings linked by a carbon–carbon bond and has a sulphur bridge between adjacent carbon atoms on the benzene ring. Crude oil contains a diverse mixture of such compounds with a variety of substitutions on the benzene rings.

oil and nutrient addition to beaches, but it is evident that the overall community response is not completely predictable. The bacterial communities in microcosms that were treated with different amounts of nutrients were distinct, and bacterial-community profiles in repli-cate samples taken from a single microcosm were highly reproducible, indicating that different nutrient-addition regimens seem to select for different microbial communi-ties in oil-contaminated sediments. However, when the bacterial communities present in replicate microcosms treated with the same amount of nutrients, rather than replicate samples from a single microcosm, were analysed, it became clear that the overall community response to even a single bioremediation treatment is variable17. This indicates that heterogeneity in the extent of oiling or small local differences in nutrient concentration could have a marked effect on the composition of the developing bac-terial community. Interestingly, despite the considerable variation observed in bacterial communities that develop in response to oiling and bioremediation, the efficacy of hydrocarbon removal is similar, indicating that oil-spill bioremediation should be a robust technology and that there are many combinations of different bacteria that can either directly or indirectly promote effective removal of hydrocarbons and other non-hydrocarbon components of crude oil.

Although the overall bacterial-community response is variable, it has become evident from several studies that, within this background of variation, there are core themes. For example, oiling and bioremediation treat-ments markedly reduce the diversity of the bacterial communities in the short-term, and the decrease in bac-terial diversity can be accounted for by strong selection for particular specialist hydrocarbon-degrading bacteria, such as Alcanivorax spp.17,18,25 (FIG. 3).

Can the results of small-scale laboratory experiments be extrapolated to the field, and how do community dynamics compare in different environmental settings? Laboratory studies have shown that the overall bacterial-community response to hydrocarbon contamination is variable, but certain hydrocarbon-degrading taxa do become prevalent in oil-impacted environments. Laboratory studies have also shown that Archaea do not have an important role in hydrocarbon degradation on contaminated beaches and, indeed, because of their sensitivity to oil pollution, could be useful markers of ecosystem recovery.

Before drawing wider conclusions, however, it is important to know whether the results obtained in labora-tory microcosms can be extrapolated to the field18,36. Field studies have shown that many of the features observed in laboratory microcosms are also observed under field con-ditions. In beach sediments collected in the field, bacterial communities have limited diversity and are homogeneous in composition in space and time. The overall response of the bacterial community in the field to oiling and bio-remediation treatments is variable, similar to the response in laboratory microcosms, although in field studies using oil treatment alone or in situations in which added nutri-ents were not retained in interstitial waters, the bacterial

communities did not seem to respond and were similar to those in uncontaminated beach sediment18. In com-mon with laboratory experiments, specific hydrocarbon-degrading organisms (for example, Alcanivorax spp.) were selected in the field under conditions in which nutrient addition resulted in an increase in nutrient concentrations in interstitial waters18.

Despite these important common features of labora-tory and field experiments, important differences have been observed. In the field, the rate and the extent of oil degradation have been found to be lower, prob-ably as a result of the lower mean temperature in these experiments, which ranged from more than 20°C to less than 5°C. In laboratory microcosms, phenanthrene and dibenzothiophenes were degraded17, but such degrada-tion was not observed in the above field experiment18. In addition, a marked increase in members of the α-proteobacteria has been observed during the later stages of oil-spill bioremediation treatments on a beach in Delaware28. This increase has also been observed in laboratory-microcosm experiments17 but, curiously, not in other field trials, in the United Kingdom18 and Japan29. In the Japanese study, a strong dominance by bacteria related to Pseudomonas putida was observed after 12 weeks29, and in the UK field trial, Pseudomonas spp. most closely related to Pseudomonas stutzeri, which was not found in corresponding laboratory studies17, were detected only transiently early in the experiment18.

Perhaps one of the greatest contrasts observed between field and laboratory experiments has been the effect of oil on archaeal communities. In laboratory microcosms, oil was observed to have an unequivocal negative effect on Archaea36. If this were the case in the field, then Archaea could be excellent markers of eco-system recovery. However, the data from field experi-ments are much more variable, and it is evident that regular tidal inundation returned sufficient numbers of Archaea to the beach sediments for them to be detected readily in oil-contaminated samples36.

So, although some broad similarity is observed in the dynamics of microbial communities in response to spilled oil and bioremediation (for example, selection for Alcanivorax and Cycloclasticus spp.), the specific response can vary from beach to beach, and bac terial-community composition does not converge after oil contamination and nutrient amendment. A similar observation has been made for diesel-contaminated soil39. In fact, even at the same beach location, similar treatments can result in heterogeneity in the dynamics of the bacterial com-munities as a whole18,28. Because bac terial-community composition is consistent across a beach before any perturbation induced by oil contamination18, it seems that the lack of convergence in bacterial communities does not reflect the initial composition of the bacterial communities present.

Our understanding of bacterial-community dynamics during bioremediation is still in its infancy, and many stud-ies have used different experimental and sampling pro-cedures (for example, different sampling times), thereby complicating comparisons between studies. Monitoring of 16S-rRNA genes has been widely applied in studies

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Phylogenetic microarray A microarray ‘printed’ with a range of marker genes or oligonucleotides (often including ribosomal-RNA-gene sequences) that is diagnostic for specific microbial taxa. Such arrays can be used to track the simultaneous presence of many microbial taxa.

of hydrocarbon-degrading bacterial communities. By supplementing phylogenetic-based community-dynamics tools with analysis of community dynamics using specific catabolic genes as markers, clearer informa-tion can be obtained on the behaviour of the organisms that are specifically responsible for degrading hydro-carbons, free from variations in members of the com-munity that do not directly participate in hydro carbon degradation. With respect to oil-spill bioremediation, the dynamics of alkane-degrading bacteria have been fol-lowed by quantifying three groups of alkane- hydroxylase genes40. This showed that a group of bacteria that degrade short alkanes (that is, C11–C12) and carry group I alkane-hydroxylase genes was enriched in the first 3 days of the experiment, and it was suggested that these bac teria might correspond to Alcanivorax spp., although no sequence analysis was carried out to confirm this idea. Bacteria containing group II alkane hydroxylases were not detected until day 10 of the experiment, corresponding to the degradation of higher-molecular-weight alkanes. Interestingly, group III alkane hydroxylases were initially undetectable, but after 3 days, they were present, although at a lower abundance than group I alkane hydroxylases. However, group III alkane hydroxylases were more abun-dant than group II alkane hydroxylases, indicating that organisms that carry group III alkane-hydroxylase genes have an important role in the degradation of higher-molecular-weight alkanes throughout the period of alkane degradation. This finding indicates that — rather than the sequential removal of alkanes according to molecular

weight (that is, lower-molecular-weight alkanes followed by higher-molecular-weight alkanes), which is the gen-erally accepted order of alkane degradation — there is simultaneous degradation of all alkanes irrespective of molecular weight, and it is simply the lower rates of deg-radation of the high-molecular-weight alkanes that causes the illusion of sequential degradation.

The application of DNA microarrays that combine phylogenetic markers with functional markers is likely to aid further in unravelling general trends in successful bioremediation at the community level. With respect to a phylogenetic microarray, one can envisage a nested approach ranging from detection of the phylum down to the species, as has been used in a DNA microarray developed to analyse communities of sulphate -reducing prokaryotes41. A functional gene array that includes DNA from a wide range of genes involved in anaerobic and aerobic degradation of pollutants has recently been developed42, and this array could provide opportunities to associate the occurrence of particular catabolic pathways in microbial communities with environmental condi-tions and with removal of different classes of compound during the course of bioremediation.

Ecology of in situ hydrocarbon biodegradationBioremediation studies tend to focus on the micro-organisms that degrade the contaminants. However, these microorganisms form part of an ecological network, which involves many direct and indirect interactions with other community members and the environment (and therefore is influenced by environmental variables such as nutrient availability or physicochemical parameters). Such interac-tions include competition for limiting nutrients, predation by protozoa, lysis by phage and cooperative interactions that increase degradation (FIG. 4). Integration of biologi-cal theory, mathematical modelling and experimental testing should allow better insights into the functioning of these ecological networks and should help to identify the species or processes that actually control contaminant degradation, as well as help to determine how to tailor bioremediation.

Systems biology. Systems-biology approaches, which are mainly applied at the cellular level at present, can be extended to ecosystems and used to detect, or to predict, the controlling species or processes. For example, in the 1970s, the development of metabolic-control analysis and its application to enzymatic pathways in biochemistry showed that the concept of a single rate-limiting enzyme is all-too-often incorrect. The control of metabolite flux and concentration is often distributed between several enzymes and might not even be carried out by an enzyme that was previously assumed to catalyse ‘the rate-limiting step’43,44. By analogy to enzymatic pathways, control in biodegradation can be distributed between several func-tional groups of microorganisms and might not be carried out by the microorganisms that degrade the pollutants. The importance of understanding which microorganisms substantially control environmental fluxes or concentra-tions of metabolic intermediates lies in the possibility of specifically targeting those physiological groups

Figure 4 | A microbial degradation network. The network indicates that oil biodegradation involves more biological components than just the microorganisms that directly attack oil (the primary oil degraders) and shows that the primary oil degraders interact with these components. Oil-degrading bacteria are shown in green. Solid arrows indicate material fluxes, and broken arrows indicate direct interactions (for example, lysis by phage and predation by protozoa). For simplicity, only one function is assigned to a microorganism in this schema. However, it should be noted that a microorganism can have more than one function or ability (for example, to weather minerals to release phosphate (P), and to degrade oil). It should also be noted that primary oil degraders need to compete with other microorganisms for limiting nutrients (such as P) and that non-oil-degrading microorganisms (shown in yellow) can be affected by metabolites and other compounds that are released by oil-degrading bacteria and vice versa.

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Eutrophication Enrichment of an environment with nutrients such that algal blooms and other negative environmental effects occur.

and increasing their activity to effect faster bioremedia-tion. After the identification of key organisms, in-depth studies can then be used to focus on their ecophysiology, and their importance in situ can be tested by specific stimulation and molecular monitoring.

Resource-ratio theory. The bioavailability of growth- and biomass-limiting nutrients, especially nitrogen and phosphorus, is a key factor in rapid and successful bio remediation of petroleum hydrocarbons4, and the addition of these nutrients is a common practice in bio-remediation. However, care must be exercised to avoid the addition of excess nutrients, because this can have detrimental effects, such as eutrophication. So, understand-ing the basis of competition for nutrients between target hydrocarbon-degrading bacteria and other org anisms that can use inorganic nutrients, such as eukaryotic algae and cyanobacteria, could be important.

Competition for — and partition of — resources has been formalized in resource-ratio theory45. This theory predicts that the structure and function of biological communities are influenced not only by the absolute amounts of limiting nutrients but also by the relative availability of these nutrients with respect to the meta-bolic needs of different organisms. An important control on hydrocarbon degradation in the environment is the availability of limiting nutrients, so resource-ratio theory might help to predict the composition and properties of hydrocarbon-degrading microbial communities. Initial studies indicate that nutrient concentrations might affect the relative degradation of polycyclic aromatic hydrocarbons and saturated hydrocarbons, and these changes in degradation could be related to differences in bacterial-community composition46. Examination of bacterial-community dynamics in response to different amounts of nutrients in oiled sediments indicates that the situation is more complex than can be predicted by resource-ratio theory in its basic form17. Nevertheless, there are some tantalizing signs that nutrient availability could have an important role in dictating the nature of hydrocarbon-degrading communities.

Although there is no clear signal to relate bacterial-community composition to nutrient concentrations17, there is some indication that different genotypes of Alcanivorax can become prevalent under conditions of high and low nutrient input. Two distinct Alcanivorax genotypes were identified in laboratory and field oil-spill bioremediation experiments, and the patterns of the relative abundance of 16S-rRNA sequences in clone libraries from sediments that were treated with different amounts of nutrients indi-cate that the different genotypes are adapted to higher or lower concentrations of nutrients (FIG. 5). Alcanivorax sp. type 1 seems to favour higher nutrient concentrations and was detected with high relative abundance in 16S-rRNA-clone libraries in beach sediments that were treated with 4% nitrogen and 0.4% phosphorus, as well as early in the experiment in microcosm sediments that were treated with oil alone. Alcanivorax sp. type 2 favoured lower nutri-ent concentrations and was detected in microcosms that were treated with 1% nitrogen and 0.1% phosphorus, as well as in later samples that were treated with oil alone

and in field samples that were treated with slow-release fertilizer (FIG. 5). Different Alcanivorax spp. have differ-ent physio logical properties10,21,23,47, and the selection of different Alcanivorax genotypes during bioremediation might affect the kinetics and patterns of biodegradation that occur.

The influence of predation. Stimulation of intrinsic recycling of nutrients, by increasing the turnover of bio-mass, could be an alternative to the addition of nutrients to stimulate bioremediation. The turnover of biomass can be increased by phage-mediated lysis or predation. Hydrocarbon pollution can induce prophage (possibly by functioning as a general stressor), resulting in lysis of a large proportion of the bacterial community48,49, and this might explain changes in community composition that are independent of extensive oil degradation17. Under both aerobic50 and anaerobic51 conditions, the number of protozoa that prey on bacteria tends to increase in response to a pollution event, owing to the increase in the number of bacteria that occurs as a result of the

Figure 5 | The effect of nutrients on Alcanivorax spp. The figure shows the abundance of different Alcanivorax genotypes identified in libraries of 16S-rRNA-gene clones from beach microcosms that were treated with different amounts of nutrients, as well as in samples from a field study of oil-spill bioremediation in which slow-release fertilizer was added to stimulate hydrocarbon biodegradation17,18. With the exception of one microcosm that was treated with oil alone and no fertilizer, Alcanivorax sp. type 1 was not detected in samples that were treated with small amounts of nutrients. Alcanivorax sp. type 2 was only detected in microcosms that were treated with oil and small amounts of fertilizer (including slow release (SR) fertilizer in the field) and not in microcosms that were treated with higher amounts of nutrients. This indicates that different genotypes of Alcanivorax might be adapted to environments with different concentrations of nutrients. Nitrogen (N) was added as sodium nitrate, phosphorus (P) as potassium phosphate. The label above each bar indicates the concentration of fertilizer used: 0% represents 0% N and 0% P; 1% represents 1% N and 0.1% P; 4% represents 4% N and 0.4% P; and SR represents treatment with slow-release fertilizer in the field. The time of sampling in days is indicated in parentheses. rRNA, ribosomal RNA.

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Syntrophy A close metabolic interaction between different groups of organisms. The term is usually used with respect to interspecies hydrogen transfer in anaerobic systems.

Surfactant A surface-active agent that reduces the surface tension of two liquids: for example, an agent that functions as a dispersant or emulsifier of oil and water.

Bioaugmentation The addition of non-native microorganisms to polluted sites to clean up toxic wastes.

higher concentration of available substrates. Predators indirectly affect contaminant biodegradation, owing to their ability to selectively graze on, and control the biomass of, bacteria.

The influence of protozoa on biodegradation is often negative, because it decreases the number of degradative bacteria in comparison to the situation in the absence of predators52. However, predation has been shown to stimulate bacterial degradation of toluene and benzene considerably on a per-cell basis53,54. In addition, pred-ation creates a nutritional loop, because predators can remineralize nutrients, which in turn increases bacterial growth. Recent modelling showed that under nitrogen or phosphorus limitation, the overall rate of biodegra-dation can be increased compared with a situation in the absence of predators, whereas this was not the case under electron-acceptor or carbon limitation (W.F.M.R., unpublished observations). Stimulation of carbon min-eralization by predation has also been observed in the environment55; however, this has not yet been shown in relation to hydrocarbon degradation.

The importance of interactions. A clear example of the requirement for interaction between microorgan-isms is syntrophic interspecies hydrogen transfer in the degradation of organic matter. Interspecies hydrogen transfer seems to be an absolute requirement under methanogenic conditions56,57. So far, there has been no description of natural consortia that depend on hydro-gen transfer between a fermentative microorganism and a terminal-electron-accepting microorganism during anaerobic hydrocarbon degradation under nitrate-, iron- or sulphate-reducing conditions58, although syntrophic relationships between fermentative bacteria and metha-nogens have been shown59. Syntrophic degradation coupled to different electron acceptors can occur, and syntrophic co-cultures constructed in the laboratory from toluene-degrading, iron- or sulphate-reducing bacteria with nitrate-reducing Wolinella succinogenes (a strain incapable of toluene degradation) degraded toluene with nitrate as the terminal electron acceptor60.

Another mutualistic interaction during biodegrada-tion, which has been described more recently, relates to the production of surfactants that promote pollutant bio-availability and degradation. Iwabuchi et al.61 observed that in oil-polluted sea water supplemented with nitro-gen, phosphorus and iron, significant biodegradation of an aromatic fraction of crude oil occurred only when extracellular polysaccharides from Rhodococcus rhodo-chrous were present. The extracellular poly saccharides resulted in emulsification of oil, followed by changes in the composition of the bacterial community, with Cycloclasticus spp. becoming dominant. Changes in microbial communities can depend on the amount of surfactant present, and in hydrocarbon-contaminated soil that was amended with different concentrations of the non-ionic surfactant Witconol SN70, Rhodococcus and Nocardia populations were replaced by Pseudomonas and Alcaligenes populations at increased concentrations62. In part, this was due to consumption of the surfactant by the Pseudomonas and Alcaligenes populations.

In a benzo[a]pyrene-mineralizing bacterial consortium recovered from soil, a Rhodanobacter strain that was unable to grow on benzo[a]pyrene in pure culture grew on metabolites produced by other consortium members and strongly contributed to benzo[a]pyrene mineralization by increasing its bioavailability63.

These examples show that the success of biodegra-dation is not just determined by the degradative micro-organisms, possibly explaining why bioaugmentation often fails to increase biodegradation rates, and they indicate that an alternative way to increase bioremediation could be to inoculate the environment with, or promote the growth of, organisms that carry out important second-ary functions. An additional reason why the addition of pollutant-degrading microorganisms frequently fails is that the inoculated strain has poor survival or low activ-ity in the environment as a result of stresses that are not encountered under laboratory conditions. An alterna-tive approach to the introduction of specific organisms involves the introduction of plasmid-borne catabolic genes from a donor strain into the indigenous bacterial population. This approach has been used to stimulate 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil. Although the host quickly disappeared, native transconjugants that had acquired the genes encod-ing 2,4-D-catabolizing enzymes proliferated, and the pollutant was quickly degraded64.

Hydrocarbon degradation in the genomic ageTraditional molecular genetics has already offered many valuable insights into the genetic basis of hydrocarbon degradation, and in a short review, it is not possible to do justice to this vast body of elegant work. The genomes of several hydrocarbon-degrading bacteria have now been sequenced, and these include aerobic and anaerobic organisms that can degrade simple aromatic hydrocarbons (for example, P. putida KT2440 (REF. 65), Geobacter metallireducens GS-15 and Azoarcus sp. EbN1 (REF. 66)) and the aerobic saturated-hydrocarbon-degrading bacterium Alcanivorax borku-mensis SK2 (REF. 67). Interestingly, it is evident from the screening of completed and partial genome sequences that many organisms that are not usually considered to be hydrocarbon-degrading bacteria contain genes that are homologous to alkane-hydroxylase genes. Van Beilen et al.68 report that Burkholderia mallei, Burkholderia pseudomallei, Burkholderia fungorum, Pseudomonas aeruginosa, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium avium, M. avium subsp. paratuberculosis and Silicibacter pomeroyi contain homologues of alkane-hydroxylase genes, and it has been shown for several of these spe-cies that the proteins encoded are functional alkane hydroxylases.

Despite this wealth of genomic information, several mysteries remain regarding alkane-degrading enzyme systems. All of the alkane-hydroxylase systems that have been examined oxidize only a small range of relatively low-molecular-weight alkanes, and knockout mutants of known alkane-hydroxylase systems in alkane-degrading

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Metagenomics The study of microbial genome fragments that are recovered from environmental samples, in contrast to genomes that are isolated from clonal cultures.

bacteria do not always lose the ability to oxidize alkanes. This indicates that there are alternative, as-yet-undiscovered alkane-hydroxylation systems69. Furthermore, a novel dioxygenase has been implicated in the oxidation of high-molecular-weight alkanes70. The prevalence of such systems in the environment has yet to be established.

The growing genomic-data resource on hydrocarbon-degrading bacteria, from both conventional, targeted molecular studies and genome sequencing68, now pro-vides a framework within which metagenomic studies of hydrocarbon-degrading prokaryotic communities can be carried out. Metagenomic analysis of hydrocarbon-degrading communities is attractive for two main reasons. First, there is a wealth of relevant genomic data and biochemical information available to facilitate robust functional interpretation of data from the hydrocarbon-degradation metagenome. Second, effective sampling of the metagenome of microbial communities that show high diversity requires a huge sequencing effort. The sequencing effort required to sample the metagenome effectively is reduced when the diversity of the commu-nity is lower71. The dominance of particular bacterial taxa, which can constitute up to 90% of the prokaryotic community in a hydrocarbon-impacted environment16, lends itself well to comprehensive comparative meta-genomic studies. The complexity of the hydrocarbon-degrading metagenome can also be constrained by using methods such as stable-isotope probing to selectively enrich the DNA from components of the community that are involved in the degradation of hydrocarbons and their metabolites72. This approach is particularly attractive because it overcomes one of the potential problems of analyses based on stable-isotope probing. Addition of an isotopically labelled substrate can itself

perturb the environment, which places restrictions on how the results are interpreted because, following the perturbation, it can be argued that the results no longer relate to in situ conditions. In the context of hydrocarbon degradation, it is generally the organisms that respond to high inputs of hydrocarbons to an environment that are of most interest. So, any perturbation caused by addi-tion of labelled substrate is likely to be small compared with that resulting from contaminating hydrocarbons.

Indications that different Alcanivorax genotypes are selected in the presence of different amounts of nutrients (FIG. 5) also pave the way for hypothesis-driven meta-genomic studies. Metagenomic analysis in this context could allow us to answer specific questions that relate to the causes of selection of a particular bacterium under a given set of environmental conditions. Furthermore, with comprehensive metagenome sampling, it becomes possible to apply approaches such as proteomics with a degree of confidence73. Interesting novel proteins that are detected by two-dimensional gel electrophoresis of proteins recovered from a complex environmental sample can be confidently associated with a DNA sequence only if the sequence has been recovered from the metagenome. In the absence of such information, environmental proteomics is likely to be particularly challenging. Metagenome-enabled environ-mental proteomics opens the door to meaningful temporal analysis of gene-expression patterns, a luxury that might not always be available for environments for which the metagenome has been poorly sampled.

Intelligent application of genomic technologies, in conjunction with more conventional biochemical and microbial-community analyses, now provides many exciting opportunities for increasing our understanding of the biology that underpins hydrocarbon degradation in a relevant environmental context.

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AcknowledgementsWe are grateful to the Biotechnology and Biological Sciences Research Council (United Kingdom) and to our colleagues at AEA Technology plc (Didcot, Oxfordshire, United Kingdom), who have supported our work on hydrocarbon-degrading microbial communities. W.F.M.R. is supported by The Netherlands BSIK (Besluit Subsidies Investeringen Kennisinfrastructuur) Ecogenomics Research Programme. Information on the thickness of the oil layer that could form on the oceans from oil released from natural seeps (see Abstract) comes from REF. 2.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:Entrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=genomeprjAlcanivorax borkumensis | Burkholderia fungorum | Burkholderia mallei | Burkholderia pseudomallei | Geobacter metallireducens | Legionella pneumophila | M. avium subsp. paratuberculosis | Mycobacterium avium | Mycobacterium bovis | Mycobacterium smegmatis | Mycobacterium tuberculosis | Pseudomonas aeruginosa | Pseudomonas putida | Silicibacter pomeroyi | Wolinella succinogenesEntrez Nucleotide: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=NucleotideAB166953 | AF062642

FURTHER INFORMATIONMicrobial ecology, at the University of Newcastle upon Tyne : http://www.ceg.ncl.ac.uk/activities/microbial/microbial.htmThe Netherlands BSIK Ecogenomics Research Programme: http://www.ecogenomics.nlAccess to this interactive links box is free online.

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marine microorganisms make a meal of oil

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