The problem of marine oil pollution

Our seas, oceans and coastal zones are under great stress; and pollution, particularly by crude oil, remains a major threat to the sustainability of planet Earth [1]. An estimated 1.3 million tonnes of petroleum enters the marine environment each year [2]. Acute pollution incidents cause great public concern, notably ~600,000 tonnes of crude oil released after the Deepwater Horizon explosion in the Gulf of Mexico [3] and ~63,000 tonnes from the Prestige oil-tanker [4] off the coast of north-west Spain. The fate of crude oil spilled at sea (Figure 1) depends on both the prevailing weather and the composition of the oil; but its environmental impact is exacerbated on reaching the shoreline, especially in low-energy habitats, such as lagoons and salt marshes. Acute pollution events can result in mass mortality; for example, more than 66% of total species richness (including polychaetes, molluscs, crustaceans and insects) was lost in the worst affected beaches following the Prestige spill [5]. Hydrocarbons also contaminate the feathers and fur of marine birds and mammals, resulting in the loss of hydrophobic properties, leading to death from hypothermia [6], or lethal doses following ingestion of oil during preening.

Figure 1
figure 1

Fate of a marine oil spill (for a more detailed explanation, seehttp://www.itopf.com/marine-spills/fate/weathering-process/). Spreading is affected by the action of winds, waves, water currents, oil type and temperature, and enhances evaporation of the volatile fractions such as low molecular weight alkanes and monoaromatic hydrocarbons. Spilt oil is broken into droplets and dispersed through the water column, enhancing the biodegradation of hydrocarbons and dissolution of water-soluble fractions of oil. Turbulent seas cause water droplets to be suspended in the oil, resulting in water-in-oil emulsions, alternatively known as chocolate mousse, which is difficult to degrade because of its high viscosity and reduced surface area. Photo-oxidation is the process by which hydrocarbons, especially PAHs, react with oxygen in the presence of sunlight, resulting in structural changes that can on the one hand lead to increased water solubility or, conversely, increased recalcitrance to biodegradation. Sedimentation will general only occur when oil adsorbs to particles owing to nearly all crude oils having a lower density than seawater.

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Moreover, the impact of hydrocarbons, especially polycyclic aromatic hydrocarbons (PAHs), on wildlife and fisheries may be long-lasting; for example the Fisheries Exclusion Zone imposed after the Braer spill (Shetland Islands, United Kingdom, 1993) due to contaminated fish and shellfish, remained in place for over 6 years. Chronic pollution can cause physiological or behavioural damage at sub-lethal concentrations; and genetic damage and decreases in both growth and fecundity have been observed in fish [7, 8]. Deep-sea sediments and associated biota are also chronically affected by drilling, which deposits vast amounts of oil-contaminated drill cuttings on the seafloor [9]. Even when oil-contaminated coastal sediments appear to be clean (e.g. Prince William Sound that was contaminated by the Exxon Valdez spill in 1989), toxic oil components, such as high molecular weight (HMW) PAHs, may remain buried and sorbed to sediment particles, and can be released to the environment by bioturbation or human activities such as dredging [10].

Crude oil is a natural, heterogeneous mixture of hydrocarbons, with potentially 20,000 chemical components [11], consisting mainly of alkanes with different chain lengths and branch points, cycloalkanes, mono-aromatic and polycyclic aromatic hydrocarbons (Figure 2; [12]). Some compounds contain nitrogen, sulfur and oxygen [12]; while trace amounts of phosphorus, and heavy metals such as nickel and vanadium are also found [13]. Its composition varies widely, and each oil component has different physico-chemical properties, including viscosity, solubility and capacity to absorb (Table 1), as well as varying in its bioavailability and toxicity. Crude oil, released naturally from the geosphere to the biosphere (e.g. from cold seeps [14]) may supply up to half of the oil in the sea [2]. Although hydrocarbons are relatively stable molecules, their “fuel value” and presence in the environment for millions of years have led to the evolution of many microbes able to activate and use them as a major or sole source of carbon and energy, including at least 175 genera of Bacteria [15]. Several haloarchaeal genera [16] and many Eukarya can grow on or transform hydrocarbons [17]. Biodegradation of crude oil to carbon dioxide and water is the major process by which hydrocarbon-contaminated environments are remediated.

Figure 2
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Structure of selected components of petroleum.

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Table 1 Selected hydrocarbons and their solubility in deionised water at 25°C and hydrophobicity indicated as Log Kow
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The principal marine hydrocarbon degraders

The starting point in elucidating potential complex interactions involved in hydrocarbon biodegradation is to identify the microbes primarily responsible for biodegradation, and their catabolic pathways. It has long been known that the enzymatic activation of hydrocarbons by oxygen is a pivotal step in their biodegradation, and several mechanisms have been elucidated for aromatic [12, 18, 19] and aliphatic [12, 20] compounds. However, our understanding of the catabolic processes for HMW PAHs [21] and anaerobic activation mechanisms and pathways, e.g. fumarate addition, carboxylation and O2-independent hydroxylation, have emerged only recently [22–25].

The microbial response to an oil spill at sea is dependent on numerous factors, including the oil composition and degree of weathering, as well as environmental conditions, particularly temperature and nutrient concentrations. Nevertheless, there are some typical patterns; most notable is the large increase in abundance of Alcanivorax spp., which degrade straight-chain and branched alkanes [26–32], followed by Cycloclasticus spp., which degrade PAHs [26–30, 33–36].

Since the cultivation of Alcanivorax borkumensis[37], functional genomic, biochemical and physiological analyses have revealed the underlying basis of its success [28, 38–40]. While it lacks catabolic versatility, utilising alkanes almost exclusively as carbon and energy sources, it has multiple alkane-catabolism pathways, with key enzymes including alkane hydoxylases (a non-haem diiron monooxygenase; AlkB1 and AlkB2) and three cytochrome P450-dependent alkane monooxygenases [38]. Their relative expression is influenced by the type of alkane supplied as carbon and energy source and phase of growth [38]. Alcanivorax borkumensis also possesses a multitude of other adaptations to access oil (e.g. synthesis of emulsifiers and biofilm formation [38]) and to survive in open marine environments (e.g. scavenging nutrients and resistance to ultraviolet light [38, 40]). Acinetobacter spp., which are commonly isolated from oil-contaminated marine environments [41], also have a diverse array of alkane hydroxylase systems enabling them to metabolize both short- and long-chain alkanes [20, 42]. For example, Acinetobacter strain DSM 17874 contains a flavin-binding monooxygenase, AlmA, which allows it to utilize C32 and C36n-alkanes [43]. The almA gene has also been found in Alcanivorax dieselolei B-5 and is induced by long-chain n-alkanes of C22 - C36[44]. A diverse array of alkB gene sequences, encoding alkane hydroxylase, has been detected in the environment [45, 209]. However, there are few studies investigating the effects on hydrocarbon mineralisation, and the outcomes are sometimes conflicting, perhaps as a consequence of environmental differences or technical approaches. Using eukaryote inhibitors, Tso and Taghon [212] showed that grazing had a beneficial effect on naphthalene degradation in estuarine sediments, possibly because the protozoa selectively grazed those bacteria that were not attached to naphthalene, thus allowing attached naphthalene-degrading bacteria to flourish by reducing competition for nutrients and other resources. Mattison and Harayama [213] reported a four-fold increase in toluene mineralization by a Pseudomonas sp. in the presence of the bacterivorous flagellate Heteromita globosa than in its absence, though Pseudomonas numbers reduced to 60% of the original biomass in the presence of the flagellate. In this case it was suggested that, in addition to selectively grazing the less-active bacteria, H. globosa enhanced naphthalene degradation by excreting growth-stimulating metabolites or ammonium and phosphate. Rogerson and Berger [214] proposed that stimulation of crude-oil degradation by Colpidium colpoda may additionally have been due to increasing oxygen flow caused by the swimming action of the ciliate and/or production of oil-emulsifying mucus that may have enhanced hydrocarbon bioavailability. Stoeck and Edgcomb [209] provide examples of other indirect benefits of protozoa to oil biodegradation. In contrast, Näslund et al. [215] found that meiofaunal grazers reduced naphthalene degradation in marine sediments. By reducing the number of larger grazers, oil pollution can result in microalgal blooms [216, 217]. Although the benefits of phototrophs have been outlined earlier, such a bloom may be disadvantageous because of algal competition for nutrients with hydrocarbon-degrading bacteria. More systematic studies investigating the role of different types of grazers under defined scenarios with varying levels of complexity are required to provide a clearer understanding of the nature of the interactions involved and the impact of grazers on hydrocarbon degradation.

Bacteriovorax spp. are obligate predatory bacteria that prey on other bacteria, but information regarding their potential role in oil-degrading communities is limited and conflicting. During hydrocarbon-degradation mesocosm experiments, Bacteriovorax were detected in microbial communities between days 21 and 35 [218] and days 21 and 28 [210]. However, in a similar experiment Bacteriovorax represented 11% of the bacterial community at day 0, but by day 15 none were detected [219].

Bacteriophages might also affect microbial oil degradation either positively or negatively. Pollutants can induce prophage [27, 220], and the resultant bacteriophage-induced lysis of bacterial cells, unlike grazing, releases all cellular components back into the marine environment for reuse by other microbes. Such a phage-driven microbial-loop was implicated in enhancing total organic carbon removal in reactors treating oil-contaminated waters [221]. Rosenberg et al. [221] found extremely high densities of bacteria and phages in these reactors, and they isolated phages, including one that infected a strain of Marinobacter cultured from the same location. Using the GeoChip-based high-throughput microarray, Lu et al. [222] observed significantly higher numbers of bacteriophage replication genes in the Deepwater Horizon deep-sea oil plume samples than in non-plume control samples collected at the same depth. Because previous studies had reported a significant increase in biomass in the plume samples [223], it was surmised that the bacteriophages provided a constant supply of nutrients needed for bacterial hydrocarbon degradation through phage-mediated biomass turnover. Furthermore, phages, together with various mobile genetic elements, are important in dissemination of valuable genetic material, including hydrocarbon-degradation genes and in the generation of new catabolic pathways via lateral gene transfer [224, 225].

A brief overview of microbial interactions with macrofauna and plants

There exists substantial evidence that bioturbation by larger fauna has a significant impact on the degradation of petroleum hydrocarbons in oil-contaminated sediments. By selective-removal experiments, Cuny et al. [226] found that the marine polychaete, Nereis diversicolor, increased the abundance of bacteria known to play important roles in aerobic hydrocarbon degradation. It was suggested that digestive solubilizers produced by the polychaete via feeding might have enhanced the bioavailability of the hydrocarbons and/or burrowing activities enhanced oxygen transfer to hydrocarbon-degrading bacteria. Gilbert et al. [227] had demonstrated previously that the digestive process of the polychaete Nereis virens altered the composition and reduced the concentration of ingested aliphatic hydrocarbons. It was therefore surmised that surfactant production in the gut of the worm led to these changes in the hydrocarbons. In addition to aerating deeper sediments, burrowing animals may transport pollutants or degrading bacteria deeper into sediments or return buried pollutants back to the surface [228, 229].

Plant roots oxygenate their rhizosphere and provide sugars and other compounds that stimulate microbial activity; and ultimately their major polymers, such as lignin, upon entering the soil will be attacked by a suite of (fungal) extracellular enzymes, which will initiate fungal degradation of PAHs. Phytoremediation, which exploits these features, has been employed in terrestrial soils, but only trials have been carried out in coastal zones [230]. For example, Lin and Mendelssohn [231] investigated both tolerance limit to crude oil and phytoremediation potentials of the salt-marsh grass Spartina patens. It could survive at concentrations up to 320 mg oil g-1 dry sediment, and at oil doses of between 40 and 160 mg g-1 oil degradation was significantly higher than in unplanted sediments. The rhizopheres of mangrove species were shown to harbour a variety of bacteria that both degraded oil and potentially stimulated plant growth [208]. As with algal-bacterial interactions, a more complete understanding of the molecular interactions between plants and associated bacteria and fungi will only improve the possibility of this technology being rationally applied to remove oil in the coastal zone [232].

Concluding remarks and prospects for using interacting microbes for oil-spill cleanup

There has been a lot of debate about the validity of bioaugmentation, specifically supplementing the environment with microbes to enhance biodegradation or detoxification of pollutants. Examples of success and failure abound. The key reasons for failure include: use of a single organism, focus on biodegrading strains only, microbes not adapted to the environment, inadequate dispersion/ access to the pollutant, lack of protection (e.g. from grazers), other factors limiting biodegradation (e.g. nutrients). Now, there is overwhelming evidence that using a consortium of microbes rather than a single strain greatly enhances the chances of successful bioaugmentation.

A well designed microbial consortium will have complementary catabolic pathways, as well as the potential to disperse and make the hydrocarbons readily bioavailable. Gallego et al. [233], for example, demonstrated the vastly superior efficacy of a designed four-species consortium over individual species in the bioremediation of oil-tank sludge. A six-species manufactured consortium, including a fungus, Fusarium sp., mineralised 78% of the PAHs from soil in 70 days, compared with negligible mineralization in an uninoculated control, and much lower degradation with single-species inocula [234]. Successful bioaugmentation is also a function of the competition between the introduced microorganisms and the autochthonous microbial community, and the study of this biotic pressure requires more attention.

Despite the improved biodegradation of hydrocarbons in bacterial co-cultures with microalgae, there have been few attempts to exploit this in the remediation of petroleum contamination. Munoz and Guieysse [235] describe ex-situ bioremediation using photobioreactors, but for marine pollution an in-situ approach is preferred owing to the large volume of polluted material. The critical phase of crude-oil contamination of the shoreline is the first few days. If the oil is not rapidly degraded then it will start to sink into the sediment where it can remain for decades. While it is true that hydrocarbonoclastic microbes will emerge from the native community, this process may take days. Thus, there is a role for bioaugmentation to bolster the in-situ hydrocarbon-degrading community in this crucial period. The potential to apply relevant hydrocarbonoclastic bacteria with or without associated microalgae should be investigated further.

Clearly there are many fundamental gaps in our understanding of microbial interactions; however, by a combination of reductionist experiments through to modelling the co-occurrence of microbial communities on a large scale, the field is advancing. The nature of interactions can be captured by single-cell and in-situ-metabolism imaging techniques such as Raman-FISH [236] and Nano-SIMS [237], as well as co-localisation studies using Magneto-FISH [238]. The requisite tools are constantly being developed, such that we can characterise and analyse in more depth the function of diverse components of DOM or the epimetabolome, as well as the volatile organic compounds, including the all-important signalling molecules. It is essential to make greater sense of metabolomics and protein and gene expression analyses in microbial consortia via the tools of systems biology [86, 239]. A better understanding of microbial community metabolic networks will arise from recreating natural consortia in which modifications can be made a gene at a time. The result will be a clearer picture of microbial interactions and thus the functioning of global biogeochemical cycles, with potential practical offshoots, not least a more rational approach to the remediation of marine pollution.