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ScienceDirect Hydrocarbon biodegradation in intertidal wetland sediments Terry J McGenity Intertidal wetlands, primarily salt marsh, mangrove and mudflats, which provide many essential ecosystem services, are under threat on numerous fronts; a situation that is made worse by crude-oil pollution. Microbes are the main vehicle for remediation of such sediments, and new discoveries, such as novel biodegradation pathways, means of accessing oil, multispecies interactions, and community-level responses to oil addition, are helping us to understand, predict and monitor the fate of oil. Despite this, there are many challenges, not least because of the heterogeneity of these ecosystems and the complexity of crude oil. For example, there is growing awareness about the toxicity of the oxygenated products that result from crude-oil weathering, which are difficult to degrade. This review highlights how developments in areas as diverse as systems biology, microbiology, ecology, biogeochemistry and analytical chemistry are enhancing our understanding of hydrocarbon biodegradation and thus bioremediation of oilpolluted intertidal wetlands. Addresses School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK Corresponding author: McGenity, Terry J ([email protected])
Current Opinion in Biotechnology 2014, 27:46–54 This review comes from a themed issue on Environmental biotechnology Edited by Hauke Harms and Howard Junca
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erosion caused by oil-induced destruction of salt-marsh plants [3]. The acute/chronic, direct/indirect impacts of oil pollution on salt-marsh ecosystems and the services they provide are nicely reviewed [1]. The fine-grained sediment of intertidal wetlands is testament to the low tidal and wave energy, which is often sufficient to carry oil slicks onto the shore, but after trapping by sediment and vegetation, is too low to flush away the oil. The abundance of clay minerals coupled with high organic loads encourage sorption of the most hydrophobic hydrocarbons, and sedimentary accretion and bioturbation can result in burial of hydrocarbons in zones of low redox potential. Hydrocarbons have been found in fine-grained coastal sediments many decades after a spill due to slow anaerobic biodegradation, for example the Florida spill at West Falmouth, MA [4] (Figure 1). However, these hydrocarbons can become accessible by processes such as resuspension, bioturbation, erosion and dredging. Such accessibility is a doubleedged sword – increasing the potential for biodegradation while enhancing the risk to the biota [5]. Microbes have had millions of years to adapt to the stress imposed by hydrocarbons and to exploit their ‘fuel value’ [6], resulting in an array of microorganisms from the three domains of life that have evolved to use them as a source of both energy and carbon [7–9]. Before focussing on (microbial) ecology, which underpins bioremediation, it is important to consider hydrocarbon bioavailability and catabolism, which help us to understand when and where degradation of different hydrocarbons can occur, informs remediation strategies, and provides important tools (e.g. in the form of oligonucleotide primers and metabolites) that allow biodegradation processes to be studied in situ.
Introduction
Bioavailability of crude oil
Intertidal wetlands, in particular temperate-zone salt marshes, tropical mangrove and mudflats are environments with high primary productivity that provide many important environmental, economic and societal benefits, such as protection from wave and storm damage, removal of excess nutrients from water, provision of food and habitats for fish, livestock and diverse wildlife [1], and they are generally considered to be a net sink for greenhouse gases [2]. However, they are very susceptible to oil pollution, owing to their frequent proximity to river outflows, oil refineries and industrial plants, as well as receiving marine-derived oil from shipping, natural seeps, oildrilling and blow-outs (Figure 1). These globally important habitats, already threatened by sea-level rise and diverse human activities, are made more susceptible to
More than 10,000 hydrocarbons and other molecules are present in crude oil, with diverse physico-chemical properties and correspondingly disparate stress effects and potential for biodegradation (Figure 2). With increasing carbon number, hydrocarbons are less bioavailable, volatile and soluble (more hydrophobic), and tend to adsorb to surfaces more readily, although the degree of aromaticity, ring positions and branching may cause deviations from this general rule [10]. Microbes have evolved numerous mechanisms to enhance bioavailability. First and foremost is the capacity to uptake and degrade the hydrocarbon thereby lowering the concentration in the immediate vicinity of the cell and driving diffusive flux of poorly soluble compounds towards the cell [10]. Second is the ability to increase the surface area
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Oil biodegradation in intertidal wetlands McGenity 47
Figure 1
Sea Empress 1996 72,860 t Exxon Valdez * 1981 38,500 t
Deepwater Horizon 2010 530,000 t
Amoco Cadiz 1978 227,000 t Hebei Spirit 2007 10,500 t
Erika 1999 19,800 t
Florida 1969 550 t Tank rupture 1986 7000 t
Arabian Gulf
Prestige* 2002 63,000 t
Solar 2002 800 t
Niger Delta
Katina 1992 72,000 t Metula 1974 51,000 t Current Opinion in Biotechnology
Location of the world’s salt marshes and mangroves, and selected crude-oil spills that have affected them. Salt marsh (green), dominated by shrubs and grasses, occur at high latitudes, whereas mangrove (blue), dominated by trees, occur in tropical and subtropical zones. Regions with both types of intertidal wetland (brown) are found in some subtropical regions, such as the Gulf of Mexico. Mudflats are more seaward unvegetated zones, rich in microbial mats, often associated with salt marsh and mangrove. The figure, from [76], is indicative only, and excludes many smaller islands fringed by mangrove. The name of the ship involved in the oil spill (or blowout information) is given italics, followed by the date of the spill and the amount released in tons. Those spills marked with * were large but had only a relatively minor impact on salt marsh and mangrove. The two locations marked with a gold star are the scenes of countless spills: the Arabian Gulf, which, in addition to shipping accidents, was the site of the largest release of oil into the sea, during the Gulf War. In the Niger Delta, oil exploration and transport have left a legacy of large-scale habitat destruction, especially of mudflats and mangrove, and ecological and health problems that exceed those caused by the Deepwater Horizon spill [77]. The oil spill data was derived from http:// mapsof.net/map/oil-spills-world-map-fr and [1].
of the bulk-phase hydrocarbon available to the microbe by synthesis of emulsifying agents/biosurfactants [10,11,12]. Thereafter, many hydrocarbon-degrading microbes modify their cell envelope to enable attachment to hydrocarbons (see [11]). In particular, experimental proteomic studies show that Marinobacter hydrocarbonoclasticus is well adapted to forming a biofilm on alkanes, accumulating carbon reserves in the form of intracellular wax esters, which can then fuel detachment and movement to other locations, where detached cells have acclimated to reattach to fresh alkane resources [13]. Alcanivorax borkumensis is similarly well adapted to life in a biofilm [14,15] (Figure 3), and transcriptomic analysis has revealed outer-membrane lipoprotein Blc as a candidate for the enigmatic process of alkane uptake [14]. In a few microbes, sensing of, and chemotaxis towards, hydrocarbons has been shown to enhance bioavailability and biodegradation [16] and the alkane-sensory system has recently been described in Alcanivorax dieselolei strain B-5 [17], but hydrocarbon-taxis is undoubtedly much more widespread. www.sciencedirect.com
Fate of oil in the open sea and intertidal wetlands The chemical composition and physical nature of crude oil evolve when released into the sea (a process referred to as weathering), depending on winds, waves, water currents, oil type, light exposure, nutrient availability and temperature (see [11]), with implications for its fate. The main process responsible for mineralisation of crude oil hydrocarbons is microbial biodegradation [18,19]. Nevertheless, oil frequently reaches intertidal zones with different degrees of modification depending on the oil source, prevailing environmental conditions and proximity of the spill to land. The oil spill on 10 April 2010 caused by the explosion at the Deepwater Horizon rig (herein referred to as DWH spill) released 636 million litres of Macondo (Light Louisiana) crude oil into the Gulf of Mexico over 3 months [20] – a disaster that spurred a large-scale remediation and research effort. Most models of the fate of oil in the sea are based on surface spills, but further complexities are introduced when oil derives from the deep sea Current Opinion in Biotechnology 2014, 27:46–54
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Figure 2
Crude oil n-hexane
Precipitates
Dissolves Column separation
toluene Dissolves
n-hexane Saturates (15-60%)
toluene / methanol
toluene Aromatics (3-30%)
Resins
Asphaltenes (mean 6%) CH3
S
CH3
CH3
S
R (CH2)nCO2H
N CH3 S CH3
Cyclo-alkanes (naphthenes), branched and straight-chain alkanes (paraffins). Shown are (left to right): cyclohexane, n-decane, pristane.
Mono- and polycyclic aromatic hydrocarbons, including alkylated forms. Shown are (left to right): benzene, methylnaphthalene, benzo [a]pyrene.
Polar & heterocyclic N, S & O-containing hydrocarbons. Shown are (top to bottom): dibenzothiophene, naphthenic acid (generic structure of an alkyl-substituted alicyclic carboxylic acid).
CH3
Large, heterocyclic N, S & O-containing compounds, with traces of heavy metals such as vanadium. The structure shown is illustrative.
Current Opinion in Biotechnology
Operational classification of crude-oil components based on the SARA system (Saturates-Aromatics-Resins-Asphaltenes), with some representative structures. The SARA system, shown here and modified from [78], provides a useful operational breakdown of crude oil. Alkanes, including straightchain, branched and cyclic, generally constitute the main mass of oil, followed by PAHs. Several descriptors exist, such as ‘light’ and ‘heavy’, for crude oils containing low-molecular-weight alkanes and polycyclic aromatic hydrocarbons (PAHs), compared to those consisting of high-molecular-weight hydrocarbons (>C20), resins and asphaltenes, respectively. Naphthenic acids (comprising mainly of alkyl-substituted alicyclic carboxylic acids), are a major and highly toxic component of oil-sand process-affected waters, and also constitute 1% of crude oils and up to 4% of North-Sea crude oils [79].
and is rich in gaseous hydrocarbons and water-soluble aromatics, such as benzene, toluene, xylene, ethylbenzene (BTEX) [21]. Reddy et al. [21] detected little biodegradation of BTEX in the early plume, supported by metatranscriptomic data [22]; while Hazen et al. [23] reported half lives of n-alkanes of 1 to 6 days at the deepsea temperature of 5 8C, and implicated a new group in the order Oceanospirillales (94% 16S rRNA sequence similarity to Oleispira antarctica) as the main enriched microbe. Further single-cell genomic analysis revealed that this dominant bacterium had genes for alkane and cycloalkane degradation that were expressed in the plume [22]. Other abundant microbes in the plume are Current Opinion in Biotechnology 2014, 27:46–54
the pyschrophilic short-chain-alkane-degrading Colwellia and PAH-degrading Cycloclasticus spp. [24]. Despite deep-sea degradation and other attenuating processes, slicks appeared on the sea surface and oil reached the Gulf-of-Mexico shoreline. Tar balls and water-in-oil emulsions (chocolate mousse), which are difficult to degrade because of their high viscosity and reduced surface area, were particularly common after the DWH spill. By comparing original Macondo oil to samples taken 500 days after the spill from beaches surrounding the Gulf of Mexico, Aeppli et al. [25] found that the proportion of oxygenated hydrocarbons increased tenfold, forming the www.sciencedirect.com
Oil biodegradation in intertidal wetlands McGenity 49
Figure 3
alkS
alkB1 alkG alkH
alkJ 0.5 µm
AlkB OH Current Opinion in Biotechnology
Hydrocarbon degradation in intertidal wetlands: from genome to ecosystem. Gene information, for example from the alkane hydroxylase gene (alkB) cluster in Alcanivorax borkumensis; one of several over-expressed in the presence of alkanes [14], informs on mechanisms of degradation (e.g. terminal oxidation of alkanes [54]) and vice versa. A. borkumensis must first access hydrocarbons and transport them across the membrane – the image shows a small (1 mm diameter) oil droplet emulsified by surrounding cells of A. borkumensis (copyright: Dr. Jessica Beddow). Populations of A. borkumensis form part of a wider community, as illustrated by this false-colour image showing photosynthetic organisms, in this case diverse benthic diatoms, on the surface of a mudflat in which A. borkumensis and other microbes were degrading hydrocarbons (field of view is 60 mm 40 mm). These benthic phototrophs probably contribute to hydrocarbon degradation by generating oxygen [15]. The image of a salt-marsh tidal creek connected to the North Sea (copyright: Prof. Graham Underwood) demonstrates the heterogeneity of the habitat, particularly the different degrees and types of algal and plant cover. Vertical heterogeneity (not shown) is equally pronounced, with oxygen diffusing no more than a few millimetres below the surface and different TEAs becoming available at depth. Finally, the understanding and knowledge acquired in less complex bio-systems provide complementary tools for investigating the ecology and biogeochemistry of ecosystems.
majority of hydrocarbons in heavily weathered samples. Most oxygenated hydrocarbons derived from partial biodegradation and photo-oxidation rather than simply an enrichment of these difficult-to-degrade compounds. Advanced analytical tools such as two-dimensional gaschromatography and direct immersion ionization techniques together with ultra-high resolution mass spectrometry are beginning to shed further light on the composition of such compounds [26]. Investigating the fate of such seemingly recalcitrant and toxic crude-oil components should be a priority of future research, as the oxidation of aromatic hydrocarbons with transition metals entrained in tar balls can result in toxic environmentally persistent free radicals, some of which have only recently been identified [27]. www.sciencedirect.com
Bioavailable PAHs in the water column at four coastal locations increased 7–14 weeks after the DWH spill, with PAHs persisting at elevated levels for a year at one location [28]. Other studies have shown how Macondo oil, enriched in high-molecular-weight hydrocarbons, increased the organic load by one to two orders of magnitude at several centimetres depth in sediments months after the spill, while surface water concentrations were the same as in pristine sites [29], thereby emphasising that intertidal finegrained sediments are major collecting points for spilled oil that will be impacted over the long term. The volume of the dispersant, COREXIT-9500, added to increase the surface-area-to-volume ratio of DWH oil, was almost the same as the crude oil itself (see [28]). The Current Opinion in Biotechnology 2014, 27:46–54
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balance of evidence suggests that such action reduced the amount of oil washed ashore [30,31,32], and there is good evidence that the surfactant within COREXIT is biodegraded in the sea [32]. However, potential disadvantages of using dispersants in the coastal zone must be considered; for example, sediment-column experiments showed that COREXIT (6 ppm) increased the mobility of PAHs from crude oil by up to two orders of magnitude in saturated permeable sediments [33]. If COREXIT also accelerates PAH penetration into fine-grained sediments, PAHs would persist owing to slower biodegradation in the anaerobic zone.
Anaerobic hydrocarbon biodegradation – from genetics and biochemistry to ecology and biogeochemistry Fine-grained intertidal sediments have steep vertical gradients of terminal electron acceptors (TEAs), with oxygen often absent below the top millimetre [34]. Hydrocarbon biodegradation has been demonstrated with all of the most common TEAs (nitrate, iron III, sulphate) and under methanogenic conditions, but the mechanisms by which the apolar and very stable C–H bond is activated in the absence of molecular oxygen have been much debated [6]. Direct carboxylation appears to be the main activation mechanism for non-alkylated aromatic hydrocarbons, although alternative mechanisms such as methylation followed by addition to fumarate are feasible [35]. For both alkyl-substituted aromatics and alkanes, the addition to fumarate, catalysed by glycyl-radical-containing enzymes, is the primary mode of hydrocarbon activation [36,37]. However, absence of alkyl succinate metabolites suggests that this may not be the case for alkanes under methanogenic conditions [38]. Also, gammaproteobacterial strain HdN1 has been proposed to employ nitrite (NO2 ) to activate alkanes while using nitrate as a TEA [39]. It is also feasible that dismutation of NO (from NO2 ) generates O2 (and N2), with the resultant oxygen activating the alkanes [40]. The presence of oxygenase genes in strain HdN1, and more complete evidence for this mechanism being responsible for oxidation of methane coupled to nitrate reduction, lend supports to the ‘dark oxygen mechanism’, a process akin to that proposed for hydrocarbon-oxidising (per)chlorate-reducing bacteria [39,40]. In marine sediments, where sulphate is abundant, sulphate-reducing bacteria (SRB) are commonly implicated in hydrocarbon degradation. Many pure cultures of hydrocarbon-degrading SRB have been reported [41]. Also, crude oil addition frequently leads to enhanced sulphate reduction in intertidal sediments and microcosms [29,42– 44], and addition of inhibitors of sulphate-reduction prevents hydrocarbon degradation [44]. The improved identification of signature metabolites from fumarateaddition pathways [37], and the understanding of enzyme diversity, for example benzyl succinate synthase (BSS), Current Opinion in Biotechnology 2014, 27:46–54
are providing important tools for identification and quantification of the relevant processes in the environment [45]. Comparison between deep-sea sediments close to the DWH blow-out and a remote uncontaminated sediment revealed an increase in: Deltaproteobacteria (including genera of SRBs), genes coding for enzymes with potential for both aliphatic and aromatic hydrocarbon activation via fumarate addition, and of anaerobic hydrocarbon metabolites [46]. While such studies implicate SRBs in hydrocarbon degradation and show that anaerobic degradation of hydrocarbons is occurring, it is sometimes difficult to disentangle whether enhanced SRB activity is due to their direct involvement in hydrocarbon degradation or an indirect effect, that is, resulting from hydrocarbon-induced cell lysis and release of organic matter. SRBs are not necessarily the primary ring-opening microbes in situ; they form syntrophic partnerships whereby their consumption of fermentation products makes the preceding reactions more energetically favourable [6,47]. To further confound interpretation, some SRBs switch metabolism to act as syntrophs, for example in cooperation with methanogens in medium lacking sulphate [47,48]. Members of the genus Smithella (Deltaproteobacteria in the family Syntrophaceae) have been implicated as the key anaerobic alkane-degrading syntrophs in association with methanogens in estuarine enrichments [49]. The perception of anoxic horizons beneath oxic zones in intertidal sediments is an over-simplification. Bioturbation introduces oxygen deeper into sediments. Anoxic microsites exist in surface layers. Light-induced variations in oxygenic photosynthesis result in differential diel oxygen exposure, further complicated by tidal cover that affects oxygen diffusion into the sediment. Thus, aerobes and anaerobes co-habit, facultative anaerobes abound, and the effect of all of these processes on in situ and ex situ bioremediation is starting to be considered [50]. In microcosms derived from marine sediments, hydrocarbon degradation was significantly enhanced by sequential anaerobic–aerobic degradation (via sulphate reduction in the anaerobic step), compared to degradation under purely aerobic or anaerobic conditions [51]. In situ, redox oscillations facilitate nutrient and TEA regeneration, and may serve to encourage more efficient cycling of metabolites [50]. It is worth speculating on other processes that may influence hydrocarbon consumption. Pfeffer et al. [52] proposed that cm-long filamentous Desulfobulbaceae bacteria, vertically orientated in marine sediments, used sulphide as an electron donor in the anoxic zone and transported electrons through their periplasm to oxygen (the TEA) in the upper sediment. Consumption of toxic sulphide is likely to benefit many (including anaerobic www.sciencedirect.com
Oil biodegradation in intertidal wetlands McGenity 51
hydrocarbon-degrading) microbes. It is conceivable that electron-shuttling microbes have a direct role in taking electrons from buried hydrocarbons – thereby using anoxic activation coupled with aerobic degradation. After all, representatives of the Desulfobulbaceae possess benzylsuccinate synthase a-subunit (bssA) genes [53].
Aerobic hydrocarbon biodegradation – from genetics and biochemistry to ecology and biogeochemistry Oil tends to float and so, at least in the early stages of a spill, remains in the sediment’s oxic zone. Biodegradation of hydrocarbons is faster under oxic compared with anoxic conditions [6], and aerobic hydrocarbon degradation is the prevalent process in salt-marsh sediments [42]. Oxygen not only serves as a high-energy-yielding TEA, but is also a reactant, which, catalysed by oxygenases, activates hydrocarbons [6]. There exists a lot of information about the variety and structure of oxygenases, their associated enzymes, the nature of the activation products and their pathways to central metabolism, as well as regulatory and transport mechanisms [17,54,55]. Essentially, for lowmolecular-weight alkanes (C2 to C5), soluble non-haem iron monooxygenases are employed, while for mid-range alkanes (C6 to C20), microbes use integral-membrane non-haem iron monooxygenases related to AlkB (alkane hydroxylases) and the haem-containing cytochrome P450 enzymes [54]. Far less is known about the mechanisms of degradation of long-chain alkanes (i.e. with >20 carbons), and diverse underlying biochemistries seem to be involved [17,54]. The flavin-binding monooxygenase, AlmA, for example, is found in many hydrocarbonoclastic genera such as Acinetobacter and Alcanivorax [17,54,56]. The differential expression of multiple alkane-degrading enzymes in the same species is a recurring theme [14,17,19,54,56]. Relative increased gene expressions for Alcanivorax dieselolei, for example, are: p450 with C8–C16 alkanes, almA with C22–C36, alkB1 and alkB2 with C12–C26, and both alkB1 and almB with branched alkanes [56]. Parallel developments in understanding aerobic degradation of aromatic hydrocarbons are emerging [55], particularly for high-molecular-weight PAHs (i.e. with more than three aromatic rings) that are very poorly soluble, adsorb to sediment particles and are highly toxic [57]. Their degradation by fungal extracellular nonspecific enzymes, principally produced to initiate breakdown of complex biopolymers, is well known [58,59]. But our understanding of bacterially mediated catabolism of high-molecular-weight PAHs has been advanced by a systems-biology-based investigation of Mycobacterium vanbaalenii PYR-1, originally isolated from oil-contaminated estuarine sediment, which resulted in a metabolic network starting with 7 PAHs, leading to 224 chemical reactions linking 183 metabolites ultimately funnelled into the central b-ketoadipate pathway [60]. www.sciencedirect.com
Fundamental knowledge about aromatic and aliphatic hydrocarbon catabolic pathways is important for understanding the ecology of oil-polluted intertidal wetlands [61,62] (Figure 3), because environmental gene analysis and quantification currently rely on a limited number of genes, mostly alkB for aliphatics, which may not always provide a faithful picture of the alkane-degrading potential or capacity in the environment, as was found when rates of degradation and gene expression were compared in coastal sediment microcosms [63]. The application of degenerate primers based on almA genes demonstrated their presence in several genera of coastal marine bacteria enriched on crude oil, especially Alcanivorax and Marinobacter, and two distinct phylogenetic groups [64]. Novel and iterative approaches [61,62] with isolates, consortia and natural communities will give a more complete understanding of pathways and networks, and capture the full diversity within and beyond gene families. When oil lands on a salt marsh it is useful to discern the microbial activity due to petroleum consumption compared with consumption of other organic matter, such as plant detritus. Pearson et al. [65] took advantage of the difference in d13C of the dominant plant, Spartina alterniflora ( 13%) and crude oil ( 27%), by isotopic analysis of rRNA from two-week incubated sediment samples, to reveal that 26% of the assimilated carbon derived from oil. Surprisingly, given their prevalence in oil-polluted marine habitats [18,19], Alcanivorax and Cycloclasticus were not found, whereas Idiomarina 16S rRNA genes were abundantly detected in the oiled sediment [65]. Intertidal mudflat cores, subjected to crude oil and a simulated tide, revealed an increase in aerobic obligately hydrocarbonoclastic bacterial genera (notably Alcanivorax, Cycloclasticus, Oleibacter and Oceanospirillales ME113-related sequences) that constituted 7.8% of sequences in the oiled sediment after 12 days [66]. Two of these genera were also abundant in biofilms that detached from the sediment and floated on the water surface on day 22 (Alcanivorax and Oceanospirillales ME113 constituted 48.5% and 10.6% of sequences, respectively). This enrichment of aerobic hydrocarbonoclastic bacteria in tidal biofilms, which also consisted of diatoms, suggests that they are active zones of hydrocarbon degradation. Thus, aerobic hydrocarbonoclatic bacteria, such as Alcanivorax and Cycloclasticus, are important in fine-grained intertidal sediments, just as they are in sandy sediments [18,67] and the open sea [18]. However, they are not always detected, and the roles of other microbes, both uncultivated and cultivated, for example Oleibacter sp. [66,68], should be investigated. Also, there is increasing awareness that interactions between oxygenic phototrophs and hydrocarbonoclastic bacteria may be important for hydrocarbon degradation [11,15,66,69], and hydrocarbons, like isoprene, released from marine and coastal microalgae may even Current Opinion in Biotechnology 2014, 27:46–54
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contribute to the survival of such hydrocarbonoclastic bacteria in the absence of an oil spill [70].
in Louisiana salt marshes after the BP Deepwater Horizon oil spill. Proc Natl Acad Sci USA 2012, 109:11234-11239. 4.
Bioremediation A key question that arises from the foregoing discussion is whether to clean up intertidal fine-grained sediments. Many (e.g. [71]) argue that the best strategy is not to intervene but allow the native microbes to perform the task. Nutrient addition, although successful in other environments [18,30], has limited impact in fine-grained sediments [71,72], in which oxygen rather than nutrients is often the limiting factor. While introduction of oxygen would stimulate biodegradation of buried hydrocarbons [5], the overall environmental impact together with disruption of the chemocline and phototrophic interactions with hydrocarbonoclastic bacteria would be detrimental. If bioremediation is to be considered, the role of environmental factors influencing hydrocarbon degradation have to be accounted for, especially temperature [73] and salinity [7]. Bioaugmentation, although largely disregarded owing to poor performance in the past [71], is receiving renewed interest, and with relevant indigenous microbes and carrier materials, may be valuable for accelerating biodegradation of fresh, near-shore oil spills, thereby reducing the quantity of oil reaching the coast. When salt-marsh and mangrove plants have been so heavily oiled that they are unlikely to recover, phytoremediation offers a means to stabilise the sediment. Additionally, some plants and their associated microbes have led to increased oil biodegradation in trials, for example Scirpus maritimus in salt marshes [74] and Avicennia schaueriana in mangrove [75]. Naturally, due consideration must be given to the impact on the local ecology, and native mixtures of species should be used.
Acknowledgements Funding from the UK Natural Environmental Research Council (NERC) is gratefully acknowledged, particularly grants: NE/J01561X/1 (a hierarchical approach to the examination of the relationship between biodiversity and ecosystem service flows across coastal margins) and NE/J009555/1 (microbial degradation of isoprene in the terrestrial environment).
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34. Duran R, Gon˜i-Urriza MS: Impact of pollution on microbial mats. In Handbook of Hydrocarbon and Lipid Microbiology. Edited by Timmis KN, McGenity TJ, van der Meer JR, de Lorenzo V. Berlin Heidelberg: Springer; 2010:2339-2348. 35. Meckenstock RU, Mouttaki H: Anaerobic degradation of nonsubstituted aromatic hydrocarbons. Curr Opin Biotechnol 2011, 22:406-414. 36. Callaghan AV: Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins. Front Microbiol 2013, 4:89.
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25. Aeppli C, Carmichael CA, Nelson RK, Lemkau KL, Graham WM, Redmond MC, Valentine DL, Reddy CM: Oil weathering after the Deepwater Horizon disaster led to the formation of oxygenated residues. Environ Sci Technol 2012, 46:8799-8807. This study demonstrates how oxygenated hydrocarbons become so abundant in coastal sediments, and why more research is needed to understand their fate and toxicity. 26. McKenna AM, Nelson RK, Reddy CM, Savory JJ, Kaiser NK, Fitzsimmons JE, Marshall AG, Rodgers RP: Expansion of the analytical window for oil spill characterization by ultrahigh resolution mass spectrometry: beyond gas chromatography. Environ Sci Technol 2013, 47:7530-7539. 27. Kiruri LW, Dellinger B, Lomnick S: Tar balls from Deep Water Horizon oil spill: environmentally persistent free radicals (EPFR) formation during crude weathering. Environ Sci Technol 2013, 47:4220-4226. This study provides evidence that oxygenated hydrocarbons, resulting from weathering of crude oil, are a source of a new type of environmentally persistent free radicals. 28. Allan SE, Smith BW, Anderson KA: Impact of the Deepwater Horizon oil spill on bioavailable polycyclic aromatic hydrocarbons in Gulf of Mexico coastal waters. Environ Sci Technol 2012, 46:2033-2039. The beauty of this study is that it demonstrates the value of good-quality long-term monitoring programmes, which in this case allowed a comparison of PAH contamination in coastal waters before and after the DWH oil spill. 29. Natter M, Keevan J, Wang Y, Keimowitz AR, Okeke BC, Son A, Lee M-K: Level and degradation of Deepwater Horizon spilled oil in coastal marsh sediments and pore-water. Environ Sci Technol 2012, 46:5744-5755. 30. Atlas R, Hazen TC: Oil biodegradation and bioremediation: a tale of the two worst spills in U.S. history. Environ Sci Technol 2011, 45:6709-6715. This illustrates how oil spills can have very different outcomes depending on: oil type, proximity to the shore, type of coastline, latitude and temperature etc.; and how responses must be tailored accordingly. 31. Lee K, Nedwed T, Prince RC, Palandro D: Lab tests on the biodegradation of chemically dispersed oil should consider the rapid dilution that occurs at sea. Mar Pollut Bull 2013, 73:314-318. The authors provide a well-argued reminder that faithful reproduction of environmental conditions is required in order to extrapolate from microcosms and meso-cosms; in this case by applying relevant concentrations of the dispersant COREXIT. 32. Campo P, Venosa AD, Suidan MT: Biodegradability of Corexit 9500 and dispersed South Louisiana crude oil at 5 and 25 -C. Environ Sci Technol 2013, 47:1960-1967. This study demonstarates biodegradation at 25 and 5 8C of dioctyl sodium sulfosuccinate (DOSS), the biosurfactant in Corexit 9500, which was used to emulsify oil during the DWH spill. 33. Zuijdgeest A, Huettel M: Dispersants as used in response to the MC252-spill lead to higher mobility of polycyclic aromatic hydrocarbons in oil-contaminated Gulf of Mexico sand. PLoS ONE 2012, 7:e50549. www.sciencedirect.com
39. Zedelius J, Rabus R, Grundmann O, Werner I, Brodkorb D, Schreiber F, Ehrenreich P, Behrends A, Wilkes H, Kube M et al.: Alkane degradation under anoxic conditions by a nitratereducing bacterium with possible involvement of the electron acceptor in substrate activation. Environ Microbiol Rep 2011, 3:125-135. The authors observe absence of relevant genes and metabolites for the fumarate-addition mechanism of alkane activation in gammaproteobacterial strain HdN1. Then follow this up with experimental manipulation of the nitrogen species in culture medium, which leads to two hypothesised mechanisms: activation by nitrite or by oxygen (generated by dismutation of nitric oxide). 40. Ettwig KF, Speth DR, Reimann J, Wu ML, Jetten MSM, Keltjens JT: Bacterial oxygen production in the dark. Front Microbiol 2012, 3:273. 41. Widdel F, Knittel K, Galushko A: Anaerobic hydrocarbondegrading microorganisms: an overview. In Handbook of Hydrocarbon and Lipid Microbiology. Edited by Timmis KN, McGenity TJ, van der Meer JR, de Lorenzo V. Berlin Heidelberg: Springer; 2010:1997-2021. 42. Shin WS, Pardue JH, Jackson WA: Oxygen demand and sulfate reduction in petroleum hydrocarbon contaminated salt marsh soils. Water Res 2000, 34:1345-1353. 43. Sua´rez-Sua´rez A, Lo´pez-Lo´pez A, Tovar-Sa´nchez A, Yarza P, Orfila A, Terrados J, Arnds J, Marque´s S, Niemann H et al.: Response of sulfate-reducing bacteria to an artificial oil-spill in a coastal marine sediment. Environ Microbiol 2011, 13:14881499. 44. Sherry A, Gray ND, Ditchfield AK, Aitken CM, Jones DM, Ro¨ling WFM, Hallmann C, Larter SR, Bowler BFJ, Head IM: Anaerobic biodegradation of crude oil under sulphatereducing conditions leads to only modest enrichment of recognized sulphate-reducing taxa. Int Biodeter Biodeg 2013, 81:105-113. 45. von Netzer F, Pilloni G, Kleindienst S, Kru¨ger M, Knittel K, Gru¨ndger F, Lueders T: Enhanced gene detection assays for fumarate-adding enzymes allow uncovering anaerobic hydrocarbon degraders in terrestrial and marine systems. Appl Environ Microbiol 2012, 79:543-552. 46. Kimes NE, Callaghan AV, Aktas DF, Smith WL, Sunner J, Golding B, Drozdowska M, Hazen TC, Suflita JM, Morris PJ: Metagenomic analysis and metabolite profiling of deep-sea sediments from the Gulf of Mexico following the Deepwater Horizon oil spill. Front Microbiol 2013, 4:50. 47. McInerney MJ, Sieber JR, Gunsalus RP: Syntrophy in anaerobic global carbon cycles. Curr Opin Biotechnol 2009, 20:623-632. 48. Callaghan AV, Morris BEL, Pereira IAC, McInerney MJ, Austin RN, Groves JT, Kukor JJ, Suflita JM, Young LY, Zylstra GJ, Wawrik B: The genome sequence of Desulfatibacillum alkenivorans AK01: a blueprint for anaerobic alkane oxidation. Environ Microbiol 2012, 14:101-113. 49. Gray ND, Sherry A, Grant RJ, Rowan AK, Hubert CRJ, Callbeck CM, Aitken CM, Jones DM, Adams JJ, Larter SR, Current Opinion in Biotechnology 2014, 27:46–54
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This large-scale survey of almA genes showed that they are widespread in marine and coastal environments and in alkane-degrading genera, which adds to the evidence suggesting that AlmA is the main enzyme involved in degradation of long-chain alkanes. 65. Pearson A, Kraunz KS, Sessions AL, Dekas AE, Leavitt WD, Edwards KJ: Quantifying microbial utilization of marsh sediments by using the C-13 petroleum hydrocarbons in salt content of bacterial rRNA. Appl Environ Microb 2008, 74:11571166. 66. Coulon F, Chronoupolou P-M, Fahy A, Paı¨sse´ S, Gon˜i-Urriza MS, Peperzak L, Acun˜a-Alvarez L, McKew BA, Brussard C, Underwood GJC et al.: Central role of dynamic tidal biofilms dominated by aerobic hydrocarbonoclastic bacteria and diatoms in the biodegradation of hydrocarbons in coastal mudflats. Appl Environ Microbiol 2012, 78:3638-3648. This study highlights the significance of hydrocarbon biodegradation by Alcanivorax borkumensis and other oil-degraders attached to tidal biofilms containing oxygenic phototrophic diatoms. It also demonstrates niche partitioning in Alcanivorax species, with sediment and tidal-biofilm ecotypes. 67. Kostka JE, Prakash O, Overholt WA, Green SJ, Freyer G, Canion A, Delgardio J, Norton N, Hazen TC, Huettel M: Hydrocarbon-degrading bacteria and the bacterial community response in Gulf of Mexico beach sands impacted by the Deepwater Horizon oil spill. Appl Environ Microbiol 2011, 77:7962-7974. 68. Teramoto M, Suzuki M, Okazaki F, Hatmanti A, Harayama S: Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic hydrocarbons in the tropical marine environment. Microbiology 2009, 155: 3362-3370.
56. Liu C, Wang W, Wu Y, Zhou Z, Lai Q: Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B5. Environ Microbiol 2011, 13:1168-1178.
69. Al-Bader D, Eliyas M, Rayan R, Radwan S: Subsurface associations of Acaryochloris-related Picocyanobacteria with oil-utilizing bacteria in the Arabian Gulf water body: promising consortia in oil sediment bioremediation. Microb Ecol 2013, 65:555-565.
57. Kanaly RA, Harayama S: Advances in the field of high molecular-weight polycyclic aromatic hydrocarbon biodegradation by bacteria. Microb Biotechnol 2010, 3:136-164.
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59. Harms H, Schlosser D, Wick LY: Untapped potential: exploiting fungi in bioremediation of hazardous chemicals. Nat Rev Microbiol 2011, 9:177-192. This review highlights the value of fungi, not only in degradation of organic pollutants, but also their role as a conduit for bacteria to access pollutants. 60. Kweon O, Kim S-J, Holland RD, Chen H, Kim D-W, Gao Y, Yu LR, Baek S, Baek D-H, Ahn H, Cerniglia CE: Polycyclic aromatic hydrocarbon metabolic network in Mycobacterium vanbaalenii PYR-1. J Bacteriol 2011, 193:4326-4337. Catabolism of high molecular weight PAHs has received scant attention, but by this systems-biology approach the authors have provided a detailed understanding of the network of interactions involved. 61. Vilchez-Vargas R, Junca H, Pieper DH: Metabolic networks, microbial ecology and ‘omics’ technologies: towards understanding in situ biodegradation processes. Environ Microbiol 2010, 12:3089-3104. 62. Jeon CO, Madsen EL: In situ microbial metabolism of aromatichydrocarbon environmental pollutants. Curr Opin Biotechnol 2013, 24:474-481. 63. Paı¨sse´ S, Gon˜i-Urriza MS, Coulon F, Duran R: Are alkane hydroxylase genes (alkB) relevant to assess petroleum bioremediation processes in chronically polluted coastal sediments? Appl Microbiol Biotechnol 2011, 92:835-844. 64. Wang WP, Shao ZZ: Diversity of flavin-binding monooxygenase genes (almA) in marine bacteria capable of degradation long-chain alkanes. FEMS Microbiol Ecol 2012, 80:523-533.
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ScienceDirect Hydrocarbon biodegradation in intertidal wetland sediments Terry J McGenity Intertidal wetlands, primarily salt marsh, mangrove and mudflats, which provide many essential ecosystem services, are under threat on numerous fronts; a situation that is made worse by crude-oil pollution. Microbes are the main vehicle for remediation of such sediments, and new discoveries, such as novel biodegradation pathways, means of accessing oil, multispecies interactions, and community-level responses to oil addition, are helping us to understand, predict and monitor the fate of oil. Despite this, there are many challenges, not least because of the heterogeneity of these ecosystems and the complexity of crude oil. For example, there is growing awareness about the toxicity of the oxygenated products that result from crude-oil weathering, which are difficult to degrade. This review highlights how developments in areas as diverse as systems biology, microbiology, ecology, biogeochemistry and analytical chemistry are enhancing our understanding of hydrocarbon biodegradation and thus bioremediation of oilpolluted intertidal wetlands. Addresses School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK Corresponding author: McGenity, Terry J ([email protected])
Current Opinion in Biotechnology 2014, 27:46–54 This review comes from a themed issue on Environmental biotechnology Edited by Hauke Harms and Howard Junca
0958-1669/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.copbio.2013.10.010
erosion caused by oil-induced destruction of salt-marsh plants [3]. The acute/chronic, direct/indirect impacts of oil pollution on salt-marsh ecosystems and the services they provide are nicely reviewed [1]. The fine-grained sediment of intertidal wetlands is testament to the low tidal and wave energy, which is often sufficient to carry oil slicks onto the shore, but after trapping by sediment and vegetation, is too low to flush away the oil. The abundance of clay minerals coupled with high organic loads encourage sorption of the most hydrophobic hydrocarbons, and sedimentary accretion and bioturbation can result in burial of hydrocarbons in zones of low redox potential. Hydrocarbons have been found in fine-grained coastal sediments many decades after a spill due to slow anaerobic biodegradation, for example the Florida spill at West Falmouth, MA [4] (Figure 1). However, these hydrocarbons can become accessible by processes such as resuspension, bioturbation, erosion and dredging. Such accessibility is a doubleedged sword – increasing the potential for biodegradation while enhancing the risk to the biota [5]. Microbes have had millions of years to adapt to the stress imposed by hydrocarbons and to exploit their ‘fuel value’ [6], resulting in an array of microorganisms from the three domains of life that have evolved to use them as a source of both energy and carbon [7–9]. Before focussing on (microbial) ecology, which underpins bioremediation, it is important to consider hydrocarbon bioavailability and catabolism, which help us to understand when and where degradation of different hydrocarbons can occur, informs remediation strategies, and provides important tools (e.g. in the form of oligonucleotide primers and metabolites) that allow biodegradation processes to be studied in situ.
Introduction
Bioavailability of crude oil
Intertidal wetlands, in particular temperate-zone salt marshes, tropical mangrove and mudflats are environments with high primary productivity that provide many important environmental, economic and societal benefits, such as protection from wave and storm damage, removal of excess nutrients from water, provision of food and habitats for fish, livestock and diverse wildlife [1], and they are generally considered to be a net sink for greenhouse gases [2]. However, they are very susceptible to oil pollution, owing to their frequent proximity to river outflows, oil refineries and industrial plants, as well as receiving marine-derived oil from shipping, natural seeps, oildrilling and blow-outs (Figure 1). These globally important habitats, already threatened by sea-level rise and diverse human activities, are made more susceptible to
More than 10,000 hydrocarbons and other molecules are present in crude oil, with diverse physico-chemical properties and correspondingly disparate stress effects and potential for biodegradation (Figure 2). With increasing carbon number, hydrocarbons are less bioavailable, volatile and soluble (more hydrophobic), and tend to adsorb to surfaces more readily, although the degree of aromaticity, ring positions and branching may cause deviations from this general rule [10]. Microbes have evolved numerous mechanisms to enhance bioavailability. First and foremost is the capacity to uptake and degrade the hydrocarbon thereby lowering the concentration in the immediate vicinity of the cell and driving diffusive flux of poorly soluble compounds towards the cell [10]. Second is the ability to increase the surface area
Current Opinion in Biotechnology 2014, 27:46–54
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Oil biodegradation in intertidal wetlands McGenity 47
Figure 1
Sea Empress 1996 72,860 t Exxon Valdez * 1981 38,500 t
Deepwater Horizon 2010 530,000 t
Amoco Cadiz 1978 227,000 t Hebei Spirit 2007 10,500 t
Erika 1999 19,800 t
Florida 1969 550 t Tank rupture 1986 7000 t
Arabian Gulf
Prestige* 2002 63,000 t
Solar 2002 800 t
Niger Delta
Katina 1992 72,000 t Metula 1974 51,000 t Current Opinion in Biotechnology
Location of the world’s salt marshes and mangroves, and selected crude-oil spills that have affected them. Salt marsh (green), dominated by shrubs and grasses, occur at high latitudes, whereas mangrove (blue), dominated by trees, occur in tropical and subtropical zones. Regions with both types of intertidal wetland (brown) are found in some subtropical regions, such as the Gulf of Mexico. Mudflats are more seaward unvegetated zones, rich in microbial mats, often associated with salt marsh and mangrove. The figure, from [76], is indicative only, and excludes many smaller islands fringed by mangrove. The name of the ship involved in the oil spill (or blowout information) is given italics, followed by the date of the spill and the amount released in tons. Those spills marked with * were large but had only a relatively minor impact on salt marsh and mangrove. The two locations marked with a gold star are the scenes of countless spills: the Arabian Gulf, which, in addition to shipping accidents, was the site of the largest release of oil into the sea, during the Gulf War. In the Niger Delta, oil exploration and transport have left a legacy of large-scale habitat destruction, especially of mudflats and mangrove, and ecological and health problems that exceed those caused by the Deepwater Horizon spill [77]. The oil spill data was derived from http:// mapsof.net/map/oil-spills-world-map-fr and [1].
of the bulk-phase hydrocarbon available to the microbe by synthesis of emulsifying agents/biosurfactants [10,11,12]. Thereafter, many hydrocarbon-degrading microbes modify their cell envelope to enable attachment to hydrocarbons (see [11]). In particular, experimental proteomic studies show that Marinobacter hydrocarbonoclasticus is well adapted to forming a biofilm on alkanes, accumulating carbon reserves in the form of intracellular wax esters, which can then fuel detachment and movement to other locations, where detached cells have acclimated to reattach to fresh alkane resources [13]. Alcanivorax borkumensis is similarly well adapted to life in a biofilm [14,15] (Figure 3), and transcriptomic analysis has revealed outer-membrane lipoprotein Blc as a candidate for the enigmatic process of alkane uptake [14]. In a few microbes, sensing of, and chemotaxis towards, hydrocarbons has been shown to enhance bioavailability and biodegradation [16] and the alkane-sensory system has recently been described in Alcanivorax dieselolei strain B-5 [17], but hydrocarbon-taxis is undoubtedly much more widespread. www.sciencedirect.com
Fate of oil in the open sea and intertidal wetlands The chemical composition and physical nature of crude oil evolve when released into the sea (a process referred to as weathering), depending on winds, waves, water currents, oil type, light exposure, nutrient availability and temperature (see [11]), with implications for its fate. The main process responsible for mineralisation of crude oil hydrocarbons is microbial biodegradation [18,19]. Nevertheless, oil frequently reaches intertidal zones with different degrees of modification depending on the oil source, prevailing environmental conditions and proximity of the spill to land. The oil spill on 10 April 2010 caused by the explosion at the Deepwater Horizon rig (herein referred to as DWH spill) released 636 million litres of Macondo (Light Louisiana) crude oil into the Gulf of Mexico over 3 months [20] – a disaster that spurred a large-scale remediation and research effort. Most models of the fate of oil in the sea are based on surface spills, but further complexities are introduced when oil derives from the deep sea Current Opinion in Biotechnology 2014, 27:46–54
48 Environmental biotechnology
Figure 2
Crude oil n-hexane
Precipitates
Dissolves Column separation
toluene Dissolves
n-hexane Saturates (15-60%)
toluene / methanol
toluene Aromatics (3-30%)
Resins
Asphaltenes (mean 6%) CH3
S
CH3
CH3
S
R (CH2)nCO2H
N CH3 S CH3
Cyclo-alkanes (naphthenes), branched and straight-chain alkanes (paraffins). Shown are (left to right): cyclohexane, n-decane, pristane.
Mono- and polycyclic aromatic hydrocarbons, including alkylated forms. Shown are (left to right): benzene, methylnaphthalene, benzo [a]pyrene.
Polar & heterocyclic N, S & O-containing hydrocarbons. Shown are (top to bottom): dibenzothiophene, naphthenic acid (generic structure of an alkyl-substituted alicyclic carboxylic acid).
CH3
Large, heterocyclic N, S & O-containing compounds, with traces of heavy metals such as vanadium. The structure shown is illustrative.
Current Opinion in Biotechnology
Operational classification of crude-oil components based on the SARA system (Saturates-Aromatics-Resins-Asphaltenes), with some representative structures. The SARA system, shown here and modified from [78], provides a useful operational breakdown of crude oil. Alkanes, including straightchain, branched and cyclic, generally constitute the main mass of oil, followed by PAHs. Several descriptors exist, such as ‘light’ and ‘heavy’, for crude oils containing low-molecular-weight alkanes and polycyclic aromatic hydrocarbons (PAHs), compared to those consisting of high-molecular-weight hydrocarbons (>C20), resins and asphaltenes, respectively. Naphthenic acids (comprising mainly of alkyl-substituted alicyclic carboxylic acids), are a major and highly toxic component of oil-sand process-affected waters, and also constitute 1% of crude oils and up to 4% of North-Sea crude oils [79].
and is rich in gaseous hydrocarbons and water-soluble aromatics, such as benzene, toluene, xylene, ethylbenzene (BTEX) [21]. Reddy et al. [21] detected little biodegradation of BTEX in the early plume, supported by metatranscriptomic data [22]; while Hazen et al. [23] reported half lives of n-alkanes of 1 to 6 days at the deepsea temperature of 5 8C, and implicated a new group in the order Oceanospirillales (94% 16S rRNA sequence similarity to Oleispira antarctica) as the main enriched microbe. Further single-cell genomic analysis revealed that this dominant bacterium had genes for alkane and cycloalkane degradation that were expressed in the plume [22]. Other abundant microbes in the plume are Current Opinion in Biotechnology 2014, 27:46–54
the pyschrophilic short-chain-alkane-degrading Colwellia and PAH-degrading Cycloclasticus spp. [24]. Despite deep-sea degradation and other attenuating processes, slicks appeared on the sea surface and oil reached the Gulf-of-Mexico shoreline. Tar balls and water-in-oil emulsions (chocolate mousse), which are difficult to degrade because of their high viscosity and reduced surface area, were particularly common after the DWH spill. By comparing original Macondo oil to samples taken 500 days after the spill from beaches surrounding the Gulf of Mexico, Aeppli et al. [25] found that the proportion of oxygenated hydrocarbons increased tenfold, forming the www.sciencedirect.com
Oil biodegradation in intertidal wetlands McGenity 49
Figure 3
alkS
alkB1 alkG alkH
alkJ 0.5 µm
AlkB OH Current Opinion in Biotechnology
Hydrocarbon degradation in intertidal wetlands: from genome to ecosystem. Gene information, for example from the alkane hydroxylase gene (alkB) cluster in Alcanivorax borkumensis; one of several over-expressed in the presence of alkanes [14], informs on mechanisms of degradation (e.g. terminal oxidation of alkanes [54]) and vice versa. A. borkumensis must first access hydrocarbons and transport them across the membrane – the image shows a small (1 mm diameter) oil droplet emulsified by surrounding cells of A. borkumensis (copyright: Dr. Jessica Beddow). Populations of A. borkumensis form part of a wider community, as illustrated by this false-colour image showing photosynthetic organisms, in this case diverse benthic diatoms, on the surface of a mudflat in which A. borkumensis and other microbes were degrading hydrocarbons (field of view is 60 mm 40 mm). These benthic phototrophs probably contribute to hydrocarbon degradation by generating oxygen [15]. The image of a salt-marsh tidal creek connected to the North Sea (copyright: Prof. Graham Underwood) demonstrates the heterogeneity of the habitat, particularly the different degrees and types of algal and plant cover. Vertical heterogeneity (not shown) is equally pronounced, with oxygen diffusing no more than a few millimetres below the surface and different TEAs becoming available at depth. Finally, the understanding and knowledge acquired in less complex bio-systems provide complementary tools for investigating the ecology and biogeochemistry of ecosystems.
majority of hydrocarbons in heavily weathered samples. Most oxygenated hydrocarbons derived from partial biodegradation and photo-oxidation rather than simply an enrichment of these difficult-to-degrade compounds. Advanced analytical tools such as two-dimensional gaschromatography and direct immersion ionization techniques together with ultra-high resolution mass spectrometry are beginning to shed further light on the composition of such compounds [26]. Investigating the fate of such seemingly recalcitrant and toxic crude-oil components should be a priority of future research, as the oxidation of aromatic hydrocarbons with transition metals entrained in tar balls can result in toxic environmentally persistent free radicals, some of which have only recently been identified [27]. www.sciencedirect.com
Bioavailable PAHs in the water column at four coastal locations increased 7–14 weeks after the DWH spill, with PAHs persisting at elevated levels for a year at one location [28]. Other studies have shown how Macondo oil, enriched in high-molecular-weight hydrocarbons, increased the organic load by one to two orders of magnitude at several centimetres depth in sediments months after the spill, while surface water concentrations were the same as in pristine sites [29], thereby emphasising that intertidal finegrained sediments are major collecting points for spilled oil that will be impacted over the long term. The volume of the dispersant, COREXIT-9500, added to increase the surface-area-to-volume ratio of DWH oil, was almost the same as the crude oil itself (see [28]). The Current Opinion in Biotechnology 2014, 27:46–54
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balance of evidence suggests that such action reduced the amount of oil washed ashore [30,31,32], and there is good evidence that the surfactant within COREXIT is biodegraded in the sea [32]. However, potential disadvantages of using dispersants in the coastal zone must be considered; for example, sediment-column experiments showed that COREXIT (6 ppm) increased the mobility of PAHs from crude oil by up to two orders of magnitude in saturated permeable sediments [33]. If COREXIT also accelerates PAH penetration into fine-grained sediments, PAHs would persist owing to slower biodegradation in the anaerobic zone.
Anaerobic hydrocarbon biodegradation – from genetics and biochemistry to ecology and biogeochemistry Fine-grained intertidal sediments have steep vertical gradients of terminal electron acceptors (TEAs), with oxygen often absent below the top millimetre [34]. Hydrocarbon biodegradation has been demonstrated with all of the most common TEAs (nitrate, iron III, sulphate) and under methanogenic conditions, but the mechanisms by which the apolar and very stable C–H bond is activated in the absence of molecular oxygen have been much debated [6]. Direct carboxylation appears to be the main activation mechanism for non-alkylated aromatic hydrocarbons, although alternative mechanisms such as methylation followed by addition to fumarate are feasible [35]. For both alkyl-substituted aromatics and alkanes, the addition to fumarate, catalysed by glycyl-radical-containing enzymes, is the primary mode of hydrocarbon activation [36,37]. However, absence of alkyl succinate metabolites suggests that this may not be the case for alkanes under methanogenic conditions [38]. Also, gammaproteobacterial strain HdN1 has been proposed to employ nitrite (NO2 ) to activate alkanes while using nitrate as a TEA [39]. It is also feasible that dismutation of NO (from NO2 ) generates O2 (and N2), with the resultant oxygen activating the alkanes [40]. The presence of oxygenase genes in strain HdN1, and more complete evidence for this mechanism being responsible for oxidation of methane coupled to nitrate reduction, lend supports to the ‘dark oxygen mechanism’, a process akin to that proposed for hydrocarbon-oxidising (per)chlorate-reducing bacteria [39,40]. In marine sediments, where sulphate is abundant, sulphate-reducing bacteria (SRB) are commonly implicated in hydrocarbon degradation. Many pure cultures of hydrocarbon-degrading SRB have been reported [41]. Also, crude oil addition frequently leads to enhanced sulphate reduction in intertidal sediments and microcosms [29,42– 44], and addition of inhibitors of sulphate-reduction prevents hydrocarbon degradation [44]. The improved identification of signature metabolites from fumarateaddition pathways [37], and the understanding of enzyme diversity, for example benzyl succinate synthase (BSS), Current Opinion in Biotechnology 2014, 27:46–54
are providing important tools for identification and quantification of the relevant processes in the environment [45]. Comparison between deep-sea sediments close to the DWH blow-out and a remote uncontaminated sediment revealed an increase in: Deltaproteobacteria (including genera of SRBs), genes coding for enzymes with potential for both aliphatic and aromatic hydrocarbon activation via fumarate addition, and of anaerobic hydrocarbon metabolites [46]. While such studies implicate SRBs in hydrocarbon degradation and show that anaerobic degradation of hydrocarbons is occurring, it is sometimes difficult to disentangle whether enhanced SRB activity is due to their direct involvement in hydrocarbon degradation or an indirect effect, that is, resulting from hydrocarbon-induced cell lysis and release of organic matter. SRBs are not necessarily the primary ring-opening microbes in situ; they form syntrophic partnerships whereby their consumption of fermentation products makes the preceding reactions more energetically favourable [6,47]. To further confound interpretation, some SRBs switch metabolism to act as syntrophs, for example in cooperation with methanogens in medium lacking sulphate [47,48]. Members of the genus Smithella (Deltaproteobacteria in the family Syntrophaceae) have been implicated as the key anaerobic alkane-degrading syntrophs in association with methanogens in estuarine enrichments [49]. The perception of anoxic horizons beneath oxic zones in intertidal sediments is an over-simplification. Bioturbation introduces oxygen deeper into sediments. Anoxic microsites exist in surface layers. Light-induced variations in oxygenic photosynthesis result in differential diel oxygen exposure, further complicated by tidal cover that affects oxygen diffusion into the sediment. Thus, aerobes and anaerobes co-habit, facultative anaerobes abound, and the effect of all of these processes on in situ and ex situ bioremediation is starting to be considered [50]. In microcosms derived from marine sediments, hydrocarbon degradation was significantly enhanced by sequential anaerobic–aerobic degradation (via sulphate reduction in the anaerobic step), compared to degradation under purely aerobic or anaerobic conditions [51]. In situ, redox oscillations facilitate nutrient and TEA regeneration, and may serve to encourage more efficient cycling of metabolites [50]. It is worth speculating on other processes that may influence hydrocarbon consumption. Pfeffer et al. [52] proposed that cm-long filamentous Desulfobulbaceae bacteria, vertically orientated in marine sediments, used sulphide as an electron donor in the anoxic zone and transported electrons through their periplasm to oxygen (the TEA) in the upper sediment. Consumption of toxic sulphide is likely to benefit many (including anaerobic www.sciencedirect.com
Oil biodegradation in intertidal wetlands McGenity 51
hydrocarbon-degrading) microbes. It is conceivable that electron-shuttling microbes have a direct role in taking electrons from buried hydrocarbons – thereby using anoxic activation coupled with aerobic degradation. After all, representatives of the Desulfobulbaceae possess benzylsuccinate synthase a-subunit (bssA) genes [53].
Aerobic hydrocarbon biodegradation – from genetics and biochemistry to ecology and biogeochemistry Oil tends to float and so, at least in the early stages of a spill, remains in the sediment’s oxic zone. Biodegradation of hydrocarbons is faster under oxic compared with anoxic conditions [6], and aerobic hydrocarbon degradation is the prevalent process in salt-marsh sediments [42]. Oxygen not only serves as a high-energy-yielding TEA, but is also a reactant, which, catalysed by oxygenases, activates hydrocarbons [6]. There exists a lot of information about the variety and structure of oxygenases, their associated enzymes, the nature of the activation products and their pathways to central metabolism, as well as regulatory and transport mechanisms [17,54,55]. Essentially, for lowmolecular-weight alkanes (C2 to C5), soluble non-haem iron monooxygenases are employed, while for mid-range alkanes (C6 to C20), microbes use integral-membrane non-haem iron monooxygenases related to AlkB (alkane hydroxylases) and the haem-containing cytochrome P450 enzymes [54]. Far less is known about the mechanisms of degradation of long-chain alkanes (i.e. with >20 carbons), and diverse underlying biochemistries seem to be involved [17,54]. The flavin-binding monooxygenase, AlmA, for example, is found in many hydrocarbonoclastic genera such as Acinetobacter and Alcanivorax [17,54,56]. The differential expression of multiple alkane-degrading enzymes in the same species is a recurring theme [14,17,19,54,56]. Relative increased gene expressions for Alcanivorax dieselolei, for example, are: p450 with C8–C16 alkanes, almA with C22–C36, alkB1 and alkB2 with C12–C26, and both alkB1 and almB with branched alkanes [56]. Parallel developments in understanding aerobic degradation of aromatic hydrocarbons are emerging [55], particularly for high-molecular-weight PAHs (i.e. with more than three aromatic rings) that are very poorly soluble, adsorb to sediment particles and are highly toxic [57]. Their degradation by fungal extracellular nonspecific enzymes, principally produced to initiate breakdown of complex biopolymers, is well known [58,59]. But our understanding of bacterially mediated catabolism of high-molecular-weight PAHs has been advanced by a systems-biology-based investigation of Mycobacterium vanbaalenii PYR-1, originally isolated from oil-contaminated estuarine sediment, which resulted in a metabolic network starting with 7 PAHs, leading to 224 chemical reactions linking 183 metabolites ultimately funnelled into the central b-ketoadipate pathway [60]. www.sciencedirect.com
Fundamental knowledge about aromatic and aliphatic hydrocarbon catabolic pathways is important for understanding the ecology of oil-polluted intertidal wetlands [61,62] (Figure 3), because environmental gene analysis and quantification currently rely on a limited number of genes, mostly alkB for aliphatics, which may not always provide a faithful picture of the alkane-degrading potential or capacity in the environment, as was found when rates of degradation and gene expression were compared in coastal sediment microcosms [63]. The application of degenerate primers based on almA genes demonstrated their presence in several genera of coastal marine bacteria enriched on crude oil, especially Alcanivorax and Marinobacter, and two distinct phylogenetic groups [64]. Novel and iterative approaches [61,62] with isolates, consortia and natural communities will give a more complete understanding of pathways and networks, and capture the full diversity within and beyond gene families. When oil lands on a salt marsh it is useful to discern the microbial activity due to petroleum consumption compared with consumption of other organic matter, such as plant detritus. Pearson et al. [65] took advantage of the difference in d13C of the dominant plant, Spartina alterniflora ( 13%) and crude oil ( 27%), by isotopic analysis of rRNA from two-week incubated sediment samples, to reveal that 26% of the assimilated carbon derived from oil. Surprisingly, given their prevalence in oil-polluted marine habitats [18,19], Alcanivorax and Cycloclasticus were not found, whereas Idiomarina 16S rRNA genes were abundantly detected in the oiled sediment [65]. Intertidal mudflat cores, subjected to crude oil and a simulated tide, revealed an increase in aerobic obligately hydrocarbonoclastic bacterial genera (notably Alcanivorax, Cycloclasticus, Oleibacter and Oceanospirillales ME113-related sequences) that constituted 7.8% of sequences in the oiled sediment after 12 days [66]. Two of these genera were also abundant in biofilms that detached from the sediment and floated on the water surface on day 22 (Alcanivorax and Oceanospirillales ME113 constituted 48.5% and 10.6% of sequences, respectively). This enrichment of aerobic hydrocarbonoclastic bacteria in tidal biofilms, which also consisted of diatoms, suggests that they are active zones of hydrocarbon degradation. Thus, aerobic hydrocarbonoclatic bacteria, such as Alcanivorax and Cycloclasticus, are important in fine-grained intertidal sediments, just as they are in sandy sediments [18,67] and the open sea [18]. However, they are not always detected, and the roles of other microbes, both uncultivated and cultivated, for example Oleibacter sp. [66,68], should be investigated. Also, there is increasing awareness that interactions between oxygenic phototrophs and hydrocarbonoclastic bacteria may be important for hydrocarbon degradation [11,15,66,69], and hydrocarbons, like isoprene, released from marine and coastal microalgae may even Current Opinion in Biotechnology 2014, 27:46–54
52 Environmental biotechnology
contribute to the survival of such hydrocarbonoclastic bacteria in the absence of an oil spill [70].
in Louisiana salt marshes after the BP Deepwater Horizon oil spill. Proc Natl Acad Sci USA 2012, 109:11234-11239. 4.
Bioremediation A key question that arises from the foregoing discussion is whether to clean up intertidal fine-grained sediments. Many (e.g. [71]) argue that the best strategy is not to intervene but allow the native microbes to perform the task. Nutrient addition, although successful in other environments [18,30], has limited impact in fine-grained sediments [71,72], in which oxygen rather than nutrients is often the limiting factor. While introduction of oxygen would stimulate biodegradation of buried hydrocarbons [5], the overall environmental impact together with disruption of the chemocline and phototrophic interactions with hydrocarbonoclastic bacteria would be detrimental. If bioremediation is to be considered, the role of environmental factors influencing hydrocarbon degradation have to be accounted for, especially temperature [73] and salinity [7]. Bioaugmentation, although largely disregarded owing to poor performance in the past [71], is receiving renewed interest, and with relevant indigenous microbes and carrier materials, may be valuable for accelerating biodegradation of fresh, near-shore oil spills, thereby reducing the quantity of oil reaching the coast. When salt-marsh and mangrove plants have been so heavily oiled that they are unlikely to recover, phytoremediation offers a means to stabilise the sediment. Additionally, some plants and their associated microbes have led to increased oil biodegradation in trials, for example Scirpus maritimus in salt marshes [74] and Avicennia schaueriana in mangrove [75]. Naturally, due consideration must be given to the impact on the local ecology, and native mixtures of species should be used.
Acknowledgements Funding from the UK Natural Environmental Research Council (NERC) is gratefully acknowledged, particularly grants: NE/J01561X/1 (a hierarchical approach to the examination of the relationship between biodiversity and ecosystem service flows across coastal margins) and NE/J009555/1 (microbial degradation of isoprene in the terrestrial environment).
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