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Chapter 18 Bioremediation of marine oil spills
Studies in Surface Science and Catalysis 151 R. Vazquez-Duhalt and R. Quintero-Ramirez (Editors) © 2004 Published by Elsevier B.V.
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Chapter 18
Bioremediation of marine oil spills R. C. P r i n c e ~ a n d J. R. C l a r k 2
~ExxonMobil Research & Engineering Co. Annandale, NJ 08801 2ExxonMobil Research & Engineering Co. Fairfax, VA 22037
1. I N T R O D U C T I O N Crude oils are the liquid fossil residues of aquatic algae, sometimes with minor contributions from terrestrial plants, that grew in the distant past. Then as now, we can imagine that most of this material was biodegraded and recycled on an essentially annual timescale, but a small fraction became buried and underwent diagenesis and catagenesis to become oil [1]. This process usually took millions of years, and was dependent on the depth of burial and the temperature. Some oil dates from the Precambrian (>570 million years ago), but most is rather younger; the average age of commercially important oil is about 100 million years, with the majority being from the Jurassic and Cretaceous (180 to 85 million years ago) [2]. Commercially important oil has migrated from its source rock to a reservoir, and it is not unusual for these reservoirs to leak. If the leak reaches the surface, it is known as an oil seep. Humans have used material from such seeps for thousands of years. Early uses include hafting stone axes to their handles, as an embalming agent, and as a medical nostrum. Genesis (11,3) says that bitumen was used as the mortar for the Tower of Babel, and Exodus (2,3) that Moses' basket was made waterproof with a bitumen daub. It seems likely that several religions started around natural gas seeps, either as eternal flames or as sources of hallucinogenic vapors [3]. But these were only very minor uses, and it is only in the last century and a half that oil has come to play a truly central role in modem society [4]. Terrestrial seeps were the first locations to be drilled when oil production began in earnest in the nineteenth century, such as the 1860 Drake well in Pennsylvania, but marine seeps were drilled by the end of the century.
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Marine seeps are widespread, as shown in Figure l, and are a major source of oil into the World's oceans [5]. Even with today's enormous commerce in oil, seeps provide about 62% of the total releases into the coastal marine environment of North America, and 47% of the world. Such seeps must have been occurring for millions of years, providing an important input of degradable carbon for local ecosystems and perhaps even major fisheries [6]. A diverse group of microorganisms exploits this natural input. Oil-degrading microbes have been found in all marine environments where they have been looked for, and more than seventy genera of eubacteria and archaea, and a hundred genera of fungi, have been shown capable of degrading petroleum hydrocarbons [7]. It is these organisms that remove oil seepage and spilled oil from the marine and terrestrial environment, and underpin the bioremediation strategies for dealing with spilled oil that we will describe here. 2. A N T H R O P O G E N I C INPUT OF OIL INTO THE W O R L D ' S OCEANS Oil fuels the modem world on an enormous scale; annual consumption is of the order of 3.5 billion US gallons (1.2 x 10 ~~ liters) per day [8]. Much of this is produced at sea; more than 25% of US production is from the Continental Shelf, and 25% of Saudi Arabia's production, 80% of Nigeria's, and 100% of Angola's, Australia's, Brazil's and Malaysia's production is offshore. This production is associated with some oil and grease discharges into the marine environment associated with the produced water; some 2,700 tonnes per year in the US, and more than ten times this in the rest of the world [5]. This input is dwarfed, however, by oil in municipal run-off from the land, and from the standard operation of marine shipping (Figure 2). Catastrophic spills from tankers and other ships are well known, but in fact their contribution to total oil input into the oceans is relatively small, some 8% of the global input, 2% of North American input. Nevertheless, since such spills are large on the local scale, they often require environmentally appropriate and cost-effective responses. Fortunately, despite the increasing volume of transported oil, the amount spilled from catastrophic spills has been generally decreasing over the last few decades [9]. The major exception to this decline was the appalling environmental crime in the Arabian Gulf in 1991, where Iraqi forces deliberately released more than a million tonnes (about 260 million gallons or a billion liters) of oil into the sea near Kuwait [ 10]. An additional 350 million gallons (1.2 x l 09 1) were deposited in the Gulf as fallout from the smoke plumes of the > 700 oil well fires in the Kuwait oil fields [ 11 ], making this by far the largest man-made release of oil into the marine environment to date.
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3. COMPOSITION OF CRUDE OIL
Crude oils are complex mixtures of hydrocarbons with significant quantities (typically about 15%) of compounds containing additional heteroatoms such as oxygen, sulfur or nitrogen. The hydrogen to carbon ratio of the hydrocarbons is typically between 1.5 and 2, indicating a mixture of aliphatic (predominant carbons are -CH2-, either as linear molecules, known as paraffins, or as rings known as naphthenes) and aromatic species (principal carbons are-HC=CH - in rings). Alkenes and alkynes, linear unsaturated molecules, are rare in crude oils, although they can be abundant in some refined products such as gasoline. Tissot and Welte [2] calculate the average composition of more than 525 crude oils as 58.2% saturates, 28.6% aromatics and 14.2% polar compounds, noting that the absolute values vary widely in different oils. On average, there is rough parity between paraffins, naphthenes and aromatic hydrocarbons in crude oils [2]. Paraffins in crude oils may start with methane, and extend to waxes with more than seventy carbons. Their total content varies widely, from essentially undetectable to as high as 35%, depending on source and reservoir conditions, but they typically make up 15-20% of an undegraded crude oil. There are also branched alkanes; especially in the C6 to C8 range, but pristane (C19H40) and phytane (C20H42), molecular relics of the phytol chains of chlorophylls and perhaps other biomolecules, are usually the most abundant individual branched alkanes. Pristane is thought to be the result of initial partial degradation of phytol in the presence of oxygen, while phytane is thought to be the result of diagenesis in the absence of oxygen [2].
Marine oil seeps
Fig. 1. Map of the major oil seeps into the World's oceans. Data taken from reference [5].
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Annual input of oil to the seas World
North America
Natural Seeps Extraction !--I Transportation Consumption
347 million gallons (US)
69.4 million gallons (US)
Fig. 2. Oil input into the World's oceans. Data taken from reference [5]. The naphthenes include parent compounds, such as cyclopentane, cyclohexane and decalin, together with their alkylated congeners. Tissot and Welte [2] quote the average composition of the naphthene fraction of 299 crude oils as 54.9% one and two ring naphthenes, 20.4% tricyclic naphthenes, and 24.0% tetra and pentacyclic naphthenes. These latter molecules are amongst the better understood molecular biomarkers in crude oils, and they are used extensively in correlating reservoirs and source rocks [12], in assigning the depositional environment of source rocks [12], and more recently as conserved internal markers during biodegradation [ 13]. Because of the separation procedures used in the analysis of crude oils, any molecule containing at least one aromatic ring is included in the "aromatic" fraction, regardless of the presence of saturated rings and/or alkyl substituents. Aromatic heterocycles containing sulfur, such as thiophenes, benzothiophenes and dibenzothiophenes, or nitrogen, such as the indoles, carbazoles and quinolines, also fall into the aromatic category. Alkylated aromatic species are more abundant than their parent compounds, with mono-, di- and tri-methyl derivatives usually being most abundant. Nevertheless, the median aromatic structure probably has one or two methyl groups and a long-chain alkyl substituent [ 14]. The polar molecules are the most difficult to characterize because they typically cannot be analyzed by gas chromatography, the method of choice for the molecular characterization of hydrocarbons. Petroleum polar compounds contain heteroatoms such as nitrogen, oxygen and/or sulfur, and the category includes the porphyrins, typically with nickel or vanadium in the tetrapyrole,
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naphthenic acids and large molecules known as asphaltenes. Some asphaltenes have molecular weights into the thousands and even higher, and many are suspended in the oil rather than dissolved in it [15]. The polar fraction of the oil contains the majority of the color centers in crude oil, and in isolation these materials are difficult to distinguish from more recent biological residues, such as the humic and fulvic acids [16], except with sophisticated tools such as isotope analysis. Recent developments in electrospray mass spectrometry promise significant progress in determining the molecular structure of these compounds [ 17, 18]. Several hundred different crude oils are being produced today, and their chemical composition and properties vary quite widely. They are typically classified by their density. The oil industry uses a unit known as API (American Petroleum Institute) gravity, which is defined as [142.5/(specific gravity)] 131.5, and expressed as degrees (~ Thus water has an API gravity of 10 ~ and denser fluids will have lower API gravities. For convenience, oils with API gravities greater than 40 ~ are said to be light oils, while those with API gravities of less than about 17 ~ are said to be heavy. Note that even these typically float on water, especially seawater. Light oils have higher proportions of hydrocarbons; heavy oils are rich in polars and asphaltenes. Viscosity is roughly inversely proportional to API gravity, but it is also dependent on the physical state of the polar compounds and longer alkanes in the oil, and is highly dependent on the temperature. Among petroleum products in commerce, crude oil is transported in the largest volumes, both in undersea pipelines and in tankers. Refined products are also shipped, and of course all ships contain large amounts of fuel for their own propulsion. Refining crude oils tbr commercial applications starts with distillation, and the simplest distinction of the various refined products can be related to this process. The most volatile liquid product is aviation gasoline, followed by automobile gasoline, jet fuels, diesel and heating oils, and then the heavy oils that are used for fueling ships and some electrical generation. Most of the molecules in gasoline have between four and ten carbons, most in diesel have between nine and twenty, and heavy fuel oils typically have very few molecules with less than fifteen carbon atoms except for some added as a diluent to achieve the appropriate viscosity to facilitate distribution. As an aside it is appropriate to note that fuels are valued based on their combustion properties, and not chemical composition. Fuels with the same name may have very different chemical compositions if they come from different refineries [19]. 4. PHYSICAL FATE OF SPILLED OIL When oil gets into the oceans from a seep, urban runoff or a spill from a production facility, pipeline or a tanker, it becomes subject to a group of phenomena that are usually grouped under the term "weathering" [20]. Almost all oils float, allowing the smallest molecules to evaporate [21, 22]. These
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molecules are either degraded photochemically [23] or are washed from the atmosphere in rain and then biodegraded [24], likely far from the spill site. Under particularly aggressive aeration in water, such as in surf, evaporation may extend to molecules with >30 carbon atoms [25], but evaporation is more usually limited to molecules with less than about 15 carbon atoms [21]. Thus evaporation is the likely fate of most of a gasoline spill, three-quarters or more of a diesel spill, and perhaps 20-40% of a typical crude oil. Heavy fuels, such as the Bunker fuels used in ships, and bitumen emulsions (Orimulsion| [26]) do not contain a significant volatile fraction. Two competing emulsification processes occur as water and oil mix; water can become entrained in the oil to form an emulsion known as mousse [27], or oil can disperse into the water column as a suspension of small droplets, as happened during the 1993 Braer spill off the Shetland Islands [28]. Mousses are remarkably persistent, and are thought to be the precursors of tarballs that can last for decades [29]. As we shall discuss below, chemical dispersants that break emulsions and stimulate the natural dispersion process are effective tools in the oil spill response "toolkit". Oil also interacts with small mineral particles in a process originally termed "Clay-oil flocculation" [30], and now termed "Oil-Mineral Fines Interactions" [31 ]. Like dispersion, this dramatically increases the surface area of the oil. Aliphatic hydrocarbons are remarkably insoluble, but small aromatics, particularly the notorious BTEX (benzene, toluene, ethylbenzene and the xylenes) and small polar molecules such as naphthenic acids dissolve from floating slicks or dispersed oil, and even from oils immobilized on shoreline sediments and particles [32]. Again, their eventual fate is biodegradation. Aromatic hydrocarbons can be photochemically oxidized [33], converting them to polar products that are probably polymerized species. The process is most effective on the larger and more alkylated forms, and although such hydrocarbons are only a minor component of crude oils [2], they have important toxicological properties [34], and are on the USEPA list of priority pollutants [35] and the EU list of priority substances in the field of water policy [36]. Since light cannot penetrate very far into a dark oil slick, photooxidation has little effect on the bulk properties of spilled oil. Nevertheless it may be important in generating a polymerized "skin" that enhances the stability of tarballs and "pavements" on beaches. Layers of immobile, hardened oil and sediment, termed pavements, form when oil reaches a shoreline as a heavy, thick slick. Oil becomes trapped in the sediment, and the oil and the sediment become saturated with each other [37]. Oil incorporated into such pavements is effectively preserved from weathering processes until this heavy, solidified material is physically disrupted, so a major goal of spill clean-up operations is to prevent the formation of pavements.
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5. EVENTUAL FATE OF SPILLED OIL The weathering processes described above distribute and change the oil in various ways, but they do not actually remove oil from the environment. Only two processes, combustion and biodegradation, actually eliminate oil by converting it to carbon dioxide and water. Some spills do spontaneously ignite, as happened to the Haven spill in the Mediterranean [38], and deliberate ignition is an accepted response option in some situations, such as that of the New Carissa off the coast of Oregon [39]. Under optimal conditions burning may consume >90% of a spill, but there is usually only a small window of opportunity for success [40]. Far more generally, it is biodegradation that removes oil from the environment. As mentioned above, a diverse group of microorganisms has evolved to degrade hydrocarbons, many able to live with hydrocarbons as their sole source of carbon. They are probably ubiquitous, having been found in almost all natural environments where they have been searched for, and obviously very effective, since they have been consuming the vast majority of the oil entering the world's oceans from natural seeps for millions of years (600,000 tonnes, >600 million liters, per year). What separates these organisms from other heterotrophs is their ability to transform hydrocarbons into organic alcohols and acids that enter cellular metabolism. Under aerobic conditions the most common microbial activation of hydrocarbons involves the addition of one or both atoms of molecular oxygen. Alternatively, the activation may involve the addition of hydrogen peroxide. The activation of aromatic hydrocarbons is discussed by Foght in chapter 5, and many pathways are available in the University of Minnesota Biocatalysis/Biodegradation Database [41] and in a recent encyclopedia article [42]. Here it suffices to say that the vast majority of hydrocarbons are biodegradable under aerobic conditions. Thus refined products, such as gasoline, diesels and jet fuels, that are almost entirely hydrocarbons, are essentially completely biodegradable. McMillen et al. [43] examined the short-term biodegradability of 17 crude oils in soil microcosms, and found that the API gravity was the most useful predictor of biodegradability. At 0.5 wt% oil in soil with appropriate nutrients, moisture and aeration, more than 61% of the most degradable oil (API = 46 ) was lost in four weeks, while only 10% of the least degradable oil (API - 15 ) was consumed under the same conditions. Further degradation occurred on a longer timescale, and the literature reports biodegradation potentials as high as 97% for particularly light oils [44]. An important distinction between hydrocarbon-degrading microorganisms and animals and plants is that many microbes degrade polycyclic aromatic hydrocarbons to carbon dioxide, water and biomass. Animals and plants can also activate these molecules, but they do so with enzyme systems that form stable
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permutations of the polycyclic hydrocarbons (see chapter 3). The enzyme systems are known as Cytochrome P450s because of their prominent absorption band when treated with carbon monoxide [45]. These enzymes generate epoxides that are excreted as adducts with sugars, anions, etc., but which may alternatively intercalate and form adducts with DNA [46]. It is thus clearly preferable that polycyclic aromatic hydrocarbons be degraded by bacteria rather than eukaryotes, and facilitating such a preference is one of the advantages of a successful bioremediation protocol. Aerobic biodegradation of hydrocarbons occurs over a wide range of environmental conditions [47]. Although no hyperthermophilic oil-degraders have yet been found, extreme thermophiles such as Thermus and Bacillus species degrade polycyclic aromatic hydrocarbons and long chain alkanes at 6070 ~ [48]. Significant biodegradation occurs below 0~ [49] and extremely halophilic oil-degrading organisms have been described [50] tl'iat degrade hydrocarbons in the presence of several molar salt. In the last decade it has become clear that hydrocarbons are also degraded under anoxic anaerobic conditions. Small water-soluble aromatic compounds, such as benzene and toluene, have been shown to undergo biodegradation under sulfate-reducing, nitrate-reducing, perchlorate-reducing, ferric ion reducing, humic acid-reducing and methanogenic conditions [51 ], and this phenomenon is proving important in remediating terrestrial spills where these compounds have reached groundwater [52]. Larger hydrocarbons, such as n-alkanes up to nC34H70 [53] and two- three- and four-ring aromatic hydrocarbons [54] are also biodegraded under anaerobic conditions. This may be important if oil spills contaminate anaerobic environments, such as marshes, and in the degradation of the traces of oil that become entrained in sediments in harbors. Wherever oil is biodegraded, it is important to bear in mind the fact that crude oil and refined products provide a rather unusual "food" for heterotrophs. While hydrocarbons are excellent sources of carbon and energy, they do not provide any of the other nutrients essential for life; there are no significant amounts of biologically available nitrogen, phosphorus or other elements. Of course most environments have at least trace amounts of these essential nutrients, but most marine environments offer meager reserves to sustain new growth. It is thus likely that any significant input of hydrocarbon is likely to overwhelm the background levels of nutrients, and their availability soon limits that biodegradation. As we shall see below, alleviating this limitation forms the basis of the simplest forms of shoreline bioremediation.
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6. SPILL R E S P O N S E 6.1. At sea:
When oil is spilled at sea, deployment of mechanical equipment designed for containment and recovery is often a slow and inefficient, if not ineffective, response. The rapid spreading of the oil, the slow rate at which mechanical equipment (once deployed) can encounter spreading oil, and interference from waves and currents often limits recovery effectiveness to less than 20% of the oil spilled, significantly less under conditions of severe wind and weather [55, 56]. Unrecovered oil remains in the environment, and undergoes the weathering processes described above, with the most severe environmental consequences resulting when oil strands on shorelines [57, 55]. Beached oil increases the likelihood of contamination for habitats and animals found in subtidal, intertidal and supratidal areas, which include some of the most productive and diverse portions of the marine environment. Burning spilled oil in a contained and controlled manor, so as not to jeopardize the bulk of remaining cargo or other response assets, can rapidly remove bulk oil from the water surface. However, it is a logistical and operational challenge to contain the oil, arrange and control its placement out of the immediate area of spill response activity, and ensure sufficient oil thickness to sustain an efficient burn [40]. Many of the logistical and physical constraints working against efficient mechanical containment and recovery also confound attempts to collect and burn oil on water. When the oil does burn, the unburned residue is comprised mostly of the heavy, longer chain hydrocarbons, which are relatively resistant to ready microbial degradation [58]. Dispersants are widely recognized by many regulatory agencies as an effective at-sea response that provides a net environmental benefit when compared to reliance on mechanical recovery alone (see chapter 9). Application of chemical dispersants facilitates the breakup of the oil slick, moving oil from the water surface into the water column as neutrally buoyant oil droplets ranging from 1 to 100 micrometers in diameter, due to the mixing action of waves and currents. Subsequently, this plume of oil droplets rapidly distributes throughout the water column, mixing into lateral and deeper water masses and reducing oil concentrations below levels of concern for marine life. The rate and effectiveness of this process depends on the nature of the spilled oil (its API gravity and viscosity, degree of weathering, extent of emulsification, and pour point) and the ability of the dispersant formulation to mix with the oil. Dispersants have been an effective aspect of oil spill response over the past 30 years, with applications to major and smaller oil spill incidents in many of the world's oceans (Fig. 3). From 1970 through 1998, dispersants have been used on approximately 37% of oil spills covered in a worldwide database by the Oil Spill Intelligence Reporter [59]. In addition to countless small-scale tests
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that have been conducted in laboratories around the world, critical assessments of dispersant performance have been organized by private and government research organizations, often cooperatively, using controlled releases of large volumes of oil and dispersant applications under real world conditions (Fig. 3). These studies have led to modem dispersant formulations with improved effectiveness and greater environmental safety. A range of dispersant products are stockpiled around the world for spill response, and a few have been shown to be effective over a broad range of oil types and environmental conditions [60]. An important environmental consideration associated with dispersant use is assessing the environmental tradeoff between intentionally exposing water column plants and animals to dispersed oil and the often significant effects of unrecovered oil left to drift at sea to potentially strand on a shoreline. In most cases, these considerations demonstrate a net environmental benefit to the use of dispersants because the short-term, transient exposure of water column communities has much less ecological effect than the prolonged, wide-spread contamination of oil reaching shorelines [57, 55, 61 ]. The environmental risks of dispersed oil are further decreased by the rapid degradation of the small, dispersed oil droplets moving through the water column, compared to the persistence observed for bulk oil stranded on shorelines and incorporated into sediments. The large surface to volume ratio characteristic of micron-sized dispersed oil droplets provides a colonizing substrate for oil degrading bacteria and a source of degradable hydrocarbon to support their growth. And, because the small oil droplets are widely dispersed in the water column, the supply of nitrogen and phosphorus nutrients needed to support bacterial degradation of the diluted oil is sufficient to maintain a viable degrader community in association with the oil droplet. Furthermore, laboratory studies have shown that some dispersants can enhance the initial rate of oil degradation due to the presence of constituents that serve as initial substrates for nascent bacterial growth [62, 63]. Laboratory studies of the fate of dispersed oil droplets have characterized the process by which it becomes a physical substrate for supporting a microbial community as well as a chemical substrate to support their growth. Within 2 to 4 days, the dispersed oil droplet becomes colonized by oil degrading microbes [63-65]. Subsequently, this can become a full floating heterotrophic community consisting of oil, bacteria, protozoa and even nematodes. Macnaughton et al. [65] reported that by day 16, the size of the clusters increased and sank in test microcosms, most likely the result of reduced buoyancy due to oil biodegradation and increased biomass associated with the droplets.
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Dispersant demonstrations and use
Fig. 3. Dispersant response to oil spills. Data taken from reference 59.
6.2. On shore:
If oil reaches shore then the first response is to collect it [66]. Oil typically lands on only the upper portion of the intertidal zone, and on sandy beaches it may be possible to collect the oiled sand with mechanical equipment. This was done, for example, with the spill from the Sea Empress [67]. Particularly heavy oils may be best picked up by hand, as in the case of the spill from the Prestige [68]. On rocky beaches it may be possible to wash oil back into the sea where it can be collected with skimmers, as was done following the spill from the Exxon Valdez [69]. Once the bulk oil has been removed by physical techniques, residual oil is eventually naturally biodegraded. Bioremediation aims to stimulate the rate of natural biodegradation, without causing any additional adverse impact, by at least partially alleviating whatever is limiting microbial growth. In most porous, and therefore aerobic shorelines, the most likely limitation is biologically available nitrogen and phosphorus, and effective bioremediation protocols have applied various forms of fertilizers to deliver these nutrients. Research on this topic has been going on for decades in many parts of the world (Fig. 4; reviewed in 42, 44, 70-77). The simplest approach is to alleviate the nutrient limitation of oil biodegradation by adding fertilizers. This was the basis for the successful bioremediation of the spill from the Exxon Valdez [78-
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80]. Two different fertilizers were used, an oleophilic fertilizer, Inipol EAP22| designed to adhere to oil and deliver nutrients at the oil-water interface [81 ] and a slow-release granular agricultural product (Customblen| that would release nutrients over many weeks through the beach gravel. Inipol EAP22 is a microemulsion with an external oil phase of oleic acid and trilaureth-4phosphate, containing an internal phase of urea in aqueous solution, cosolubilized with butoxy-ethanol to adjust the viscosity. It contains 7.4% nitrogen and 0.7% phosphorus by weight, and was applied with airless sprayers transported on small shallow-draft catamarans. Customblen| is a high quality agricultural fertilizer designed to release its nutrients over several weeks. It consists primarily of ammonium nitrate, calcium phosphate and ammonium phosphate, encapsulated in polymerized linseed oil. Customblen contains 28% nitrogen and 3.5% phosphorus by weight, and was applied by workers walking the beaches with broadcast spreaders. An extensive monitoring program demonstrated that the fertilizer applications were successful at stimulating the rate ofbiodegradation some two- to five-fold [78-80] A quite similar approach was used on a limited scale following the spill from the Sea Empress [82]. Much of this spill was treated with dispersants while at sea, and most of the residue that landed on shore was collected by work crews, but some oil landed on a relatively steep (gradient 10-12.5%) shingle and pebble beach at Bulwell Bay. Because the beach was so steep, slow-release fertilizer was placed in 1 m mesh bags, and secured to the beach with steel pegs. Again, the rate of biodegradation was stimulated more than two-fold on the fertilized part of the beach. To our knowledge, these are the only two occasions when bioremediation by the addition of relatively simple fertilizers was used following a spill, but there have been field and laboratory tests all over the world that have found similar results (see Figure 4). All sorts of fertilizers have been used, usually with success, including soluble and slow release forms of inorganic and organic nitrogen. Our most recent experiments were on Spitsbergen, the largest island of Svalbard, Norway, (approximately 78 ~ N, 17' E.) [83, 84]. Slow release and soluble fertilizers were applied in much the same way they were in Alaska, and they led to an approximate doubling of the rate of biodegradation, even in this cold, Arctic environment. A slightly more complex approach has been championed by Rosenberg and colleagues [71, 85, 86]. In this case the fertilizer is an insoluble polymer of urea and formaldehyde, and it is applied together with an oil-degrading bacterial inoculum that can use this nitrogen source. The approach was apparently able to stimulate the biodegradation of a small spill (100 tons) of a heavy crude oil on a sandy beach between Haifa and Acre in Israel in the early 1990's [71, 86].
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Marine bioremediation demonstrations and use
Fig. 4. Bioremediation response to oil spills. Data taken from references 7 0 - 80.
Others have suggested that what really limits oil biodegradation in the environment is the absence of effective oil degrading microorganisms, and they therefore seek to add such organisms. Most recently this has been attempted on heavy oil spilled by the Nakhodka in the Sea of Japan [87, 88]. Assessing this work is problematic. The published analyses of the field work rely on digital photography of representative oiled rocks, and no detailed chemical analyses have been presented that can be compared with what has been found in other spills. Earlier microbial inocula did not perform well in standardized tests [89]. An important corollary to any oil spill remediation is that it should have a net environmental benefit [90]. By aiming to stimulate natural processes, bioremediation is likely to have minimal adverse effects if carried out carefully and conscientiously, but there are obvious potential risks that must be evaluated. Potential adverse impacts include the possibility that the fertilizer applications might be acutely toxic to marine biota, might stimulate nearshore algal blooms, might cause the production of biosurfactants that could result in increased removal of oil from the shorelines by tidal flushing and lead to broader shoreline impacts, or might generate toxic by-products. Careful monitoring following the spill from the Exxon Valdez [91] and a field trial in the Arctic [92] failed to detect any adverse environmental impact from the careful application of fertilizers, while the rate of hydrocarbon biodegradation was stimulated two- to five-fold.
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7. CONCLUSIONS
Oil spill bioremediation technologies epitomize modem environmental techniques: working with natural processes to remove spilled oil from the environment while minimizing undesirable environmental impacts. If a floating oil slick cannot be collected or burnt, chemical dispersants will cause the oil to move into the water column as tiny droplets with a dramatically increased surface area that allows rapid biodegradation. If oil reaches a shoreline and cannot be removed physically, the careful addition of fertilizers will stimulate oil biodegradation without adverse environmental impact. These two tools are thus an important part of the toolkit for dealing with accidental and deliberate releases of oil into the marine environment.
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J.M. Hunt, Petroleum Geochemistry and Geology, 2nd edition. W.H. Freeman, New York. 1996. B.P Tissot and D.H. Welte, Petroleum Formation and Occurrence. Springer-Verlag, Berlin. 1984. J.Z. De Boer, J.R. Hale, and J. Chanton, New evidence of the geological origins of the ancient Delphic oracle (Greece). Geology, 29 (2001) 707. D. Yergin, The Prize, the epic quest for oil, money and power. Simon and Schuster, New York. 1992. National Research Council, Oil in the Sea III: Inputs, Fates and Effects, National Academy Press, Washington DC, 2002. E.M Levy and K. Lee, Can. J. Fish. Aquat. Sci. 45 (1988) 349. R.C. Prince, Petroleum and other hydrocarbons, biodegradation of. In Encyclopedia of Environmental Microbiology (G. Bitton, ed.) John Wiley, New York, 2002 pp. 24022416. Anonymous. Industry at a glance. World Oil 224 (2003) 75. D.S. Etkin, Historical overview of oil spills from all sources. In: Proceedings of the 1999 International Oil Spill Conference. American Petroleum Institute, Washington DC. 1999 pp. 1097-1102. H.J. Barth, Mar. Poll. Bull. 46 (2003) 1245. A.N. A1Ghadban, F. Abdali, and M.S. Massoud, Environ. International 24 (1998) 23. K. Peters and J.M. Moldowan, The Biomarker Guide; Interpreting molecular fossils in petroleum and ancient sediments. Prentice-Hall, Englewood Cliffs, NJ. 1993. R.C. Prince, D.L. Elmendorf, J.R. Lute, C.S. Hsu, C.E. Haith, J.D. Senius, G.J. Dechert, G.S. Douglas, and E.L. Butler, Environ. Sci. Technol. 28 (1994) 142. W.K. Robbins and C.S. Hsu, Petroleum Composition. In: Kirk-Othmer Encyclopedia of Chemical Technology, Fourth Edition. John Wiley & Sons, New York. 1996 pp. 352370. Y.A. Ibrahim, M.A. Abdelhameed, T.A. A1-Sahhaf, and M.A. Fahim, Petrol. Sci. Technol. 21 (2003) 825. J Burdon, Soil Science, 166 (2001) 752.
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[17] K. Qian, R.P. Rodgers, C.L. Hendrickson, M.R. Emmett and A.G. Marshall, Energy & Fuels, 15 (2001) 492. [18] K. Qian, W.K. Robbins, C.A. Hughey, H.J. Cooper, R.P. Rodgers and A.G. Marshall, Energy & Fuels, 15 (2001) 1505. [19] A.D. Uhler, S.A. Stout, K.J. McCarthy, S. Emsbo-Mattingly, G.S. Douglas and P.W. Beall, Contaminated Soil Sediment and Water, April/May (2002) 20. [20] R.C. Prince, R.M. Garrett, R.E. Bare, M.J. Grossman, T. Townsend, J.M. Suflita, K. Lee, E.H. Owens, G.A. Sergy, J.F. Braddock, J. E Lindstrom. and R.R. Lessard, Spill Sci. Technol. Bull, 8 (2003) 145. [21] M.F. Fingas, The evaporation of oil spills: development and implementation of new prediction methodology. In Proceedings of the 1999 International Oil Spill Conference, American Petroleum Institute, Washington DC, 1999 pp. 185-194. [22] Y. Wang and Y. Huang, Appl. Geochem. 18 (2003) 1641. [23] M.D. Hurley, O. Sokolov, T.J. Wallington, H. Takekawa, M. Karasawa, B. Klotz, I. Barnes, and K.H. Becker, Environ. Sci. Technol. 35 (2001) 1358. [24] K.M. Arzayus, R.M. Dickhut, and E.A. Canuel, Environ. Sci. Technol. 35 (2001) 2178. [25] R.C. Prince, R.T. Stibrany, J. Hardenstine, G.S. Douglas and E.H. Owens, Environ. Sci. Technol. 36 (2002) 2822. [26] Bitor Corporation (2004) www.orimulsionfilel.com/ [27] M.F. Fingas, B. Fieldhouse, J. Lane and J.V. Mullin, What causes the formation of water-in-oil emulsions? In Proceedings of the 2001 International Oil Spill Conference, American Petroleum Institute, Washington DC, 2001 pp. 109-114. [28] R. Thomas and T. Lunel, The Braer Incident; dispersion in action. In Proceedings of the Sixteenth Arctic Marine Oilspill Program Technical Seminar, Edmonton, Alberta, 1993 pp. 843-859. [29] R. Goodman, Spill Sci. Technol. Bull. 8 (2003) 117. [30] J.R. Bragg and E.H. Owens. Clay-oil flocculation as a natural cleansing process following oil spills: Part l-studies of shoreline sediments and residues from past spills. In: Proceedings of the Seventeenth Arctic and Marine Oilspill Program (AMOP) Technical Seminar. Ottawa, Canada. 1994 pp. 1-24. [31 ] E.H. Owens and K. Lee, Mar. Poll. Bull. 47 (2003) 397. [32] E. Lafargue and P. Le Thiez, Org. Geochem. 24 (1996) 1141. [33] R.M. Garrett, I.J. Picketing, C.E. Haith, and R.C. Prince, Environ. Sci. Technol. 32 (1998) 3719. [34] International Agency for Research on Cancer. IARC Monograph on the evaluation of the carcinogenic risk of chemicals to humans. Polynuclear aromatic hydrocarbons, Part 1, Chemical, environmental and experimental data, Vol. 32. World Health Organization, Geneva. 1983 [35] L.H. Keith and W.A. Telliard, Environ. Sci. Technol. 13 (1979) 416. [36] European Commission, DECISION No 2455/2001/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 20 November 2001 establishing the list of priority substances in the field of water policy and amending Directive 2000/60/EC, Official Journal of the European Union L331, 1-5, 15 December 2001. [37] E.H. Owens, J.R. Harper, W. Robson and P.D. Boehm, Arctic 40 (1987) 109. [38] M. Martinelli, A. Luise, E. Tromellini, T.C. Sauer, J.M. Neff and G.S. Douglas, The M/C Haven oil spill: Environmental assessment of exposure pathways and resource injury. Proceedings of the 1995 Oil Spill Conference, American Petroleum Institute, Washington, D.C. 1995 pp. 679-685.
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[39] J.J. Gallagher, H.B. Hile and J.A. Miller, The old New Carissa; a study in patience. Proceedings of the 2001 Oil Spill Conference, American Petroleum Institute, Washington, D.C. 2001 pp. 85-90. [40] I. Buist, Spill Sci. Technol. Bull. 8 (1993) 341. [41 ] The University of Minnesota Biocatalysis/Biodegradation Database (2004) htlp://umbbd.ahc.unm.edu/ [42] R.C. Prince, Crude oil biodegradation In The Encyclopedia of Environmental Analysis and Remediation, Volume 2, 1327-1342. John Wiley, New York. 1998. [43] S.J. McMillen, A.G. Requejo, G.N. Young, P.S. Davis, P.D. Cook, J.M. Kerr, and N.R. Gray, Bioremediation potential of crude oil spilled on soil. In: Microbial Processes for Bioremediation (R.E. Hinchee, C.M. Vogel, and F.J. Brockman, eds.). Battelle Press, OH. 1995 pp, 91-99. [44] R.C. Prince, Crit. Rev. MicrobioI. 19 (1993) 217. [45] C.J. Omiecinski, R.P. Remmel and V.P.. Hosagrahara, Toxicol. Sci. 48 (1999) 151. [46] V.J. Melendez-Colon, A. Luch, A. Seidel, and W.M. Baird, Cancer Res. 59 (1999)1412. [47] R. Margesin and F. Schinner, Appl. Microbiol. Biotechnol. 56 (2001) 650. [48] H. Feitkenhauer, R. Muller and H. M~irkl, Biodegradation 14 (2003) 367. [49] A.G. Rike, K.B. Haugen, M. Borresen, B. Engene. and P. Kolstad, Cold Regions Sci. Technol. 37 (2003) 97. [50] M.J. Gauthier, B. Lafay, R. Christen, L. Fernandez, M. Acquaviva, P., Bonin and J.C. Bertrand, Int. J. System. Bacteriol. 42 (1992) 568. [51 ] A.M. Spormann and F. Widdel, Biodegradation 11 (2000) 85. [52] J.A. Cunningham, H. Rahme, G.D. Hopkins, C. Lebron, and M. Reinhard, Environ. Sci. Technol. 35 (2000) 1663. [53] M.E. Caldwell, R.M. Garrett, R.C. Prince and J.M. Suflita, Environ. Sci. Technol. 32 (1998)2191. [54] J.D. Coates, J. Woodward, J. Allen, P. Philp and D.R. Lovley, Appl. Environ. Microbiol. 63 (1997) 3589. [55] A. Lewis and D. Aurand, Putting Dispersants to Work: Overcoming Obstacles. API Technical Report IOSC-004. 1997 International Oil Spill Conference Issue Paper. API. Washington, DC. 1997. [56] D.S. Etkin and P. Tebeau, Assessing progress and benefits of oil spill response technology development since Exxon Valdez. Proceedings of the 2003 International Oil Spill Conference, American Petroleum Institute, Washington, DC, 2003 pp. 843-850. [57] National Research Council, Using Oil Dispersants on the Sea. National Academy Press. Washington, DC. 1989. [58] R.M. Garrett, C.C. Guanette, C.E. Haith and R.C. Prince, Environ. Sci. Technol. 34 (2000) 1934. [59] D.S. Etkin, Factors in the Dispersant use decision-making process" historical overview and look to the future, Proceedings of the twenty-first Arctic and Marine Oil Spill Program (AMOP) Technical Seminar. Environment Canada. Ottawa. 1998 pp. 281-304 [60] R.R. Lessard and G. DeMarco. Spill Sci. Technol. Bull. 6 (2000) 59. [61 ] International Petroleum Industry Environmental Conservation Association (IPIECA), Oil Spill Preparedness and Response. Volume 5" Dispersants and their Role in Oil Spill Response. London. 2000. [62] R. Varadaraj, M.L. Robbins, J. Bock, S. Pace and D. MacDonald, Dispersion and biodegradation of oil spills on Water. In: Proceedings of the 1995 International Oil Spill Conference, American Petroleum Institute, Washington, DC. 1995 pp. 101-106.
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[63] R. Swannell and F. Daniel, Effect of dispersants on oil biodegradation under simulated marine conditions. Proceedings of the 1999 International Oil Spill Conference, American Petroleum Institute, Washington, DC, 1999, pp. 169-176. [64] J.E. Lindstrom and J.F. Braddock, Mar. Poll. Bull. 44 (2002) 739. [65] S.J. Macnaughton, R. Swannell, F. Daniel and L. Bristow, Spill Sci. Technol. Bull. 8 (2003) 179. [66] B.E. Ornitz and M.A. Champ, Oil Spills First Principles. Elsevier, New York. 2002. [67] T. Lunel, K. Lee, R. Swannell, P. Wood, J. Rusin, N. Bailey, C. Halliwell, L. Davies, M. Sommerville, A. Dobie, D. Mitchell and M. McDonagh, Shoreline clean up during the Sea Empress incident: the role of surf washing (clay oil flocculation), dispersants and bioremediation. In Proceedings of the Nineteenth Arctic and Marine Oil Spill Program Seminar. Environment Canada, 1996 pp. 1521-1540. [68] J. Bohannon, X. Bosch and J. Withgott, Science 298 (2002) 1695. [69] S.A. Nauman, Shoreline clean-up: equipment and operations. In Proceedings of the 1991 International Oil Spill Conference, American Petroleum Institute, Washington DC, 1991 pp. 141-148. [70] R.M. Atlas, Crit. Rev. Microbiol. 5 (1977) 371. [71 ] E. Rosenberg, R. Legman, A. Kushmaro, R. Taube, E. Adler and E.Z. Ron, Biodegradation 3 (1992) 337. [72] R.P.J. Swannell, K. Lee and M, McDonagh, Microbiol. Rev. 60 (1996) 342. [73] Q. Lin, I.A. Mendelssohn, C.B. Henry, P.O. Roberts, M.M. Walsh, E.B. Overton and R.J. Portier, Environ. Tech. 20 (1999) 825. [74] M. Mathew, J.P. Obbard, Y.P. Ting, Y.H. Gin and H.M. Tan, Acta Biotechnol. 19 (1999) 225. [75] K. Lee and S. deMora, Environ. Tech. 20 (1999) 783. [76] I. Head and R.P.J. Swannell, Curr. Opin. Biotechnol. 10 (1999) 234. [77] S. Harayama, H. Kishira, Y. Kasai and K. Shutsubo, J Mol Microbiol Biotechnol. 1 (1999) 63. [78] J.R. Bragg, R.C. Prince, E.J. Harner, and R.M. Atlas, Nature 368 (1994) 413. [79] R.C. Prince, J.R. Clark, J.E Lindstrom, E.L. Butler, E.J. Brown, G. Winter, M.J. Grossman, R.R. Parrish, R.E. Bare, J.F. Braddock, W.G. Steinhauer, G.S. Douglas, J.M. Kennedy, P.J. Barter, J.R. Bragg, E.J. Harner and R.M. Atlas, Bioremediation of the Exxon Valdez oil spill: monitoring safety and efficacy. In Hydrocarbon Remediation(R. E. Hinchee, B. C. Alleman, R. E. Hoeppel and R. N. Miller, eds.) Lewis Publishers, Boca Raton, FL., 1994 pp. 107-124. [80] R.C. Prince and J.R. Bragg, Bioremediation J. 1 (1997) 97. [81] A. Ladousse and B. Tramier, Results of 12 years of research in spilled oil bioremediation: Inipol EAP22. In: Proceedings of the 1991 International Oil Spill Conference, American Petroleum Institute, Washington DC, 1991 pp. 577-582. [82] R.P.J. Swannell, D. Mitchell, G. Lethbridge, D. Jones, D. Heath, M. Hagley, M. Jones, S. Petch, R. Milne, R. Croxford and K. Lee, Environmental Technol. 20 (1999) 863. [83] C.C. Gu6nette, G.A. Sergy, E.H. Owens, R.C. Prince and K. Lee, Spill Sci. Technol. Bull. 8 (2003), 245. [84] R.C. Prince, R.E. Bare, R.M. Garrett, M.J. Grossman, C.E. Haith, L.G. Keim, K Lee, G.J. Holtom, P. Lambert, G.A. Sergy, E.H. Owens and C.C. Gu6nette, Spill Sci. Technol. Bull. 8 (2003) 303. [85] E. Rosenberg, R. Legman, A. Kushmaro, E. Adler, H. Abir and E.Z. Ron, J Biotechnol. 51 (1996) 273.
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[86] E. Rosenberg and E.Z. Ron, Non-polluting compositions to degrade hydrocarbons and microorganisms for use thereof. US Patent 5780290 1998 [87] T. Hozumi, H. Tsutsumi, and M. Kono, Mar. Poll. Bull. 40 (2000) 308. [88] H. Tsutsumi, M. Kono, K. Takai, T. Manabe, M. Haraguchi, I. Yamamoto, and C. Oppenheimer, Mar. Poll. Bull. 40 (2000) 320. [89] A.D. Venosa, J.R. Haines and D.M. Allen, J. Ind. Microbiol. 10 (1992) 1. [90] J.M. Baker, Net environmental Benefit Analysis for oil spill response. In Proceedings of the 1995 International Oil Spill Conference, American Petroleum Institute, Washington DC, 1995 pp. 611-614. [91 ] R.C. Prince, J.R. Clark and K. Lee, Bioremediation effectiveness; removing hydrocarbons while minimizing environmental impact. In Proceedings of the Ninth International Petroleum Environmental Conference, Albuquerque, NM. 2002,available at: http://ipec.utulsa.edu/lpec/Conf2002/prince clark l e e 109.pdf [92] K. Lee, G. Wohlgeschaffen, G.H. Tremblay, B.T. Johnson, G.A. Sergy, R.C. Prince, C.C. Gurnette and E.H. Owens, Spill Sci. Technol. Bull, 8 (2003) 273.
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Chapter 18
Bioremediation of marine oil spills R. C. P r i n c e ~ a n d J. R. C l a r k 2
~ExxonMobil Research & Engineering Co. Annandale, NJ 08801 2ExxonMobil Research & Engineering Co. Fairfax, VA 22037
1. I N T R O D U C T I O N Crude oils are the liquid fossil residues of aquatic algae, sometimes with minor contributions from terrestrial plants, that grew in the distant past. Then as now, we can imagine that most of this material was biodegraded and recycled on an essentially annual timescale, but a small fraction became buried and underwent diagenesis and catagenesis to become oil [1]. This process usually took millions of years, and was dependent on the depth of burial and the temperature. Some oil dates from the Precambrian (>570 million years ago), but most is rather younger; the average age of commercially important oil is about 100 million years, with the majority being from the Jurassic and Cretaceous (180 to 85 million years ago) [2]. Commercially important oil has migrated from its source rock to a reservoir, and it is not unusual for these reservoirs to leak. If the leak reaches the surface, it is known as an oil seep. Humans have used material from such seeps for thousands of years. Early uses include hafting stone axes to their handles, as an embalming agent, and as a medical nostrum. Genesis (11,3) says that bitumen was used as the mortar for the Tower of Babel, and Exodus (2,3) that Moses' basket was made waterproof with a bitumen daub. It seems likely that several religions started around natural gas seeps, either as eternal flames or as sources of hallucinogenic vapors [3]. But these were only very minor uses, and it is only in the last century and a half that oil has come to play a truly central role in modem society [4]. Terrestrial seeps were the first locations to be drilled when oil production began in earnest in the nineteenth century, such as the 1860 Drake well in Pennsylvania, but marine seeps were drilled by the end of the century.
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Marine seeps are widespread, as shown in Figure l, and are a major source of oil into the World's oceans [5]. Even with today's enormous commerce in oil, seeps provide about 62% of the total releases into the coastal marine environment of North America, and 47% of the world. Such seeps must have been occurring for millions of years, providing an important input of degradable carbon for local ecosystems and perhaps even major fisheries [6]. A diverse group of microorganisms exploits this natural input. Oil-degrading microbes have been found in all marine environments where they have been looked for, and more than seventy genera of eubacteria and archaea, and a hundred genera of fungi, have been shown capable of degrading petroleum hydrocarbons [7]. It is these organisms that remove oil seepage and spilled oil from the marine and terrestrial environment, and underpin the bioremediation strategies for dealing with spilled oil that we will describe here. 2. A N T H R O P O G E N I C INPUT OF OIL INTO THE W O R L D ' S OCEANS Oil fuels the modem world on an enormous scale; annual consumption is of the order of 3.5 billion US gallons (1.2 x 10 ~~ liters) per day [8]. Much of this is produced at sea; more than 25% of US production is from the Continental Shelf, and 25% of Saudi Arabia's production, 80% of Nigeria's, and 100% of Angola's, Australia's, Brazil's and Malaysia's production is offshore. This production is associated with some oil and grease discharges into the marine environment associated with the produced water; some 2,700 tonnes per year in the US, and more than ten times this in the rest of the world [5]. This input is dwarfed, however, by oil in municipal run-off from the land, and from the standard operation of marine shipping (Figure 2). Catastrophic spills from tankers and other ships are well known, but in fact their contribution to total oil input into the oceans is relatively small, some 8% of the global input, 2% of North American input. Nevertheless, since such spills are large on the local scale, they often require environmentally appropriate and cost-effective responses. Fortunately, despite the increasing volume of transported oil, the amount spilled from catastrophic spills has been generally decreasing over the last few decades [9]. The major exception to this decline was the appalling environmental crime in the Arabian Gulf in 1991, where Iraqi forces deliberately released more than a million tonnes (about 260 million gallons or a billion liters) of oil into the sea near Kuwait [ 10]. An additional 350 million gallons (1.2 x l 09 1) were deposited in the Gulf as fallout from the smoke plumes of the > 700 oil well fires in the Kuwait oil fields [ 11 ], making this by far the largest man-made release of oil into the marine environment to date.
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3. COMPOSITION OF CRUDE OIL
Crude oils are complex mixtures of hydrocarbons with significant quantities (typically about 15%) of compounds containing additional heteroatoms such as oxygen, sulfur or nitrogen. The hydrogen to carbon ratio of the hydrocarbons is typically between 1.5 and 2, indicating a mixture of aliphatic (predominant carbons are -CH2-, either as linear molecules, known as paraffins, or as rings known as naphthenes) and aromatic species (principal carbons are-HC=CH - in rings). Alkenes and alkynes, linear unsaturated molecules, are rare in crude oils, although they can be abundant in some refined products such as gasoline. Tissot and Welte [2] calculate the average composition of more than 525 crude oils as 58.2% saturates, 28.6% aromatics and 14.2% polar compounds, noting that the absolute values vary widely in different oils. On average, there is rough parity between paraffins, naphthenes and aromatic hydrocarbons in crude oils [2]. Paraffins in crude oils may start with methane, and extend to waxes with more than seventy carbons. Their total content varies widely, from essentially undetectable to as high as 35%, depending on source and reservoir conditions, but they typically make up 15-20% of an undegraded crude oil. There are also branched alkanes; especially in the C6 to C8 range, but pristane (C19H40) and phytane (C20H42), molecular relics of the phytol chains of chlorophylls and perhaps other biomolecules, are usually the most abundant individual branched alkanes. Pristane is thought to be the result of initial partial degradation of phytol in the presence of oxygen, while phytane is thought to be the result of diagenesis in the absence of oxygen [2].
Marine oil seeps
Fig. 1. Map of the major oil seeps into the World's oceans. Data taken from reference [5].
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Annual input of oil to the seas World
North America
Natural Seeps Extraction !--I Transportation Consumption
347 million gallons (US)
69.4 million gallons (US)
Fig. 2. Oil input into the World's oceans. Data taken from reference [5]. The naphthenes include parent compounds, such as cyclopentane, cyclohexane and decalin, together with their alkylated congeners. Tissot and Welte [2] quote the average composition of the naphthene fraction of 299 crude oils as 54.9% one and two ring naphthenes, 20.4% tricyclic naphthenes, and 24.0% tetra and pentacyclic naphthenes. These latter molecules are amongst the better understood molecular biomarkers in crude oils, and they are used extensively in correlating reservoirs and source rocks [12], in assigning the depositional environment of source rocks [12], and more recently as conserved internal markers during biodegradation [ 13]. Because of the separation procedures used in the analysis of crude oils, any molecule containing at least one aromatic ring is included in the "aromatic" fraction, regardless of the presence of saturated rings and/or alkyl substituents. Aromatic heterocycles containing sulfur, such as thiophenes, benzothiophenes and dibenzothiophenes, or nitrogen, such as the indoles, carbazoles and quinolines, also fall into the aromatic category. Alkylated aromatic species are more abundant than their parent compounds, with mono-, di- and tri-methyl derivatives usually being most abundant. Nevertheless, the median aromatic structure probably has one or two methyl groups and a long-chain alkyl substituent [ 14]. The polar molecules are the most difficult to characterize because they typically cannot be analyzed by gas chromatography, the method of choice for the molecular characterization of hydrocarbons. Petroleum polar compounds contain heteroatoms such as nitrogen, oxygen and/or sulfur, and the category includes the porphyrins, typically with nickel or vanadium in the tetrapyrole,
499
naphthenic acids and large molecules known as asphaltenes. Some asphaltenes have molecular weights into the thousands and even higher, and many are suspended in the oil rather than dissolved in it [15]. The polar fraction of the oil contains the majority of the color centers in crude oil, and in isolation these materials are difficult to distinguish from more recent biological residues, such as the humic and fulvic acids [16], except with sophisticated tools such as isotope analysis. Recent developments in electrospray mass spectrometry promise significant progress in determining the molecular structure of these compounds [ 17, 18]. Several hundred different crude oils are being produced today, and their chemical composition and properties vary quite widely. They are typically classified by their density. The oil industry uses a unit known as API (American Petroleum Institute) gravity, which is defined as [142.5/(specific gravity)] 131.5, and expressed as degrees (~ Thus water has an API gravity of 10 ~ and denser fluids will have lower API gravities. For convenience, oils with API gravities greater than 40 ~ are said to be light oils, while those with API gravities of less than about 17 ~ are said to be heavy. Note that even these typically float on water, especially seawater. Light oils have higher proportions of hydrocarbons; heavy oils are rich in polars and asphaltenes. Viscosity is roughly inversely proportional to API gravity, but it is also dependent on the physical state of the polar compounds and longer alkanes in the oil, and is highly dependent on the temperature. Among petroleum products in commerce, crude oil is transported in the largest volumes, both in undersea pipelines and in tankers. Refined products are also shipped, and of course all ships contain large amounts of fuel for their own propulsion. Refining crude oils tbr commercial applications starts with distillation, and the simplest distinction of the various refined products can be related to this process. The most volatile liquid product is aviation gasoline, followed by automobile gasoline, jet fuels, diesel and heating oils, and then the heavy oils that are used for fueling ships and some electrical generation. Most of the molecules in gasoline have between four and ten carbons, most in diesel have between nine and twenty, and heavy fuel oils typically have very few molecules with less than fifteen carbon atoms except for some added as a diluent to achieve the appropriate viscosity to facilitate distribution. As an aside it is appropriate to note that fuels are valued based on their combustion properties, and not chemical composition. Fuels with the same name may have very different chemical compositions if they come from different refineries [19]. 4. PHYSICAL FATE OF SPILLED OIL When oil gets into the oceans from a seep, urban runoff or a spill from a production facility, pipeline or a tanker, it becomes subject to a group of phenomena that are usually grouped under the term "weathering" [20]. Almost all oils float, allowing the smallest molecules to evaporate [21, 22]. These
500
molecules are either degraded photochemically [23] or are washed from the atmosphere in rain and then biodegraded [24], likely far from the spill site. Under particularly aggressive aeration in water, such as in surf, evaporation may extend to molecules with >30 carbon atoms [25], but evaporation is more usually limited to molecules with less than about 15 carbon atoms [21]. Thus evaporation is the likely fate of most of a gasoline spill, three-quarters or more of a diesel spill, and perhaps 20-40% of a typical crude oil. Heavy fuels, such as the Bunker fuels used in ships, and bitumen emulsions (Orimulsion| [26]) do not contain a significant volatile fraction. Two competing emulsification processes occur as water and oil mix; water can become entrained in the oil to form an emulsion known as mousse [27], or oil can disperse into the water column as a suspension of small droplets, as happened during the 1993 Braer spill off the Shetland Islands [28]. Mousses are remarkably persistent, and are thought to be the precursors of tarballs that can last for decades [29]. As we shall discuss below, chemical dispersants that break emulsions and stimulate the natural dispersion process are effective tools in the oil spill response "toolkit". Oil also interacts with small mineral particles in a process originally termed "Clay-oil flocculation" [30], and now termed "Oil-Mineral Fines Interactions" [31 ]. Like dispersion, this dramatically increases the surface area of the oil. Aliphatic hydrocarbons are remarkably insoluble, but small aromatics, particularly the notorious BTEX (benzene, toluene, ethylbenzene and the xylenes) and small polar molecules such as naphthenic acids dissolve from floating slicks or dispersed oil, and even from oils immobilized on shoreline sediments and particles [32]. Again, their eventual fate is biodegradation. Aromatic hydrocarbons can be photochemically oxidized [33], converting them to polar products that are probably polymerized species. The process is most effective on the larger and more alkylated forms, and although such hydrocarbons are only a minor component of crude oils [2], they have important toxicological properties [34], and are on the USEPA list of priority pollutants [35] and the EU list of priority substances in the field of water policy [36]. Since light cannot penetrate very far into a dark oil slick, photooxidation has little effect on the bulk properties of spilled oil. Nevertheless it may be important in generating a polymerized "skin" that enhances the stability of tarballs and "pavements" on beaches. Layers of immobile, hardened oil and sediment, termed pavements, form when oil reaches a shoreline as a heavy, thick slick. Oil becomes trapped in the sediment, and the oil and the sediment become saturated with each other [37]. Oil incorporated into such pavements is effectively preserved from weathering processes until this heavy, solidified material is physically disrupted, so a major goal of spill clean-up operations is to prevent the formation of pavements.
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5. EVENTUAL FATE OF SPILLED OIL The weathering processes described above distribute and change the oil in various ways, but they do not actually remove oil from the environment. Only two processes, combustion and biodegradation, actually eliminate oil by converting it to carbon dioxide and water. Some spills do spontaneously ignite, as happened to the Haven spill in the Mediterranean [38], and deliberate ignition is an accepted response option in some situations, such as that of the New Carissa off the coast of Oregon [39]. Under optimal conditions burning may consume >90% of a spill, but there is usually only a small window of opportunity for success [40]. Far more generally, it is biodegradation that removes oil from the environment. As mentioned above, a diverse group of microorganisms has evolved to degrade hydrocarbons, many able to live with hydrocarbons as their sole source of carbon. They are probably ubiquitous, having been found in almost all natural environments where they have been searched for, and obviously very effective, since they have been consuming the vast majority of the oil entering the world's oceans from natural seeps for millions of years (600,000 tonnes, >600 million liters, per year). What separates these organisms from other heterotrophs is their ability to transform hydrocarbons into organic alcohols and acids that enter cellular metabolism. Under aerobic conditions the most common microbial activation of hydrocarbons involves the addition of one or both atoms of molecular oxygen. Alternatively, the activation may involve the addition of hydrogen peroxide. The activation of aromatic hydrocarbons is discussed by Foght in chapter 5, and many pathways are available in the University of Minnesota Biocatalysis/Biodegradation Database [41] and in a recent encyclopedia article [42]. Here it suffices to say that the vast majority of hydrocarbons are biodegradable under aerobic conditions. Thus refined products, such as gasoline, diesels and jet fuels, that are almost entirely hydrocarbons, are essentially completely biodegradable. McMillen et al. [43] examined the short-term biodegradability of 17 crude oils in soil microcosms, and found that the API gravity was the most useful predictor of biodegradability. At 0.5 wt% oil in soil with appropriate nutrients, moisture and aeration, more than 61% of the most degradable oil (API = 46 ) was lost in four weeks, while only 10% of the least degradable oil (API - 15 ) was consumed under the same conditions. Further degradation occurred on a longer timescale, and the literature reports biodegradation potentials as high as 97% for particularly light oils [44]. An important distinction between hydrocarbon-degrading microorganisms and animals and plants is that many microbes degrade polycyclic aromatic hydrocarbons to carbon dioxide, water and biomass. Animals and plants can also activate these molecules, but they do so with enzyme systems that form stable
502
permutations of the polycyclic hydrocarbons (see chapter 3). The enzyme systems are known as Cytochrome P450s because of their prominent absorption band when treated with carbon monoxide [45]. These enzymes generate epoxides that are excreted as adducts with sugars, anions, etc., but which may alternatively intercalate and form adducts with DNA [46]. It is thus clearly preferable that polycyclic aromatic hydrocarbons be degraded by bacteria rather than eukaryotes, and facilitating such a preference is one of the advantages of a successful bioremediation protocol. Aerobic biodegradation of hydrocarbons occurs over a wide range of environmental conditions [47]. Although no hyperthermophilic oil-degraders have yet been found, extreme thermophiles such as Thermus and Bacillus species degrade polycyclic aromatic hydrocarbons and long chain alkanes at 6070 ~ [48]. Significant biodegradation occurs below 0~ [49] and extremely halophilic oil-degrading organisms have been described [50] tl'iat degrade hydrocarbons in the presence of several molar salt. In the last decade it has become clear that hydrocarbons are also degraded under anoxic anaerobic conditions. Small water-soluble aromatic compounds, such as benzene and toluene, have been shown to undergo biodegradation under sulfate-reducing, nitrate-reducing, perchlorate-reducing, ferric ion reducing, humic acid-reducing and methanogenic conditions [51 ], and this phenomenon is proving important in remediating terrestrial spills where these compounds have reached groundwater [52]. Larger hydrocarbons, such as n-alkanes up to nC34H70 [53] and two- three- and four-ring aromatic hydrocarbons [54] are also biodegraded under anaerobic conditions. This may be important if oil spills contaminate anaerobic environments, such as marshes, and in the degradation of the traces of oil that become entrained in sediments in harbors. Wherever oil is biodegraded, it is important to bear in mind the fact that crude oil and refined products provide a rather unusual "food" for heterotrophs. While hydrocarbons are excellent sources of carbon and energy, they do not provide any of the other nutrients essential for life; there are no significant amounts of biologically available nitrogen, phosphorus or other elements. Of course most environments have at least trace amounts of these essential nutrients, but most marine environments offer meager reserves to sustain new growth. It is thus likely that any significant input of hydrocarbon is likely to overwhelm the background levels of nutrients, and their availability soon limits that biodegradation. As we shall see below, alleviating this limitation forms the basis of the simplest forms of shoreline bioremediation.
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6. SPILL R E S P O N S E 6.1. At sea:
When oil is spilled at sea, deployment of mechanical equipment designed for containment and recovery is often a slow and inefficient, if not ineffective, response. The rapid spreading of the oil, the slow rate at which mechanical equipment (once deployed) can encounter spreading oil, and interference from waves and currents often limits recovery effectiveness to less than 20% of the oil spilled, significantly less under conditions of severe wind and weather [55, 56]. Unrecovered oil remains in the environment, and undergoes the weathering processes described above, with the most severe environmental consequences resulting when oil strands on shorelines [57, 55]. Beached oil increases the likelihood of contamination for habitats and animals found in subtidal, intertidal and supratidal areas, which include some of the most productive and diverse portions of the marine environment. Burning spilled oil in a contained and controlled manor, so as not to jeopardize the bulk of remaining cargo or other response assets, can rapidly remove bulk oil from the water surface. However, it is a logistical and operational challenge to contain the oil, arrange and control its placement out of the immediate area of spill response activity, and ensure sufficient oil thickness to sustain an efficient burn [40]. Many of the logistical and physical constraints working against efficient mechanical containment and recovery also confound attempts to collect and burn oil on water. When the oil does burn, the unburned residue is comprised mostly of the heavy, longer chain hydrocarbons, which are relatively resistant to ready microbial degradation [58]. Dispersants are widely recognized by many regulatory agencies as an effective at-sea response that provides a net environmental benefit when compared to reliance on mechanical recovery alone (see chapter 9). Application of chemical dispersants facilitates the breakup of the oil slick, moving oil from the water surface into the water column as neutrally buoyant oil droplets ranging from 1 to 100 micrometers in diameter, due to the mixing action of waves and currents. Subsequently, this plume of oil droplets rapidly distributes throughout the water column, mixing into lateral and deeper water masses and reducing oil concentrations below levels of concern for marine life. The rate and effectiveness of this process depends on the nature of the spilled oil (its API gravity and viscosity, degree of weathering, extent of emulsification, and pour point) and the ability of the dispersant formulation to mix with the oil. Dispersants have been an effective aspect of oil spill response over the past 30 years, with applications to major and smaller oil spill incidents in many of the world's oceans (Fig. 3). From 1970 through 1998, dispersants have been used on approximately 37% of oil spills covered in a worldwide database by the Oil Spill Intelligence Reporter [59]. In addition to countless small-scale tests
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that have been conducted in laboratories around the world, critical assessments of dispersant performance have been organized by private and government research organizations, often cooperatively, using controlled releases of large volumes of oil and dispersant applications under real world conditions (Fig. 3). These studies have led to modem dispersant formulations with improved effectiveness and greater environmental safety. A range of dispersant products are stockpiled around the world for spill response, and a few have been shown to be effective over a broad range of oil types and environmental conditions [60]. An important environmental consideration associated with dispersant use is assessing the environmental tradeoff between intentionally exposing water column plants and animals to dispersed oil and the often significant effects of unrecovered oil left to drift at sea to potentially strand on a shoreline. In most cases, these considerations demonstrate a net environmental benefit to the use of dispersants because the short-term, transient exposure of water column communities has much less ecological effect than the prolonged, wide-spread contamination of oil reaching shorelines [57, 55, 61 ]. The environmental risks of dispersed oil are further decreased by the rapid degradation of the small, dispersed oil droplets moving through the water column, compared to the persistence observed for bulk oil stranded on shorelines and incorporated into sediments. The large surface to volume ratio characteristic of micron-sized dispersed oil droplets provides a colonizing substrate for oil degrading bacteria and a source of degradable hydrocarbon to support their growth. And, because the small oil droplets are widely dispersed in the water column, the supply of nitrogen and phosphorus nutrients needed to support bacterial degradation of the diluted oil is sufficient to maintain a viable degrader community in association with the oil droplet. Furthermore, laboratory studies have shown that some dispersants can enhance the initial rate of oil degradation due to the presence of constituents that serve as initial substrates for nascent bacterial growth [62, 63]. Laboratory studies of the fate of dispersed oil droplets have characterized the process by which it becomes a physical substrate for supporting a microbial community as well as a chemical substrate to support their growth. Within 2 to 4 days, the dispersed oil droplet becomes colonized by oil degrading microbes [63-65]. Subsequently, this can become a full floating heterotrophic community consisting of oil, bacteria, protozoa and even nematodes. Macnaughton et al. [65] reported that by day 16, the size of the clusters increased and sank in test microcosms, most likely the result of reduced buoyancy due to oil biodegradation and increased biomass associated with the droplets.
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Dispersant demonstrations and use
Fig. 3. Dispersant response to oil spills. Data taken from reference 59.
6.2. On shore:
If oil reaches shore then the first response is to collect it [66]. Oil typically lands on only the upper portion of the intertidal zone, and on sandy beaches it may be possible to collect the oiled sand with mechanical equipment. This was done, for example, with the spill from the Sea Empress [67]. Particularly heavy oils may be best picked up by hand, as in the case of the spill from the Prestige [68]. On rocky beaches it may be possible to wash oil back into the sea where it can be collected with skimmers, as was done following the spill from the Exxon Valdez [69]. Once the bulk oil has been removed by physical techniques, residual oil is eventually naturally biodegraded. Bioremediation aims to stimulate the rate of natural biodegradation, without causing any additional adverse impact, by at least partially alleviating whatever is limiting microbial growth. In most porous, and therefore aerobic shorelines, the most likely limitation is biologically available nitrogen and phosphorus, and effective bioremediation protocols have applied various forms of fertilizers to deliver these nutrients. Research on this topic has been going on for decades in many parts of the world (Fig. 4; reviewed in 42, 44, 70-77). The simplest approach is to alleviate the nutrient limitation of oil biodegradation by adding fertilizers. This was the basis for the successful bioremediation of the spill from the Exxon Valdez [78-
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80]. Two different fertilizers were used, an oleophilic fertilizer, Inipol EAP22| designed to adhere to oil and deliver nutrients at the oil-water interface [81 ] and a slow-release granular agricultural product (Customblen| that would release nutrients over many weeks through the beach gravel. Inipol EAP22 is a microemulsion with an external oil phase of oleic acid and trilaureth-4phosphate, containing an internal phase of urea in aqueous solution, cosolubilized with butoxy-ethanol to adjust the viscosity. It contains 7.4% nitrogen and 0.7% phosphorus by weight, and was applied with airless sprayers transported on small shallow-draft catamarans. Customblen| is a high quality agricultural fertilizer designed to release its nutrients over several weeks. It consists primarily of ammonium nitrate, calcium phosphate and ammonium phosphate, encapsulated in polymerized linseed oil. Customblen contains 28% nitrogen and 3.5% phosphorus by weight, and was applied by workers walking the beaches with broadcast spreaders. An extensive monitoring program demonstrated that the fertilizer applications were successful at stimulating the rate ofbiodegradation some two- to five-fold [78-80] A quite similar approach was used on a limited scale following the spill from the Sea Empress [82]. Much of this spill was treated with dispersants while at sea, and most of the residue that landed on shore was collected by work crews, but some oil landed on a relatively steep (gradient 10-12.5%) shingle and pebble beach at Bulwell Bay. Because the beach was so steep, slow-release fertilizer was placed in 1 m mesh bags, and secured to the beach with steel pegs. Again, the rate of biodegradation was stimulated more than two-fold on the fertilized part of the beach. To our knowledge, these are the only two occasions when bioremediation by the addition of relatively simple fertilizers was used following a spill, but there have been field and laboratory tests all over the world that have found similar results (see Figure 4). All sorts of fertilizers have been used, usually with success, including soluble and slow release forms of inorganic and organic nitrogen. Our most recent experiments were on Spitsbergen, the largest island of Svalbard, Norway, (approximately 78 ~ N, 17' E.) [83, 84]. Slow release and soluble fertilizers were applied in much the same way they were in Alaska, and they led to an approximate doubling of the rate of biodegradation, even in this cold, Arctic environment. A slightly more complex approach has been championed by Rosenberg and colleagues [71, 85, 86]. In this case the fertilizer is an insoluble polymer of urea and formaldehyde, and it is applied together with an oil-degrading bacterial inoculum that can use this nitrogen source. The approach was apparently able to stimulate the biodegradation of a small spill (100 tons) of a heavy crude oil on a sandy beach between Haifa and Acre in Israel in the early 1990's [71, 86].
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Marine bioremediation demonstrations and use
Fig. 4. Bioremediation response to oil spills. Data taken from references 7 0 - 80.
Others have suggested that what really limits oil biodegradation in the environment is the absence of effective oil degrading microorganisms, and they therefore seek to add such organisms. Most recently this has been attempted on heavy oil spilled by the Nakhodka in the Sea of Japan [87, 88]. Assessing this work is problematic. The published analyses of the field work rely on digital photography of representative oiled rocks, and no detailed chemical analyses have been presented that can be compared with what has been found in other spills. Earlier microbial inocula did not perform well in standardized tests [89]. An important corollary to any oil spill remediation is that it should have a net environmental benefit [90]. By aiming to stimulate natural processes, bioremediation is likely to have minimal adverse effects if carried out carefully and conscientiously, but there are obvious potential risks that must be evaluated. Potential adverse impacts include the possibility that the fertilizer applications might be acutely toxic to marine biota, might stimulate nearshore algal blooms, might cause the production of biosurfactants that could result in increased removal of oil from the shorelines by tidal flushing and lead to broader shoreline impacts, or might generate toxic by-products. Careful monitoring following the spill from the Exxon Valdez [91] and a field trial in the Arctic [92] failed to detect any adverse environmental impact from the careful application of fertilizers, while the rate of hydrocarbon biodegradation was stimulated two- to five-fold.
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7. CONCLUSIONS
Oil spill bioremediation technologies epitomize modem environmental techniques: working with natural processes to remove spilled oil from the environment while minimizing undesirable environmental impacts. If a floating oil slick cannot be collected or burnt, chemical dispersants will cause the oil to move into the water column as tiny droplets with a dramatically increased surface area that allows rapid biodegradation. If oil reaches a shoreline and cannot be removed physically, the careful addition of fertilizers will stimulate oil biodegradation without adverse environmental impact. These two tools are thus an important part of the toolkit for dealing with accidental and deliberate releases of oil into the marine environment.
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