Biostimulation Strategies for Enhanced Bioremediation of Marine Oil Spills Including Chronic Pollution

  • Maria NikolopoulouEmail author
  • Nicolas Kalogerakis
Living reference work entry
Part of the Handbook of Hydrocarbon and Lipid Microbiology book series (HHLM)


Biostimulation offers an excellent strategy for combating oil spills following first response actions. Bioremediation rates can be enhanced significantly through the successful stimulation of indigenous degraders with suitable nutrients. The conditions under which biostimulation leads to increased effectiveness are reviewed and strategies for successful biostimulation applications to open sea and near shore environments including chronically polluted sites are suggested.

1 Introduction

As long as society keeps on relying on petroleum hydrocarbons to cover its energy needs, despite the stricter environmental regulations that have been adopted by most countries, oil spills will remain a serious risk to marine ecosystems. Although conventional methods , such as physical removal with booms, skimmers, and absorbent materials, are the first response option, they rarely achieve complete cleanup of oil spills and must be deployed soon after the spill occurs. Chemical methods, particularly dispersants, although they have been routinely used in many countries as a response action, are only allowed when the coastline depth is more than 15 m. In addition, due to their potential toxicity effects on marine organisms they can be applied only under certain conditions. On the other hand, enhanced bioremediation has emerged as a promising technology for combating marine oil spills following first response actions. Intrinsic bioremediation can be enhanced by either of the two complementary approaches, bioaugmentation and biostimulation. In bioaugmentation, the addition of oil-degrading bacteria boosts bioremediation rates whereas in biostimulation, the growth of indigenous hydrocarbon degraders is stimulated by the addition of nutrients (mainly N and P) or other growth-limiting nutrients. Increased addition of N and P sources can result in eutrophication of the marine environment. The common strategy is to design biostimulants which target the oil droplets in the sea water and are not readily diluted and washed out by the wave action. In this work, we present a quick review of existing approaches and current research actions for open sea, near shore, or chronically polluted marine areas by petroleum hydrocarbons.

2 Oil Spill Weathering Processes

Whenever oil is introduced into the marine environment, a series of physical, chemical, and biological processes start taking place that alter the composition and properties of the original oil. As bioremediation is a rather slow process used after conventional cleanup has been applied, the residual oil is often highly weathered before enhanced bioremediation strategies are applied.

The weathering processes include spreading, evaporation, dissolution, photo-oxidation, dispersion, emulsification, biodegradation as well as adsorption onto suspended particulates, sedimentation, and tar ball formation. The combined result of these processes creates a high variability in field studies and difficulties in the evaluation of the efficacy of bioremediation enhancing agents.

This problem can be overcome through the use of biomarkers – practically nonbiodegradable components present in crude oil. The extent of biodegradation is estimated by evaluating the concentration ratio of a target hydrocarbon to one of these recalcitrant biomarkers. Several substances have been used as biomarkers (e.g., pristine and phytane; hopanes and alkylated PAHs isomers) although hopanes have emerged as the best choice (Prince et al. 1994). Hopane normalization is an effective way to distinguish biodegradation from the effects of the physical washout and sand/sediment exchange (Venosa et al. 1996).

3 Biostimulation

Biostimulation refers to the addition of one or more rate-limiting nutrients to accelerate contaminant biodegradation rates. In most shoreline ecosystems that have been heavily contaminated with hydrocarbons, nutrients are likely the limiting factors in oil biodegradation. Spilled petroleum hydrocarbons represent a large carbon source whereas in most marine environments the presence of nitrogen and phosphorous is limited. Oxygen represents another very significant and potentially rate limiting nutrient that should be kept in mind before embarking on a biostimulation field application.

3.1 Water Soluble Inorganic Nutrients

Most laboratory experiments have shown that addition of growth limiting nutrients, namely nitrogen and phosphorus, enhances the rate of oil biodegradation and the optimum ratio of carbon to nitrogen to phosphorus is about 100:10:1. The actual amount of N and P needed for biodegradation of the released hydrocarbons is site-specific as it is associated with the type of oil components and the background value of nutrients in the marine environment.

Xia et al. (2006) studied the effects of different forms of N in seawater polluted by diesel. They found that the addition of NO3-N was more successful than that of NH4-N in accordance with previous studies by Wrenn et al. (1994) where in poorly buffered seawater polluted with Arabian light crude oil, nitrate was found as a better nitrogen source than ammonia. This is attributed to acid production associated with ammonia metabolism which inhibits oil biodegradation. When the culture pH is controlled, the performance of oil biodegradation is similar for both amendments with a shorter lag time for ammonia. With no control of pH, nitrate was found to have the most pronounced effect in stimulating oil degradation when using pristane as a biomarker (Ramstad and Sveum 1995).

Prevailing seawater temperature affects oil biodegradation. Coulon et al. (2007) found that when increasing temperature from 4 °C to 20 °C had a significant effect in all microcosm treatments and the maximum degradation of TPH was observed at 20 °C. Furthermore, addition of N and P resulted in the greatest hydrocarbon degradation. However, these results do not exclude bioremediation as a treatment in polluted arctic environments, as Wrabel and Peckol (2000) showed the effectiveness of nutrients application at coastline temperatures of the western North Atlantic. Biostimulation has been tested and applied successfully to enhance oil biodegradation in cold Arctic, alpine, and Antarctic environments where psychrophilic bacteria are plentiful (Margesin and Schinner 1999).

Commonly used water-soluble nutrient products include mineral nutrient salts (e.g., KNO3, NaNO3, NH3NO3, K2HPO4, MgNH4PO4, Ca(H2PO4)2, Na5P3O10) and many commercial inorganic fertilizers (e.g., the 23:2 N:P garden fertilizer used in the Exxon Valdez case). Typically, they are applied in the field by spraying aqueous nutrient solutions or by spreading dry granules. This approach has been effective in enhancing oil biodegradation in many field trials (Roling et al. 2004; Swannell et al. 1996; Venosa et al. 1996) including Arctic environments (Prince et al. 2003). However, the problem that still remains is that water soluble nutrients are easily washed by wave and tide action, and thus enhanced biodegradation is difficult to achieve in nonsheltered marine environments or medium to high energy shorelines.

3.2 Slow Release Fertilizers

Considerable effort has been devoted to the development of nutrient delivery systems that overcome the washout problems characteristic of open sea and intertidal environments. Use of slow release fertilizers can provide a continuous source of nutrients to oil contaminated areas overcoming the requirement for multiple nutrient applications in the field and resulting in cost benefits compared to water-soluble nutrients due to less frequent application. Slow release fertilizers consist typically of inorganic nutrients in solid form coated with a hydrophobic compound like paraffin or vegetable oil. The most well-known slow-release fertilizer Customblen (vegetable oil coated calcium phosphate, ammonium phosphate, and ammonium nitrate) performed well on some of the shorelines of Prince William Sound, particularly in combination with an oleophilic fertilizer (Atlas 1995; Swannell et al. 1996).

Kasai et al. (2002) investigated the effects of slow release fertilizers (solid granular nitrogen fertilizer (Super IB) and slow-release solid granular phosphorous fertilizer (Linstar 30)) on oil biodegradation. The addition of fertilizers promoted the degradation of certain components of crude oil: more than 90% of n-alkanes (C15–C30) and more than 60% of (alkyl)naphthalenes were degraded within 30 days, whereas the degradation of three-ring aromatics (phenanthrene, anthracene, fluorene, and their alkylsubstituted derivatives) was less extensive, being between 30% and 40%. In contrast, Maki et al. (2002, 2003) found that alkanes degraded to a lesser extent than naphthalenes or fluorenes and to almost the same extent as dibenzothiophenes and phenanthrenes in field experiments performed in sand and cobble stone beaches of Japan after Nakhodka oil spill. However, in both laboratory and field experiments the final degradation efficiencies for each oil component in the fertilized sections were not significantly different from those in the unfertilized sections, and the degradation of each oil component had almost ceased after 6 weeks. It was concluded that excessive amounts of macronutrients are required to accelerate oil biodegradation and under these conditions fertilization is only effective in the early stages.

The challenge that still remains in applying slow release fertilizers is to control the release rates so that suitable nutrient concentrations can be maintained over longer periods of time in the marine environment. Fast release rates do not provide a long-term source of nutrients, whereas very slow release rates are insufficient to enhance biodegradation rates. For example, Sveum and Ramstad (1995) tested Max Bac, a slow release fertilizer similar to Customblen, and found that it failed to enhance oil biodegradation significantly due to its slow release rate. On the other hand, if one uses a mixture of water soluble and slow release fertilizers in one application better results are obtained.

3.3 Oleophilic Fertilizers

An alternative strategy to overcome the problem of quick dilution and wash out of water-soluble nutrients containing nitrogen and phosphorus is the use of oleophilic biostimulants . The application of N and P sources in oleophilic form is considered to be a more effective nutrient application method, since oleophilic additives remain dissolved in the oil phase and thus are available at the oil-water or oil-sediment interface where they enhance bacterial growth and metabolism (Santas and Santas 2000).

The most well-known oleophilic fertilizer is Inipol EAP22 , a microemulsion containing urea as N-source, lauryl phosphate as P-source, 2-butoxy-1-ethanol as a surfactant, and oleic acid to give the mixture its hydrophobicity. This fertilizer has been subjected to extensive studies under various shoreline conditions and was successfully used in oil bioremediation on the shorelines of Prince William Sound (Swannell et al. 1996; Zhu et al. 2001). Another oleophilic fertilizer that was used extensively at the Prestige heavy fuel oil spill is S200 which differs from Inipol EAP22 only in the formulation of the surfactant component (Díez et al. 2005; Jiménez et al. 2006). Díez et al. (2005) observed enhanced biodegradation of the Prestige fuel oil in microcosms containing S200 compared with those containing inorganic phosphorous and nitrogenous salts. These results led to a bioremediation field assay at a cobblestone mixed with sand and gravel beach on the Cantabrian coast (north Spain) using S200. A rigorous control of biodegradation of aliphatic and aromatic hydrocarbons using internal conservative molecular markers for 220 days showed an acceleration of biodegradation at 30–60 days and an enhancement of biodegradation, especially of the heavier n-alkanes (C25–C35) and the alkylated PAHs (Jiménez et al. 2006). Other oleophilic fertilizers include polymerized urea and formaldehyde, and organic fertilizers derived from natural products such as fishmeal, meat meal, lecithin, and uric acid . Uric acid which is the major component of guano fertilizer has been effectively used for crude oil degradation in a simulated open system resulting in 70% petroleum degradation (Knezevich et al. 2006). In a recent study, Gertler et al. (2015) investigated metabolic processes and microbial community changes in a series of microcosms where it was observed that about 80% of uric acid was converted to ammonium within the first few days of the experiment. Experimental data suggested that strains related to Halomonas spp. converted uric acid into ammonium, which stimulated the growth of hydrocarbon degrading microbial consortia.

The effectiveness of oleophilic fertilizers depends on the characteristics of the site such as type of sediment or high/low energy wave action and tide. From early on it was shown that oleophilic fertilizers can be more effective than water-soluble fertilizers when the spilled oil resided in the intertidal zone (Sveum et al. 1994); however, no enhancement of biodegradation rates was observed in zones of limited water transport. Variable results have also been produced regarding the persistence of oleophilic fertilizers. Some studies showed that Inipol EAP22 can persist in a sandy beach for a long time under simulated tide and wave actions (Santas and Santas 2000; Swannell et al. 1995); however, experience from very high energy shorelines even oleophilic fertilizers can be rapidly washed out. It is noted that addition of rhamnolipid biosurfactants alone had little effect on biodegradation; however, in combination with water soluble nutrient additions, provoked a significant increase (McKew et al. 2007; Nikolopoulou et al. 2013a) and even greater increase when combined with oleophilic fertilizers (Nikolopoulou et al. 2013b). Sole biosurfactant addition is warranted only to increase bioavailability of weathered petroleum components in situations where background levels of N and P are relatively high.

Many researches have compared the effectiveness of these nutrient products to stimulate oil biodegradation rates. The variable results from laboratory and filed studies indicate the importance of prevailing local conditions. Water-soluble fertilizers are likely more cost-effective in low-energy and fine-grained shorelines and generally sheltered sites where wash out is limited. On the other hand, slow-release fertilizers may be ideal nutrient sources if the nutrient release rates can be well controlled and the nondissolved particles cannot be washed out by the wave action. Finally, oleophilic fertilizers may be more suitable for use in higher-energy, coarse-grained beaches and generally exposed sites and open sea environments. Biostimulation with nutrients and biosurfactants enables naturally occurring microbes to adapt better and faster to the oil spill environment resulting in shorter lag phase and faster crude oil degradation (Nikolopoulou and Kalogerakis 2008), thus making it an effective tool for combating oil spills.

3.4 Oxygen as a Rate Limiting Substrate

Despite the apparent effectiveness of oleophilic fertilizers or mixed products, no enhancement of oil biodegradation rates should be expected if they are added to an anoxic marine environment. In several instances, the concentration of dissolved oxygen can be close to zero leading to practically zero aerobic biodegradation rates. It should be noted that although anaerobic biodegradation of hydrocarbons has been documented in marine environments, the actual rate is particularly low. Although oxygen can be successfully delivered (in various forms) to hydrocarbon-contaminated soils and groundwater enhancing biodegradation rates, this is not the case in marine environments as it is very difficult to implement such technologies in the field. Tiling is essentially the only option in aerating the top layers of contaminated sediments during low tide.

4 Biostimulation Strategies for Chronically Polluted Sites

What should our action be when faced with a chronically polluted marine site? Before proceeding with the addition of slow release or oleophilic fertilizers, one should make sure that such an action will lead to an increase in biodegradation rates . To answer this question, one must consider the following cases:
  1. 1.

    Is it an old pollution event or is there continuous seeping of petroleum products into the chronically polluted site? The later is often encountered near petroleum refineries or crude/refined oil storage tanks sites located on the shoreline where we may have intermittent seeping (following rainfall patterns and changes in groundwater levels) of petroleum products that are present as free product (typically as LNAPLs) on top of contaminated groundwater bodies into the seawater through seeps at the bottom of the sea in close proximity to the shore. If this area is not exposed to high waves, there is little mixing action, and the petroleum contamination appears to be permanent. In such cases, addition of fertilizers is expected to reduce the visible pollution; however, it will not solve the problem. The source of the contamination should be contained and the free product recovered using well known and tested technologies (e.g., funnel and gate systems, bioslurping and hydraulic walls) before it reaches the seawater. Then, biostimulation with mixture of water soluble and slow release fertilizers should help in the complete bioremediation of the site. Such sites are often low energy environments, and hence there is no need for oleophilic formulations.

    Another situation that needs to be considered is the continuous release of transportation fuel from a shipwreck at the bottom of the sea. Again, if we have a chronically polluted site, it must be a low energy environment where the floating oil phase is not quickly dispersed by the wave action and it may accumulate on the shoreline. In such an environment, again biostimulation with water soluble slow release fertilizers is a good option to increase biodegradation rates.

  2. 2.

    If the chronic pollution is due to an old incident, there is no continuous source of hydrocarbons and the site appears to have very minimal recovery, then one must consider (i) lack of a rate limiting nutrient , (ii) excessive toxicity due to the presence of other organic compounds or heavy metals, or (iii) limited bioavailability of the contaminants. The second is often related to sheltered marine environments (low energy wave areas) like commercial harbors near industrial zones. The management of contaminated sediments from a variety of chemicals is a complex issue that should not aim on just meeting simple chemical thresholds, but instead it should aim at a basin-scale good ecological status and should include ecological risk assessment of sediment-bound chemicals on aquatic biota (Apitz et al. 2005). Bioremediation of contaminated sediments is beyond the scope of this review. However, biostimulation can be an important tool when dealing with chronically polluted sediments from petroleum hydrocarbons due to an oil spill. The role of biosurfactants can be significant to increase the bioavailability of the heavier hydrocarbons (Banat et al. 2010; Antoniou et al. 2015).

    In addition, one should consider the type of shoreline in question, e.g., fine-grained or coarse-grained sand beach, gravel beach, exposed tidal flat, sheltered rocky shore, sheltered tidal flat, or the most sensitive salt marshes and mangroves. The least sensitive exposed rocky shores tend to recover from oil spills within a few months, whereas the very sensitive salt marshes can maintain petroleum contamination for many years. Tidal pumping enhances oil penetration into the sediments. Permeability and porosity of the sediments dictate the rate and depth of oil penetration. On coarse-grained beaches, oil can penetrate deeper and remain longer (when it is trapped below the limit of wave action) compared to finer grained sediments such as silts and clay. However, oil is more easily removed by water flushing from coarse-grained sediments (Zhu et al. 2001). From the above, we can conclude that chronic petroleum pollution can only occur on beaches where the wave/tidal action is not strong. The addition of nutrients is only warranted when oxygen is not a limiting substrate. If oxygen is limiting, no significant gains are expected through biostimulation.


There are no viable options available to oxygenate sediments except simple tilling which is a physical method used routinely in “landfarming” for the management of petroleum sludges. As an alternative, solid peroxygen materials like calcium or magnesium peroxide (CaO2 and MgO2) or calcium superoxide (CaO4) which contains higher percentage of stored oxygen than CaO2 can be applied. These techniques appear to be the only low cost means of aeration of the top layer of sediments. If measured concentrations of N and P are very low, biostimulation should also be employed.

The distribution of hydrocarbon degraders is strongly related to the historical exposure of the environment to hydrocarbons. Marine environments with chronic oil contamination will have a much higher percentage of hydrocarbon degraders than nonpolluted sites. In pristine environments, hydrocarbon degraders are extremely low in concentration; however, several studies have shown that starting from pristine seawater, addition of petroleum hydrocarbons with suitable addition of N and P sources leads to an explosion in the population of hydrocarbon degraders in a short number of days [e.g., Cappello et al. 2007].

5 Research Needs

Although the problem of combating marine oil spills has been studied extensively for several decades, there are several areas that could benefit from further research developments. These are:
  • Development of low cost oxygenation systems for aerobic bioremediation of contaminated anoxic sediments.

  • Development of novel biostimulants that are nontoxic to the marine environment, for example, by increasing in situ production of rhamnolipids at the oil-water interface.

  • Development of novel oleophilic amendments with better transport characteristics for application in cold shoreline environments.

  • Further increase our understanding of the function of microbial biofilms that develop around an oil droplet and its relationship to different types of biostimulants.



This work was funded by EU FP-7 PROJECT KILL•SPILL – “Integrated Biotechnological Solutions for Combating marine Oil Spills” (Grant agreement No. 312139).


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Copyright information

© Springer International Publishing AG 2016

Authors and Affiliations

  1. 1.School of Environmental EngineeringTechnical University of CreteChaniaGreece

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