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5.2.2 Accelerated Biodegradation

Naturally occurring hydrocarbon-degrading bacteria are found in all environments.  Although these bacteria are capable of initiating the biodegradation of spilled crude oil, attempts have been made to increase removal efficiency through bioremediation strategies.  Most bioremediation attempts have focused on developing good biostimulation strategies, typically by applying degradation rate-limiting nutrients, or the combination of these and other treatments, to accelerate the natural biodegradation processes.  Bioremediation processes, if successful, are cost-effective and reduce the environmental impacts of marine oil spills (Prince 1993; Swannell et al. 1996; Prince and Clark 2004; Prince 2005; Prince and Atlas 2005).  An alternative to biostimulation is bioaugmentation which involves the inoculation of indigenous or exogenous microbial cultures with high biodegradation potentials for contaminants.  Bioaugmentation approaches have been reported to improve biodegradation of hydrocarbons from oil spills in cold soil or marine sediments, and can be used in combination with fertilizers (Margesin and Schinner 1997; Ruberto et al. 2003).  Bacterial mats from marine oil-contaminated sites have also been suggested for use in the degradation of coastal oil spills, although these are of greater relevance for spills in temperate areas (Cohen 2002).  Various methods and strategies for bioremediation have been reviewed (e.g. Lee and Merlin 1999; Prince 2010).

To date, most experimental oil bioremediation studies in Arctic or Antarctic environments have been conducted on stranded oils, employing the application of fertilizers to stimulate the indigenous flora, and often in combination with mechanical treatments which improve oxygen and nutrient availability (e.g. Sveum and Ladousse 1989; Prince et al. 2003a; Obbard et al. 2004; Pelletier et al. 2004). Bioremediation strategies have also been applied to real oil spill situations such as the Exxon Valdez accident in 1989, where it formed part of a beach cleaning strategy (Bragg et al. 1994).

Also the use of chemical dispersants is regarded as an effective way of stimulating biodegradation of oil.  The dispersants work as surfactants, changing the surface characteristics of the oil, and reducing the droplet size.  Chemical dispersants have been used primarily on surface oil spills, but were also injected directly (Corexit 9500) at the wellhead during the Deep Water Horizon (DWH) spill (Atlas and Hazen 2011). Biostimulation

Biostimulation includes the addition of nutrients or other methods to enhance the capability of the indigenous microbial communities to degrade pollution components.  Biostimulation has been regarded as a cost-effective strategy for secondary cleanup mainly of stranded oil pollution.  However, other methods may also be regarded as biostimulatory actions, for instance the use of chemical dispersants for oil spills in seawater, since this may aid in oil biodegradation by increasing the oil surface area accessible to the oil-degrading microbes. Shoreline sediments

Most biostimulation activities have focused on stranded oil, with application of fertilizers to increase natural degradation by the indigenous microbial flora.  Biostimulation treatment is often combined with mechanical treatment to improve oxygen and nutrient availability.

In marine environments some growth- and biomass-stimulating factors are essential for oil biodegradation, especially nitrogen and phosphorous and the addition of these nutrients are common practice in bioremediation.  Balanced nutrient availability is important for biodegradation and the composition of hydrocarbon-degrading communities, since nutrient amendments, in some instances, can inhibit microbial activity (Braddock et al. 1997).  It is therefore important to avoid excess nutrients, which can cause detrimental effects, such as eutrophication.  During biostimulation, molar carbon/nitrogen/phosphorous ratios of 100/10/1 have often been used (e.g. Bouchez et al. 1995; Obbard et al. 2004).  However, results from laboratory studies have also shown that certain microbial populations may require different N/P ratios for optimal degradation of different hydrocarbons (Smith et al. 1998). Nutrient products are available as briquettes, granules or liquid fertilizers.  Liquid inorganic fertilizers have proven effective but require frequent application, and therefore oleophilic slow-release nutrient formulations have been developed, which promote hydrocarbon degraders at the oil-water interface.

For improved results bioremediation may be combined with other clean-up procedures.  Surf washing and the use of surfactants may increase the surface area of the oil and hence increase oil degradation. Ex situ technologies like land farming (spreading the polluted sediments over a larger area for better oxygenation), composting and biopiling may be used for treating oily waste during spill treatment (Lynch and Moffat 2005) although these approaches have seen limited application in polar environments.

Several field biostimulation trials have been conducted on Arctic beaches at Spitzbergen, either in Ny Ålesund (78° 55' N, 11° 56' E) or in the Van Mijen fjord close to the small mining society Svea (77°56’N, 16°43’E).  Experiments performed by SINTEF and the oil company ELF in the 1980s in Ny Ålesund with the slow-release oleophilic fertilizer Inipol EA22 indicated that application of the fertilizer to oil in beach sediments resulted in increased biodegradation in coarse sediments, but not on oil in finer sediments (Sveum and Ladousse 1989).  During a full-scale trial at the ITOSS (In Situ Treatment of Sediment) program in 1997 several remediation processes were tested on intermediate fuel oil (IF-30) artificially stranded on mixed (sand and pebble) intertidal shorelines.  The remediation methods included sediment relocation (surf washing), mixing (tilling) and bioremediation (Guenette et al. 2003; Sergy et al. 2003).  The fertilizers used included both soluble (prilled ammonium nitrate and superphosphate [Ca(H2PO4)2]) and commercial slow-release (Inipol SP1) chemicals, applied to the top of the sediments during the first two months of the experiment.  The introduction of fertilizers resulted in elevated levels of bioavailable nitrogen and phosphorous in oiled sediments.  The biodegradation rates were approximately doubled over a period of one year in the oiled sediments that received fertilizers when compared to non-treated oiled sediments, and no acute toxicity was associated with the bioremediation treatment (Prince et al. 2003).  Mixing/tilling also seemed to result in increased microbial activity for limited periods by increasing sediment permeability (Owens et al. 2003).

Biostimulation field experiments have also been conducted in Antarctic environments. An Arabian crude oil was added to several 1 m2 enclosures on intertidal sandy beaches on the main island of the Kerguelen Archipelago (49°19’S, 69°42.5’E).  Different fertilizers were added to the top of the oil, including the slow-release Inipol EAP 22 and various experimental mixtures consisting of dry fish compost, with or without supplements of urea, phosphate and charged or neutral surfactants (Pelletier et al. 2004).  During a 300-day experiment the oil was eventually depleted in both untreated and treated sediments in this cold environment (seawater temperatures 3-4°C), but the various fertilizers accelerated the biodegradation rates.  It was also observed that a fertilizer with a neutral surfactant reduced the toxicity of the oil during the last three months of the experiment (Delille et al. 2002).

Bioremediation was used as an oiled-beach cleaning technology on a full-scale oil spill in Arctic environments during the Exxon Valdez accident in March 1989.  This spill in Prince William Sound, Alaska, resulted in the release of 41 million liters of Alaskan North Slope crude oil.  Bioremediation was used extensively, employing the fertilizers Inipol EAP 22 and Customblen (slow-release granulated fertilizer).  Approximately 50,000 kg nitrogen and 5,000 kg phosphorous were applied to the shorelines over the summers of 1989-1992 (Bragg et al. 1994).  For a low-energy beach containing both surface and subsurface oil and treated with both fertilizers, it was estimated that the fertilizers enhanced oil biodegradation by 5.5 times over non-treated controls (Bragg et al. 1994).

Results from this bioremediation field experiment, supplemented with several laboratory experiments have been summarized by Pritchard et al. (1992):

  • Fertilizers with slow release nutrients were recommended in tidal zones to avoid rapid wash away of the fertilizer components.  Solid granulated fertilizers were easier to apply over large areas, and adhered well to the oiled beach material. These fertilizers persisted over periods of 2-3 weeks after application.  Also fertilizer briquettes were tested with outcomes similar to the granules.  Burial of fertilizer material parallel to the water line has also been suggested.
  • Components of the nitrogen and phosphorous from liquid Inipol EAP 22 seemed to be released rapidly when submerged in seawater.  However, residual nutrients remained and were available for oil-degrading bacteria.  Laboratory experiments indicated that the nitrogen part of the oleophilic fertilizer was more important than the phosphorous compounds for biodegradation.
  • Inspection of areas treated with Inipol EAP 22 showed faster visual disappearance of oil from cobble surfaces than untreated surfaces, and laboratory experiments indicated biodegradation as the cause for disappearance rather than washing.  However, oil under the cobbles remained. It was also suggested that Inipol could work as a surfactant by chemically removing aliphatic hydrocarbons from samples.
  • Measurements of bioremediation effectiveness should include analyses of several hydrocarbon groups, in addition to the aliphatic hydrocarbons.  Measuring compositional changes in the aromatic fraction by GCMS was therefore added as a further dimension to biodegradation.  Bioremediation should also result in removal of oil residues, measured as a reduction in total mass of the oil. 
  • The use of common indicators of biodegradation like nC18/Phytane was problematic since phytane in some instances disappeared at the same rate as nC18 alkane (phytane-degrading microbes were actually isolated from beach material).  For aromatics a number of methyl-substituted homologues close in mass number to their parent structures were selected, and these were normalized to hopane, which are resistant to biodegradation.
  • Results from oil compound analyses showed that reduction in nC18/Phytane ratios resulted in corresponding changes in aromatic and heterocyclic hydrocarbons.  Samples with reduced nC18/Phytane ratios also showed increased concentrations of compounds associated with asphaltenes and polar materials, which may be partially biodegraded material.  Further, there was a positive correlation between nC18/Phytane reduction and removal of residual oil, determined by mass.  It was therefore suggested that reduction in oil residues could be used as a measure of biodegradation.

Interactions between stranded oil and mineral fines have been used to reduce oil adhesion to solid surfaces and generate stable oil droplets released to the water column.  In this way the increased surface area of oil became more accessible to nutrients, oxygen and bacteria, resulting in increased microbial activity and oil biodegradation (Lee et al. 1996). Seawater

Efforts to stimulate crude oil biodegradation in seawater and ice (see next section) have not been investigated to the same extent as for stranded oil.  Most remediation strategies in oiled seawater have focused on mechanical removal methods such as the use of oil booms and skimmers.  In addition to mechanical methods, chemical dispersants have also been widely used as an alternative treatment. Dispersants are mixtures of surface-active chemicals that reduce the surface tension of the oil, resulting in the formation of oil droplets that are smaller than those initially generated by mechanical wave action (Brandvik 1997). These chemicals are primarily used to disperse oil spilled on the seawater surface into the water column.  This approach aims to reduce the impacts of oil spills on seabird and mammal populations in the vicinity of the spill and help prevent the oil reaching the coastline.  In addition, this process increases the oil-water interface and generates more bioavailable surface area for microorganisms.

The efficiencies of chemical dispersants on crude oil degradation at low seawater temperatures have shown conflicting results. In a seawater mesocosm experiment (3.5 m3 flow-through tanks with natural seawater from the St. Lawrence Estuary, Quebec) with temperatures of ‑1.8 to 5.5°C, Forties and Western Sweet Blend crude oils were treated with the dispersant Corexit® 9527 or different surfactant mixtures.  Over a 63-day period at water temperatures >0°C, chemical dispersal was found to result in higher biodegradation rates than in untreated oil samples (Siron et al. 1995).  In microcosm studies with a seawater temperature of 8°C, MacNaughton et al. (2003) added the dispersant Corexit 9500 to Alaskan North Slope (ANS) crude oil.  The dispersant resulted in rapid colonization of oil droplets by bacteria, and heterotrophic and oil-degrading microbes proliferated in the microcosms.  However, when the total hydrocarbons, C11 to C35 n-alkanes, or the sum of selected aromatics were assessed over a degradation period of 35 days, the addition of dispersant resulted in only slow or negligible biodegradation of the oil when compared to naturally dispersed oil.  In another biodegradation experiment, Lindstrom and Braddock (2002) exposed cultures of oil-degrading microbes to ANS (fresh or evaporated and spiked with radiolabelled hydrocarbons) dispersed with Corexit® 9500 over a 2 month period at 8°C.  Respirometric analyses were conducted to determine 14CO2 mineralization of 14C-labelled dodecane, hexadecane, 2-methyl-naphthalene and phenanthrene. The dispersant was found to inhibit degradation of some of the hydrocarbons (hexadecane and phenanthrene), while others (dodecane and 2-methyl-naphthalene) were unaffected when compared to mineralization of the oil without dispersant.  It was suggested that carbon mineralization, at least initially, was the result of dispersant mineralization rather than degradation of the oil compounds. In a biodegradation study comparing two dispersants, Corexit® 9500 and JD2000, fresh Prudhoe Bay crude oil was mixed with the dispersants and a microbial culture originating from the shorelines contaminated during the Exxon Valdez accident.  Biodegradation of n-alkanes and PAHs was measured over a period of 46 days at 5°C.  The first-order degradation rates of most n-alkanes and PAHs were found to be higher with dispersants than without dispersants, although these data were not statistically significant.  When the same experiment was performed at 20°C the degradation rates of n-alkanes and PAHs were more rapid than at 5°C, showing temperature-related biodegradation rates both in non-dispersed and dispersed oil. Furthermore, the influence of the two dispersants on biodegradation differed between the two degradation temperatures (Venosa and Holder 2007).  Correlation studies between the droplet surface area of dispersed oil and the resulting degradation indicated that both dispersed area and dispersant chemistry controlled the degradation and that the surfactant blend hydrophile-lipophile balance and treatment levels were also significant controlling factors (Varadaraj et al. 1995).  Recently, Prince and Butler (2013) suggested that the variability in results may result from low oil concentrations in a physically dynamic experimental system which would act to form only slightly larger oil droplets than dispersed oil treatment.  Additionally, the authors indicate that an accelerated rate of biodegradation occurs when a dispersed oil treatment is compared to biodegradation of a surfaced oil, principally due to relatively small droplet size.

A variety of bacteria and yeasts also produce biosurfactants like rhamonolipids, sophorolipids and surfactin.  Biosurfactants consists of fatty acid hydrophobic parts and carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid or alcohol as hydrophilic part (Mulligan 2005). Most biosurfactants are produced from hydrocarbon substrates (Syldatk and Wagner 1987).  It has been suggested that rhamnolipis addition can enhance biodegradation of hydrocarbon mixtures in liquid systems and soil (Maier and Soberon-Chevez 2000).  Two mechanisms for enhanced biodegradation were proposed; enhanced substrate solubility and interactions with the cell surface to increase the hydrophobicity of the cell surface, allowing improving association of hydrophobic hydrocarbons (Mulligan 2005).  Combined use of rhamnolipids and slow-release fertilizers (Inipol EAp-22) also enhanced biodegradation of aromatic and aliphatic hydrocarbons in liquid phase and soil (Churchill et al. 1995).  Studies withRhodococcus sp. Q15 grown on hexadecane or diesel fuel at 5°C showed production of biosurfactants at low temperature (5°C), indicating that the cell surfaces became more hydrophobic  (Whyte et al.1999).

In addition to chemical dispersants use of fertilizers to increase oil compound biodegradation has been investigated to some extent.  As early as in the 1970s enhanced biodegradation of oil spills by lipophilic slow-release fertilizers was investigated (Olivieri et al. 1975).  In a study with hydrocarbon-degrading bacteria (Alcanivorax sp.) and an open seawater-based system at 30°C guano was used as fertilizer.  It was demonstrated that commercial guano was an effective source of nitrogen and phosphorus for the growth of bacteria on crude oil, and that the guano resulted in extensive biodegradation of crude oil (n-alkanes (C10- C36) and polyaromatics at (Knezevich et al. 2007). Marine ice

Bioremediation of oil in ice is an intriguing prospect. If biodegradation of crude oil could be stimulated in ice, especially for the most toxic compounds migrating out of the ice through the brine channels, this would be of benefit for organisms inhabiting the polluted ice or nearby areas.   Studies have shown that fertilizers can stimulate biodegradation of crude oils in cold seawater under controlled experimental conditions (Delille et al. 1998). The slow-release oleophilic fertilizer Inipol EAP 22 was added to Antarctic seawater contaminated with “Arabian light” crude oil in a mesocosm study.  The experiment was completed over 5 weeks during the Austral summers of 1992/1993 and 1993/1994. In both ice-covered and ice-free seawater, the addition of the fertilizer enhanced both the concentrations of heterotrophic and hydrocarbon-degrading bacteria and increased the rate of biodegradation during the experiments, measured as n-C17/Pristane and n-C18/Phytane ratios.

A winter field experiment was conducted at Svalbard in 2004 as part of the Arctic Operational Platform (ARCOP) program.  Crude Statfjord oil with and without fertilizers (mixture of Inipol EAP 22 and fish meal) was placed in fjord ice (Van Mijen Fjord, Svea) for a period of 6 months (December 2004-June 2005).  At sub-zero temperatures no significant degradation of oil hydrocarbons occurred with the addition of nutrients, but at 0°C melt pool samples fertilized with inorganic nutrients showed a significant change in bacterial diversity (Gerdes and Dieckmann 2006).  Importantly, many of the available slow-release fertilizers are not suitable for use in Arctic regions as they will solidify if used in ice at very low temperatures. For example, the pour point of Inipol EAP 22 is 11°C which makes it difficult to use effectively under Arctic conditions.  As a result slow-release fertilizers will require reformulation or new products will need to be developed specifically for use at very low temperatures.

A few studies have also been conducted to determine the impacts on ice protist communities after oil contamination and subsequent fertilizer treatment.  One of these studies formed part of the ARCOP field trial on Svalbard.  In oil-contaminated ice (no fertilizers) the protist communities were destroyed through complete ice coverage.  Upon addition of fertilizers a less pronounced decline of organisms in the ice interior was observed. Thus, the use of fertilizers (Inipol and fish meal) helped to maintain higher diversity and biomass of protists in the ice. In a separate study, heterotrophic flagellates appeared to escape or avoid the oil contamination by downward migration (Ikävelko et al. 2005).  In an Antarctic field experiment conducted during the Austral winter of 1993, land-fast ice on the continental shelf of Terre Adélie was contaminated with crude oil (Arabian light) or diesel fuel, and negative effects on the ice microalgae were determined by chlorophyll A measurements.  In crude oil-contaminated ice, negative effects were induced which lasted throughout the ice coverage period.  The diesel contamination studies were found to cause an even more rapid effect on the algae than the crude oil. However, the addition of the fertilizer Inipol EAP 22 resulted in clearly favorable effects on the sea ice microalgae (Fiala and Delille 1999). Bioaugmentation

Bioaugmentation has been proposed as a bioremediation method for soil and sediments, often as a supplement to biostimulation treatments. Introduction of exogenous hydrocarbonoclastic bacteria for detoxification of hydrocarbon-polluted cold environments has been reported, with variable success. A number of commercial products exist, which include microbial inocula.  These products are often lacking essential information about the bacterial content.  National authorities may also be skeptical about using these products without proper product information.

In a study of diesel oil-contaminated Alpine soil, a psychrophilic diesel oil-degrading inoculum was added to the contaminated soil, but biostimulation with fertilizers proved more efficient than the bioaugmentation for improved biodegradation activity (Margesin and Schinner 1997).  In microcosm experiments performed in Antarctic gas-oil polluted soil (Jubany Station, King George Island, South Shetland Islands) inoculation of the psychrotolerant strain B-2-2 resulted in 75% hydrocarbon removal, whereas 35% hydrocarbon removal was observed by biostimulation methods when compared to abiotic controls (Ruberto et al. 2003).

Several bioaugmentation studies from marine environments have been reported, although none of these are from cold waters.  A laboratory biodegradation and toxicity study of 12 commercially available bioaugmentation products applied to weathered oil (Alaska North Slope) in seawater at 20°C showed that 3 of the products enhanced biodegradation more than nutrient-amended controls, but only one product resulted in reduced toxicity (Aldrett et al. 1997).  In a marine sediment microcosm study the aromatic-degrading bacterial strain Cycloclasticus sp. E2 was shown to play an important role during degradation of naphthalene in combination with biostimulation treatment (Miyasaka et al. 2006).  Interestingly, bacteria from this genus were also abundant during bioremediation treatment of Arctic oiled beaches at Svalbard (Grossman et al. 2000).

Bioaugmentation has often proved inferior to biostimulation.  One plausible explanation for this may be that the introduced bacteria will have an immediate effect due to the biomass added, but these exogenous microbes may gradually be outcompeted by the indigenous microbes adapted to the local environment.   In a study with small- and mesoscale systems, addition of inorganic nutrients was more efficient at enhancing oil biodegradation in sediments than a commercial product consisting of nutrients and bacterial inocula.  The use of the bioaugmentation product suppressed both the rate and the extent of oil loss by tidal activity and biodegradation, when compared to the periodic addition of inorganic nutrients (Lee et al. 1997). Understanding Processes in Accelerated Biodegradation

Purposeful acceleration of biodegradation through, for example, ecological engineering requires an understanding of the microorganisms that are present in the local environment and their responses to stimuli such as those outlined in the previous sections.  Advances have been made in this area, including development of techniques and submersible vehicles that allow evaluation of biodegradation in situ, improvements in laboratory equipment to simulate the natural environment, maturation of molecular methods to track population dynamics during the degradation process, and development of techniques that allow better understanding of functions carried out by those populations.  These topics were dealt with in more detail in the analytical methods subsection of this section entitle Determining Biodegradation section.  The –omics approaches are of particular importance to accelerated biodegradation.  The effects of management practices on microbial populations and functions, with resulting effects on the components of degraded oil and rates of degradation, can begin to be understood through the emerging fields of metagenomics, metaproteomics, and metabolomics.  These techniques promise to provide much greater understanding of microbial responses, which in turn will allow evaluation and management of oil degradation processes in the Arctic environment.