Bioremediation Of Marine Oil Spills Research Articles

Performance evaluation of rhamnolipid biosurfactant produced by Pseudomonas aeruginosa and its effect on marine oil-spill remediation.

This study aimed to reveal that the effect of biosurfactant on the dispersion and degradation of crude oil. Whole genome analysis showed that Pseudomonas aeruginosa GB-3 contained abundant genes involved in biosurfactant synthesis and metabolic processes and had the potential to degrade oil. The biosurfactant produced by strain GB-3 was screened by various methods. The results showed that the surface tension reduction activity was 28.6 mN·m-1 and emulsification stability was exhibited at different pH, salinity and temperature. The biosurfactant was identified as rhamnolipid by LC-MS and FTIR. The fermentation conditions of strain GB-3 were optimized by response surface methodology, finally the optimal system (carbon source: glucose, nitrogen source: ammonium sulfate, C/N ratio:16:1, pH: 7, temperature: 30-35°C) was determined. Compared with the initial fermentation, the yield of biosurfactant increased by 4.4 times after optimization. In addition, rhamnolipid biosurfactant as a dispersant could make the dispersion of crude oil reach 38% within seven days, which enhanced the bioavailability of crude oil. As a biostimulant, it could also improve the activity of indigenous microorganism and increase the degradation rate of crude oil by 10-15%. This study suggested that rhamnolipid biosurfactant had application prospect in bioremediation of marine oil-spill.

Bioaugmentation enhance the bioremediation of marine crude oil pollution: Microbial communities and metabolic pathways.

Bioaugmentation is an effective strategy used to speed up the bioremediation of marine oil spills. In the present study, a highly efficient petroleum degrading bacterium (Pseudomonas aeruginosa ZS1) was applied to the bioremediation of simulated crude oil pollution in different sampling sites in the South China Sea. The metabolic pathways of ZS1 to degrade crude oil, the temporal dynamics of the microbial community response to crude oil contamination, and the biofortification process were investigated. The results showed that the abundance and diversity of the microbial community decreased sharply after the occurrence of crude oil contamination. The best degradation rate of crude oil, which was achieved in the samples from the sampling site N3 after the addition of ZS1 bacteria, was 50.94% at 50 days. C13 alkanes were totally oxidized by ZS1 in the 50 days. The degradation rate of solid n-alkanes (C18-C20) was about 70%. Based on the whole genome sequencing and the metabolites analysis of ZS1, we found that ZS1 degraded n-alkanes through the terminal oxidation pathway and aromatic compounds through the catechol pathway. This study provides data support for further research on biodegradation pathways of crude oil and contributes to the subsequent development of more reasonable bioremediation strategies.

Field studies on monitoring the marine oil spill bioremediation site in Chennai

Oil spills have become a threat to the ecosystem by releasing the petroleum hydrocarbons and gained substantial public concern in the Chennai coast. This study assesses the effectiveness of bioremediation and its impact on the environment due to remedial operations in the site. Soil and water samples were collected from the bio remediation site at regular intervals of the pit from the topsoil and 20 cm below the ground level from June 2017 to Nov 2019. The average TPH concentration present in the bottom and top soil of bioremediation pit were vary in the range of 21,238.4–46,600 mg/kg and 17577–26910 mg/kg. Central Pollution Control Board (CPCB) allowable limit for TPH concentration present in the soil should be 5000 mg/kg. We have also observed that the mixing was not uniform in the pit and major amount of oil has been penetrated deep inside the soil. Results on gravimetric analysis showed that there was still a large amount of untreated long-chain hydrocarbons are there in the pit. From the results, we can conclude that nC30-nC40 and lower carbon range alkane intermediates have to be treated with additional treatment like thermal smoldering and pyrolysis.

Bioremediation of marine oil spills by immobilized oil-degrading bacteria and nutrition emulsion.

The combination of bioaugmentation and biostimulation was used to speed up the bioremediation of marine oil spills. A novel carrier material that consisted of puffed panicum miliaceum (PPM), calcium alginate and chitosan was prepared. The porous structure and low density of PPM ensured this carrier material not only had appropriate physical and biological properties for the aggregation of microorganisms but also was biodegradable and floating on the seawater surface for bioremediation of oil pollution. An oil-degrading bacterial consortium was immobilized via adsorption on the carrier material. The immobilized bacteria were observed with scanning electron microscopy. The number of viable cells immobilized on the material was approximately 1.12 × 108CFU/g. To solve the problem of nutrients supplementation in seawater, an emulsion formed with urea solution, soybean lecithin, alcohol and oleic acid was prepared as oleophilic fertilizer. The results from laboratory and field mesocosm experiments showed that the combination of immobilized bacteria and the emulsion achieved a higher oil removal efficiency compared with the use of them separately. The results of field mesocosm experiments conducted in the coastal seawater showed that most of the petroleum pollutant (> 98%) was removed from the surface of seawater in 24h. GC-MS analysis showed that most components of petroleum pollutants had been removed. This formula with immobilized bacteria and emulsion can be exploited further for the bioremediation of marine oil spills.

Microbial-induced biosurfactant-mediated biocatalytic approach for the bioremediation of simulated marine oil spill

Marine crude oil spill is the most alarming environmental issue and the dreadful instance of petroleum hydrocarbon pollution around the world. Successful remediation of these toxic pollutants demands for the ample production of biosurfactant and biocatalysts by the native hydrocarbon-degrading microbes. The main focus of this investigation is to determine the potential of a halo-tolerant and biosurfactant-producing hydrocarbonoclastic bacterium, Enterobacter hormaechei, for the effective bioremediation of marine oil spill. The strain had the ability to produce biosurfactant with excellent surface and emulsification activities along with biocatalysts. The activity of the extracellular enzymes such as lipase and laccase was found to be 160 U/ml and 38 U/ml, respectively, whereas the intracellular enzymes like alkane hydroxylase, alcohol dehydrogenase and esterase showed the corresponding activities of 48 U/ml, 86 U/ml and 102 U/ml. Overall, E. hormaechei could be able to degrade nearly 85% of petroleum hydrocarbons present in crude oil within 10 days of incubation. The biosurfactant was characterized to be an anionic, high molecular weight (48 kDa) lipoprotein-type biosurfactant. The biosurfactant was further characterized by Fourier transform infrared spectroscopy. The E. hormaechei was employed for the treatment of simulated marine oil spill and the degradation followed pseudo-second-order kinetics with rate constant k2 0.2775 and R2 0.9923. The crude oil degradation was confirmed by gas chromatography–mass spectrometry. The study suggested that the E. hormaechei is a potential biosurfactant and biocatalysts producer with the effective management of accidental marine oil spills.

Bioremediation of diesel oil in marine environment

Natural gas emissions from oil spill ensue changes to microbial consortia in oceans which might cause ecotoxicological impacts on marine life. Gas flaring, a technique in the clean-up of oil spill, is a major source of greenhouse gas emission and possess high risk of fire hazard. It is of utmost importance to avoid flaring and resort to cleaner techniques such as bioremediation. The study focuses on bioremediation of marine oil spill by indigenous bacterial consortia using beeswax as a biostimulant which supplements the limiting nutrients such as nitrate and phosphate. The experimental study was conducted by adding diesel oil in marine water with beeswax for bioremediation. The vital parameters such as dissolved oxygen, pH, diesel range organics, total microbial count, nitrate and phosphate contents were measured at intervals of 5 days. The indigenous bacteria utilized oil as carbon source and beeswax as nutrient source for growth and metabolism. The results showed 87% removal of oil content in treatment sample while only 59% reduction was achieved in the corresponding control sample. Evaporation of oil results in formation of aerosols and black carbon which can lead to climate change. The study proves that bioremediation of marine oil spill is an environmentally benign clean-up technique for oil spill which can reduce carbon emission.

Study on immobilization of marine oil-degrading bacteria by carrier of algae materials.

This study investigated the immobilizations with of bacteria two kinds of algal materials, Enteromorpha residue and kelp residue. The lipophilicity of them were compared by diesel absorption rates. The immobilization efficiency of Bacillus sp. E3 was measured to evaluate whether these carriers would satisfy the requirement for biodegradation of oil spills. The bacteria were immobilized through adsorption with the sterilized and non-sterilized carriers to compare the differences between the two treatments. Oil degradation rates were determined using gravimetric and GC-MS methods. Results showed the absorption rates of Enteromorpha residue and kelp residue for diesel were 411 and 273% respectively and remained approximately 105 and 120% after 2h of erosion in simulated seawater system. After immobilized of Bacillus sp. E3, the oil degradation rates of them were higher than 65% after 21 days biodegradations. GC-MS analysis showed that two immobilizations degraded higher than 70% of the total alkane and the total PAHs, whereas the free bacteria degraded 63% of the total alkane and 66% the total PAHs. And the bacteria immobilized with the carriers degraded more HMW-alkanes and HMW-PAHs than the free bacteria. The bacteria immobilized by non-sterilized kelp residue showed a considerably higher degradation rate than that using sterilized kelp residue. A considerably higher cells absorption rate of immobilization was obtained when using kelp residue, and the preparation of immobilization was low cost and highly efficient. The experiments show the two algae materials, especially the kelp residue, present potential application in bioremediation of marine oil spills.

Marine Oil-Degrading Microorganisms and Biodegradation Process of Petroleum Hydrocarbon in Marine Environments: A Review.

Due to the toxicity of petroleum compounds, the increasing accidents of marine oil spills/leakages have had a significant impact on our environment. Recently, different remedial techniques for the treatment of marine petroleum pollution have been proposed, such as bioremediation, controlled burning, skimming, and solidifying. (Hedlund and Staley in Int J Syst Evol Microbiol 51:61-66, 2001). This review introduces an important remedial method for marine oil pollution treatment-bioremediation technique-which is considered as a reliable, efficient, cost-effective, and eco-friendly method. First, the necessity of bioremediation for marine oil pollution was discussed. Second, this paper discussed the species of oil-degrading microorganisms, degradation pathways and mechanisms, the degradation rate and reaction model, and the factors affecting the degradation. Last, several suggestions for the further research in the field of marine oil spill bioremediation were proposed.

An insight into the growth of Alcanivorax borkumensis under different inoculation conditions

Abstract Alcanivorax borkumensis is a hydrocarbon degrading bacterium found to dominate bacterial communities in marine regions containing high levels of hydrocarbons. It has been linked to oil degradation around oil spill sites; thus, it has potential to be used actively in oil spill remediation. Here, we investigate the effect of solution and interfacial conditions on the growth of A. borkumensis . We show that providing A. borkumensis with dissolved organic nitrogen as an additional nutrient in solution leads to shorter lag times prior to hydrocarbon utilization at the oil–water interface. Hence, A. borkumensis can be encouraged to utilize n -alkanes present at the surface of the system quicker by supplementing the system with dissolved organic nitrogen. For fixed oil–water interfacial areas, the growth rates of bacteria show weak dependence on the initial bacteria concentrations; however, increasing bulk interfacial area leads to higher bacterial growth rates due to an increased amount of available surface area for degradation. To our knowledge, this is the first study to offer quantitative insight into how A. borkumensis can be actively supported in their utilization and degradation of oil for the bioremediation of marine oil spills.

Perspective: what is known, and not known, about the connections between alkane oxidation and metal uptake in alkanotrophs in the marine environment.

Should iron and copper be added to the environment to stimulate the natural bioremediation of marine oil spills? The key enzymes that catalyze the oxidation of alkanes require either iron or copper, and the concentration of these ions in seawater is vanishingly low. Nevertheless, the dependence of alkane oxidation activity on external metal concentrations remains unclear. This perspective will summarize what is known about the co-regulation of alkane oxidation and metal acquisition and pose a series of critical questions to which, for the most part, we do not yet have answers. The paucity of answers points to the need for additional studies to illuminate the cellular biology connecting microbial growth on alkanes to the acquisition of metal ions.

Montmorillonite mitigates the toxic effect of heavy oil on hydrocarbon-degrading bacterial growth: implications for marine oil spill bioremediation

AbstractThe ability of montmorillonite to mitigate the toxic effect of heavy oil from theNakhodkaoil spill, by growth of hydrocarbon-degrading bacteria and enable bioremediation was studied. Montmorillonite enhanced the bacterial growth significantly (P< 0.05) in the main treatment containing heavy oil+bacteria+montmorillonite (OBM), because the specific growth rate (μ) was greater than that in the biotic control treatment containing heavy oil+bacteria (OB). Significant amounts of Si and Al (major constituents of montmorillonite) were not released in the aqueous phase over the ∽24-day experiment (P> 0.05). Transmission electron microscopic observation showed that the hydrocarbon-degrading bacterial cells were covered and encrusted with montmorillonite particles. Scanning transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (STEM-EDS) also showed that the surrounding of the bacterial cells was frequently rich in Si but not in Al. Fourier transform infrared (FTIR) spectroscopy indicated that the heavy oil-bacterial cell-montmorillonite particle complex retained the composition of both water and heavy oil. X-ray powder diffractrometery (XRD) analysis revealed that heavy oil and heavy oil-bacteria did not change the basal spacing of montmorillonite over a period of 24 days. The enhancement of hydrocarbon-degrading bacterial growth is attributed to montmorillonite likely serving as both bacterial growth-supporting carrier and protective outer layer against high concentrations of heavy oil that inhibit growth. These results shed light on the interactions in oil-bacteria-clay complexes and could potentially be used in marine oil spill bioremediation.

Rhamnolipids enhance marine oil spill bioremediation in laboratory system

This paper presents a simulated marine oil spill bioremediation experiment using a bacterial consortium amended with rhamnolipids. The role of rhamnolipids in enhancing hydrocarbon biodegradation was evaluated via GC–FID and GC–MS analysis. Rhamnolipids enhanced total oil biodegradation efficiency by 5.63%, with variation in normal alkanes, polyaromatic hydrocarbons (PAHs) and biomakers biodegradation. The hydrocarbons biodegradation by bacteria consortium overall follows a decreasing order of PAHs>n-alkanes>biomarkers, while in different order of PAHs>biomarkers>n-alkanes when rhamnolipids was used, and the improvement in the removal efficiency by rhamnolipids follows another order of biomarkers>n-alkanes>PAHs. Rhamnolipids played a negative role in degradation of those hydrocarbons with relatively volatile property, such as n-alkanes with short chains, PAHs and sesquiterpenes with simple structure. As to the long chain normal alkanes and PAHs and biomakers with complex structure, the biosurfactant played a positive role in these hydrocarbons biodegradation.

Presence of Catechol Metabolizing Enzymes in Virgibacillus Salarius

Hydrocarbon contamination of marine ecosystems has been a major environmental concern. Hydrocarbon metabolizing capacity of four halotolerant bacteria (Bacillus atrophaeus, Halomonas shengliensis, Halomonas koreensis, and Virgibacillus sala- rius) isolated from saline soil of Khambhat, India was investigated. Presence of catechol metabolizing enzymes (catechol 2,3 dio- xygenase, chlorocatechol 1,2 dioxygenase, and protocatechuate 3,4 dioxygenase) was checked in V. salarius, as only this among all the test organisms could grow on the hydrocarbon substrates used, and compared with Pseudomonas oleovorans. Effect of salinity of the growth medium on activity of catechol metabolizing enzymes was also studied. Catechol 2,3 dioxygenase activity in both the organisms was more susceptible to increase in salinity of the growth medium than chlorocatechol 1,2-dioxygenase activity. To the best of our awareness, this is the first report of catechol metabolism in V. salarius. V. salarius was found to be capable of weak biofilm formation. As V. salarius is capable of growing at high salt concentration, alkaline pH, hydrocarbon degradation, and also of growth in presence of various metal ions, it can be an attractive candidate for bioremediation of marine oil spills. Organisms like V. salarius can also serve as a model for multiple stress tolerance in prokaryotes.

MICROBIAL COMMUNITY DURING BIOREMEDIATION EXPERIMENTAL ON OIL SPILL IN COASTAL OF PARI ISLAND

There is an information how to identify hydrocarbon degrading bacteria for bioremediation of marine oil spill. We have Bioremediation treatment for degradation of oil spill on Pari island and need two kind of experiment there are tanks experiment (sampling 0 to 90 days) and semi enclosed system (sampling 0 to 150 days). Biostimulation with nutrients (N and P) was done to analyze biodegradation of hydrocarbon compounds. Experiment design using fertilizer Super IB and Linstar will stimulate bacteria can degrade oil, n-alkane, and alkane as poly aromatic hydrocarbon. The bacteria communities were monitored and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) and Clone Library; oil chemistry was analyzed by Gas Chromatography Mass Spectrometry (GCMS). DNA (deoxyribonucleic acid) was extracted from colonies of bacteria and sequence determination of the 16S rDNA was amplified by primers U515f and U1492r. Strains had been sequence and had similarity about 90-99% to their closest taxa by homology Blast search and few of them suspected as new species. The results showed that fertilizers gave a significant effect on alkane, PAH and oil degradation in tanks experiment but not in the field test. Dominant of the specific bacteria on this experiment were Alcanivorax, Marinobacter and Prosthecochloris.
 
 Keywords: Bioremediation, Biostimulation, DGGE, PAH, Pari Island

MICROBIAL COMMUNITY DURING BIOREMEDIATION EXPERIMENTAL ON OIL SPILL IN COASTAL OF PARI ISLAND

There is an information how to identify hydrocarbon degrading bacteria for bioremediation of marine oil spill. We have Bioremediation treatment for degradation of oil spill on Pari island and need two kind of experiment there are tanks experiment (sampling 0 to 90 days) and semi enclosed system (sampling 0 to 150 days). Biostimulation with nutrients (N and P) was done to analyze biodegradation of hydrocarbon compounds. Experiment design using fertilizer Super IB and Linstar will stimulate bacteria can degrade oil, n-alkane, and alkane as poly aromatic hydrocarbon. The bacteria communities were monitored and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) and Clone Library; oil chemistry was analyzed by Gas Chromatography Mass Spectrometry (GCMS). DNA (deoxyribonucleic acid) was extracted from colonies of bacteria and sequence determination of the 16S rDNA was amplified by primers U515f and U1492r. Strains had been sequence and had similarity about 90-99% to their closest taxa by homology Blast search and few of them suspected as new species. The results showed that fertilizers gave a significant effect on alkane, PAH and oil degradation in tanks experiment but not in the field test. Dominant of the specific bacteria on this experiment were Alcanivorax, Marinobacter and Prosthecochloris.
 
 Keywords: Bioremediation, Biostimulation, DGGE, PAH, Pari Island

Bioremediation of marine oil spills: when and when not – theExxon Valdezexperience

SummaryIn this article we consider what we have learned from the Exxon Valdez oil spill (EVOS) in terms of when bioremediation should be considered and what it can accomplish. We present data on the state of oiling of Prince William Sound shorelines 18 years after the spill, including the concentration and composition of subsurface oil residues (SSOR) sampled by systematic shoreline surveys conducted between 2002 and 2007. Over this period, 346 sediment samples were analysed by GC‐MS and extents of hydrocarbon depletion were quantified. In 2007 alone, 744 sediment samples were collected and extracted, and 222 were analysed. Most sediment samples from sites that were heavily oiled by the spill and physically cleaned and bioremediated between 1989 and 1991 show no remaining SSOR. Where SSOR does remain, it is for the most part highly weathered, with 82% of 2007 samples indicating depletion of total polycyclic aromatic hydrocarbon (Total PAH) of > 70% relative to EVOS oil. This SSOR is sequestered in patchy deposits under boulder/cobble armour, generally in the mid‐to‐upper intertidal zone. The relatively high nutrient concentrations measured at these sites, the patchy distribution and the weathering state of the SSOR suggest that it is in a form and location where bioremediation likely would be ineffective at increasing the rate of hydrocarbon removal.

Efficacy of intervention strategies for bioremediation of crude oil in marine systems and effects on indigenous hydrocarbonoclastic bacteria

There is little information on how different strategies for the bioremediation of marine oil spills influence the key indigenous hydrocarbon-degrading bacteria (hydrocarbonoclastic bacteria, HCB), and hence their remediation efficacy. Therefore, we have used quantitative polymerase chain reaction to analyse changes in concentrations of HCB in response to intervention strategies applied to experimental microcosms. Biostimulation with nutrients (N and P) produced no measurable increase in either biodegradation or concentration of HCB within the first 5 days, but after 15 days there was a significant increase (29%; P < 0.05) in degradation of n-alkanes, and an increase of one order of magnitude in concentration of Thalassolituus (to 10(7) cells ml(-1)). Rhamnolipid bioemulsifier additions alone had little effect on biodegradation, but, in combination with nutrient additions, provoked a significant increase: 59% (P < 0.05) more n-alkane degradation by 5 days than was achieved with nutrient additions alone. The very low Alcanivorax cell concentrations in the microcosms were hardly influenced by addition of nutrients or bioemulsifier, but strongly increased after their combined addition, reflecting the synergistic action of the two types of biostimulatory agents. Bioaugmentation with Thalassolituus positively influenced hydrocarbon degradation only during the initial 5 days and only of the n-alkane fraction. Bioaugmentation with Alcanivorax was clearly much more effective, resulting in 73% greater degradation of n-alkanes, 59% of branched alkanes, and 28% of polynuclear aromatic hydrocarbons, in the first 5 days than that obtained through nutrient addition alone (P < 0.01). Enhanced degradation due to augmentation with Alcanivorax continued throughout the 30-day period of the experiment. In addition to providing insight into the factors limiting oil biodegradation over time, and the competition and synergism between HCB, these results add weight to the use of bioaugmentation in oil pollution mitigation strategies.

English

Pollution in marine environment due to heavier petroleum products such as high-speed diesel is known to take from days to months for complete natural remediation owing to its low volatility. For the survival of marine flora and fauna, it is important to control pollution caused by such recalcitrant and xenobiotic substances. Several petroleum hydrocarbons found in nature are toxic and recalcitrant. Therefore, pollution due to high-speed diesel is a cause of concern. The natural dispersion of high-speed diesel, a slow process, is attributed to an overall combined effect of physico-chemical and biological processes which take months for complete dispersion. History of marine oil spill bioremediation indicates limited laboratory studies. But experiences from various oil spill management and field trials indicate important role of bioremediation, where, biodegradation of hydrocarbons through microbial mediators plays a major role in pollutant oil dispersion. These microbial mediators such as bioemulsifiers and fimbrae, help in emulsification, dispersion, allowing attachment of bacteria to oil layers, followed by substrate-specific enzymatic biodegradation in water.

Bioremediation of Marine Oil Spills

In the long run, biodegradation is the eventual fate of oil spilled at sea that cannot be collected or burnt. Stimulating this biodegradation is thus an important option for maximizing the removal of oil from the environment, and minimizing the environmental impact of a spill. For handling oil while it is still floating on the sea surface, dispersants are advantageous because they maximize the surface area available for microbial attack, and stimulate biodegradation. If oil beaches on a shoreline, it is likely that biodegradation is limited by nutrients such as nitrogen and phosphorus, and the careful application of fertilizers stimulates the biodegradation of residual beached oil. These approaches epitomize modern environmental technologies; working with natural phenomena to achieve a more rapid clean-up while minimizing undesirable environmental impacts of a spill. For handling oil while it is still floating on the sea surface, dispersants are advantageous because they maximize the surface area available for microbial attack, and stimulate biodegradation. If oil beaches on a shoreline, it is likely that biodegradation is limited by nutrients such as nitrogen and phosphorus, and the careful application of fertilizers stimulates the biodegradation of residual beached oil. These approaches epitomize modern environmental technologies; working with natural phenomena to achieve a more rapid clean-up while minimizing undesirable environmental impacts.

Environment

Hydrocarbon-degrading microorganisms are ubiquitously distributed in soil and aquatic environments. Populations of hydrocarbon-degraders normally constitute less than 1% of the total microbial communities, but when oil pollutants are present these hydrocarbon-degrading populations increase, typically to 10% of the community. With regard to rates of natural degradation, these typically have been found to be low and limited by environmental factors. Rates reported for pristine marine waters typically are less than 0·03 g/m3/day. In adapted communities rates of hydrocarbon degradation of 0·5–50 g/m3/day have been reported. Bioremediation tries to raise the rates of degradation found naturally to significantly higher rates. The two general approaches that have been tested for the bioremediation of marine oil spills are the application of fertilizer to enhance the abilities of the indigenous hydrocarbon-utilizing bacteria and the addition of naturally occurring adapted microbial hydrocarbon-degraders by seeding. Bioremediation, accomplished by the application of fertilizer to enhance the abilities of the indigenous hydrocarbon-utilizing bacteria, was successfully applied for the treatment of the 1989 Alaskan oil spill in Prince William Sound, Alaska. Seeding with adapted nonindigenous microbial hydrocarbon degraders was tested on smaller spills in Texas—such as the Mega Borg spill—but bioremediation to remove petroleum pollutants by seeding has yet to be demonstrated as efficacious in field trials. The spill of more than 200,000 barrels of crude oil from the oil tanker Exxon Valdez in Prince William Sound, Alaska, as well as smaller spills in Texas—such as the Mega Borg spill—have been treated by bioremediation to remove petroleum pollutants. The Exxon Valdez spill formed the basis for a major study on bioremediation through fertilizer application and the largest application of this emerging technology. Three types of nutrient supplementation were tested: water-soluble (23:2 N:P garden fertilizer formulation): slow-release (Customblen); and oleophilic (Inipol EAP 22). Each fertilizer was tested in laboratory simulations and in field demonstration plots to determine the efficacy of nutrient supplementation. The use of Inipol EAP22 (oleophilic microemulsion with urea as a nitrogen source, laureth phosphate as a phosphate source, and oleic acid as a carbon source) and Customblen (slow-release calcium phosphate, ammonium phosphate, and ammonium nitrate within a polymerized vegetable oil coating) was approved for shoreline treatment and was used as a major part of the cleanup effort. Multiple regression models showed that nitrogen applications were effective in stimulating the rates of biodegradation.