Microbial degradation of oil spills enhanced by a slow-release fertilizer.

The improved cleanup of marine oil spills by stimulating biodegradation through the use of a slow-release fertilizer is reported. A paraffin-supported fertilizer containing MgNH4PO4 as active ingredient was developed and evaluated in laboratory and field experiments using quantitative infrared spectrometry and chromatographic techniques. The biodegradation of Sarir crude oil in the sea was considerably enhanced by paraffin-supported fertilizer. After 21 days 63% had disappeared as compared to 40% in the control area.     

Application of biosurfactant produced from peanut oil cake by Lactobacillus delbrueckii in biodegradation of crude oil

Lactobacillus delbrueckii cultured with peanut oil cake as the carbon source yielded 5.35 mg ml(-1) of biosurfactant production. Five sets of microcosm biodegradation experiments were carried out with crude oil as follows: set 1 - bacterial cells+crude oil, set 2 - bacterial cells+crude oil+fertilizer, set 3 - bacterial cells+crude oil+biosurfactant, set 4 - bacterial cells+crude oil+biosurfactant+fertilizer, set 5 - with no bacterial cells, fertilizer and biosurfactant (control). Maximum degradation of crude oil was observed in set 4 (75%). Interestingly, when biosurfactant and bacterial cells were used (set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in set 4 was 7% higher degradation level in microcosm experiments. It is evident from the results that biosurfactants alone is capable of promoting biodegradation to a large extent without added fertilizers.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Incorporation of Petroleum Carbon Into Soil Organic Matter During Biodegradation

A procedure, based on measurement of the stable carbon isotope ¹³C, has been developed for determining the extent to which petroleum carbon is incorporated into soil organic matter (SOM) by humification of biomass produced during biodegradation of the petroleum in soil. We have shown that a crude oil having a δ¹³C of-27.4%, when biodegraded in a soil containing SOM with a δ¹³C of-15.7%, resulted in a change of the δ¹³C of the bound SOM reflecting that of petroleum carbon. Comparison of five soil biodegradation tests using different amounts and types of fertilizer to stimulate biodegradation of the oil in this soil showed that the extent of the δ¹³C change in the bound SOM varied with the extent of oil biodegradation observed. To obtain ¹³C data on the SOM, the residual petroleum was first removed by rigorous extraction with dichloromethane using a Soxhlet apparatus. The extracted soil was then combusted to release bound carbon as CO₂, which was analyzed for ¹³C. Where the SOM has a δ¹³C similar to that of petroleum, ¹⁴C measurements of SOM would give similar results. This type of data, referred to as the petroleum “footprint” in the SOM, could be useful in identifying or confirming intrinsic biodegradation of petroleum in contaminated soil.     

Oil spill bioremediation: experiences,lessons and results from the Exxon Valdez oil spill in Alaska

The use of bioremediation as a supplemental cleanup technology in the Exxon Valdez oil spill, in Prince William Sound, Alaska, has proven to be a good example of the problems and successes associated with the practical application of this technology. Field studies conducted by scientists from the U.S. Environmental Protection Agency have demonstrated that oil degradation by indigenous microflora on the beaches of Prince William Sound was accelerated by adding fertilizer directly to the surfaces of oil-contaminated beaches. Although several types of fertilizers were used in the studies, only the results from the application of an oleophilic fertilizer are presented. The fertilizer enhanced biodegradation of the oil, as measured by changes in hydrocarbon composition and bulk oil weight per unit of beach material, by approximately two-fold relative to untreated controls. Laboratory studies verified the usefulness of the oleophilic fertilizer as a nutrient source, but the contribution of its oleophilic components towards enhancing biodegradation is still unclear.These studies supported bioremediation as a useful cleanup strategy that was subsequently used by Exxon on a large scale. The Exxon Valdez experience has also provided a number of informative lessons that have significant relevance to future oil bioremediation efforts. This paper discusses these lessons and the difficulties in assessing the effectiveness of bioremediation in the field.     

Potential of vetiver (vetiveria zizanioides (L.) Nash) for phytoremediation of petroleum hydrocarbon-contaminated soils in Venezuela

Venezuela is one of the largest oil producers in the world. For the rehabilitation of oil-contaminated sites, phytoremediation represents a promising technology whereby plants are used to enhance biodegradation processes in soil. A greenhouse study was conducted to determine the tolerance of vetiver (Vetiveria zizanioides (L.) Nash) to a Venezuelan heavy crude oil in soil. Additionally, the plant's potential for stimulating the biodegradation processes of petroleum hydrocarbons was tested under the application of two fertilizer levels. In the presence of contaminants, biomass and plant height were significantly reduced. As for fertilization, the lower fertilizer level led to higher biomass production. The specific root surface area was reduced under the effects of petroleum. However, vetiver was found to tolerate crude-oil contamination in a concentration of 5% (w/w). Concerning total oil and grease content in soil, no significant decrease under the influence of vetiver was detected when compared to the unplanted control. Thus, there was no evidence of vetiver enhancing the biodegradation of crude oil in soil under the conditions of this trial. However, uses of vetiver grass in relation to petroleum-contaminated soils are promising for amelioration of slightly polluted sites, to allow other species to get established and for erosion control.     

Shoreline Bioremediation Following the Exxon Valdez Oil Spill in Alaska

Bioremediation played an important role in the cleanup following the oil spill from the Exxon Valdez in Prince William Sound, Alaska. The initial spill response included washing the beaches and collecting the oil with skimmers, but while this was in progress a joint program between Exxon and the United States Environmental Protection Agency investigated the feasibility of using bioremediation to remove residual oil. Early experiments demonstrated that oil biodegradation on affected shorelines was nutrient-limited, and several fertilizer application strategies were tested to overcome this limitation. Two fertilizers, the liquid oleophilic Inipol EAP22 and the solid slow-release Customblen™, were chosen for wide-scale application. Field monitoring convincingly demonstrated that the fertilizer strategy effectively stimulated oil biodegradation severalfold, with no adverse environmental impacts.     

Bioremediation of Soil Artificially Contaminated with Petroleum Hydrocarbon Oil Mixtures: Evaluation of the Use of Animal Manure and Chemical Fertilizer

The search for cheaper and environmentally friendly options of enhancing petroleum hydrocarbon degradation has continued to elicit research interest. One of such options is the use of animal manure as biostimulating agents. A combination of treatments consisting of the application of poultry manure, piggery manure, goat manure, and chemical fertilizer was evaluated in situ during a period of 4 weeks of remediation. Each treatment contained petroleum hydrocarbon mixture (kerosene, diesel oil, and gasoline mixtures) (10% w/w) in soil as a sole source of carbon and energy. After 4 weeks of remediation, the results showed that poultry manure, piggery manure, goat manure, and NPK (nitrogen, phosphorous, and potash [potassium]) fertilizer exhibited 73%, 63%, 50%, and 39% total petroleum hydrocarbon degradation, respectively. Thus, all the biostimulating treatment strategies showed the ability to enhance petroleum hydrocarbon microbial degradation. However, poultry manure, piggery manure, and goat manure treatments showed greater petroleum hydrocarbon reductions than NPK fertilizer treatment. A first-order kinetic equation was fitted to the biodegradation data and the specific degradation rate constant (k) values obtained showed that the order of effectiveness of these biostimulating strategies in the cleanup of soil contaminated with petroleum hydrocarbon mixtures (mixture of kerosene, diesel oil, and gasoline) is NPK fertilizer < goat manure < piggery manure < poultry manure. Therefore, this present work has indicated that the application of poultry manure, piggery manure, goat manure, and chemical fertilizer could enhance petroleum hydrocarbon degradation with poultry manure, showing a greater effectiveness and thus could be one of the severally sought environmentally friendly ways of remediating natural ecosystem contaminated with crude oil.     

Effects of nutrient and temperature on degradation of petroleum hydrocarbons in sub-Antarctic coastal seawater

In an attempt to evaluate the potential of petroleum bioremediation at high latitudes environments, microcosm studies using Antarctic coastal seawater contaminated with diesel or crude oil were conducted in Kerguelen Archipelago (49°22′S, 70°12′E). Microcosms were incubated at three different temperatures (4, 10 and 20°C). During experiments, changes observed in microbial assemblages (total direct count, heterotrophic cultivable microorganisms and hydrocarbon-degrading microorganisms) were generally similar for all incubation temperatures, but chemical data showed only some slight changes in biodegradation indices [Σ(C12–C20)/Σ(C21–C32) and C17/pristane]. The complete data set provided strong evidence of the presence of indigenous hydrocarbon-degrading bacteria in Antarctic seawater and their high potential for hydrocarbon bioremediation. The rate of oil degradation could be increased by the addition of a commercial fertilizer, but water temperature had little effects on biodegradation efficiency which is in conflict with the typical temperature-related assumption predicting 50% rate reduction when temperature is reduced by 10°C. Global warming of Antarctic seawater should not increase significantly the rate of oil biodegradation in these remote regions.     

Effects of nitrogen source on crude oil biodegradation

Summary The effects of NH₄Cl and KNO₃ on biodegradation of light Arabian crude oil by an oil-degrading enrichment culture were studied in respirometers. In poorly buffered sea salts medium, the pH decreased dramatically in cultures that contained NH₄Cl, but not in those supplied with KNO₃. The ammonia-associated pH decline was severe enough to completely stop oil biodegradation as measured by oxygen uptake. Regular adjustment of the culture pH allowed oil biodegradation to proceed normally. A small amount of nitrate accumulated in all cultures that contained ammonia, but nitrification accounted for less than 5% of the acid that was observed. The nitrification inhibitor, nitrapyrin, had no effect on the production of nitrate or acid in ammonia-containing cultures. When the culture pH was controlled, either by regular adjustment of the culture pH or by supplying adequate buffering capacity in the growth medium, the rate and extent of oil biodegradation were similar in NH₄Cl- and KNO₃-containing cultures. the lag time was shorter in pH-controlled cultures supplied with ammonia than in nitrate-containing cultures.     

Effect of iron on the biodegradation of petroleum in seawater.

The biodegradation of South Louisiana (SL) crude oil and the effects of nitrogen, phosphorus, and iron supplements on this process were compared in a polluted (10,900 oil degraders per liter) and in a relatively clean (750 oil degraders per liter) littoral seawater sample taken along the New Jersey coast. Without supplements, the biodegradation of SL crude oil was negligible in both seawater samples. Addition of nitrogen and phosphorus allowed very rapid biodegradation (72% in 3 days) in polluted seawater. Total iron in this seawater sample was high (5.2 muM), and the addition of iron did not increase the biodegradation rate further. In the less polluted and less iron-rich (1.2 muM) seawater sample, biodegradation of SL crude oil was considerably slower (21% in 3 days) and the addition of chelated iron had a stimulating effect. Ferric octoate was shown to have a similar stimulating effect on SL crude oil biodegradation as chelated iron. Ferric octoate, in combination with paraffinized urea and octylphosphate, is suitable for treatment of floating oil slicks. We conclude that spills of SL crude and similar oils can be cleaned up rapidly and efficiently by stimulated biodegradation, provided the water temperatures are favorable.     

Biodegradation of Pyrene in Marine Beach Sediments

The potential of chitosan (0.1% dry weight equivalent) as a bioremediation additive for removal of the recalcitrant polycyclic aromatic hydrocarbon (PAH) pyrene in marine beach sediments was investigated using an open irrigation system over a 63-day period. Osmocote, a slow release fertilizer, was used as the key nutrient supplement at a concentration of 1% in sediment (dry weight equivalent). Osmocote significantly (p < .05) enhanced nutrient levels, and the metabolic activity of the indigenous microbial biomass. Both additives were comparable in stimulating pyrene biodegradation rates; with chitosan (0.062 day^?1) being slightly more effective as an amendment than Osmocote (0.051 day^?1). Loss of pyrene in a control sediment (i.e., pyrene, without additives) was 66.6% over a 63-day period. The concurrent application of additives yielded the greatest biodegradation rates (0.072day^?1), resulting in a 98.2% loss of pyrene over 63 days. The treatment of oil contaminated beach sediments with both osmocote (1%) and chitosan (0.1%) is therefore recommended as an effective treatment for the intrinsic biodegradation of recalcitrant PAHs in oil-contaminated beach sediments.     

Enhanced Biodegradation of Diesel Oil in Seawater Supplemented with Nutrients

The imbalance of C, N, and P caused by the spilled oil could be regulated by the addition of nitrogen and phosphorous. Moreover, different kinds of N and P sources were used in order to stimulate oil biodegradation under laboratory and field conditions, but the results were conflicting. To evaluate the effectiveness of nutrient supplementation, N sources (NO₃‐N and NH₄‐N) and P sources (PO₄‐P) were applied to the simulated diesel‐polluted seawater in the N/P ratio of 10:1 and 20:1, respectively. The results showed that the addition of nutrients increased the oil biodegradation rate and the counts of petroleum degrading bacteria (PDB) and heterotrophic bacteria (HB). A strongly positive correlation existed (the interrelated coefficient was nearly 0.9) between the percentage ratio of PDB/HB and the oil biodegradation rates, and therefore the percentage ratio of PDB/HB could be used as a good indicator to predict oil biodegradation. Among the four samples treated with nutrients, the biodegradation efficiency of the group amended with NO₃‐N and PO₄‐P in the ratio of 10:1 (10NO₃‐P group) was as much as 75.8 %, while in the 10NH₄‐P, 20NO₃‐P and 20NH₄‐P groups this value was 61.3 %, 52.4 % and 40.5, respectively. It would take natural degradation without nutrient supplementation about 78 days to achieve the result obtained within 14 days with 10NO₃‐P amendment . Chemical and microbiological analyses confirmed that the addition of nutrients in the same N/P ratio remarkably enhanced the biodegradation rate and the counts of microorganisms in the NO₃‐N treated groups, indicating that the microorganisms tend to utilize NO₃‐N rather than NH₄‐N as their growth N source. When the same kind of N source was added to the system, the promoted efficiency in the 10:1 (N/P ratio) groups was notable compared to the 20:1 groups, i.e., adding nutrients in the ratio of 10:1 is superior in the stimulation of oil biodegradation to the ratio of 20:1.     

Biodegradation and adsorption of C1- and C2-phenanthrenes and C1- and C2-dibenzothiophenes in the presence of clay minerals: effect on forensic diagnostic ratios

The impact of modified montmorillonites on adsorption and biodegradation of crude oil C1-phenanthrenes, C1-dibenzothiophenes, C2-phenanthrenes and C2-dibenzothiophenes was investigated in aqueous clay/oil microcosm experiments with a hydrocarbon degrading microorganism community. Consequently, the effect on C1-dibenzothiophenes/C1-phenanthrenes, C2-dibenzothiophenes/C2-phenanthrenes, 2+3-methyldibenzothiophene/4-methyldibenzothiophene and 1-methyldibenzothiophene/4-methyldibenzothiophene ratios commonly used as diagnostic ratios for oil forensic studies was evaluated. The clay mineral samples were treated to produce acid activated montmorillonite, organomontmorillonite and homoionic montmorillonite which were used in this study. The different clay minerals (modified and unmodified) showed varied degrees of biodegradation and adsorption of the C1-phenanthrenes, C1-dibenzothiophenes, C2-phenanthrenes and C2-dibenzothiophenes. The study indicated that as opposed to biodegradation, adsorption has no effect on the diagnostic ratios. Among the diagnostic ratios reviewed, only C2-dibenzothiophenes/C2-phenanthrenes ratio was neither affected by adsorption nor biodegradation making this ratio very useful in forensic studies of oil spills and oil–oil correlation.     

Influence of chemical surfactants on the biodegradation of crude oil by a mixed bacterial culture.

The effects of surfactant physicochemical properties, such as the hydrophile-lipophile balance (HLB) and molecular structure, on the biodegradation of 2% w/v Bow River crude oil by a mixed-bacterial culture were examined. Viable counts increased 4.6-fold and total petroleum hydrocarbon (TPH) biodegradation increased 57% in the presence of Igepal CO-630, a nonylphenol ethoxylate (HLB 13, 0.625 g/L). Only the nonylphenol ethoxylate with an HLB value of 13 substantially enhanced biodegradation. The surfactants from other chemical classes with HLB values of 13 (0.625 g/L) had no effect or were inhibitory. TPH biodegradation enhancement by Igepal CO-630 occurred at concentrations above the critical micelle concentration. When the effect of surfactant on individual oil fractions was examined, the biodegradation enhancement for the saturate and aromatic fractions was the same. In all cases, biodegradation resulted in increased resin and asphaltene concentrations. Optimal surfactant concentrations for TPH biodegradation reduced resin and asphaltene formation. Chemical surfactants have the potential to improve crude oil biodegradation in complex microbial systems, and surfactant selection should consider factors such as molecular structure, HLB, and surfactant concentration.     

Bioremediation and Biorestoration of a Crube Oil-Contaminated Freshwater Wetland on the St.Lawrence River

Biostimulation by nutrient enrichment and phytoremediation were studied for the restoration of an acutely stressed freshwater wetland experimentally exposed to crude oil. The research was carried out along the shores of the St. Lawarence River at Ste. Croix, Quebec, Canada. The research determined the effectiveness of fertilizer addition in enhancing the biodegradation rates of residual oil. It further examined the rate at which the stressed ecosystem recovered with and without the addition of inorganic fertilizers and the role of nutrients in enhancing wetland restoration in the absence of healthy wetland plants. Chemical analysis of integrated sediment core samples to the depth of oil penetration within the experimental plots indicated that addition of inorganic nutrients did not enhance the disappearance of alkanes or PAHs. In surface samples, however, hydrocarbon disappearance rates were higher when the metabolic activity of wetland plants was suppressed by the removal of emergent plant growth. These results suggest that oxygen limitation plays a major role in preventing rapid biodegradation of hydrocarbons in anoxic wetland sediment.     

Petroleum Bioremediation in Seawater Using Guano as the Fertilizer

The biodegradation of hydrocarbon pollutants in open systems, such as oceans, is generally limited by the availability of utilizable nitrogen and phosphorus sources. Here the authors demonstrate the potential of overcoming this problem with guano as the fertilizer. In the first set of experiments, the principle and conditions for growing bacteria on a water insoluble fertilizer was established, using uric acid as the nitrogen source and a pure culture of an isolated hydrocarbon-degrading bacterium, Alcanivorax sp. OK2. Using a simulated open system, it was demonstrated that uric acid (the major nitrogen component of guano) binds to crude oil and is available for the growth of strain OK2 and petroleum degradation. In the second set of experiments, using a simulated open system, it was demonstrated that commercial guano was an effective source of nitrogen and phosphorus for the growth of marine bacteria on crude oil. Bacterial cultures reached over 10⁸ cells per ml and 70% of the crude oil was degraded. Controls using ammonium sulfate and phosphate in place of guano in the simulated open system reached only 10⁶ cells per ml and showed no detectable hydrocarbon degradation. Isolation and characterization of the bacteria in the crude oil/guano cultures indicated that they were primarily strains of Alcanivorax and Alteromonas.     

Long-Term Evolution of Biodegradation and Volatilization Rates in a Crude Oil-Contaminated Aquifer

Volatilization and subsequent biodegradation near the water Table make up a coupled natural attenuation pathway that results in significant mass loss of hydrocarbons. Rates of biodegradation and volatilization were documented twice 12 years apart at a crude-oil spill site near Bemidji, Minnesota. Biodegradation rates were determined by calibrating a gas transport model to O₂, CO₂, and CH₄ gas-concentration data in the unsaturated zone. Reaction stoichiometry was assumed in converting O₂ and CO₂ gas-flux estimates to rates of aerobic biodegradation and CH₄ gas-flux estimates to rates of methanogenesis. Model results indicate that the coupled pathway has resulted in significant hydrocarbon mass loss at the site, and it was estimated that approximately 10.52 kg/day were lost in 1985 and 1.99 kg/day in 1997. In 1985 3% of total volatile hydrocarbons diffusing from the floating oil were biodegraded in the lower 1 m of the unsaturated zone and increased to 52% by 1997. Rates of hydrocarbon biodegradation above the center of the floating oil were relatively stable from 1985 to 1997, as the primary metabolic pathway shifted from aerobic to methanogenic biodegradation. Model results indicate that in 1997 biodegradation under methanogenenic conditions represented approximately one-half of total hydrocarbon biodegradation in the lower 1 m of the unsaturated zone. Further downgradient, where substrate concentrations have greatly increased, total biodegradation rates increased by greater than an order of magnitude from 0.04 to 0.43 g/m²-day. It appears that volatilization is the primary mechanism for attenuation in early stages of plume evolution, while biodegradation dominates in later stages.     

Oil bioremediation in a high and a low phosphorus soil

Phosphorus (P) content may influence bioremediation of soils contaminated with crude oil. A soil testing high in plant available P (Weswood, 194 mg P kg^?1 soil) and one testing low in plant available P (Lufkin, 2 mg P kg^?1 soil) were selected for laboratory experiments on oil biodegradation. Plant available P content was determined using acidified ammonium acetate at pH 4.2 as the soil extractant. Soils were amended with 3, 6, and 9% crude oil by weight and incubated for 120 d at 25°C. Treatments consisted of a factorial arrangement, with soil, N, P, and oil concentration as factors. Addition of P without N generally did not enhance biodegradation. Addition of N without P approximately tripled the quantity of oil degraded. Addition of P and N together did not increase biodegradation of oil more than addition of N alone when oil concentration was 3%. At 6 and 9% oil concentrations, CO₂ evolution increased for both soils by adding P and N together in comparison to adding N alone, and total petroleum hydrocarbon (TPH) bio‐degradation increased by 30% for the Weswood soil by 60 d and at least 25% for the Lufkin soil by 30 d. The quantity of plant‐available P or total P in soil was not very useful in predicting need for supplemental P. Addition of P to soil to enhance oil degradation was only beneficial for oil concentrations above 3% and the positive effect for higher concentrations was transitory.     

N-alkane biodegradation by a marine bacterium in the presence of an oleophilic nutriment

Summary Hexadecane biodegradation by a marine bacterium has been investigated in the presence of an oleophilic nutriment (INIPOL EAP 22). Hydrocarbon attack was only observed after metabolism of the fatty acids present in the fertilizer. The bacterium used up 95 % fatty acids in the first 24 hours. Hexadecane biodegradation took place after 50 h incubation and reached 40 % after 360 h.