Biodegradability of dispersed crude oil at two different temperatures

Laboratory experiments were initiated to study the biodegradability of oil after dispersants were applied. Two experiments were conducted, one at 20 degrees C and the other at 5 degrees C. In both experiments, only the dispersed oil fraction was investigated. Each experiment required treatment flasks containing 3.5% artificial seawater and crude oil previously dispersed by either Corexit 9500 or JD2000 at a dispersant-to-oil ratio of 1:25. Two different concentrations of dispersed oil were prepared, the dispersed oil then transferred to shake flasks, which were inoculated with a bacterial culture and shaken on a rotary shaker at 200 rpm for several weeks. Periodically, triplicate flasks were removed and sacrificed to determine the residual oil concentration remaining at that time. Oil compositional analysis was performed by gas chromatography/mass spectrometry (GC/MS) to quantify the biodegradability. Dispersed oil biodegraded rapidly at 20 degrees C and less rapidly at 5 degrees C, in line with the hypothesis that the ultimate fate of dispersed oil in the sea is rapid loss by biodegradation.     

Isolation and characterization of two crude oil-degrading yeast strains, Yarrowia lipolytica PG-20 and PG-32, from the Persian Gulf

Among six crude oil-degrading yeasts that were isolated from an oil-polluted area in the Persian Gulf, two yeast strains showed high degradation activity of aliphatic hydrocarbons. From an analysis of 18S rRNA sequences and biochemical characteristics, these strains were identified as Yarrowia lipolytica strains PG-20 and PG-32. Gas Chromatography (GC) analysis of the crude oil remaining in the culture medium after 1 week at 30°C showed that the strains PG-20 and PG-32 degraded 68% and 58% of crude oil, respectively. The optimal growth condition and biodegradation of hydrocarbons was in ONR medium with an acidic pH (pH5). These two strains may degrade aliphatic hydrocarbons more efficiently than aromatic hydrocarbons, although strain PG-20 had better degradation than strain PG-32. The two Y. lipolytica strains reduce surface tension when cultured on hydrocarbon substrates (1% v/v). These strains showed a cell surface hydrophobicity higher than 70%. These results suggested that Y. lipolytica strains PG-20 and PG-32 have high crude oil degrading activity due to their high emulsifying activity and cell hydrophobicity. In conclusion, these yeast strains can be useful for the bioremediation process in the Persian Gulf and decreasing oil pollution in this marine ecosystem.     

Biodegradation of Crude Oil by Individual Bacterial Strains and a Mixed Bacterial Consortium Isolated from Hydrocarbon Contaminated Areas

A preliminary study was undertaken to determine the optimal conditions for the biodegradation of a crude oil. Among 57 oil‐degrading bacterial cultures isolated from oil‐contaminated soil samples, Bacillus sp. IOS1‐7, Corynebacterium sp. BPS2‐6, Pseudomonas sp. HPS2‐5, and Pseudomonas sp. BPS1‐8 were selected for the study based on the efficiency of crude oil utilization. Along with the selected individual strains, a mixed bacterial consortium prepared using the above strains was also used for degradation studies. The mixed bacterial consortium showed more growth and degradation than did individual strains. At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 77% of the crude oil. This was followed by 69% by Pseudomonas sp. BPS1‐8, 64% by Bacillus sp. IOS1‐7, 45% by Pseudomonas sp. HPS2‐5, and 41% by Corynebacterium sp. BPS2‐6. The percentage of degradation by the mixed bacterial consortium decreased from 77 to 45% as the concentration of crude oil was increased from 1 to 12%. Temperature of 35°C and pH 7 were found to be optimum for maximum degradation.     

Natural cleanup of heavy fuel oil on rocks: an in situ experiment

Changes in the chemical composition of a heavy fuel oil, Bunker C, exposed to the elements for 556 days in the vicinity of Brest Harbour (France, (48 degrees 18(') N, 4 degrees 32(') W)) have been studied. Samples with exposure to full or reflected sunlight, and in the dark, were analysed by thin layer chromatography and gas chromatography coupled with mass spectrometry and compared with the initial oil. Using hopane as a conserved internal standard, an average of more than 56% of the total hydrocarbon in the residual stranded oil had been removed in the 556 days. The results indicate that dissolution, biodegradation and photooxidation all play important roles in the weathering process, with their respective contributions depending on the exposure.     

Bioremediation of petroleum hydrocarbons in anoxic marine sediments: Consequences on the speciation of heavy metals

We investigated the effects of biostimulation and bioagumentation strategies applied to harbor sediments displaying reducing conditions and high concentrations of petroleum hydrocarbons and heavy metals. We compared the microbial efficiency of hydrocarbon removal from sediments maintained for 60 days in anoxic conditions and inoculated with acetate, sulfate-reducing bacterial strains and acetate and sulfate-reducing bacteria. All treatments determined a significant increase in the microbial growth and significant decreases of hydrocarbon contents and of redox potential values. The addition of sulfate-reducing bacterial strains to the sediment was the most efficient treatment for the hydrocarbon removal. In all experiments, significant changes of the heavy metals’ phase repartition were observed. The results reported here suggest that the biodegradation of petroleum hydrocarbons in anoxic marine sediments may be enhanced by stimulating microbial anaerobic metabolism, but care should be applied to monitor the potential changes in the mobility and bioavailability of heavy metals induced by bio-treatments.     

Toxicogenomic analysis of immune system-related genes in Japanese flounder (Paralichthys olivaceus) exposed to heavy oil

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19-20 degrees C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.     

Plasmid incidence in marine bacteria isolated from petroleum polluted sites on different petroleum hydrocarbons

Oil-degrading bacteria isolated from oil spills, an industrial bay, and an offshore oil field by liquid enrichment on crude oils and polynuclear aromatic hydrocarbon compounds were screened for extra-chromosomal DNA. Plasmids were detected in 21% of the strains isolated on whole crude oil and in 17% of the strains isolated on polynuclear aromatic hydrocarbons. Multiple plasmids were observed in 50% of the plasmid-containing strains. Pseudomonas was the predominant genus isolated during the study. Plasmids do not appear to be of importance to these strains during degradation of freshly introduced oil at a nonpolluted site such as might be the case in an ocean oil spill. Plasmids do appear to be significant in the adaptation of Pseudomonas species to chronic petroleum pollution.     

Oxidation of fugitive methane in ground water linked to bacterial sulfate reduction

When fugitive methane migrates upward along boreholes of oil and gas wells, it may migrate into shallow ground water or pass through overlying soil to the atmosphere. Prior to this study, there was little information on the fate of fugitive methane that migrates into ground water. In a field study near Lloydminster, Alberta, Canada, we found hydrogeochemical evidence that fugitive methane from an oil well migrated into a shallow aquifer but has been attenuated by dissimilatory bacterial sulfate reduction at low temperature ( approximately 5 degrees C) under anaerobic conditions. Evidence includes spatial and temporal trends in concentrations of methane and sulfate in ground water and associated trends in concentrations of bicarbonate and sulfide. Within 10 m of the oil well, sulfate concentrations were low, and sulfate was enriched in both 34S and 18O. Sulfate concentrations had a strong positive correlation with delta13C values of bicarbonate, and sulfide was depleted in 34S compared to sulfate. These data indicate that bacterial sulfate reduction occurred near the production well. Near the oil well, elevated concentrations of bicarbonate were observed, and the bicarbonate was depleted in 13C. Modeling indicates that the main source of this excess 13C-depleted bicarbonate is oxidized methane. In concert with the sulfate concentration and isotope data, these results support an interpretation that in situ bacterial oxidation of methane has occurred, linked to bacterial sulfate reduction. Bacterial sulfate reduction may play a major role in bioattenuation of fugitive natural gas in ground water in western Canada.     

Long-term chemical effects of petroleum in south Louisiana wetlands—I. Organic carbon in sediments and waters

The chemical effects of chronic petroleum input into a shallow water marsh were examined by measuring hydrocarbon levels and dissolved organic carbon content of sediments associated with two active oil fields in south Louisiana. Annual levels of total organic carbon in the surface waters of the oil fields were higher by 1 mg C/l. in the salt marsh and 5 mg C/l. in the fresh marsh than the respective controlsites. Average dissolved organic carbon concentrations in the interstitial waters of cores taken within the oil field environments were 105% higher than the control in the salt marsh and 43% higher than the control in the fresh marsh. Significantly lower ratios of C₁₇ to pristane occurred in both oil field sediments; however, average odd-even predominance values were not indicative of petroleum contaminated sediments. The results indicate that microbial processes are responsible for dissolution of petroleum into dissolved organic carbon and that dissolved organic carbon concentrations may be a more significant measure of chronic petroleum input than hydrocarbon distribution.     

Biodegradation and environmental behavior of biodiesel mixtures in the sea: An initial study

Biodiesel, a mixture of fatty acid methyl esters (FAMEs) derived from animal fats or vegetable oils, is rapidly moving towards the mainstream as an alternative source of energy. However, the behavior of biodiesel, or blends of biodiesel with fossil diesel, in the marine environment have yet to be fully understood. Hence, we performed a series of initial laboratory experiments and simple calculations to evaluate the microbial and environmental fate of FAMEs. Aerobic seawater microcosms spiked with biodiesel or mixtures of biodiesel and fossil diesel revealed that the FAMEs were degraded at roughly the same rate as n-alkanes, and more rapidly than other hydrocarbon components. The residues extracted from these different microcosms became indistinguishable within weeks. Preliminary results from physical-chemical calculations suggest that FAMEs in biodiesel mixtures will not affect the evaporation rates of spilled petroleum hydrocarbons but may stabilize oil droplets in the water column and thereby facilitate transport.     

Geochemical evolution during the cracking of crude oil into gas under different pressure systems

Two comparative simulation experiments(a normal atmospheric-pressure opening system and a 20 MPa closed system)were conducted to study the geochemical evolution of n-alkane,sterane,and terpane biomarkers in the process of oil cracking into gas under different pressures.With an initial experimental temperature set at 300°C,the temperature was increased to 650°C at a heating rate of 30°C/h.The products were tested every 50°C starting at 300°C,and a pressure of 20 MPa was achieved using a water column.The low-maturity crude oil sample was from the Paleogene system in the Dongying sag in eastern China.The threshold temperature obtained for the primary oil cracking process in both pressure systems was 450°C.Before the oil was cracked into gas,some components,including macromolecular n-alkanes,were cracked into medium-or small-sized n-alkanes.The secondary oil cracking of heavy hydrocarbon gases of C2–5to methane mainly occurred between 550°C to 650°C,and the parameters Ln(C1/C2)and Ln(C1/C3),as well as the dry coefficients,increased.Overpressure inhibited the oil cracking process.In the 20 MPa system,the oil conversion rate decreased,the temperature threshold for gas generation rose,and oil cracking was inhibited.Compared with the normal pressure system,high-carbon n-alkanes and other compounds in the 20 MPa pressure system were reserved.Furthermore,the parameters∑C21-/∑22+,Ln(C1/C2),and Ln(C1/C3),as well as the dry coefficients,decreased within the main temperature range.During secondary oil cracking(550°C to 600°C),the Ph/nC18and Pr/nC17decreased.High pressure influenced the evolution of the biomarkers Ts and Tm,C31homohopane,C29sterane,and their related maturity parameters to different extents during oil cracking under different temperature ranges.     

Oil refinery wastewater treatment using coupled electrocoagulation and fixed film biological processes

Oil refinery wastewater was treated using a coupled treatment process including electrocoagulation (EC) and a fixed film aerobic bioreactor. Different variables were tested to identify the best conditions using this procedure. After EC, the effluent was treated in an aerobic biofilter. EC was capable to remove over 88% of the overall chemical oxygen demand (COD) in the wastewater under the best working conditions (6.5 V, 0.1 M NaCl, 4 electrodes without initial pH adjustment) with total petroleum hydrocarbon (TPH) removal slightly higher than 80%. Aluminum release from the electrodes to the wastewater was found an important factor for the EC efficiency and closely related with several operational factors. Application of EC allowed to increase the biodegradability of the sample from 0.015, rated as non-biodegradable, up to 0.5 widely considered as biodegradable. The effluent was further treated using an aerobic biofilter inoculated with a bacterial consortium including gram positive and gram negative strains and tested for COD and TPH removal from the EC treated effluent during 30 days. Cell count showed the typical bacteria growth starting at day three and increasing up to a maximum after eight days. After day eight, cell growth showed a plateau which agreed with the highest decrease on contaminant concentration. Final TPHs concentration was found about 600 mgL⁻¹ after 30 days whereas COD concentration after biological treatment was as low as 933 mgL⁻¹. The coupled EC-aerobic biofilter was capable to remove up to 98% of the total TPH amount and over 95% of the COD load in the oil refinery wastewater.     

Screening of biosurfactant producers from petroleum hydrocarbon contaminated sources in cold marine environments

An overview of literature about isolating biosurfactant producers from marine sources indicated no such producers have been reported form North Atlantic Canada. Water and sediment samples were taken from petroleum hydrocarbon contaminated coastal and offshore areas in this region. Either n-hexadecane or diesel was used as the sole carbon source for the screening. A modified colony-based oil drop collapsing test was used to cover sessile biosurfactant producers. Fifty-five biosurfactant producers belong to genera of Alcanivorax, Exiguobacterium, Halomonas, Rhodococcus, Bacillus, Acinetobacter, Pseudomonas, and Streptomyces were isolated. The first three genera were established after 1980s with interesting characteristics and limited relevant publications. Some of the 55 isolated strains were found with properties such as greatly reducing surface tension, stabilizing emulsion and producing flocculant. Isolates P6-4P and P1-5P were selected to demonstrate the performance of biosurfactant production, and were found to reduce the surface tension of water to as low as 28 dynes/cm.     

Biodegradation of crude oil using an efficient microbial consortium in a simulated marine environment

Ochrobactrum sp. N1, Brevibacillus parabrevis N2, B. parabrevis N3 and B. parabrevis N4 were selected when preparing a mixed bacterial consortium based on the efficiency of crude oil utilization. A crude oil degradation rate of the N-series microbial consortium reached upwards of 79% at a temperature of 25 °C in a 3.0% NaCl solution in the shake flask trial. In the mesocosm experiment, a specially designed device was used to simulate the marine environment. The internal tank size was 1.5 m (L)×0.8 m (W)×0.7 m (H). The microbial growth conditions, nutrient utilization and environmental factors were thoroughly investigated. Over 51.1% of the crude oil was effectively removed from the simulated water body. The escalation process (from flask trials to the mesocosm experiment), which sought to represent removal under conditions more similar to the field, proved the high efficiency of using N-series bacteria in crude oil degradation.     

The scientific connotation of oil and gas formations under deep fluids and organic-inorganic interaction

As a relatively stable craton block in the earth system, the petroliferous basin is influenced by the evolution of the earth system from the early development environment of source rocks, hydrocarbon formation, and reservoir dissolution to hydrocarbon accumulation or destruction. As a link between the internal and external factors of the basin, deep fluids run through the whole process of hydrocarbon formation and accumulation through organic-inorganic interaction. The nutrients carried by deep fluids promote the bloom of hydrocarbon-generating organisms and extra addition of carbon and hydrogen source, which are beneficial to the development of high-quality source rock and enhancement of the hydrocarbon generation potential. The energy carried by the deep fluid promotes the early maturation of the source rock and facilitates the hydrocarbon generation by activation and hydrogenation in high-mature hydrocarbon sources. The dissolution alteration of carbonate rocks and clastic reservoirs by CO_2-rich deep fluids improves the deep reservoir space, thus extending the oil and gas reservoir space into greater depth. The extraction of deeply retained crude oil by deep supercritical CO_2 and the displacement of CH_4 in shale have both improved the hydrocarbon fluidity in deep and tight reservoirs. Simultaneously, the energy and material carried by deep fluids(C, H, and catalytic substances) not only induce inorganic CH_4 formation by Fischer-Tropsch(F-T) synthesis and "hydrothermal petroleum" generation from organic matter by thermal activity but also cause the hydrothermal alteration of crude oil from organic sources. Therefore, from the perspective of the interaction of the earth's sphere, deep fluids not only input a significant amount of exogenous C and H into sedimentary basins but also improve the reservoir space for oil and gas, as well as their enrichment and accumulation efficiencies.     

Effects of three oil spill dispersants on marine bacterial populations: I. Preliminary Study. Quantitative Evolution of Aerobes

The effects of three hydrocarbon dispersant agents (Corexit 9527, Hydrogamosol LT and OSR LT 126) on the bacterial flora of the marine environment are analysed in 20-square-metre basins filled with lagoon seawater.Four months after the first treatment, oil slicks were no longer visible, whereas the appearance of the untreated reference slick had hardly changed. The treatment of 10-litre crude-oil slicks causes an appreciable and long lasting increase in the bacterial population.     

Biodegradation of petroleum products in experimental plots in Antarctic marine sediments is location dependent

Clean sediment collected from O'Brien Bay, East Antarctica, was artificially contaminated with a mix of Special Antarctic Blend diesel fuel and lubricating oil and deployed in two uncontaminated locations (O'Brien and Sparkes Bays) and a previously contaminated bay (Brown Bay) to evaluate whether a history of prior contamination would influence the biodegradation process. Detailed analysis of the hydrocarbon composition in the sediment after 11 weeks revealed different patterns of degradation in each bay. Biodegradation indices showed that hydrocarbon biodegradation occurred in all three bays but was most extensive in Brown Bay. This study shows that even within a relatively small geographical area, the longevity of hydrocarbons in Antarctic marine sediments can be variable. Our results are consistent with faster natural attenuation of spilt oil at sites with previous exposure to oil but further work is needed to confirm this. Such information would be useful when evaluating the true risk and longevity of oils spills.     

Occurrence of MTBE in Heating Oil and Diesel Fuel in Connecticut

The objective of this study was to confirm if MTBE, which is not intentionally added to fuels other than gasoline, is a contaminant in heating oil and diesel fuel. The study entailed conducting a statewide sampling program of heating oil and diesel fuel in Connecticut. An analytical method was developed to conduct analyses of heating oil and diesel fuel for MTBE in the milligram per liter (mg/L) range. The method involved equilibrating product with water to extract MTBE followed by static head-space analysis on aliquots of the water. Analyses were conducted using a gas chromatograph with a MTBE specific column. The statewide sampling program confirmed the widespread occurrence of MTBE in heating oil and diesel fuel. MTBE was detected in all samples collected during our sampling program at concentrations ranging from 9.7 to 906 mg/L in heating oil (26 samples), and from 74 to 120 mg/L in diesel fuel (five samples). Based on these ranges. MTBE concentrations in ground water in the vicinity of heating oil and diesel fuel releases could exceed thousands of micrograms per liter. Our analysis would suggest that the levels of heating oil and diesel fuel contamination observed could result from the commingling of only a few parts gasoline with thousands of parts of these fuels. The extent to which MTBE occurs in heating oil and diesel fuel nationwide is not known, but our data suggests that it may be widespread.     

Biodegradative potential and characterization of psychrotolerant polychlorinated biphenyl-degrading marine bacteria isolated from a coastal station in the Terra Nova Bay (Ross Sea, Antarctica)

Antarctic marine bacteria were screened for their ability to degrade polychlorinated biphenyls (PCB) as the sole carbon and energy source at both 4 degrees C and 15 degrees C. PCB-degrading isolates (7.1%) were identified by sequencing their 16S rDNA as Pseudoalteromonas, Psychrobacter and Arthrobacter members. One representative isolate per genera was selected for evaluating the biodegradative potential under laboratory scale and phenotypically characterized. Removal of individual PCB congeners was between 35.6% and 79.8% at 4 degrees C and between 0.4% and 82.8% at 15 degrees C. Differences in the removal patterns of PCB congeners were observed in relation to the phylogenetic affiliation: Arthrobacter isolate showed similar biodegradation efficiencies when growing at 4 degrees C and 15 degrees C, while Pseudoalteromonas better degraded PCBs at 15 degrees C. No biodegradation was detected for Psychrobacter isolate at 4 degrees C. Results obtained highlight the occurrence of PCB-degrading bacteria in Antarctic seawater and suggest the potential exploitation of autochthonous bacteria for PCB bioremediation in cold marine environments.     

Effects of chitin on microbial emulsification, mineralization potential, and toxicity of bunker C fuel oil

Bunker C, one of the most frequently spilled petroleum products in the US, is difficult to remove from oiled surfaces and is relatively recalcitrant to biodegradation; therefore, emulsification and biodegradability must be optimized before bioremediation can be considered a viable treatment option. Sand from a freshly oiled beach near Dutch Harbor, Alaska, was incubated at 10 degrees C with nutrients (Bushnell-Haas (BH)) or nutrients with crab shell chitin (BH-C). BH-C amendment resulted in greater numbers of bunker C emulsifiers and greater mineralization potentials for hexadecane, phenanthrene, and fluorene than with BH only. Compared to BH alone, mineralization potentials for bunker C also were higher in BH-C, with an estimated 8% of fuel oil mineralized after 6 weeks. Microbially emulsified oil was more toxic than in uninoculated controls (p < 0.05) as measured by Microtox assays. However, toxicity was significantly lower in BH-C than BH after 4 and 6 weeks incubation (p < 0.05).