A Model for diffusion controlled bioavailability of crude oil components

Crude oil is a complex mixture of several different structural classes of compounds including alkanes, aromatics, heterocyclic polar compounds, and asphaltenes. The rate and extent of microbial degradation of crude oil depends on the interaction between the physical and biochemical properties of the biodegradable compounds and their interactions with the non-biodegradable fraction. In this study we have systematically altered the concentration of non-biodegradable material in the crude oil and analyzed its impact on transport of the biodegradable components of crude oil to the microorganisms. We have also developed a mathematical model that explains and accounts for the dependence of biodegradation of crude oil through a putative bioavailability parameter. Experimental results indicate that as the asphaltene concentration in oil increases, the maximum oxygen uptake in respirometers decreases. The mathematically fitted bioavailability parameter of degradable components of oil also decreases as the asphaltene concentration increases.     

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.     

Laboratory evaluation of crude oil biodegradation with commercial or natural microbial inocula.

Experiments have been performed to screen eight microbial commercial products that, according to the manufacturers, are able to degrade crude oil. This study compared the crude oil biodegradation activity of commercial inocula with that of natural inocula (activated sludge and tropical aquarium water). Some of the latter were previously adapted to the crude oil as the only carbon source. Nutrients and sorbents in the commercial formulations were eliminated, and each inoculum was precultured on marine yeast extract medium. Crude oil biodegradability tests were conducted with close initial substrate concentration to initial bacterial concentration ratios (S0/X0) of 0.94 g of crude oil/10(9) CFU, which allowed a comparison of biodegradation activity. The inocula oxidized the crude oil after a short lag time of less than 3-18 days. After that time, the rate of oxidation varied between 45 and 244 mg O2/(L.day). Crude oil biodegradation after a 28-day test was effective only for 10 out of 12 inocula (from 0.1 to 25% in weight). Biodegradation mainly corresponded to the saturated fraction of the crude oil; the asphaltene fraction was never significantly biodegraded. Our results led to the conclusion that natural inocula, either adapted or not adapted to crude oil, were the most active (from 16 to 25% of loss in crude oil weight) and only one commercial inoculum was able to degrade 18% of the crude oil. Other inocula had a biodegradation activity ranging from 0.1 to 14%.     

Probing the Carbonyl Functionality of a Petroleum Resin and Asphaltene through Oximation and Schiff Base Formation in Conjunction with N-15 NMR

Despite recent advances in spectroscopic techniques, there is uncertainty regarding the nature of the carbonyl groups in the asphaltene and resin fractions of crude oil, information necessary for an understanding of the physical properties and environmental fate of these materials. Carbonyl and hydroxyl group functionalities are not observed in natural abundance ¹³C nuclear magnetic resonance (NMR) spectra of asphaltenes and resins and therefore require spin labeling techniques for detection. In this study, the carbonyl functionalities of the resin and asphaltene fractions from a light aliphatic crude oil that is the source of groundwater contamination at the long term USGS study site near Bemidji, Minnesota, have been examined through reaction with ¹⁵N-labeled hydroxylamine and aniline in conjunction with analysis by solid and liquid state ¹⁵N NMR. Ketone groups were revealed through ¹⁵N NMR detection of their oxime and Schiff base derivatives, and esters through their hydroxamic acid derivatives. Anilinohydroquinone adducts provided evidence for quinones. Some possible configurations of the ketone groups in the resin and asphaltene fractions can be inferred from a consideration of the likely reactions that lead to heterocyclic condensation products with aniline and to the Beckmann reaction products from the initially formed oximes. These include aromatic ketones and ketones adjacent to quaternary carbon centers, β-hydroxyketones, β-diketones, and β-ketoesters. In a solid state cross polarization/magic angle spinning (CP/MAS) ¹⁵N NMR spectrum recorded on the underivatized asphaltene as a control, carbazole and pyrrole-like nitrogens were the major naturally abundant nitrogens detected.     

Biological upgrading of heavy crude oil

Heavy crudes (bitumen) are extremely viscous and contain high concentrations of asphaltene, resins, nitrogen and sulfur containing heteroaromatics and several metals, particularly nickel and vanadium. These properties of heavy crude oil present serious operational problems in heavy oil production and downstream processing. There are vast deposits of heavy crude oils in many parts of the world. In fact, these reserves are estimated at more than seven times the known remaining reserves of conventional crude oils. It has been proven that reserves of conventional crude oil are being depleted, thus there is a growing interest in the utilization of these vast resources of unconventional oils to produce refined fuels and petrochemicals by upgrading. Presently, the methods used for reducing viscosity and upgradation is cost intensive, less selective and environmentally reactive. Biological processing of heavy crudes may provide an ecofriendly alternative or complementary process with less severe process conditions and higher selectivity to specific reactions to upgrade heavy crude oil. This review describes the prospects and strengths of biological processes for upgrading of heavy crude oil.     

Crude oil effects on microsomal mixed-function oxidase system components in the striped mullet (Mugil cephalus)

The effects of exposure to two types of crude oil on microsomal mixed-function oxidase system components in livers of juvenile striped mullet ($\text{Mugil cephalus}$) were investigated. Mullet were exposed for 4 days to emulsified Empire Mix or Saudi Arabian crude oils at an initial concentration of 75 ppm and an average of 1 ppm in the water column. Liver size was increased by about 50% following exposure to both oils. Since neither total hepatic protein nor microsomal protein increased as rapidly as did liver size, the concentrations of both were reduced following oil exposures. The proportion of microsomal protein to total hepatic protein or wet weight was not altered following crude oil exposure. Both cytochromes P-450 and $\text{b}$₅ were induced following oil treatment. NADPH-dependent enzymes assayed with cytochrome $\text{c}$ and dichlorophenolindophenol as substrates showed increases in activity after exposure to Empire Mix crude oil but only the latter enzyme activity was increased on a microsomal protein basis following Saudi Arabian crude oil treatment. Activities of NADH cytochrome $\text{c}$ and NADH cytochrome $\text{b}$₅ reductases appeared to vary with the protein level. However, since liver size was increased, oil-treated mullet had more of all parameters measured than did control mullet. Although the acute toxicity of Saudi Arabian crude oil to mullet is greater than that of Empire Mix crude oil, Empire Mix crude oil had greater inductive effects on microsomal oxidase components.     

Petroleum biodegradation in marine environments

Petroleum-based products are the major source of energy for industry and daily life. Petroleum is also the raw material for many chemical products such as plastics, paints, and cosmetics. The transport of petroleum across the world is frequent, and the amounts of petroleum stocks in developed countries are enormous. Consequently, the potential for oil spills is significant, and research on the fate of petroleum in a marine environment is important to evaluate the environmental threat of oil spills, and to develop biotechnology to cope with them. Crude oil is constituted from thousands of components which are separated into saturates, aromatics, resins and asphaltenes. Upon discharge into the sea, crude oil is subjected to weathering, the process caused by the combined effects of physical, chemical and biological modification. Saturates, especially those of smaller molecular weight, are readily biodegraded in marine environments. Aromatics with one, two or three aromatic rings are also efficiently biodegraded; however, those with four or more aromatic ring are quite resistant to biodegradation. The asphaltene and resin fractions contain higher molecular weight compounds whose chemical structures have not yet been resolved. The biodegradability of these compounds is not yet known. It is known that the concentrations of available nitrogen and phosphorus in seawater limit the growth and activities of hydrocarbon-degrading microorganisms in a marine environment. In other words, the addition of nitrogen and phosphorus fertilizers to an oil-contaminated marine environment can stimulate the biodegradation of spilled oil. This notion was confirmed in the large-scale operation for bioremediation after the oil spill from the Exxon Valdez in Alaska. Many microorganisms capable of degrading petroleum components have been isolated. However, few of them seem to be important for petroleum biodegradation in natural environments. One group of bacteria belonging to the genus Alcanivorax does become predominant in an oil-contaminated marine environment, especially when nitrogen and phosphorus fertilizers are added to stimulate the growth of endogenous microorganisms.     

Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil

Pseudomonas aeruginosa strain SP4, isolated from petroleum-contaminated soil in Thailand, was used to produce a biosurfactant from a nutrient broth with palm oil as the carbon source. The key components of the crude biosurfactant were fractionated by using HPLC-ELSD technique. With the use of ATR-FTIR spectroscopy, in combination with (1)H NMR and MS analyses, chemical structures of the fractionated components of the crude biosurfactant were identified as rhamnolipid species. When compared to synthetic surfactants, including Pluronic F-68, which is a triblock nonionic surfactant containing poly(ethylene oxide) and poly(propylene oxide), and sodium dodecyl sulfate, the crude biosurfactant showed comparable physicochemical properties, in terms of the surface activities. The crude biosurfactant reduced the surface tension of pure water to 29.0 mN/m with a critical micelle concentration of approximately 200 mg/l, and it exhibited good thermal and pH stability. The crude biosurfactant also formed stable water-in-oil microemulsions with crude oil and various types of vegetable oils, but not with short-chain hydrocarbons.     

Sphingobacterium bambusaue及其紫外诱变菌株的石油降解功能

【目的】研究Sphingobacterium bambusaue及其紫外诱变菌株的石油降解功能。【方法】紫外诱变后筛选石油降解高效菌株; 以不同石油浓度、pH值及盐浓度优化培养条件, 用重量法检测高效菌株石油降解率。【结果】发现菌株S. bambusaue在石油降解培养基中培养5 d的石油降解率为25.86%, UV诱变高效菌株IBFC2009-S3培养5 d的石油降解为42.85%, 比始发菌株提高65.7%; UV诱变菌株IBFC2009-S3的优化培养条件为石油浓度0.5 g/L、pH值7.0以及NaCl质量浓度为10 g/L, 其石油降解率可达50.51%。【结论】首次报道S. bambusaue具有石油降解功能; 紫外诱变获得的菌株S3的石油降解能力较强。     

Microbial degradation kinetics of solid alkane dissolved in nondegradable oil phase

An Acinetobacter species was isolated and found to be able to grow on crude oil n-alkanes and solid alkanes at room temperature as the sole carbon source. The growth of the isolate on n-heneicosane dissolved in non-biodegradable pristane has been studied. A kinetic model of the growth of microorganism on the hydrophobic substrate dissolved in non-biodegradable oil droplet assuming direct contact of cell with oil droplet was developed and validated with a model system of crude oil biodegradation. The model was focused on the substrate transport to the cell being contact with the surface of droplet. The high value of saturation constant of n-heneicosane, K_s = 0.086 kg m⁻³, and the maximum specific growth rate, μ_m = 0.60 h⁻¹, were obtained. The transport limitation was considered and estimated. The high value of attached cell fraction was reasonable to explain the observed growth rate by the direct contact model and varied with time till it reached a plateau at the stationary growth phase. By considering the direct contact of the cells with the surface of pristane and the transport of n-heneicosane to the cell, the degradation of hydrophobic substrate in the oil phase could be elucidated.     

Variability in Pseudomonas aeruginosa lipopolysaccharide expression during crude oil degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Evidence for in situ crude oil biodegradation after the Prestige oil spill

In November 2002, the oil tanker Prestige sank off the Spanish coast after releasing approximately 17,000 tones of heavy fuel, coating several hundred kilometers of coastline in oil sludge. In December 2002 and February 2003, samples were collected from the shore of the Galician coast to analyse the indigenous population ability to carry out crude oil degradation in situ. Carbon isotopic ratio of the dissolved inorganic carbon (DIC) in seawater samples was used as a rapid method to directly assess activity of microbes on the oil components. 12CO2/13CO2 ratio in samples from certain locations along the coast revealed degradation of a very delta13C-negative source such as the Prestige crude oil (-30.6 per thousand). Putative biodegradation processes taking place at areas with high income of fresh seawater could not be detected with this technique. Laboratory-scale biostimulation processes carried out in samples with the highest oil biodegradation activity showed that N/P deficiency in seawater is a limiting factor for crude oil degradation. The most probable number (MPN) of crude oil component degraders was estimated for several aromatic compounds (naphthalene, anthracene, phenanthrene, pyrene) and for undecane. Our results clearly show that bacteria present in the contaminated water are readily able to transform components of the crude oil into inorganic carbon.     

陕北地区石油污染土壤中不动杆菌属的筛选、鉴定及降解性能

从陕北地区石油污染土壤中分离鉴定得到两株不动杆菌属(Acinetobacter sp.)的高效石油降解菌A.sp 1和A.sp 2,分别从盐浓度、pH值、氮源、磷源和接种量等因素进行研究以确定其最佳石油降解条件,并进一步通过GC-MS(Gas ChromatographyMass Spectrometer)方法分析其在最佳条件下对原油组分的不同降解性能。结果显示:A.sp 1在盐浓度为1%、pH值为6—7、磷源为KH2PO4和K2HPO4、氮源为尿素和接种量为4%的条件下,最高降解率可达到60%。A.sp 2在盐浓度为1%、pH值为7—9、磷源为KH2PO4和K2HPO4、氮源为硝酸铵和接种量为8%的条件下,最高降解率可达到67%。GC-MS分析结果表明,菌株A.sp 1对石油烃类C21—C25有明显的降解效果,菌株A.sp 2对石油烃类C20—C30的降解效果较好。     

Effect of Environmental Parameters on Bacterial Degradation of Bunker C Oil, Crude Oils, and Hydrocarbons

Mixed microbial cultures, previously enriched on Bunker C fuel oil, grew on and degraded Bunker C fuel oil at temperatures ranging from 5 to 28 C. At 15 C, 41 to 85% of the benzene-soluble components of Bunker C disappeared after incubation for 7 days; at 5 C the values ranged from 21 to 52% after 14 days of incubation. A Nocardia sp. isolated from a culture enriched on Bunker C oil grew on Venezuelan crude oil, Bunker C, hexadecane, and a hydrocarbon mixture at temperatures of 5 and 15 C. The 10-C decrease in temperature resulted in an average 2.2-fold decrease in generation time of the bacteria. Gas-liquid chromatographic measurements of Venezuelan and Arabian crude oils which had been incubated with the Nocardia sp. showed significant degradation of the n-alkane portion and the chromatographically unresolved components of the oils. The concentration of elemental nitrogen required to bring about the disappearance of 1 mg of hexadecane by the Nocardia sp. was 0.5 mg. The results confirm suggestions that the rate of natural biodegradation of oil in marine temperate-to-polar zones is probably limited by low temperatures and phosphorus concentrations, but suggest that the concentrations of nitrogen occurring naturally are probably not rate-limiting factors.     

Biological souring of crude oil under anaerobic conditions

Seawater injection into oil reservoirs for purposes of secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil-field water are believed to be produced by microbial degradation of crude oil. The objective of this research was to investigate mechanisms of souring, focusing specifically on VFA production via crude oil biodegradation. To this end, a microbial consortium collected from an oil–water separator was suspended in seawater; crude oil or liquid n-alkane mixture was added to the culture medium as the sole carbon source, and the culture was incubated under anaerobic conditions for 190 days. Physicochemical analysis showed that preferential toluene degradation and sulfate reduction occurred concomitantly in the culture containing crude oil. Sulfide concentrations were much lower in the alkane-supplemented culture than in the crude oil-supplemented culture. These observations suggest that SRB are related to the toluene activation and VFA consumption steps of crude oil degradation. Therefore, the electron donors for SRB are not only VFA, but many components of crude oil, especially toluene. Alkanes were also degraded by microorganisms, but did not contribute to reservoir souring.     

Effects of visible and UV light on the characteristics and properties of crude oil-in-water (O/W) emulsions

The effects of visible and UV light on the characteristics and properties of Prudhoe Bay (PB) and South Louisiana (SL) emulsions were investigated to better understand the role of sunlight on the fate of spilled crude oils that form emulsions with a dispersant in the aquatic environment. Before irradiation, crude oil emulsions showed the presence of dispersed crude oil micelles in a continuous water phase and crude oil components floating on the surface. The crude oil micelles decreased in size with irradiation, but emulsions retained their high degree of polydispersity. UV irradiation reduced the stability of emulsions more effectively than visible light. The reduction of micelles size caused the viscosity of emulsions to increase and melting point to decrease. Further, irradiation increased acid concentrations and induced ion formation which lowered the pH and increased the conductivity of emulsions, respectively. Ni and Fe in PB emulsions were extracted from crude oil with UV irradiation, which may provide an efficient process for metal removal. The emulsions were stable toward freeze/thaw cycles and their melting temperatures generally decreased with irradiation. Evidence of ˙OH production existed when emulsions were exposed to UV but not to visible light. The presence of H(2)O(2) enhanced the photodegradation of crude oil. Overall, the changes in emulsion properties were attributed to direct photodegradation and photooxidation of crude oil components.     

Isolation, identification, and structure of a potent alkyl-peroxyl radical scavenger in crude canola oil, canolol

Alkylhydroperoxides in oxidized oil are undesirable components because they become alkylperoxyl radicals (ROO*) in the presence of heme, hemoglobin, or myoglobin in red meat. ROO* are biochemically reactive and damage nucleic acids and proteins, thereby harming living cells. We isolated a component, a highly potent ROO* scavenger, from crude canola oil (rapeseed), which we designated canolol, and identified its chemical structure, 4-vinyl-2,6-dimethoxyphenol. The canolol content of crude canola oil greatly increased after the seed was roasted as compared with that from unroasted seed, but it decreased in highly purified oil. This anti-ROO* activity was highest in crude oil, deceased after each refining step, and was lowest in highly purified refined oil. Canolol was thus generated during roasting. As shown previously, canolol is one of the most potent anti-ROO* components in crude canola oil and its potency is much greater than that of well-known antioxidants, including alpha-tocopherol, vitamin C, beta-carotene, rutin, and quercetin.     

Variability in Pseudomonas aeruginosa Lipopolysaccharide Expression during Crude Oil Degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Unexpected Interaction with Dispersed Crude Oil Droplets Drives Severe Toxicity in Atlantic Haddock Embryos

The toxicity resulting from exposure to oil droplets in marine fish embryos and larvae is still subject for debate. The most detailed studies have investigated the effects of water-dissolved components of crude oil in water accommodated fractions (WAFs) that lack bulk oil droplets. Although exposure to dissolved petroleum compounds alone is sufficient to cause the characteristic developmental toxicity of crude oil, few studies have addressed whether physical interaction with oil micro-droplets are a relevant exposure pathway for open water marine speices. Here we used controlled delivery of mechanically dispersed crude oil to expose pelagic embryos and larvae of a marine teleost, the Atlantic haddock (Melanogrammus aeglefinus). Haddock embryos were exposed continuously to two different concentrations of dispersed crude oil, high and low, or in pulses. By 24 hours of exposure, micro-droplets of oil were observed adhering and accumulating on the chorion, accompanied by highly elevated levels of cyp1a, a biomarker for exposure to aromatic hydrocarbons. Embryos from all treatment groups showed abnormalities representative of crude oil cardiotoxicity at hatch (5 days of exposure), such as pericardial and yolk sac edema. Compared to other species, the frequency and severity of toxic effects was higher than expected for the waterborne PAH concentrations (e.g., 100% of larvae had edema at the low treatment). These findings suggest an enhanced tissue uptake of PAHs and/or other petroleum compounds from attached oil droplets. These studies highlight a novel property of haddock embryos that leads to greater than expected impact from dispersed crude oil. Given the very limited number of marine species tested in similar exposures, the likelihood of other species with similar properties could be high. This unanticipated result therefore has implications for assessing the ecological impacts of oil spills and the use of methods for dispersing oil in the open sea.     

Molecular Simulation and the Aggregation of the Heavy Fractions in Crude Oils

The use of Molecular Simulation in the study of aggregates of the molecules of the heavy fractions of crude oils is reviewed. Molecular Mechanics calculations of aggregates of asphaltenes having a single large aromatic region (continental type) and others having smaller aromatic regions connected by alkyl chains (archipelago type) are discussed in terms of the molecular recognition processes present in petroleum. Stacking of the aromatic regions was the most important process in the formation of aggregates of asphaltenes of the continental type with some unfavorable contributions from its saturated rings and alkyl side chains. The steric interference of these groups limits the growth of the aggregates to a small number of molecules. The asphaltenes of the archipelago type showed more complex aggregates because some molecules act as bridges and tangling between them may occur. The interaction of the asphaltene aggregates with resin molecules was analyzed and it was found that the high selectivity for some sites of the asphaltenes explains the specificity of the resins for its own crude oil.