Microbial petroleum degradation: application of computerized mass spectrometry.

An analytical procedure is presented for obtaining detailed characterization of petroleum hydrocarbons which undergo microbial degradation. The procedure includes column chromatographic separation and characterization of the resulting fractions by mass spectrometry and gas chromatography. The use of computerized low-resolution mass spectrometry is offered as a method for assessing microbial degradation of petroleum. This method provides information which cannot, at the present time, be obtained by other available analytical methods. Use of this method to evaluate degradation of a South Louisiana crude oil by a mixed culture of estuarine bacteria revealed that asphaltenes and resins increased by 28% after degradation, while saturates and aromatics decreased by 83.4% and 70.5%, respectively. Most of the normal and branched-chain alkanes were degraded (96.4%), but an increase in long-chain alkanes (C28-C32) after degradation was observed by gas-liquid chromatography. Susceptibility of cycloalkanes to degradation was less as the structure varied, i.e., 6-ring greater than 1-ring greater than 2-ring greater than 3-ring greater than 5-ring greater than 4-ring. Susceptibility of aromatic components to degradation decreased with increase in the number of rings, viz., monoaromatics greater than diaromatics greater than triaromatics greater than tetraaromatics greater than pentaaromatics. Aromatic nuclei containing sulfur were twice as refractory as non-sulfur analogs.     

Characterization of Petroleum-Contaminated Soils by Thin-Layer Chromatography with Flame Ionization Detection

Petroleum hydrocarbons from 20 soils from refineries or other industrial sites were extracted with a mixture of chloroform and methanol (1:1, v/v), and the extracts were analyzed by thin layer chromatography with flame ionization detection (TLC/FID). The TLC/FID procedure has been used widely in biological and medical research but generally has been underutilized in environmental chemistry. The analysis method involved spotting a small volume of sample extract (typically 1 to 3?µl) on ten silica-coated quartz rods, and chromatographically separating constituents in the spots using solvent systems of increasing polarities (hexane, toluene, and dichloromethane + methanol). We achieved complete separation of saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes from the hydrocarbon-contaminated soils with this method. Analysis of the separated constituents by TLC/FID also allowed quantification of aromatic and aliphatic hydrocarbons without interference from soil biogenic lipids. A simplified version of the method permitted excellent separation of aliphatics +aromatics (forming a single peak) from resins and asphaltenes. The procedure is rapid (complete analysis of ten samples in about 1?h after extraction). Thus, the method seems well suited for synoptic surveys or screening and characterizing numerous samples prior to using more detailed and costly analyses.     

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.     

Characteristics of bioemulsifier V2-7 synthesized in culture media added of hydrocarbons: chemical composition, emulsifying activity and rheological properties

The bioemulsifier V2-7 is an exopolysaccharide (EPS) synthesized by strain F2-7 of Halomonas eurihalina and it has the property of emulsifying a wide range of hydrocarbons i.e. n-tetradecane, n-hexadecane, n-octane, xylene mineral light and heavy oils, petrol and crude oil. Characteristics of exopolysaccharide V2-7 produced in media supplemented with various hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol or crude oil) were studied. Yield production varied from 0.54 to 1.45 g L(-1) according to the hydrocarbon added, in the same way chemical composition, viscosity and emulsifying activity of EPS varied with the culture conditions. Respect to chemical composition, percentage of uronic acids found in exopolymers produced in hydrocarbon media was always higher than that described for V2-7 EPS (1.32%) obtained with glucose. This large amount of uronic acid present could be useful in biodetoxification and waste water treatment. On the other hand, the highest amount of biopolymer was synthesized with mineral light oil, while the most active emulsifiers were those obtained from media added with petrol and n-octane. Furthermore, all EPS were capable of emulsifying crude oil more efficiently than the three chemical surfactants tested as control (Tween 20, Tween 80 and Triton X-100). The capacity of strain F2-7 to grow and produce bioemulsifier in presence of oil hydrocarbons together with the high emulsifying activity and low viscosity power of the biopolymers synthesized in hydrocarbons media could be considered highly beneficial for application of both bioemulsifier and producing strain in bioremediation of oil pollutants.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

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.     

Alasan, a new bioemulsifier from Acinetobacter radioresistens.

Acinetobacter radioresistens KA53, isolated by enrichment culture, was found to produce an extracellular, nondialyzable emulsifying agent (referred to as alasan) when grown on ethanol medium in a batch-fed reactor. The crude emulsifier was concentrated from the cell-free culture fluid by ammonium sulfate precipitation to yield 2.2 g of emulsifier per liter. Alasan stabilized a variety of oil-in-water emulsions, including n-alkanes with chain lengths of 10 or higher, alkyl aromatics, liquid paraffin, soybean and coconut oils, and crude oil. Alasan was 2.5 to 3.0 times more active after being heated at 100 degrees C under neutral or alkaline conditions. Emulsifying activity was observed over the entire pH range studied (pH 3.3 to 9.2), with a clear maximum at pH 5.0. Magnesium ions stimulated the activity both below (pH 3.3 to 4.5) and above (pH 5.5 to 9.3) the pH optimum. Alasan activity was higher in 20 mM citrate than in 20 mM acetate or Tris-HCl buffer. Preliminary chemical characterization of alasan indicated that it is a complex of an anionic, high-molecular-weight, alanine-containing heteropolysaccharide and protein.     

A renewable energy source — hydrocarbon gases resulting from pyrolysis of the marine nanoplanktonic alga Emiliania huxleyi

The marine coccolithophore, Emiliania huxleyi, grown in the laboratory was subjected to vacuum pyrolysis at various temperatures from 100 to 500 °C. The highest yield of pyrolytic gases (183 mL g⁻¹ dry cells) was obtained at 400 °C. The amount of total hydrocarbon gas produced at 400 °C was 129 mL, about 10 times higher than at 300 °C. CH₄ was the major component at the high gas-production stage (400–500 °C). The great increase in hydrocarbon gases at 400 °C was accompanied by a marked decrease in liquid saturates and aromatics. The results indicate that the liquid hydrocarbons (oil) produced by pyrolysis at lower temperature is a direct source for the formation of the hydrocarbon gases. Due to its large potential for the production of biomass and hydrocarbons with low energy input, E. huxleyi is suggested as one of candidates for the production of renewable fuels. This revised version was published online in July 2006 with corrections to the Cover Date.     

A microbial consortium isolated from a crude oil sample that uses asphaltenes as a carbon and energy source

A microbial consortium capable of mineralizing asphaltenes was obtained from the Maya crude oil. The enrichment system was built with a glass column reactor containing mineral medium supplied with asphaltenes as energy and carbon source. The consortium growth was evaluated in Casoy agar during 40 weeks. The steady-state phase of the enriched bacterial community was observed after 10 weeks when the culture reach 10(5) to 10(6) CFU ml(-1). The isolates belong to bacterial genus reported for degradation of other hydrocarbons and they were identified as Corynebacterium sp., Bacillus sp., Brevibacillus sp. and Staphylococcus sp. The bacterial consortium growth was evaluated by a viable counts during 14 days exposed to different aeration, temperature, salinity, and pH conditions. The ability of the consortium to mineralize asphaltenes was evaluated using the method of ISO 9439 in glass column reactors of 20 x 3.2 cm during 13 days. Temperatures of 55 degrees C and salinity of 1.8% were growth limiting. The respiration of the microbial consortium using asphaltenes as a sole carbon source (800 micromoles CO2 in 13 days) was significantly higher than those of the samples containing only the microbial consortium (200 micromoles CO2) or only asphaltenes (300 micromoles CO2). These results indicated the existence of asphaltenes-degradating microbes in the crude oil and confirmed that the consortium could mineralize asphaltenes in conditions of room temperature, salinity of 100 ppm, aeration of 1 l min(-1) and pH of 7.4.     

Biodegradability of Erika fuel oil

The biodegradation of the fuel oil resulting from the Erika wreck was studied by computerized gas chromatography in laboratory cultures over 80 days. The total extent of biodegradation was around 11%. The degraded compounds were the molecules of the light cracking fraction used to dilute the distillation residue, as well as n-alkanes and part of the branched alkanes. Part of the polycyclic aromatic hydrocarbons PAH and alkyl PAH was also degraded. The very low biodegradability of the Erika fuel is attributable to its chemical composition. The product is rich in components that are inherently resistant or refractory to microbial metabolism such as resins, asphaltenes and polycyclic saturated and aromatic hydrocarbons.     

Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria

The susceptibility to bioremediation of the hydrocarbons contained in a waste from crude oil extraction was examined. Laboratory scale batch reactors were inoculated with indigenous bacteria and biodegradation was followed for 45 days. The total hydrocarbon content was reduced to 70% of its initial value at the end of the experiments. Saturated and aromatic hydrocarbons were the most readily degraded fractions with, respectively, 70% and 60% of the fraction remaining at the end of the experiment. A minor degradation was observed in the resins fraction (20%), whereas the asphaltenes fraction remained almost constant.The substrate preferences of the natural population towards various fractions of the crude oil were determined by both the length of the lag phase and the slope of the exponential growth in a mineral salt-base medium containing either of the different hydrocarbon fraction as the sole source of carbon. The highest consumption rate for every fraction during the time course experiments was in agreement with the shortest lag phase and the greatest exponential growth slope in the corresponding selective media, indicating changes in the population composition.     

Effects of HCO-3 on Surface Calcification and CO2 Fixation in Marine Emiliania huxleyi

Emiliania huxleyi is a ubiquitous species with the largest biomass in marine planktonics. When cells of E. huxleyi were grown in ESM with additional 20 mmol/L HC03- , coccolith scales were formed on cell surface and the weight ratio of calcium carbonate fixed in coecoliths to organic substance in cells was about 2.47: 1. It was only about 0.05: 1 in cells grown in ESM without HCO3- addition, where no coccoliths were observed under scanning electron microscope, and the content of lipid reached 18.1% of dry. cell weight. It was demonstrated that the HCO3- concentration was the key factor to control the calcification on cell surface. Therefore, in addition to the pathway of photosynthesis for CO2 fixation, calcification on cell surface forming coccoliths is an alternative pathway for fixing dissolved inorganic carbon in E. huxleyi. Moreover, being rich in lipids, E. huxleyi cells produced high content of hydrocarbons including extractable organic matter, saturates and aromatics under pyrolysis at 300℃. Among those, the yield of saturates from E. huxleyi reached as high as 2.8%, 6-15 times that from other algae. All these suggest that E. huxleyi is a good experimental system for studies on the optimization of environment through carbon cycle and renewable energy in algal biomass.     

Selective substrate utilization by marine hydrocarbonoclastic bacteria

Several strains of bacteria, isolated from marine environments, were characterized for their hydrocarbon oxidizing abilities using a complex synthetic mixture of hydrocarbons. Attempts were made at a broad classification of these organisms on the basis of their behavior towards four major groups of hydrocarbons, normal paraffins, iso-paraffins, cyclo-paraffins, and aromatics, known to be present in crude oils. Although bacteria appear to be able to oxidize hydrocarbons at random, this study has shown that it may be possible to recognize a rudimental pattern if we view their oxidative abilities in terms of groups of hydrocarbons rather than individual compounds. A study of the action of combined strains on the synthetic hydrocarbon mixture was performed. It was found that no particular benefit could be derived as compared to the use of single strains.     

Low-temperature microbial degradation of crude oil products differing in the extent of condensation

Out of the 30 strains capable of oil degradation at 4-6 degrees C, four were selected by the ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol-benzene resins) at 4-6 degrees C. The maximum degrees of degradation of masut and ethanol-benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). The strains provide a basis for developing biodegrader preparations applicable to bioremediation of oil-polluted sites under the conditions of cold climate.     

Production of alkanes (C7–C29) from different part of poplar tree via direct deoxy-liquefaction

Poplar leaves, poplar bark and poplar wood were deoxy-liquefied directly in an air-proof stainless steel reactor at different temperatures. The oils from leaves at 350 °C, from bark at 400 °C and from wood at 450 °C, at which the liquid product yields were the maximum, were analyzed by GC–MS. The oils obtained from three parts of poplar tree were quite different from each other in the relative contents of their compositions. The oil from leaves was rich in hydrocarbons (alkanes: C₇–C₂₉; aromatics) and poor in phenolics, while oil from wood was rich in phenolics and poor in hydrocarbons. The oil from bark was moderate. Relative contents of hydrocarbons in the leaves oil were as high as 60.01% but decreased to 29.71% in bark oil and 11.43% in wood oil. GC analysis of gases and FT-IR, GC–MS and elemental analysis of oils were performed in this study.     

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.     

Aerobic Biodegradation of Crude Oil Components by Acidophilic Mycobacteria

Biodegradation of crude oil components by strain AG_S10, an acidophilic member of the genus Mycobacterium, was studied under extremely acidic conditions (pH 2.5). The degree of degradation of the same hydrocarbons in different kinds of oil was found to be different. The direction of biodegradation was, however, the same: the share of n-alkanes in oxidized oil decreased, while the share of branched alkanes increased. At the same time, the degree of redistribution of methane hydrocarbons in degraded oil varied significantly for different oils, although no strict dependence on the type of oil was found. After 28 days of incubation at 30°C and pH 2.5, the degradation of n- and iso-alkanes was 99 and 44%, respectively for the light, low-viscosity oil of the Nizhnevartovsk deposit, 58 and 32%, respectively for the medium-density oil of the Moscow oil-procesing plant, and 80 and 16% and 99 and 69%, respectively for the heavy, viscous oils of the Cheremukhovskoe and Usinkoye oil fields. Moreover, after extended cultivation time strain AG_S10 completely utilized alkanes, as well as a significant part of the naphthene component of the aliphatic fraction. The studied strain was characterized by ability to oxidize a broad spectrum of methane hydrocarbons, including high-molecular C₁₇–C₃₀n-alkanes, in oils of different properties and composition. Apart from its scientific interest, farther investigation of biodegradation of high-paraffin oils and viscous oils with elevated paraffin content by strain AG_S10 may be useful in view of the technical issues associated with paraffin accumulation in the course of recovery and transportation of these oils.     

Degradation of Petroleum by an Alga, Prototheca Zopfii

Prototheca zopfii is an achlorophyllous alga which degrades oil. It has been found to degrade 10 and 40% of a motor oil and crude oil, respectively, when tested under appropriate conditions. Degradation of the crude oil observed in this study compares well with the amount of degradation accomplished by bacteria. P. zopfii was found to degrade a greater percentage of the aromatic hydrocarbons in motor oil than of the saturated hydrocarbons and a greater percentage of saturated hydrocarbons in crude oil than of aromatic hydrocarbons. Resins and asphaltens were produced during degradation of motor oil, whereas these fractions in crude oil were degraded. P. zopfii did not demonstrate preferential utilization of lower homologues of cycloalkanes and aromatics as has been observed with bacteria.     

Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B.

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.