Kathleen Semple

Comparison of oil composition changes due to biodegradation and physical weathering in different oils

The well-characterized Alberta Sweet Mixed Blend oil and several other oils which are commonly transported in Canada were physically weathered and then incubated with a defined microbial inoculum. The purpose was to produce quantitative data on oil components and component groups which are more susceptible or resistant to biodegradation, and to determine how oils rank in relation to each other in terms of biodegradation potential. The biodegraded oils were characterized by quantitative determination of changes in important hydrocarbon groups including the total petroleum hydrocarbons, total saturates and aromatics, and also by quantitation of more than 100 individual target aliphatic, aromatic and biomarker components. The study reveals a pattern of distinct oil composition changes due to biodegradation, which is significantly different from the pattern due to physical or short-term weathering. It is important to be able to distinguish between these two forms of loss, so that loss due to weathering is not interpreted as loss due to biodegradation in the laboratory or in the field. Based on these findings, the oil composition changes due to biodegradation can be readily differentiated from those due to physical weathering. To rank the tested oils with respect to biodegradability, losses in total petroleum hydrocarbons and aromatics were used to calculate biodegradation potential indices, employing equations proposed by Environment Canada and the US National Oceanic and Atmospheric Administration. The different methods produced very similar biodegradation trends, confirming that patterns of oil biodegradability do exist.

Anaerobic biodegradation of longer-chain n-alkanes coupled to methane production in oil sands tailings.

Extraction of bitumen from mined oil sands ores produces enormous volumes of tailings that are stored in settling basins (current inventory ≥ 840 million m(3)). Our previous studies revealed that certain hydrocarbons (short-chain n-alkanes [C(6)-C(10)] and monoaromatics [toluene, o-xylene, m-xylene]) in residual naphtha entrained in the tailings are biodegraded to CH(4) by a consortium of microorganisms. Here we show that higher molecular weight n-alkanes (C(14), C(16), and C(18)) are also degraded under methanogenic conditions in oil sands tailings, albeit after a lengthy lag (~180 d) before the onset of methanogenesis. Gas chromatographic analyses showed that the longer-chain n-alkanes each added at ~400 mg L(-1) were completely degraded by the resident microorganisms within ~440 d at ~20 °C. 16S rRNA gene sequence analysis of clone libraries implied that the predominant pathway of longer-chain n-alkane metabolism in tailings is through syntrophic oxidation of n-alkanes coupled with CO(2) reduction to CH(4). These studies demonstrating methanogenic biodegradation of longer-chain n-alkanes by microbes native to oil sands tailings may be important for effective management of tailings and greenhouse gas emissions from tailings ponds.

Chloroperoxidase-mediated modifications of petroporphyrins and asphaltenes

Abstract Octaethylporphine, nickel octaethylporphine, vanadyl octaethylporphine, and a petroporphyrin-rich, low-molecular-weight fraction of asphaltenes from Cold Lake heavy oil were treated with chloroperoxidase from Caldariomyces fumago . Reactions in aqueous phosphate buffer (pH 3.0) or in a ternary solvent system of toluene, isopropanol, and water (3 m m phosphate buffer, pH 3.0) were absolutely dependent on the presence of hydrogen peroxide and chloride. Enzyme treatment resulted in reduction of the absorption of the Soret peak. The porphyrins and asphaltenes were insoluble in the aqueous buffer system; thus, mass transfer limited the reactions. These substrates were more soluble in the ternary system and the reactions were more complete, yielding decreases in metal recovery associated with the methylene chloride-soluble porphyrin-containing material. These decreases were: 93% of the Ni from nickel octaethylporphine, 53% of the V from vanadyl octaethylporphine, and 20% of the total Ni and V from the asphaltene fraction. This work clearly demonstrated that an extracellular enzyme, chloroperoxidase, can alter components in the asphaltene fraction of petroleum. Because of the requirement for chloride, the enzyme-mediated reactions likely yield chlorinated products which would be undesirable in a refinery feedstock if this enzymatic process was used for the demetallation of petroleum.

Effect of water-miscible organic solvents on the catalytic activity of cytochrome c

The effect of five water-miscible organic solvents (tetrahydrofuran, N,N-dimethylformamide, acetonitrile, 2-propanol, and methanol) on the oxidation of pinacyanol chloride (Quinaldine Blue) by horse heart cytochrome c was determined. Hydrogen peroxide was used as the oxidant, and a change in catalytic property of the dissolved protein was observed after a certain threshold concentration of the organic solvent had been reached. The maximum specific activity was correlated with the Dimroth-Reichardt parameter for the solvents, which is directly related to the free energy of the solvation process. The kinetic constants for the oxidation of pinacyanol chloride were determined in systems containing different proportions of tetrahydrofuran. The best catalytic efficiency (kcat/KM,app) was obtained in a system containing 50% tetrahydrofuran in phosphate buffer. In a mixture containing 90% tetrahydrofuran, cytochrome c showed 18% of its maximum activity. The inactivation of cytochrome c was mainly due to the presence of hydrogen peroxide, and a direct correlation was found between the inactivation constant and the concentration of hydrogen peroxide in the system. The chemical modifications and immobilization of cytochrome c were able to change its biocatalytic activity and stability in the organic solvent system. The kinetic constants and the inactivation of three other type c cytochromes, from Saccharomyces cerevisiae, Pseudomonas aeruginosa, and Desulfovibrio vulgaris Hildenborough in a system containing 90% tetrahydrofuran were compared with those of cytochrome c from horse heart. Cytochrome c551 from P. aeruginosa showed the best stability against hydrogen peroxide and a higher catalytic efficiency than that of horse heart cytochrome c.

A statistical comparison of two culturing methods for enumerating sulfate-reducing bacteria

Abstract The addition of alkaline pyrogallol-soaked cotton wool plugs has been used by other workers to remove oxygen from the headspace gas in culture tubes for the growth of sulfate-reducing bacteria (SRB). This study compares the enumeration fo SRB using the most probable number (MPN) method in tubes with and without the pyrogallol plugs. In both cases, the liquid medium contained two iron finishing nails which help reduce the redox potential of the medium. For each of the 25 samples from oil fields, cooling water systems and natural environments, the time-consuming method using pyrogallol plugs in screw cap tubes yielded virtually the same MPN values as the same method without the plugs and using Kaput® closures on the tubes. However, the pyrogallol plug method, which requires approximately 10-fold more technician time, was superior for pure culture enumeration and in cases where SRB greatly outnumber heterotrophs.

Anaerobic alkane biodegradation by cultures enriched from oil sands tailings ponds involves multiple species capable of fumarate addition

A methanogenic short-chain alkane-degrading culture (SCADC) was enriched from oil sands tailings and transferred several times with a mixture of C6, C7, C8 and C10 n-alkanes as the predominant organic carbon source, plus 2-methylpentane, 3-methylpentane and methylcyclopentane as minor components. Cultures produced ∼40% of the maximum theoretical methane during 18 months incubation while depleting the n-alkanes, 2-methylpentane and methylcyclopentane. Substrate depletion correlated with detection of metabolites characteristic of fumarate activation of 2-methylpentane and methylcyclopentane, but not n-alkane metabolites. During active methanogenesis with the mixed alkanes, reverse-transcription PCR confirmed the expression of functional genes (assA and bssA) associated with hydrocarbon addition to fumarate. Pyrosequencing of 16S rRNA genes amplified during active alkane degradation revealed enrichment of Clostridia (particularly Peptococcaceae) and methanogenic Archaea (Methanosaetaceae and Methanomicrobiaceae). Methanogenic cultures transferred into medium containing sulphate produced sulphide, depleted n-alkanes and produced the corresponding succinylated alkane metabolites, but were slow to degrade 2-methylpentane and methylcyclopentane; these cultures were enriched in Deltaproteobacteria rather than Clostridia. 3-Methylpentane was not degraded by any cultures. Thus, nominally methanogenic oil sands tailings harbour dynamic and versatile hydrocarbon-degrading fermentative syntrophs and sulphate reducers capable of degrading n-, iso- and cyclo-alkanes by addition to fumarate.

Development of a standard bacterial consortium for laboratory efficacy testing of commercial freshwater oil spill bioremediation agents

Six crude oil-degrading bacterial strains isolated from different soil and water environments were combined to create a defined consortium for use in standardized efficacy testing of commercial oil spill bioremediation agents (OSBA). The isolates were cryopreserved in individual aliquots at pre-determined cell densities, stored at −70°C, and thawed for use as standardized inocula as needed. Aliquots were prepared with precision (typically within 10% of the mean) ensuring reproducible inoculation. Five of the six strains displayed no appreciable loss of viability during cryopreservation exceeding 2.5 years, and five isolates demonstrated stable hydrocarbon-degrading phenotypes during inoculum preparation and storage. When resuscitated, the defined consortium reproducibly biodegraded Alberta Sweet Mixed Blend crude oil (typically ± 7% of the mean of triplicate cultures), as determined by quantitative gas chromatography–mass spectrometry of various analyte classes. Reproducible biodegradation was observed within a batch of inoculum in trials spanning 2.5 years, and among three batches of inoculum prepared more than 2 years apart. Biodegradation was comparable after incubation for 28 days at 10°C or 14 days at 22°C, illustrating the temperature tolerance of the bacterial consortium. The results support the use of the synthetic consortium as a reproducible, predictable inoculum to achieve standardized efficacy tests for evaluating commercial OSBA.

Biodegradation of C7 and C8 iso-alkanes under methanogenic conditions

Iso-alkanes comprise a substantial proportion of petroleum and refined products that impact the environment, but their fate is cryptic under methanogenic conditions. We investigated methanogenic biodegradation of C7 and C8 iso-alkanes found in naphtha, specifically 2-methylhexane, 3-methylhexane, 2-methylheptane, 4-methylheptane and 3-ethylhexane. These were incubated as a mixture or individually with enrichment cultures derived from oil sands tailings ponds that generate methane from naphtha components; substrate depletion and methane production were monitored for up to 663 days. 3-Methylhexane and 4-methylheptane were degraded both singly and in the mixture, whereas 2-methylhexane and 2-methylheptane resisted degradation as single substrates but were depleted in the iso-alkane mixture, suggesting co-metabolism. 3-Ethylhexane was degraded neither singly nor with co-substrates. Putative metabolites consistent with succinylated C7 and C8 were detected, suggesting activation by addition of iso-alkanes to fumarate and corresponding to detection of alkylsuccinate synthase-like genes. 454 pyrotag sequencing, cloning and terminal restriction fragment length polymorphism of 16S rRNA genes revealed predominance of a novel member of the family Peptococcaceae (order Clostridiales) and Archaea affiliated with Methanoregula and Methanosaeta. We report here isomer-specific metabolism of C7 -C8 iso-alkanes under methanogenic conditions and propose their activation by a novel Peptococcaceae via addition to fumarate.

Long-Term Incubation Reveals Methanogenic Biodegradation of C5 and C6 iso-Alkanes in Oil Sands Tailings.

iso-Alkanes are major components of petroleum and have been considered recalcitrant to biodegradation under methanogenic conditions. However, indigenous microbes in oil sands tailings ponds exposed to solvents rich in 2-methylbutane, 2-methylpentane, 3-methylpentane, n-pentane, and n-hexane produce methane in situ. We incubated defined mixtures of iso- or n-alkanes with mature fine tailings from two tailings ponds of different ages historically exposed to different solvents: one, ~10 years old, receiving C5-C6 paraffins and the other, ~35 years old, receiving naphtha. A lengthy incubation (>6 years) revealed iso-alkane biodegradation after lag phases of 900-1800 and ~280 days, respectively, before the onset of methanogenesis, although lag phases were shorter with n-alkanes (~650-1675 and ~170 days, respectively). 2-Methylpentane and both n-alkanes were completely depleted during ~2400 days of incubation, whereas 2-methylbutane and 3-methylpentane were partially depleted only during active degradation of 2-methylpentane, suggesting co-metabolism. In both cases, pyrotag sequencing of 16S rRNA genes showed codominance of Peptococcaceae with acetoclastic (Methanosaeta) and hydrogenotrophic (Methanoregula and Methanolinea) methanogens. These observations are important for predicting long-term greenhouse-gas emissions from oil sands tailings ponds and extend the known range of hydrocarbons susceptible to methanogenic biodegradation in petroleum-impacted anaerobic environments.