Aerobic biodegradation of cycloalkanes in non-aqueous extracted oil sands tailings.

Management of growing volumes of fluid fine tailings (FFT) is a significant challenge for oil sands industry. A potential alternative non-aqueous solvent extraction (NAE) process uses cycloalkane solvent such as cyclohexane or cyclopentane with very little water and generates smaller volumes of 'dry' solids (NAES) with residual solvent. Here we investigate remediation of NAES in a simulated bench-scale upland reclamation scenario. In the first study, microcosms with nutrient medium plus FFT as inoculum were amended with cyclohexane and incubated for ∼1 year, monitoring for cyclohexane biodegradation under aerobic conditions. Biodegradation of cyclohexane occurred under aerobic conditions with no metabolic intermediates detected. A second study using NAES mixed with FFT spiked with cyclohexane and cyclopentane, with or without additional nutrients (nitrogen and phosphorus), showed complete and rapid aerobic biodegradation of both cycloalkanes in NAES inoculated with FFT and supplemented with nutrients. 16S rRNA gene sequencing revealed dominance of Rhodoferax and members of Burkholderiaceae during aerobic cyclohexane biodegradation in FFT, and Hydrogenophaga, Acidovorax, Defluviimonas and members of Porticoccaceae during aerobic biodegradation of cyclohexane and cyclopentane in NAES inoculated with FFT and supplemented with nutrients. The findings indicate that biodegradation of cycloalkanes from NAES is possible under aerobic condition, which will contribute to the successful reclamation of oil sands tailings for land closure.

Methanogenic Biodegradation of iso-Alkanes by Indigenous Microbes from Two Different Oil Sands Tailings Ponds.

iso-Alkanes, a major fraction of the solvents used in bitumen extraction from oil sand ores, are slow to biodegrade in anaerobic tailings ponds. We investigated methanogenic biodegradation of iso-alkane mixtures comprising either three (2-methylbutane, 2-methylpentane, 3-methylpentane) or five (2-methylbutane, 2-methylpentane, 2-methylhexane, 2-methylheptane, 2-methyloctane) iso-alkanes representing paraffinic and naphtha solvents, respectively. Mature fine tailings (MFT) collected from two tailings ponds, having different residual solvents (paraffinic solvent in Canadian Natural Upgrading Limited (CNUL) and naphtha in Canadian Natural Resources Limited (CNRL)), were amended separately with the two mixtures and incubated in microcosms for ~1600 d. The indigenous microbes in CNUL MFT produced methane from the three-iso-alkane mixture after a lag of ~200 d, completely depleting 2-methylpentane while partially depleting 2-methylbutane and 3-methylpentane. CNRL MFT exhibited a similar degradation pattern for the three iso-alkanes after a lag phase of ~700 d, but required 1200 d before beginning to produce methane from the five-iso-alkane mixture, preferentially depleting components in the order of decreasing carbon chain length. Peptococcaceae members were key iso-alkane-degraders in both CNUL and CNRL MFT but were associated with different archaeal partners. Co-dominance of acetoclastic (Methanosaeta) and hydrogenotrophic (Methanolinea and Methanoregula) methanogens was observed in CNUL MFT during biodegradation of three-iso-alkanes whereas CNRL MFT was enriched in Methanoregula during biodegradation of three-iso-alkanes and in Methanosaeta with five-iso-alkanes. This study highlights the different responses of indigenous methanogenic microbial communities in different oil sands tailings ponds to iso-alkanes.

Methanogenic biodegradation of iso-alkanes and cycloalkanes during long-term incubation with oil sands tailings.

Microbes indigenous to oil sands tailings ponds methanogenically biodegrade certain hydrocarbons, including n-alkanes and monoaromatics, whereas other hydrocarbons such as iso- and cycloalkanes are more recalcitrant. We tested the susceptibility of iso- and cycloalkanes to methanogenic biodegradation by incubating them with mature fine tailings (MFT) collected from two depths (6 and 31 m below surface) of a tailings pond, representing different lengths of exposure to hydrocarbons. A mixture of five iso-alkanes and three cycloalkanes was incubated with MFT for 1700 d. Iso-alkanes were completely biodegraded in the order 3-methylhexane > 4-methylheptane > 2-methyloctane > 2-methylheptane, whereas 3-ethylhexane and ethylcyclopentane were only partially depleted and methylcyclohexane and ethylcyclohexane were not degraded during incubation. Pyrosequencing of 16S rRNA genes showed enrichment of Peptococcaceae (Desulfotomaculum) and Smithella in amended cultures with acetoclastic (Methanosaeta) and hydrogenotrophic methanogens (Methanoregula and Methanoculleus). Bioaugmentation of MFT by inoculation with MFT-derived enrichment cultures reduced the lag phase before onset of iso-alkane and cycloalkane degradation. However, the same enrichment culture incubated without MFT exhibited slower biodegradation kinetics and less CH4 production, implying that the MFT solid phase (clay minerals) enhanced methanogenesis. These results help explain and predict continued emissions of CH4 from oil sands tailings repositories in situ.

Genomic insights into temperature-dependent transcriptional responses of Kosmotoga olearia, a deep-biosphere bacterium that can grow from 20 to 79 °C.

Temperature is one of the defining parameters of an ecological niche. Most organisms thrive within a temperature range that rarely exceeds ~30 °C, but the deep subsurface bacterium Kosmotoga olearia can grow over a temperature range of 59 °C (20-79 °C). To identify genes correlated with this flexible phenotype, we compared transcriptomes of K. olearia cultures grown at its optimal 65 °C to those at 30, 40, and 77 °C. The temperature treatments affected expression of 573 of 2224 K. olearia genes. Notably, this transcriptional response elicits re-modeling of the cellular membrane and changes in metabolism, with increased expression of genes involved in energy and carbohydrate metabolism at high temperatures and up-regulation of amino acid metabolism at lower temperatures. At sub-optimal temperatures, many transcriptional changes were similar to those observed in mesophilic bacteria at physiologically low temperatures, including up-regulation of typical cold stress genes and ribosomal proteins. Comparative genomic analysis of additional Thermotogae genomes indicates that one of K. olearia's strategies for low-temperature growth is increased copy number of some typical cold response genes through duplication and/or lateral acquisition. At 77 °C one-third of the up-regulated genes are of hypothetical function, indicating that many features of high-temperature growth are unknown.

The microbiology of oil sands tailings: past, present, future.

Surface mining of enormous oil sands deposits in northeastern Alberta, Canada since 1967 has contributed greatly to Canada's economy but has also received negative international attention due largely to environmental concerns and challenges. Not only have microbes profoundly affected the composition and behavior of this petroleum resource over geological time, they currently influence the management of semi-solid tailings in oil sands tailings ponds (OSTPs) and tailings reclamation. Historically, microbial impacts on OSTPs were generally discounted, but next-generation sequencing and biogeochemical studies have revealed unexpectedly diverse indigenous communities and expanded our fundamental understanding of anaerobic microbial functions. OSTPs that experienced different processing and management histories have developed distinct microbial communities that influence the behavior and reclamation of the tailings stored therein. In particular, the interactions of Deltaproteobacteria and Firmicutes with methanogenic archaea impact greenhouse gas emissions, sulfur cycling, pore water toxicity, sediment biogeochemistry and densification, water usage and the trajectory of long-term mine waste reclamation. This review summarizes historical data; synthesizes current understanding of microbial diversity and activities in situ and in vitro; predicts microbial effects on tailings remediation and reclamation; and highlights knowledge gaps for future research.

Trace metal mobilization from oil sands froth treatment thickened tailings exhibiting acid rock drainage.

Froth treatment thickened tailings (TT) are a waste product of bitumen extraction from surface-mined oil sands ores. When incubated in a laboratory under simulated moist oxic environmental conditions for ~450d, two different types of TT (TT1 and TT2) exhibited the potential to generate acid rock drainage (ARD) by producing acid leachate after 250 and 50d, respectively. We report here the release of toxic metals from TT via ARD, which could pose an environmental threat if oil sands TT deposits are not properly managed. Trace metal concentrations in leachate samples collected periodically revealed that Mn and Sr were released immediately even before the onset of ARD. Spikes in Co and Ni concentrations were observed both pre-ARD and during active ARD, particularly in TT1. For most elements measured (Fe, Cr, V, As, Cu, Pb, Zn, Cd, and Se), leaching was associated with ARD production. Though equivalent acidification (pH2) was achieved in leachate from both TT types, greater metal release was observed from TT2 where concentrations reached 10,000ppb for Ni, 5000ppb for Co, 3000ppb for As, 2000ppb for V, and 1000ppb for Cr. Generally, metal concentrations decreased in leachate with time during ARD and became negligible by the end of incubation (~450d) despite appreciable metals remaining in the leached TT. These results suggest that using TT for land reclamation purposes or surface deposition for volume reduction may unfavorably impact the environment, and warrants application of appropriate strategies for management of pyrite-enriched oil sands tailings streams.

Co-occurrence of methanogenesis and N2 fixation in oil sands tailings.

Oil sands tailings ponds in northern Alberta, Canada have been producing biogenic gases via microbial metabolism of hydrocarbons for decades. Persistent methanogenic activity in tailings ponds without any known replenishment of nutrients such as fixed nitrogen (N) persuaded us to investigate whether N2 fixation or polyacrylamide (PAM; used as a tailings flocculant) could serve as N sources. Cultures comprising mature fine tailings (MFT) plus methanogenic medium supplemented with or deficient in fixed N were incubated under an N2 headspace. Some cultures were further amended with citrate, which is used in oil sands processing, as a relevant carbon source, and/or with PAM. After an initial delay, N-deficient cultures with or without PAM produced methane (CH4) at the same rate as N-containing cultures, indicating a mechanism of overcoming apparent N-deficiency. Acetylene reduction and (15)N2 incorporation in all N-deficient cultures (with or without PAM) suggested active N2 fixation concurrently with methanogenesis but inability to use PAM as a N source. 16S rRNA gene pyrosequencing revealed little difference between archaeal populations regardless of N content. However, bacterial sequences in N-deficient cultures showed enrichment of Hyphomicrobiaceae and Clostridium members that might contain N2-fixing species. The results are important in understanding long-term production of biogenic greenhouse gases in oil sands tailings.

Preferential methanogenic biodegradation of short-chain n-alkanes by microbial communities from two different oil sands tailings ponds.

Oil sands tailings ponds harbor diverse anaerobic microbial communities capable of methanogenic biodegradation of solvent hydrocarbons entrained in the tailings. Mature fine tailings (MFT) from two operators (Albian and CNRL) that use different extraction solvents were incubated with mixtures of either two (n-pentane and n-hexane) or four (n-pentane, n-hexane, n-octane and n-decane) n-alkanes under methanogenic conditions for ~600 d. Microbes in Albian MFT began methane production by ~80 d, achieving complete depletion of n-pentane and n-hexane in the two-alkane mixture and their preferential biodegradation in the four-alkane mixture. Microbes in CNRL MFT preferentially metabolized n-octane and n-decane in the four-alkane mixture after a ~80 d lag but exhibited a lag of ~360 d before commencing biodegradation of n-pentane and n-hexane in the two-alkane mixture. 16S rRNA gene pyrosequencing revealed Peptococcaceae members as key bacterial n-alkane degraders in all treatments except CNRL MFT amended with the four-alkane mixture, in which Anaerolineaceae, Desulfobacteraceae (Desulfobacterium) and Syntrophaceae (Smithella) dominated during n-octane and n-decane biodegradation. Anaerolineaceae sequences increased only in cultures amended with the four-alkane mixture and only during n-octane and n-decane biodegradation. The dominant methanogens were acetoclastic Methanosaetaceae. These results highlight preferential n-alkane biodegradation by microbes in oil sands tailings from different producers, with implications for tailings management and reclamation.

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.

Microbial metabolism alters pore water chemistry and increases consolidation of oil sands tailings.

Tailings produced during bitumen extraction from surface-mined oil sands ores (tar sands) comprise an aqueous suspension of clay particles that remain dispersed for decades in tailings ponds. Slow consolidation of the clays hinders water recovery for reuse and retards volume reduction, thereby increasing the environmental footprint of tailings ponds. We investigated mechanisms of tailings consolidation and revealed that indigenous anaerobic microorganisms altered porewater chemistry by producing CO and CH during metabolism of acetate added as a labile carbon amendment. Entrapped biogenic CO decreased tailings pH, thereby increasing calcium (Ca) and magnesium (Mg) cations and bicarbonate (HCO) concentrations in the porewater through dissolution of carbonate minerals. Soluble ions increased the porewater ionic strength, which, with higher exchangeable Ca and Mg, decreased the diffuse double layer of clays and increased consolidation of tailings compared with unamended tailings in which little microbial activity was observed. These results are relevant to effective tailings pond management strategies.

Oil sands thickened froth treatment tailings exhibit acid rock drainage potential during evaporative drying.

Bitumen extraction from oil sands ores after surface mining produces different tailings waste streams: 'froth treatment tailings' are enriched in pyrite relative to other streams. Tailings treatment can include addition of organic polymers to produce thickened tailings (TT). TT may be further de-watered by deposition into geotechnical cells for evaporative drying to increase shear strength prior to reclamation. To examine the acid rock drainage (ARD) potential of TT, we performed predictive analyses and laboratory experiments on material from field trials of two types of thickened froth treatment tailings (TT1 and TT2). Acid-base accounting (ABA) of initial samples showed that both TT1 and TT2 initially had net acid-producing potential, with ABA values of -141 and -230 t CaCO3 equiv. 1000 t(-1) of TT, respectively. In long-term kinetic experiments, duplicate ~2-kg samples of TT were incubated in shallow trays and intermittently irrigated under air flow for 459 days to simulate evaporative field drying. Leachates collected from both TT samples initially had pH~6.8 that began decreasing after ~50 days (TT2) or ~250 days (TT1), stabilizing at pH~2. Correspondingly, the redox potential of leachates increased from 100-200 mV to 500-580 mV and electrical conductivity increased from 2-5 dS m(-1) to 26 dS m(-1), indicating dissolution of minerals during ARD. The rapid onset and prolonged ARD observed with TT2 is attributed to its greater pyrite (13.4%) and lower carbonate (1.4%) contents versus the slower onset of ARD in TT1 (initially 6.0% pyrite and 2.5% carbonates). 16S rRNA gene pyrosequencing analysis revealed rapid shift in microbial community when conditions became strongly acidic (pH~2) favoring the enrichment of Acidithiobacillus and Sulfobacillus bacteria in TT. This is the first report showing ARD potential of TT and the results have significant implications for effective management of pyrite-enriched oil sands tailings streams/deposits.

Microbially-accelerated consolidation of oil sands tailings. Pathway I: changes in porewater chemistry.

Dispersed clay particles in mine tailings and soft sediments remain suspended for decades, hindering consolidation and challenging effective management of these aqueous slurries. Current geotechnical engineering models of self-weight consolidation of tailings do not consider microbial contribution to sediment behavior, however, here we show that microorganisms indigenous to oil sands tailings change the porewater chemistry and accelerate consolidation of oil sands tailings. A companion paper describes the role of microbes in alteration of clay chemistry in tailings. Microbial metabolism in mature fine tailings (MFT) amended with an organic substrate (hydrolyzed canola meal) produced methane (CH4) and carbon dioxide (CO2). Dissolution of biogenic CO2 lowered the pH of amended MFT to pH 6.4 vs. unamended MFT (pH 7.7). About 12% more porewater was recovered from amended than unamended MFT during 2 months of active microbial metabolism, concomitant with consolidation of tailings. The lower pH in amended MFT dissolved carbonate minerals, thereby releasing divalent cations including calcium (Ca(2+)) and magnesium (Mg(2+)) and increasing bicarbonate (HCO(-) 3) in porewater. The higher concentrations increased the ionic strength of the porewater, in turn reducing the thickness of the diffuse double layer (DDL) of clay particles by reducing the surface charge potential (repulsive forces) of the clay particles. The combination of these processes accelerated consolidation of oil sands tailings. In addition, ebullition of biogenic gases created transient physical channels for release of porewater. In contrast, saturating the MFT with non-biogenic CO2 had little effect on consolidation. These results have significant implications for management and reclamation of oil sands tailings ponds and broad importance in anaerobic environments such as contaminated harbors and estuaries containing soft sediments rich in clays and organics.

Microbially-accelerated consolidation of oil sands tailings. Pathway II: solid phase biogeochemistry.

Consolidation of clay particles in aqueous tailings suspensions is a major obstacle to effective management of oil sands tailings ponds in northern Alberta, Canada. We have observed that microorganisms indigenous to the tailings ponds accelerate consolidation of mature fine tailings (MFT) during active metabolism by using two biogeochemical pathways. In Pathway I, microbes alter porewater chemistry to indirectly increase consolidation of MFT. Here, we describe Pathway II comprising significant, direct and complementary biogeochemical reactions with MFT mineral surfaces. An anaerobic microbial community comprising Bacteria (predominantly Clostridiales, Synergistaceae, and Desulfobulbaceae) and Archaea (Methanolinea/Methanoregula and Methanosaeta) transformed Fe(III) minerals in MFT to amorphous Fe(II) minerals during methanogenic metabolism of an added organic substrate. Synchrotron analyses suggested that ferrihydrite (5Fe2O3. 9H2O) and goethite (α-FeOOH) were the dominant Fe(III) minerals in MFT. The formation of amorphous iron sulfide (FeS) and possibly green rust entrapped and masked electronegative clay surfaces in amended MFT. Both Pathways I and II reduced the surface charge potential (repulsive forces) of the clay particles in MFT, which aided aggregation of clays and formation of networks of pores, as visualized using cryo-scanning electron microscopy (SEM). These reactions facilitated the egress of porewater from MFT and increased consolidation of tailings solids. These results have large-scale implications for management and reclamation of oil sands tailings ponds, a burgeoning environmental issue for the public and government regulators.

The EmhABC efflux pump in Pseudomonas fluorescens LP6a is involved in naphthalene tolerance but not efflux.

The EmhABC efflux pump in Pseudomonas fluorescens LP6a effluxes polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene and anthracene but not naphthalene. We previously showed that the presence of EmhABC decreased the efficiency of phenanthrene biodegradation. In this study, we determined whether P. fluorescens LP6a tolerance to naphthalene is a function of the EmhABC efflux pump and how its presence affects the efficiency of naphthalene biodegradation. Growth, membrane fatty acid (FA) composition, and cell morphology showed that 5-mmol L(-1) naphthalene is inhibitory to P. fluorescens LP6a strains. The deleterious effect of naphthalene is suppressed in the presence of EmhABC, which suggests that, although naphthalene is not effluxed by EmhABC, this efflux pump is involved in tolerance of naphthalene toxicity. LP6a mutants lacking the EmhB efflux pump were unable to convert cis-unsaturated FAs to cyclopropane FAs, indicating that naphthalene interferes with the formation of cyclopropane FAs and supporting the proposal that EmhABC is involved in FA turnover in P. fluorescens LP6a strains. The EmhABC efflux pump increases the efficiency of naphthalene metabolism in strain LP6a, which may make naphthalene efflux unnecessary. Thus, the activity of hydrocarbon efflux pumps may be an important factor to consider when selecting bacterial strains for bioremediation or biocatalysis of PAHs.

Microbial communities involved in methane production from hydrocarbons in oil sands tailings.

Microbial metabolism of residual hydrocarbons, primarily short-chain n-alkanes and certain monoaromatic hydrocarbons, in oil sands tailings ponds produces large volumes of CH(4) in situ. We characterized the microbial communities involved in methanogenic biodegradation of whole naphtha (a bitumen extraction solvent) and its short-chain n-alkane (C(6)-C(10)) and BTEX (benzene, toluene, ethylbenzene, and xylenes) components using primary enrichment cultures derived from oil sands tailings. Clone libraries of bacterial 16S rRNA genes amplified from these enrichments showed increased proportions of two orders of Bacteria: Clostridiales and Syntrophobacterales, with Desulfotomaculum and Syntrophus/Smithella as the closest named relatives, respectively. In parallel archaeal clone libraries, sequences affiliated with cultivated acetoclastic methanogens (Methanosaetaceae) were enriched in cultures amended with n-alkanes, whereas hydrogenotrophic methanogens (Methanomicrobiales) were enriched with BTEX. Naphtha-amended cultures harbored a blend of these two archaeal communities. The results imply syntrophic oxidation of hydrocarbons in oil sands tailings, with the activities of different carbon flow pathways to CH(4) being influenced by the primary hydrocarbon substrate. These results have implications for predicting greenhouse gas emissions from oil sands tailings repositories.

The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a.

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹4C-phenanthrene to radiolabeled polar metabolites and ¹4CO2. The emhB⁻ WEN strain produced approximately twice as much ¹4CO2 and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹4C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.

An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a.

BACKGROUND: Efflux pumps belonging to the resistance-nodulation-division (RND) superfamily in bacteria are involved in antibiotic resistance and solvent tolerance but have an unknown physiological role. EmhABC, a RND-type efflux pump in Pseudomonas fluorescens strain cLP6a, extrudes hydrophobic antibiotics, dyes and polycyclic aromatic hydrocarbons including phenanthrene. The effects of physico-chemical factors such as temperature or antibiotics on the activity and expression of EmhABC were determined in order to deduce its physiological role(s) in strain cLP6a in comparison to the emhB disruptant strain, cLP6a-1. RESULTS: Efflux assays conducted with (14)C-phenanthrene showed that EmhABC activity is affected by incubation temperature. Increased phenanthrene efflux was measured in cLP6a cells grown at 10°C and decreased efflux was observed at 35°C compared with cells grown at the optimum temperature of 28°C. Membrane fatty acids in cLP6a cells were substantially altered by changes in growth temperature and in the presence of tetracycline. Changed membrane fatty acids and increased membrane permeability were associated with ~30-fold increased expression of emhABC in cLP6a cells grown at 35°C, and with increased extracellular free fatty acids. Growth of P. fluorescens cLP6a at supra-optimal temperature was enhanced by the presence of EmhABC compared to strain cLP6a-1. CONCLUSIONS: Combined, these observations suggest that the EmhABC efflux pump may be involved in the management of membrane stress effects such as those due to unfavourable incubation temperatures. Efflux of fatty acids replaced as a result of membrane damage or phospholipid turnover may be the primary physiological role of the EmhABC efflux pump in P. fluorescens cLP6a.

Influence of adhesion on aerobic biodegradation and bioremediation of liquid hydrocarbons.

Biodegradation of poorly water-soluble liquid hydrocarbons is often limited by low availability of the substrate to microbes. Adhesion of microorganisms to an oil-water interface can enhance this availability, whereas detaching cells from the interface can reduce the rate of biodegradation. The capability of microbes to adhere to the interface is not limited to hydrocarbon degraders, nor is it the only mechanism to enable rapid uptake of hydrocarbons, but it represents a common strategy. This review of the literature indicates that microbial adhesion can benefit growth on and biodegradation of very poorly water-soluble hydrocarbons such as n-alkanes and large polycyclic aromatic hydrocarbons dissolved in a non-aqueous phase. Adhesion is particularly important when the hydrocarbons are not emulsified, giving limited interfacial area between the two liquid phases. When mixed communities are involved in biodegradation, the ability of cells to adhere to the interface can enable selective growth and enhance bioremediation with time. The critical challenge in understanding the relationship between growth rate and biodegradation rate for adherent bacteria is to accurately measure and observe the population that resides at the interface of the hydrocarbon phase.

Biological souring and mitigation in oil reservoirs.

Souring in oil field systems is most commonly due to the action of sulfate-reducing prokaryotes, a diverse group of anaerobic microorganisms that respire sulfate and produce sulfide (the key souring agent) while oxidizing diverse electron donors. Such biological sulfide production is a detrimental, widespread phenomenon in the petroleum industry, occurring within oil reservoirs or in topside processing facilities, under low- and high-temperature conditions, and in onshore or offshore operations. Sulfate reducers can exist either indigenously in deep subsurface reservoirs or can be "inoculated" into a reservoir system during oil field development (e.g., via drilling operations) or during the oil production phase. In the latter, souring most commonly occurs during water flooding, a secondary recovery strategy wherein water is injected to re-pressurize the reservoir and sweep the oil towards production wells to extend the production life of an oil field. The water source and type of production operation can provide multiple components such as sulfate, labile carbon sources, and sulfate-reducing communities that influence whether oil field souring occurs. Souring can be controlled by biocides, which can non-specifically suppress microbial populations, and by the addition of nitrate (and/or nitrite) that directly impacts the sulfate-reducing population by numerous competitive or inhibitory mechanisms. In this review, we report on the diversity of sulfate reducers associated with oil reservoirs, approaches for determining their presence and effects, the factors that control souring, and the approaches (along with the current understanding of their underlying mechanisms) that may be used to successfully mitigate souring in low-temperature and high-temperature oil field operations.