Draft genome sequence of Pseudomonas sp. ER28, a cyclohexane pentanoic acid degrader isolated from oil sands process-affected water from Alberta, Canada.

We report the draft genome sequence of Pseudomonas sp. ER28, capable of utilizing the model naphthenic acid, cyclohexane pentanoic acid, as its sole carbon source. It was recovered from oil sands process-affected water containing cyclic and acyclic naphthenic acids. The genome size is 5.7 Mbp, and the G + C content is 60%.

The Mystery of Piezophiles: Understudied Microorganisms from the Deep, Dark Subsurface.

Microorganisms that can withstand high pressure within an environment are termed piezophiles. These organisms are considered extremophiles and inhabit the deep marine or terrestrial subsurface. Because these microorganisms are not easily accessed and require expensive sampling methods and laboratory instruments, advancements in this field have been limited compared to other extremophiles. This review summarizes the current knowledge on piezophiles, notably the cellular and physiological adaptations that such microorganisms possess to withstand and grow in high-pressure environments. Based on existing studies, organisms from both the deep marine and terrestrial subsurface show similar adaptations to high pressure, including increased motility, an increase of unsaturated bonds within the cell membrane lipids, upregulation of heat shock proteins, and differential gene-regulation systems. Notably, more adaptations have been identified within the deep marine subsurface organisms due to the relative paucity of studies performed on deep terrestrial subsurface environments. Nevertheless, similar adaptations have been found within piezophiles from both systems, and therefore the microbial biogeography concepts used to assess microbial dispersal and explore if similar organisms can be found throughout deep terrestrial environments are also briefly discussed.

Biodegradation of Polymers Used in Oil and Gas Operations: Towards Enzyme Biotechnology Development and Field Application.

Linear and crosslinked polymers are commonly used in the oil and gas industry. Guar-derived polymers have been extensively utilized in hydraulic fracturing processes, and recently polyacrylamide and cellulose-based polymers have also found utility. As these polymers are used during various phases of the hydraulic fracturing process, they can accumulate at formation fracture faces, resulting in undesired filter cakes that impede oil and gas recovery. Although acids and chemical oxidizers are often added in the fracturing fluids to degrade or 'break' polymer filter cakes, the constant use of these chemicals can be hazardous and can result in formation damage and corrosion of infrastructure. Alternately, the use of enzymes is an attractive and environmentally friendly technology that can be used to treat polymer accumulations. While guar-linkage-specific enzyme breakers isolated from bacteria have been shown to successfully cleave guar-based polymers and decrease their molecular weight and viscosity at reservoir conditions, new enzymes that target a broader range of polymers currently used in hydraulic fracturing operations still require research and development for effective application. This review article describes the current state-of-knowledge on the mechanisms and enzymes involved in biodegradation of guar gum, polyacrylamide (and hydrolyzed polyacrylamide), and carboxymethyl cellulose polymers. In addition, advantages and challenges in the development and application of enzyme breaker technologies are discussed.

Assessing Microbial Corrosion Risk on Offshore Crude Oil Production Topsides under Conditions of Nitrate and Nitrite Treatment for Souring.

Oilfield souring is a detrimental effect caused by sulfate-reducing microorganisms that reduce sulfate to sulfide during their respiration process. Nitrate or nitrite can be used to mitigate souring, but may also impart a corrosion risk. Produced fluids sampled from the topside infrastructure of two floating, production, storage, and offloading (FPSO) vessels (Platform A and Platform B) were assessed for microbial corrosion under nitrate and nitrite breakthrough conditions using microcosm tests incubated at 54 °C. Microbial community compositions on each individual FPSO were similar, while those between the two FPSO vessels differed. Platform B microbial communities responded as expected to nitrate breakthrough conditions, where nitrate-reducing activity was enhanced and sulfate reduction was inhibited. In contrast, nitrate treatments of Platform A microbial communities were not as effective in preventing sulfide production. Nitrite breakthrough conditions had the strongest sulfate reduction inhibition in samples from both platforms, but exhibited the highest pitting density. Live experimental replicates with no nitrate or nitrite additive yielded the highest general corrosion rates in the study (up to 0.48 mm/year), while nitrate- or nitrite-treated fluids revealed general corrosion rates that are considered low or moderate (<0.12 mm/year). Overall, the results of this study provide a description of nitrogen- and sulfur-based microbial activities under thermophilic conditions, and their risk for MIC that can occur along fluid processing lines on FPSO topsides that process fluids during offshore oil production operations.

Preparation and identification of carboxymethyl cellulose-degrading enzyme candidates for oilfield applications.

Carboxymethyl cellulose (CMC) is often used during hydraulic fracturing (fracking) operations as a fluid viscosifier to facilitate proppant delivery. However, the accumulation of residual CMC at fracture faces can result in formation damage, thereby impeding oil and gas recovery. Whereas harsh chemical oxidizers are typically added to disrupt these polymer accumulations, there is now industrial interest in developing clean, biological approaches for the degradation of CMC under fracking conditions. Using a methanogenic culture known to utilize CMC under conditions typically found in oil fields, we developed an efficient method to isolate and purify CMC-degrading enzymes. Initial purification and concentration of cellular components produced an increase in exo-ß-(1,4)-exoglucanase and ß-(1,4)-glucosidase activities by 9-fold and 26-fold, respectively. Partially purified extracts provided substantial degradation of CMC as monitored by viscosity reduction within three hours at 50 °C, an improvement over the untreated cell-free extract which required 48 h to achieve similar viscosity values, outperforming a commercially-available cellulase preparation. Putative cellulases were identified within the isolated enzyme population, with endo-ß-(1,4)-xylanase from Caldicoprobacter faecalis hypothesized to be an important contributor to CMC degradation. This study demonstrates that enzyme technology holds great promise as a viable approach to degrade CMC accumulations under field conditions.

Differential protein expression during growth on model and commercial mixtures of naphthenic acids in Pseudomonas fluorescens Pf-5.

Naphthenic acids (NAs) are carboxylic acids with the formula (Cn H2n+Z O2 ) and are among the most toxic, persistent constituents of oil sands process-affected waters (OSPW), produced during oil sands extraction. Currently, the proteins and mechanisms involved in NA biodegradation are unknown. Using LC-MS/MS shotgun proteomics, we identified proteins overexpressed during the growth of Pseudomonas fluorescens Pf-5 on a model NA (4'-n-butylphenyl)-4-butanoic acid (n-BPBA) and commercial NA mixture (Acros). By day 11, >95% of n-BPBA was degraded. With Acros, a 17% reduction in intensity occurred with 10-18 carbon compounds of the Z family -2 to -14 (major NA species in this mixture). A total of 554 proteins (n-BPBA) and 631 proteins (Acros) were overexpressed during growth on NAs, including several transporters (e.g., ABC transporters), suggesting a cellular protective response from NA toxicity. Several proteins associated with fatty acid, lipid, and amino acid metabolism were also overexpressed, including acyl-CoA dehydrogenase and acyl-CoA thioesterase II, which catalyze part of the fatty acid beta-oxidation pathway. Indeed, multiple enzymes involved in the fatty acid oxidation pathway were upregulated. Given the presumed structural similarity between alkyl-carboxylic acid side chains and fatty acids, we postulate that P. fluorescens Pf-5 was using existing fatty acid catabolic pathways (among others) during NA degradation.

Denitrification Biokinetics: Towards Optimization for Industrial Applications.

Denitrification is a microbial process that converts nitrate (NO3 -) to N2 and can play an important role in industrial applications such as souring control and microbially enhanced oil recovery (MEOR). The effectiveness of using NO3 - in souring control depends on the partial reduction of NO3 - to nitrite (NO2 -) and/or N2O while in MEOR complete reduction of NO3 - to N2 is desired. Thauera has been reported as a dominant taxon in such applications, but the impact of NO3 - and NO2 - concentrations, and pH on the kinetics of denitrification by this bacterium is not known. With the goal of better understanding the effects of such parameters on applications such as souring and MEOR, three strains of Thauera (K172, NS1 and TK001) were used to study denitrification kinetics when using acetate as an electron donor. At low initial NO3 - concentrations (∼1 mmol L-1) and at pH 7.5, complete NO3 - reduction by all strains was indicated by non-detectable NO3 - concentrations and near-complete recovery (> 97%) of the initial NO3-N as N2 after 14 days of incubation. The relative rate of denitrification by NS1 was low, 0.071 mmol L-1 d-1, compared to that of K172 (0.431 mmol L-1 d-1) and TK001 (0.429 mmol L-1 d-1). Transient accumulation of up to 0.74 mmol L-1 NO2 - was observed in cultures of NS1 only. Increased initial NO3 - concentrations resulted in the accumulation of elevated concentrations of NO2 - and N2O, particularly in incubations with K172 and NS1. Strain TK001 had the most extensive NO3 - reduction under high initial NO3 - concentrations, but still had only ∼78% of the initial NO3-N recovered as N2 after 90 days of incubation. As denitrification proceeded, increased pH substantially reduced denitrification rates when values exceeded ∼ 9. The rate and extent of NO3 - reduction were also affected by NO2 - accumulation, particularly in incubations with K172, where up to more than a 2-fold rate decrease was observed. The decrease in rate was associated with decreased transcript abundances of denitrification genes (nirS and nosZ) required to produce enzymes for reduction of NO2 - and N2O. Conversely, high pH also contributed to the delayed expression of these gene transcripts rather than their abundances in strains NS1 and TK001. Increased NO2 - concentrations, N2O levels and high pH appeared to cause higher stress on NS1 than on K172 and TK001 for N2 production. Collectively, these results indicate that increased pH can alter the kinetics of denitrification by Thauera strains used in this study, suggesting that liming could be a way to achieve partial denitrification to promote NO2 - and N2O production (e.g., for souring control) while pH buffering would be desirable for achieving complete denitrification to N2 (e.g., for gas-mediated MEOR).

Enzyme biotechnology development for treating polymers in hydraulic fracturing operations.

Carboxymethyl cellulose (CMC) is a polymer used in many different industrial sectors. In the oil and gas industry, CMC is often used during hydraulic fracturing (fracking) operations as a thickening agent for effective proppant delivery. Accumulations of CMC at fracture faces (known as filter cakes) can impede oil and gas recovery. Although chemical oxidizers are added to disrupt these accumulations, there is industrial interest in developing alternative, enzyme-based treatments. Little is known about CMC biodegradation under fracking conditions. Here, we enriched a methanogenic CMC-degrading culture and demonstrated its ability to enzymatically utilize CMC under the conditions that typify oil fields. Using the extracellular enzyme fraction from the culture, significant CMC viscosity reduction was observed between 50 and 80˚C, at salinities up to 20% (w/v) and at pH 5-8 compared to controls. Similar levels of viscosity reduction by extracellular enzymes were observed under oxic and anoxic conditions. This proof-of-concept study demonstrates that enzyme biotechnology holds great promise as a viable approach to treating CMC filter cakes under oilfield conditions.

The Effect of an Adsorbent Matrix on Recovery of Microorganisms from Hydrocarbon-Contaminated Groundwater.

The microbial degradation of recalcitrant hydrocarbons is an important process that can contribute to the remediation of oil and gas-contaminated environments. Due to the complex structure of subsurface terrestrial environments, it is important to identify the microbial communities that may be contributing to biodegradation processes, along with their abilities to metabolize different hydrocarbons in situ. In this study, a variety of adsorbent materials were assessed for their ability to trap both hydrocarbons and microorganisms in contaminated groundwater. Of the materials tested, a porous polymer resin (Tenax-TA) recovered the highest diversity of microbial taxa in preliminary experiments and was selected for additional (microcosm-based) testing. Oxic and anoxic experiments were prepared with groundwater collected from a contaminated aquifer to assess the ability of Tenax-TA to adsorb two environmental hydrocarbon contaminants of interest (toluene and benzene) while simultaneously providing a surface for microbial growth and hydrocarbon biodegradation. Microorganisms in oxic microcosms completely degraded both targets within 14 days of incubation, while anoxically-incubated microorganisms metabolized toluene but not benzene in less than 80 days. Community analysis of Tenax-TA-associated microorganisms revealed taxa highly enriched in sessile hydrocarbon-degrading treatments, including Saprospiraceae, Azoarcus, and Desulfoprunum, which may facilitate hydrocarbon degradation. This study showed that Tenax-TA can be used as a matrix to effectively trap both microorganisms and hydrocarbons in contaminated environmental systems for assessing and studying hydrocarbon-degrading microorganisms of interest.

Preserving Microbial Community Integrity in Oilfield Produced Water.

Determining a representative microbial signature from any given location is dependent on robust sample collection and handling. Different sampling locations and hence sample properties can vary widely; for example, soil would be collected and handled differently compared to liquid samples. In the event that sample material has a low concentration of biomass, large quantities need to be collected for microbial community analysis. This is certainly the case when investigating the microbiology of oilfield systems, wherein produced water (PW) is one of the most common sources for microbial sampling. As the detrimental effects of microbial metabolism within these industrial milieus are becoming increasingly well-established, the characterization of microbial community composition using molecular biological analyses is becoming more commonplace for accurate monitoring. As this field continues to develop, the importance for standardized operating protocols cannot be understated, so that industry can make the most informed operational decisions possible. Accurately identifying oilfield microbial communities is paramount, as improper preservation and storage following sample collection is known to lead to erroneous microbial identifications. Preserving oilfield PW can be challenging, as many locations are remote, requiring lengthy periods of time before samples can be processed and analyzed. While previous studies have characterized the effects of various preservatives on concentrated, filtered, or purified microbial samples, to the best of our knowledge, no such study has been undertaken on low biomass liquid samples. To this end, we investigated the effectiveness of nine different preservation conditions on PW collected from the same sampling location within a heavy-oil producing field, and monitored how the microbial community changed over the period of a month. Our results reveal that the choice of preservative drastically affects microbial community, and should be selected with careful consideration before sampling occurs.

Combined Use of Diagnostic Fumarate Addition Metabolites and Genes Provides Evidence for Anaerobic Hydrocarbon Biodegradation in Contaminated Groundwater.

The widespread use of hydrocarbon-based fuels has led to the contamination of many natural environments due to accidental spills or leaks. While anaerobic microorganisms indigenous to many fuel-contaminated groundwater sites can play a role in site remediation (e.g., monitored natural attenuation, MNA) via hydrocarbon biodegradation, multiple lines of evidence in support of such bioremediation are required. In this study, we investigated two fuel-contaminated groundwater sites for their potential to be managed by MNA. Microbial community composition, biogeochemical indicators, fumarate addition metabolites, and genes diagnostic of both alkane and alkyl-monoaromatic hydrocarbon activation were assessed. Fumarate addition metabolites and catabolic genes were detected for both classes of hydrocarbon biodegradation at both sites, providing strong evidence for in situ anaerobic hydrocarbon biodegradation. However, relevant metabolites and genes did not consistently co-occur within all groundwater samples. Using newly designed mixtures of quantitative polymerase chain reaction (qPCR) primers to target diverse assA and bssA genes, we measured assA gene abundances ranging from 105-108 copies/L, and bssA gene abundances ranging from 105-1010 copies/L at the sites. Overall, this study demonstrates the value of investigating fuel-contaminated sites using both metabolites and genes diagnostic of anaerobic hydrocarbon biodegradation for different classes of hydrocarbons to help assess field sites for management by MNA.

Bioelectrochemical remediation of phenanthrene in a microbial fuel cell using an anaerobic consortium enriched from a hydrocarbon-contaminated site.

Polycyclic aromatic hydrocarbons (PAH) are organic pollutants that require remediation due to their detrimental impact on human and environmental health. In this study, we used a novel approach of sequestering a model PAH, phenanthrene, onto a solid carbon matrix bioanode in a microbial fuel cell (MFC) to assess its biodegradation coupled with power generation. Here, the bioanode serves as a site for enrichment of electroactive and hydrocarbon-degrading microorganisms, which can simultaneously act to biodegrade a pollutant and generate power. Carbon cloth electrodes loaded with two rates of phenanthrene (2 and 20 mg cm-2) were compared using dual chamber MFCs that were operated for 50 days. The lower loading rate of 2 mg cm-2 was most efficient in the degradation of phenanthrene and had higher power production capacities (37 mW m-2) as compared to the higher loading rate of 20 mg cm-2 (power production of 19.2 mW m-2). FTIR (Fourier-Transform Infrared Spectroscopy) analyses showed a depletion in absorbance peak signals associated with phenanthrene. Microbes known to have electroactive properties or phenanthrene biodegradation abilities like Pseudomonas, Rhodococcus, Thauera and Ralstonia were enriched over time in the MFCs, substantiating the electrochemical and FTIR analyses. The MFC approach taken here thus offers great promise towards PAH bioelectroremediation.

High temperature utilization of PAM and HPAM by microbial communities enriched from oilfield produced water and activated sludge.

Non-hydrolyzed polyacrylamide (PAM) and partially hydrolyzed polyacrylamide (HPAM) are commonly used polymers in various industrial applications, including in oil and gas production operations. Understanding the microbial utilization of such polymers can contribute to improved recovery processes and help to develop technologies for polymer remediation. Microbial communities enriched from oilfield produced water (PW) and activated sludge from Alberta, Canada were assessed for their ability to utilize PAM and HPAM as nitrogen and carbon sources at 50 °C. Microbial growth was determined by measuring CO2 production, and viscosity changes and amide concentrations were used to determine microbial utilization of the polymers. The highest CO2 production was observed in incubations wherein HPAM was added as a nitrogen source for sludge-derived enrichments. Our results showed that partial deamination of PAM and HPAM occurred in both PW and sludge microbial cultures after 34 days of incubation. Whereas viscosity changes were not observed in cultures when HPAM or PAM was provided as the only carbon source, sludge enrichment cultures amended with HPAM and glucose showed significant decreases in viscosity. 16S rRNA gene sequencing analysis indicated that microbial members from the family Xanthomonadaceae were enriched in both PW and sludge cultures amended with HPAM or PAM as a nitrogen source, suggesting the importance of this microbial taxon in the bio-utilization of these polymers. Overall, our results demonstrate that PAM and HPAM can serve as nitrogen sources for microbial communities under the thermophilic conditions commonly found in environments such as oil and gas reservoirs.

Anaerobic degradation of hexadecane and phenanthrene coupled to sulfate reduction by enriched consortia from northern Gulf of Mexico seafloor sediment.

To advance understanding of the fate of hydrocarbons released from the Deepwater Horizon oil spill and deposited in marine sediments, this study characterized the microbial populations capable of anaerobic hydrocarbon degradation coupled with sulfate reduction in non-seep sediments of the northern Gulf of Mexico. Anaerobic, sediment-free enrichment cultures were obtained with either hexadecane or phenanthrene as sole carbon source and sulfate as a terminal electron acceptor. Phylogenetic analysis revealed that enriched microbial populations differed by hydrocarbon substrate, with abundant SSU rRNA gene amplicon sequences from hexadecane cultures showing high sequence identity (up to 98%) to Desulfatibacillum alkenivorans (family Desulfobacteraceae), while phenanthrene-enriched populations were most closely related to Desulfatiglans spp. (up to 95% sequence identity; family Desulfarculaceae). Assuming complete oxidation to CO2, observed stoichiometric ratios closely resembled the theoretical ratios of 12.25:1 for hexadecane and 8.25:1 for phenanthrene degradation coupled to sulfate reduction. Phenanthrene carboxylic acid was detected in the phenanthrene-degrading enrichment cultures, providing evidence to indicate carboxylation as an activation mechanism for phenanthrene degradation. Metagenome-assembled genomes (MAGs) revealed that phenanthrene degradation is likely mediated by novel genera or families of sulfate-reducing bacteria along with their fermentative syntrophic partners, and candidate genes linked to the degradation of aromatic hydrocarbons were detected for future study.

Stable Isotope and Metagenomic Profiling of a Methanogenic Naphthalene-Degrading Enrichment Culture.

Polycyclic aromatic hydrocarbons (PAH) such as naphthalene are widespread, recalcitrant pollutants in anoxic and methanogenic environments. A mechanism catalyzing PAH activation under methanogenic conditions has yet to be discovered, and the microbial communities coordinating their metabolism are largely unknown. This is primarily due to the difficulty of cultivating PAH degraders, requiring lengthy incubations to yield sufficient biomass for biochemical analysis. Here, we sought to characterize a new methanogenic naphthalene-degrading enrichment culture using DNA-based stable isotope probing (SIP) and metagenomic analyses. 16S rRNA gene sequencing of fractionated DNA pinpointed an unclassified Clostridiaceae species as a putative naphthalene degrader after two months of SIP incubation. This finding was supported by metabolite and metagenomic evidence of genes predicted to encode for enzymes facilitating naphthalene carboxylic acid CoA-thioesterification and degradation of an unknown arylcarboxyl-CoA structure. Our findings also suggest a possible but unknown role for Desulfuromonadales in naphthalene degradation. This is the first reported functional evidence of PAH biodegradation by a methanogenic consortium, and we envision that this approach could be used to assess carbon flow through other slow growing enrichment cultures and environmental samples.

Microbial community analyses of produced waters from high-temperature oil reservoirs reveal unexpected similarity between geographically distant oil reservoirs.

As a preliminary investigation for the development of microbial-enhanced oil recovery strategies for high-temperature oil reservoirs (~70 to 90°C), we have investigated the indigenous microbial community compositions of produced waters from five different high-temperature oil reservoirs near Segno, Texas, U.S. (~80 to 85°C) and Crossfield, Alberta, Canada (~75°C). The DNA extracted from these low-biomass-produced water samples were analysed with MiSeq amplicon sequencing of partial 16S rRNA genes. These sequences were analysed along with additional sequence data sets available from existing databases. Despite the geographical distance and difference in the physicochemical properties, the microbial compositions of the Segno and Crossfield produced waters exhibited unexpectedly high similarity, as indicated by the results of beta diversity analyses. The major operational taxonomic units included acetoclastic and hydrogenotrophic methanogens (Methanosaetaceae, Methanobacterium and Methanoculleus), as well as bacteria belonging to the families Clostridiaceae and Thermotogaceae, which have been recognized to include thermophilic, thermotolerant, and/or spore-forming subtaxa. The sequence data retrieved from the databases exhibited different clustering patterns, as the communities from close geographical locations invariably had low beta diversity and the physicochemical properties and conditions of the reservoirs apparently did not have a substantial role in shaping of microbial communities.

Methanogenic Paraffin Biodegradation: Alkylsuccinate Synthase Gene Quantification and Dicarboxylic Acid Production.

Paraffinic n-alkanes (>C17) that are solid at ambient temperature comprise a large fraction of many crude oils. The comparatively low water solubility and reactivity of these long-chain alkanes can lead to their persistence in the environment following fuel spills and pose serious problems for crude oil recovery operations by clogging oil production wells. However, the degradation of waxy paraffins under the anoxic conditions characterizing contaminated groundwater environments and deep subsurface energy reservoirs is poorly understood. Here, we assessed the ability of a methanogenic culture enriched from freshwater fuel-contaminated aquifer sediments to biodegrade the model paraffin n-octacosane (C28H58). Compared with that in controls, the consumption of n-octacosane was coupled to methane production, demonstrating its biodegradation under these conditions. Smithella was postulated to be an important C28H58 degrader in the culture on the basis of its high relative abundance as determined by 16S rRNA gene sequencing. An identified assA gene (known to encode the α subunit of alkylsuccinate synthase) aligned most closely with those from other Smithella organisms. Quantitative PCR (qPCR) and reverse transcription qPCR assays for assA demonstrated significant increases in the abundance and expression of this gene in C28H58-degrading cultures compared with that in controls, suggesting n-octacosane activation by fumarate addition. A metabolite analysis revealed the presence of several long-chain α,ω-dicarboxylic acids only in the C28H58-degrading cultures, a novel observation providing clues as to how methanogenic consortia access waxy hydrocarbons. The results of this study broaden our understanding of how waxy paraffins can be biodegraded in anoxic environments with an application toward bioremediation and improved oil recovery.IMPORTANCE Understanding the methanogenic biodegradation of different classes of hydrocarbons has important applications for effective fuel-contaminated site remediation and for improved recovery from oil reservoirs. Previous studies have clearly demonstrated that short-chain alkanes (C17) that comprise many fuel mixtures. Using an enrichment culture derived from a freshwater fuel-contaminated site, we demonstrate that the model waxy alkane n-octacosane can be biodegraded under methanogenic conditions by a presumed Smithella phylotype. Compared with that of controls, we show an increased abundance and expression of the assA gene, which is known to be important for anaerobic n-alkane metabolism. Metabolite analyses revealed the presence of a range of α,ω-dicarboxylic acids found only in n-octacosane-degrading cultures, a novel finding that lends insight as to how anaerobic communities may access waxes as growth substrates in anoxic environments.

Benzene and Naphthalene Degrading Bacterial Communities in an Oil Sands Tailings Pond.

Oil sands process-affected water (OSPW), produced by surface-mining of oil sands in Canada, is alkaline and contains high concentrations of salts, metals, naphthenic acids, and polycyclic aromatic compounds (PAHs). Residual hydrocarbon biodegradation occurs naturally, but little is known about the hydrocarbon-degrading microbial communities present in OSPW. In this study, aerobic oxidation of benzene and naphthalene in the surface layer of an oil sands tailings pond were measured. The potential oxidation rates were 4.3 µmol L-1 OSPW d-1 for benzene and 21.4 µmol L-1 OSPW d-1 for naphthalene. To identify benzene and naphthalene-degrading microbial communities, metagenomics was combined with stable isotope probing (SIP), high-throughput sequencing of 16S rRNA gene amplicons, and isolation of microbial strains. SIP using 13C-benzene and 13C-naphthalene detected strains of the genera Methyloversatilis and Zavarzinia as the main benzene degraders, while strains belonging to the family Chromatiaceae and the genus Thauera were the main naphthalene degraders. Metagenomic analysis revealed a diversity of genes encoding oxygenases active against aromatic compounds. Although these genes apparently belonged to many phylogenetically diverse taxa, only a few of these taxa were predominant in the SIP experiments. This suggested that many members of the community are adapted to consuming other aromatic compounds, or are active only under specific conditions. 16S rRNA gene sequence datasets have been submitted to the Sequence Read Archive (SRA) under accession number SRP109130. The Gold Study and Project submission ID number in Joint Genome Institute IMG/M for the metagenome is Gs0047444 and Gp0055765.

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.

Embryonic exposure to model naphthenic acids delays growth and hatching in the pond snail Lymnaea stagnalis.

Naphthenic acids (NAs), a class of structurally diverse carboxylic acids with often complex ring structures and large aliphatic tail groups, are important by-products of many petrochemical processes including the oil sands mining activity of Northern Alberta. While it is evident that NAs have both acute and chronic harmful effects on many organisms, many aspects of their toxicity remain to be clarified. Particularly, while substantive data sets have been collected on NA toxicity in aquatic prokaryote and vertebrate model systems, to date, nothing is known about the toxic effects of these compounds on the embryonic development of aquatic invertebrate taxa, including freshwater mollusks. This study examines under laboratory conditions the toxicity of NAs extracted from oil sands process water (OSPW) and the low-molecular weight model NAs cyclohexylsuccinic acid (CHSA), cyclohexanebutyric acid (CHBA), and 4-tert-butylcyclohexane carboxylic acid (4-TBCA) on embryonic development of the snail Lymnaea stagnalis, a common freshwater gastropod with a broad Palearctic distribution. Evidence is provided for concentration-dependent teratogenic effects of both OSPW-derived and model NAs with remarkably similar nominal threshold concentrations between 15 and 20 mg/L and 28d EC50 of 31 mg/L. In addition, the data provide evidence for substantial toxicokinetic differences between CHSA, CHBA and 4-TBCA. Together, our study introduces Lymnaea stagnalis embryonic development as an effective model to assay NA-toxicity and identifies molecular architecture as a potentially important toxicokinetic parameter in the toxicity of low-molecular weight NA in embryonic development of aquatic gastropods.