Use of propionic acid additions to enhance zinc removal from mine drainage in short residence time, flow-through sulfate-reducing bioreactors.

The effectiveness of liquid carbon additions to enhance zinc removal in laboratory-scale short hydraulic residence time (19 h) compost bioreactors receiving synthetic mine water with a high influent zinc concentration (45 mg/L) was investigated. Effective removal of such elevated zinc concentrations could not be sustained by sulfate reduction and/or other attenuation processes without carbon supplementation. Propionic acid addition resulted in improved and sustained performance by promoting the activities of sulfate reducing bacteria, leading to efficient zinc removal (mean 99%) via bacterial sulfate reduction. In contrast, cessation of propionic acid addition led to carbon limitation and the growth of sulfur oxidising bacteria, compromising zinc removal by bacterial sulfate reduction. These research findings demonstrate the potential for modest liquid carbon additions to compost-based passive treatment systems to engineer microbial responses which enhance rates of zinc attenuation in a short hydraulic residence time, enabling remediation of highly polluting mine drainage at sites with limited land availability.

Biogeochemical consequences of a changing Arctic shelf seafloor ecosystem.

Unprecedented and dramatic transformations are occurring in the Arctic in response to climate change, but academic, public, and political discourse has disproportionately focussed on the most visible and direct aspects of change, including sea ice melt, permafrost thaw, the fate of charismatic megafauna, and the expansion of fisheries. Such narratives disregard the importance of less visible and indirect processes and, in particular, miss the substantive contribution of the shelf seafloor in regulating nutrients and sequestering carbon. Here, we summarise the biogeochemical functioning of the Arctic shelf seafloor before considering how climate change and regional adjustments to human activities may alter its biogeochemical and ecological dynamics, including ecosystem function, carbon burial, or nutrient recycling. We highlight the importance of the Arctic benthic system in mitigating climatic and anthropogenic change and, with a focus on the Barents Sea, offer some observations and our perspectives on future management and policy.

An Unexpectedly Broad Thermal and Salinity-Tolerant Estuarine Methanogen Community.

Moderately thermophilic (Tmax, ~55 °C) methanogens are identified after extended enrichments from temperate, tropical and low-temperature environments. However, thermophilic methanogens with higher growth temperatures (Topt ≥ 60 °C) are only reported from high-temperature environments. A microcosm-based approach was used to measure the rate of methane production and methanogen community structure over a range of temperatures and salinities in sediment from a temperate estuary. We report short-term incubations (<48 h) revealing methanogens with optimal activity reaching 70 °C in a temperate estuary sediment (in situ temperature 4-5 °C). While 30 °C enrichments amended with acetate, H2 or methanol selected for corresponding mesophilic trophic groups, at 60 °C, only hydrogenotrophs (genus Methanothermobacter) were observed. Since these methanogens are not known to be active under in situ temperatures, we conclude constant dispersal from high temperature habitats. The likely provenance of the thermophilic methanogens was studied by enrichments covering a range of temperatures and salinities. These enrichments indicated that the estuarine sediment hosted methanogens encompassing the global activity envelope of most cultured species. We suggest that estuaries are fascinating sink and source environments for microbial function study.

Organic complexation of U(VI) in reducing soils at a natural analogue site: Implications for uranium transport.

Understanding the long-term fate, stability, and bioavailability of uranium (U) in the environment is important for the management of nuclear legacy sites and radioactive wastes. Analysis of U behavior at natural analogue sites permits evaluation of U biogeochemistry under conditions more representative of long-term equilibrium. Here, we have used bulk geochemical and microbial community analysis of soils, coupled with X-ray absorption spectroscopy and µ-focus X-ray fluorescence mapping, to gain a mechanistic understanding of the fate of U transported into an organic-rich soil from a pitchblende vein at the UK Needle's Eye Natural Analogue site. U is highly enriched in the Needle's Eye soils (∼1600 mg kg-1). We show that this enrichment is largely controlled by U(VI) complexation with soil organic matter and not U(VI) bioreduction. Instead, organic-associated U(VI) seems to remain stable under microbially-mediated Fe(III)-reducing conditions. U(IV) (as non-crystalline U(IV)) was only observed at greater depths at the site (>25 cm); the soil here was comparatively mineral-rich, organic-poor, and sulfate-reducing/methanogenic. Furthermore, nanocrystalline UO2, an alternative product of U(VI) reduction in soils, was not observed at the site, and U did not appear to be associated with Fe-bearing minerals. Organic-rich soils appear to have the potential to impede U groundwater transport, irrespective of ambient redox conditions.

Transformation of organic matter in a Barents Sea sediment profile: coupled geochemical and microbiological processes.

Process-based, mechanistic investigations of organic matter transformation and diagenesis directly beneath the sediment-water interface (SWI) in Arctic continental shelves are vital as these regions are at greatest risk of future change. This is in part due to disruptions in benthic-pelagic coupling associated with ocean current change and sea ice retreat. Here, we focus on a high-resolution, multi-disciplinary set of measurements that illustrate how microbial processes involved in the degradation of organic matter are directly coupled with inorganic and organic geochemical sediment properties (measured and modelled) as well as the extent/depth of bioturbation. We find direct links between aerobic processes, reactive organic carbon and highest abundances of bacteria and archaea in the uppermost layer (0-4.5 cm depth) followed by dominance of microbes involved in nitrate/nitrite and iron/manganese reduction across the oxic-anoxic redox boundary (approx. 4.5-10.5 cm depth). Sulfate reducers dominate in the deeper (approx. 10.5-33 cm) anoxic sediments which is consistent with the modelled reactive transport framework. Importantly, organic matter reactivity as tracked by organic geochemical parameters (n-alkanes, n-alkanoic acids, n-alkanols and sterols) changes most dramatically at and directly below the SWI together with sedimentology and biological activity but remained relatively unchanged across deeper changes in sedimentology. This article is part of the theme issue 'The changing Arctic Ocean: consequences for biological communities, biogeochemical processes and ecosystem functioning'.

Beyond N and P: The impact of Ni on crude oil biodegradation.

N and P are the key limiting nutrients considered most important for the stimulation of crude oil degradation but other trace nutrients may also be important. Experimental soil microcosms were setup to investigate crude oil degradation in the context of Ni amendments. Amended Nickel as NiO, NiCl2, or, a porphyrin complex either inhibited, had no effect, or, enhanced aerobic hydrocarbon degradation in an oil-contaminated soil. Biodegradation was significantly (95% confidence) enhanced (70%) with low levels of Ni-Porph (12 mg/kg) relative to an oil-only control; whereas, NiO (200 and 350 mg/kg) significantly inhibited (36 and 87%) biodegradation consistent with oxide particle induced reactive oxygen stress. Microbial community compositions were also significantly affected by Ni. In 16S rRNA sequence libraries, the enriched hydrocarbon degrading genus, Rhodococcus, was partially replaced by a Nocardia sp. in the presence of low levels of NiO (12 and 50 mg/kg). In contrast, the highest relative and absolute Rhodococcus abundances were coincident with the maximal rates of oil degradation observed in the Ni-Porph-amended soils. Growth dependent constitutive requirements for Ni-dependent urease or perhaps Ni-dependent superoxide dismutase enzymes (found in Rhodococcus genomes) provided a mechanistic explanation for stimulation. These results suggest biostimulation technologies, in addition to N and P, should also consider trace nutrients such as Ni tacitly considered adequately supplied and available in a typical soil.

Understanding drivers of antibiotic resistance genes in High Arctic soil ecosystems.

Soils in tropical and temperate locations are known to be a sink for the genetic potential of anthropogenic-driven acquired antibiotic resistance (AR). In contrast, accumulation of acquired AR is less probable in most Polar soils, providing a platform for characterizing background resistance and establishing a benchmark for assessing AR spread. Here, high-throughput qPCR and geochemistry were used to quantify the abundance and diversity of both antibiotic resistance genes (ARGs) and selected mobile genetic elements (MGEs) across eight soil clusters in the Kongsfjorden region of Svalbard in the High Arctic. Relative ARG levels ranged by over two orders of magnitude (10-6 to 10-4 copies/16S rRNA gene copy), and showed a gradient of potential human and wildlife impacts across clusters as evidenced by altered geochemical conditions and increased "foreign" ARG abundances (i.e., allochthonous), including blaNDM-1. Impacted clusters exhibited 100× higher total ARGs and MGEs in tandem with elevated secondary nutrients, especially available P that is typically low and limiting in Arctic soils. In contrast, ARGs in less-impacted clusters correlated strongly to local soil lithology. The most plausible source of exogenous P and allochthonous ARGs in this region is bird and other wildlife guano, disseminated either by local human wastes or via direct carriage and deposition. Regardless of pathway, accumulation of apparent allochthonous ARGs and MGEs in High Arctic soils is concerning, highlighting the importance of characterizing Arctic sites now to establish benchmarks for tracking AR spread around the world.

In situ arsenic oxidation and sorption by a Fe-Mn binary oxide waste in soil.

The ability of a Fe-Mn binary oxide waste to adsorb arsenic (As) in a historically contaminated soil was investigated. Initial laboratory sorption experiments indicated that arsenite [As(III)] was oxidized to arsenate [As(V)] by the Mn oxide component, with concurrent As(V) sorption to the Fe oxide. The binary oxide waste had As(III) and As(V) adsorption capacities of 70mgg-1 and 32mgg-1 respectively. X-ray Absorption Near-Edge Structure and Extended X-ray Absorption Fine Structure at the As K-edge confirmed that all binary oxide waste surface complexes were As(V) sorbed by mononuclear bidentate corner-sharing, with 2 Fe at ∼3.27Ǻ. The ability of the waste to perform this coupled oxidation-sorption reaction in real soils was investigated with a 10% by weight addition of the waste to an industrially As contaminated soil. Electron probe microanalysis showed As accumulation onto the Fe oxide component of the binary oxide waste, which had no As innately. The bioaccessibility of As was also significantly reduced by 7.80% (p<0.01) with binary oxide waste addition. The results indicate that Fe-Mn binary oxide wastes could provide a potential in situ remediation strategy for As and Pb immobilization in contaminated soils.

How to access and exploit natural resources sustainably: petroleum biotechnology.

As we transition from fossil fuel reliance to a new energy future, innovative microbial biotechnologies may offer new routes to maximize recovery from conventional and unconventional energy assets; as well as contributing to reduced emission pathways and new technologies for carbon capture and utilization. Here we discuss the role of microbiology in petroleum biotechnologies in relation to addressing UN Sustainable Development Goal 12 (ensure sustainable consumption and production patterns), with a focus on microbially-mediated energy recovery from unconventionals (heavy oil to methane), shale gas and fracking, bioelectrochemical systems for the production of electricity from fossil fuel resources, and innovations in synthetic biology. Furthermore, using wastes to support a more sustainable approach to fossil fuel extraction processes is considered as we undertake the move towards a more circular global economy.

Methanotroph-derived bacteriohopanepolyol signatures as a function of temperature related growth, survival, cell death and preservation in the geological record.

Interpretation of bacteriohopanepolyol (BHP) biomarkers tracing microbiological processes in modern and ancient sediments relies on understanding environmental controls of production and preservation. BHPs from methanotrophs (35-aminoBHPs) were studied in methane-amended aerobic river-sediment incubations at different temperatures. It was found that: (i) With increasing temperature (4°C-40°C) a 10-fold increase in aminopentol (associated with Crenothrix and Methylobacter spp. growth) occurred with only marginal increases in aminotriol and aminotetrol; (ii) A further increase in temperature (50°C) saw selection for the thermophile Methylocaldum and mixtures of aminopentol and C-3 methylated aminopentol, again, with no increase in aminotriol and aminotetrol. (iii) At 30°C, more aminopentol and an aminopentol isomer and unsaturated aminopentol were produced after methanotroph growth and the onset of substrate starvation/oxygen depletion. (iv) At 50°C, aminopentol and C-3 methylated aminopentol, only accumulated during growth but were clearly resistant to remineralization despite cell death. These results have profound implications for the interpretation of aminoBHP distributions and abundances in modern and past environments. For instance, a temperature regulation of aminopentol production but not aminotetrol or aminotriol is consistent with and, corroborative of, observed aminopentol sensitivity to climate warming recorded in a stratigraphic sequence deposited during the Paleocene-Eocene thermal maximum (PETM).

Microbial Biotechnology 2020; microbiology of fossil fuel resources.

This roadmap examines the future of microbiology research and technology in fossil fuel energy recovery. Globally, the human population will be reliant on fossil fuels for energy and chemical feedstocks for at least the medium term. Microbiology is already important in many areas relevant to both upstream and downstream activities in the oil industry. However, the discipline has struggled for recognition in a world dominated by geophysicists and engineers despite widely known but still poorly understood microbially mediated processes e.g. reservoir biodegradation, reservoir souring and control, microbial enhanced oil recovery. The role of microbiology is even less understood in developing industries such as shale gas recovery by fracking or carbon capture by geological storage. In the future, innovative biotechnologies may offer new routes to reduced emissions pathways especially when applied to the vast unconventional heavy oil resources formed, paradoxically, from microbial activities in the geological past. However, despite this potential, recent low oil prices may make industry funding hard to come by and recruitment of microbiologists by the oil and gas industry may not be a high priority. With regards to public funded research and the imperative for cheap secure energy for economic growth in a growing world population, there are signs of inherent conflicts between policies aimed at a low carbon future using renewable technologies and policies which encourage technologies which maximize recovery from our conventional and unconventional fossil fuel assets.

Microbial Communities in a High Arctic Polar Desert Landscape.

The High Arctic is dominated by polar desert habitats whose microbial communities are poorly understood. In this study, we used next generation sequencing to describe the α- and ß-diversity of microbial communities in polar desert soils from the Kongsfjorden region of Svalbard. Ten phyla dominated the soils and accounted for 95% of all sequences, with the Proteobacteria, Actinobacteria, and Chloroflexi being the major lineages. In contrast to previous investigations of Arctic soils, relative Acidobacterial abundances were found to be very low as were the Archaea throughout the Kongsfjorden polar desert landscape. Lower Acidobacterial abundances were attributed to characteristic circumneutral soil pHs in this region, which has resulted from the weathering of underlying carbonate bedrock. In addition, we compared previously measured geochemical conditions as possible controls on soil microbial communities. Phosphorus, pH, nitrogen, and calcium levels all significantly correlated with ß-diversity, indicating landscape-scale lithological control of available nutrients, which in turn, significantly influenced soil community composition. In addition, soil phosphorus and pH significantly correlated with α-diversity, particularly with the Shannon diversity and Chao 1 richness indices.

A temperate river estuary is a sink for methanotrophs adapted to extremes of pH, temperature and salinity.

River Tyne (UK) estuarine sediments harbour a genetically and functionally diverse community of methane-oxidizing bacteria (methanotrophs), the composition and activity of which were directly influenced by imposed environmental conditions (pH, salinity, temperature) that extended far beyond those found in situ. In aerobic sediment slurries methane oxidation rates were monitored together with the diversity of a functional gene marker for methanotrophs (pmoA). Under near in situ conditions (4-30°C, pH 6-8, 1-15 g l(-1) NaCl), communities were enriched by sequences affiliated with Methylobacter and Methylomonas spp. and specifically a Methylobacter psychrophilus-related species at 4-21°C. More extreme conditions, namely high temperatures ≥ 40°C, high ≥ 9 and low ≤ 5 pH, and high salinities ≥ 35 g l(-1) selected for putative thermophiles (Methylocaldum), acidophiles (Methylosoma) and haloalkaliphiles (Methylomicrobium). The presence of these extreme methanotrophs (unlikely to be part of the active community in situ) indicates passive dispersal from surrounding environments into the estuary.

Response of Methanogens in Arctic Sediments to Temperature and Methanogenic Substrate Availability.

Although cold environments are major contributors to global biogeochemical cycles, comparatively little is known about their microbial community function, structure, and limits of activity. In this study a microcosm based approach was used to investigate the effects of temperature, and methanogenic substrate amendment, (acetate, methanol and H2/CO2) on methanogen activity and methanogen community structure in high Arctic wetlands (Solvatnet and Stuphallet, Svalbard). Methane production was not detected in Stuphallet sediment microcosms (over a 150 day period) and occurred within Solvatnet sediments microcosms (within 24 hours) at temperatures from 5 to 40°C, the maximum temperature being at far higher than in situ maximum temperatures (which range from air temperatures of -1.4 to 14.1°C during summer months). Distinct responses were observed in the Solvatnet methanogen community under different short term incubation conditions. Specifically, different communities were selected at higher and lower temperatures. At lower temperatures (5°C) addition of exogenous substrates (acetate, methanol or H2/CO2) had no stimulatory effect on the rate of methanogenesis or on methanogen community structure. The community in these incubations was dominated by members of the Methanoregulaceae/WCHA2-08 family-level group, which were most similar to the psychrotolerant hydrogenotrophic methanogen Methanosphaerula palustris strain E1-9c. In contrast, at higher temperatures, substrate amendment enhanced methane production in H2/CO2 amended microcosms, and played a clear role in structuring methanogen communities. Specifically, at 30°C members of the Methanoregulaceae/WCHA2-08 predominated following incubation with H2/CO2, and Methanosarcinaceaeand Methanosaetaceae were enriched in response to acetate addition. These results may indicate that in transiently cold environments, methanogen communities can rapidly respond to moderate short term increases in temperature, but not necessarily to the seasonal release of previously frozen organic carbon from thawing permafrost soils. However, as temperatures increase such inputs of carbon will likely have a greater influence on methane production and methanogen community structure. Understanding the action and limitations of anaerobic microorganisms within cold environments may provide information which can be used in defining region-specific differences in the microbial processes; which ultimately control methane flux to the atmosphere.

Remediation of a historically Pb contaminated soil using a model natural Mn oxide waste.

A natural Mn oxide (NMO) waste was assessed as an in situ remediation amendment for Pb contaminated sites. The viability of this was investigated using a 10 month lysimeter trial, wherein a historically Pb contaminated soil was amended with a 10% by weight model NMO. The model NMO was found to have a large Pb adsorption capacity (qmax 346±14 mg g(-1)). However, due to the heterogeneous nature of the Pb contamination in the soils (3650.54-9299.79 mg kg(-1)), no treatment related difference in Pb via geochemistry could be detected. To overcome difficulties in traditional geochemical techniques due to pollutant heterogeneity we present a new method for unequivocally proving metal sorption to in situ remediation amendments. The method combines two spectroscopic techniques; namely electron probe microanalysis (EPMA) and X-ray photoelectron spectroscopy (XPS). Using this we showed Pb immobilisation on NMO, which were Pb free prior to their addition to the soils. Amendment of the soil with exogenous Mn oxide had no effect on microbial functioning, nor did it perturb the composition of the dominant phyla. We conclude that NMOs show excellent potential as remediation amendments.

Life in the slow lane; biogeochemistry of biodegraded petroleum containing reservoirs and implications for energy recovery and carbon management.

Our understanding of the processes underlying the formation of heavy oil has been transformed in the last decade. The process was once thought to be driven by oxygen delivered to deep petroleum reservoirs by meteoric water. This paradigm has been replaced by a view that the process is anaerobic and frequently associated with methanogenic hydrocarbon degradation. The thermal history of a reservoir exerts a fundamental control on the occurrence of biodegraded petroleum, and microbial activity is focused at the base of the oil column in the oil water transition zone, that represents a hotspot in the petroleum reservoir biome. Here we present a synthesis of new and existing microbiological, geochemical, and biogeochemical data that expands our view of the processes that regulate deep life in petroleum reservoir ecosystems and highlights interactions of a range of biotic and abiotic factors that determine whether petroleum is likely to be biodegraded in situ, with important consequences for oil exploration and production. Specifically we propose that the salinity of reservoir formation waters exerts a key control on the occurrence of biodegraded heavy oil reservoirs and introduce the concept of palaeopickling. We also evaluate the interaction between temperature and salinity to explain the occurrence of non-degraded oil in reservoirs where the temperature has not reached the 80-90°C required for palaeopasteurization. In addition we evaluate several hypotheses that might explain the occurrence of organisms conventionally considered to be aerobic, in nominally anoxic petroleum reservoir habitats. Finally we discuss the role of microbial processes for energy recovery as we make the transition from fossil fuel reliance, and how these fit within the broader socioeconomic landscape of energy futures.

Kinetic parameters for nutrient enhanced crude oil biodegradation in intertidal marine sediments.

Availability of inorganic nutrients, particularly nitrogen and phosphorous, is often a primary control on crude oil hydrocarbon degradation in marine systems. Many studies have empirically determined optimum levels of inorganic N and P for stimulation of hydrocarbon degradation. Nevertheless, there is a paucity of information on fundamental kinetic parameters for nutrient enhanced crude oil biodegradation that can be used to model the fate of crude oil in bioremediation programmes that use inorganic nutrient addition to stimulate oil biodegradation. Here we report fundamental kinetic parameters (Ks and qmax) for nitrate- and phosphate-stimulated crude oil biodegradation under nutrient limited conditions and with respect to crude oil, under conditions where N and P are not limiting. In the marine sediments studied, crude oil degradation was limited by both N and P availability. In sediments treated with 12.5 mg/g of oil but with no addition of N and P, hydrocarbon degradation rates, assessed on the basis of CO2 production, were 1.10 ± 0.03 µmol CO2/g wet sediment/day which were comparable to rates of CO2 production in sediments to which no oil was added (1.05 ± 0.27 µmol CO2/g wet sediment/day). When inorganic nitrogen was added alone maximum rates of CO2 production measured were 4.25 ± 0.91 µmol CO2/g wet sediment/day. However, when the same levels of inorganic nitrogen were added in the presence of 0.5% P w/w of oil (1.6 µmol P/g wet sediment) maximum rates of measured CO2 production increased more than four-fold to 18.40 ± 1.04 µmol CO2/g wet sediment/day. Ks and qmax estimates for inorganic N (in the form of sodium nitrate) when P was not limiting were 1.99 ± 0.86 µmol/g wet sediment and 16.16 ± 1.28 µmol CO2/g wet sediment/day respectively. The corresponding values for P were 63 ± 95 nmol/g wet sediment and 12.05 ± 1.31 µmol CO2/g wet sediment/day. The qmax values with respect to N and P were not significantly different (P < 0.05). When N and P were not limiting Ks and qmax for crude oil were 4.52 ± 1.51 mg oil/g wet sediment and 16.89 ± 1.25 µmol CO2/g wet sediment/day. At concentrations of inorganic N above 45 µmol/g wet sediment inhibition of CO2 production from hydrocarbon degradation was evident. Analysis of bacterial 16S rRNA genes indicated that Alcanivorax spp. were selected in these marine sediments with increasing inorganic nutrient concentration, whereas Cycloclasticus spp. were more prevalent at lower inorganic nutrient concentrations. These data suggest that simple empirical estimates of the proportion of nutrients added relative to crude oil concentrations may not be sufficient to guarantee successful crude oil bioremediation in oxic beach sediments. The data we present also help define the maximum rates and hence timescales required for bioremediation of beach sediments.

Volatile hydrocarbons inhibit methanogenic crude oil degradation.

Methanogenic degradation of crude oil in subsurface sediments occurs slowly, but without the need for exogenous electron acceptors, is sustained for long periods and has enormous economic and environmental consequences. Here we show that volatile hydrocarbons are inhibitory to methanogenic oil biodegradation by comparing degradation of an artificially weathered crude oil with volatile hydrocarbons removed, with the same oil that was not weathered. Volatile hydrocarbons (nC5-nC10, methylcyclohexane, benzene, toluene, and xylenes) were quantified in the headspace of microcosms. Aliphatic (n-alkanes nC12-nC34) and aromatic hydrocarbons (4-methylbiphenyl, 3-methylbiphenyl, 2-methylnaphthalene, 1-methylnaphthalene) were quantified in the total hydrocarbon fraction extracted from the microcosms. 16S rRNA genes from key microorganisms known to play an important role in methanogenic alkane degradation (Smithella and Methanomicrobiales) were quantified by quantitative PCR. Methane production from degradation of weathered oil in microcosms was rapid (1.1 ± 0.1 µmol CH4/g sediment/day) with stoichiometric yields consistent with degradation of heavier n-alkanes (nC12-nC34). For non-weathered oil, degradation rates in microcosms were significantly lower (0.4 ± 0.3 µmol CH4/g sediment/day). This indicated that volatile hydrocarbons present in the non-weathered oil inhibit, but do not completely halt, methanogenic alkane biodegradation. These findings are significant with respect to rates of biodegradation of crude oils with abundant volatile hydrocarbons in anoxic, sulphate-depleted subsurface environments, such as contaminated marine sediments which have been entrained below the sulfate-reduction zone, as well as crude oil biodegradation in petroleum reservoirs and contaminated aquifers.

Improving PCR efficiency for accurate quantification of 16S rRNA genes.

Quantitative real-time PCR is a valuable tool for microbial ecologists. To obtain accurate absolute quantification it is essential that PCR efficiency for pure standards is close to amplification efficiency for test samples. Counter to normal expectation that PCR efficiency might be lower in environmental DNA, due to the presence of PCR inhibitors, we report the counterintuitive observation that PCR efficiency of pure standards can be lower than for environmental DNA. This can lead to overestimation of gene abundances if not corrected. SYBR green-based qPCR assays of 16S rRNA genes targeting Bacteria, Syntrophus and Smithella spp., Marinobacter spp., Methanomicrobiales, Methanosarcinaceae, and Methanosaetaceae in samples from methanogenic crude oil biodegradation enrichments were tested. In five out of the six assays, PCR efficiency was lower with pure standards than with environmental DNA samples. We developed a solution to this problem based on amending pure clone standards with a background of non-target environmental 16S rRNA genes which significantly improved PCR efficiency of standards in the qPCR assays that exhibited this phenomenon. Overall this method of qPCR standard preparation achieved a more reliable and robust quantification of 16S rRNA genes. We believe this may be a potentially common issue in microbial ecology that often goes unreported, as intuitively one would not expect standards to have poorer PCR efficiency than samples.

Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil.

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO(2) -reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, > 99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n = 382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n = 96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense - an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.