An underappreciated DIET for anaerobic petroleum hydrocarbon-degrading microbial communities.

Direct interspecies electron transfer (DIET) via electrically conductive minerals can play a role in the anaerobic oxidation of petroleum hydrocarbons in contaminated sites and can be exploited for the development of new, more effective bioremediation approaches.

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs.

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 µmol CH4 g-1 oil d-1 , orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC-MS and FTICR-MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.

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.

Bridging spatially segregated redox zones with a microbial electrochemical snorkel triggers biogeochemical cycles in oil-contaminated River Tyne (UK) sediments.

Marine sediments represent an important sink for a number of anthropogenic organic contaminants, including petroleum hydrocarbons following an accidental oil spill. Degradation of these compounds largely depends on the activity of sedimentary microbial communities linked to biogeochemical cycles, in which abundant elements such as iron and sulfur are shuttled between their oxidized and reduced forms. Here we show that introduction of a small electrically conductive graphite rod ("the electrochemical snorkel") into an oil-contaminated River Tyne (UK) sediment, so as to create an electrochemical connection between the anoxic contaminated sediment and the oxygenated overlying water, has a large impact on the rate of metabolic reactions taking place in the bulk sediment. The electrochemical snorkel accelerated sulfate reduction processes driven by organic contaminant oxidation and suppressed competitive methane-producing reactions. The application of a comprehensive suite of chemical, spectroscopic, biomolecular and thermodynamic analyses suggested that the snorkel served as a scavenger of toxic sulfide via a redox interaction with the iron cycle. Taken as a whole, the results of this work highlight a new strategy for controlling biological processes, such as bioremediation, through the manipulation of the electron flows in contaminated sediments.

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.

Succession in the petroleum reservoir microbiome through an oil field production lifecycle.

Subsurface petroleum reservoirs are an important component of the deep biosphere where indigenous microorganisms live under extreme conditions and in isolation from the Earth's surface for millions of years. However, unlike the bulk of the deep biosphere, the petroleum reservoir deep biosphere is subject to extreme anthropogenic perturbation, with the introduction of new electron acceptors, donors and exogenous microbes during oil exploration and production. Despite the fundamental and practical significance of this perturbation, there has never been a systematic evaluation of the ecological changes that occur over the production lifetime of an active offshore petroleum production system. Analysis of the entire Halfdan oil field in the North Sea (32 producing wells in production for 1-15 years) using quantitative PCR, multigenic sequencing, comparative metagenomic and genomic bins reconstruction revealed systematic shifts in microbial community composition and metabolic potential, as well as changing ecological strategies in response to anthropogenic perturbation of the oil field ecosystem, related to length of time in production. The microbial communities were initially dominated by slow growing anaerobes such as members of the Thermotogales and Clostridiales adapted to living on hydrocarbons and complex refractory organic matter. However, as seawater and nitrate injection (used for secondary oil production) delivered oxidants, the microbial community composition progressively changed to fast growing opportunists such as members of the Deferribacteres, Delta-, Epsilon- and Gammaproteobacteria, with energetically more favorable metabolism (for example, nitrate reduction, H2S, sulfide and sulfur oxidation). This perturbation has profound consequences for understanding the microbial ecology of the system and is of considerable practical importance as it promotes detrimental processes such as reservoir souring and metal corrosion. These findings provide a new conceptual framework for understanding the petroleum reservoir biosphere and have consequences for developing strategies to manage microbiological problems in the oil industry.

Electrobioremediation of oil spills.

Annually, thousands of oil spills occur across the globe. As a result, petroleum substances and petrochemical compounds are widespread contaminants causing concern due to their toxicity and recalcitrance. Many remediation strategies have been developed using both physicochemical and biological approaches. Biological strategies are most benign, aiming to enhance microbial metabolic activities by supplying limiting inorganic nutrients, electron acceptors or donors, thus stimulating oxidation or reduction of contaminants. A key issue is controlling the supply of electron donors/acceptors. Bioelectrochemical systems (BES) have emerged, in which an electrical current serves as either electron donor or acceptor for oil spill bioremediation. BES are highly controllable and can possibly also serve as biosensors for real time monitoring of the degradation process. Despite being promising, multiple aspects need to be considered to make BES suitable for field applications including system design, electrode materials, operational parameters, mode of action and radius of influence. The microbiological processes, involved in bioelectrochemical contaminant degradation, are currently not fully understood, particularly in relation to electron transfer mechanisms. Especially in sulfate rich environments, the sulfur cycle appears pivotal during hydrocarbon oxidation. This review provides a comprehensive analysis of the research on bioelectrochemical remediation of oil spills and of the key parameters involved in the process.

Complementary Microorganisms in Highly Corrosive Biofilms from an Offshore Oil Production Facility.

Offshore oil production facilities are frequently victims of internal piping corrosion, potentially leading to human and environmental risks and significant economic losses. Microbially influenced corrosion (MIC) is believed to be an important factor in this major problem for the petroleum industry. However, knowledge of the microbial communities and metabolic processes leading to corrosion is still limited. Therefore, the microbial communities from three anaerobic biofilms recovered from the inside of a steel pipe exhibiting high corrosion rates, iron oxide deposits, and substantial amounts of sulfur, which are characteristic of MIC, were analyzed in detail. Bacterial and archaeal community structures were investigated by automated ribosomal intergenic spacer analysis, multigenic (16S rRNA and functional genes) high-throughput Illumina MiSeq sequencing, and quantitative PCR analysis. The microbial community analysis indicated that bacteria, particularly Desulfovibrio species, dominated the biofilm microbial communities. However, other bacteria, such as Pelobacter, Pseudomonas, and Geotoga, as well as various methanogenic archaea, previously detected in oil facilities were also detected. The microbial taxa and functional genes identified suggested that the biofilm communities harbored the potential for a number of different but complementary metabolic processes and that MIC in oil facilities likely involves a range of microbial metabolisms such as sulfate, iron, and elemental sulfur reduction. Furthermore, extreme corrosion leading to leakage and exposure of the biofilms to the external environment modify the microbial community structure by promoting the growth of aerobic hydrocarbon-degrading organisms.

Biodegradation of crude oil saturated fraction supported on clays.

The role of clay minerals in crude oil saturated hydrocarbon removal during biodegradation was investigated in aqueous clay/saturated hydrocarbon microcosm experiments with a hydrocarbon degrading microorganism community. The clay minerals used for this study were montmorillonite, palygorskite, saponite and kaolinite. The clay mineral samples were treated with hydrochloric acid and didecyldimethylammonium bromide to produce acid activated- and organoclays respectively which were used in this study. The production of organoclay was restricted to only montmorillonite and saponite because of their relative high CEC. The study indicated that acid activated clays, organoclays and unmodified kaolinite, were inhibitory to biodegradation of the hydrocarbon saturates. Unmodified saponite was neutral to biodegradation of the hydrocarbon saturates. However, unmodified palygorskite and montmorillonite were stimulatory to biodegradation of the hydrocarbon saturated fraction and appears to do so as a result of the clays' ability to provide high surface area for the accumulation of microbes and nutrients such that the nutrients were within the 'vicinity' of the microbes. Adsorption of the saturated hydrocarbons was not significant during biodegradation.

Compositional changes of crude oil SARA fractions due to biodegradation and adsorption on colloidal support such as clays using Iatroscan.

The compositional changes of saturates, aromatics, resins and asphaltenes (SARA) fractions in aqueous clay/oil microcosm experiments with a hydrocarbon-degrading microorganism community were analysed using Iatroscan. The clay mineral samples used in this study were organomontmorillonite, acid-activated montmorillonite and K, Ca, Zn and Cr montmorillonites produced by modifying the original montmorillonite sample. The evaluation and quantification of biodegradation and adsorption were carried out using a combination of the Iatroscan and gravimetric analysis. The SARA compositions in the presence of organomontmorillonite and acid-activated montmorillonite after incubation follow the same pattern in which the aromatic fraction is higher than the other fractions unlike in the presence of unmodified, K, Ca and Zn montmorillonites, where the saturates fraction is higher than the other fractions. Changes in SARA fractions due to biodegradation seemed to occur most in the presence of unmodified and calcium montmorillonites; hence, the removal of SARA fractions due to biodegradation was significant and enhanced in the presence of these two clay samples. However, biodegradation in the presence of organomontmorillonite and acid-activated and Cr montmorillonites was hindered. The study indicated that Cr montmorillonite adsorbed resins most, whereas Zn and K montmorillonites adsorbed aromatics most after incubation.

Biofuel components change the ecology of bacterial volatile petroleum hydrocarbon degradation in aerobic sandy soil.

We tested the hypothesis that the biodegradation of volatile petroleum hydrocarbons (VPHs) in aerobic sandy soil is affected by the blending with 10 percent ethanol (E10) or 20 percent biodiesel (B20). When inorganic nutrients were scarce, competition between biofuel and VPH degraders temporarily slowed monoaromatic hydrocarbon degradation. Ethanol had a bigger impact than biodiesel, reflecting the relative ease of ethanol compared to methyl ester biodegradation. Denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA genes revealed that each fuel mixture selected for a distinct bacterial community, each dominated by Pseudomonas spp. Despite lasting impacts on soil bacterial ecology, the overall effects on VHP biodegradation were minor, and average biomass yields were comparable between fuel types, ranging from 0.40 ± 0.16 to 0.51 ± 0.22 g of biomass carbon per gram of fuel carbon degraded. Inorganic nutrient availability had a greater impact on petroleum hydrocarbon biodegradation than fuel composition.

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.

Methods for recovery of microorganisms and intact microbial polar lipids from oil-water mixtures: laboratory experiments and natural well-head fluids.

Most of the world's remaining petroleum resource has been altered by in-reservoir biodegradation which adversely impacts oil quality and production, ultimately making heavy oil. Analysis of the microorganisms in produced reservoir fluid samples is a route to characterization of subsurface biomes and a better understanding of the resident and living microorganisms in petroleum reservoirs. The major challenges of sample contamination with surface biota, low abundances of microorganisms in subsurface samples, and viscous emulsions produced from biodegraded heavy oil reservoirs are addressed here in a new analytical method for intact polar lipids (IPL) as taxonomic indicators in petroleum reservoirs. We have evaluated the extent to which microbial cells are removed from the free water phase during reservoir fluid phase separation by analysis of model reservoir fluids spiked with microbial cells and have used the resultant methodologies to analyze natural well-head fluids from the Western Canada Sedimentary Basin (WCSB). Analysis of intact polar membrane lipids of microorganisms using liquid chromatography-mass spectrometry (LC-MS) techniques revealed that more than half of the total number of microorganisms can be recovered from oil-water mixtures. A newly developed oil/water separator allowed for filtering of large volumes of water quickly while in the field, which reduced the chances of contamination and alterations to the composition of the subsurface microbial community after sample collection. This method makes the analysis of IPLs (or indirectly microorganisms) from well-head fluids collected in remote field settings possible and reliable. To the best of our knowledge this is the first time that IPLs have been detected in well-head oil-water mixtures.

Thermodynamic constraints on methanogenic crude oil biodegradation.

Methanogenic degradation of crude oil hydrocarbons is an important process in subsurface petroleum reservoirs and anoxic environments contaminated with petroleum. There are several possible routes whereby hydrocarbons may be converted to methane: (i) complete oxidation of alkanes to H2 and CO2, linked to methanogenesis from CO2 reduction; (ii) oxidation of alkanes to acetate and H2, linked to acetoclastic methanogenesis and CO2 reduction; (iii) oxidation of alkanes to acetate and H2, linked to syntrophic acetate oxidation and methanogenesis from CO2 reduction; (iv) oxidation of alkanes to acetate alone, linked to acetoclastic methanogenesis and (v) oxidation of alkanes to acetate alone, linked to syntrophic acetate oxidation and methanogenesis from CO2 reduction. We have developed the concept of a 'window of opportunity' to evaluate the range of conditions under which each route is thermodynamically feasible. On this basis the largest window of opportunity is presented by the oxidation of alkanes to acetate alone, linked to acetoclastic methanogenesis. This contradicts field-based evidence that indicates that in petroleum rich environments acetoclastic methanogenesis is inhibited and that methanogenic CO2 reduction is the predominant methanogenic process. Our analysis demonstrates that under those biological constraints oxidation of alkanes to acetate and H2, linked to syntrophic acetate oxidation and methanogenesis from CO2 reduction offers a greater window of opportunity than complete oxidation of alkanes to H2 and CO2 linked to methanogenic CO2 reduction, and hence is the process most likely to occur.

Marine microorganisms make a meal of oil.

Hundreds of millions of litres of petroleum enter the environment from both natural and anthropogenic sources every year. The input from natural marine oil seeps alone would be enough to cover all of the world's oceans in a layer of oil 20 molecules thick. That the globe is not swamped with oil is testament to the efficiency and versatility of the networks of microorganisms that degrade hydrocarbons, some of which have recently begun to reveal the secrets of when and how they exploit hydrocarbons as a source of carbon and energy.

Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil.

A field-scale experiment with a complete randomized block design was performed to study the degradation of buried oil on a shoreline over a period of almost 1 year. The following four treatments were examined in three replicate blocks: two levels of fertilizer treatment of oil-treated plots, one receiving a weekly application of liquid fertilizer and the other treated with a slow-release fertilizer; and two controls, one not treated with oil and the other treated with oil but not with fertilizer. Oil degradation was monitored by measuring carbon dioxide evolution and by chemical analysis of the oil. Buried oil was degraded to a significantly greater extent in fertilized plots, but no differences in oil chemistry were observed between the two different fertilizer treatments, although carbon dioxide production was significantly higher in the oil-treated plots that were treated with slow-release fertilizer during the first 14 days of the experiment. Bacterial communities present in the beach sediments were profiled by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments and 16S rRNA amplified by reverse transcriptase PCR. Similarities between the DGGE profiles were calculated, and similarity matrices were subjected to statistical analysis. These analyses showed that although significant hydrocarbon degradation occurred both in plots treated with oil alone and in the plots treated with oil and liquid fertilizer, the bacterial community structure in these plots was, in general, not significantly different from that in the control plots that were not treated with oil and did not change over time. In contrast, the bacterial community structure in the plots treated with oil and slow-release fertilizer changed rapidly, and there were significant differences over time, as well as between blocks and even within plots. The differences were probably related to the higher concentrations of nutrients measured in interstitial water from the plots treated with slow-release fertilizer. Bacteria with 16S rRNA sequences closely related (>99.7% identity) to Alcanivorax borkumensis and Pseudomonas stutzeri sequences dominated during the initial phase of oil degradation in the plots treated with slow-release fertilizer. Field data were compared to the results of previous laboratory microcosm experiments, which revealed significant differences.

Biological activity in the deep subsurface and the origin of heavy oil.

At temperatures up to about 80 degrees C, petroleum in subsurface reservoirs is often biologically degraded, over geological timescales, by microorganisms that destroy hydrocarbons and other components to produce altered, denser 'heavy oils'. This temperature threshold for hydrocarbon biodegradation might represent the maximum temperature boundary for life in the deep nutrient-depleted Earth. Most of the world's oil was biodegraded under anaerobic conditions, with methane, a valuable commodity, often being a major by-product, which suggests alternative approaches to recovering the world's vast heavy oil resource that otherwise will remain largely unproduced.

The microbiology of hydrocarbon degradation in subsurface petroleum reservoirs: perspectives and prospects.

The majority of the Earth's petroleum resource is partly biodegraded. This is of considerable practical significance and can limit economic exploitation of petroleum reserves and lead to problems during petroleum production. Knowledge of the microorganisms present in petroleum reservoirs, their physiological properties and the biochemical potential for hydrocarbon degradation benefits successful petroleum exploration. Anaerobic conditions prevail in petroleum reservoirs and biological hydrocarbon degradation is apparently inhibited at temperatures above 80-90 degrees C. We summarise available knowledge and conjecture on the dominant biological processes active during subsurface petroleum biodegradation.

Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation.

Degradation of oil on beaches is, in general, limited by the supply of inorganic nutrients. In order to obtain a more systematic understanding of the effects of nutrient addition on oil spill bioremediation, beach sediment microcosms contaminated with oil were treated with different levels of inorganic nutrients. Oil biodegradation was assessed respirometrically and on the basis of changes in oil composition. Bacterial communities were compared by numerical analysis of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Nutrient amendment over a wide range of concentrations significantly improved oil degradation, confirming that N and P limited degradation over the concentration range tested. However, the extent and rate of oil degradation were similar for all microcosms, indicating that, in this experiment, it was the addition of inorganic nutrients rather than the precise amount that was most important operationally. Very different microbial communities were selected in all of the microcosms. Similarities between DGGE profiles of replicate samples from a single microcosm were high (95% +/- 5%), but similarities between DGGE profiles from replicate microcosms receiving the same level of inorganic nutrients (68% +/- 5%) were not significantly higher than those between microcosms subjected to different nutrient amendments (63% +/- 7%). Therefore, it is apparent that the different communities selected cannot be attributed to the level of inorganic nutrients present in different microcosms. Bioremediation treatments dramatically reduced the diversity of the bacterial community. The decrease in diversity could be accounted for by a strong selection for bacteria belonging to the alkane-degrading Alcanivorax/Fundibacter group. On the basis of Shannon-Weaver indices, rapid recovery of the bacterial community diversity to preoiling levels of diversity occurred. However, although the overall diversity was similar, there were considerable qualitative differences in the community structure before and after the bioremediation treatments.