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1.  Metagenomic analysis and metabolite profiling of deep–sea sediments from the Gulf of Mexico following the Deepwater Horizon oil spill 
Marine subsurface environments such as deep-sea sediments, house abundant and diverse microbial communities that are believed to influence large-scale geochemical processes. These processes include the biotransformation and mineralization of numerous petroleum constituents. Thus, microbial communities in the Gulf of Mexico are thought to be responsible for the intrinsic bioremediation of crude oil released by the Deepwater Horizon (DWH) oil spill. While hydrocarbon contamination is known to enrich for aerobic, oil-degrading bacteria in deep-seawater habitats, relatively little is known about the response of communities in deep-sea sediments, where low oxygen levels may hinder such a response. Here, we examined the hypothesis that increased hydrocarbon exposure results in an altered sediment microbial community structure that reflects the prospects for oil biodegradation under the prevailing conditions. We explore this hypothesis using metagenomic analysis and metabolite profiling of deep-sea sediment samples following the DWH oil spill. The presence of aerobic microbial communities and associated functional genes was consistent among all samples, whereas, a greater number of Deltaproteobacteria and anaerobic functional genes were found in sediments closest to the DWH blowout site. Metabolite profiling also revealed a greater number of putative metabolites in sediments surrounding the blowout zone relative to a background site located 127 km away. The mass spectral analysis of the putative metabolites revealed that alkylsuccinates remained below detection levels, but a homologous series of benzylsuccinates (with carbon chain lengths from 5 to 10) could be detected. Our findings suggest that increased exposure to hydrocarbons enriches for Deltaproteobacteria, which are known to be capable of anaerobic hydrocarbon metabolism. We also provide evidence for an active microbial community metabolizing aromatic hydrocarbons in deep-sea sediments of the Gulf of Mexico.
PMCID: PMC3598227  PMID: 23508965
Deepwater Horizon; metagenomics; metabolomics; oil-degradation
2.  Recent Advances in Petroleum Microbiology 
Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times (<60 s) are being used effectively. Microbes are being injected into partially spent petroleum reservoirs to enhance oil recovery. However, these microbial processes have not exhibited consistent and effective performance, primarily because of our inability to control conditions in the subsurface environment. Microbes may be exploited to break stable oilfield emulsions to produce pipeline quality oil. There is interest in replacing physical oil desulfurization processes with biodesulfurization methods through promotion of selective sulfur removal without degradation of associated carbon moieties. However, since microbes require an environment containing some water, a two-phase oil-water system must be established to optimize contact between the microbes and the hydrocarbon, and such an emulsion is not easily created with viscous crude oil. This challenge may be circumvented by application of the technology to more refined gasoline and diesel substrates, where aqueous-hydrocarbon emulsions are more easily generated. Molecular approaches are being used to broaden the substrate specificity and increase the rates and extents of desulfurization. Bacterial processes are being commercialized for removal of H2S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.
PMCID: PMC309048  PMID: 14665675
3.  Isolation and Identification of hydrocarbon degrading bacteria from Ennore creek 
Bioinformation  2013;9(3):150-157.
The widespread problem caused due to petroleum products, is their discharge and accidental spillage in marine environment proving to be hazardous to the surroundings as well as life forms. Thus remediation of these hydrocarbons by natural decontamination process is of utmost importance. Bioremediation is a non-invasive and cost effective technique for the clean-up of these petroleum hydrocarbons. In this study we have investigated the ability of microorganisms present in the sediment sample to degrade these hydrocarbons, crude oil in particular, so that contaminated soils and water can be treated using microbes. Sediments samples were collected once in a month for a period of twelve months from area surrounding Ennore creek and screened for hydrocarbon degrading bacteria. Of the 113 crude oil degrading isolates 15 isolates were selected and cultivated in BH media with 1% crude oil as a sole carbon and energy source. 3 efficient crude oil bacterial isolates Bacillus subtilis I1, Pseudomonas aeruginosa I5 and Pseudomonas putida I8 were identified both biochemically and phylogenetically. The quantitative analysis of biodegradation is carried out gravimetrically and highest degradation rate, 55% was recorded by Pseudomonas aeruginosa I5 isolate.
PMCID: PMC3569603  PMID: 23424279
4.  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.
PMCID: PMC3990054  PMID: 24782848
oil spill; bioremediation; kinetics; Ks; half saturation constant; maximal rates; Alcanivorax; Cycloclasticus
5.  Microbial degradation of hydrocarbons in the environment. 
Microbiological Reviews  1990;54(3):305-315.
The ecology of hydrocarbon degradation by microbial populations in the natural environment is reviewed, emphasizing the physical, chemical, and biological factors that contribute to the biodegradation of petroleum and individual hydrocarbons. Rates of biodegradation depend greatly on the composition, state, and concentration of the oil or hydrocarbons, with dispersion and emulsification enhancing rates in aquatic systems and absorption by soil particulates being the key feature of terrestrial ecosystems. Temperature and oxygen and nutrient concentrations are important variables in both types of environments. Salinity and pressure may also affect biodegradation rates in some aquatic environments, and moisture and pH may limit biodegradation in soils. Hydrocarbons are degraded primarily by bacteria and fungi. Adaptation by prior exposure of microbial communities to hydrocarbons increases hydrocarbon degradation rates. Adaptation is brought about by selective enrichment of hydrocarbon-utilizing microorganisms and amplification of the pool of hydrocarbon-catabolizing genes. The latter phenomenon can now be monitored through the use of DNA probes. Increases in plasmid frequency may also be associated with genetic adaptation. Seeding to accelerate rates of biodegradation has been shown to be effective in some cases, particularly when used under controlled conditions, such as in fermentors or chemostats.
PMCID: PMC372779  PMID: 2215423
6.  Effect of Pyocyanin on a Crude-Oil-Degrading Microbial Community 
Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 μM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C29 and C30 hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.
PMCID: PMC444818  PMID: 15240276
7.  Recent studies in microbial degradation of petroleum hydrocarbons in hypersaline environments 
Many hypersaline environments are often contaminated with petroleum compounds. Among these, oil and natural gas production sites all over the world and hundreds of kilometers of coastlines in the more arid regions of Gulf countries are of major concern due to the extent and magnitude of contamination. Because conventional microbiological processes do not function well at elevated salinities, bioremediation of hypersaline environments can only be accomplished using high salt-tolerant microorganisms capable of degrading petroleum compounds. In the last two decades, there have been many reports on the biodegradation of hydrocarbons in moderate to high salinity environments. Numerous microorganisms belonging to the domain Bacteria and Archaea have been isolated and their phylogeny and metabolic capacity to degrade a variety of aliphatic and aromatic hydrocarbons in varying salinities have been demonstrated. This article focuses on our growing understanding of bacteria and archaea responsible for the degradation of hydrocarbons under aerobic conditions in moderate to high salinity conditions. Even though organisms belonging to various genera have been shown to degrade hydrocarbons, members of the genera Halomonas Alcanivorax, Marinobacter, Haloferax, Haloarcula, and Halobacterium dominate the published literature. Despite rapid advances in understanding microbial taxa that degrade hydrocarbons under aerobic conditions, not much is known about organisms that carry out similar processes in anaerobic conditions. Also, information on molecular mechanisms and pathways of hydrocarbon degradation in high salinity is scarce and only recently there have been a few reports describing genes, enzymes and breakdown steps for some hydrocarbons. These limited studies have clearly revealed that degradation of oxygenated and non-oxygenated hydrocarbons by halophilic and halotolerant microorganisms occur by pathways similar to those found in non-halophiles.
PMCID: PMC4005966  PMID: 24795705
hypersaline environments; biodegradation; oxygenated and non-oxygenated hydrocarbons; halophilic and halotolerant bacteria and archaea; molecular mechanism of degradation
8.  Field evaluations of marine oil spill bioremediation. 
Microbiological Reviews  1996;60(2):342-365.
Bioremediation is defined as the act of adding or improving the availability of materials (e.g., nutrients, microorganisms, or oxygen) to contaminated environments to cause an acceleration of natural biodegradative processes. The results of field experiments and trials following actual spill incidents have been reviewed to evaluate the feasibility of this approach as a treatment for oil contamination in the marine environment. The ubiquity of oil-degrading microorganisms in the marine environment is well established, and research has demonstrated the capability of the indigenous microflora to degrade many components of petroleum shortly after exposure. Studies have identified numerous factors which affect the natural biodegradation rates of oil, such as the origin and concentration of oil, the availability of oil-degrading microorganisms, nutrient concentrations, oxygen levels, climatic conditions, and sediment characteristics. Bioremediation strategies based on the application of fertilizers have been shown to stimulate the biodegradation rates of oil in aerobic intertidal sediments such as sand and cobble. The ratio of oil loading to nitrogen concentration within the interstitial water has been identified to be the principal controlling factor influencing the success of this bioremediation strategy. However, the need for the seeding of natural environments with hydrocarbon-degrading bacteria has not been clearly demonstrated under natural environmental conditions. It is suggested that bioremediation should now take its place among the many techniques available for the treatment of oil spills, although there is still a clear need to set operational limits for its use. On the basis of the available evidence, we have proposed preliminary operational guidelines for bioremediation on shoreline environments.
PMCID: PMC239447  PMID: 8801437
9.  Conversion of crude oil to methane by a microbial consortium enriched from oil reservoir production waters 
The methanogenic biodegradation of crude oil is an important process occurring in petroleum reservoirs and other oil-containing environments such as contaminated aquifers. In this process, syntrophic bacteria degrade hydrocarbon substrates to products such as acetate, and/or H2 and CO2 that are then used by methanogens to produce methane in a thermodynamically dependent manner. We enriched a methanogenic crude oil-degrading consortium from production waters sampled from a low temperature heavy oil reservoir. Alkylsuccinates indicative of fumarate addition to C5 and C6 n-alkanes were identified in the culture (above levels found in controls), corresponding to the detection of an alkyl succinate synthase encoding gene (assA/masA) in the culture. In addition, the enrichment culture was tested for its ability to produce methane from residual oil in a sandstone-packed column system simulating a mature field. Methane production rates of up to 5.8 μmol CH4/g of oil/day were measured in the column system. Amounts of produced methane were in relatively good agreement with hydrocarbon loss showing depletion of more than 50% of saturate and aromatic hydrocarbons. Microbial community analysis revealed that the enrichment culture was dominated by members of the genus Smithella, Methanosaeta, and Methanoculleus. However, a shift in microbial community occurred following incubation of the enrichment in the sandstone columns. Here, Methanobacterium sp. were most abundant, as were bacterial members of the genus Pseudomonas and other known biofilm forming organisms. Our findings show that microorganisms enriched from petroleum reservoir waters can bioconvert crude oil components to methane both planktonically and in sandstone-packed columns as test systems. Further, the results suggest that different organisms may contribute to oil biodegradation within different phases (e.g., planktonic vs. sessile) within a subsurface crude oil reservoir.
PMCID: PMC4017130  PMID: 24829563
crude oil; hydrocarbon methanogenesis; crude oil reservoir; pyrotag sequencing; alkylsuccinates
10.  Central Role of Dynamic Tidal Biofilms Dominated by Aerobic Hydrocarbonoclastic Bacteria and Diatoms in the Biodegradation of Hydrocarbons in Coastal Mudflats 
Applied and Environmental Microbiology  2012;78(10):3638-3648.
Mudflats and salt marshes are habitats at the interface of aquatic and terrestrial systems that provide valuable services to ecosystems. Therefore, it is important to determine how catastrophic incidents, such as oil spills, influence the microbial communities in sediment that are pivotal to the function of the ecosystem and to identify the oil-degrading microbes that mitigate damage to the ecosystem. In this study, an oil spill was simulated by use of a tidal chamber containing intact diatom-dominated sediment cores from a temperate mudflat. Changes in the composition of bacteria and diatoms from both the sediment and tidal biofilms that had detached from the sediment surface were monitored as a function of hydrocarbon removal. The hydrocarbon concentration in the upper 1.5 cm of sediments decreased by 78% over 21 days, with at least 60% being attributed to biodegradation. Most phylotypes were minimally perturbed by the addition of oil, but at day 21, there was a 10-fold increase in the amount of cyanobacteria in the oiled sediment. Throughout the experiment, phylotypes associated with the aerobic degradation of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) (Cycloclasticus) and alkanes (Alcanivorax, Oleibacter, and Oceanospirillales strain ME113), substantively increased in oiled mesocosms, collectively representing 2% of the pyrosequences in the oiled sediments at day 21. Tidal biofilms from oiled cores at day 22, however, consisted mostly of phylotypes related to Alcanivorax borkumensis (49% of clones), Oceanospirillales strain ME113 (11% of clones), and diatoms (14% of clones). Thus, aerobic hydrocarbon biodegradation is most likely to be the main mechanism of attenuation of crude oil in the early weeks of an oil spill, with tidal biofilms representing zones of high hydrocarbon-degrading activity.
PMCID: PMC3346363  PMID: 22407688
11.  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.
PMCID: PMC3982060  PMID: 24765087
methanogenic; oil biodegradation; volatile hydrocarbons; non-weathered oil; weathered oil; n-alkanes
12.  Effective bioremediation strategy for rapid in situ cleanup of anoxic marine sediments in mesocosm oil spill simulation 
The purpose of present study was the simulation of an oil spill accompanied by burial of significant amount of petroleum hydrocarbons (PHs) in coastal sediments. Approximately 1000 kg of sediments collected in Messina harbor were spiked with Bunker C furnace fuel oil (6500 ppm). The rapid consumption of oxygen by aerobic heterotrophs created highly reduced conditions in the sediments with subsequent recession of biodegradation rates. As follows, after 3 months of ageing, the anaerobic sediments did not exhibit any significant levels of biodegradation and more than 80% of added Bunker C fuel oil remained buried. Anaerobic microbial community exhibited a strong enrichment in sulfate-reducing PHs-degrading and PHs-associated Deltaproteobacteria. As an effective bioremediation strategy to clean up these contaminated sediments, we applied a Modular Slurry System (MSS) allowing the containment of sediments and their physical–chemical treatment, e.g., aeration. Aeration for 3 months has increased the removal of main PHs contaminants up to 98%. As revealed by CARD-FISH, qPCR, and 16S rRNA gene clone library analyses, addition of Bunker C fuel oil initially affected the activity of autochthonous aerobic obligate marine hydrocarbonoclastic bacteria (OMHCB), and after 1 month more than the third of microbial population was represented by Alcanivorax-, Cycloclasticus-, and Marinobacter-related organisms. In the end of the experiment, the microbial community composition has returned to a status typically observed in pristine marine ecosystems with no detectable OMHCB present. Eco-toxicological bioassay revealed that the toxicity of sediments after treatment was substantially decreased. Thus, our studies demonstrated that petroleum-contaminated anaerobic marine sediments could efficiently be cleaned through an in situ oxygenation which stimulates their self-cleaning potential due to reawakening of allochtonous aerobic OMHCB.
PMCID: PMC3995047  PMID: 24782850
marine anoxic sediments; crude oil pollution; hydrocarbonoclastic bacteria; in situ bioremediation; aerated slurry system
13.  Impact of Oil on Bacterial Community Structure in Bioturbated Sediments 
PLoS ONE  2013;8(6):e65347.
Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions – with tidal cycles and natural seawater – was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g−1 wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by Gammaproteobacteria and Deltaproteobacteria. In the oiled-microcosms, the addition of H. diversicolor reduced the phylotype-richness, sequences associated to Actinobacteria, Firmicutes and Plantomycetes were not detected. These observations highlight the influence of the bioturbation on the bacterial community structure without affecting the biodegradation capacities.
PMCID: PMC3677869  PMID: 23762350
14.  Understanding Plant-Microbe Interactions for Phytoremediation of Petroleum-Polluted Soil 
PLoS ONE  2011;6(3):e17961.
Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of petroleum pollution, however relatively little is known about how these interactions are influenced by petroleum pollution. In this experimental study using a microcosm approach, we examined how plant ecophysiological traits, soil nutrients and microbial activities were influenced by petroleum pollution in Phragmites australis, a phytoremediating species. Generally, petroleum pollution reduced plant performance, especially at early stages of plant growth. Petroleum had negative effects on the net accumulation of inorganic nitrogen from its organic forms (net nitrogen mineralization (NNM)) most likely by decreasing the inorganic nitrogen available to the plants in petroleum-polluted soils. However, abundant dissolved organic nitrogen (DON) was found in petroleum-polluted soil. In order to overcome initial deficiency of inorganic nitrogen, plants by dint of high colonization of arbuscular mycorrhizal fungi might absorb some DON for their growth in petroleum-polluted soils. In addition, through using a real-time polymerase chain reaction method, we quantified hydrocarbon-degrading bacterial traits based on their catabolic genes (i.e. alkB (alkane monooxygenase), nah (naphthalene dioxygenase) and tol (xylene monooxygenase) genes). This enumeration of target genes suggests that different hydrocarbon-degrading bacteria experienced different dynamic changes during phytoremediation and a greater abundance of alkB was detected during vegetative growth stages. Because phytoremediation of different components of petroleum is performed by different hydrocarbon-degrading bacteria, plants’ ability of phytoremediating different components might therefore vary during the plant life cycle. Phytoremediation might be most effective during the vegetative growth stages as greater abundances of hydrocarbon-degrading bacteria containing alkB and tol genes were observed at these stages. The information provided by this study enhances our understanding of the effects of petroleum pollution on plant-microbe interactions and the roles of these interactions in the phytoremediation of petroleum-polluted soil.
PMCID: PMC3060916  PMID: 21437257
15.  In situ detection of anaerobic alkane metabolites in subsurface environments 
Alkanes comprise a substantial fraction of crude oil and refined fuels. As such, they are prevalent within deep subsurface fossil fuel deposits and in shallow subsurface environments such as aquifers that are contaminated with hydrocarbons. These environments are typically anaerobic, and host diverse microbial communities that can potentially use alkanes as substrates. Anaerobic alkane biodegradation has been reported to occur under nitrate-reducing, sulfate-reducing, and methanogenic conditions. Elucidating the pathways of anaerobic alkane metabolism has been of interest in order to understand how microbes can be used to remediate contaminated sites. Alkane activation primarily occurs by addition to fumarate, yielding alkylsuccinates, unique anaerobic metabolites that can be used to indicate in situ anaerobic alkane metabolism. These metabolites have been detected in hydrocarbon-contaminated shallow aquifers, offering strong evidence for intrinsic anaerobic bioremediation. Recently, studies have also revealed that alkylsuccinates are present in oil and coal seam production waters, indicating that anaerobic microbial communities can utilize alkanes in these deeper subsurface environments. In many crude oil reservoirs, the in situ anaerobic metabolism of hydrocarbons such as alkanes may be contributing to modern-day detrimental effects such as oilfield souring, or may lead to more beneficial technologies such as enhanced energy recovery from mature oilfields. In this review, we briefly describe the key metabolic pathways for anaerobic alkane (including n-alkanes, isoalkanes, and cyclic alkanes) metabolism and highlight several field reports wherein alkylsuccinates have provided evidence for anaerobic in situ alkane metabolism in shallow and deep subsurface environments.
PMCID: PMC3671572  PMID: 23761789
anaerobic; metabolites; alkylsuccinates; alkanes; subsurface
16.  Bioreactor-based bioremediation of hydrocarbon-polluted Niger Delta marine sediment, Nigeria 
3 Biotech  2011;2(1):53-66.
Crude oil-polluted marine sediment from Bonny River loading jetty Port Harcourt, Nigeria was treated in seven 2.5 l stirred-tank bioreactors designated BNPK, BNK5, BPD, BNO3, BUNa, BAUT, and BUK over a 56-day period. Five bioreactors were biostimulated with either K2HPO4, NH4NO3, (NH4)2SO4, NPK, urea or poultry droppings while unamended (BUNa) and heat-killed (BAUT) treatments were controls. For each bioreactor, 1 kg (wet weight) sediment amended with 1 l seawater were spiked with 20 ml and 20 mg of crude oil and anthracene which gave a total petroleum hydrocarbons (TPH) range of 106.4–116 ppm on day 0. Polycyclic aromatic hydrocarbons (PAH) in all spiked sediment slurry ranged from 96.6 to 104.4 ppm. TPH in each treatment was ≤14.9 ppm while PAH was ≤6.8 ppm by day 56. Treatment BNO3 recorded highest heterotrophic bacterial count (9.8 × 108 cfu/g) and hydrocarbon utilizers (1.15 × 108 cfu/g). By day 56, the percentages of biodegradation of PAHs, as measured with GC–FID were BNK5 (97.93%), BNPK (98.38%), BUK (98.82%), BUNa (98.13%), BAUT (93.08%), BPD (98.92%), and BNO3 (98.02%). BPD gave the highest degradation rate for PAH. TPH degradation rates were as follows: BNK5 (94.50%), BNPK (94.77%), BUK (94.10%), BUNa (94.77%), BAUT (75.04%), BPD (95.35%), BNO3 (95.54%). Fifty-six hydrocarbon utilizing bacterial isolates obtained were Micrococcus spp. 5 (9.62%), Staphylococcus spp. 3 (5.78%), Pseudomonas spp. 7 (13.46%), Citrobacter sp. 1 (1.92%), Klebsiella sp. 1 (1.92%), Corynebacterium spp. 5 (9.62%), Bacillus spp. 5 (9.62%), Rhodococcus spp. 7 (13.46%), Alcanivorax spp. 7 (13.46%), Alcaligenes sp. 1 (1.92%), Serratia spp. 2 (3.85%), Arthrobacter spp. 7 (13.46%), Nocardia spp. 2 (3.85%), Flavobacterium sp. 1 (1.92%), Escherichia sp. 1 (1.92%), Acinetobacter sp. 1 (1.92%), Proteus sp. 1 (1.92%) and unidentified bacteria 10 (17%). These results indicate that the marine sediment investigated is amenable to bioreactor-based bioremediation and that abiotic factors also could contribute to hydrocarbon attenuation as recorded in the heat-killed (BAUT) control.
PMCID: PMC3339588  PMID: 22582157
Niger Delta; Marine sediment; Bioreactor; Crude oil; Bonny loading jetty; Chemistry; Bioinformatics; Agriculture; Stem Cells; Biomaterials; Biotechnology; Cancer Research
17.  Bioreactor-based bioremediation of hydrocarbon-polluted Niger Delta marine sediment, Nigeria 
3 Biotech  2011;2(1):53-66.
Crude oil-polluted marine sediment from Bonny River loading jetty Port Harcourt, Nigeria was treated in seven 2.5 l stirred-tank bioreactors designated BNPK, BNK5, BPD, BNO3, BUNa, BAUT, and BUK over a 56-day period. Five bioreactors were biostimulated with either K2HPO4, NH4NO3, (NH4)2SO4, NPK, urea or poultry droppings while unamended (BUNa) and heat-killed (BAUT) treatments were controls. For each bioreactor, 1 kg (wet weight) sediment amended with 1 l seawater were spiked with 20 ml and 20 mg of crude oil and anthracene which gave a total petroleum hydrocarbons (TPH) range of 106.4–116 ppm on day 0. Polycyclic aromatic hydrocarbons (PAH) in all spiked sediment slurry ranged from 96.6 to 104.4 ppm. TPH in each treatment was ≤14.9 ppm while PAH was ≤6.8 ppm by day 56. Treatment BNO3 recorded highest heterotrophic bacterial count (9.8 × 108 cfu/g) and hydrocarbon utilizers (1.15 × 108 cfu/g). By day 56, the percentages of biodegradation of PAHs, as measured with GC–FID were BNK5 (97.93%), BNPK (98.38%), BUK (98.82%), BUNa (98.13%), BAUT (93.08%), BPD (98.92%), and BNO3 (98.02%). BPD gave the highest degradation rate for PAH. TPH degradation rates were as follows: BNK5 (94.50%), BNPK (94.77%), BUK (94.10%), BUNa (94.77%), BAUT (75.04%), BPD (95.35%), BNO3 (95.54%). Fifty-six hydrocarbon utilizing bacterial isolates obtained were Micrococcus spp. 5 (9.62%), Staphylococcus spp. 3 (5.78%), Pseudomonas spp. 7 (13.46%), Citrobacter sp. 1 (1.92%), Klebsiella sp. 1 (1.92%), Corynebacterium spp. 5 (9.62%), Bacillus spp. 5 (9.62%), Rhodococcus spp. 7 (13.46%), Alcanivorax spp. 7 (13.46%), Alcaligenes sp. 1 (1.92%), Serratia spp. 2 (3.85%), Arthrobacter spp. 7 (13.46%), Nocardia spp. 2 (3.85%), Flavobacterium sp. 1 (1.92%), Escherichia sp. 1 (1.92%), Acinetobacter sp. 1 (1.92%), Proteus sp. 1 (1.92%) and unidentified bacteria 10 (17%). These results indicate that the marine sediment investigated is amenable to bioreactor-based bioremediation and that abiotic factors also could contribute to hydrocarbon attenuation as recorded in the heat-killed (BAUT) control.
PMCID: PMC3339588  PMID: 22582157
Niger Delta; Marine sediment; Bioreactor; Crude oil; Bonny loading jetty
18.  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.
PMCID: PMC4227522  PMID: 25426105
biogeochemistry; oil reservoirs; microbial ecology; energy; hydrocarbon biodegradation
19.  Fate of Nitrogen-Fixing Bacteria in Crude Oil Contaminated Wetland Ultisol 
The effect of crude oil on the growth of legumes (Calopogonium muconoides and Centrosema pubescens) and fate of nitrogen-fixing bacteria in wetland ultisol was investigated using standard cultural techniques. The results revealed observable effects of oil on soil physico-chemistry, plant growth and nodulation as well as on densities of heterotrophic, hydrocarbonoclastic and nitrogen fixing bacteria. The effects however varied with different levels (0.5%, 1%, 5%, 10%, 15% and 20%) of pollution. Ammonium and nitrate levels were high in the unpolluted soil but decreased with increase in pollution levels. Nitrite was not detected in contaminated soil probably due to the reduction in numbers of nitrogen fixers, from 5.26 ± 0.23 × l06cfu/g in unpolluted soil to 9.0 ± 0.12 × 105 and 2.2 ± 0.08 × l05 cfu/g in soils with 5% and 20% levels of pollution respectively. The contaminated soil also exhibited gross reduction in the nodulation of legumes. A range of 13–57 nodules was observed in legumes from polluted soil against 476 nodules recorded for plants cultured on unpolluted soil. The heterogeneity of the microbial loads between oil-polluted and unpolluted soil were statistically significant (p < 0.05, ANOVA). Positive significant relationships were observed between the levels of hydrocarbons and the densities of heterotrophic bacteria (r = 0.91) and that of hydrocarbon utilizing bacteria (r = 0.86). On the other hand, relationships between the densities of nitrogen fixing bacteria and total hydrocarbons content was negative (r = −0.30) while positive relationships were recorded between the densities of different microbial groups and treatment periods except at 15% and 20% pollution levels. The LSD tests revealed highly significant differences (p < 0.001) in the physiological groups of soil microorganisms at all levels of pollution. The results imply that crude oil seriously affects rhizosphere microbial growth in legumes. Among the bacterial species isolated, Clostridium pasteurianum, Bacillus polymyxa and Pseudomonas aeruginosa exhibited greater ability to degrade hydrocarbons than Azotobacter sp, Klebsiella pneumoniae and Derxia gummusa while Nitrosomonas and Nitrobacter had the least degradability. A continuous monitoring of the environment is advocated to prevent extinction of nitrogen-fixing bacteria and total loss of soil fertility attributable to petroleum hydrocarbon contamination in the Niger Delta ultisol.
PMCID: PMC3155754  PMID: 21755289
Fate; Nitrogen-fixing; Bacteria; Crude oil polluted wetland
20.  Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil 
Applied and Environmental Microbiology  2005;71(11):7008-7018.
Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.
PMCID: PMC1287751  PMID: 16269736
21.  Practical Considerations and Challenges Involved in Surfactant Enhanced Bioremediation of Oil 
BioMed Research International  2013;2013:328608.
Surfactant enhanced bioremediation (SEB) of oil is an approach adopted to overcome the bioavailability constraints encountered in biotransformation of nonaqueous phase liquid (NAPL) pollutants. Fuel oils contain n-alkanes and other aliphatic hydrocarbons, monoaromatics, and polynuclear aromatic hydrocarbons (PAHs). Although hydrocarbon degrading cultures are abundant in nature, complete biodegradation of oil is rarely achieved even under favorable environmental conditions due to the structural complexity of oil and culture specificities. Moreover, the interaction among cultures in a consortium, substrate interaction effects during the degradation and ability of specific cultures to alter the bioavailability of oil invariably affect the process. Although SEB has the potential to increase the degradation rate of oil and its constituents, there are numerous challenges in the successful application of this technology. Success is dependent on the choice of appropriate surfactant type and dose since the surfactant-hydrocarbon-microorganism interaction may be unique to each scenario. Surfactants not only enhance the uptake of constituents through micellar solubilization and emulsification but can also alter microbial cell surface characteristics. Moreover, hydrocarbons partitioned in micelles may not be readily bioavailable depending on the microorganism-surfactant interactions. Surfactant toxicity and inherent biodegradability of surfactants may pose additional challenges as discussed in this review.
PMCID: PMC3857904  PMID: 24350261
22.  Metagenomic analysis of microbial consortium from natural crude oil that seeps into the marine ecosystem offshore Southern California 
Standards in Genomic Sciences  2014;9(3):1259-1274.
Crude oils can be major contaminants of the marine ecosystem and microorganisms play a significant role in the degradation of its main constituents. To increase our understanding of the microbial hydrocarbon degradation process in the marine ecosystem, we collected crude oil from an active seep area located in the Santa Barbara Channel (SBC) and generated a total of about 52 Gb of raw metagenomic sequence data. The assembled data comprised ~500 Mb, representing ~1.1 million genes derived primarily from chemolithoautotrophic bacteria. Members of Oceanospirillales, a bacterial order belonging to the Deltaproteobacteria, recruited less than 2% of the assembled genes within the SBC metagenome. In contrast, the microbial community associated with the oil plume that developed in the aftermath of the Deepwater Horizon (DWH) blowout in 2010, was dominated by Oceanospirillales, which comprised more than 60% of the metagenomic data generated from the DWH oil plume. This suggests that Oceanospirillales might play a less significant role in the microbially mediated hydrocarbon conversion within the SBC seep oil compared to the DWH plume oil. We hypothesize that this difference results from the SBC oil seep being mostly anaerobic, while the DWH oil plume is aerobic. Within the Archaea, the phylum Euryarchaeota, recruited more than 95% of the assembled archaeal sequences from the SBC oil seep metagenome, with more than 50% of the sequences assigned to members of the orders Methanomicrobiales and Methanosarcinales. These orders contain organisms capable of anaerobic methanogenesis and methane oxidation (AOM) and we hypothesize that these orders – and their metabolic capabilities – may be fundamental to the ecology of the SBC oil seep.
PMCID: PMC4149020  PMID: 25197496
Bioremediation; hydrocarbon-degradation; marine ecosystem; crude oil; natural oil seeps; anaerobic methane oxidation; bacteria; archaea; metagenomics
23.  Oil degradation in soil. 
The environmental effects of adding certain selected petroleum products to field soils at widely separated geographical locations under optimum conditions for biodegradation were studied. The locations selected for study of soil biodegradation of six oils (used crankcase oil from cars, used crankcase oil from trucks, an Arabian Heavy crude oil, a Coastal Mix crude oil, a home heating oil no. 2, and a residual fuel oil no. 6) were Marcus Hook, Pennsylvania, Tulsa, Oklahoma, and Corpus Christi, Texas. The investigative process, covering a period of 1 year at each location, was conducted in 14 fields plots (1.7 by 3.0 m) to which the oils were added in a single application at a rate of 11.9 m3/4 X 10(3) m2. One-half of the plots at each location were fertilized, and the incorporation of the oils and fertilizers was accomplished with rototillers to a depth of 10 to 15 cm. Concentrations of all oils decreased significantly at all locations. The average reduction ranged from 48.5 to 90.0% depending upon the type of oil and location. Rates of degradation did not exceed 2.4 m3/4 X 10(3) m2 per month. Compositional changes in the oil with time were investigated using silica gel fractionation, gas chromatography, and ultraviolet absorbance. With the possible exception of the two fuel oils, the compositional changes were generally in the same direction for all of the oils. The silica gel fractionation and gravimetric data on residual oils show that all classes of compounds were degraded, but the more polar type degrade more slowly. Analysis of runoff water, leachate, and soils indicated that at the concentration applied no oil less was observed from these plots via water movement. No significant movement of lead compounds added to the soils in the used crankcase oils was observed. Significant increases in hydrocarbon-utilizing microorganisms were demonstrated in all treated plots using either the pure hydrocarbon, n-hexadecane, or the applied oils as the growth substrate. These increases were usually sustained throughout the year. Significant increases in hydrocarbon-utilizing fungi were not demonstrated by the plating technique used. The concentrations of residual oils or their oxidation products were of sufficient magnitude in the treated plots, 9 months after application, to cause significant inhibition of plant growth. From the data obtained, it was not possible to determine the type of compounds causing this inhibition or their long-term environmental effects.
PMCID: PMC169815  PMID: 1267448
24.  Physical and Metabolic Interactions of Pseudomonas sp. Strain JA5-B45 and Rhodococcus sp. Strain F9-D79 during Growth on Crude Oil and Effect of a Chemical Surfactant on Them 
Applied and Environmental Microbiology  2001;67(10):4874-4879.
Methods to enhance crude oil biodegradation by mixed bacterial cultures, for example, (bio)surfactant addition, are complicated by the diversity of microbial populations within a given culture. The physical and metabolic interactions between Rhodococcus sp. strain F9-D79 and Pseudomonas sp. strain JA5-B45 were examined during growth on Bow River crude oil. The effects of a nonionic chemical surfactant, Igepal CO-630 (nonylphenol ethoxylate), also were evaluated. Strain F9-D79 grew attached to the oil-water interface and produced a mycolic acid-containing capsule. Crude oil emulsification and surface activity were associated with the cellular fraction. Strain JA5-B45 grew in the aqueous phase and was unable to emulsify oil, but cell-free supernatants mediated kerosene-water emulsion formation. In coculture, stable emulsions were formed and strain JA5-B45 had an affinity for the capsule produced by strain F9-D79. Igepal CO-630 inhibited F9-D79 cells from adhering to the interface, and cells grew dispersed in the aqueous phase as 0.5-μm cocci rather than 2.5-μm rods. The surfactant increased total petroleum hydrocarbon removal by strain JA5-B45 from 4 to 22% and included both saturated compounds and aromatics. In coculture, TPH removal increased from 13 to 40% following surfactant addition. The culture pH normally increased from 7.0 to between 7.5 and 8.5, although addition of Igepal CO-630 to F9-D79 cultures resulted in a drop to pH 5.5. We suggest a dual role for the nonylphenol ethoxylate surfactant in the coculture: (i) to improve hydrocarbon uptake by strain JA5-B45 through emulsification and (ii) to prevent strain F9-D79 from adhering to the oil-water interface, indirectly increasing hydrocarbon availability. These varied effects on hydrocarbon biodegradation could explain some of the known diversity of surfactant effects.
PMCID: PMC93243  PMID: 11571196
25.  Progressive Degradation of Crude Oil n-Alkanes Coupled to Methane Production under Mesophilic and Thermophilic Conditions 
PLoS ONE  2014;9(11):e113253.
Although methanogenic degradation of hydrocarbons has become a well-known process, little is known about which crude oil tend to be degraded at different temperatures and how the microbial community is responded. In this study, we assessed the methanogenic crude oil degradation capacity of oily sludge microbes enriched from the Shengli oilfield under mesophilic and thermophilic conditions. The microbial communities were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes combined with cloning and sequencing. Enrichment incubation demonstrated the microbial oxidation of crude oil coupled to methane production at 35 and 55°C, which generated 3.7±0.3 and 2.8±0.3 mmol of methane per gram oil, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that crude oil n-alkanes were obviously degraded, and high molecular weight n-alkanes were preferentially removed over relatively shorter-chain n-alkanes. Phylogenetic analysis revealed the concurrence of acetoclastic Methanosaeta and hydrogenotrophic methanogens but different methanogenic community structures under the two temperature conditions. Candidate divisions of JS1 and WWE 1, Proteobacteria (mainly consisting of Syntrophaceae, Desulfobacteraceae and Syntrophorhabdus) and Firmicutes (mainly consisting of Desulfotomaculum) were supposed to be involved with n-alkane degradation in the mesophilic conditions. By contrast, the different bacterial phylotypes affiliated with Caldisericales, “Shengli Cluster” and Synergistetes dominated the thermophilic consortium, which was most likely to be associated with thermophilic crude oil degradation. This study revealed that the oily sludge in Shengli oilfield harbors diverse uncultured microbes with great potential in methanogenic crude oil degradation over a wide temperature range, which extend our previous understanding of methanogenic degradation of crude oil alkanes.
PMCID: PMC4237390  PMID: 25409013

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