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.

Enhanced crude oil biodegradative potential of natural phytoplankton-associated hydrocarbonoclastic bacteria.

Phytoplankton have been shown to harbour a diversity of hydrocarbonoclastic bacteria (HCB), yet it is not understood how these phytoplankton-associated HCB would respond in the event of an oil spill at sea. Here, we assess the diversity and dynamics of the bacterial community associated with a natural population of marine phytoplankton under oil spill-simulated conditions, and compare it to that of the free-living (non phytoplankton-associated) bacterial community. While the crude oil severely impacted the phytoplankton population and was likely conducive to marine oil snow formation, analysis of the MiSeq-derived 16S rRNA data revealed dramatic and differential shifts in the oil-amended communities that included blooms of recognized HCB (e.g., Thalassospira, Cycloclasticus), including putative novel phyla, as well as other groups with previously unqualified oil-degrading potential (Olleya, Winogradskyella, and members of the inconspicuous BD7-3 phylum). Notably, the oil biodegradation potential of the phytoplankton-associated community exceeded that of the free-living community, and it showed a preference to degrade substituted and non-substituted polycyclic aromatic hydrocarbons. Our study provides evidence of compartmentalization of hydrocarbon-degrading capacity in the marine water column, wherein HCB associated with phytoplankton are better tuned to degrading crude oil hydrocarbons than that by the community of planktonic free-living bacteria.

Characterization of the surface-active exopolysaccharide produced by Halomonas sp TGOS-10: Understanding its role in the formation of marine oil snow.

In this study, we characterize the exopolymer produced by Halomonas sp. strain TGOS-10 -one of the organisms found enriched in sea surface oil slicks during the Deepwater Horizon oil spill. The polymer was produced during the early stationary phase of growth in Zobell's 2216 marine medium amended with glucose. Chemical and proton NMR analysis showed it to be a relatively monodisperse, high-molecular-mass (6,440,000 g/mol) glycoprotein composed largely of protein (46.6% of total dry weight of polymer). The monosaccharide composition of the polymer is typical to that of other marine bacterial exopolymers which are generally rich in hexoses, with the notable exception that it contained mannose (commonly found in yeast) as a major monosaccharide. The polymer was found to act as an oil dispersant based on its ability to effectively emulsify pure and complex oils into stable oil emulsions-a function we suspect to be conferred by the high protein content and high ratio of total hydrophobic nonpolar to polar amino acids (52.7:11.2) of the polymer. The polymer's chemical composition, which is akin to that of other marine exopolymers also having a high protein-to-carbohydrate (P/C) content, and which have been shown to effect the rapid and non-ionic aggregation of marine gels, appears indicative of effecting marine oil snow (MOS) formation. We previously reported the strain capable of utilising aromatic hydrocarbons when supplied as single carbon sources. However, here we did not detect biodegradation of these chemicals within a complex (surrogate Macondo) oil, suggesting that the observed enrichment of this organism during the Deepwater Horizon spill may be explained by factors related to substrate availability and competition within the complex and dynamic microbial communities that were continuously evolving during that spill.

Effect of ocean acidification on the growth, response and hydrocarbon degradation of coccolithophore-bacterial communities exposed to crude oil.

Hydrocarbon-degrading bacteria, which can be found living with eukaryotic phytoplankton, play a pivotal role in the fate of oil spillage to the marine environment. Considering the susceptibility of calcium carbonate-bearing phytoplankton under future ocean acidification conditions and their oil-degrading communities to oil exposure under such conditions, we investigated the response of non-axenic E. huxleyi to crude oil under ambient versus elevated CO2 concentrations. Under elevated CO2 conditions, exposure to crude oil resulted in the immediate decline of E. huxleyi, with concomitant shifts in the relative abundance of Alphaproteobacteria and Gammaproteobacteria. Survival of E. huxleyi under ambient conditions following oil enrichment was likely facilitated by enrichment of oil-degraders Methylobacterium and Sphingomonas, while the increase in relative abundance of Marinobacter and unclassified Gammaproteobacteria may have increased competitive pressure with E. huxleyi for micronutrient acquisition. Biodegradation of the oil was not affected by elevated CO2 despite a shift in relative abundance of known and putative hydrocarbon degraders. While ocean acidification does not appear to affect microbial degradation of crude oil, elevated mortality responses of E. huxleyi and shifts in the bacterial community illustrates the complexity of microalgal-bacterial interactions and highlights the need to factor these into future ecosystem recovery projections.

Enrichment and Characterisation of a Mixed-Source Ethanologenic Community Degrading the Organic Fraction of Municipal Solid Waste Under Minimal Environmental Control.

The utilisation of the organic fraction of municipal solid waste as feedstock for bioethanol production could reduce the need for disposal of the ever-increasing amounts of municipal solid waste, especially in developing countries, and fits with the integrated goals of climate change mitigation and transport energy security. Mixed culture fermentation represents a suitable approach to handle the complexity and variability of such waste, avoiding expensive and vulnerable closed-control operational conditions. It is widely accepted that the control of pH in these systems can direct the fermentation process toward a desired fermentation product, however, little empirical evidence has been provided in respect of lignocellulosic waste substrates and different environmental inocula sources. We evaluated ethanol production from the organic fraction of municipal solid waste using five different inocula sources where lignocellulose degradation putatively occurs, namely, compost, woodland soil, rumen, cow faeces and anaerobic granular sludge, when incubated in batch microcosms at either initially neutral or acidic pH and under initially aerobic or anaerobic conditions. Although ethanol was produced by all the inocula tested, their performance was different in response to the imposed experimental conditions. Rumen and anaerobic granular sludge produced significantly the highest ethanol concentrations (∼30 mM) under initially neutral and acidic pH, respectively. A mixed-source community formed by mixing rumen and sludge (R + S) was then tested over a range of initial pH. In contrast to the differences observed for the individual inocula, the maximal ethanol production of the mixed community was not significantly different at initial pH of 5.5 and 7. Consistent with this broader functionality, the microbial community analyses confirmed the R + S community enriched comprised bacterial taxa representative of both original inocula. It was demonstrated that the interaction of initial pH and inocula source dictated ethanologenic activity from the organic fraction of municipal solid waste. Furthermore, the ethanologenic mixed-source community enriched, was comprised of taxa belonging to the two original inocula sources (rumen and sludge) and had a broader functionality. This information is relevant when diverse inocula sources are combined for mix culture fermentation studies as it experimentally demonstrates the benefits of diversity and function assembled from different inocula.

Hydrocarbon-degradation and MOS-formation capabilities of the dominant bacteria enriched in sea surface oil slicks during the Deepwater Horizon oil spill.

A distinctive feature of the Deepwater Horizon (DwH) oil spill was the formation of significant quantities of marine oil snow (MOS), for which the mechanism(s) underlying its formation remain unresolved. Here, we show that Alteromonas strain TK-46(2), Pseudoalteromonas strain TK-105 and Cycloclasticus TK-8 - organisms that became enriched in sea surface oil slicks during the spill - contributed to the formation of MOS and/or dispersion of the oil. In roller-bottle incubations, Alteromonas cells and their produced EPS yielded MOS, whereas Pseudoalteromonas and Cycloclasticus did not. Interestingly, the Cycloclasticus strain was able to degrade n-alkanes concomitantly with aromatics within the complex oil mixture, which is atypical for members of this genus. Our findings, for the first time, provide direct evidence on the hydrocarbon-degrading capabilities for these bacteria enriched during the DwH spill, and that bacterial cells of certain species and their produced EPS played a direct role in MOS formation.

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.