Markus Bill

Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill

The Deepwater Horizon oil spill resulted in a massive influx of hydrocarbons into the Gulf of Mexico (the Gulf). To better understand the fate of the oil, we enriched and isolated indigenous hydrocarbon-degrading bacteria from deep, uncontaminated waters from the Gulf with oil (Macondo MC252) and dispersant used during the spill (COREXIT 9500). During 20 days of incubation at 5°C, CO(2) evolution, hydrocarbon concentrations and the microbial community composition were determined. Approximately 60% to 25% of the dissolved oil with or without COREXIT, respectively, was degraded, in addition to some hydrocarbons in the COREXIT. FeCl(2) addition initially increased respiration rates, but not the total amount of hydrocarbons degraded. 16S rRNA gene sequencing revealed a succession in the microbial community over time, with an increase in abundance of Colwellia and Oceanospirillales during the incubations. Flocs formed during incubations with oil and/or COREXIT in the absence of FeCl(2) . Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy revealed that the flocs were comprised of oil, carbohydrates and biomass. Colwellia were the dominant bacteria in the flocs. Colwellia sp. strain RC25 was isolated from one of the enrichments and confirmed to rapidly degrade high amounts (approximately 75%) of the MC252 oil at 5°C. Together these data highlight several features that provide Colwellia with the capacity to degrade oil in cold, deep marine habitats, including aggregation together with oil droplets into flocs and hydrocarbon degradation ability.

Succession of Hydrocarbon-Degrading Bacteria in the Aftermath of the Deepwater Horizon Oil Spill in the Gulf of Mexico

The Deepwater Horizon oil spill produced large subsurface plumes of dispersed oil and gas in the Gulf of Mexico that stimulated growth of psychrophilic, hydrocarbon degrading bacteria. We tracked succession of plume bacteria before, during and after the 83-day spill to determine the microbial response and biodegradation potential throughout the incident. Dominant bacteria shifted substantially over time and were dependent on relative quantities of different hydrocarbon fractions. Unmitigated flow from the wellhead early in the spill resulted in the highest proportions of n-alkanes and cycloalkanes at depth and corresponded with dominance by Oceanospirillaceae and Pseudomonas. Once partial capture of oil and gas began 43 days into the spill, petroleum hydrocarbons decreased, the fraction of aromatic hydrocarbons increased, and Colwellia, Cycloclasticus, and Pseudoalteromonas increased in dominance. Enrichment of Methylomonas coincided with positive shifts in the δ(13)C values of methane in the plume and indicated significant methane oxidation occurred earlier than previously reported. Anomalous oxygen depressions persisted at plume depths for over six weeks after well shut-in and were likely caused by common marine heterotrophs associated with degradation of high-molecular-weight organic matter, including Methylophaga. Multiple hydrocarbon-degrading bacteria operated simultaneously throughout the spill, but their relative importance was controlled by changes in hydrocarbon supply.

Environmental and biological controls on methyl halide emissions from southern California coastal salt marshes

Methyl bromide (CH3Br) and methyl chloride(CH3Cl) emission rates from southernCalifornia coastal salt marshes show largespatial and temporal variabilities that arestrongly linked to biological and environmentalfactors. Here we discuss biogeochemical linesof evidence pointing to vegetation as theprimary source of CH3Br and CH3Clemissions from salt marshes. Sediments andmacroalgae do not appear to be major producersof these compounds, based on observations thatthe highest fluxes are not inhibited by soilinundation; their emissions are not correlatedwith those of certain gases produced in soils;and emissions from mudflat- andmacroalgae-dominated sites are relativelysmall. In contrast, the seasonal and spatialvariabilities of methyl halide fluxes in thesesalt marshes are consistent with the productionof these compounds by vascular plants, althoughthe possibility of production by microflora orfungi associated with the salt marsh vegetationis not ruled out. Flux chamber measurements ofemission rates are largely correlated to theoverall plant biomass enclosed in the chamber,but appear also to be highly dependent on thepredominant plant species. Emission ratesfollow a diurnal trend similar to the trends ofambient air temperature and photosyntheticallyactive radiation, but not surface soiltemperature. Diurnal variabilities in thecarbon isotope compositions of CH3Cl andCH3Br and their relative ratios ofemissions are consistent with simultaneouslycompeting mechanisms of uptake andproduction.

Carbon Isotope Fractionation during Aerobic Biodegradation of Trichloroethene by Burkholderia cepacia G4: a Tool To Map Degradation Mechanisms

ABSTRACT The strain Burkholderia cepacia G4 aerobically mineralized trichloroethene (TCE) to CO2 over a time period of ∼20 h. Three biodegradation experiments were conducted with different bacterial optical densities at 540 nm (OD540s) in order to test whether isotope fractionation was consistent. The resulting TCE degradation was 93, 83.8, and 57.2% (i.e., 7.0, 16.2, and 42.8% TCE remaining) at OD540s of 2.0, 1.1, and 0.6, respectively. ODs also correlated linearly with zero-order degradation rates (1.99, 1.11, and 0.64 μmol h−1). While initial nonequilibrium mass losses of TCE produced only minor carbon isotope shifts (expressed in per mille δ13CVPDB), they were 57.2, 39.6, and 17.0‰ between the initial and final TCE levels for the three experiments, in decreasing order of their OD540s. Despite these strong isotope shifts, we found a largely uniform isotope fractionation. The latter is expressed with a Rayleigh enrichment factor, ε, and was −18.2 when all experiments were grouped to a common point of 42.8% TCE remaining. Although, decreases of ε to −20.7 were observed near complete degradation, our enrichment factors were significantly more negative than those reported for anaerobic dehalogenation of TCE. This indicates typical isotope fractionation for specific enzymatic mechanisms that can help to differentiate between degradation pathways.

Field evidence for co-metabolism of trichloroethene stimulated by addition of electron donor to groundwater.

For more than 10 years, electron donor has been injected into the Snake River aquifer beneath the Test Area North site of the Idaho National Laboratory for the purpose of stimulating microbial reductive dechlorination of trichloroethene (TCE) in groundwater. This has resulted in significant TCE removal from the source area of the contaminant plume and elevated dissolved CH(4) in the groundwater extending 250 m from the injection well. The delta(13)C of the CH(4) increases from -56 per thousand in the source area to -13 per thousand with distance from the injection well, whereas the delta(13)C of dissolved inorganic carbon decreases from 8 per thousand to -13 per thousand, indicating a shift from methanogenesis to methane oxidation. This change in microbial activity along the plume axis is confirmed by PhyloChip microarray analyses of 16S rRNA genes obtained from groundwater microbial communities, which indicate decreasing abundances of reductive dechlorinating microorganisms (e.g., Dehalococcoides ethenogenes) and increasing CH(4)-oxidizing microorganisms capable of aerobic co-metabolism of TCE (e.g., Methylosinus trichosporium). Incubation experiments with (13)C-labeled TCE introduced into microcosms containing basalt and groundwater from the aquifer confirm that TCE co-metabolism is possible. The results of these studies indicate that electron donor amendment designed to stimulate reductive dechlorination of TCE may also stimulate co-metabolism of TCE.

Incomplete Wood–Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi

Significance We have studied the functionality of an incomplete acetyl-CoA “Wood–Ljungdahl” pathway in a strictly organohalide-respiring bacterium, Dehalococcoides mccartyi. We found that in addition to its ability to incorporate exogenous formate, this pathway cleaves acetyl-CoA to generate methyl-tetrahydrofolate for methionine biosynthesis, serving as a unique substitute of the missing methylene-tetrahydrofolate reductase function. We also found that accumulation of carbon monoxide (CO), an obligate by-product from acetyl-CoA cleavage, inhibits D. mccartyi axenic cultures, but can be ameliorated by the presence of a CO-oxidizing organism, resulting in an unusual syntrophic association. The understanding of the products and biosynthetic functions of this incomplete Wood–Ljungdahl pathway improves our knowledge of alternate central metabolic strategies used by environmental microorganisms. The acetyl-CoA “Wood–Ljungdahl” pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood–Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood–Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities.

Carbon isotope fractionation during abiotic reductive dehalogenation of trichloroethene (TCE)

Dehalogenation of trichloroethene (TCE) in the aqueous phase, either on palladium catalysts with hydrogen as the reductant or on metallic iron, was associated with strong changes in delta13C. In general, the delta13C of product phases were more negative than those of the parent compound and were enriched with time and fraction of TCE remaining. For dehalogenation with iron, the delta13C of TCE and products varied from -42/1000 to +5/1000. For the palladium experiments, the final product, ethane, reached the initial delta13C of TCE at completion of the dehalogenation reaction. During dehalogenation, the carbon isotope fractionation between TCE and product phases was not constant. The variation in delta13C of TCE and products offers a new monitoring tool that operates independently of the initial concentration of pollutants for abiotic degradation processes of TCE in the subsurface, and may be useful for evaluation of remediation efficiency.

Carbon isotope fractionation during reductive dechlorination of TCE in batch experiments with iron samples from reactive barriers

Reductive dechlorination of trichloroethene (TCE) by zero-valent iron produces a systematic enrichment of 13C in the remaining substrate that can be described using a Rayleigh model. In this study, fractionation factors for TCE dechlorination with iron samples from two permeable reactive barriers (PRBs) were established in batch experiments. Samples included original unused iron as well as material from a barrier in Belfast after almost 4 years of operation. Despite the variety of samples, carbon isotope fractionations of TCE were remarkably similar and seemed to be independent of iron origin, reaction rate, and formation of precipitates on the iron surfaces. The average enrichment factor for all experiments was -10.1 per thousand (+/- 0.4 per thousand). These results indicate that the enrichment factor provides a powerful tool to monitor the reaction progress, and thus the performance, of an iron-reactive barrier over time. The strong fractionation observed may also serve as a tool to distinguish between insufficient residence time in the wall and a possible bypassing of the wall by the plume, which should result in an unchanged isotopic signature of the TCE. Although further work is necessary to apply this stable isotope method in the field, it has potential to serve as a unique monitoring tool for PRBs based on zero-valent iron.

Identification and characterization of high methane-emitting abandoned oil and gas wells

Significance Millions of abandoned oil and gas wells exist across the United States and around the world. Our study analyzes historical and new field datasets to quantify the number of abandoned wells in Pennsylvania, individual and cumulative methane emissions, and the attributes that help explain these emissions. We show that (i) methane emissions from abandoned wells persist over multiple years and likely decades, (ii) high emitters appear to be unplugged gas wells and plugged/vented gas wells, as required in coal areas, and (iii) the number of abandoned wells may be as high as 750,000 in Pennsylvania alone. Knowing the attributes of high emitters will lead to cost-effective mitigation strategies that target high methane-emitting wells. Recent measurements of methane emissions from abandoned oil/gas wells show that these wells can be a substantial source of methane to the atmosphere, particularly from a small proportion of high-emitting wells. However, identifying high emitters remains a challenge. We couple 163 well measurements of methane flow rates; ethane, propane, and n-butane concentrations; isotopes of methane; and noble gas concentrations from 88 wells in Pennsylvania with synthesized data from historical documents, field investigations, and state databases. Using our databases, we (i) improve estimates of the number of abandoned wells in Pennsylvania; (ii) characterize key attributes that accompany high emitters, including depth, type, plugging status, and coal area designation; and (iii) estimate attribute-specific and overall methane emissions from abandoned wells. High emitters are best predicted as unplugged gas wells and plugged/vented gas wells in coal areas and appear to be unrelated to the presence of underground natural gas storage areas or unconventional oil/gas production. Repeat measurements over 2 years show that flow rates of high emitters are sustained through time. Our attribute-based methane emission data and our comprehensive estimate of 470,000–750,000 abandoned wells in Pennsylvania result in estimated state-wide emissions of 0.04–0.07 Mt (1012 g) CH4 per year. This estimate represents 5–8% of annual anthropogenic methane emissions in Pennsylvania. Our methodology combining new field measurements with data mining of previously unavailable well attributes and numbers of wells can be used to improve methane emission estimates and prioritize cost-effective mitigation strategies for Pennsylvania and beyond.

Temperature and injection water source influence microbial community structure in four Alaskan North Slope hydrocarbon reservoirs.

A fundamental knowledge of microbial community structure in petroleum reservoirs can improve predictive modeling of these environments. We used hydrocarbon profiles, stable isotopes, and high-density DNA microarray analysis to characterize microbial communities in produced water from four Alaskan North Slope hydrocarbon reservoirs. Produced fluids from Schrader Bluff (24–27°C), Kuparuk (47–70°C), Sag River (80°C), and Ivishak (80–83°C) reservoirs were collected, with paired soured/non-soured wells sampled from Kuparuk and Ivishak. Chemical and stable isotope data suggested Schrader Bluff had substantial biogenic methane, whereas methane was mostly thermogenic in deeper reservoirs. Acetoclastic methanogens (Methanosaeta) were most prominent in Schrader Bluff samples, and the combined δD and δ13C values of methane also indicated acetoclastic methanogenesis could be a primary route for biogenic methane. Conversely, hydrogenotrophic methanogens (e.g., Methanobacteriaceae) and sulfide-producing Archaeoglobus and Thermococcus were more prominent in Kuparuk samples. Sulfide-producing microbes were detected in all reservoirs, uncoupled from souring status (e.g., the non-soured Kuparuk samples had higher relative abundances of many sulfate-reducers compared to the soured sample, suggesting sulfate-reducers may be living fermentatively/syntrophically when sulfate is limited). Sulfate abundance via long-term seawater injection resulted in greater relative abundances of Desulfonauticus, Desulfomicrobium, and Desulfuromonas in the soured Ivishak well compared to the non-soured well. In the non-soured Ivishak sample, several taxa affiliated with Thermoanaerobacter and Halomonas predominated. Archaea were not detected in the deepest reservoirs. Functional group taxa differed in relative abundance among reservoirs, likely reflecting differing thermal and/or geochemical influences.