To better understand reaction pathways of pyrite oxidation and biogeochemical controls on δ
18O and δ
34S values of the generated sulfate in acid mine drainage (AMD) and other natural environments, ...we conducted a series of pyrite oxidation experiments in the laboratory. Our biological and abiotic experiments were conducted under aerobic conditions by using O
2 as an oxidizing agent and under anaerobic conditions by using dissolved Fe(III)
aq as an oxidant with varying δ
18O
H
2O
values in the presence and absence of
Acidithiobacillus ferrooxidans. In addition, aerobic biological experiments were designed as short- and long-term experiments where the final pH was controlled at ∼2.7 and 2.2, respectively. Due to the slower kinetics of abiotic sulfide oxidation, the aerobic abiotic experiments were only conducted as long term with a final pH of ∼2.7. The δ
34S
SO
4
values from both the biological and abiotic anaerobic experiments indicated a small but significant sulfur isotope fractionation (∼−0.7‰) in contrast to no significant fractionation observed from any of the aerobic experiments. Relative percentages of the incorporation of water-derived oxygen and dissolved oxygen (O
2) to sulfate were estimated, in addition to the oxygen isotope fractionation between sulfate and water, and dissolved oxygen. As expected, during the biological and abiotic anaerobic experiments all of the sulfate oxygen was derived from water. The percentage incorporation of water-derived oxygen into sulfate during the oxidation experiments by O
2 varied with longer incubation and lower pH, but not due to the presence or absence of bacteria. These percentages were estimated as 85%, 92% and 87% from the short-term biological, long-term biological and abiotic control experiments, respectively. An oxygen isotope fractionation effect between sulfate and water
(
ε
18
O
SO
4
–
H
2
O
)
of ∼3.5‰ was determined for the anaerobic (biological and abiotic) experiments. This measured
ε
18
O
SO
4
2
-
–
H
2
O
value was then used to estimate the oxygen isotope fractionation effects
(
ε
18
O
SO
4
2
-
–
O
2
)
between sulfate and dissolved oxygen in the aerobic experiments which were −10.0‰, −10.8‰, and −9.8‰ for the short-term biological, long-term biological and abiotic control experiments, respectively. Based on the similarity between δ
18O
SO
4
values in the biological and abiotic experiments, it is suggested that δ
18O
SO
4
values cannot be used to distinguish biological and abiotic mechanisms of pyrite oxidation. The results presented here suggest that Fe(III)
aq is the primary oxidant for pyrite at pH
<
3, even in the presence of dissolved oxygen, and that the main oxygen source of sulfate is water–oxygen under both aerobic and anaerobic conditions.
Bacteria belonging to the newly classified candidate phylum "Atribacteria" (formerly referred to as "OP9" and "JS1") are common in anoxic methane-rich sediments. However, the metabolic functions and ...biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica's Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
Interpretation of the origins of iron-bearing minerals preserved in modern and ancient rocks based on measured iron isotope ratios depends on our ability to distinguish between biological and ...non-biological iron isotope fractionation processes. In this study, we compared
56Fe/
54Fe ratios of coexisting aqueous iron (Fe(II)
aq, Fe(III)
aq) and iron oxyhydroxide precipitates (Fe(III)
ppt) resulting from the oxidation of ferrous iron under experimental conditions at low pH (<3). Experiments were carried out using both pure cultures of
Acidothiobacillus ferrooxidans and sterile controls to assess possible biological overprinting of non-biological fractionation, and both SO
4
2− and Cl
− salts as Fe(II) sources to determine possible ionic/speciation effects that may be associated with oxidation/precipitation reactions. In addition, a series of ferric iron precipitation experiments were performed at pH ranging from 1.9 to 3.5 to determine if different precipitation rates cause differences in the isotopic composition of the iron oxyhydroxides. During microbially stimulated Fe(II) oxidation in both the sulfate and chloride systems,
56Fe/
54Fe ratios of residual Fe(II)
aq sampled in a time series evolved along an apparent Rayleigh trend characterized by a fractionation factor
α
Fe(III)aq–Fe(II)aq
∼
1.0022. This fractionation factor was significantly less than that measured in our sterile control experiments (∼1.0034) and that predicted for isotopic equilibrium between Fe(II)
aq and Fe(III)
aq (∼1.0029), and thus might be interpreted to reflect a biological isotope effect. However, in our biological experiments the measured difference in
56Fe/
54Fe ratios between Fe(III)
aq, isolated as a solid by the addition of NaOH to the final solution at each time point under N
2-atmosphere, and Fe(II)
aq was in most cases and on average close to 2.9‰ (
α
Fe(III)aq–Fe(II)aq
∼
1.0029), consistent with isotopic equilibrium between Fe(II)
aq and Fe(III)
aq. The ferric iron precipitation experiments revealed that
56Fe/
54Fe ratios of Fe(III)
aq were generally equal to or greater than those of Fe(III)
ppt, and isotopic fractionation between these phases decreased with increasing precipitation rate and decreasing grain size. Considered together, the data confirm that the iron isotope variations observed in our microbial experiments are primarily controlled by non-biological equilibrium and kinetic factors, a result that aids our ability to interpret present-day iron cycling processes but further complicates our ability to use iron isotopes alone to identify biological processing in the rock record.
Despite accounting for the majority of sedimentary methane, the physiology and relative abundance of subsurface methanogens remain poorly understood. We combined intact polar lipid and metagenome ...techniques to better constrain the presence and functions of methanogens within the highly reducing, organic-rich sediments of Antarctica's Adélie Basin. The assembly of metagenomic sequence data identified phylogenic and functional marker genes of methanogens and generated the first Methanosaeta sp. genome from a deep subsurface sedimentary environment. Based on structural and isotopic measurements, glycerol dialkyl glycerol tetraethers with diglycosyl phosphatidylglycerol head groups were classified as biomarkers for active methanogens. The stable carbon isotope (δ
C) values of these biomarkers and the Methanosaeta partial genome suggest that these organisms are acetoclastic methanogens and represent a relatively small (0.2%) but active population. Metagenomic and lipid analyses suggest that Thaumarchaeota and heterotrophic bacteria co-exist with Methanosaeta and together contribute to increasing concentrations and δ
C values of dissolved inorganic carbon with depth. This study presents the first functional insights of deep subsurface Methanosaeta organisms and highlights their role in methane production and overall carbon cycling within sedimentary environments.
Laboratory experiments were conducted to simulate chalcopyrite oxidation under anaerobic and aerobic conditions in the absence or presence of the bacterium
Acidithiobacillus ferrooxidans. Experiments ...were carried out with 3 different oxygen isotope values of water (
δ
18O
H2O) so that approach to equilibrium or steady-state isotope fractionation for different starting conditions could be evaluated. The contribution of dissolved O
2 and water-derived oxygen to dissolved sulfate formed by chalcopyrite oxidation was unambiguously resolved during the aerobic experiments. Aerobic oxidation of chalcopyrite showed 93
±
1% incorporation of water oxygen into the resulting sulfate during the biological experiments. Anaerobic experiments showed similar percentages of water oxygen incorporation into sulfate, but were more variable. The experiments also allowed determination of sulfate–water oxygen isotope fractionation,
ε
18O
SO4–H2O, of ~
3.8‰ for the anaerobic experiments. Aerobic oxidation produced apparent ε
SO4–H2O values (6.4‰) higher than the anaerobic experiments, possibly due to additional incorporation of dissolved O
2 into sulfate.
δ
34S
SO4 values are ~
4‰ lower than the parent sulfide mineral during anaerobic oxidation of chalcopyrite, with no significant difference between abiotic and biological processes. For the aerobic experiments, a small depletion in
δ
34S
SO4 of ~−
1.5
±
0.2‰ was observed for the biological experiments. Fewer solids precipitated during oxidation under aerobic conditions than under anaerobic conditions, which may account for the observed differences in sulfur isotope fractionation under these contrasting conditions.
Sulfide-mediated anoxygenic photosynthesis (SMAP) carried out by anaerobic phototrophic bacteria may have played an important role in sulfur cycling, formation of sulfate, and, perhaps, primary ...production in the Earth’s early oceans. Determination of ε34SSO4-Sulfide- and ε18OSO4-H2O values for bacterial sulfide oxidation will permit more refined interpretation of the δ34S and δ18OSO4 values measured in modern anoxic environments, such as meromictic lakes where sulfide commonly extends into the photic zone, and in the ancient rock record, particularly during periods of the Precambrian when anoxic and sulfidic (euxinic) conditions were believed to be more pervasive than today. Laboratory experiments with anaerobic purple and green sulfur phototrophs, Allochromatium vinosum and Chlorobaculum tepidum, respectively, were conducted to determine the sulfur and oxygen isotope fractionation during the oxidation of sulfide to sulfate. Replicate experiments were conducted at 25°C for A. vinosum and 45°C for C. tepidum, and in duplicate at three different starting oxygen isotope values for water to determine sulfate-water oxygen isotope fractionations accurately (ε18OSO4-H2O). ε18OSO4-H2O values of 5.6±0.2‰ and 5.4±0.1‰ were obtained for A. vinosum and C. tepidum, respectively. Temperature had no apparent effect on the ε18OSO4-H2O values. By combining all data from both cultures, an average ε18OSO4-H2O value of 5.6±0.3‰ was obtained for SMAP. This value falls between those previously reported for bacterial oxidation of sphalerite and elemental sulfur (7–9‰) and abiotic and biotic oxidation of pyrite and chalcopyrite (2–4‰). Sulfur isotope fractionation between sulfide and sulfate formed by A.vinosum was negligible (0.1±0.2‰) during all experiments. For C. tepidum an apparent fractionation of −2.3±0.5‰ was observed during the earlier stages of oxidation based on bulk δ34S measurements of sulfate and sulfide and became smaller (−0.7±0.3‰) when sulfate concentrations rose above 0.5mM and sulfide concentrations had became negligible.
Vast reserves of coal represent a largely untapped resource that can be used to produce methane gas, a cleaner energy alternative compared to burning oil or coal. Microorganisms are able to utilize ...coal as a carbon source, producing biogenic methane. The conversion of coal to methane by microorganisms has been demonstrated experimentally by a number of research groups, but coal handling and treatment prior to incubation often goes unreported and may impact biogenic methane production. Microcosm experiments were designed to assess how prior exposure of coal to oxygen might influence methane production (e.g., as in a dewatered coal-bed natural gas system). Microcosms containing oxidized and un-oxidized coal samples from the Powder River Basin were incubated with and without inoculation with an enrichment culture derived from coal. Gas chromatography and pyrosequencing of the 16S rRNA gene were used to assess how coal oxidation affects methane production and microbial community structure within microcosm samples. Although the magnitude of methane production differed between experiments, the oxidized coal microcosms consistently produced between 50 and 100 micromoles less methane per gram of coal than the un-oxidized microcosms. Additionally, un-inoculated microcosms produced levels of methane comparable to their inoculated counterparts, demonstrating the importance of native, coal-associated microbial assemblages in biogenic methane production. Specific methanogens were identified in the different treatments and their relative prevalence supported the relative level of methane production. Common coal-associated bacterial groups such as δ- and γ-Proteobacteria, Spirochaetes and Firmicutes were prevalent in different microcosms, though the presence of specific bacteria was not correlated with methane production. These data suggest that while coal oxidation decreased methane production, oxidation was not a primary factor in the variation between microcosm community structures.
•In microcosm experiments, coal oxidation resulted in decreased methane production.•Native microbial assemblages were more important to methane formation than inocula.•Methanogen prevalence reflected extent of methane generation.•Variable bacterial prevalence verified need for diverse microbes to degrade coal.
A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. ...Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This "Henderson candidate division" dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and FIRMICUTES: Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.
Independent soil microcosm experiments were used to investigate the effects of the fungicides mancozeb and chlorothalonil, and the herbicide prosulfuron, on N
2O and NO production by nitrifying and ...denitrifying bacteria in fertilized soil. Soil cores were amended with NH
4NO
3 or NH
4NO
3 and pesticide, and the N
2O and NO concentrations were monitored periodically for approximately 48
h following amendment. Nitrification is the major source of N
2O and NO in these soils at soil moistures relevant to those observed at the field site where the cores were collected. At pesticide concentrations from 0.02 to 10 times that of a standard single application on a corn crop, N
2O and NO production was inhibited by all three pesticides. Generally N
2O production was inhibited by the pesticides from 10 to 62% and 20 to 98% at the lowest and highest dosages, respectively. Nitric oxide production was generally inhibited from about 5 to 47% and by 20 to 97% at the lowest and highest dosages, respectively. Nitrous oxide and nitric oxide production by nitrification was more susceptible to inhibition by these pesticides than denitrification. Production of both N
2O and NO by nitrification was inhibited by as much as 99%, at the highest concentration of pesticide applied. The net production of N
2O increased as soil moisture increased. The rate of NO production was greatest at the intermediate moistures investigated, between 14 and 19% gravimetric soil moisture, suggestive that nitrification is the dominant source of NO.
A series of carefully controlled laboratory studies was carried out to investigate oxygen and iron isotope fractionation during the intracellular production of magnetite (Fe$_3$O$_4$) by two ...different species of magnetotactic bacteria at temperatures between 4° and 35°C under microaerobic and anaerobic conditions. No detectable fractionation of iron isotopes in the bacterial magnetites was observed. However, oxygen isotope measurements indicated a temperature-dependent fractionation for Fe$_3$O$_4$ and water that is consistent with that observed for Fe$_3$O$_4$ produced extracellularly by thermophilic Fe$^{3+}$-reducing bacteria. These results contrast with established fractionation curves estimated from either high-temperature experiments or theoretical calculations. With the fractionation curve established in this report, oxygen-18 isotope values of bacterial Fe$_3$O$_4$ may be useful in paleoenvironmental studies for determining the oxygen-18 isotope values of formation waters and for inferring paleotemperatures.
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