Isotopic evidence of enhanced carbonate dissolution at a coal mine drainage site in Allegheny County, Pennsylvania, USA

18 O SO4 isotopic signatures of the mine drainage and the presence of presumptive SO 4 -reducing bacteria suggest that SO 4 reduction activity also contributes C depleted in 13 C isotope to the total DIC pool. With distance downstream from the mine portal, C isotope signatures in the drainage increased , accompanied by decreased total DIC concentrations and increased pH. These data are consistent with H 2 SO 4 dissolution of carbonate rocks, enhanced by cation exchange, and C release to the atmosphere via CO 2 outgassing

Isotopic evidence of enhanced carbonate dissolution at a coal mine drainage site in Allegheny County, Pennsylvania, USA

18 O SO4 isotopic signatures of the mine drainage and the presence of presumptive SO 4 -reducing bacteria suggest that SO 4 reduction activity also contributes C depleted in 13 C isotope to the total DIC pool. With distance downstream from the mine portal, C isotope signatures in the drainage increased , accompanied by decreased total DIC concentrations and increased pH. These data are consistent with H 2 SO 4 dissolution of carbonate rocks, enhanced by cation exchange, and C release to the atmosphere via CO 2 outgassing

Expanding And Refining A Model For Denitrification In Rhodobacter Sphaeroides 2.4.3

Rhodobacter sphaeroides strain 2.4.3 is a photoheterotroph of the αproteobacteria. It is metabolically versatile and in addition to anoxygenic photosynthesis and aerobic respiration, it is capable of denitrification. The denitrification system of strain 2.4.3 has been studied in detail by our lab. Here, we expand on our model for denitrification. The genome sequence of strain 2.4.3 revealed the presence of two periplasmic nitrate reductases. Using expression and mutant studies, we show that one of these enzymes is expressed aerobically and is involved in aerobic redox homeostasis. The second enzyme is expressed under low oxygen conditions and is the nitrate reductase involved in denitrification. We provide evidence for oxygen sensitivity of the denitrification regulator NnrR in a mutant lacking the high oxygen affinity cytochrome oxidase. NnrR is a member of the FNR/CFP family of transcriptional regulators that has been shown to regulate denitrification genes in response to the denitrification intermediate nitric oxide. Using the predicted DNA binding motif of NnrR we analyzed the genome for genes potentially under NnrR control. We identified five new genes and demonstrate that expression is dependent on NnrR. One of these genes encodes pseudoazurin, which is shown to be an electron donor to nitrite reductase along with a cytochrome. Two other genes identified encode NorEF, putative additional subunits of the nitric oxide reductase enzyme complex. Inactivation of these genes decreases denitrification activity and cells show greater sensitivity to nitric oxide. We also investigated denitrification-independent nitric oxide signaling and metabolism. The Sphaeroides Heme Protein (SHP) has been shown to have nitric oxide dioxygenase activity. Here, we show that shp is induced under stationary phase photo-growth and that the shp mutant is not impaired in growth under these conditions. Finally, we attempt to characterize a nitric oxide binding protein that may mediate signaling through a diguanylate cyclase. This may link nitric oxide into the cellular cyclic-diguanosine monophosphate signaling pathway

Identification, Functional Studies, and Genomic Comparisons of New Members of the NnrR Regulon in Rhodobacter sphaeroides▿

Analysis of the Rhodobacter sphaeroides 2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated paz, norEF, and cdgA, in the NnrR regulon, while the status of the fourth sequence, within the tat operon promoter, remains uncertain. nnrV, under control of a previously identified NnrR site, was also identified. paz encodes a pseudoazurin that is a donor of electrons to nitrite reductase. paz inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome c2 together reduced nitrite reduction. Inactivation of norEF reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of norEF increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of R. sphaeroides with norEF, even though all four of the strains whose genomes have been sequenced have the norCBQD operon and nnrR. norEF was shown to provide resistance to nitrite when it was mobilized into R. sphaeroides strain 2.4.1 containing nirK. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in R. sphaeroides revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions

Agrobacterium tumefaciens C58 Uses ActR and FnrN To Control nirK and nor Expression▿

Agrobacterium tumefaciens can grow anaerobically via denitrification. To learn more about how cells regulate production of nitrite and nitric oxide, experiments were carried out to identify proteins involved in regulating expression and activity of nitrite and nitric oxide reductase. Transcription of NnrR, required for expression of these two reductases, was found to be under control of FnrN. Insertional inactivation of the response regulator actR significantly reduced nirK expression and Nir activity but not nnrR expression. Purified ActR bound to the nirK promoter but not the nor or nnrR promoter. A putative ActR binding site was identified in the nirK promoter region using mutational analysis and an in vitro binding assay. A nirK promoter containing mutations preventing the binding of ActR showed delayed expression but eventually reached about 65% of the activity of an equivalent wild-type promoter lacZ fusion. Truncation of the nirK promoter revealed that truncation up to and within the ActR binding site reduced expression, but fragments lacking the ActR binding site and retaining the NnrR binding site showed expression as high as or higher than the full-length fragment. Additional experiments revealed that expression of paz, encoding the copper protein pseudoazurin, was highly reduced in the actR or fnrN mutants and that ActR binds to the paz promoter. Inactivation of paz reduced Nir activity by 55%. These results help explain why Nir activity is very low in the actR mutant even though a nirK promoter with mutations in the ActR binding site showed significant expression

Isotopic evidence of enhanced carbonate dissolution at a coal mine drainage site in Allegheny County, Pennsylvania, USA

Stable isotopes were used to determine the sources and fate of dissolved inorganic C (DIC) in the circumneutral pH drainage from an abandoned bituminous coal mine in western Pennsylvania. The C isotope signatures of DIC (δ{sup 13}C{sub DIC}) were intermediate between local carbonate and organic C sources, but were higher than those of contemporaneous Pennsylvanian age groundwaters in the region. This suggests a significant contribution of C enriched in {sup 13}C due to enhanced carbonate dissolution associated with the release of H{sub 2}SO{sub 4} from pyrite oxidation. The Sr isotopic signature of the drainage was similar to other regional mine waters associated with the same coal seam and reflected contributions from limestone dissolution and cation exchange with clay minerals. The relatively high δ{sup 34}S{sub SO4} and δ{sup 18}O{sub SO4} isotopic signatures of the mine drainage and the presence of presumptive SO{sub 4}-reducing bacteria suggest that SO{sub 4} reduction activity also contributes C depleted in {sup 13}C isotope to the total DIC pool. With distance downstream from the mine portal, C isotope signatures in the drainage increased, accompanied by decreased total DIC concentrations and increased pH. These data are consistent with H{sub 2}SO{sub 4} dissolution of carbonate rocks, enhanced by cation exchange, and C release to the atmosphere via CO{sub 2} outgassing

Temporal Changes in Microbial Ecology and Geochemistry in Produced Water from Hydraulically Fractured Marcellus Shale Gas Wells

Microorganisms play several important roles in unconventional gas recovery, from biodegradation of hydrocarbons to souring of wells and corrosion of equipment. During and after the hydraulic fracturing process, microorganisms are subjected to harsh physicochemical conditions within the kilometer-deep hydrocarbon-bearing shale, including high pressures, elevated temperatures, exposure to chemical additives and biocides, and brine-level salinities. A portion of the injected fluid returns to the surface and may be reused in other fracturing operations, a process that can enrich for certain taxa. This study tracked microbial community dynamics using pyrotag sequencing of 16S rRNA genes in water samples from three hydraulically fractured Marcellus shale wells in Pennsylvania, USA over a 328-day period. There was a reduction in microbial richness and diversity after fracturing, with the lowest diversity at 49 days. Thirty-one taxa dominated injected, flowback, and produced water communities, which took on distinct signatures as injected carbon and electron acceptors were attenuated within the shale. The majority (>90%) of the community in flowback and produced fluids was related to halotolerant bacteria associated with fermentation, hydrocarbon oxidation, and sulfur-cycling metabolisms, including heterotrophic genera <i>Halolactibacillus, Vibrio, Marinobacter, Halanaerobium,</i> and <i>Halomonas,</i> and autotrophs belonging to <i>Arcobacter</i>. Sequences related to halotolerant methanogenic genera <i>Methanohalophilus</i> and <i>Methanolobus</i> were detected at low abundance (<2%) in produced waters several months after hydraulic fracturing. Five taxa were strong indicators of later produced fluids. These results provide insight into the temporal trajectory of subsurface microbial communities after “fracking” and have important implications for the enrichment of microbes potentially detrimental to well infrastructure and natural gas fouling during this process

Microbial Community Changes in Hydraulic Fracturing Fluids and Produced Water from Shale Gas Extraction

Microbial communities associated with produced water from hydraulic fracturing are not well understood, and their deleterious activity can lead to significant increases in production costs and adverse environmental impacts. In this study, we compared the microbial ecology in prefracturing fluids (fracturing source water and fracturing fluid) and produced water at multiple time points from a natural gas well in southwestern Pennsylvania using 16S rRNA gene-based clone libraries, pyrosequencing, and quantitative PCR. The majority of the bacterial community in prefracturing fluids constituted aerobic species affiliated with the class <i>Alphaproteobacteria</i>. However, their relative abundance decreased in produced water with an increase in halotolerant, anaerobic/facultative anaerobic species affiliated with the classes <i>Clostridia</i>, <i>Bacilli</i>, <i>Gammaproteobacteria</i>, <i>Epsilonproteobacteria</i>, <i>Bacteroidia</i>, and <i>Fusobacteria</i>. Produced water collected at the last time point (day 187) consisted almost entirely of sequences similar to <i>Clostridia</i> and showed a decrease in bacterial abundance by 3 orders of magnitude compared to the prefracturing fluids and produced water samplesfrom earlier time points. Geochemical analysis showed that produced water contained higher concentrations of salts and total radioactivity compared to prefracturing fluids. This study provides evidence of long-term subsurface selection of the microbial community introduced through hydraulic fracturing, which may include significant implications for disinfection as well as reuse of produced water in future fracturing operations