Agricultural soil denitrifiers possess extensive nitrite reductase gene diversity.

Denitrification transforms nitrogen applied as fertilizer and emits N2 O, which is a potent greenhouse gas. Very little is known about the identities of abundant and active denitrifiers in agricultural soils. We coupled DNA stable-isotope probing (DNA-SIP) with flow-through reactors (FTRs) to detect active agricultural soil denitrifiers. The FTRs were incubated with nitrate and 13 C6 -glucose under anoxic conditions and sampled at multiple time points. Labelled DNA from active microorganisms was analyzed by 16S rRNA gene fingerprinting, amplicon and shotgun metagenomic sequencing. Taxonomic representation of heavy fractions was consistent across sites and time points, including Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus and Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas) and Actinobacteria (4%; Streptomycetaceae). Most nitrite-reductase reads from heavy DNA annotated to the copper-containing form (nirK). Assigned taxonomies of active denitrifiers based on reads matching the nirK gene were comparable to those obtained through nitric oxide (norB) and RNA polymerase (rpoB) annotations but not the nitrous oxide reductase gene (nosZ). Analysis of recovered metagenomes from heavy DNA demonstrated extensive nirK sequence family diversity, including novel taxonomic groups that are not captured by existing primers.

Applying Machine Learning to investigate metal isotope variations at the watershed scale: A case study with lithium isotopes across the Yukon River Basin.

Constraining the multiple climatic, lithological, topographic, and geochemical variables controlling isotope variations in large rivers is often challenging with standard statistical methods. Machine learning (ML) is an efficient method for analyzing multidimensional datasets, resolving correlated processes, and exploring relationships between variables simultaneously. We tested four ML algorithms to elucidate the controls of riverine δ7Li variations across the Yukon River Basin (YRB). We compiled (n = 102) and analyzed new samples (n = 21), producing a dataset of 123 river water samples collected across the basin during the summer including δ7Li and extracted environmental, climatological, and geological characteristics of the drainage area for each sample from open-access geospatial databases. The ML models were trained, tuned, and tested under multiple scenarios to avoid issues such as overfitting. Random Forests (RF) performed best at predicting δ7Li across the basin, with the median model explaining 62 % of the variance. The most important variables controlling δ7Li across the basin are elevation, lithology, and past glacial coverage, which ultimately influence weathering congruence. Riverine δ7Li has a negative dependence on elevation. This reflects congruent weathering in kinetically-limited mountain zones with short residence times. The consistent ranking of lithology, specifically igneous and metamorphic rock cover, as a top feature controlling riverine δ7Li modeled by the RFs is unexpected. Further study is required to validate this finding. Rivers draining areas that were extensively covered during the last glacial maximum tend to have lower δ7Li due to immature weathering profiles resulting in short residence times, less secondary mineral formation and therefore more congruent weathering. We demonstrate that ML provides a fast, simple, visualizable, and interpretable approach for disentangling key controls of isotope variations in river water. We assert that ML should become a routine tool, and present a framework for applying ML to analyze spatial metal isotope data at the catchment scale.

Prevalence of anaerobic ammonium-oxidizing bacteria in contaminated groundwater.

Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: anaerobic oxidation of ammonium and reduction of nitrite to form dinitrogen gas (N(2)). Anammox organisms appear to be widely distributed in natural and artificial environments. However, their roles in groundwater ammonium attenuation remain unclear and only limited biomarker-based data confirmed their presence prior to this study. We used complementary molecular and isotope-based methods to assess anammox diversity and activity occurring at three ammonium-contaminated groundwater sites: quantitative PCR, denaturing gradient gel electrophoresis, sequencing of 16S rRNA genes, and (15)N-tracer incubations. Here we show that anammox performing organisms were abundant bacterial community members. Although all sites were dominated by Candidatus Brocadia-like sequences, the community at one site was particularly diverse, possessing four of five known genera of anammox bacteria. Isotope data showed that anammox produced up to 18 and 36% of N(2) at these sites. By combining molecular and isotopic results we have demonstrated the diversity, abundance, and activity of these autotrophic bacteria. Our results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.

Quantifying natural source zone depletion at petroleum hydrocarbon contaminated sites: A comparison of 14C methods.

Surficial CO2 efflux surveys have been used to delineate hydrocarbon source zones in contaminated aquifers and provide estimates of hydrocarbon biodegradation rates. This approach requires distinguishing between CO2 derived from petroleum degradation and CO2 produced from natural soil respiration. To this end, radiocarbon has been used to differentiate between 14C-depleted CO2 from hydrocarbon degradation and 14C-enriched CO2 from natural soil respiration to effectively quantify the contribution of each source to total CO2 efflux, and by deduction natural source zone depletion (NSZD) rates. In this study, a systematic method comparison has been conducted to evaluate available approaches for collecting CO2 gas samples for radiocarbon analysis used to correct total CO2 efflux measurements for quantifying natural source zone depletion rates. Gas samples for radiocarbon analysis were sampled from (i) dynamic closed chambers (located at ground surface), (ii) static chambers (also at ground surface), (iii) shallow soil gas probes (0.3 m bgs), and (iv) soil gas monitoring wells (~0.6 m below ground surface) during a CO2 efflux survey conducted at the site of a historical pipeline rupture near Bemidji, MN. The mean fraction of radiocarbon (F14C) obtained from samples overlying the source zone were (i) 0.93 ± 0.01, (ii) 0.73 ± 0.03, (iii) 0.71 ± 0.04, and (iv) 0.41 ± 0.06, for the four methods respectively. These F14C values were used to apportion total CO2 efflux measurements into contributions of contaminant-derived CO2 efflux and natural soil respiration to evaluate natural source zone depletion processes. Results suggest that the method of radiocarbon sampling has a significant effect on the calculated fraction of the CO2 efflux originating from contaminant-related soil respiration, with contributions ranging between 27% and 59% of total soil respiration. Results indicate that radiocarbon sampled from static chambers and shallow soil gas probes methods offer the best compromise between CO2 sample yield and sample representativeness, providing the most reliable estimates of CO2 effluxes originating from contaminant degradation. However, the results also show that for this study, all methods agree within a factor of <2.3 regarding the inferred NSZD rates.

Sources of solutes and carbon cycling in perennially ice-covered Lake Untersee, Antarctica.

Perennially ice-covered lakes that host benthic microbial ecosystems are present in many regions of Antarctica. Lake Untersee is an ultra-oligotrophic lake that is substantially different from any other lakes on the continent as it does not develop a seasonal moat and therefore shares similarities to sub-glacial lakes where they are sealed to the atmosphere. Here, we determine the source of major solutes and carbon to Lake Untersee, evaluate the carbon cycling and assess the metabolic functioning of microbial mats using an isotope geochemistry approach. The findings suggest that the glacial meltwater recharging the closed-basin and well-sealed Lake Untersee largely determines the major solute chemistry of the oxic water column with plagioclase and alumino-silicate weathering contributing < 5% of the Ca2+-Na+ solutes to the lake. The TIC concentration in the lake is very low and is sourced from melting of glacial ice and direct release of occluded CO2 gases into the water column. The comparison of δ13CTIC of the oxic lake waters with the δ13C in the top microbial mat layer show no fractionation due to non-discriminating photosynthetic fixation of HCO3- in the high pH and carbon-starved water. The 14C results indicate that phototrophs are also fixing respired CO2 from heterotrophic metabolism of the underlying microbial mats layers. The findings provide insights into the development of collaboration in carbon partitioning within the microbial mats to support their growth in a carbon-starved ecosystem.

The seasonal fluctuations and accumulation of iodine-129 in relation to the hydrogeochemistry of the Wolf Creek Research Basin, a discontinuous permafrost watershed.

The long lived radioisotope (129)I is a uranium fission product, and an environmental contaminant of the nuclear age. Consequently, it can trace anthropogenic releases of (129)I in watersheds, and has been identified as a potential means to distinguish water sources in discharge (Nimz, 1998). The purpose of this work was to identify the sources and mass input of (129)I and trace the transport, partitioning and mass balance of (129)I over time in a remote watershed. We monitored (129)I and other geochemical and isotope tracers (e.g. δ(14)CDIC, δ(13)CDIC, δ(2)H, δ(18)O, etc.) in precipitation and discharge from the Wolf Creek Research Basin (WCRB), a discontinuous permafrost watershed in the Yukon Territory, Canada, and evaluated the use of (129)I as a water end-member tracer. Radiocarbon and geochemical tracers of weathering show that discharge is comprised of (i) groundwater baseflow that has recharged under open system conditions, (ii) spring freshet meltwater that has derived solutes through closed-system interaction with saturated soils, and (iii) active layer drainage. The abundance of (129)I and the (129)I/(127)I ratio correlated with geochemical tracers suggests varying contributions of these three water end-members to discharge. The (129)I concentration was highest at the onset of freshet, reaching 17.4×10(6) atoms/L, and likely reflects the lack of interaction between meltwater and organic matter at that time. This peak in (129)I was followed by a decline over the summer to its lowest value. Mass balance calculations of the (129)I budget show that the input to the watershed via precipitation is nearly one order of magnitude higher than the output suggesting that such arctic watersheds accumulate nearly 90% of the annual input, primarily in soil organic matter. Temporal variations in discharge (129)I concentrations correlated with changes in discharge water sources suggesting that (129)I is a promising hydrologic tracer, particularly when used in concert with other stable and radioisotopes.

Sources and circulation of water and arsenic in the Giant Mine, Yellowknife, NWT, Canada.

Recovery of gold from arsenopyrite-hosted ore in the Giant Mine camp, Yellowknife, NWT, Canada, has left a legacy of arsenic contamination that poses challenges for mine closure planning. Seepage from underground chambers storing some 237,000 tonnes of arsenic trioxide dust, has As concentrations exceeding 4000 ppm. Other potential sources and sinks of As also exist. Sources and movement of water and arsenic are traced using the isotopes of water and sulphate. Mine waters (16 ppm As; AsV/AsIII approximately 150) are a mixture of two principal water sources--locally recharged, low As groundwaters (0.5 ppm As) and Great Slave Lake (GSL; 0.004 ppm As) water, formerly used in ore processing and discharged to the northwest tailings impoundment (NWTP). Mass balance with delta18O shows that recirculation of NWTP water to the underground through faults and unsealed drillholes contributes about 60% of the mine water. Sulphate serves to trace direct infiltration to the As2O3 chambers. Sulphate in local, low As groundwaters (0.3-0.6 ppm As; delta34SSO4 approximately 4% and delta18OSO4 approximately -10%) originates from low-temperature aqueous oxidation of sulphide-rich waste rock. The high As waters gain a component of 18O-enriched sulphate derived from roaster gases (delta18OSO4) = + 3.5%), consistent with their arsenic source from the As2O3 chambers. High arsenic in NWTP water (approximately 8 ppm As; delta18OSO4 = -2%) derived from mine water, is attenuated to close to 1 ppm during infiltration back to the underground, probably by oxidation and sorption by ferrihydrite.

Extraction of (129)I and (127)I via combustion from organic rich samples using (125)I as a quantitative tracer.

Iodine-129 ((129)I) is a biophilic, naturally occurring radioisotope (half-life: 1.57 × 10(7) years) that has been released in large quantities by nuclear fuel reprocessing. This iodine has cycled throughout the globe and chiefly the northern hemisphere and can be found in a wide variety of environmental materials, particularly organic rich soil and organic matter. Extracting iodine reliably from solid samples has been done by a variety of methods, however, pyrohydrolysis has been the most widely used. There is a wide variation between existing pyrohydrolysis techniques and this raises questions about the quantitative recovery of iodine from method to method. In order to quantify iodine recovery from pyrohydrolysis we have spiked samples with an iodine-125 radiotracer prior to combustion and trapping in an alkaline solution. Inorganic (125)I tracer was used as well as humic acid labeled with (125)I to simulate the behavior of (129)I and (127)I in complex organic substances and extract iodine regardless of how it is partitioned. Using these tracers we explored the effect on recovery of (125)I under a variety of combustion parameters. These include carrier gas flow rate and iodine volatilization temperature. We observed that the best recoveries of (125)I were at flow rates between 400 and 800 mL/min and most (125)I recoveries were above 85%. The experiment to determine the temperature at which iodine volatilizes from the sample showed two distinct trends for the release of iodine. One trend showed that most iodine is released at approximately 525 °C, while the other trend showed that the samples needed to reach 800 °C and remain there for at least an hour. These findings illustrate the usefulness and importance of using a quantitative recovery tracer for every iodine extraction. We then combusted and precipitated several Atlantic Ocean seaweed and standard reference materials for AMS analysis as AgI. The (129)I concentration of the seaweed ranged between 4.4-5.5 × 10(9) atoms/g and the (129)I/(127)I ratio was 2.3-2.9 × 10(-9), both of which compare well to published values for Atlantic seaweed. The results for the standard reference materials also agree with specified values indicating that this technique is reliable. By optimizing pyrohydrolysis conditions and testing the recovery of iodine with a (125)I tracer it is possible to quantify and maximize recovery from organic samples. This will allow for the investigation of variations in the (129)I concentration and (129)I/(127)I ratio with a high degree of precision in complex, organic rich samples.

Microbial diversity in endostromatolites (cf. Fissure Calcretes) and in the surrounding permafrost landscape, Haughton impact structure region, Devon Island, Canada.

In recent years, endostromatolites, which consist of finely laminated calcite columns that grow orthogonally within millimeter- to centimeter-thick fissures in limestone bedrock outcrops, have been discovered in dolomitic outcrops in the Haughton impact structure region, Devon Island, Canada. The growth mechanism of the endostromatolites is believed to be very slow and possibly intertwined with biotic and abiotic processes. Therefore, to discern how endostromatolites form in this polar desert environment, the composition of the microbial community of endostromatolites was determined by means of molecular phylogenetic analysis and compared to the microbial communities found in the surrounding soils. The microbial community present within endostromatolites can be inferred to be (given the predominant metabolic traits of related organisms) mostly aerobic and chemoheterotrophic, and belongs in large part to the phylum Actinobacteria and the subphylum Alphaproteobacteria. The identification of these bacteria suggests that the conditions within the fissure were mostly oxidizing during the growth of endostromatolite. The DNA sequences also indicate that a number of bacteria that closely resemble Rubrobacter radiotolerans are abundant in the endostromatolites as well as other Actinobacteria and Alphaproteobacteria. Some of these taxa have been associated with calcite precipitation, which suggests that the endostromatolites might in fact be microbially mediated. Bacterial communities from nearby permanently frozen soils were more diverse and harbored all the phyla found in the endostromatolites with additional taxa. This study on the microbial communities preserved in potentially microbially mediated secondary minerals in the Arctic could help in the search for evidence of life-forms near the edge of habitability on other planetary bodies.

Sorption of strontium onto bacteriogenic iron oxides.

Bacteriogenic iron oxides (BIOS) were obtained from a dilute, circumneutral groundwater seep, characterized with respect to mineralogy, and examined for their ability to sorb aqueous Sr2+. BIOS were composed of microbial sheaths encrusted in 2-line ferrihydrite. Sorption experiments indicated that Sr remained completely unbound at pH < 4.5, but sorption increased with increasing pH (maximum of 95% at pH > 7.6). EXAFS analysis of Sr-loaded BIOS failed to elucidate whether Sr sorption occurred on sites specific to the mineral or microbial fraction, but indicated that sorption likely occurred by outer-sphere complexation between BIOS and hydrated Sr2+. Sorption experiments showed that at low ionic strength (I = 0.001 M), sorption followed a Langmuir isotherm (S(max) = 3.41 mol Sr (g of Fe)(1-), K(ads) = 1.26). At higher ionic strength (I = 0.1 M), there was significant inhibition of Sr sorption (S(max) = 1.06 mol Sr (g of Fe)(1-), K(ads) = 1.23), suggesting that sorption to BIOS occurs by outer-sphere complexation. The results suggest that, under dilute circumneutral conditions, BIOS deposits should efficiently sorb dissolved Sr from groundwater flow systems where such deposits exist. This finding has particular relevance to sites impacted by radioactive 90Sr groundwater contamination.

Using a simple high-performance liquid chromatography separation and fraction collection methodology to achieve compound-specific isotopic analysis for dissolved organic compounds.

A new application for the quantitative and isotopic analyses of dissolved inorganic and dissolved organic carbon compounds has been developed. Dissolved organic matter (DOM) in natural water samples can be separated on a high-performance liquid chromatography (HPLC) column and collected as fractions. Each discrete fraction can then be analyzed using the technique of St-Jean (Rapid Commun. Mass Spectrom. 2003; 17: 419-428) with a total inorganic carbon/total organic carbon (TIC/TOC) analyzer interfaced with a continuous-flow isotope ratio mass spectrometer. Experimental data using short-chain fatty acid standards (formic, acetic, and propionic acids) show that fraction recoveries of 100% are possible and that sample integrity is maintained. 13C-isotopic analyses of products prior to and subsequent to extraction and collection show no isotopic effects associated with the methodology, and errors are well within the accepted analytical uncertainty of the IRMS. Comparison of data from pure standards and organic-rich natural waters shows that quantitative analyses still need to be done with standards that more closely imitate the matrices of the samples, in order to acquire an appropriate calibration curve. Injections of organic-rich matrices on the HPLC column did not affect fraction recovery, nor did they create high background of partially retained organic compounds slowly released from the HPLC column, and hence 13C-isotopic results are relatively unaffected. The specific limitation on this methodology is the required use of carbon-free carrier solvents due to potential memory effects associated with the TIC/TOC analyzer. Further developments of this application could make routine compound-specific isotopic analyses (CSIA) for a wider range of organic materials possible.