Oxygen Isotope Analysis of Nanomole Phosphate Using PO3- Fragment in ESI-Orbitrap-MS.

The oxygen isotope composition of phosphate is a useful tool for studying biogeochemical phosphorus cycling. However, the current Ag3PO4 method is not only tedious in PO43- extraction and purification but also requires a large-sized sample at the micromole level, thereby limiting its application. Here, we present an approach to measuring the oxygen isotope composition, δ18O, of dissolved phosphate at the nanomole level using electrospray ionization Orbitrap mass spectrometry (ESI-Orbitrap-MS). We compared the reproducibility of δ18O measurements using the H2PO4- ions (m/z = 97 and 99 for H2P16O4- and H2P18O16O3-, respectively) and using the PO3- fragment ions (m/z = 79 and 81 for P16O3- and P18O16O2-, respectively) generated by source fragmentation and by higher-energy collisional dissociation, respectively. The results demonstrate that phosphate δ18O can be more reliably measured by the PO3- ions than by the H2PO4- ions. PO3- generated by source fragmentation at 40 V achieved the highest reproducibility for δ18O based on precision tests. Furthermore, the mass spectrum for a 50:50 µM mixed solution of phosphate and sulfate revealed that PO3- ions resulting from source fragmentation at 40 V are the predominant species in the Orbitrap analyzer. Notably, P16O3- ions (m/z: 79) are not interfered with by 32S16O3- (m/z: 80) ions. This is in contrast to the case for 1H2P16O4- ions, which share the same m/z value with 1H32S16O4- ions and exhibit much lower signal intensity than HSO4- ions. Using the PO3- fragment method and six phosphate standards with a wide range of δ18O values, we obtained a calibration line with a slope of 0.94 (R2 = 0.98). The overall uncertainty for ESI-Orbitrap-MS phosphate δ18O measurement was 0.8‰ (n = 30; 1 SD). With much room for improvement, the PO3- fragment method presents a better approach to measuring the phosphate oxygen isotope composition, applicable to nanomole sample sizes in a liquid phase.

Sulfur assimilation using gaseous carbonyl sulfide by the soil fungus Trichoderma harzianum.

Fungi have the capacity to assimilate a diverse range of both inorganic and organic sulfur compounds. It has been recognized that all sulfur sources taken up by fungi are in soluble forms. In this study, we present evidence that fungi can utilize gaseous carbonyl sulfide (COS) for the assimilation of a sulfur compound. We found that the filamentous fungus Trichoderma harzianum strain THIF08, which has constitutively high COS-degrading activity, was able to grow with COS as the sole sulfur source. Cultivation with 34S-labeled COS revealed that sulfur atom from COS was incorporated into intracellular metabolites such as glutathione and ergothioneine. COS degradation by strain THIF08, in which as much of the moisture derived from the agar medium as possible was removed, indicated that gaseous COS was taken up directly into the cell. Escherichia coli transformed with a COS hydrolase (COSase) gene, which is clade D of the ß-class carbonic anhydrase subfamily enzyme with high specificity for COS but low activity for CO2 hydration, showed that the COSase is involved in COS assimilation. Comparison of sulfur metabolites of strain THIF08 revealed a higher relative abundance of reduced sulfur compounds under the COS-supplemented condition than the sulfate-supplemented condition, suggesting that sulfur assimilation is more energetically efficient with COS than with sulfate because there is no redox change of sulfur. Phylogenetic analysis of the genes encoding COSase, which are distributed in a wide range of fungal taxa, suggests that the common ancestor of Ascomycota, Basidiomycota, and Mucoromycota acquired COSase at about 790-670 Ma.IMPORTANCEThe biological assimilation of gaseous CO2 and N2 involves essential processes known as carbon fixation and nitrogen fixation, respectively. In this study, we found that the fungus Trichoderma harzianum strain THIF08 can grow with gaseous carbonyl sulfide (COS), the most abundant and ubiquitous gaseous sulfur compound, as a sulfur source. When the fungus grew in these conditions, COS was assimilated into sulfur metabolites, and the key enzyme of this assimilation process is COS hydrolase (COSase), which specifically degrades COS. Moreover, the pathway was more energy efficient than the typical sulfate assimilation pathway. COSase genes are widely distributed in Ascomycota, Basidiomycota, and Mucoromycota and also occur in some Chytridiomycota, indicating that COS assimilation is widespread in fungi. Phylogenetic analysis of these genes revealed that the acquisition of COSase in filamentous fungi was estimated to have occurred at about 790-670 Ma, around the time that filamentous fungi transitioned to a terrestrial environment.

Isotopic Evidence for Microbial Nitrogen Cycling in a Glacier Interior of High-Mountain Asia.

Glaciers are now acknowledged as an important biome globally, but biological processes in the interior of the glacier (englacial) are thought to be slow and to play only a minor role in biogeochemical cycles. In this study, we demonstrate extensive, microbially driven englacial nitrogen cycling in an Asian glacier using the stable isotopes (δ15N, δ18O, and Δ17O values) of nitrate. Apparent decreases in Δ17O values of nitrate in an 8 m shallow firn core from the accumulation area indicate that nitrifiers gradually replaced ∼80% of atmospheric nitrate with nitrate from microbial nitrification on a decadal scale. Nitrate concentrations did not increase with depth in this core, suggesting the presence of nitrate sinks by microbial assimilation and denitrification within the firn layers. The estimated englacial metabolic rate using isotopic mass balance was classified as growth metabolism, which is approximately 2 orders of magnitude more active than previously known cold-environment metabolisms. In a 56 m ice core from the interior of the ablation area, we found less nitrification but continued microbial nitrate consumption, implying that organic matter is microbially accumulated over centuries before appearing on the ablating surface. Such englacial microbial products may support supraglacial microbes, potentially promoting glacial darkening and melting. With predicted global warming and higher nitrogen loads, englacial nutrient cycling and its roles may become increasingly important in the future.

Discovering Nature's Fingerprints: Isotope Ratio Analysis on Bioanalytical Mass Spectrometers.

For a generation or more, the mass spectrometry that developed at the frontier of molecular biology was worlds apart from isotope ratio mass spectrometry, a label-free approach done on optimized gas-source magnetic sector instruments. Recent studies show that electrospray-ionization Orbitraps and other mass spectrometers widely used in the life sciences can be fine-tuned for high-precision isotope ratio analysis. Since isotope patterns form everywhere in nature based on well-understood principles, intramolecular isotope measurements allow unique insights into a fascinating range of research topics. This Perspective introduces a wider readership to current topics in stable isotope research with the aim of discussing how soft-ionization mass spectrometry coupled with ultrahigh mass resolution can enable long-envisioned progress. We highlight novel prospects of observing isotopes in intact polar compounds and speculate on future directions of this adventure into the overlapping realms of biology, chemistry, and geology.

Role of Dust and Iron Solubility in Sulfate Formation during the Long-Range Transport in East Asia Evidenced by 17O-Excess Signatures.

Numerical models have been developed to elucidate air pollution caused by sulfate aerosols (SO42-). However, typical models generally underestimate SO42-, and oxidation processes have not been validated. This study improves the modeling of SO42- formation processes using the mass-independent oxygen isotopic composition [17O-excess; Δ17O(SO42-)], which reflects pathways from sulfur dioxide (SO2) to SO42-, at the background site in Japan throughout 2015. The standard setting in the Community Multiscale Air Quality (CMAQ) model captured SO42- concentration, whereas Δ17O(SO42-) was underestimated, suggesting that oxidation processes were not correctly represented. The dust inline calculation improved Δ17O(SO42-) because dust-derived increases in cloud-water pH promoted acidity-driven SO42- production, but Δ17O(SO42-) was still overestimated during winter as a result. Increasing solubilities of the transition-metal ions, such as iron, which are a highly uncertain modeling parameter, decreased the overestimated Δ17O(SO42-) in winter. Thus, dust and high metal solubility are essential factors for SO42- formation in the region downstream of China. It was estimated that the remaining mismatch of Δ17O(SO42-) between the observation and model can be explained by the proposed SO42- formation mechanisms in Chinese pollution. These accurately modeled SO42- formation mechanisms validated by Δ17O(SO42-) will contribute to emission regulation strategies required for better air quality and precise climate change predictions over East Asia.

Sunlight-driven nitrate loss records Antarctic surface mass balance.

Standard proxies for reconstructing surface mass balance (SMB) in Antarctic ice cores are often inaccurate or coarsely resolved when applied to more complicated environments away from dome summits. Here, we propose an alternative SMB proxy based on photolytic fractionation of nitrogen isotopes in nitrate observed at 114 sites throughout East Antarctica. Applying this proxy approach to nitrate in a shallow core drilled at a moderate SMB site (Aurora Basin North), we reconstruct 700 years of SMB changes that agree well with changes estimated from ice core density and upstream surface topography. For the under-sampled transition zones between dome summits and the coast, we show that this proxy can provide past and present SMB values that reflect the immediate local environment and are derived independently from existing techniques.

Isotopic evidence for acidity-driven enhancement of sulfate formation after SO2 emission control.

After the 1980s, atmospheric sulfate reduction is slower than the dramatic reductions in sulfur dioxide (SO2) emissions. However, a lack of observational evidence has hindered the identification of causal feedback mechanisms. Here, we report an increase in the oxygen isotopic composition of sulfate ([Formula: see text]) in a Greenland ice core, implying an enhanced role of acidity-dependent in-cloud oxidation by ozone (up to 17 to 27%) in sulfate production since the 1960s. A global chemical transport model reproduces the magnitude of the increase in observed [Formula: see text] with a 10 to 15% enhancement in the conversion efficiency from SO2 to sulfate in Eastern North America and Western Europe. With an expected continued decrease in atmospheric acidity, this feedback will continue in the future and partially hinder air quality improvements.

A GC-IRMS method for measuring sulfur isotope ratios of carbonyl sulfide from small air samples.

A new system was developed for measuring sulfur isotopes δ 33S and δ 34S from atmospheric carbonyl sulfide (COS) on small air samples of several liters, using pre-concentration and gas chromatography - isotope ratio mass spectrometry (GC-IRMS). Measurements of COS isotopes provide a tool for quantifying the COS budget, which will help towards better understanding climate feedback mechanisms. For a 4 liter sample at ambient COS mixing ratio, ~500 parts per trillion (ppt), we obtain a reproducibility error of 2.1 ‰ for δ 33S and 0.4 ‰ for δ 34S. After applying corrections, the uncertainty for an individual ambient air sample measurement is 2.5 ‰ for δ 33S and 0.9 ‰ for δ 34S. The ability to measure small samples allows application to a global-scale sampling program with limited logistical effort. To illustrate the application of this newly developed system, we present a timeseries of ambient air measurements, during the fall and winter of 2020 and 2021 in Utrecht, the Netherlands. The observed background values were δ 33S = 1.0 ± 3.4 ‰ and δ 34S = 15.5 ± 0.8 ‰ (VCDT). The maximum observed COS mixing ratios was only 620 ppt. This, in combination with the relatively high δ 34S suggests that the Netherlands receives little COS-containing anthropogenic emissions. We observed a change in COS mixing ratio and δ 34S with different air mass origin, as modelled with HYSPLIT backward trajectory analyses. An increase of 40 ppt in mean COS mixing ratio was observed between fall and winter, which is consistent with the expected seasonal cycle in the Netherlands. Additionally, we present the results of samples from a highway tunnel to characterize vehicle COS emissions and isotopic composition. The vehicle emissions were small, with COS/CO 2 being 0.4 ppt/ppm; the isotopic signatures are depleted relatively to background atmospheric COS.

Development of Microdroplet Generation Method for Organic Solvents Used in Chemical Synthesis.

Recently, chemical operations with microfluidic devices, especially droplet-based operations, have attracted considerable attention because they can provide an isolated small-volume reaction field. However, analysis of these operations has been limited mostly to aqueous-phase reactions in water droplets due to device material restrictions. In this study, we have successfully demonstrated droplet formation of five common organic solvents frequently used in chemical synthesis by using a simple silicon/glass-based microfluidic device. When an immiscible liquid with surfactant was used as the continuous phase, the organic solvent formed droplets similar to water-in-oil droplets in the device. In contrast to conventional microfluidic devices composed of resins, which are susceptible to swelling in organic solvents, the developed microfluidic device did not undergo swelling owing to the high chemical resistance of the constituent materials. Therefore, the device has potential applications for various chemical reactions involving organic solvents. Furthermore, this droplet generation device enabled control of droplet size by adjusting the liquid flow rate. The droplet generation method proposed in this work will contribute to the study of organic reactions in microdroplets and will be useful for evaluating scaling effects in various chemical reactions.

Temperature control on wastewater and downstream nitrous oxide emissions in an urbanized river system.

Although eutrophic urban rivers receiving loads of wastewater represent an important anthropogenic source of N2O, little is known as to how temperature and other environmental factors affect temporal variations in N2O emissions from wastewater treatment plants (WWTPs) and downstream rivers. Two-year monitoring at a WWTP and five river sites was complemented with available water quality data, laboratory incubations, and stable isotopes in N2O and NO3- to explore how wastewater effluents interact with seasonal changes in environmental conditions to affect downstream metabolic processes and N2O emissions from the lower Han River traversing the megacity Seoul. Water quality data from four WWTPs revealed significant inverse relationships between water temperature and the concentrations or fluxes of total N (TN) in effluents. Increased TN fluxes at low temperatures concurred with N2O surges in WWTP effluents and downstream rivers, counteracting the long-term decline in TN fluxes resulting from enhanced wastewater treatments. Incubation experiments with river water and sediment, in isolation or combined, implied the hypoxic winter sediment as a large source of N2O, whereas the anoxic summer sediment produced a smaller amount of N2O only when it was added with oxic water. For both WWTP effluents and downstream rivers, bulk isotope ratios and intramolecular distribution of 15N in N2O distinctly differed between summer and winter, indicating incomplete denitrification in the hypoxic sediment at low temperatures as a primary downstream source adding to WWTP-derived N2O. Winter surges in wastewater TN and sediment N2O release highlight temperature variability as an underappreciated control over anthropogenic N2O emissions from increasingly urbanized river systems worldwide.

Isotopic constraints on the formation pathways and sources of atmospheric nitrate in the Mt. Everest region.

Inorganic particulate nitrate (p-NO3-), gaseous nitric acid (HNO3(g)) and nitrogen oxides (NOx = NO + NO2), as main atmospheric pollutants, have detrimental effects on human health and aquatic/terrestrial ecosystems. Referred to as the 'Third Pole' and the 'Water Tower of Asia', the Tibetan Plateau (TP) has attracted wide attention on its environmental changes. Here, we evaluated the oxidation processes of atmospheric nitrate as well as traced its potential sources by analyzing the isotopic compositions of nitrate (δ15N, δ18O, and Δ17O) in the aerosols collected from the Mt. Everest region during April to September 2018. Over the entire sampling campaigns, the average of δ15N(NO3-), δ18O(NO3-), and Δ17O(NO3-) was -5.1 ± 2.3‰, 66.7 ± 10.2‰, and 24.1 ± 3.9‰, respectively. The seasonal variation in Δ17O(NO3-) indicates the relative importance of O3 and HO2/RO2/OH in NOx oxidation processes among different seasons. A significant correlation between NO3- and Ca2+ and frequent dust storms in the Mt. Everest region indicate that initially, the atmospheric nitrate in this region might have undergone a process of settling; subsequently, it got re-suspended in the dust. Compared with the Δ17O(NO3-) values in the northern TP, our observed significantly higher values suggest that spatial variations in atmospheric Δ17O(NO3-) exist within the TP, and this might result from the spatial variations of the atmospheric O3 levels, especially the stratospheric O3, over the TP. The observed δ15N(NO3-) values predicted remarkably low δ15N values in the NOx of the sources and the N isotopic fractionation plays a crucial role in the seasonal changes of δ15N(NO3-). Combined with the results from the backward trajectory analysis of air mass, we suggest that the vehicle exhausts and agricultural activities in South Asia play a dominant role in determining the nitrate levels in the Mt. Everest region.

Constraining the atmospheric OCS budget from sulfur isotopes.

Carbonyl sulfide (OCS), the most abundant sulfur-containing gas in the atmosphere, is used as a proxy for photosynthesis rate estimation. However, a large missing source of atmospheric OCS has been inferred. Sulfur isotope measurements (34S/32S ratio and δ34S) on OCS are a feasible tool to distinguish OCS sources from oceanic and anthropogenic emissions. Here we present the latitudinal (north-south) observations of OCS concentration and [Formula: see text]S within Japan. The observed [Formula: see text]S of OCS of 9.7 to 14.5‰ reflects source and sink effects. Particularly in winter, latitudinal decreases in [Formula: see text]S values of OCS were found to be correlated with increases in OCS concentrations, resulting an intercept of (4.7 ± 0.8)‰ in the Keeling plot approach. This result implies the transport of anthropogenic OCS emissions from the Asian continent to the western Pacific by the Asian monsoon outflow. The estimated background [Formula: see text]S of OCS in eastern Asia is consistent with the [Formula: see text]S of OCS previously reported in Israel and the Canary Islands, suggesting that the background [Formula: see text]S of OCS in the Northern Hemisphere ranges from 12.0 to 13.5‰. Our constructed sulfur isotopic mass balance of OCS revealed that anthropogenic sources, not merely oceanic sources, account for much of the missing source of atmospheric OCS.

Microdroplet synthesis of azo compounds with simple microfluidics-based pH control.

Conventional solution-phase synthesis of azo compounds is complicated by the need for precise pH and temperature control, high concentrations of pH control reagents, and by-product removal. In this work, we exploited the advantages of microdroplet chemistry to realize the simple and highly efficient synthesis of an azo compound using microfluidics-based pH control. Owing to the small size of microdroplets, heat exchange between a microdroplet and its environment is extremely fast. Furthermore, chemical reactions in microdroplets occur rapidly due to the short diffusion distance and vortex flow. Formation of the azo compound reached completion in less than 3 s at room temperature, compared with 1 h at 0 °C under conventional conditions. pH control was simple, and the pH control reagent concentration could be reduced to less than one-tenth of that used in the conventional method. No by-products were generated, and thus this procedure did not require a recrystallization step. The time course of the chemical reaction was elucidated by observing the growth of azo compound microcrystals in droplets at various locations along the channel corresponding to different mixing times.

Homogeneous sulfur isotope signature in East Antarctica and implication for sulfur source shifts through the last glacial-interglacial cycle.

Sulfate aerosol (SO42-) preserved in Antarctic ice cores is discussed in the light of interactions between marine biological activity and climate since it is mainly sourced from biogenic emissions from the surface ocean and scatters solar radiation during traveling in the atmosphere. However, there has been a paradox between the ice core record and the marine sediment record; the former shows constant non-sea-salt (nss-) SO42- flux throughout the glacial-interglacial changes, and the latter shows a decrease in biogenic productivity during glacial periods compared to interglacial periods. Here, by ensuring the homogeneity of sulfur isotopic compositions of atmospheric nss-SO42- (δ34Snss) over East Antarctica, we established the applicability of the signature as a robust tool for distinguishing marine biogenic and nonmarine biogenic SO42-. Our findings, in conjunction with existing records of nss-SO42- flux and δ34Snss in Antarctic ice cores, provide an estimate of the relative importance of marine biogenic SO42- during the last glacial period to be 48 ± 10% of nss-SO42-, slightly lower than 59 ± 11% during the interglacial periods. Thus, our results tend to reconcile the ice core and sediment records, with both suggesting the decrease in marine productivity around Southern Ocean under the cold climate.

Isotopic evidence for seasonality of microbial internal nitrogen cycles in a temperate forested catchment with heavy snowfall.

The Hokuriku district of central Japan receives high levels of precipitation during winter, largely in the form of snow. This study aimed to elucidate the internal nitrogen dynamics in this temperate forested region with heavy snowfall using the triple oxygen and nitrogen isotopic compositions of NO3-. The isotopic compositions of NO3- in atmospheric depositions (P and Tf), with terrestrial components of the soil layer (A0, S25, S55, and S90), ground water (G), and output (St) were measured from 2015 to 2016 in a forested catchment located in the southern area of the Ishikawa Prefecture, Japan. Seasonal distributions of Δ17O(NO3-) showed a decreasing trend from the inputs to outputs of the ecosystem. We found relatively constant Δ17O(NO3-) values in the output components (G and St), but found highly fluctuating Δ17O(NO3-) values resulting from the seasonal variations in the nitrification activity within soil waters. Specifically, we observed a lower nitrifying activity in the top soil layer throughout cold periods, presumably due to the input of cold melted snow water. The general trend of increasing δ15N(NO3-) value from the input to output components, with the changes in denitrification hotspots from shallow to deeper soil layer, can be observed between warm and cold periods. Thus, the seasonal changes of hotspots related to microbial nitrification and denitrification could be noted due to the seasonal changes in the isotopic compositions of nitrate. The estimated ecosystem-scale gross nitrification and denitrification rates are low; however, the output components are relatively stable with low concentrations of nitrate, indicating that the plant uptake of nitrogen most probably occurs at greater rates and scales in this forested ecosystem. Future nitrogen deposition and the vulnerable dynamics of snow melting are likely to have impactful consequences on such localities.

Deep-biosphere methane production stimulated by geofluids in the Nankai accretionary complex.

Microbial life inhabiting subseafloor sediments plays an important role in Earth's carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (102 to 103 cells cm-3) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.

A simple and reliable method reducing sulfate to sulfide for multiple sulfur isotope analysis.

RATIONALE: Precise analysis of four sulfur isotopes of sulfate in geological and environmental samples provides the means to extract unique information in wide geological contexts. Reduction of sulfate to sulfide is the first step to access such information. The conventional reduction method suffers from a cumbersome distillation system, long reaction time and large volume of the reducing solution. We present a new and simple method enabling the process of multiple samples at one time with a much reduced volume of reducing solution. METHODS: One mL of reducing solution made of HI and NaH2 PO2 was added to a septum glass tube with dry sulfate. The tube was heated at 124°C and the produced H2 S was purged with inert gas (He or N2 ) through gas-washing tubes and then collected by NaOH solution. The collected H2 S was converted into Ag2 S by adding AgNO3 solution and the co-precipitated Ag2 O was removed by adding a few drops of concentrated HNO3 . RESULTS: Within 2-3 h, a 100% yield was observed for samples with 0.2-2.5 µmol Na2 SO4 . The reduction rate was much slower for BaSO4 and a complete reduction was not observed. International sulfur reference materials, NBS-127, SO-5 and SO-6, were processed with this method, and the measured against accepted δ34 S values yielded a linear regression line which had a slope of 0.99 ± 0.01 and a R2 value of 0.998. CONCLUSIONS: The new methodology is easy to handle and allows us to process multiple samples at a time. It has also demonstrated good reproducibility in terms of H2 S yield and for further isotope analysis. It is thus a good alternative to the conventional manual method, especially when processing samples with limited amount of sulfate available.

Isotopic Fractionation of Sulfur in Carbonyl Sulfide by Carbonyl Sulfide Hydrolase of Thiobacillus thioparus THI115.

Carbonyl sulfide (COS) is one of the major sources of stratospheric sulfate aerosols, which affect the global radiation balance and ozone depletion. COS-degrading microorganisms are ubiquitous in soil and important for the global flux of COS. We examined the sulfur isotopic fractionation during the enzymatic degradation of COS by carbonyl sulfide hydrolase (COSase) from Thiobacillus thioparus THI115. The isotopic fractionation constant (34ɛ value) was -2.2±0.2‰. Under experimental conditions performed at parts per million by volume level of COS, the 34ɛ value for intact cells of T. thioparus THI115 was -3.6±0.7‰, suggesting that, based on Rees' model, the 34ɛ value mainly depended on COS transport into the cytoplasm. The 34ɛ value for intact cells of T. thioparus THI115 was similar to those for Mycobacterium spp. and Williamsia sp., which are known to involve the conserved region of nucleotide sequences encoding the clade D of ß-class carbonic anhydrase (ß-CA) including COSase. On the other hand, the 34ɛ value was distinct from those for bacteria in the genus Cupriavidus. These results provide an insight into biological COS degradation, which is indispensable for estimating the COS global budget based on the isotope because of the significant contribution of COS degradation by microorganisms harboring ß-CA family enzymes.

Relative Contribution of nirK- and nirS- Bacterial Denitrifiers as Well as Fungal Denitrifiers to Nitrous Oxide Production from Dairy Manure Compost.

The relative contribution of fungi, bacteria, and nirS and nirK denirifiers to nitrous oxide (N2O) emission with unknown isotopic signature from dairy manure compost was examined by selective inhibition techniques. Chloramphenicol (CHP), cycloheximide (CYH), and diethyl dithiocarbamate (DDTC) were used to suppress the activity of bacteria, fungi, and nirK-possessing denitrifiers, respectively. Produced N2O were surveyed to isotopocule analysis, and its 15N site preference (SP) and δ18O values were compared. Bacteria, fungi, nirS, and nirK gene abundances were compared by qPCR. The results showed that N2O production was strongly inhibited by CHP addition in surface pile samples (82.2%) as well as in nitrite-amended core samples (98.4%), while CYH addition did not inhibit the N2O production. N2O with unknown isotopic signature (SP = 15.3-16.2‰), accompanied by δ18O (19.0-26.8‰) values which were close to bacterial denitrification, was also suppressed by CHP and DDTC addition (95.3%) indicating that nirK denitrifiers were responsible for this N2O production despite being less abundant than nirS denitrifiers. Altogether, our results suggest that bacteria are important for N2O production with different SP values both from compost surface and pile core. However, further work is required to decipher whether N2O with unknown isotopic signature is mostly due to nirK denitrifiers that are taxonomically different from the SP-characterized strains and therefore have different SP values rather than also being interwoven with the contribution of the NO-detoxifying pathway and/or of co-denitrification.