Isotopomeric Peak Assignment for N2O in Cross-Labeling Experiments by Fiber-Enhanced Raman Multigas Spectroscopy.

Human intervention in nature, especially fertilization, greatly increased the amount of N2O emission. While nitrogen fertilizer is used to improve nitrogen availability and thus plant growth, one negative side effect is the increased emission of N2O. Successful regulation and optimization strategies require detailed knowledge of the processes producing N2O in soil. Nitrification and denitrification, the main processes responsible for N2O emissions, can be differentiated using isotopic analysis of N2O. The interplay between these processes is complex, and studies to unravel the different contributions require isotopic cross-labeling and analytical techniques that enable tracking of the labeled compounds. Fiber-enhanced Raman spectroscopy (FERS) was exploited for sensitive quantification of N2O isotopomers alongside N2, O2, and CO2 in multigas compositions and in cross-labeling experiments. FERS enabled the selective and sensitive detection of specific molecular vibrations that could be assigned to various isotopomer peaks. The isotopomers 14N15N16O (2177 cm-1) and 15N14N16O (2202 cm-1) could be clearly distinguished, allowing site-specific measurements. Also, isotopomers containing different oxygen isotopes, such as 14N14N17O, 14N14N18O, 15N15N16O, and 15N14N18O could be identified. A cross-labeling showed the capability of FERS to disentangle the contributions of nitrification and denitrification to the total N2O fluxes while quantifying the total sample headspace composition. Overall, the presented results indicate the potential of FERS for isotopic studies of N2O, which could provide a deeper understanding of the different pathways of the nitrogen cycle.

Comprehensive multi-gas study by means of fiber-enhanced Raman spectroscopy for the investigation of nitrogen cycle processes.

The extensive use of synthetic fertilizers has led to a considerable increase in reactive nitrogen input into agricultural and natural systems, resulting in negative effects in multiple ecosystems, the so-called nitrogen cascade. Since the global population relies on fertilization for food production, synthetic fertilizer use needs to be optimized by balancing crop yield and reactive nitrogen losses. Fiber-enhanced Raman spectroscopy (FERS) is introduced as a unique method for the simultaneous quantification of multiple gases to the study processes related to the nitrogen cycle. By monitoring changes in the headspace gas concentrations, processes such as denitrification, nitrification, respiration, and nitrogen fixation, as well as fertilizer addition were studied. The differences in concentration between the ambient and prepared process samples were evident in the Raman spectra, allowing for differentiation of process-specific spectra. Gas mixture concentrations were quantified within a range of low ppm to 100% for the gases N2, O2, CO2, N2O, and NH3. Compositional changes were attributed to processes of the nitrogen cycle. With help of multivariate curve resolution, it was possible to quantify N2O and CO2 simultaneously. The impact of fertilizers on N-cycle processes in soil was simulated and analyzed for identifying active processes. Thus, FERS was proven to be a suitable technique to optimize fertilizer composition and to quantify N2O and NH3 emissions, all with a single device and without further sample preparation.

Activity and electron donor preference of two denitrifying bacterial strains identified by Raman gas spectroscopy.

Human activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H2, O2, CO2, 14N2, 15N2 and 15N2O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes.

Thiosulfate- and hydrogen-driven autotrophic denitrification by a microbial consortium enriched from groundwater of an oligotrophic limestone aquifer.

Despite its potentially high relevance for nitrate removal in freshwater environments limited in organic carbon, chemolithoautotrophic denitrification has rarely been studied in oligotrophic groundwater. Using thiosulfate and H2 as electron donors, we established a chemolithoautotrophic enrichment culture from groundwater of a carbonate-rock aquifer to get more insight into the metabolic repertoire, substrate turnover, and transcriptional activity of subsurface denitrifying consortia. The enriched consortium was dominated by representatives of the genus Thiobacillus along with denitrifiers related to Sulfuritalea hydrogenivorans, Sulfuricella denitrificans, Dechloromonas sp. and Hydrogenophaga sp., representing the consortium's capacity to use multiple inorganic electron donors. Microcosm experiments coupled with Raman gas spectroscopy demonstrated complete denitrification driven by reduced sulfur compounds and hydrogen without formation of N2O. The initial nitrate/thiosulfate ratio had a strong effect on nosZ transcriptional activity and on N2 formation, suggesting similar patterns of the regulation of gene expression as in heterotrophic denitrifiers. Sequence analysis targeting nirS and nosZ transcripts identified Thiobacillus denitrificans-related organisms as the dominant active nirS-type denitrifiers in the consortium. An additional assessment of the nirS-type denitrifier community in the groundwaterclearly confirmed the potential for sulfur- and hydrogen-dependent chemolithoautotrophic denitrification as important metabolic feature widely spread among subsurface denitrifiers at the Hainich Critical Zone Exploratory.