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.

Hydrogen and C2-C6 Alkane Sensing in Complex Fuel Gas Mixtures with Fiber-Enhanced Raman Spectroscopy.

Power-to-gas is a heavily discussed option to store surplus electricity from renewable sources. Part of the generated hydrogen could be fed into the gas grid and lead to fluctuations in the composition of the fuel gas. Consequently, both operators of transmission networks and end users would need to frequently monitor the gas to ensure safety as well as optimal and stable operation. Currently, gas chromatography-based analysis methods are the state of the art. However, these methods have several downsides for time-resolved and distributed application and Raman gas spectroscopy is favorable for future point-of-use monitoring. Here, we demonstrate that fiber-enhanced Raman gas spectroscopy (FERS) enables the simultaneous detection of all relevant gases, from major (methane, CH4; hydrogen, H2) to minor (C2-C6 alkanes) fuel gas components. The characteristic peaks of H2 (585 cm-1), CH4 (2917 cm-1), isopentane (765 cm-1), i-butane (798 cm-1), n-butane (830 cm-1), n-pentane (840 cm-1), propane (869 cm-1), ethane (993 cm-1), and n-hexane (1038 cm-1) are well resolved in the broadband spectra acquired with a compact spectrometer. The fiber enhancement achieved in a hollow-core antiresonant fiber enables highly sensitive measurements with limits of detection between 90 and 180 ppm for different hydrocarbons. Both methane and hydrogen were quantified with high accuracy with average relative errors of 1.1% for CH4 and 1.5% for H2 over a wide concentration range. These results show that FERS is ideally suited for comprehensive fuel gas analysis in a future, where regenerative sources lead to fluctuations in the composition of gas.

Fiber-Enhanced Raman Gas Spectroscopy for the Study of Microbial Methanogenesis.

Microbial methanogenesis is a key biogeochemical process in the carbon cycle that is responsible for 70% of global emissions of the potent greenhouse gas methane (CH4). Further knowledge about microbial methanogenesis is crucial to mitigate emissions, increase climate model accuracy, or advance methanogenic biogas production. The current understanding of the substrate use of methanogenic microbes is limited, especially regarding the methylotrophic pathway. Here, we present fiber-enhanced Raman spectroscopy (FERS) of headspace gases as an alternate tool to study methanogenesis and substrate use in particular. The optical technique is nondestructive and sensitive to CH4, hydrogen (H2), and carbon dioxide with a large dynamic range from trace levels (demonstrated LoDs: CH4, 3 ppm; H2, 49 ppm) to pure gases. In addition, the portable FERS system can provide quantitative information about methanol concentration in the liquid phase of microbial cultures through headspace gas sampling (LoD 25 ppm). We demonstrate how FERS gas sensing could enable us to track substrate and product levels of microbial methanogenesis with just one instrument. The versatility of Raman gas spectroscopy could moreover help us to elucidate links between nitrogen and carbon cycle in microbial communities in the near future.

Characterization of fuel gases with fiber-enhanced Raman spectroscopy.

Common gaseous fuels are mixtures of several components. As the properties of the fuels can vary with the composition, but combustion needs to be stable, reliable analytical methods are highly sought after. Raman spectroscopic methods have proved their suitability for the characterization of diverse gaseous mixtures. They have the potential to overcome existing limitations of established technologies, since they are fast, non-consumptive, and accurate. Here, we demonstrate a gas sensor based on fiber-enhanced Raman spectroscopy (FERS) for fuel gas monitoring. Online detection of all gas components, including alkanes, carbon dioxide (CO2), nitrogen (N2), and hydrogen sulfide (H2S), for varying concentration ranges from tens of vol% down to the ppm level enables a comprehensive characterization of the fuels. The developed sensor system features a pinhole assembly which sufficiently reduces the background signal from the fiber to enable the detection of C2-C4 alkanes occurring in low concentrations. Detection limits in the low ppm region were achieved for the minor components of fuel gases, which allow the online monitoring of necessary purification steps, e.g., for biogas. The obtained results indicate that fiber-enhanced Raman sensors have the potential for comprehensive online and onsite gas sensing for fuel gas quality control. Graphical abstract.

Fiber-Enhanced Raman Gas Spectroscopy for 18O-13C-Labeling Experiments.

Stable isotopes are used in ecology to track and disentangle different processes and pathways. Especially for studies focused on the gas exchange of plants, sensing techniques that offer oxygen (O2) and carbon dioxide (CO2) sensitivity with isotopic discrimination are highly sought after. Addressing this challenge, fiber-enhanced Raman gas spectroscopy is introduced as a fast optical technique directly combining 13CO2 and 12CO2 as well as 18O2 and 16O2 measurements in one instrument. We demonstrate how a new type of optical hollow-core fiber, the so-called revolver fiber, is utilized for enhanced Raman gas sensing. Carbon dioxide and oxygen isotopologues were measured at concentrations expected when using 13C- and 18O-labeled gases in plant experiments. Limits of detection have been determined to be 25 ppm for CO2 and 150 ppm for O2. The combination of measurements with different integration times allows the creation of highly resolved broadband spectra. With the help of calculations based on density functional theory, the line at 1512 cm-1 occurring in the oxygen spectrum is assigned to 18O16O. The relative abundances of the isotopologues 18O16O and nitrogen 15N14N were in good agreement with typical values. For CO2, fiber-enhanced Raman spectra show the Fermi diad and hotbands of 12C16O2, 13C16O2, and 12C18O16O. Several weak lines were observed, and the line at 1426 cm-1 was identified as originating from the (0 4 0 2) → (0 2 0 2) transition of 12C16O2. With the demonstrated sensitivity and discriminatory power, fiber-enhanced Raman spectroscopy is a possible alternative means to investigate plant metabolism, directly combining 13CO2 and 12CO2 measurements with 18O2 and 16O2 measurements in one instrument. The presented method thus has large potential for basic analytical investigations as well as for applications in the environmental sciences.

In vivo imaging reveals reduced activity of neuronal circuits in a mouse tauopathy model.

Pathological alterations of tau protein play a significant role in the emergence and progression of neurodegenerative disorders. Tauopathies are characterized by detachment of the tau protein from neuronal microtubules, and its subsequent aberrant hyperphosphorylation, aggregation and cellular distribution. The exact nature of tau protein species causing neuronal malfunction and degeneration is still unknown. In the present study, we used mice transgenic for human tau with the frontotemporal dementia with parkinsonism-associated P301S mutation. These mice are prone to develop fibrillar tau inclusions, especially in the spinal cord and brainstem. At the same time, cortical neurons are not as strongly affected by fibrillar tau forms, but rather by soluble tau forms. We took advantage of the possibility to induce formation of neurofibrillary tangles in a subset of these cortical neurons by local injection of preformed synthetic tau fibrils. By using chronic in vivo two-photon calcium imaging in awake mice, we were able for the first time to follow the activity of individual tangle-bearing neurons and compare it to the activity of tangle-free neurons over the disease course. Our results revealed strong reduction of calcium transient frequency in layer 2/3 cortical neurons of P301S mice, independent of neurofibrillary tangle presence. These results clearly point to the impairing role of soluble, mutated tau protein species present in the majority of the neurons investigated in this study.

Spectral behavior of second harmonic signals from organic and non-organic materials in multiphoton microscopy.

Multimodal nonlinear microscopy allows imaging of highly ordered biological tissue due to spectral separation of nonlinear signals. This requires certain knowledge about the spectral distribution of the different nonlinear signals. In contrast to several publications we demonstrate a factor of [Formula: see text] relating the full width at half maximum of a gaussian laser pulse spectrum to the corresponding second harmonic pulse spectrum in the spatial domain by using a simple theoretical model. Experiments on monopotassium phosphate crystals (KDP-crystals) and on porcine corneal tissue support our theoretical predictions. Furthermore, no differences in spectral width were found for epi- and trans-detection of the second harmonic signal. Overall, these results may help to build an optimized multiphoton setup for spectral separation of nonlinear signals.

Molecular orientation sensitive second harmonic microscopy by radially and azimuthally polarized light.

We demonstrate the possibility to switch the z-polarization component of the illumination in the vicinity of the focus of high-NA objective lenses by applying radially and azimuthally polarized incident light. The influence of the field distribution on nonlinear effects was first investigated by the means of simulations. These were performed for high-NA objective lenses commonly used in nonlinear microscopy. Special attention is paid to the influence of the polarization of the incoming field. For linearly, circularly and radially polarized light a considerable polarization component in z-direction is generated by high NA focusing. Azimuthal polarization is an exceptional case: even for strong focusing no z-component arises. Furthermore, the influence of the input polarization on the intensity contributing to the nonlinear signal generation was computed. No distinct difference between comparable input polarization states was found for chosen thresholds of nonlinear signal generation. Differences in signal generation for radially and azimuthally polarized vortex beams were experimentally evaluated in native collagen tissue (porcine cornea). The findings are in good agreement with the theoretical predictions and display the possibility to probe the molecular orientation along the optical axis of samples with known nonlinear properties. The combination of simulations regarding the nonlinear response of materials and experiments with different sample orientations and present or non present z-polarization could help to increase the understanding of nonlinear signal formation in yet unstudied materials.