Fast and highly sensitive fiber-enhanced Raman spectroscopic monitoring of molecular H2 and CH4 for point-of-care diagnosis of malabsorption disorders in exhaled human breath.

Breath gas analysis is a novel powerful technique for noninvasive, early-stage diagnosis of metabolic disorders or diseases. Molecular hydrogen and methane are biomarkers for colonic fermentation, because of malabsorption of oligosaccharides (e.g., lactose or fructose) and for small intestinal bacterial overgrowth. Recently, the presence of these gases in exhaled breath was also correlated with obesity. Here, we report on the highly selective and sensitive detection of molecular hydrogen and methane within a complex gas mixture (consisting of H2, CH4, N2, O2, and CO2) by means of fiber-enhanced Raman spectroscopy (FERS). An elaborate FERS setup with a microstructured hollow core photonic crystal fiber (HCPCF) provided a highly improved analytical sensitivity. The simultaneous monitoring of H2 with all other gases was achieved by a combination of rotational (H2) and vibrational (other gases) Raman spectroscopy within the limited spectral transmission range of the HCPCF. The HCPCF was combined with an adjustable image-plane aperture pinhole, in order to separate the H2 rotational Raman bands from the silica background signal and improve the sensitivity down to a limit of detection (LOD) of 4.7 ppm (for only 26 fmol H2). The ability to monitor the levels of H2 and CH4 in a positive hydrogen breath test (HBT) was demonstrated. The FERS sensor possesses a high dynamic range (∼5 orders of magnitude) with a fast response time of few seconds and provides great potential for miniaturization. We foresee that this technique will pave the way for fast, noninvasive, and painless point-of-care diagnosis of metabolic diseases in exhaled human breath.

Online investigation of respiratory quotients in Pinus sylvestris and Picea abies during drought and shading by means of cavity-enhanced Raman multi-gas spectrometry.

Photosynthesis and respiration are major components of the plant carbon balance. During stress, like drought, carbohydrate supply from photosynthesis is reduced and the Krebs cycle respiration must be fueled with other stored carbon compounds. However, the dynamics of storage use are still unknown. The respiratory quotient (RQ, CO2 released per O2 consumed during respiration) is an excellent indicator of the nature of the respiration substrate. In plant science, however, online RQ measurements have been challenging or even impossible so far due to very small gas exchange fluxes during respiration. Here we apply cavity-enhanced multi-gas Raman spectrometry (CERS) for online in situ RQ measurements in drought-tolerant pine (Pinus sylvestris [L.]) and drought-intolerant spruce (Picea abies [L. H. Karst]). Two different treatments, drought and shading, were applied to reduce photosynthesis and force dependency on stored substrates. Changes in respiration rates and RQ values were continuously monitored over periods of several days with low levels of variance. The results show that both species switched from COH-dominated respiration (RQ = 1.0) to a mixture of substrates during shading (RQ = 0.77-0.81), while during drought only pine did so (RQ = 0.75). The gas phase measurements were complemented by concentration measurements of non-structural carbohydrates and lipids. These first results suggest a physiological explanation for greater drought tolerance in pine. CERS was proven as powerful technique for non-consumptive and precise real-time monitoring of respiration rates and respirational quotients for the investigation of plant metabolism under drought stress conditions that are predicted to increase with future climate change.

Raman spectroscopic investigation of 13CO 2 labeling and leaf dark respiration of Fagus sylvatica L. (European beech).

An important issue, in times of climate change and more extreme weather events, is the investigation of forest ecosystem reactions to these events. Longer drought periods stress the vitality of trees and promote mass insect outbreaks, which strongly affect ecosystem processes and services. Cavity-enhanced Raman gas spectrometry was applied for online multi-gas analysis of the gas exchange rates of O2 and CO2 and the labeling of Fagus sylvatica L. (European beech) seedlings with (13)CO2. The rapid monitoring of all these gases simultaneously allowed for the separation of photosynthetic uptake of CO2 by the beech seedlings and a constant (12)CO2 efflux via respiration and thus for a correction of the measured (12)CO2 concentrations in course of the labeling experiment. The effects of aphid infestation with the woolly beech aphid (Phyllaphis fagi L.) as well as the effect of a drought period on the respirational gas exchange were investigated. A slightly decreased respirational activity of drought-stressed seedlings in comparison to normally watered seedlings was found already for a low drought intensity. Cavity-enhanced Raman gas monitoring of O2, (12)CO2, and (13)CO2 was proven to be a powerful new tool for studying the effect of drought stress and aphid infestation on the respirational activity of European beech seedlings as an example of important forest species in Central Europe.

Fiber-enhanced Raman multigas spectroscopy: a versatile tool for environmental gas sensing and breath analysis.

Versatile multigas analysis bears high potential for environmental sensing of climate relevant gases and noninvasive early stage diagnosis of disease states in human breath. In this contribution, a fiber-enhanced Raman spectroscopic (FERS) analysis of a suite of climate relevant atmospheric gases is presented, which allowed for reliable quantification of CH4, CO2, and N2O alongside N2 and O2 with just one single measurement. A highly improved analytical sensitivity was achieved, down to a sub-parts per million limit of detection with a high dynamic range of 6 orders of magnitude and within a second measurement time. The high potential of FERS for the detection of disease markers was demonstrated with the analysis of 27 nL of exhaled human breath. The natural isotopes (13)CO2 and (14)N(15)N were quantified at low levels, simultaneously with the major breath components N2, O2, and (12)CO2. The natural abundances of (13)CO2 and (14)N(15)N were experimentally quantified in very good agreement to theoretical values. A fiber adapter assembly and gas filling setup was designed for rapid and automated analysis of multigas compositions and their fluctuations within seconds and without the need for optical readjustment of the sensor arrangement. On the basis of the abilities of such miniaturized FERS system, we expect high potential for the diagnosis of clinically administered (13)C-labeled CO2 in human breath and also foresee high impact for disease detection via biologically vital nitrogen compounds.

Enhanced Raman multigas sensing - a novel tool for control and analysis of (13)CO(2) labeling experiments in environmental research.

Cavity-enhanced Raman multigas spectrometry is introduced as a versatile technique for monitoring of (13)CO2 isotope labeling experiments, while simultaneously quantifying the fluxes of O2 and other relevant gases across a wide range of concentrations. The multigas analysis was performed in a closed cycle; no gas was consumed, and the gas composition was not altered by the measurement. Isotope labeling of plant metabolites via photosynthetic uptake of (13)CO2 enables the investigation of resource flows in plants and is now an important tool in ecophysiological studies. In this experiment the (13)C labeling of monoclonal cuttings of Populus trichocarpa was undertaken. The high time resolution of the online multigas analysis allowed precise control of the pulse labeling and was exploited to calculate the kinetics of photosynthetic (13)CO2 uptake and to extrapolate the exact value of the (13)CO2 peak concentration in the labeling chamber. Further, the leaf dark respiration of immature and mature leaves was analyzed. The quantification of the photosynthetic O2 production rate as a byproduct of the (13)CO2 uptake correlated with the amount of available light and the leaf area of the plants in the labeling chamber. The ability to acquire CO2 and O2 respiration rates simultaneously also simplifies the determination of respiratory quotients (rate of O2 uptake compared to CO2 release) and thus indicates the type of combusted substrate. By combining quantification of respiration quotients with the tracing of (13)C in plants, cavity enhanced Raman spectroscopy adds a valuable new tool for studies of metabolism at the organismal to ecosystem scale.

Investigation of gas exchange processes in peat bog ecosystems by means of innovative Raman gas spectroscopy.

Highly sensitive Raman gas spectroscopy is introduced for simultaneous real time analysis of O(2), CO(2), CH(4), and N(2) in order to elucidate the dynamics of greenhouse gases evolving from climate-sensitive ecosystems. The concentrations and fluxes of this suite of biogenic gases were quantified in the head space of a water-saturated, raised peat bog ecotron. The intact peat bog, exhibiting various degradation stages of peat and sphagnum moss, was exposed to various light regimes in order to determine important ecosystem parameters such as the maximum photosynthesis rate of the sphagnum as well as the extent of soil and plant respiration. Miniaturized Raman gas spectroscopy was proven to be an extremely versatile analytical technique that allows for onsite multigas analysis in high temporal resolution. Therefore it is an urgently needed tool for elucidation of complex biochemical processes especially in climate-sensitive ecosystems and consequently for the estimation of climate-relevant gas budgets.

Raman spectroscopy-an innovative and versatile tool to follow the respirational activity and carbonate biomineralization of important cave bacteria.

Raman gas spectrometry is introduced as a unique tool for the investigation of the respiratory activity that is indicative for growth of bacteria involved in biomineralization. Growth of these bacteria cannot be monitored using conventional turbidity-based optical density measurements due to concomitant mineral formation in the medium. The respiratory activity of carbonate-precipitating Arthrobacter sulfonivorans , isolated from the recently discovered Herrenberg Cave, was investigated during its lifecycle by means of innovative cavity-enhanced Raman gas analysis. This method allowed rapid and nonconsumptive online quantification of CO2 and O2 in situ in the headspace of the bacterial culture. Carbon dioxide production rates of A. sulfonivorans showed two maxima due to its pleomorphic growth lifecycle. In contrast, only one maximum was observed in control organism Pseudomonas fluorescens with a one-stage lifecycle. Further insight into the biomineralization process over time was provided by a combination of Raman macro- and microspectroscopy. With the help of this spatially resolved chemical imaging of the different types of calcium carbonate minerals, it was elucidated that the surface of the A. sulfonivorans bacterial cells served as nuclei for biomineralization of initially spherical vaterite precipitates. These vaterite biominerals continued growing as chemically stable rock-forming calcite crystals with rough edges. Thus, the utilization of innovative Raman multigas spectroscopy, combined with Raman mineral analysis, provided novel insights into microbial-mediated biomineralization and, therefore, provides a powerful methodology in the field of environmental sciences.

Calcite biomineralization by bacterial isolates from the recently discovered pristine karstic herrenberg cave.

Karstic caves represent one of the most important subterranean carbon storages on Earth and provide windows into the subsurface. The recent discovery of the Herrenberg Cave, Germany, gave us the opportunity to investigate the diversity and potential role of bacteria in carbonate mineral formation. Calcite was the only mineral observed by Raman spectroscopy to precipitate as stalactites from seepage water. Bacterial cells were found on the surface and interior of stalactites by confocal laser scanning microscopy. Proteobacteria dominated the microbial communities inhabiting stalactites, representing more than 70% of total 16S rRNA gene clones. Proteobacteria formed 22 to 34% of the detected communities in fluvial sediments, and a large fraction of these bacteria were also metabolically active. A total of 9 isolates, belonging to the genera Arthrobacter, Flavobacterium, Pseudomonas, Rhodococcus, Serratia, and Stenotrophomonas, grew on alkaline carbonate-precipitating medium. Two cultures with the most intense precipitate formation, Arthrobacter sulfonivorans and Rhodococcus globerulus, grew as aggregates, produced extracellular polymeric substances (EPS), and formed mixtures of calcite, vaterite, and monohydrocalcite. R. globerulus formed idiomorphous crystals with rhombohedral morphology, whereas A. sulfonivorans formed xenomorphous globular crystals, evidence for taxon-specific crystal morphologies. The results of this study highlighted the importance of combining various techniques in order to understand the geomicrobiology of karstic caves, but further studies are needed to determine whether the mineralogical biosignatures found in nutrient-rich media can also be found in oligotrophic caves.

Rapid monitoring of intermediate states and mass balance of nitrogen during denitrification by means of cavity enhanced Raman multi-gas sensing.

The comprehensive investigation of changes in N cycling has been challenging so far due to difficulties with measuring gases such as N2 and N2O simultaneously. In this study we introduce cavity enhanced Raman gas spectroscopy as a new analytical methodology for tracing the stepwise reduction of (15)N-labelled nitrate by the denitrifying bacteria Pseudomonas stutzeri. The unique capabilities of Raman multi-gas analysis enabled real-time, continuous, and non-consumptive quantification of the relevant gases ((14)N2, (14)N2O, O2, and CO2) and to trace the fate of (15)N-labeled nitrate substrate ((15)N2, (15)N2O) added to a P. stutzeri culture with one single measurement. Using this new methodology, we could quantify the kinetics of the formation and degradation for all gaseous compounds (educts and products) and thus study the reaction orders. The gas quantification was complemented with the analysis of nitrate and nitrite concentrations for the online monitoring of the total nitrogen element budget. The simultaneous quantification of all gases also enabled the contactless and sterile online acquisition of the pH changes in the P. stutzeri culture by the stoichiometry of the redox reactions during denitrification and the CO2-bicarbonate equilibrium. Continuous pH monitoring - without the need to insert an electrode into solution - elucidated e.g. an increase in the slope of the pH value coinciding with an accumulation of nitrite, which in turn led to a temporary accumulation of N2O, due to an inhibition of nitrous oxide reductase. Cavity enhanced Raman gas spectroscopy has a high potential for the assessment of denitrification processes and can contribute substantially to our understanding of nitrogen cycling in both natural and agricultural systems.