The combination of single-cell Raman microspectroscopy (SCRM) and stable isotope probing (SIP) enables
tracking of carbon or hydrogen fluxes into microorganisms at the single-cell level. Therefore, ...it has high potential for the analysis of metabolic processes and biogeochemical cycles. However, especially for high throughput applications such as imaging or cell sorting, it is hampered by low Raman scattering intensities (and therefore long acquisition times). In order to overcome these limitations, this study brings forward a systematic investigation of Resonance Raman (RR) enhanced SCRM for SIP of bacterial carotenoids. Dynamic carbon uptake from
C-glucose was successfully monitored and quantified utilizing
C stable isotope-induced red-shifts of RR signals. High single-cell phenotypic heterogeneity was revealed in terms of carbon uptake and, unlike in previous studies, clear evidence for
synthesis of carotenoids was found. For the first time, hydrogen uptake into carotenoids was systematically investigated by deuterium labeling (providing a direct probe for metabolic activity of single cells). In carotenoid single-cell Resonance Raman (SCRR) spectra, a unique pattern of signal red-shifts and apparent blue-shifts was observed and quantitatively evaluated. Finally, a novel combined approach for simultaneous monitoring of carbon and hydrogen uptake revealed complementary effects in carotenoid SCRR spectra that can be analyzed in parallel. Overall, it was shown that the high RR intensity, simplicity of spectral features and straightforward signal processing make microbial carotenoids an ideal target for quantitative multi-element SIP, with great potential for high throughput applications.
Since the EU demands the use of renewable energy sources, also in the transport sector, the usage of biodiesel fuels increases continuously. As the physicochemical properties of soot are important ...for the oxidation behavior during the regeneration of diesel particle filters (DPF) and literature provides contradictory information about the reactivity and the structure of biodiesel soot, the aim of this work was to investigate the reactivity of soot produced by a diesel engine operated with fuels of different biodiesel content at varying engine operating parameters.
By temperature-programmed oxidation (TPO) an increasing reactivity with increasing biofuel ratio, except for the conditions with lowest injection pressure was determined. This implies that soot generated with 100% biofuel (consisting of rapeseed oil methyl ester) is more reactive than soot generated with commercial gasoline station fuel containing up to 7% biodiesel, while soot from fossil fuel is even less reactive. In addition, reactivity increases with increasing injection and boost pressure. Raman microspectroscopy (RM) analysis gave very similar spectra for the received soot samples, i.e. all generated soot samples possess a similar graphitic nanostructure. Additionally, a trend in reactivity with the particle size as well as the content of Fe, Zn, and Cu in the soot, which was determined by inductively coupled plasma mass spectrometry (ICP-MS), could be revealed. The soot reactivity is therefore not only determined by one parameter but a product of many soot properties like nanostructure, particle size and/or inorganic components as impurities.
When following the structural change in the DPF during regeneration, a decreased structural order was observed in the beginning. After a certain time (ca. 40 s) a homogeneous oxidation of the soot takes place, which does not change the structure of the soot inside the DPF. This coincides with findings of a constant oxidation rate after a certain time. Overall, it can be ascertained that no graphitization of the soot takes place inside the DPF during regeneration.
Raman microspectroscopy (RM), temperature-programmed oxidation (TPO), high-resolution transmission electron microscopy (HRTEM), and electron energy loss spectroscopy (EELS) were combined to get ...comprehensive information on the relationship between structure and reactivity of soot in samples of spark discharge (GfG), heavy duty engine diesel (EURO VI and IV) soot, and graphite powder upon oxidation by oxygen at increasing temperatures. GfG soot and graphite powder represent the higher and lower reactivity limits. Raman microspectroscopic analysis was conducted by determination of spectral parameters using a five band fitting procedure (G, D1−D4) as well as by evaluation of the dispersive character of the D mode. The analysis of spectral parameters shows a higher degree of disorder and a higher amount of molecular carbon for untreated GfG soot samples than for samples of untreated EURO VI and EURO IV soot. The structural analysis based on the dispersive character of the D mode revealed substantial differences in ordering descending from graphite powder, EURO IV, VI to GfG soot. HRTEM images and EELS analysis of EURO IV and VI samples indicated a different morphology and a higher structural order as compared to GfG soot in full agreement with the Raman analysis. These findings are also confirmed by the reactivity of soot during oxidation (TPO), where GfG soot was found to be the most reactive and EURO IV and VI soot samples exhibited a moderate reactivity.
In this study, the applicability of Raman microscopy (RM) for nondestructive chemical analysis of biofilm matrix, including microbial constituents and extracellular polymeric substances (EPS), has ...been assessed. The examination of a wide range of reference samples such as biofilm-specific polysaccharides, proteins, microorganisms, and encapsulated bacteria revealed characteristic frequency regions and specific marker bands for different biofilm constituents. Based on received data, the assignment of Raman bands in spectra of multispecies biofilms was performed. The study of different multispecies biofilms showed that RM can correlate various structural appearances within the biofilm to variations in their chemical composition and provide chemical information about a complex biofilm matrix. The results of RM analysis of biofilms are in good agreement with data obtained by confocal laser scanning microscopy (CLSM). Thus, RM is a promising tool for a label-free chemical characterization of different biofilm constituents. Moreover, the combination of RM with CLSM analysis for the study of biofilms grown under different environmental conditions can provide new insights into the complex structure/function correlations in biofilms.
Plastic particles in marine and freshwater environments span from macroscopic to microscopic size classes. Each may have a different impact on individuals, populations and ecosystems, but still the ...wide variety of methods used in beach sediment sampling inhibit comparisons among studies and therefore hampers a risk assessment. A large portion of the uncertainties is due to differing sampling strategies.
By quantifying the alongshore distribution of macro- and microplastic particles within five beaches of Lake Garda, we aim to shed light on the accumulation behavior of microplastic particles at an exemplary lake which might give indications for potential sampling zones. The identification of plastic at the single particle level with a spatial resolution down to 1μm was performed by Raman microspectroscopy. Given the time consuming approach we reduced the number of samples in the field but increased the spatial area where a single sample was taken, by utilizing a transect approach in combination with sediment cores (5cm depth).
The study revealed that, in comparison to the water line and the high-water line, the drift line of all five beaches always contained plastic particles. Since the drift line accumulate particulate matter on a relatively distinct zone, it will enable a comparable sampling of microplastic particles. The applied sampling approach provided a representative method for quantifying microplastic down to 1μm on a shore consisting of pebbles and sand. Hence, as first step towards a harmonization of beach sediment sampling we suggest to perform sampling at the drift line, although further methodological improvements are still necessary.
Display omitted
•Plastic abundance varies at natural lake beach (accumulation) zones.•Drift line might be an appropriate beach sediment sampling zone for microplastic.•Sample strategy adaptations are necessary to enable microplastic identification down to 1μm.
Raman microspectroscopy is a prime tool to characterize the molecular and isotopic composition of microbial cells. However, low sensitivity and long acquisition times limit a broad applicability of ...the method in environmental analysis. In this study, we explore the potential, the applicability, and the limitations of stable isotope Raman microspectroscopy (SIRM), resonance SIRM, and SIRM in combination with surface-enhanced Raman scattering (SERS) for the characterization of single bacterial cells. The latter two techniques have the potential to significantly increase sensitivity and decrease measurement times in SIRM, but to date, there are no (SERS-SIRM) or only a limited number (resonance SIRM) of studies in environmental microbiology. The analyzed microorganisms were grown with substrates fully labeled with the stable isotopes 13C or 2H and compounds with natural abundance of atomic isotopes (12C 98.89% or 1H 99.9844%, designated as 12C or 1H, respectively). Raman bands of bacterial cell compounds in stable isotope-labeled microorganisms exhibited a characteristic red-shift in the spectra. In particular, the sharp phenylalanine band was found to be an applicable marker band for SIRM analysis of the Deltaproteobacterium strain N47 growing anaerobically on 13C-naphthalene. The study of G. metallireducens grown with 13C- and 2H-acetate showed that the information on the chromophore cytochrome c obtained by resonance SIRM at 532 nm excitation wavelength can be successfully complemented by whole-organism fingerprints of bacteria cells achieved by regular SIRM after photobleaching. Furthermore, we present here for the first time the reproducible SERS analysis of microbial cells labeled with stable isotopes. Escherichia coli strain DSM 1116 cultivated with 12C- or 13C-glucose was used as a model organism. Silver nanoparticles synthesized in situ were applied as SERS media. We observed a reproducible red-shift of an adenine-related marker band from 733 to 720 cm–1 in SERS spectra for 13C-labeled cells. Additionally, Raman measurements of 12C/13C-glucose and -phenylalanine mixtures were performed to elucidate the feasibility of SIRM for nondestructive quantitative and spatially resolved analysis. The performed analysis of isotopically labeled microbial cells with SERS-SIRM and resonance SIRM paves the way toward novel approaches to apply Raman microspectroscopy in environmental process studies.
Microplastics in food is a relatively new research field with only few studies available so far. Scientists have been pointing out that some of these studies apply questionable analytical methods. ...Nevertheless, media often use such results to gain attention of the readers. It is therefore of particular significance, that only those scientific studies are published, clearly presenting valid data on the content of microplastics in food. Unfortunately, the study by Zuccarello et al. shows very critical aspects regarding analytical methods used and conclusions made. The applied procedure is not described and, therefore, does not allow any assessment by other groups, which is indispensable prerequisite of any scientific publication. Moreover, the analytical method used for the identification and quantification of microplastic particles – SEM-EDX – is not sound and not validated. Therefore, in our opinion the results on the contamination of bottled mineral water with microplastics published by Zuccarello et al. are more than questionable.
The increasing production and use of engineered inorganic nanoparticles (EINPs) escalate the risk for their unintended release into the environment. Coating of nanoparticles like natural organic ...matter (NOM) plays an important role in the stability, toxicity, and transport of nanoparticles. Surface‐enhanced Raman scattering (SERS) has been proven to be a promising method to detect and characterize the coating on noble metal nanoparticles. Here, we report on the synthesis of core‐shell Au–Ag nanoparticles (NPs) with a high SERS enhancement factor to study the exchange and competition of different coating agents with different binding abilities to simulate the release of NPs into a receiving environment with a number of potential coating agents. Suwannee River natural organic matter (SRNOM), 4‐mercaptobenzoic acid (4‐MBA), and 4‐mercaptopyridine (4‐MPy) were selected molecules for the experiments. The SERS experimental setup parameters such as aggregation size, laser excitation wavelength, and laser power were optimized before further experiments were conducted. It was shown that 4‐MPy has a higher binding affinity than SRNOM and 4‐MBA through the presence of simultaneous S and N atoms, which leads to dominating the coating process when two coating agents are present in the media at the same time. In addition, it was observed that 4‐MPy and 4‐MBA can replace the SRNOM coating on Au–Ag NPs.
We report the development of a new technique for label-free microarray readout based on surface-enhanced Raman scattering (SERS). In doing so, an investigation on optimized SERS substrates for the ...application to in situ microorganism analysis by Raman microscopy was carried out. Chemically synthesized nanoparticles were successfully applied to an immunoassay for label-free detection of single microorganisms. In this way, species specific, reproducible, and strong SERS spectra were collected from different bacteria immobilized on a chip. Furthermore, quantitative analysis of the microorganisms was performed using Raman mapping. Unlike conventional SERS detection of bacteria, which requires dehydration prior to analysis, our system enables us to detect and quantify microorganisms in an aqueous environment in situ. Hence, the nondestructive analysis of living bacteria cells is possible. Moreover, the “whole-organism fingerprint” SERS spectra can be adopted for further chemical characterization of microorganisms.
In the environment the weathering of plastic debris is one of the main sources of secondary microplastic (MP). It is distinct from primary MP, as it is not intentionally engineered, and presents a ...highly heterogeneous analyte composed of plastic fragments in the size range of 1 μm-1 mm. To detect secondary MP, methods must be developed with appropriate reference materials. These should share the characteristics of environmental MP which are a broad size range, multitude of shapes (fragments, spheres, films, fibers), suspensibility in water, and modified particle surfaces through aging (additional OH, C=O, and COOH). To produce such a material, we bring forward a rapid sonication-based fragmentation method for polystyrene (PS), polyethylene terephthalate (PET), and polylactic acid (PLA), which yields up to 10
/15 mL dispersible, high purity MP particles in aqueous media. To satisfy the claim of a reference material, the key properties-composition and size distribution to ensure the homogeneity of the samples, as well as shape, suspensibility, and aging -were analyzed in replicates (
= 3) to ensure a robust production procedure. The procedure yields fragments in the range of 100 nm-1 mm (<20 μm, 54.5 ± 11.3% of all particles). Fragments in the size range 10 μm-1 mm were quantitatively characterized via Raman microspectroscopy (particles = 500-1,000) and reflectance micro Fourier transform infrared analysis (particles = 10). Smaller particles 100 nm-20 μm were qualitatively characterized by scanning electron microcopy (SEM). The optical microscopy and SEM analysis showed that fragments are the predominant shape for all polymers, but fibers are also present. Furthermore, the suspensibility and sedimentation in pure MilliQ water was investigated using ultraviolet-visible spectroscopy and revealed that the produced fragments sediment according to their density and that the attachment to glass is avoided. Finally, a comparison of the infrared spectra from the fragments produced through sonication and naturally aged MP shows the addition of polar groups to the surface of the particles in the OH, C=O, and COOH region, making these particles suitable reference materials for secondary MP.
PDF
