Valerian Ciobotă

The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy

AbstractPrevious studies dealing with bacterial identification by means of Raman spectroscopy have demonstrated that micro-Raman is a suitable technique for single-cell microbial identification. Raman spectra yield fingerprint-like information about all chemical components within one cell, and combined with multivariate methods, differentiation down to species or even strain level is possible. Many microorganisms may accumulate high amounts of polyhydroxyalkanoates (PHA) as carbon and energy storage materials within the cell and the Raman bands of PHA might impede the identification and differentiation of cells. To date, the identification by means of Raman spectroscopy have never been tested on bacteria which had accumulated PHA. Therefore, the aim of this study is to investigate the effect of intracellular polymer accumulation on the bacterial identification rate. Combining fluorescence imaging and Raman spectroscopy, we identified polyhydroxybutyrate (PHB) as a storage polymer accumulating in the investigated cells. The amount of energy storage material present within the cells was dependent on the physiological status of the microorganisms and strongly influenced the identification results. Bacteria in the stationary phase formed granules of crystalline PHB, which obstructed the Raman spectroscopic identification of bacterial species. The Raman spectra of bacteria in the exponential phase were dominated by signals from the storage material. However, the bands from proteins, lipids, and nucleic acids were not completely obscured by signals from PHB. Cells growing under either oxic or anoxic conditions could also be differentiated, suggesting that changes in Raman spectra can be interpreted as an indicator of different metabolic pathways. Although the presence of PHB induced severe changes in the Raman spectra, our results suggest that Raman spectroscopy can be successfully used for identification as long as the bacteria are not in the stationary phase. FigureStained bacteria with or without PHB within the cells, and the corresponding Raman spectra.

The effect of antimonate, arsenate, and phosphate on the transformation of ferrihydrite to goethite, hematite, feroxyhyte, and tripuhyite

Iron oxides, typical constituents of many soils, represent a natural immobilization mechanism for toxic elements. Most iron oxides are formed during the transformation of poorly crystalline ferrihydrite to more crystalline iron phases. The present study examined the impact of well known contaminants, such as P(V), As(V), and Sb(V), on the ferrihydrite transformation and investigated the transformation products with a set of bulk and nano-resolution methods. Irrespective of the pH, P(V) and As(V) favor the formation of hematite (α-Fe2O3) over goethite (α-FeOOH) and retard these transformations at high concentrations. Sb(V), on the other hand, favors the formation of goethite, feroxyhyte (d’-FeOOH), and tripuhyite (FeSbO4) depending on pH and Sb(V) concentration. The elemental composition of the transformation products analyzed by inductively coupled plasma optical emission spectroscopy show high loadings of Sb(V) with molar Sb:Fe ratios of 0.12, whereas the molar P:Fe and As:Fe ratios do not exceed 0.03 and 0.06, respectively. The structural similarity of feroxyhyte and hematite was resolved by detailed electron diffraction studies, and feroxyhyte was positively identified in a number of the samples examined. These results indicate that, compared to P(V) and As(V), Sb(V) can be incorporated into the structure of certain iron oxides through Fe(III)-Sb(V) substitution, coupled with other substitutions. However, the outcome of the ferrihydrite transformation (hematite, goethite, feroxyhyte, or tripuhyite) depends on the Sb(V) concentration, pH, and temperature.

Raman investigations of Upper Cretaceous phosphorite and black shale from Safaga District, Red Sea, Egypt.

The mineral composition of the Upper Cretaceous Duwi phosphorite deposits and underlying Quseir Variegated Shale from Safaga district, Red Sea Range, Egypt, was investigated by dispersive and Fourier transformed Raman spectroscopy. The only phosphorous containing mineral detected in the phosphorite deposits was carbonate fluorapatite. Often carbonate fluorapatite appears associated with calcium sulfate and seldom with calcium carbonate in the investigated samples. Iron is present in the form of goethite and pyrite in the phosphorite layer, while pyrite, marcasite and hematite were identified in the Quseir Shale samples. Also, a high amount of disordered carbon was detected in the black shale layers. The Raman results confirm the hypothesis that the formation of the phosphorites took place in a marine environment. During the formation of black shale, the redox conditions changed, with the pH reaching values of 4 or even lower. Diagenetic and weathering transformations had taken place in the phosphorite deposits, calcium sulfate and goethite being products of these types of processes.

Handheld Raman spectroscopy for the early detection of plant diseases: Abutilon mosaic virus infecting Abutilon sp.

Plant diseases have a direct impact on the productivity of crops, and therefore the early detection of diseases is crucial. Abutilon mosaic virus (AbMV) (family Geminiviridae; genus Begomovirus) is an important virus infecting ornamental crops throughout the world. Abutilon hybridum showing bright yellow mosaic symptoms were observed in gardens in Tumbaco, Ecuador. The infection was confirmed by Polymerase Chain Reaction (PCR) using degenerate begomovirus primers. The amplicon (∼500 bp) was sequenced, submitted to NCBI (KP877621) and showed 68.7–100% and 72.7–100% sequence identity with other begomoviruses at nucleotide and amino acid levels, respectively. In order to evaluate Raman spectroscopy as a diagnostic tool for AbMV infection, spectra of leaves from healthy and infected plants were recorded. Raman signals of carotenoids are the most important features and there is a significant decrease in the intensity of these bands when the plant is infected. The difference in the intensity of the bands, particularly the one at 1526 cm−1, is proposed as a basis for the early detection of viral infection in plants.

Reactions of Alkaline Minerals in the Atmosphere

The excessive exploitation of watercourses for human activities affects the water balance of many lakes around the world. A well-known dramatic example is the desiccation of the Aral Sea, which currently has almost disappeared. One of the consequences of the desiccation of saline water bodies as the Aral Sea is the phenomenon known as “salt storms”, which transport huge amounts of salt over long distances. Some “soda lakes” (alkaline lakes), for example, the Owens Lake (USA), are also affected by desiccation processes, resulting in the wind transport of the alkaline dust. In spite of the remarkable scientific efforts to study heterogeneous reactions taking place in the atmosphere, the reactions between solid species under the influence of humid air have not received the necessary attention. To our knowledge, the chemical reaction between CaCO3 and (NH4)2SO4 in the presence of humid air is the only report of reactions taking place between solid salts in the presence of humid air. However, there are other salts which should also be considered; Na2CO3·H2O (thermonatrite) and Na3(HCO3)(CO3) ·2 H2O (trona) were identified in the alkaline dust transported from the Owens (dry) Lake by wind. Trona and Na6(CO3)(SO4)2 (burkeite) were identified in the salt efflorescences of playas of the Mojave Desert (USA), which are periodically removed (and transported) by wind. Depending on the size of the emitted particles, they can sediment or remain suspended for many days. The suspended material (fine particles) undergoes different atmospheric processes, one of them is coagulation (two particles combining to form one). Coagulation processes can produce internally mixed particles. This means that different solid substances are in contact in single particles (each particle is a mixture). If there is a chemical reaction between two solid compounds present in a single micrometer particle, this reaction takes also place on a macroscale, that is, in a bulk powder mixture. Kinetic differences between experiments on microand macroscales can be expected, fundamentally because of the surface area and gas diffusion. Since the reaction rate in a bulk solid mixture may be lower than that in a single particle, it is more likely to detect intermediate products. This is the reason why we use bulk powder mixtures in this work. We report here, for the first time, solid-state reactions of the alkaline salts thermonatrite, trona, and burkeite with (NH4)2SO4 in the presence of humid air (about 70% relative humidity) at ordinary temperatures (21–23 8C). These solidstate reactions can be attributed to the interaction of ions in a liquid film formed on the solids. The observation of changes in the relative amount of reactants over time, as well as the identification of the reaction products were achieved using Raman spectroscopy. We also demonstrate that the reactivity between solid salts is not restricted to alkaline salts. Considering both the experiments reported by Mori et al. and our own results, the chemical reactivity of the solid powder mixtures seems to be strongly linked to the humidity of the air. When the solid mixtures are exposed to ordinary conditions, that is, temperatures ranging between 21 and 23 8C and a relative humidity (RH) of air below 50 %, the chemical reaction rate is very slow; the Raman spectra of mixtures exposed to ambient conditions during 24 h show no or only weak bands of the reaction products (see Figure S1 in the Supporting Information). Remarkable reactivity was observed consistently on exposing the solid powder mixtures to higher RH values. An unequivocal sign that reactions were taking place in the mixtures was the release of NH3, which is a product of acid–base reactions involving CO3 2 and/or HCO3 , and NH4 + ions. The formation of CO2 can be deduced from these reactions. Figure 1a shows the Raman spectra recorded at different times from an equimolar mixture of (NH4)2SO4 and Na2CO3·H2O (thermonatrite) exposed to humid air (70% RH). After 5 minutes two new bands at 1079 and 996 cm 1 are observed, which belong to anhydrous Na2CO3 and Na2SO4 in phase III, respectively. Na2SO4 in phase III (a high-temperature phase) was observed in earlier crystallization experiments of single droplets 6] and it is known to transform slowly to phase V (room-temperature phase). After 30 minutes a small band emerges at 1045 cm 1 (which can be assigned to NaHCO3 and/or NH4HCO3), while the band of anhydrous Na2CO3 decreases, obviously as a consequence of the watervapor absorption to regenerate Na2CO3·H2O. After 3 h the bands belonging to anhydrous Na2CO3 and Na2CO3·H2O (1069 cm ) disappear, instead a band at 984 cm 1 (lecontite, NH4NaSO4·2 H2O) appears. A band emerges and grows gradually at 993 cm 1 (Na2SO4 in phase V), while the band of Na2SO4 in phase III decreases and finally disappears. There is evidence of a phase transition Na2SO4(III)!Na2SO4(V), probably because of the contact with humid air, since the stability of phase III has been associated to dry conditions. [*] P. Vargas Jentzsch, V. Ciobotă, Dr. P. Rçsch, Prof. Dr. J. Popp Institut f r Physikalische Chemie and Abbe Center of Photonics Friedrich-Schiller-Universit t Jena Helmholtzweg 4, 07743 Jena (Germany) E-mail: juergen.popp@uni-jena.de

Distinction of Ecuadorian varieties of fermented cocoa beans using Raman spectroscopy

Cocoa (Theobroma cacao) is a crop of economic importance. In Ecuador, there are two predominant cocoa varieties: National and CCN-51. The National variety is the most demanded, since its cocoa beans are used to produce the finest chocolates. Raman measurements of fermented, dried and unpeeled cocoa beans were performed using a handheld spectrometer. Samples of the National and CCN-51 varieties were collected from different provinces and studied in this work. For each sample, 25 cocoa beans were considered and each bean was measured at 4 different spots. The most important Raman features of the spectra were assigned and discussed. The spectroscopic data were processed using chemometrics, resulting in a distinction of varieties with 91.8% of total accuracy. Differences in the average Raman spectra of cocoa beans from different sites but within the same variety can be attributed to environmental factors affecting the cocoa beans during the fermentation and drying processes.

Applications of Raman Spectroscopy to Virology and Microbial Analysis

This chapter reports from the utilization of Raman spectroscopic techniques like Raman microscopy, Raman optical activity (ROA), UV-resonance Raman (UVRR)-spectroscopy, surface enhanced Raman spectroscopy (SERS), and tip-enhanced Raman spectroscopy (TERS) for the investigation of viruses and microorganisms, especially bacteria and yeasts for medical and pharmaceutical applications. The application of these Raman techniques allows for the analysis of chemical components of cells and subcellular regions, as well as the monitoring of chemical differences occurring as a result of the growth of microorganisms. In addition, the interaction of microorganisms with active pharmaceutical agents can be investigated. In combination with chemometric methods Raman spectroscopy can also be applied to identify microorganisms both in micro colonies and even on single cells.