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Fungal melanins are distinctive markers of rock-inhabiting ascomycetes. These complex polyphenols play important roles in stress tolerance while being essential components of fungal cell walls and useful biomarkers. Here we report signatures of melanins and carotenoids in pigment mutant strains of the black yeast Knufia petricola A95 using Raman spectroscopy
Two main types of inks coexisted in Early Medieval times: carbon inks and iron-gall inks. Many variations within these types existed, depending on the source of raw materials and their proportion. In addition to these, an intermediate mixed type of ink, with both iron and carbon as colouring agents, was sometimes used, especially in early Arab manuscripts.
Two projects are being conducted currently at the Bundesanstalt für Materialforschung und -prüfung (BAM) concerning the analysis of inks with Raman spectroscopy. The first one aims at comparing the spectra of different carbon inks from Antiquity to the Middle Ages with Raman spectroscopy, while the second one focuses on the identification and comparison of mixed inks.
For the first project, several carbon inks were made using different sources of carbon (pine soot, olive oil soot, date seeds charcoal etc.) and various binders (fish glue, gum Arabic, proteinic binder etc.), following antique recipes. These inks were then analysed with Raman spectroscopy and fitted with the “two-peaks” and the “three-peaks” models following the procedure described by Goler et al., to help spotting differences between the spectra, depending on the recipe. Some samples were then disposed in an ageing chamber at 80°C and 60% RH and analysed again in order to determine whether changed in the spectra are observable. The procedure proposed by Goler et al. to date inks with Raman spectroscopy was also evaluated during this process. Although several carbon inks could be distinguished from their spectra, no differences were seen between aged and fresh samples. Furthermore, experimental conditions were found to have such a significant influence that reliable dating of inks through Raman spectroscopy seems dubious.
The growing need to implement sensors such as NIR or Raman spectroscopy for the in-situ monitoring of bioprocesses which follows the standards of Quality by Design is either restricted by the impact of the huge water signal or by a disturbing fluorescence background originating from compounds in the culture media. Furthermore, the characterization of the bioprocess samples is challenging due to changing conditions in course of cultivation.
Here we evaluate two different process-suitable Raman spectroscopic approaches, namely time-gated Raman which bears the potential to extract the Raman signal from the fluorescence background, and cw- Raman with NIR excitation in combination with Surface Enhanced Raman Spectroscopy- (SERS) to investigate cell-free supernatants of Escherichia coli sampled over the course of a cultivation. A confocal Raman microscope was used as a reference for the process devices. The concentration of the analytes, glucose, acetate as well as metabolites such as cAMP, AMP and amino-acids were determined by offline by High-Performance Liquid Chromatography (HPLC) to serve as reference for the calibration of the Raman and SERS spectral data.
Multivariate evaluation of the Raman and SERS spectra by Partial Least Squares Regression (PLSR) yielded for most of the analytes robust correlations at each sampling point. Repeated investigation of the off-line samples over a larger experimental period suggested not only a high reliability of the Raman data in general but also a high repeatability of the SERS experiments. Similar spectral features in different quality and signal/noise ratios were measured with all three set-ups. Major results of the comparison of the different Raman spectroscopic approaches and their combination with SERS are summarized and conclusions are drawn on which approach provides the most accurate concentration data among the target analytes.
Acknowledgement
The authors kindly thank Mario Birkholz (IHP, Frankfurt (Oder), Germany) for the opportunity to use a confocal Raman microscope, Alex Bunker and Tapani Viitala (Division of Pharmaceutical Biosciences, Centre for Drug Research, University of Helsinki, Finland).