Quantitative Raman Spectroscopy Analysis of Polyhydroxyalkanoates Produced by Cupriavidus necator H16

Abstract

We report herein on the application of Raman spectroscopy to the rapid quantitative analysis of polyhydroxyalkanoates (PHAs), biodegradable polyesters accumulated by various bacteria. This theme was exemplified for quantitative detection of the most common member of PHAs, poly(3-hydroxybutyrate) (PHB) in Cupriavidus necator H16. We have identified the relevant spectral region (800–1800 cm−1) incorporating the Raman emission lines exploited for the calibration of PHB (PHB line at 1736 cm−1) and for the selection of the two internal standards (DNA at 786 cm−1 and Amide I at 1662 cm−1). In order to obtain quantitative data for calibration of intracellular content of PHB in bacterial cells reference samples containing PHB amounts—determined by gas chromatography—from 12% to 90% (w/w) were used. Consequently, analytical results based on this calibration can be used for fast and reliable determination of intracellular PHB content during biotechnological production of PHB since the whole procedure—from bacteria sampling, centrifugation, and sample preparation to Raman analysis—can take about 12 min. In contrast, gas chromatography analysis takes approximately 8 h.

Label-free, simultaneous quantification of starch, protein and triacylglycerol in single microalgal cells

“…The methodology introduced here to rationally determine minimal sampling depth for a given population is generally applicable. It should not only guide the design of future studies, but also enable retrospective assessment of past studies that either sampled at only a few cells per population [ 24 – 26 ] or offered no rationale for their choice of sampling depth for a particular system [ 19 , 22 , 23 , 27 , 28 ]. Our study also suggests that efforts to automate SCRS acquisition from a given sample will be highly valuable, as it might allow routine analysis at sampling depth of dozens or even hundreds of cells.…”

“…This is important as many factors including the potential overlaps of Raman bands assignment among compounds, choice of sample pre-treatment methods, parameters of Raman measurement and species-specific property of microalgae can all potentially limit the practicability and reliability of SCRS in generating the measurements in a quantitative and ‘landscape-like’ manner. (iii) To derive the overall content and its degree of variation for target molecules in a cellular population, most studies have either sampled cells at a very low sampling depth [ 24 – 26 ], i.e., the number of cells measured for SCRS (e.g., only three cells sampled from each population [ 24 ]), or have not provided any rationale for their choice of sampling depth [ 19 , 22 , 23 , 27 , 28 ]. In fact, the link between method performance and sample depth, an experimental parameter directly determining throughput and common to all SCRS-based experiments, has not been critically probed.…”

Monitoring Candida parapsilosis and Staphylococcus epidermidis Biofilms by a Combination of Scanning Electron Microscopy and Raman Spectroscopy

“…We used a commercial Renishaw Raman microspectrometer (Renishaw inVia, Renishaw plc., Wotton-under-Edge, UK), with a 785 nm diode laser as the excitation source. The laser beam was focused on the sample with a microscope lens (Leica, Wetzlar, Germany, 50×, NA 0.5), the laser spot diameter was 2 µm × 10 µm (note that this laser spot shape is characteristic for the Renishaw InVia instrument [51,64,65]).…”

“…Raman spectra from living microorganisms contain multiple spectral peaks corresponding to unique interatomic vibrations in biomolecules, e.g., nucleic acids, proteins, carbohydrates, and lipids [51,52,53,54]. It has been shown that Raman microspectroscopy and Raman imaging can be regarded as the methods of choice for many studies of microorganisms, cells and other biological samples [51,55,56,57,58,59,60,61,62,63,64,65]. Detailed databases of Raman spectral features encountered in biological samples had been published before [60].…”

Evaluation of nitrate as internal standard for quantitative determination of urea in urine by Raman spectroscopy

“…The use of internal standard (IS) in Raman is reported in the literature with good results for different analytes (A) and samples (S). Among the main uses are (IS/A/S): 4-mercaptopyridine/3,4-methylenedioxymethamphetamine and α-methyltryptamine hydrochloride/drugs [18]; d-nicotine/nicotine/liquids for electronic cigarettes [19], amide/poly(3-hydroxybutyrate)/bacteria [20]; OH stretching band of water/sulfate/aqueous solution [21]; 1-propanethiol/polycyclic aromatic hydrocarbons/food contact materials [22]. However, the selection of a suitable IS for a specific problem is still a challenge.…”