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.

Institute of Scientific Instruments of the CAS Brno 61264 Czech Republic

Materials Research Centre Faculty of Chemistry Brno University of Technology Brno 61200 Czech Republic

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Smith R. Biodegradable Polymers for Industrial Applications. CRC Press; Cambridge, UK: 2005. p. 245.

Tan G.-Y.A., Chen C.-L., Li L., Ge L., Wang L., Razaad I.M.N., Li Y., Zhao L., Mo Y., Wang J.-Y. Start a Research on Biopolymer Polyhydroxyalkanoate (PHA): A Review. Polymers. 2014;6:706–754. doi: 10.3390/polym6030706. DOI

Liu C.C., Zhang L.L., An J., Chen B., Yang H. Recent strategies for efficient production of polyhydroxyalkanoates by micro-organisms. Lett. Appl. Microbiol. 2015;62:9–15. doi: 10.1111/lam.12511. PubMed DOI

Koller M., Rodriguez-Contreras A. Techniques for tracing PHA-producing organisms and for qualitative and quantitative analysis of intra- and extracellular PHA. Eng. Life Sci. 2015;15:558–581. doi: 10.1002/elsc.201400228. DOI

Schie I.W., Huser T. Methods and applications of Raman microspectroscopy to single-cell analysis. Appl. Spectrosc. 2013;67:813–828. PubMed

Afseth N.K., Bloomfield M., Wold J.P., Matousek P.A. Novel approach for subsurface through-skin analysis of salmon using spatially offset raman spectroscopy (SORS) Appl. Spectrosc. 2014;68:255–262. doi: 10.1366/13-07215. PubMed DOI

De Gelder J., De Gussem K., Vandenabeele P., Moens L. Reference database of Raman spectra of biological molecules. J. Raman Spectrosc. 2007;38:1133–1147. doi: 10.1002/jrs.1734. DOI

Brauchle E., Schenke-Leyland K. Raman spectroscopy in biomedicine—Non-invasive in vitro analysis of cells and extracellular matrix components in tissues. Biotechnol. J. 2013;8:288–297. doi: 10.1002/biot.201200163. PubMed DOI PMC

Samek O., Al-Marashi J.F.M., Telle H.H. The potential of Raman spectroscopy for the identification of biofilm formation by Staphylococcus epidermidis. Laser Phys. Lett. 2010;7:378–383. doi: 10.1002/lapl.200910154. DOI

Samek O., Telle H.H., Harris L.G., Bloomfield M., Mack D. Raman spectroscopy for rapid discrimination of Staphylococcus epidermidis clones related to medical device-associated infections. Laser Phys. Lett. 2008;5:465–470. doi: 10.1002/lapl.200810011. DOI

Bernatová S., Samek O., Pilát Z., Šerý M., Ježek J., Jákl P., Šiler M., Krzyžánek V., Zemánek P., Holá V., et al. Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy. Molecules. 2013;18:13188–13199. doi: 10.3390/molecules181113188. PubMed DOI PMC

Samek O., Jonáš A., Pilát Z., Zemánek P., Nedbal L., Tříska J., Kotas P., Trtílek M. Raman microspectroscopy of individual algal cells: Sensing unsaturation of storage lipids in vivo. Sensors. 2010;10:8635–8651. doi: 10.3390/s100908635. PubMed DOI PMC

Sandt C., Smith-Palmer T., Pink J., Brennan L., Pink D. Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ. J. Appl. Microbiol. 2007;103:1808–1820. doi: 10.1111/j.1365-2672.2007.03413.x. PubMed DOI

Choo-Smith L.P., Marquelin K., van Vreeswijk T., Bruining H.A., Puppels G.J., Ngo Thi N.A., Kirchner C., Naumann D., Ami D., Villa A.M., et al. Investigating microbial (Micro)colony heterogeneity by vibrational spectroscopy. Appl. Environ. Microbiol. 2001;67:1461–1469. doi: 10.1128/AEM.67.4.1461-1469.2001. PubMed DOI PMC

Samek O., Mlynariková K., Bernatová S., Ježek J., Krzyžánek V., Šiler M., Zemánek P., Růžička F., Holá V., Mahelová M. Candida parapsilosis Biofilm Identification by Raman Spectroscopy. Int. J. Mol. Sci. 2014;15:23924–23935. doi: 10.3390/ijms151223924. PubMed DOI PMC

Maquelin K., Choo-Smith L.P., van Vreeswijk T., Endtz H.P., Smith B., Bennett R., Bruining H.A., Puppels G.J. Raman Spectroscopic Method for Identification of Clinically Relevant Microorganisms Growing on Solid Culture Medium. Anal. Chem. 2000;72:12–19. doi: 10.1021/ac991011h. PubMed DOI

Maquelin K., Choo-Smith L.P., Endtz H.P., Bruining H.A., Puppels G.J. Rapid identification of Candida species by confocal Raman microspectroscopy. J. Clin. Microbiol. 2002;40:594–600. doi: 10.1128/JCM.40.2.594-600.2002. PubMed DOI PMC

Espagnon I., Ostrovskii D., Mathey R., Dupoy M., Joly P.L., Novelli-Rousseau A., Pinston F., Gal O., Mallard F., Leroux D.F. Direct identification of clinically relevant bacterial and yeast microcolonies and macrocolonies on solid culture media by Raman spectroscopy. J. Biomed. Opt. 2014;19:027004. doi: 10.1117/1.JBO.19.2.027004. PubMed DOI

Gelder J., Willemse-Erix D., Scholtes M.J., Sanchez J.I., Maquelin K., Vandenabeele P., Boever P., Puppels J.G., Moens L., Vos P. Monitoring poly(3-hydroxybutyrate) production in Cupriavidus necator DSM 428 (H16) with Raman spectroscopy. Anal. Chem. 2008;80:2155–2160. doi: 10.1021/ac702185d. PubMed DOI

Ciobota V., Burkhardt E.M., Schumacher W., Rosch P., Kusel K., Popp J. The influence of intracellular storage material on bacterial identification by means of Raman spectroscopy. Anal. Bioanal. Chem. 2010;397:2929–2937. doi: 10.1007/s00216-010-3895-1. PubMed DOI

Izumi C.M.S., Temperini M. FT-Raman investigation of biodegradable polymers: Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Vibrat. Spectr. 2010;54:127–132. doi: 10.1016/j.vibspec.2010.07.011. DOI

Naumann D. FT-Infrared and Ft-Raman spectroscopy in biomedical research. Appl. Spectrosc. Rev. 2001;36:239–298. doi: 10.1081/ASR-100106157. DOI

Tao Z., Peng L., Zhang P., Li Y.-Q., Wang G. Probing the Kinetic Anabolism of Poly-Beta-Hydroxybutyrate in Cupriavidus necator H16 Using Single-Cell Raman Spectroscopy. Sensors. 2016;16:1257. doi: 10.3390/s16081257. PubMed DOI PMC

Peng P., Zeng C., Zhou Y., Lian S., Nie G. Rapid Determination of Turmeric Roots Quality Based on the Raman Spectrum of Curcumin. Food Anal. Methods. 2015;8:103. doi: 10.1007/s12161-014-9874-y. DOI

Obruca S., Petrik S., Benesova P., Svoboda Z., Eremka L., Marova I. Utilization of oil extracted from spent coffee grounds for sustainable production of polyhydroxyalkanoates. Appl. Microbiol. Biotechnol. 2014;98:5883–5890. doi: 10.1007/s00253-014-5653-3. PubMed DOI

Sato H., Dybal J., Murakami R., Noda I., Ozaki Y. Infrared and Raman spectroscopy and quantum chemistry calculation studies of C–H/O hydrogen bondings and thermal behavior of biodegradable polyhydroxyalkanoate. J. Mol. Struct. 2005;744–747:35–46. doi: 10.1016/j.molstruc.2004.10.069. DOI

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