Raman microspectroscopy of individual algal cells: sensing unsaturation of storage lipids in vivo

Abstract

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm(-1) (cis C═C stretching mode) and 1,445 cm(-1) (CH(2) scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.

Keywords: Raman spectroscopy; algal cells; iodine value; lipids.

Figure 1.

Visualization of the lipid bodies in algal cells. Nile Red (NR) fluorescence image of the lipid bodies (left) and Differential Interference Contrast (DIC) image (right) of the same living cell are compared. Complex internal compartmentalization is visible in the DIC image and the structures corresponding to the lipid bodies can be clearly identified. The scale bar is 5 μm.

Figure 2.

Schematic diagram of the experimental setup for Raman microspectroscopy. BF––bandpass filter, D––dichroic mirror, Exp––beam expander, FM––flipping mirror, L1,2––lenses, NDF1,2––neutral density filters, NF1,2––notch filters, PBS––polarizing beam splitter, WP––lambda-half wave plate. Inset shows the detail of the studied sample.

Figure 3.

Typical Raman scattering spectrum of Trachydiscus minutus. Individual numbered bands are assigned in Table 1. Raman bands 7 and 9 are used to calculate the degree of lipid unsaturation. Spectrum acquisition parameters: integration time 20 s, laser power at the specimen 13 mW. Red numbers indicate β-carotene vibrations.

Figure 4.

Dependence of the observed ratio ν_U/ν_S of the Raman spectral peaks at 1656 cm⁻¹ and 1445 cm⁻¹ on the molecule mass unsaturation N_C═C/N_CH2 Crosses mark experimental data obtained with pure fatty acids, straight line is a fit of this data. Circles indicate verification data points obtained with mixtures of oleic and arachidonic acids of different molar ratios; this data was not included in the fit.

Figure 5.

Calibration curve for estimating the iodine value (IV) from the Raman spectral data. The IV range of 0–430 is covered which includes virtually all biologically relevant fatty acids. Crosses mark experimental data obtained with pure fatty acids, continuous line is a parabolic fit of this data. Circles indicate verification data points obtained with mixtures of oleic and arachidonic acids of different molar ratios; this data was not included in the fit. Formula given in the top left corner of the graph was used for calculating IV of the studied algae from experimental spectroscopic data.

Figure 6.

Typical Raman scattering spectra of intracellular lipid bodies contained in three different algal species: Trachydiscus minutus (top), Botryoccocus sudeticus (middle), and Chlamydomonas sp (bottom). Raman bands used for the calculations of iodine value are highlighted with dashed vertical lines. Corresponding pictures to the left of the spectra show the lipid bodies from which the spectra were recorded (indicated by the black arrows).

Figure 7.

Raman scattering spectrum of β-carotene. The inset shows the detail of β-carotene band at 1,442 cm⁻¹.