Raman Microspectroscopic Analysis of Selenium Bioaccumulation by Green Alga Chlorella vulgaris

Abstract

Selenium (Se) is an element with many commercial applications as well as an essential micronutrient. Dietary Se has antioxidant properties and it is known to play a role in cancer prevention. However, the general population often suffers from Se deficiency. Green algae, such as Chlorella vulgaris, cultivated in Se-enriched environment may be used as a food supplement to provide adequate levels of Se. We used Raman microspectroscopy (RS) for fast, reliable, and non-destructive measurement of Se concentration in living algal cells. We employed inductively coupled plasma-mass spectrometry as a reference method to RS and we found a substantial correlation between the Raman signal intensity at 252 cm^-1 and total Se concentration in the studied cells. We used RS to assess the uptake of Se by living and inactivated algae and demonstrated the necessity of active cellular transport for Se accumulation. Additionally, we observed the intracellular Se being transformed into an insoluble elemental form, which we further supported by the energy-dispersive X-ray spectroscopy imaging.

Keywords: Chlorella vulgaris; EDX; ICP-MS; Raman spectroscopy; algae; bioaccumulation; selenium.

Figure 1

A capillary microfluidic device for observation and exchange of algal samples under stable optical settings of the Raman measurement. The microfluidic tubing was approximately 30 cm long to allow easy manipulation and simultaneously avoid unintentional movement of the slide while operating the syringe. Image parts not to scale.

Figure 2

Raman spectra of algal samples cultivated with maximal Se concentration (32 mg Se/g DW/day) or no (0 mg Se/g DW/day) added Se. The signal of Se-Se vibrations was observed at 252 cm⁻¹. Each spectrum was averaged from 20 measurements. See Supplementary Materials Figures S5 and S6 for more Raman spectra from the algal samples.

Figure 3

Comparison of Se concentrations in algal samples during the cultivation measured by ICP-MS (red/grey stripes) and RS (green). Each bar represents the mean value for specific added Se concentration (3 values for ICP-MS; 120–160 spectra for RS). The thick horizontal grey line indicates the change of scale of both Y-axes for better clarity of low Se concentration measurements. The scale for higher Se concentrations is red for further visual separation. Horizontal dashed line represents the LOQ. Error-bars: 2 standard deviations (SD).

Figure 4

Relation between the Se concentrations measured by ICP-MS and the corresponding Raman signal intensity I_RR (Se) values. Each data-point represents the mean value from all the processed Raman spectra (40–60) obtained from a single sample. Error-bars: 2 SD.

Figure 5

Se accumulation by regular (living) and heat inactivated (dead) algal cells. Only the living (dark green) and dead (black) cells on day 4 accumulated significant amounts of Se during the cultivation period. The asterisk represents the sample that was significantly (p = 0.05) different from the control when analyzed with a paired t-test. Error-bars: 2 SD.

Figure 6

(a) Se supplemented C. vulgaris cells imaged by SEM. (b) EDX scan of the identical areas with Se signal presented in red—the saturation of these spots is directly proportional to Se concentration. (c) The binary version of the EDX scan after a stringent noise removal. The black spots represent the Se signal above the threshold. The Se concentrations detected by ICP-MS and the percentages of area coverage by the Se signal are listed below the images. Removal of the background noise revealed only the biggest Se clusters. The samples were selected to cover the entire interval of Se concentrations used in our experiments. Scale bar: 2 μm.

Figure 7

Se concentrations in the selected algal samples derived from the EDX map (on Figure 6), expressed in per cent of the image area covered by the Se signal, in relation to the Se respective concentrations measured by ICP-MS. Quasilinear dependence was found between the two data-sets.