Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae

Abstract

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of the algae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. The differences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm(2) area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.

Seasonal Variations in Surface Metabolite Composition of Fucus vesiculosus and Fucus serratus from the Baltic Sea

“…Former studies observed that phenolic phlorotannins in the brown alga Cystoseira tamariscifolia [62] and the antifouling sesquiterpene caulerpenyne from Caulerpa taxifolia [30], exhibit annual cycles regulated by solar radiation, showing higher compound concentrations in months with greatest irradiance. This type of light-dependent metabolite production in macroalgae and their partial exudation in Fucus (actively by transport or passively by loss of integrity of surface cells) through its outer thallus surface as described for the pigment fucoxanthin [8, 10] or dissolved organic carbon [29] could be the main (i.e. statistically dominant) cause for the observed seasonal variance in surface metabolite composition.…”

“…For example, polyhalogenated and polyphenolic compounds were found at or near the surface of red and brown macroalgae [5–7]. Furthermore, the pigment fucoxanthin, the osmolyte dimethylsulphopropionate (DMSP) as well as the amino acid proline have been detected on the surface of the brown macroalgae Fucus vesiculosus [8–10]. Such surface-associated metabolites are also referred to as “surface metabolites”.…”

The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms

“…“Soft” surface extraction techniques (Nylund et al, 2007; Lachnit et al, 2010; Saha et al, 2011) and non-intrusive analytical techniques (e.g., confocal resonance Rahman spectroscopy; Grosser et al, 2012) allow for the isolation and analysis of compounds in the boundary layer. Bioassays should be used to verify activities of whole or fractionated extracts against foulers, consumers, pathogens.…”

Investigation of Microalgal Carotenoid Content Using Coherent Anti‐Stokes Raman Scattering (CARS) Microscopy and Spontaneous Raman Spectroscopy

“…However, Raman cross‐sections of typical molecules found in living cells are quite low necessitating longer acquisition times. The increase in scattering intensities achievable when using resonance Raman has been employed to examine metabolites and pigments on and in the vicinity of algal surfaces . However, photodegradation or photo‐bleaching and strong fluorescent background due to UV/Vis excitation wavelengths in resonance with electronic excited states limits its applicability for imaging pigment distribution inside diatoms.…”