Raman spectroscopy reveals a previously unknown extracellular cyanobacterial pigment with unique molecular signatures. The method enables precise in situ identification, expanding understanding of microbial photoprotection and supporting discovery of new pigment classes.
Study: Novel Raman signature evidences an unknown cyanobacterial pigment. Image Credit: luchschenF/Shutterstock
In a recent article published in the journal Scientific Reports, researchers investigates an extracellular pigment produced by the cyanobacterium Chamaesiphon polonicus, employing advanced optical spectroscopy techniques to identify its nature and assess whether it represents a new category of cyanobacterial UV-screening pigment.
Cyanobacterial Pigment Analysis
Cyanobacterial pigments serve essential biological roles, especially in photoprotection, which is critical for survival under intense solar ultraviolet radiation. Scytonemin and gloeocapsin are widely studied extracellular pigments that accumulate in the extracellular polymeric substances (EPS) surrounding cells, providing UV screening.
Raman microspectroscopy, an optical vibrational spectroscopy method that relies on inelastic light scattering from molecular vibrations, is a powerful, non-destructive technique that enables in situ pigment identification with micron-scale resolution. Previous Raman studies have successfully distinguished pigments like scytonemin at cellular and subcellular levels, highlighting the method's sensitivity to molecular structure and enabling pigment taxonomy.
Chamaesiphon genus cyanobacteria produce conspicuous extracellular pigments, but the pigment from C. polonicus has been enigmatic, with absorption spectra suggesting a carotenoid-like nature - though this was unconfirmed. This study harnesses Raman spectroscopy combined with chemometrics to explore this pigment’s vibrational signature and compare it to known microbial and synthetic pigments, emphasizing optical fingerprinting capacity to reveal novel pigment chemistry.
The Current Study
The cyanobacterial strain C. polonicus SAG 32.87 was cultured under controlled light and temperature conditions, providing biomass for both intact cells and isolated extracellular sheaths. Raman microspectroscopy employed multiple laser excitations in the visible-to-near-infrared range (514 nm, 633 nm, and 785 nm) to overcome fluorescence and improve spectral quality.
Spectral data were collected from intracellular and extracellular regions to isolate the pigment signature. High-resolution Raman mapping was used to spatially resolve pigment distribution, and computational techniques, including second derivative processing, distinguished overlapping bands. For comparative purposes, Raman spectra of 12 natural and two synthetic pigments from bacteria, cyanobacteria, fungi, and plants were recorded under similar conditions.
Chemometric methods, including non-metric multidimensional scaling (NMDS) and hierarchical clustering analysis (HCA), were applied to classify pigments based on spectral similarities, using spectral features as proxies for molecular structural properties. Correlations between spectral ordination and molecular parameters (e.g., molecular weight, topological polar surface area) were statistically analyzed to infer pigment complexity relative to known compounds.
Raman Spectra Reveals Novel Pigment
The Raman spectra of the C. polonicus extracellular pigment showed distinctive vibrational bands that differed markedly from the known signatures of scytonemin, gloeocapsin, carotenoids, and other microbial pigments. Contrary to prior assumptions based on absorption spectra, carotenoid-like vibrational features such as characteristic C=C stretching modes were absent.
Instead, the pigment exhibited several bands suggestive of aromatic molecular structures with potential nitrogen incorporation, pointed to by carbon-nitrogen bond vibrations, hinting at a nitrogen-containing aromatic heterocycle, possibly an indole-like ring. Notably, a strong Raman band near 1740 cm^-1 indicated the presence of carbonyl (C=O) functional groups such as ketones or esters, consistent with an aromatic backbone bearing side chains. These optical signatures and band broadening may be influenced by the pigment’s amorphous state or heterogeneous molecular interactions within the EPS matrix.
Chemometric analyses highlighted the pigment’s uniqueness: it formed a distinct cluster, separate from scytonemin, gloeocapsin, and other tested pigments, including anthraquinone derivatives and plant flavonoids like quercetin. While some spectral overlap with quercetin was observed, the overall molecular profile did not support a direct match.
The absence of carotenoid bands and the presence of nitrogenous aromatic features suggest a novel pigment class, potentially related to polyketide biosynthesis pathways, though no known bacterial analogs exist for azaphilone-type pigments implicated in fungal UV protection. Raman mapping demonstrated that this pigment localized in the extracellular sheath, consistent with its protective function.
The sensitivity of Raman spectroscopy to the molecular and electronic structure of pigments, combined with chemometric clustering, proved critical for distinguishing this novel pigment from others. The study underscores Raman microspectroscopy’s strength as a diagnostic optical tool for microbial pigment discovery, enabling in situ detection and spatial localization within biofilms.
Such optical fingerprints serve not only functional but taxonomic purposes, opening avenues for the identification of microbial lineages and their adaptive traits, both in modern environments and the fossil record, where pigment preservation can inform evolutionary histories.
Significance of the Work
The optical and vibrational characterization of the extracellular pigment produced by Chamaesiphon polonicus SAG 32.87 revealed a molecular signature distinct from previously characterized cyanobacterial pigments scytonemin and gloeocapsin. Raman microspectroscopy established the pigment as a potentially new class of extracellular cyanobacterial UV-screening compound, distinguished by unique vibrational bands indicative of nitrogenous aromatic structures and carbonyl functionalities.
These findings demonstrate the potential of optical vibrational spectroscopy combined with chemometrics to discover and classify novel microbial pigments with ecological and evolutionary significance. The work highlights the importance of further optical studies to unravel the diversity of photoprotective strategies in cyanobacteria and showcases how Raman spectroscopy can extend microbial pigment catalogs, providing new markers for modern and ancient biological systems.
Journal Reference
Lara Y.J., Lambion A., et al. (2026). Novel Raman signature evidences an unknown cyanobacterial pigment. Sci Rep (2026). https://doi.org/10.1038/s41598-026-46521-x, https://www.nature.com/articles/s41598-026-46521-x