Black stains of Lascaux Cave involve different melanin synthesis pathways as shown by Alkaline/H2O2 hydrolysis and UHPLC-ESI/QTOF analysis of pigmented fungal isolates and stains from cave walls

Abstract

Lascaux cave in southwestern France is a UNESCO landmark famous for its Paleolithic engravings and paintings, but it suffered from microbial outgrowth and black strain formation following unbalanced touristic operations and chemical treatments. Pigments synthesized by black fungi isolated from these stains were previously identified as melanin, but a limited range of black fungi was assessed and black stains themselves have been investigated by surface-enhanced Raman spectroscopy, only. This study aimed at identifying the origins and chemical features of the melanin polymers produced by 10 black fungal strains isolated from Lascaux black stains. Fungi were identified by ITS2 sequencing. Mycelial pigment extracts were analyzed by UHPLC/ESI-QTOF after alkaline H₂O₂ hydrolysis, which allowed to detect simultaneously monomers of different melanin types. Analytical markers from several biosynthetic pathways of melanin were evidenced in all fungal isolates, and some strains showed the ability to produce melanin from at least five different pathways, which to our knowledge constitutes the first report of such a variety of means to achieve black pigmentation within a single fungal strain. As proof of principle, this method was applied to black stains directly collected from the cave and revealed three to four melanin types in samples from the Apse, the Nave and the Passage. Some of these melanin types corresponded to those of the fungal isolates analyzed, namely Alternaria alternata, Acremonium nepalense or Ochroconis lascauxensis. This is the first characterization of the types of melanin synthesized on Lascaux surfaces. Results emphasize the interest of this method to understand different pigmentation patterns in caves and to analyze melanin pigments from diverse biological matrices.