Carotenoid-producing bacteria from the aphotic zones of Morca Cave (Türkiye) and the cave’s relevance as a terrestrial analogue for subsurface astrobiology

Abstract

Caves serve as terrestrial analogues for extraterrestrial subsurface environments: permanently dark, nutrient-poor and physicochemically stable on biologically relevant timescales. Morca Cave (Anamur, Mersin, Türkiye) extends to depths below 1200 m and is one of Türkiye’s deepest and least disturbed cave systems. Within our study, Gram-positive bacteria dominated at four of the five sampling sites (64.4–86.7%), while Gram-negative bacteria predominated at only one site (51.4%). Thirty-nine isolates produced visible yellow or orange pigmentation under laboratory conditions and were selected for further characterisation. Thin-layer chromatography detected xanthophyll-type pigments in 35 of these 39 isolates and a β-carotene-range spot in 22. HPLC tentatively identified a lutein-like xanthophyll in 36 of 39 isolates at concentrations of 1.34–34.65 ppb. Only two isolates yielded quantifiable HPLC peaks tentatively assigned to putative β-carotene; the remaining high-Rf TLC bands likely correspond to other apolar carotenoids. All pigment identifications are tentative, based on TLC and HPLC matching against authentic standards, and require LC-MS/MS or NMR confirmation. 16S rRNA gene sequencing assigned 31 of the 39 pigmented isolates to known taxa. Actinomycetia accounted for 20 isolates, Betaproteobacteria for six, Alphaproteobacteria for two, Bacilli for two and Gammaproteobacteria for one. Lutein-like pigment was recovered from bacteria isolated from surface samples at all three depths (−400, −620, −680 m) and from sediment samples at −400 and −620 m under standard heterotrophic cultivation; these isolates therefore retain biosynthetic capacity for carotenoid production independent of light. To our knowledge, this is the first study combining carotenoid profiling with 16S rRNA gene-based taxonomic assignment across a vertical depth gradient in a Turkish deep-karst cave system. The pigment concentration ranges and taxonomic distribution reported here provide an initial empirical reference dataset for carotenoid biosignature research in aphotic, oligotrophic subsurface environments proposed as terrestrial analogues for Mars and icy-moon habitability studies.