13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea

Marine pelagic redoxclines are zones of high dark CO2 fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO2 fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO2 incorporation in C-13-bicarbonate pulse experiments. The incorporation of C-13 into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in C-13 slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO2-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the C-13-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO2-fixing Proteobacteria within this habitat.