Microbial oxygenic photosynthesis in thermal habitats is thought to be performed by Bacteria in circumneutral to alkaline systems (pH > 6) and by Eukarya in acidic systems (pH < 3), yet the predominant oxygenic phototrophs in thermal environments with pH values intermediate to these extremes have received little attention. Sequencing of 16S and 18S rRNA genes was performed on samples from twelve hot springs in Yellowstone National Park (Wyoming, USA) with pH values from 3.0 to 5.5, revealing that Cyanobacteria of the genus Chlorogloeopsis and algae of the genus Cyanidioschyzon (phylum Rhodophyta) coexisted in ten of these springs. Cyanobacteria were detected at pH values as low as 3.0, challenging the paradigm of Cyanobacteria being excluded below pH values of 4.0. Cyanobacterial 16S rRNA genes were more abundant than rhodophyte 18S rRNA genes by up to 7 orders of magnitude, with rhodophyte template abundance approaching that of Cyanobacteria only at the most acidic sites. Light-driven carbon fixation was observed at two sites where chlorophyll a was detected but not at two other sites where chlorophyll a was not detected. Collectively, these observations suggest that many of the rhodophyte 18S rRNA gene sequences were from inactive cells. Fluctuations in the supply of meteoric water likely contributes to physicochemical variability in these springs, leading to transitions in photosynthetic community composition. Spatial, but perhaps not temporal, overlap in the habitat ranges of bacterial and eukaryal oxygenic phototrophs indicates that the notion of a sharp transition between these lineages with respect to pH is unwarranted.

In high-pH ($\text{pH}>10$) fluids that have participated in low-temperature ($<150\,^{\circ}\text{C}$) serpentinization, the dominant form of C is often methane (CH$_{4}$), but the origin of this CH$_{4}$ is uncertain. To assess CH$_{4}$ origin during low-temperature serpentinization, we pumped fluids from aquifers within the Samail Ophiolite, Oman. We determined fluid chemical compositions, analyzed taxonomic profiles of fluid-hosted microbial communities, and measured isotopic compositions of hydrocarbon gases. We found that 16S rRNA gene sequences affiliated with methanogens were widespread in the aquifer. We measured clumped isotopologue ($^{13}$CH$_{3}$D and $^{12}$CH$_{2}$D$_{2}$) relative abundances less than equilibrium, consistent with substantial microbial CH$_{4}$ production. Further, we observed an inverse relationship between dissolved inorganic C concentrations and $\delta^{13}\text{C}_{\text{CH}_{4}}$ across fluids bearing microbiological evidence of methanogenic activity, suggesting that the apparent C isotope effect of microbial methanogenesis is modulated by C availability. A second source of CH$_{4}$ is evidenced by the presence of CH$_{4}$-bearing fluid inclusions in the Samail Ophiolite and our measurement of high $\delta^{13}\text{C}$ values of ethane and propane, which are similar to those reported in studies of CH$_{4}$-rich inclusions in rocks from the oceanic lithosphere. In addition, we observed 16S rRNA gene sequences affiliated with aerobic methanotrophs and, in lower abundance, anaerobic methanotrophs, indicating that microbial consumption of CH$_{4}$ in the ophiolite may further enrich CH$_{4}$ in $^{13}$C. We conclude that substantial microbial CH$_{4}$ is produced under varying degrees of C limitation and mixes with abiotic CH$_{4}$ released from fluid inclusions. This study lends insight into the functioning of microbial ecosystems supported by water/rock reactions.