Lipid analysis of CO2-rich subsurface aquifers suggests an autotrophy-based deep biosphere with lysolipids enriched in CPR bacteria

Abstract

Sediment-hosted CO₂-rich aquifers deep below the Colorado Plateau (USA) contain a remarkable diversity of uncultivated microorganisms, including Candidate Phyla Radiation (CPR) bacteria that are putative symbionts unable to synthesize membrane lipids. The origin of organic carbon in these ecosystems is unknown and the source of CPR membrane lipids remains elusive. We collected cells from deep groundwater brought to the surface by eruptions of Crystal Geyser, sequenced the community, and analyzed the whole community lipidome over time. Characteristic stable carbon isotopic compositions of microbial lipids suggest that bacterial and archaeal CO₂ fixation ongoing in the deep subsurface provides organic carbon for the complex communities that reside there. Coupled lipidomic-metagenomic analysis indicates that CPR bacteria lack complete lipid biosynthesis pathways but still possess regular lipid membranes. These lipids may therefore originate from other community members, which also adapt to high in situ pressure by increasing fatty acid unsaturation. An unusually high abundance of lysolipids attributed to CPR bacteria may represent an adaptation to membrane curvature stress induced by their small cell sizes. Our findings provide new insights into the carbon cycle in the deep subsurface and suggest the redistribution of lipids into putative symbionts within this community.

Fig. 1. Community structure of 27 metagenomic samples from Crystal Geyser based on percent relative abundance of scaffolds carrying rpS3 sequences (clustered at 99% amino acid similarity).

Nonmetric multidimensional scaling based on the Bray–Curtis index. The connections show the trajectory of the different samples taken throughout the eruption cycle. Sample 01 was not included as it was an amplified library due to low biomass (see “Material and methods” for further details). Sample 26 was collected after the end of the major eruptions and is already part of the recovery phase (thus colored in pink). Black color indicates samples that were collected during transition between phases. Please note, that the sample was also size-fractioned into a 0.2-µm and a 0.1-µm filter. For details on individual rpS3 abundances please see Fig. S5 and Table S5.

Fig. 2. Carbon isotopic ratios and relative abundance of unsaturated intact polar lipids relative to the cycle of the geyser.

a Water pressure and temperature over the geyser cycle showing sourcing of fluids from the conduit (mixed), the deep aquifer, and the shallow aquifer from Ref. [12]. b Stable carbon isotope fractionation of archaeal lipids (phytane, released from archaeol), individual bacterial fatty acids (FA, released from diacylglycerols), bacterial lipids (weighted average of FA), and dissolved inorganic carbon (DIC) relative to CO₂ (εCO₂-Lipid) over the geyser cycle. Lines to the left of the panel show expected ranges of εCO₂-Lipid (accounting for up to 5‰ additional ¹³C-depletion of lipids relative to biomass, indicated by shaded areas) for the Calvin–Bassham–Benson (CBB; [–48]), the reductive tricarboxylic acid cycle (rTCA [46, 49, 50]), and the Wood–Ljungdahl pathway (WL, reductive acetyl-coenzyme A pathway; [39, 62, 63]). The blue dashed line indicates relative contribution of carbon fixation through the CBB cycle versus the rTCA cycle for bacterial lipids (assuming maximum fractionation due to high in situ [CO₂] and [DIC]). The red dashed line indicates the relative contribution of autotrophy versus heterotrophy (uptake of bacterial CBB/rTCA-fixed carbon) to archaeal lipid biomass, calculated from mass balance of δ¹³C values of bacterial and archaeal lipids (assuming maximum fractionation for archaeal autotrophy due to high in situ [CO₂] and [DIC]). c Relative abundance of unsaturated diacylglycerol membrane lipids (the number indicates the sum of double bonds in both acyl chains). The distribution is dominated by mono- and di-unsaturated diacylglycerols but polyunsaturated lipids (6–15 unsaturations) increase markedly in deep aquifer fluids. Grey shading indicates major eruptions, which source deep aquifer water under high pressure.

Fig 3. Correlation network analysis of relative abundances of organisms (rpS3 genes) and relative abundance of IPL signatures.

The primary lipids were defined based on a direct correlation of their relative abundance with rpS3 gene abundance (Bonferroni-corrected p value < 0.005). Secondary lipids showed a significant correlation with primary lipids and are indicative of a biological connection between the lipids (e.g., lipids from microbial symbionts or co-correlated organisms). Unspecific lipids shared primary lipids with different organism assignment. Due to visual limitations only few IPL names are displayed in the figure; all organisms to lipid correlations are provided in Table 1, raw data can be accessed in Tables S4 and S5.

Fig. 4. FTIR analysis of a small cell size fraction (post-0.2-µm filter collected onto a 0.1-µm filter).

a Field of view in FTIR, 1 × 1 mm (red square). b First five PCA loadings accounting for ~90% of the variance. They describe the directions of maximum variability of the analyzed system. The figure presets the first five vectors, that spectroscopically can be assigned, by similarity of shape and band position, to different types of lipids. c False color maps representing PCA scores PC1 and PC5, respectively. These maps show how the different lipids represented by the eigenvectors in (b), are distributed in the sample. The comparison of the spectral features of the loadings and the reference spectra in Fig. S11 allow assignment of PC1 to lysolipids and PC5 to unsaturated/branched lipids. The arrows point to a hotspot of cells indicating a particularly high distribution of lysolipids (PC1), surrounded by several smaller hotspots of unsaturated/branched lipids (PC5). Given the micrometric lateral resolution of the image (each pixel is 2.6 µm) it is possible to hypothesize that there is a small group of cells in the hotspot area, which is characterized by distinct membrane lipid composition. This can also be observed in other spots throughout the measured biomass. Loadings of the PCA over the whole 900–3700 cm⁻¹ spectral range are provided in Fig. S12. Scale bar 200 µm.

Fig. 5. Model for the acquisition and redistribution of carbon and lipids in the deep subsurface ecosystems of the Colorado Plateau (USA) accessible through Crystal Geyser.

Organic carbon and lipids are produced by Gallionella, Sulfurimonas, Altiarchaeum spp. or other autotrophs, redistributed through the ecosystem and acquired by other community members including CPR bacteria and DPANN archaea.