Quantifying genome-specific carbon fixation in a 750-meter deep subsurface hydrothermal microbial community

Abstract

Dissolved inorganic carbon has been hypothesized to stimulate microbial chemoautotrophic activity as a biological sink in the carbon cycle of deep subsurface environments. Here, we tested this hypothesis using quantitative DNA stable isotope probing of metagenome-assembled genomes (MAGs) at multiple 13C-labeled bicarbonate concentrations in hydrothermal fluids from a 750-m deep subsurface aquifer in the Biga Peninsula (Turkey). The diversity of microbial populations assimilating 13C-labeled bicarbonate was significantly different at higher bicarbonate concentrations, and could be linked to four separate carbon-fixation pathways encoded within 13C-labeled MAGs. Microbial populations encoding the Calvin-Benson-Bassham cycle had the highest contribution to carbon fixation across all bicarbonate concentrations tested, spanning 1-10 mM. However, out of all the active carbon-fixation pathways detected, MAGs affiliated with the phylum Aquificae encoding the reverse tricarboxylic acid (rTCA) pathway were the only microbial populations that exhibited an increased 13C-bicarbonate assimilation under increasing bicarbonate concentrations. Our study provides the first experimental data supporting predictions that increased bicarbonate concentrations may promote chemoautotrophy via the rTCA cycle and its biological sink for deep subsurface inorganic carbon.

Keywords: DNA stable isotope probing; deep biosphere; microbial carbon fixation; qSIP.

Figure 1.

Organic and inorganic carbon concentrations and 16S rRNA gene concentrations (A) A positive correlation between DOC and DIC in hydrothermal fluids at multiple sites in Biga Peninsula (see Fig. S1, Supporting Information, for map). The 750-m deep borehold (Bardakçılar geothermal field) chosen for the qSIP experiment is indicated. (B) Concentration of 16S rRNA genes at the beginning and end of the qSIP incubations, using hydrothermal fluids obtained from the 750-m deep borehole at Bardakçılar geothermal field.

Figure 2.

Increased DNA buoyant density of 16S rRNA genes in ¹³C-bicarbonate incubations relative to unlabeled controls, at 1 mM (A), 5 mM (B), and 10 mM (C) bicarbonate concentrations. Three separate ultracentrifugation and density-gradient fractionation replicates were performed for DNA extracted from each ¹³C (solid lines, filled symbols) and control (dashed lines, open symbols) bottle incubation at the three bicarbonate concentrations. The plots show the normalized relative abundance of 16S rRNA genes measured by qPCR (y-axis) as a function of density (x-axis) in three replicate ultracentrifugation tubes. The distribution of 16S rRNA gene densities in each replicate tube provided an estimate of the average DNA density and 90% CI (n = 3), which is represented as diamonds and error bars above each panel. The shaded area represents the density range for fractions that were selected for high-throughput Illumina sequencing of 16S rRNA genes for qSIP.

Figure 3.

Linking ¹³C-DIC-incorporating MAGs with the encoded carbon-fixation pathways and mixotrophic or facultative autotrophic potential. From top to bottom: ¹³C-labeled MAGs were grouped based on the presence or absence (heterotrophs) of a carbon-fixation pathway. MAGs encoding a carbon-fixation pathway (left side) were sorted based on the increase in mixotrophic or facultative autotrophic potential defined as encoded ABC-transporters for external organic substrates (bottom heatmap). Quantitative assimilation of ¹³C-bicarbonate by each MAG at the three substrate concentrations is shown in the middle plot. Individual points represent EAF values of specific OTUs phylogenetically linked to the MAGs (Fig. S9, Supporting Information). Error bars: 90% CI based on three separate CsCl density gradients (see Fig. 2). The lower heatmaps shows the metabolic pathway (KEGG) completeness of each MAG.

Figure 4.

Total carbon assimilation by ¹³C-labeled MAGs encoding different carbon-fixation pathways. The y-axis is the sum of MAG ¹³C-EAF values per group using MAG absolute abundance based on qPCR. Error bars: 90% CI based on three density-gradient fractionation replicates per treatment (Fig. 2). The total ¹³C-assimilation by rTCA-encoding bacteria was positively correlated with bicarbonate concentration (R² = 0.94), no positive correlations were observed for any of the other groups of organisms encoding different carbon-fixation pathways (CBB, WLP, and 3-HP).