Development of a ddPCR approach for the absolute quantification of soil microorganisms involved in atmospheric CO2 fixation

Carbon fixing microorganisms (CFMs) play a crucial role in soil carbon (C) cycling contributing to carbon uptake and sequestration through various metabolic pathways. Despite their significance, quantification of the absolute abundance of CFMs in soils remains elusive. This study employed a digital droplet PCR (ddPCR) approach to quantify the abundance of key and emerging CFM pathways in fen and bog across different depths (0-15 cm). Targeting total prokaryotes (16S rRNA gene), oxygenic phototrophs (23S rRNA gene), aerobic anoxygenic phototrophic bacteria (AAnPB, pufM gene), and chemoautotrophs (cbbL gene), we optimized ddPCR conditions to achieve absolute quantification of these genes. Overall, our results revealed that oxygenic phototrophs were the most abundant CFMs, constituting 12% of total prokaryotic abundance, followed by chemoautotrophs (10%) and AAnPBs (9%). Fen exhibited higher gene concentrations than bog. Depth variations were also observed, differing between fen and bog for all genes. Our findings highlight the abundance of oxygenic phototrophs and chemoautotrophs in peatlands, challenging previous estimations that relied solely on oxygenic phototrophs for microbial CO2 fixation assessments. Incorporating absolute gene quantification is crucial for a comprehensive understanding of microbial contributions to soil processes, shedding light on the intricate mechanisms of soil functioning in peatlands.