Metagenomic reconstruction of nitrogen cycling pathways in a CO₂-enriched grassland ecosystem

The nitrogen (N) cycle is a collection of important biogeochemical pathways mediated by microbial communities and is an important constraint in response to elevated CO₂ in many terrestrial ecosystems. Previous studies attempting to relate soil N cycling to microbial genetic data mainly focused on a few gene families by PCR, protein assays and functional gene arrays, leaving the taxonomic and functional composition of soil microorganisms involved in the whole N cycle less understood. In this study, 24 soil samples were collected from the long-term experimental site, BioCON, in 2009. A shotgun metagenome sequencing approach was employed to survey the microbial gene families involved in soil N cycle in the grassland that had been exposed to elevated CO₂ (eCO₂) for >12 years. In addition to evaluating the responses of major N cycling gene families to long-term eCO₂, we also aimed to characterize the taxonomic and functional composition of these gene families involved in soil N transformations. At the taxonomic level, organic N metabolism and nitrate reduction had the most diverse microbial species involved. The distinct taxonomic composition of different N cycling processes suggested that the complex N cycle in natural ecosystems was a result of multiple processes by many different microorganisms. Belowground microbial communities that mediate N cycling responded to eCO₂ in several different ways, including through stimulated abundances of the gene families related with organic decomposition, dissimilatory nitrate reduction, and N₂ fixation, and suppressed abundances of the gene families in glutamine synthesis and anammox. This study provides a genetic basis of the microorganisms involved in key processes in the N cycle in complex ecosystems, and shows that long-term eCO₂ selectively affects N cycling pathways instead of tuning up every process.