Metagenomic analysis of microbial community structure and metabolic diversity in experimental vernal pools with and without NO3- addition

Background/Question/Methods

Human activities have greatly increased nitrogen (N) levels in terrestrial and aquatic habitats through atmospheric deposition of N and nutrient runoff into aquatic environments. N enrichment of both aquatic and terrestrial habitats can have large effects on N cycling and ecosystem processes. Because of the significant role microorganisms have in N cycling, high inputs of nitrogenous compounds, such as nitrate (NO₃^-), into natural ecosystems could have cascading effects on microbial community structure, the metabolic processes that microbes perform, and the linkages between the two. We created two metagenomes to investigate the response of microbial communities to NO₃^- enrichment in a microcosm study. Microcosms were constructed using soil from a natural vernal pool and freshly fallen leaf litter and either were enriched with a solution of 10 mg NO₃^--N (+NO₃^-) or received distilled water as a control (–N). Denitrification was measured in each microcosm using a modified acetylene block technique. Metagenomes were created by 454 pyrosequencing on a GS-FLX platform and analyzed unassembled with a BLASTx comparison to the SEED database at the MG-RAST server. Each metagenome consisted of over 9,000 taxonomic and over 5,000 metabolic environmental gene tags (EGTs).   

Results/Conclusions

Even though the combined metagenomes contained over 15,000 metabolic EGTs, only five matched with denitrification genes, suggesting that denitrifiers were in low abundance in the microcosms. Low numbers of denitrifier EGTs were found in both metagenomes despite significantly different denitrification rates (30.3 mg N*kg^-1*d^-1 in the +NO₃^- treatment but denitrification was undetected in the –N treatment). A higher proportional representation of EGTs matching with NO₃^- and nitrite ammonification genes was seen in the –N metagenome, and EGTs matching with genes for nitrosative stress, a process which relieves the stress of nitric oxide accumulation during ammonification, were only detected in the –N metagenome. This suggests that in the absence of NO₃^- enrichment, ammonification was an important process for microbial growth. Ammonia assimilation EGTs were also proportionally higher in the –N metagenome, possibly a response to the build up of ammonium in the low NO₃^- microcosms. In addition, we found higher proportional representation of Acidobacteria and Alphaproteobacteria EGTs in the +NO₃^- metagenome, suggesting that organisms from these groups may increase with NO₃^- enrichment. Overall, our metagenomic analysis indicates that NO₃^- inputs may change the composition of microbial community structure and affect N metabolism in vernal pools.