¹⁵N- and ²H proteomic stable isotope probing links nitrogen flow to archaeal heterotrophic activity

Summary: Understanding how individual species contribute to nutrient transformations in a microbial community is critical to prediction of overall ecosystem function. We conducted microcosm experiments in which floating acid mine drainage (AMD) microbial biofilms were submerged - recapitulating the final stage in a natural biofilm life cycle. Biofilms were amended with either ¹⁵NH₄⁺ or deuterium oxide (²H₂O) and proteomic stable isotope probing (SIP) was used to track the extent to which different members of the community used these molecules in protein synthesis across anaerobic iron-reducing, aerobic iron-reducing and aerobic iron-oxidizing environments. Sulfobacillus spp. synthesized ¹⁵N-enriched protein almost exclusively under iron-reducing conditions whereas the Leptospirillum spp. synthesized ¹⁵N-enriched protein in all conditions. There were relatively few ¹⁵N-enriched archaeal proteins, and all showed low atom% enrichment, consistent with Archaea synthesizing protein using the predominantly ¹⁴N biomass derived from recycled biomolecules. In parallel experiments using ²H₂O, extensive archaeal protein synthesis was detected in all conditions. In contrast, the bacterial species showed little protein synthesis using ²H₂O. The nearly exclusive ability of Archaea to synthesize proteins using ²H₂O may be due to archaeal heterotrophy, whereby Archaea offset deleterious effects of ²H by accessing ¹H generated by respiration of organic compounds.