Non-cyanobacterial diazotrophs mediate dinitrogen fixation in biological soil crusts during early crust formation

Biological soil crusts (BSCs) are key components of ecosystem productivity in arid lands and they cover a substantial fraction of the terrestrial surface. In particular, BSC N-2-fixation contributes significantly to the nitrogen (N) budget of arid land ecosystems. In mature crusts, N-2-fixation is largely attributed to heterocystous cyanobacteria; however, early successional crusts possess few N-2-fixing cyanobacteria and this suggests that microorganisms other than cyanobacteria mediate N-2-fixation during the critical early stages of BSC development. DNA stable isotope probing with N-15(2) revealed that Clostridiaceae and Proteobacteria are the most common microorganisms that assimilate N-15(2) in early successional crusts. The Clostridiaceae identified are divergent from previously characterized isolates, though N-2-fixation has previously been observed in this family. The Proteobacteria identified share 498.5% small subunit rRNA gene sequence identity with isolates from genera known to possess diazotrophs (for example, Pseudomonas, Klebsiella, Shigella and Ideonella). The low abundance of these heterotrophic diazotrophs in BSCs may explain why they have not been characterized previously. Diazotrophs have a critical role in BSC formation and characterization of these organisms represents a crucial step towards understanding how anthropogenic change will affect the formation and ecological function of BSCs in arid ecosystems.