Abstract

Most acidic environments are characterized by low concentrations of nitrogen. However, this element is a key component in many biological macromolecules, and therefore the ability of acidophiles to fix nitrogen can be a key characteristic for sustaining the entire indigenous microbial community. Microbial analysis of PLS at the Escondida heap leaching operation in northern Chile revealed a diversity of bacteria to be present, but only one of which (Acidithiobacillus ferrooxidans) was known to be capable of fixing nitrogen. Metagenome analysis of an acid biofilm community in Iron Mountain that was dominated by Leptospirillum “Group II” (affiliated to Leptospirillum ferriphilum) had failed to find nif genes, leading to the conclusion that member of this species cannot fix nitrogen. In contrast, recent annotation of genome sequence data from a pure strain of L. ferriphilum isolated from a second copper mine in Chile revealed the presence of genes involved in nitrogen fixation. This information prompted the screening of over thirteen strains of Leptospirillum spp. for the presence of the nifH gene by PCR. In addition, the growth of several strains of L. ferriphilum was tested in nitrogen-free liquid media. As expected the nifH gene was detected in all seven L. ferrooxidans strains tested and in the type strain of “L. ferrodiazotrophum”. Surprisingly, a partial nifH gene was also amplified from three out of five strains of L. ferriphilum (including an isolate from the Escondida operation) suggesting that those strains could be able to fix nitrogen. Successful growth of these nifH-positive strains in N-free medium confirmed that they are diazotrophic. These results revealed that at least some strains of L. ferriphilum are critical to heap bioleaching operations by supporting the microbial community through nitrogen fixation, in addition to their key role as iron-oxidizers.