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Supplementary Material for: A Novel Enrichment Culture Highlights Core Features of Microbial Networks Contributing to Autotrophic Fe(II) Oxidation Coupled to Nitrate Reduction

posted on 02.07.2021, 05:40 by Huang Y.-M., Straub D., Kappler A., Smith N., Blackwell N., Kleindienst S.
Fe(II) oxidation coupled to nitrate reduction (NRFO) has been described for many environments. Yet very few autotrophic microorganisms catalysing NRFO have been cultivated and their diversity, as well as their mechanisms for NRFO in situ remain unclear. A novel autotrophic NRFO enrichment culture, named culture BP, was obtained from freshwater sediment. After more than 20 transfers, culture BP oxidized 8.22 mM of Fe(II) and reduced 2.42 mM of nitrate within 6.5 days under autotrophic conditions. We applied metagenomic, metatranscriptomic, and metaproteomic analyses to culture BP to identify the microorganisms involved in autotrophic NRFO and to unravel their metabolism. Overall, twelve metagenome-assembled genomes (MAGs) were constructed, including a dominant Gallionellaceae sp. MAG (≥71% relative abundance). Genes and transcripts associated with potential Fe(II) oxidizers in culture BP, identified as a Gallionellaceae sp., Noviherbaspirillum sp., and Thiobacillus sp., were likely involved in metal oxidation (e.g., cyc2, mtoA), denitrification (e.g., nirK/S, norBC), carbon fixation (e.g., rbcL), and oxidative phosphorylation. The putative Fe(II)-oxidizing protein Cyc2 was detected for the Gallionellaceae sp. Overall, a complex network of microbial interactions among several Fe(II) oxidizers and denitrifiers was deciphered in culture BP that might resemble NRFO mechanisms in situ. Furthermore, 16S rRNA gene amplicon sequencing from environmental samples revealed 36 distinct Gallionellaceae taxa, including the key player of NRFO from culture BP (approx. 0.13% relative abundance in situ). Since several of these in situ-detected Gallionellaceae taxa were closely related to the key player in culture BP, this suggests that the diversity of organisms contributing to NRFO might be higher than currently known.