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

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.

亚铁（Fe(II)）氧化耦合硝酸盐还原（nitrate reduction, NRFO）过程已在多种环境中被报道。然而，目前仅分离获得极少数量能催化NRFO的自养微生物，且此类微生物的多样性及其原位NRFO作用机制仍未明确。本研究从淡水沉积物中获取一株新型自养NRFO富集培养物，将其命名为BP培养物。经过20余次传代培养后，BP培养物在自养条件下可于6.5天内氧化8.22 mM的Fe(II)并还原2.42 mM的硝酸盐。本研究对BP培养物开展宏基因组学（metagenomic）、宏转录组学（metatranscriptomic）及宏蛋白质组学（metaproteomic）分析，以鉴定参与自养NRFO的微生物并解析其代谢通路。总计构建得到12个宏基因组组装基因组（metagenome-assembled genomes, MAGs），其中优势菌为相对丰度≥71%的披毛菌科（Gallionellaceae）未定种MAG。BP培养物中经鉴定的潜在Fe(II)氧化菌包括披毛菌科未定种、新草螺菌属（Noviherbaspirillum）未定种及硫杆菌属（Thiobacillus）未定种，与之相关的基因与转录本可能参与金属氧化（如cyc2、mtoA）、反硝化作用（如nirK/S、norBC）、碳固定（如rbcL）及氧化磷酸化过程。研究人员在披毛菌科未定种中检测到推定的Fe(II)氧化蛋白Cyc2。综上，BP培养物中解析得到由多种Fe(II)氧化菌与反硝化菌构成的复杂微生物互作网络，该网络或与原位NRFO作用机制相似。此外，对环境样本的16S rRNA基因扩增子测序结果显示，共存在36个不同的披毛菌科类群，其中包含BP培养物中的NRFO关键功能菌（原位相对丰度约为0.13%）。鉴于这些原位检测到的披毛菌科类群中有多个与BP培养物中的关键功能菌亲缘关系密切，这表明参与NRFO的微生物多样性可能高于当前认知。