Geobacterales is a recently proposed order comprising members who originally belonged to the well-known family Geobacteraceae, which is a key group in terrestrial ecosystems involved in biogeochemical cycles and has been widely investigated in bioelectrochemistry and bioenergy fields. Previous studies have illustrated the taxonomic structure of most members in this group based on genomic phylogeny; however, several members are still in a pendent or chaotic taxonomic status owing to the lack of genome sequences. To address this issue, we performed this taxonomic reassignment using currently available genome sequences, along with the description of two novel paddy soil-isolated strains, designated Red51^T and Red69^T, which are phylogenetically located within this order. Phylogenomic analysis based on 120 ubiquitous single-copy proteins robustly separated the species Geobacter luticola from other known genera and placed the genus Oryzomonas (fam. Geobacteraceae) into the family ‘Pseudopelobacteraceae’; thus, a novel genus Geomobilimonas is proposed, and the family ‘Pseudopelobacteraceae’ was emended. Moreover, genomic comparisons with similarity indexes, including average amino acid identity (AAI), percentage of conserved protein (POCP), and average nucleotide identity (ANI), showed proper thresholds as genera boundaries in this order with values of 70%, 65%, and 74% for AAI, POCP, and ANI, respectively. Based on this, the three species Geobacter argillaceus, Geobacter pelophilus, and Geobacter chapellei should be three novel genera, for which the names Geomobilibacter, Geoanaerobacter, and Pelotalea are proposed, respectively. In addition, the two novel isolated strains phylogenetically belonged to the genus Geomonas, family Geobacteraceae, and shared genomic similarity values higher than those of genera boundaries, but lower than those of species boundaries with each other and their neighbors. Taken together with phenotypic and chemotaxonomic characteristics similar to other Geomonas species, these two strains, Red51^T and Red69^T, represent two novel species in the genus Geomonas, for which the names Geomonas azotofigens sp. nov. and Geomonas diazotrophica sp. nov. are proposed, respectively.

In rice paddy soil, biological nitrogen fixation is important for sustaining soil nitrogen fertility and rice growth. Anaeromyxobacter and Geobacteriaceae, iron-reducing bacteria belonging to Deltaproteobacteria, are newly discovered nitrogen-fixing bacteria dominant in paddy soils. They utilize acetate, a straw-derived major carbon compound in paddy soil, as a carbon and energy source, and ferric iron compounds as electron acceptors for anaerobic respiration. In our previous paddy field experiments, a significant increase in soil nitrogen-fixing activity was observed after the application of iron powder to straw-returned paddy field soil. In addition, combining iron application with 60–80% of the conventional nitrogen fertilizer rate could maintain rice yields similar to those with the conventional nitrogen fertilization rate. It was thus suggested that iron application to paddy soil increased the amount of nitrogen fixed in the soil by enhancing nitrogen fixation by diazotrophic iron-reducing bacteria. The present study was conducted to directly verify this suggestion by ¹⁵N-IRMS analysis combined with ¹⁵N-DNA-stable isotope probing of iron-applied and no-iron-applied plot soils in an experimental paddy field. In no-iron-applied native paddy soil, atmospheric ¹⁵N₂ was incorporated into the soil by biological nitrogen fixation, in which diazotrophic iron-reducing bacteria were the most active drivers of nitrogen fixation. In iron-applied paddy soil, the amount of ¹⁵N incorporated into the soil was significantly higher due to enhanced biological nitrogen fixation, especially via diazotrophic iron-reducing bacteria, the most active drivers of nitrogen fixation in the soil. Thus, our previous suggestion was verified. This study provided a novel picture of active nitrogen-fixing microorganisms dominated by diazotrophic iron-reducing bacteria in paddy soil, and directly proved the effectiveness of iron application to enhance their nitrogen fixation and increase the incorporation of atmospheric nitrogen into soil. The enhancement of biological nitrogen fixation in paddy fields by iron application may lead to novel and unique paddy soil management strategies to increase soil nitrogen fertility and ensure rice yields with reduced nitrogen fertilizer input and lower environmental nitrogen burdens.

Bacteria of the family Geobacteraceae are particularly common and deeply involved in many biogeochemical processes in terrestrial and freshwater environments. As part of a study to understand biogeochemical cycling in freshwater sediments, three iron-reducing isolates, designated as Red96^T, Red100^T, and Red88^T, were isolated from the soils of two paddy fields and pond sediment located in Japan. The cells were Gram-negative, strictly anaerobic, rod-shaped, motile, and red-pigmented on agar plates. Growth of these three strains was coupled to the reduction of Fe(III)-NTA, Fe(III) citrate, and ferrihydrite with malate, methanol, pyruvate, and various organic acids and sugars serving as alternate electron donors. Phylogenetic analysis based on the housekeeping genes (16S rRNA gene, gyrB, rpoB, nifD, fusA, and recA) and 92 concatenated core genes indicated that all the isolates constituted a coherent cluster within the family Geobacteraceae. Genomic analyses, including average nucleotide identity and DNA–DNA hybridization, clearly differentiated the strains Red96^T, Red100^T, and Red88^T from other species in the family Geobacteraceae, with values below the thresholds for species delineation. Along with the genomic comparison, the chemotaxonomic features further helped distinguish the three isolates from each other. In addition, the lower values of average amino acid identity and percentage of conserved protein, as well as biochemical differences with their relatives, indicated that the three strains represented a novel genus in the family Geobacteraceae. Hence, we concluded that strains Red96^T, Red100^T, and Red88^T represented three novel species of a novel genus in the family Geobacteraceae, for which the names Oryzomonas japonicum gen. nov., sp. nov., Oryzomonas sagensis sp. nov., and Oryzomonas ruber sp. nov. are proposed, with type strains Red96^T (= NBRC 114286^T = MCCC 1K04376^T), Red100^T (= NBRC 114287^T = MCCC 1K04377^T), and Red88^T (= MCCC 1K03694^T = JCM 33033^T), respectively.

Dissimilatory nitrate reduction to ammonium (DNRA), driven by nitrate-ammonifying bacteria, is an increasingly appreciated nitrogen-cycling pathway in terrestrial ecosystems. This process reportedly generates nitrous oxide (N ₂ O), a strong greenhouse gas with ozone-depleting effects. However, it remains poorly understood how N ₂ O is produced by environmental nitrate-ammonifiers and how to identify DNRA-derived N ₂ O. In this study, we characterize two novel enzymatic pathways responsible for N ₂ O production in Geobacteraceae strains, which are predominant nitrate-ammonifying bacteria in paddy soils. The first pathway involves a membrane-bound nitrate reductase (Nar) and a hybrid cluster protein complex (Hcp–Hcr) that catalyzes the conversion of NO ₂ ⁻ to NO and subsequently to N ₂ O. The second pathway is observed in Nar-deficient bacteria, where the nitrite reductase (NrfA) generates NO, which is then reduced to N ₂ O by Hcp–Hcr. These enzyme combinations are prevalent across the domain Bacteria. Moreover, we observe distinctive isotopocule signatures of DNRA-derived N ₂ O from other established N ₂ O production pathways, especially through the highest ¹⁵ N-site preference (SP) values (43.0‰–49.9‰) reported so far, indicating a robust means for N ₂ O source partitioning. Our findings demonstrate two novel N ₂ O production pathways in DNRA that can be isotopically distinguished from other pathways. IMPORTANCE: Stimulation of DNRA is a promising strategy to improve fertilizer efficiency and reduce N ₂ O emission in agriculture soils. This process converts water-leachable NO ₃ ⁻ and NO ₂ ⁻ into soil-adsorbable NH ₄ ⁺ , thereby alleviating nitrogen loss and N ₂ O emission resulting from denitrification. However, several studies have noted that DNRA can also be a source of N ₂ O, contributing to global warming. This contribution is often masked by other N ₂ O generation processes, leading to a limited understanding of DNRA as an N ₂ O source. Our study reveals two widespread yet overlooked N ₂ O production pathways in Geobacteraceae , the predominant DNRA bacteria in paddy soils, along with their distinctive isotopocule signatures. These findings offer novel insights into the role of the DNRA bacteria in N ₂ O production and underscore the significance of N ₂ O isotopocule signatures in microbial N ₂ O source tracking.