No abstract

Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2−) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. 15N-labeling experiments revealed that NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH4+ oxidation with electrode as electron acceptor. Complete NH4+ oxidation to N2 without accumulation of NO2− and NO3− was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen.

To date, six candidate genera of anaerobic ammonium-oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus 'Candidatus Jettenia'. Planctomycete KSU-1 was found to be a mesophilic (20-42.5°C) and neutrophilic (pH 6.5-8.5) bacterium with a maximum growth rate of 0.0020 h(-1) . Planctomycete KSU-1 cells showed typical physiological and structural features of anammox bacteria; i.e. (29) N2 gas production by coupling of (15) NH4 (+) and (14) NO2 (-) , accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone-7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl-CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU-1 cells. On the basis of these experimental results, we proposed the name 'Ca. Jettenia caeni' sp. nov. for the bacterial clade of the planctomycete KSU-1.

In aerobic granular sludge (AGS) systems, different-sized microbial aggregates having different solids retention time (SRT) coexist in the same reactor compartment and are subjected to the same influent wastewater. Thus, the AGS system provides a unique ecosystem to study the importance of local (species sorting) and regional (immigration) processes in bacterial community assembly. The microbial communities of different-sized aggregates (flocs <0.2 mm, small granules (0.2–1.0 mm) and large granules >1.0 mm), influent wastewater, excess sludge and effluent of a full-scale AGS plant were characterized over a steady-state operation period of 6 months. Amplicon sequencing was integrated with mass balance to determine the SRT and net growth rate of operational taxonomic units (OTUs). We found strong evidence of species sorting as opposed to immigration, which was significantly higher at short SRT (i.e., flocs and small granules) than that at long SRT (large granules). Rare OTUs in wastewater belonging to putative functional groups responsible for nitrogen and phosphorus removal were progressively enriched with an increase in microbial aggregates size. In contrast, fecal- and sewage infrastructure-derived microbes progressively decreased in relative abundance with increase in microbial aggregate size. These findings highlight the importance of AGS as a unique model ecosystem to study fundamental microbial ecology concepts.

Summary Although metabolic pathways and associated enzymes of anaerobic ammonium oxidation (anammox) of ‘Ca. Kuenenia stuttgartiensis’ have been studied, those of other anammox bacteria are still poorly understood. NO2− reduction to NO is considered to be the first step in the anammox metabolism of ‘Ca. K. stuttgartiensis’, however, ‘Ca. Brocadia’ lacks the genes that encode canonical NO‐forming nitrite reductases (NirS or NirK) in its genome, which is different from ‘Ca. K. stuttgartiensis’. Here, we studied the anammox metabolism of ‘Ca. Brocadia sinica’. 15N‐tracer experiments demonstrated that ‘Ca. B. sinica’ cells could reduce NO2− to NH2OH, instead of NO, with as yet unidentified nitrite reductase(s). Furthermore, N2H4 synthesis, downstream reaction of NO2− reduction, was investigated using a purified ‘Ca. B. sinica' hydrazine synthase (Hzs) and intact cells. Both the ‘Ca. B. sinica’ Hzs and cells utilized NH2OH and NH4+, but not NO and NH4+, for N2H4 synthesis and further oxidized N2H4 to N2 gas. Taken together, the metabolic pathway of ‘Ca. B. sinica’ is NH2OH‐dependent and different from the one of ‘Ca. K. stuttgartiensis’, indicating metabolic diversity of anammox bacteria.