Okabe, Satoshi

Abstract Analysing the nitrogen (15ε) and oxygen (18ε) isotope effects of anaerobic ammonium oxidation (anammox) is essential for accurately assessing its potential contribution to fixed-N losses in the ocean, yet the 18ε of anammox remains unexplored. Here, we determined the previously unexplored 18ε of anammox using a highly enriched culture of the marine anammox species “Ca. Scalindua sp”. Because Scalindua significantly accelerated oxygen isotope exchange between NO2− and H2O, we introduced a new rate constant for anammox-mediated oxygen isotope exchange (keq, AMX = 8.44 ~ 13.56 × 10−2 h−1), which is substantially faster than abiotic oxygen isotope exchange (keq, abio = 1.13 × 10−2 h−1), into a numerical model to estimate the 18ε during anammox. Based on our experimental results, we successfully determined the 18ε associated with: (1) conversion of NO2− to N2 (18εNO2- → N2 = 10.6 ~ 16.1‰), (2) NO2− oxidation to NO3− (18εNO2- → NO3- = −2.9 ~ −11.0‰, inverse fractionation), (3) incorporation of oxygen from water during NO2− oxidation to NO3− (18εH2O = 16.4 ~ 19.2‰). Our study underscores the possibility that unique anammox oxygen isotope signals may be masked due to substantial anammox-mediated oxygen isotope exchange between NO2− and H2O. Therefore, careful consideration is required when utilizing δ18ONO3- and δ18ONO2- as geochemical markers to assess the potential contribution of anammox to fixed-N losses in the ocean.

Abstract Little is known about the cell physiology of anammox bacteria growing at extremely low growth rates. Here, “Candidatus Brocadia sinica” and “Candidatus Scalindua sp.” were grown in continuous anaerobic membrane bioreactors (MBRs) with complete biomass retention to determine maintenance energy (i.e., power) requirements at near-zero growth rates. After prolonged retentostat cultivations, the specific growth rates (μ) of “Ca. B. sinica” and “Ca. Scalindua sp.” decreased to 0.000023 h−1 (doubling time of 1255 days) and 0.000157 h−1 (184 days), respectively. Under these near-zero growth conditions, substrate was continuously utilized to meet maintenance energy demands (me) of 6.7 ± 0.7 and 4.3 ± 0.7 kJ mole of biomass-C−1 h−1 for “Ca. B. sinica” and “Ca. Scalindua sp.”, which accorded with the theoretically predicted values of all anaerobic microorganisms (9.7 and 4.4 kJ mole of biomass-C−1 h−1at 37 °C and 28 °C, respectively). These me values correspond to 13.4 × 10−15 and 8.6 × 10−15 watts cell−1 for “Ca. B. sinica” and “Ca. Scalindua sp.”, which were five orders of magnitude higher than the basal power limit for natural settings (1.9 × 10−19 watts cells−1). Furthermore, the minimum substrate concentrations required for growth (Smin) were calculated to be 3.69 ± 0.21 and 0.09 ± 0.05 μM NO2− for “Ca. B. sinica” and “Ca. Scalindua sp.”, respectively. These results match the evidence that “Ca. Scalindua sp.” with lower maintenance power requirement and Smin are better adapted to energy-limited natural environments than “Ca. B. sinica”, suggesting the importance of these parameters on ecological niche differentiation in natural environments.

Abstract Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in “Ca. Brocadia sinica” growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)−1 while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that “Ca. B. sinica” synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.

Summary Anaerobic ammonium‐oxidizing (anammox) bacteria affiliated with the genus ‘ Candidatus Scalindua’ are responsible for significant nitrogen loss in oceans, and thus their ecophysiology is of great interest. Here, we enriched a marine anammox bacterium, ‘ Ca . S. japonica’ from a Hiroshima bay sediment in Japan, and comparative genomic and proteomic analyses of ‘ Ca . S. japonica’ were conducted. Sequence of the 4.81‐Mb genome containing 4019 coding regions of genes (CDSs) composed of 47 contigs was determined. In the proteome, 1762 out of 4019 CDSs in the ‘ Ca . S. japonica’ genome were detected. Based on the genomic and proteomic data, the core anammox process and carbon fixation of ‘ Ca . S. japonica’ were further investigated. Additionally, the present study provides the first detailed insights into the genetic background responsible for iron acquisition and menaquinone biosynthesis in anammox bacterial cells. Comparative analysis of the ‘ Ca . Scalindua’ genomes revealed that the 1502 genes found in the ‘ Ca . S. japonica’ genome were not present in the ‘ Ca . S. profunda’ and ‘ Ca . S. rubra’ genomes, showing a high genomic diversity. This result may reflect a high phylogenetic diversity of the genus ‘ Ca . Scalindua’.

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. 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’. 15 N‐tracer experiments demonstrated that ‘ Ca . B. sinica’ cells could reduce to NH 2 OH, instead of NO, with as yet unidentified nitrite reductase(s). Furthermore, N 2 H 4 synthesis, downstream reaction of reduction, was investigated using a purified ‘ Ca . B. sinica' hydrazine synthase (Hzs) and intact cells. Both the ‘ Ca . B. sinica’ Hzs and cells utilized NH 2 OH and , but not NO and , for N 2 H 4 synthesis and further oxidized N 2 H 4 to N 2 gas. Taken together, the metabolic pathway of ‘ Ca . B. sinica’ is NH 2 OH‐dependent and different from the one of ‘ Ca . K. stuttgartiensis’, indicating metabolic diversity of anammox bacteria.

Summary 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 ‘ C andidatus   J ettenia’. 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 N 2 gas production by coupling of 15 NH 4 + and 14 NO 2 − , 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 ‘ C a . J ettenia caeni’ sp. nov. for the bacterial clade of the planctomycete KSU ‐1.