Kindaichi, Tomonori

Recirculating aquaculture systems (RAS) hold significant potential for sustainable aquaculture by providing a stable, controlled environment that supports optimal fish growth and welfare. In RAS, ammonium (NH4+) is biologically converted into nitrate (NO3−) via nitrite (NO2−) by nitrifying bacteria. As a result, NO3− usually accumulates in RAS and must subsequently be removed through denitrification in full RAS, or by regular water exchanges in partial RAS. The marine anammox bacteria Candidatus Scalindua can directly convert toxic NH4+ and NO2− into harmless nitrogen gas (N2) and has previously been identified as a promising alternative to the complex denitrification process or unsustainable frequent water exchanges in marine RAS. In this study, we evaluated the impact of high NO3− levels typically encountered in RAS on the performance and abundance of Ca. Scalindua in a laboratory-scale bioreactor. The bacterial composition of the granules, including the relative abundance of key nitrogen-cycling taxa, was analyzed along with the functional profile (i.e., NH4+ and NO2− removal efficiencies). For this purpose, a bioreactor was inoculated and fed a synthetic feed, enriched in NH4+, NO2−, minerals and trace elements until stabilization (Phase 1, 52 days). NO3− concentrations were then gradually increased to 400 mg·L−1 NO3−-N (Phase 2, 52 days), after which the reactor was followed for another 262 days (Phase 3). The reactor maintained high removal efficiencies; 88.0 ± 8.6% for NH4+ and 97.4 ± 1.7% for NO2− in Phase 2, and 95.0 ± 6.5% for NH4+ and 98.6 ± 2.7% for NO2− in Phase 3. The relative abundance of Ca. Scalindua decreased from 22.7% to 10.2% by the end of Phase 3. This was likely due to slower growth of Ca. Scalindua compared to heterotrophic bacteria present in the granule, which could use NO3− as a nitrogen source. Fluorescence in situ hybridization confirmed the presence of a stable population of Ca. Scalindua, which maintained high and stable NH4+ and NO2− removal efficiencies. These findings support the potential of Ca. Scalindua as an alternative filtering technology in marine RAS. Future studies should investigate pilot-scale applications under real-world conditions.

Abstract Candidatus Patescibacteria, also known as candidate phyla radiation (CPR), including the class‐level uncultured clade JAEDAM01 (formerly a subclass of Gracilibacteria/GN02/BD1‐5), are ubiquitous in activated sludge. However, their characteristics and relationships with other organisms are largely unknown. They are believed to be episymbiotic, endosymbiotic or predatory. Despite our understanding of their limited metabolic capacity, their precise roles remain elusive due to the difficulty in cultivating and identifying them. In previous research, we successfully recovered high‐quality metagenome‐assembled genomes (MAGs), including a member of JAEDAM01 from activated sludge flocs. In this study, we designed new probes to visualize the targeted JAEDAM01‐associated MAG HHAS10 and identified its host using fluorescence in situ hybridization (FISH). The FISH observations revealed that JAEDAM01 HHAS10‐like cells were located within dense clusters of Zoogloea , and the fluorescence brightness of zoogloeal cells decreased in the vicinity of the CPR cells. The Zoogloea MAGs possessed genes related to extracellular polymeric substance biosynthesis, floc formation and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The JAEDAM01 MAG HHAS10 possessed genes associated with type IV pili, competence protein EC and PHA degradation, suggesting a Zoogloea ‐dependent lifestyle in activated sludge flocs. These findings indicate a new symbiotic relationship between JAEDAM01 and Zoogloea .

Recirculating aquaculture systems (RAS) are promising candidates for the sustainable development of the aquaculture industry. A current limitation of RAS is the production and potential accumulation of nitrogenous wastes, ammonium (NH4+), nitrite (NO2−) and nitrate (NO3−), which could affect fish health and welfare. In a previous experiment, we have demonstrated that the marine anammox bacteria Candidatus Scalindua was a promising candidate to treat the wastewater (WW) of marine, cold-water RAS. However, the activity of the bacteria was negatively impacted after a direct exposure to RAS WW. In the current study, we have further investigated the potential of Ca. Scalindua to treat marine RAS WW in a three-phase experiment. In the first phase (control, 83 days), Ca. Scalindua was fed a synthetic feed, enriched in NH4+, NO2− and trace element (TE) mix. Removal rates of 98.9% and 99.6% for NH4+ and NO2−, respectively, were achieved. In the second phase (116 days), we gradually increased the exposure of Ca. Scalindua to nitrogen-enriched RAS WW over a period of about 80 days. In the last phase (79 days), we investigated the needs of TE supplementation for the Ca. Scalindua after they were fully acclimated to 100% RAS WW. Our results show that the gradual exposure of Ca. Scalindua resulted in a successful acclimation to 100% RAS WW, with maintained high removal rates of both NH4+ and NO2− throughout the experiment. Despite a slight decrease in relative abundance (from 21.4% to 16.7%), Ca. Scalindua remained the dominant species in the granules throughout the whole experiment. We conclude that Ca. Scalindua can be successfully used to treat marine RAS WW, without the addition of TE, once given enough time to acclimate to its new substrate. Future studies need to determine the specific needs for optimal RAS WW treatment by Ca. Scalindua at pilot scale.

Candidatus Saccharibacteria is a well-described candidate phylum that has not been successfully isolated. Nevertheless, its presence was suggested by 16S rRNA gene sequencing data, and it is frequently detected in natural environments and activated sludge. Because pure culture representatives of Candidatus Saccharibacteria are lacking, the specificity of primers for the determination of their abundance and diversity should be carefully evaluated. In this study, eight Candidatus Saccharibacteria-specific primers were selected from previous studies and evaluated for their coverage against a public database, annealing temperature of the combined primer sets, as well as their utilization to determine the detection frequencies and phylogenetic diversity by cloning analysis, and in quantification by quantitative polymerase chain reaction (PCR). Among the eight primers, four primers (TM7314F, TM7580F, TM7-910R, and TM7-1177R) showed high coverage. Cloning analysis showed that four primer sets (TM7314F and TM7-910R, TM7314F and TM7-1177R, TM7580F and TM7-910R, and TM7580F and TM7-1177R) yielded high detection frequencies for Candidatus Saccharibacteria in activated sludge from a wastewater treatment plant in Higashihiroshima City, Japan. Quantitative PCR results indicated that the primer set containing TM7314F and TM7-910R was superior for the specific detection of Candidatus Saccharibacteria in activated sludge.

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 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.

ABSTRACT The phylogenetic affiliation and physiological characteristics (e.g., K s and maximum specific growth rate [μ max ]) of an anaerobic ammonium oxidation (anammox) bacterium, “ Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “ Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.