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Aerobic granular sludge (AGS) at wastewater treatment plants (WWTPs) are known to produce nitrous oxide (N 2 O), a greenhouse gas which has a ∼300 times higher global warming potential than carbon dioxide. In this research, we studied N 2 O emissions from different sizes of AGS developed at a dissolved oxygen (DO) level of 2 mgO 2 /L while exposing them to disturbances at various DO concentrations ranging from 1 to 4 mgO 2 /L. Five different AGS size classes were studied: 212-600 µm, 600-1000 µm, 1000-1400 µm, 1400-2000 µm, and > 2000 µm. Metagenomic data showed N 2 O reductase genes (nosZ) were more abundant in the smaller AGS sizes which aligned with the observation of higher N 2 O reduction rates in small AGS under anaerobic conditions. However, when oxygen was present, the activity measurements of N 2 O emission showed an opposite trend compared to metagenomic data, smaller AGS (212 to 1000 µm) emitted significantly higher N 2 O (p < 0.05) than larger AGS (1000 µm to >2000 µm) at DO of 2, 3, and 4 mgO 2 /L. The N 2 O emission rate showed positive correlation with both oxygen levels and nitrification rate. This pattern indicates a connection between N 2 O emission and nitrification. In addition, the data suggested the penetration of oxygen into the anoxic zone of granules might have hindered nitrous oxide reduction, resulting in incomplete denitrification stopping at N 2 O and consequently contributing to an increase in N 2 O emissions. This work sets the stage to better understand the impacts of AGS size on N 2 O emissions in WWTPs under different disturbance of DO conditions, and thus ensure that wastewater treatment will comply with possible future regulations demanding lowering greenhouse gas emissions in an effort to combat climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Anaerobic ammonia oxidizing bacteria (Anammox) are implemented in high-efficiency wastewater treatment systems operated in two general configurations; one-stage systems combine aerobic ammonia oxidizing bacteria (AOB) and Anammox within a single aerated reactor, whereas two-stage configurations separate these processes into discrete tanks. Within both configurations heterotrophic populations that perform denitrification or dissimilatory nitrate reduction to ammonia (DNRA) compete for carbon and nitrate or nitrite and can impact reactor performance because DNRA retains nitrogen in the system. Therefore, it is important to understand how selective pressures imposed by one-stage and two-stage reactor configurations impact the microbial community structure and associated nitrogen transforming functions. We performed 16S rRNA gene and metagenomic sequencing on different biomass fractions (granules, flocs, and suspended biomass) sampled from two facilities treating sludge dewatering centrate: a one-stage treatment facility (Chambers Creek, Tacoma, WA) and a two-stage system (Rotterdam, Netherlands). Similar microbial populations were identified across the different samples, but relative abundances differed between reactor configurations and biomass sources. Analysis of metagenome assembled genomes (MAGs) indicated different lifestyles for abundant heterotrophic populations. Acidobacteria , Bacteroidetes , and Chloroflexi MAGs had varying capacity for DNRA and denitrification. Acidobacteria MAGs possessed high numbers of glycosyl hydrolases and glycosyl transferases indicating a role in biomass degradation. Ignavibacteria and Phycosphaerae MAGs contributed to the greater relative abundance of DNRA associated nrf genes in the two-stage granules and contained genomic features suggesting a preference for an anoxic or microoxic niche. In the one-stage granules a MAG assigned to Burkholderiales accounted for much of the abundant denitrification genes and had genomic features, including the potential for autotrophic denitrification using reduced sulfur, that indicate an ability to adapt its physiology to varying redox conditions. Overall, the competition for carbon substrates between denitrifying and DNRA performing heterotrophs may be impacted by configuration specific selective pressures. In one-stage systems oxygen availability in the bulk liquid and the oxygen gradient within granules would provide a greater niche space for heterotrophic populations capable of utilizing both oxygen and nitrate or nitrite as terminal electron acceptors, compared to two-stage systems where a homogeneous anoxic environment would favor heterotrophic populations primarily adapted to anaerobic metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bryson, Hunt, Stahl and Winkler.)

The discovery of anaerobic ammonia-oxidizing bacteria (Anammox) and, more recently, aerobic bacteria common in many natural and engineered systems that oxidize ammonia completely to nitrate (Comammox) have significantly altered our understanding of the global nitrogen cycle. A high affinity for ammonia (K m(app),NH3 ≈ 63nM) and oxygen place Comammox Nitrospira inopinata, the first described isolate, in the same trophic category as organisms such as some ammonia-oxidizing archaea. However, N. inopinata has a relatively low affinity for nitrite (K m,NO2 ≈ 449.2μM) suggesting it would be less competitive for nitrite than other nitrite-consuming aerobes and anaerobes. We examined the ecological relevance of the disparate substrate affinities by coupling it with the Anammox bacterium Candidatus Brocadia anammoxidans. Synthetic communities of the two were established in hydrogel granules in which Comammox grew in the aerobic outer layer to provide Anammox with nitrite in the inner anoxic core to form dinitrogen gas. This spatial organization was confirmed with FISH imaging, supporting a mutualistic or commensal relationship. The functional significance of interspecies spatial organization was informed by the hydrogel encapsulation format, broadening our limited understanding of the interplay between these two species. The resulting low nitrate formation and the competitiveness of Comammox over other aerobic ammonia- and nitrite-oxidizers sets this ecological cooperation apart and points to potential biotechnological applications. Since nitrate is an undesirable product of wastewater treatment effluents, the Comammox-Anammox symbiosis may be of economic and ecological importance to reduce nitrogen contamination of receiving waters., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

An aerobic granular sludge (AGS) pilot plant fed with a mixture of acetate amended centrate and secondary effluent was used to investigate the optimal granule size range for simultaneous nitrification and denitrification (SND) and ortho-phosphate removal. The anaerobic phase was mixed to understand how AGS will perform if integrated with a continuous flow activated sludge system that cannot feed the influent through the settled sludge bed. Five different granule size fractions were taken from the pilot (operated at DO setpoint of 2mgO 2 /L) and each size was subjected to activity tests in a well-controlled lab-scale AGS reactor at four dissolved oxygen (DO) concentrations of 1, 2, 3, and 4 mgO 2 /L. The size fractions were: 212 - 600 µm, 600 - 1000 µm, 1000 - 1400 µm, 1400 - 2000 µm, and >2000 µm. The smallest size range (212 - 600 µm) had the highest nitrification and phosphate removal rates at DO setpoints from 1 - 3 mgO 2 /L, which was attributed to the higher aerobic volume fraction in small granules and hence a higher abundance of phosphorus accumulating organisms (PAO) and ammonia oxidizing bacteria (AOB). In comparison, large granules (>1000 µm) had 1.4 - 4.7 times lower ammonia oxidation rates than the smallest size range, which aligned with their lower AOB abundance relative to granule biomass. The granules with the highest anoxic volume fraction had the highest abundance of nitrite reductase genes (nir gene) but did not show the highest specific nitrogen removal rate. Instead, smaller granules (212 - 600 and 600 - 1000 µm), which had a lower nir gene abundance, had the highest specific nitrogen removal rates (1.2 - 3.1 times higher than larger granules) across all DO values except at 4 mgO 2 /L. At a DO setpoint of 4 mgO 2 /L, nitrite production by ammonia oxidation (ammonia monooxygenase) exceeded nitrite reduction by nitrite reductase in granules smaller than 1000 µm, in addition, some denitrifying heterotrophs switched to oxygen utilization in deeper layers hence suppressing denitrification activity. At the DO range of 2 - 4 mg/L, granular size had a greater effect on nutrient removal than DO. Therefore, for AGS developed at an average DO setpoint of 2 mgO 2 /L, selecting for size fractions in the range of 212 - 1000 µm and avoiding DO values higher than 3 mgO 2 /L can achieve both a higher nitrogen removal capacity and energy savings. This study is the first to investigate the influence of different DO values on SND and biological phosphorus removal performance of different aerobic granular sludge sizes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Methane seep microbial communities perform a key ecosystem service by consuming the greenhouse gas methane prior to its release into the hydrosphere, minimizing the impact of marine methane sources on our climate. Although previous studies have examined the ecology and biochemistry of these communities, none has examined viral assemblages associated with these habitats. We employed virus particle purification, genome amplification, pyrosequencing and gene/genome reconstruction and annotation on two metagenomic libraries, one prepared for ssDNA and the other for all DNA, to identify the viral community in a methane seep. Similarity analysis of these libraries (raw and assembled) revealed a community dominated by phages, with a significant proportion of similarities to the Microviridae family of ssDNA phages. We define these viruses as the Eel River Basin Microviridae (ERBM). Assembly and comparison of 21 ERBM closed circular genomes identified five as members of a novel sister clade to the Microvirus genus of Enterobacteria phages. Comparisons among other metagenomes and these Microviridae major-capsid sequences indicated that this clade of phages is currently unique to the Eel River Basin sediments. Given this ERBM clade's relationship to the Microviridae genus Microvirus, we define this sister clade as the candidate genus Pequeñovirus., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)