In a quest for high-efficiency mainstream partial nitritation-anammox (PN/A) implementation: One-stage or two-stage?

Partial nitritation-anammox (PN/A) process is known as an energy-efficient technology for wastewater nitrogen removal, which possesses a great potential to bring wastewater treatment plants close to energy neutrality with reduced carbon footprint. To achieve this goal, various PN/A processes implemented in a single reactor configuration (one-stage system) or two separately dedicated reactors configurations (two-stage system) were explored over the past decades. Nevertheless, large-scale implementation of these PN/A processes for low-strength municipal wastewater treatment has a long way to go owing to the low efficiency and effectiveness in nitrogen removal. In this work, we provided a comprehensive analysis of one-stage and two-stage PN/A processes with a focus on evaluating their engineering application potential towards mainstream implementation. The difficulty for nitrite-oxidizing bacteria (NOB) out-selection was revealed as the critical operational challenge to achieve the desired effluent quality. Additionally, the operational strategies of low oxygen commonly adopted in one-stage systems for NOB suppression and facilitating anammox bacteria growth results in a low nitrogen removal rate (NRR). Introducing denitrification into anammox system was found to be necessary to improve the nitrogen removal efficiency (NRE) by reducing the produced nitrate with in-situ utilizing the organics from wastewater itself. However, this may lead to part of organics oxidized with additional oxygen consumed in one-stage system, further compromising the NRR. By applying a relatively high dissolved oxygen in PN reactor with residual ammonium control, and followed by a granules-based anammox reactor feeding with a small portion of raw municipal wastewater, it appeared that two-stage system could achieve a good effluent quality as well as a high NRR. In contrast to the widely studied one-stage system, this work provided a unique perspective that more effort should be devoted to developing a two-stage PN/A process to evaluate its application potential of high efficiency and economic benefits towards mainstream implementation.

Partial Nitritation/Anammox and biological phosphorus removal integration in a single bioreactor under mainstream conditions.

Anammox-based nitrogen removal and enhanced biological phosphorus removal (EBPR) are increasingly applied for nutrient removal from wastewater, but are typically operated in separate reactors. Here, a novel process for integrated partial nitritation/anammox (PN/A) and EBPR in a single reactor employing integrated fixed film activated sludge was tested. The reactor was fed with mainstream municipal wastewater (5.4 ± 1.3 g COD/g N) at 20 °C for 243 days. Robust ammonium, total inorganic nitrogen, and orthophosphate removal efficiencies of 94 ± 4 %, 87 ± 7 % and 92 ± 7 % were achieved. Nitrite-oxidizing organisms suppression and ammonia-oxidizing organisms retention were achieved via solids retention time control, intermittent aeration, and suspended versus attached biomass population segregation. The contribution of anammox to nitrogen removal increased from 24 % to 74 %. In parallel, a substantial enrichment of Tetrasphaera polyphosphate accumulating organisms was observed. This work demonstrates a novel intensified bioprocess coupling PN/A and EBPR in the same reactor for efficient nutrient removal from wastewater.

Effects of feast-famine nutrient regimes on wastewater algal biofuel communities.

Microalgae accumulate lipids in response to nutrient deprivation, and these lipids are a biodiesel fuel stock. Algal cultivation with secondary wastewater effluent is one proposed platform for biofuel production, which provides nutrients to algae while further polishing wastewater effluent. Algal bioreactors were tested using a feast-famine feeding regiment in simulated secondary wastewater effluent to evaluate the effects on lipid content and algal community structure. Algal polycultures were inoculated into reactors fed with synthetic secondary wastewater effluent at pH 7.5 and 9 and operated under a feast-famine nutrient (N, P, and BOD) supply regime in sequencing batch reactors. Fatty acid methyl ester contents of the reactors were assessed, which showed a decrease in lipid content after the feast-famine cycling (from 12.2% initially to 5.2% after four cycles at pH 9). This decrease in lipid content was not correlated with an increase in carbohydrate storage within biomass, nor an increase in bacterial biomass abundance relative to algal biomass in the reactors. The eukaryotic microbial communities from reactors operated at pH 9 diverged from reactors operated at pH 7.5 during cycling, with the pH 9 reactors becoming dominated by a single Operational Taxonomic Unit aligning to the Scenedesmus genus. These results suggest that high pH and feast-famine nutrient cycling may select for a less diverse algal community with a lower lipid content within a secondary wastewater polishing scheme.

Reduction and discharge of SARS-CoV-2 RNA in Chicago-area water reclamation plants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is commonly excreted in the feces and urine of infected individuals and is, therefore, detected in wastewaters where infection is present in the surrounding population. Water reclamation plants (WRPs) that treat these wastewaters commonly discharge treated effluents into the surrounding environment, yet little is known about the removal or persistence of SARS-CoV-2 RNA through wastewater treatment systems and potential for eventual release into the environment. We collected 361 24-hour composite influent and effluent samples from seven WRPs in the Greater Chicago Area in Illinois. Samples were collected over a period of 21 weeks for three large WRPs (with design max flows of 1.89-2.32 billion gallons per day and serving a combined population of 4.62 million people) and 11 weeks for four smaller WRPs (with design max flows of 96.3-186 million gallons per day and serving a combined population of >0.5 million people). A total of two of the larger WRPs implemented seasonal disinfection (using UV light or chlorination/dechlorination) for 8 weeks of this sampling period. SARS-CoV-2 RNA was quantified in the influent and effluent samples by reverse-transcription quantitative PCR (RT-qPCR) of the N1 and N2 targets of the nucleocapsid (N) gene. Although SARS-CoV-2 RNA was regularly detected in influent and effluent from all WRPs, viral RNA concentrations in the effluent samples were considerably lower, with mean effluent: influent gene copy concentration ratios ranging from 1:160 to 1:2.95 between WRPs. Samples collected while disinfection was active vs. inactive did not show any significant difference in the portion of RNA persisting through the treatment process (P > .05).

Role of air scouring in anaerobic/anoxic tanks providing nitrogen removal by mainstream anammox conversion in a hybrid biofilm/suspended growth full-scale WWTP in China.

A full-scale wastewater treatment plant in China experienced unintentional anammox bacterial enrichment on biofilm carriers placed in the anaerobic and anoxic zones of an anaerobic/anoxic/oxic process under ambient temperatures and without bioaugmentation. Here, we show that microaerophilic conditions resulting from air scouring needed for biofilm carrier suspension in the anaerobic/anoxic zones can support a robust nitritation/anammox process. Results from an in situ on/off air scouring test showed that air scouring strongly induced both ammonia and total inorganic nitrogen removal in the anaerobic/anoxic zones. Ammonium concentration in the anaerobic and anoxic tanks remained constant or even slightly increased when air scouring was off, indicating that air scouring made a noticeable difference in nitrogen profiles in the anaerobic/anoxic zones. Various batch tests further indicated that partial denitrification is not likely to generate nitrite for anammox bacteria. Robust nitritation, and anammox on the carriers, can occur at low dissolved oxygen conditions, as measured in the full-scale facility. The observations show that mainstream deammonification without sidestream bioaugmentation at moderate temperature is feasible and further optimization by a more dedicated design can result in improved nitrogen removal in cases when chemical oxygen demand is limited in mainstream wastewater treatment. PRACTITIONER POINTS: Microaerophilic conditions in a full-scale IFAS reactor caused mainstream anammox in moderate temperate area. Robust nitritation, and anammox on the carriers, can occur at low dissolved oxygen conditions in anaerobic/anoxic tanks with air scouring. Anammox can function well with conventional nitrification and denitrification process at mainstream conditions for stable nitrogen removal.

Insights into Nitrous Oxide Mitigation Strategies in Wastewater Treatment and Challenges for Wider Implementation.

Nitrous oxide (N2O) emissions account for the majority of the carbon footprint of wastewater treatment plants (WWTPs). Many N2O mitigation strategies have since been developed while a holistic view is still missing. This article reviews the state-of-the-art of N2O mitigation studies in wastewater treatment. Through analyzing existing studies, this article presents the essential knowledge to guide N2O mitigations, and the logics behind mitigation strategies. In practice, mitigations are mainly carried out by aeration control, feed scheme optimization, and process optimization. Despite increasingly more studies, real implementation remains rare, which is a combined result of unclear climate change policies/incentives, as well as technical challenges. Five critical technical challenges, as well as opportunities, of N2O mitigations were identified. It is proposed that (i) quantification methods for overall N2O emissions and pathway contributions need improvement; (ii) a reliable while straightforward mathematical model is required to quantify benefits and compare mitigation strategies; (iii) tailored risk assessment needs to be conducted for WWTPs, in which more long-term full-scale trials of N2O mitigation are urgently needed to enable robust assessments of the resulting operational costs and impact on nutrient removal performance; (iv) current mitigation strategies focus on centralized WWTPs, more investigations are warranted for decentralised systems, especially decentralized activated sludge WWTPs; and (v) N2O may be mitigated by adopting novel strategies promoting N2O reduction denitrification or microorganisms that emit less N2O. Overall, we conclude N2O mitigation research is reaching a maturity while challenges still exist for a wider implementation, especially in relation to the reliability of N2O mitigation strategies and potential risks to nutrient removal performances of WWTPs.

Microbial and Viral Communities and Their Antibiotic Resistance Genes Throughout a Hospital Wastewater Treatment System.

Antibiotic resistance poses a serious threat to global public health, and antibiotic resistance determinants can enter natural aquatic systems through discharge of wastewater effluents. Hospital wastewater in particular is expected to contain high abundances of antibiotic resistance genes (ARGs) compared to municipal wastewater because it contains human enteric bacteria that may include antibiotic-resistant organisms originating from hospital patients, and can also have high concentrations of antibiotics and antimicrobials relative to municipal wastewater. Viruses also play an important role in wastewater treatment systems since they can influence the bacterial community composition through killing bacteria, facilitating transduction of genetic material between organisms, and modifying the chromosomal content of bacteria as prophages. However, little is known about the fate and connections between ARGs, viruses, and their associated bacteria in hospital wastewater systems. To address this knowledge gap, we characterized the composition and persistence of ARGs, dsDNA viruses, and bacteria from influent to effluent in a pilot-scale hospital wastewater treatment system in Israel using shotgun metagenomics. Results showed that ARGs, including genes conferring resistance to antibiotics of high clinical relevance, were detected in all sampling locations throughout the pilot-scale system, with only 16% overall depletion of ARGs per genome equivalent between influent and effluent. The most common classes of ARGs detected throughout the system conferred resistance to aminoglycoside, cephalosporin, macrolide, penam, and tetracycline antibiotics. A greater proportion of total ARGs were associated with plasmid-associated genes in effluent compared to in influent. No strong associations between viral sequences and ARGs were identified in viral metagenomes from the system, suggesting that phage may not be a significant vector for ARG transfer in this system. The majority of viruses in the pilot-scale system belonged to the families Myoviridae, Podoviridae, and Siphoviridae. Gammaproteobacteria was the dominant class of bacteria harboring ARGs and the most common putative viral host in all samples, followed by Bacilli and Betaproteobacteria. In the total bacterial community, the dominant class was Betaproteobacteria for each sample. Overall, we found that a variety of different types of ARGs and viruses were persistent throughout this hospital wastewater treatment system, which can be released to the environment through effluent discharge.

Soil hydrology drives ecological niche differentiation in a native prairie microbiome.

While the impacts of soil moisture on soil microbiome diversity and composition are well characterized, the influence of hydrological regime has been overlooked. As precipitation patterns are altered by climate change, understanding the impact of soil hydrology on community structure and function is critical. In this work, water level was continuously monitored for over a year in a Midwestern prairie-wetland at 10 cm depth increments up to a depth of 120 cm in 10 locations. We analyzed microbiome composition and edaphic factors in soil cores collected from this unique spatially distributed, longitudinal data set. We demonstrate that the fraction of time that each sample was inundated explains more variability in diversity and composition across this site than other commonly assessed edaphic factors, such as soil pH or depth. Finally, we show that these compositional changes influence abundance of ammonia oxidizers. The observed patterns in community composition and diversity are fundamentally regulated by the interaction of water with a structured landscape, particularly an elevated sand ridge characterized by drier conditions and a lower-lying wetland with more clayey soils. Similar processes are generally expected to influence the biogeography of many terrestrial environments, as morphology, hydrology and soil properties generally co-vary.

Author Correction: Global diversity and biogeography of bacterial communities in wastewater treatment plants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants.

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.

Global diversity and biogeography of bacterial communities in wastewater treatment plants.

Microorganisms in wastewater treatment plants (WWTPs) are essential for water purification to protect public and environmental health. However, the diversity of microorganisms and the factors that control it are poorly understood. Using a systematic global-sampling effort, we analysed the 16S ribosomal RNA gene sequences from ~1,200 activated sludge samples taken from 269 WWTPs in 23 countries on 6 continents. Our analyses revealed that the global activated sludge bacterial communities contain ~1 billion bacterial phylotypes with a Poisson lognormal diversity distribution. Despite this high diversity, activated sludge has a small, global core bacterial community (n = 28 operational taxonomic units) that is strongly linked to activated sludge performance. Meta-analyses with global datasets associate the activated sludge microbiomes most closely to freshwater populations. In contrast to macroorganism diversity, activated sludge bacterial communities show no latitudinal gradient. Furthermore, their spatial turnover is scale-dependent and appears to be largely driven by stochastic processes (dispersal and drift), although deterministic factors (temperature and organic input) are also important. Our findings enhance our mechanistic understanding of the global diversity and biogeography of activated sludge bacterial communities within a theoretical ecology framework and have important implications for microbial ecology and wastewater treatment processes.

Exploring co-occurrence patterns between organic micropollutants and bacterial community structure in a mixed-use watershed.

Complex mixtures of low concentrations of organic micropollutants are commonly found in rivers and streams, but their relationship to the structure of native bacterial communities that underlie critical ecological goods and services in these systems is poorly understood. To address this knowledge gap, we used correlation-based network analysis to explore co-occurrence patterns between measured micropollutant concentrations and the associated surface water and sediment bacterial communities in a restored riparian zone of the Des Plaines River (DPR) in Illinois that is impacted by both wastewater treatment plant (WWTP) effluent and agricultural runoff. Over a two year period, we collected 55 grab samples at 11 sites along the DPR and one of its tributaries (48 surface water samples) and from WWTP effluent (7 samples), and screened for 126 organic micropollutants. In parallel, we used high-throughput 16S rRNA gene amplicon sequencing to characterize the bacterial community in sediment and surface water. Our results revealed quantifiable levels of 102 micropollutants in at least one surface water or WWTP effluent sample, 85 of which were detected in at least one surface water sample. While micropollutants were temporally and spatially variable in terms of both presence and concentration, 21 micropollutants were measured in over 75% of the 48 surface water samples. 16S rRNA gene sequencing documented diverse bacterial communities along the DPR transect, with highly distinct community structures observed in sediment and water. Bacterial community structure in surface water, but not in sediment, was significantly associated with concentrations of micropollutants, based on a Mantel test. Correlation-based network analyses revealed diverse strong and significant co-occurrence and co-exclusion patterns between specific bacterial OTUs and both micropollutant groups (defined based on k-means clustering on chemical substructure) and individual micropollutants. Significantly more associations were documented between micropollutants and bacterial taxa in the water compared to the sediment microbiomes. Taken together, our results document a significant link between complex mixtures of micropollutants commonly found in aquatic systems and associated bacterial community structure. Furthermore, our results suggest that micropollutants may exert a more significant impact on water-associated than on sediment-associated bacterial taxa.

Comammox Nitrospira are the dominant ammonia oxidizers in a mainstream low dissolved oxygen nitrification reactor.

Recent findings show that a subset of bacteria affiliated with Nitrospira, a genus known for its importance in nitrite oxidation for biological nutrient removal applications, are capable of complete ammonia oxidation (comammox) to nitrate. Early reports suggested that they were absent or present in low abundance in most activated sludge processes, and thus likely functionally irrelevant. Here we show the accumulation of comammox Nitrospira in a nitrifying sequencing batch reactor operated at low dissolved oxygen (DO) concentrations. Actual mainstream wastewater was used as influent after primary settling and an upstream pre-treatment process for carbon and phosphorus removal. The ammonia removal rate was stable and exceeded that of the treatment plant's parallel full-scale high DO nitrifying activated sludge reactor. 16S rRNA gene sequencing showed a steady accumulation of Nitrospira to 53% total abundance and a decline in conventional ammonia oxidizing bacteria to <1% total abundance over 400 + days of operation. After ruling out other known ammonia oxidizers, qPCR confirmed the accumulation of comammox Nitrospira beginning around day 200, to eventually comprise 94% of all detected amoA and 4% of total bacteria by day 407. Quantitative fluorescence in-situ hybridization confirmed the increasing trend and high relative abundance of Nitrospira. These results demonstrate that comammox can be metabolically relevant to nitrogen transformation in wastewater treatment, and can even dominate the ammonia oxidizing community. Our results suggest that comammox may be an important functional group in energy efficient nitrification systems designed to operate at low DO levels.

Granular activated carbon supplementation alters the metabolic flux of Clostridium butyricum for enhanced biohydrogen production.

In this work, the effects of granular activated carbon (GAC) supplementation on hydrogen fermentation were investigated in Clostridium butyricum. Maximum hydrogen production rate and ultimate hydrogen volume increased up to 1.7 and 1.9 fold, respectively, with 1 g/L GAC supplementation. Indicators of stable hydrogen production, such as acetate and butyrate formation increased with increasing GAC concentration. To identify the factors for hydrogen production enhancement, transcriptome analysis was conducted. Functional genes related to hydrogen production increased by GAC supplementation (1.75 fold for pfor and 1.48 fold for oxct). On the other hand, functional genes related to hydrogen consumption decreased (1.78 fold for ldh, 0.67 fold for adh, 1.64 fold for hbd, 2.4 fold for crt, and 0.71 fold for buk). Considered together, these results suggested that GAC supplementation increased biohydrogen production by changing the metabolic flux associated with hydrogen production and consumption pathways.

Spatially resolved abundances of antibiotic resistance genes and intI1 in wastewater treatment biofilms.

Attached growth bioprocesses that use biofilms to remove organic matter or nutrients from wastewater are known to harbor antibiotic resistance genes (ARGs). Biofilms in these processes are spatially heterogeneous, but little is known about depth stratification of ARGs in complex, mixed culture biofilms. To address this knowledge gap, we used an experimental approach combining cryosectioning and quantitative polymerase chain reaction to quantify the spatial distribution of three ARGs (sul1, ermB, and qnrS) and the class 1 integron-integrase gene intI1 in biofilms from a lab-scale rotating annular reactor fed with synthetic wastewater. We also used high throughput 16S ribosomal RNA (rRNA) gene sequencing to characterize community structure with depth in biofilms. The ARG sul1 and the integron-integrase gene intI1 were found in higher abundances in upper layers of biofilm near the fluid-biofilm interface than in lower layers and exhibited significant correlations between the distance from substratum and gene abundances. The genes ermB and qnrS were present in comparatively low relative abundances. Microbial community structure varied significantly by date of sampling and distance from the substratum. These findings highlight the genetic and taxonomic heterogeneity with distance from substratum in wastewater treatment biofilms and show that sul1 and intI1 are particularly abundant near fluid-biofilm interfaces where cells are most likely to detach and flow into downstream portions of treatment systems and can ultimately be released into the environment through effluent.

Morphological analysis of pore size and connectivity in a thick mixed-culture biofilm.

Morphological parameters are commonly used to predict transport and metabolic kinetics in biofilms. Yet, quantification of biofilm morphology remains challenging because of imaging technology limitations and lack of robust analytical approaches. We present a novel set of imaging and image analysis techniques to estimate internal porosity, pore size distributions, and pore network connectivity to a depth of 1 mm at a resolution of 10 µm in a biofilm exhibiting both heterotrophic and nitrifying activities. Optical coherence tomography (OCT) scans revealed an extensive pore network with diameters as large as 110 µm directly connected to the biofilm surface and surrounding fluid. Thin-section fluorescence in situ hybridization microscopy revealed that ammonia-oxidizing bacteria (AOB) distributed through the entire thickness of the biofilm. AOB were particularly concentrated in the biofilm around internal pores. Areal porosity values estimated from OCT scans were consistently lower than those estimated from multiphoton laser scanning microscopy, though the two imaging modalities showed a statistically significant correlation (r = 0.49, p < 0.0001). Estimates of areal porosity were moderately sensitive to gray-level threshold selection, though several automated thresholding algorithms yielded similar values to those obtained by manually thresholding performed by a panel of environmental engineering researchers (±25% relative error). These findings advance our ability to quantitatively describe the geometry of biofilm internal pore networks at length scales relevant to engineered biofilm reactors and suggest that internal pore structures provide crucial habitat for nitrifier growth.

Antibiotic resistome in a large-scale healthy human gut microbiota deciphered by metagenomic and network analyses.

The human gut microbiota is an important reservoir of antibiotic resistance genes (ARGs). A metagenomic approach and network analysis were used to establish a comprehensive antibiotic resistome catalog and to obtain co-occurrence patterns between ARGs and microbial taxa in fecal samples from 180 healthy individuals from 11 different countries. In total, 507 ARG subtypes belonging to 20 ARG types were detected with abundances ranging from 7.12 × 10-7 to 2.72 × 10-1 copy of ARG/copy of 16S-rRNA gene. Tetracycline, multidrug, macrolide-lincosamide-streptogramin, bacitracin, vancomycin, beta-lactam and aminoglycoside resistance genes were the top seven most abundant ARG types. The multidrug ABC transporter, aadE, bacA, acrB, tetM, tetW, vanR and vanS were shared by all 180 individuals, suggesting their common occurrence in the human gut. Compared to populations from the other 10 countries, the Chinese population harboured the most abundant ARGs. Moreover, LEfSe analysis suggested that the MLS resistance type and its subtype 'ermF' were representative ARGs of the Chinese population. Antibiotic inactivation, antibiotic target alteration and antibiotic efflux were the dominant resistance mechanism categories in all populations. Procrustes analysis revealed that microbial phylogeny structured the antibiotic resistome. Co-occurrence patterns obtained via network analysis implied that 12 species might be potential hosts of 58 ARG subtypes.

Microbial diversity in an intensively managed landscape is structured by landscape connectivity.

Intensively managed land increases the rate of nutrient and particle transport within a basin, but the impact of these changes on microbial community assembly patterns at the basin scale is not yet understood. The objective of this study was to investigate how landscape connectivity and dispersal impacts microbial diversity in an agricultural-dominated watershed. We characterized soil, sediment and water microbial communities along the Upper Sangamon River basin in Illinois-a 3600 km2 watershed strongly influenced by human activity, especially landscape modification and extensive fertilization for agriculture. We employed statistical and network analyses to reveal the microbial community structure and interactions in the critical zone (water, soil and sediment media). Using a Bayesian source tracking approach, we predicted microbial community connectivity within and between the environments. We identified strong connectivity within environments (up to 85.4 ± 13.3% of sequences in downstream water samples sourced from upstream samples, and 44.7 ± 26.6% in soil and sediment samples), but negligible connectivity across environments, which indicates that microbial dispersal was successful within but not between environments. Species sorting based on sample media type and environmental parameters was the dominant driver of community dissimilarity. Finally, we constructed operational taxonomic unit association networks for each environment and identified a number of co-occurrence relationships that were shared between habitats, suggesting that these are likely to be ecologically significant.

Growth of Nitrosococcus-Related Ammonia Oxidizing Bacteria Coincides with Extremely Low pH Values in Wastewater with High Ammonia Content.

Ammonia oxidation decreases the pH in wastewaters where alkalinity is limited relative to total ammonia. The activity of ammonia oxidizing bacteria (AOB), however, typically decreases with pH and often ceases completely in slightly acidic wastewaters. Nevertheless, nitrification at low pH has been reported in reactors treating human urine, but it has been unclear which organisms are involved. In this study, we followed the population dynamics of ammonia oxidizing organisms and reactor performance in synthetic fully hydrolyzed urine as the pH decreased over time in response to a decrease in the loading rate. Populations of the ß-proteobacterial Nitrosomonas europaea lineage were abundant at the initial pH close to 6, but the growth of a possibly novel Nitrosococcus-related AOB genus decreased the pH to the new level of 2.2, challenging the perception that nitrification is inhibited entirely at low pH values, or governed exclusively by ß-proteobacterial AOB or archaea. With the pH shift, nitrite oxidizing bacteria were not further detected, but nitrous acid (HNO2) was still removed through chemical decomposition to nitric oxide (NO) and nitrate. The growth of acid-tolerant γ-proteobacterial AOB should be prevented, by keeping the pH above 5.4, which is a typical pH limit for the N. europaea lineage. Otherwise, the microbial community responsible for high-rate nitrification can be lost, and strong emissions of hazardous volatile nitrogen compounds such as NO are likely.