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.

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.

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.

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.

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.

Complete Nutrient Removal Coupled to Nitrous Oxide Production as a Bioenergy Source by Denitrifying Polyphosphate-Accumulating Organisms.

Coupled aerobic-anoxic nitrous decomposition operation (CANDO) is a promising emerging bioprocess for wastewater treatment that enables direct energy recovery from nitrogen (N) in three steps: (1) ammonium oxidation to nitrite; (2) denitrification of nitrite to nitrous oxide (N2O); and (3) N2O conversion to N2 with energy generation. However, CANDO does not currently target phosphorus (P) removal. Here, we demonstrate that denitrifying polyphosphate-accumulating organism (PAO) enrichment cultures are capable of catalyzing simultaneous biological N and P removal coupled to N2O generation in a second generation CANDO process, CANDO+P. Over 7 months (>300 cycles) of operation of a prototype lab-scale CANDO+P sequencing batch reactor treating synthetic municipal wastewater, we observed stable and near-complete N removal accompanied by sustained high-rate, high-yield N2O production with partial P removal. A substantial increase in abundance of the PAO Candidatus Accumulibacter phosphatis was observed, increasing from 5% of the total bacterial community in the inoculum to over 50% after 4 months. PAO enrichment was accompanied by a strong shift in the dominant Accumulibacter population from clade IIC to clade IA, based on qPCR monitoring of polyphosphate kinase 1 (ppk1) gene variants. Our work demonstrates the feasibility of combining high-rate, high-yield N2O production for bioenergy production with combined N and P removal from wastewater, and it further suggests a putative denitrifying PAO niche for Accumulibacter clade IA.

Microbial biogeography across a full-scale wastewater treatment plant transect: evidence for immigration between coupled processes.

Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO2 (-) entering the reactor from an upstream trickling filter. Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO2 (-) production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct "Nitrosomonas-like" lineage dominated in activated sludge. Prior time series indicated that this "Nitrosomonas-like" lineage was dominant when NO2 (-) levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO2 (-) levels were high. This is consistent with the hypothesis that NO2 (-) production may cooccur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors.

General and rare bacterial taxa demonstrating different temporal dynamic patterns in an activated sludge bioreactor.

Temporal variation of general and rare bacterial taxa was investigated using pyrosequencing of 16S rRNA gene from activated sludge samples collected bimonthly for a two-year period. Most of operational taxonomic units (OTUs) were allocated to rare taxa (89.6%), but the rare taxa comprised a small portion of the community in terms of abundance of sequences analyzed (28.6%). Temporal variations in OTUs richness significantly differed between the two taxa groups in which the rare taxa showed a higher diversity and a more fluctuating pattern than the general taxa. Furthermore, the two taxa groups were constrained by different explanatory variables: influent BOD, effluent BOD, and DO were the significant (P < 0.05) parameters affecting the pattern of the general taxa, while temperature was the factor for the rare taxa. Over the test period, the general taxa persisted for a longer time (i.e., lower turnover rate) in the bioreactor than the rare taxa. In conclusion, this study demonstrated clear differences in temporal dynamic patterns for the general and rare bacterial taxa in an activated sludge bioreactor, which would be a foundation for better understanding the bacterial ecology of activated sludge.

Combined niche and neutral effects in a microbial wastewater treatment community.

It has long been assumed that differences in the relative abundance of taxa in microbial communities reflect differences in environmental conditions. Here we show that in the economically and environmentally important microbial communities in a wastewater treatment plant, the population dynamics are consistent with neutral community assembly, where chance and random immigration play an important and predictable role in shaping the communities. Using dynamic observations, we demonstrate a straightforward calibration of a purely neutral model and a parsimonious method to incorporate environmental influence on the reproduction (or birth) rate of individual taxa. The calibrated model parameters are biologically plausible, with the population turnover and diversity in the heterotrophic community being higher than for the ammonia oxidizing bacteria (AOB) and immigration into AOB community being relatively higher. When environmental factors were incorporated more of the variance in the observations could be explained but immigration and random reproduction and deaths remained the dominant driver in determining the relative abundance of the common taxa. Consequently we suggest that neutral community models should be the foundation of any description of an open biological system.

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.

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.

Three-dimensional carbon nanotube-textile anode for high-performance microbial fuel cells.

Microbial fuel cells (MFCs) harness the metabolism of microorganisms, converting chemical energy into electrical energy. Anode performance is an important factor limiting the power density of MFCs for practical application. Improving the anode design is thus important for enhancing the MFC performance, but only a little development has been reported. Here, we describe a biocompatible, highly conductive, two-scale porous anode fabricated from a carbon nanotube-textile (CNT-textile) composite for high-performance MFCs. The macroscale porous structure of the intertwined CNT-textile fibers creates an open 3D space for efficient substrate transport and internal colonization by a diverse microflora, resulting in a 10-fold-larger anolyte-biofilm-anode interfacial area than the projective surface area of the CNT-textile. The conformally coated microscale porous CNT layer displays strong interaction with the microbial biofilm, facilitating electron transfer from exoelectrogens to the CNT-textile anode. An MFC equipped with a CNT-textile anode has a 10-fold-lower charge-transfer resistance and achieves considerably better performance than one equipped with a traditional carbon cloth anode: the maximum current density is 157% higher, the maximum power density is 68% higher, and the energy recovery is 141% greater.

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.

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.

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.

Ammonia-oxidizing communities in a highly aerated full-scale activated sludge bioreactor: betaproteobacterial dynamics and low relative abundance of Crenarchaea.

Ammonia-oxidizing bacteria (AOB) have long been considered key to the removal of nitrogen in activated sludge bioreactors. Culture-independent molecular analyses have established that AOB lineages in bioreactors are dynamic, but the underlying operational or environmental factors are unclear. Furthermore, the contribution of ammonia-oxidizing archaea (AOA) to nitrogen removal in bioreactors has not been studied. To this end, we investigated the abundance of AOA and AOB as well as correlations between dynamics in AOB lineages and operational parameters at a municipal wastewater treatment plant sampled weekly over a 1 year period. Quantitative PCR measurements of bacterial and archaeal ammonia monooxygenase subunit A (amoA) genes revealed that the bacterial homologue predominated by at least three orders of magnitude in all samples. Archaeal amoA was only detectable in approximately 15% of these samples. Using terminal restriction fragment length polymorphism analysis, we monitored AOB lineages based on amoA genes. The Nitrosomonas europaea lineage and a novel Nitrosomonas-like cluster were the dominant AOB signatures, with a Nitrosospira lineage present at lower relative abundance. These lineages exhibited strong temporal oscillations, with one becoming sequentially dominant over the other. Using non-metric multidimensional scaling and redundancy analyses, we tested correlations between terminal restriction fragment length polymorphism profiles and 20 operational and environmental parameters. The redundancy analyses indicated that the dynamics of AOB lineages correlated most strongly with temperature, dissolved oxygen and influent nitrite and chromium. The Nitrosospira lineage signal had a strong negative correlation to dissolved oxygen and temperature, while the Nitrosomonas-like (negative correlations) and N. europaea lineages (positive correlations) were inversely linked (relative to one another) to influent nitrite and chromium. Overall, this study suggests that AOA may be minor contributors to ammonia oxidation in highly aerated activated sludge, and provides insight into parameters controlling the diversity and dominance of AOB lineages within bioreactors during periods of stable nitrification.

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.