Surface-layer protein is a public-good matrix exopolymer for microbial community organisation in environmental anammox biofilms.

Extracellular polymeric substances (EPS) are core biofilm components, yet how they mediate interactions within and contribute to the structuring of biofilms is largely unknown, particularly for non-culturable microbial communities that predominate in environmental habitats. To address this knowledge gap, we explored the role of EPS in an anaerobic ammonium oxidation (anammox) biofilm. An extracellular glycoprotein, BROSI_A1236, from an anammox bacterium, formed envelopes around the anammox cells, supporting its identification as a surface (S-) layer protein. However, the S-layer protein also appeared at the edge of the biofilm, in close proximity to the polysaccharide-coated filamentous Chloroflexi bacteria but distal to the anammox bacterial cells. The Chloroflexi bacteria assembled into a cross-linked network at the edge of the granules and surrounding anammox cell clusters, with the S-layer protein occupying the space around the Chloroflexi. The anammox S-layer protein was also abundant at junctions between Chloroflexi cells. Thus, the S-layer protein is likely transported through the matrix as an EPS and also acts as an adhesive to facilitate the assembly of filamentous Chloroflexi into a three-dimensional biofilm lattice. The spatial distribution of the S-layer protein within the mixed species biofilm suggests that it is a "public-good" EPS, which facilitates the assembly of other bacteria into a framework for the benefit of the biofilm community, and enables key syntrophic relationships, including anammox.

Controlling anammox speciation and biofilm attachment strategy using N-biotransformation intermediates and organic carbon levels.

Conventional nitrogen removal in wastewater treatment requires a high oxygen and energy input. Anaerobic ammonium oxidation (anammox), the single-step conversion of ammonium and nitrite to nitrogen gas, is a more energy and cost effective alternative applied extensively to sidestream wastewater treatment. It would also be a mainstream treatment option if species diversity and physiology were better understood. Anammox bacteria were enriched up to 80%, 90% and 50% relative abundance, from a single inoculum, under standard enrichment conditions with either stepwise-nitrite and ammonia concentration increases (R1), nitric oxide supplementation (R2), or complex organic carbon from mainstream wastewater (R3), respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica occurred at ammonium and nitrite concentrations > 270 mg NH4-N L-1 and 340 mg NO2-N L-1 respectively. With NO present, heterotrophic growth was inhibited, and Ca. Jettenia coexisted with Ca. B. caroliniensis before diminishing as nitrite increased to 160 mg NO2-N L-1. Organic carbon supplementation led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially formed biofilms on surfaces, whereas Ca. Brocadia sinica formed granules in suspension. Our results indicate that multiple anammox bacteria species co-exist and occupy sub-niches in anammox reactors, and that the dominant population can be reversibly shifted by, for example, changing nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis). Speciation has implications for wastewater process design, where the optimum cell immobilization strategy (i.e. carriers vs granules) depends on which species dominates.

Global warming readiness: Feasibility of enhanced biological phosphorus removal at 35 °C.

Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30 °C to be achievable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors for > 300 days at 30 °C and 35 °C, respectively, and followed the dynamics of the communities of polyphosphate accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescence in situ hybridization analyses. Stable and nearly complete phosphorus (P) removal was achieved at 30 °C; similarly, long term P removal was stable at 35 °C with effluent PO43-_P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Candidatus Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. A slow-feeding strategy effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. Comparative cycle studies revealed higher carbon and P cycling activity of Ca. Accumulibacter when the temperature was increased from 30 °C to 35 °C, implying that the lowered P removal performance at 35 °C was not a direct effect of temperature, but a result of higher metabolic rates of carbon (and/or P) utilization of PAOs and GAOs, the resultant carbon deficiency, and escalated community competition. An increase in the TOC-to-PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited PAOs. In general, a slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at 35 °C, representing basic conditions suitable for full-scale treatment plants experiencing higher water temperatures.

Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation.

This study describes the first direct functional assignment of a highly abundant extracellular protein from a key environmental and biotechnological biofilm performing an anaerobic ammonium oxidation (anammox) process. Expression levels of Brosi_A1236, belonging to a class of proteins previously suggested to be cell surface associated, were in the top one percentile of all genes in the "Candidatus Brocadia sinica"-enriched biofilm. The Brosi_A1236 structure was computationally predicted to consist of immunoglobulin-like anti-parallel ß-strands, and circular dichroism conducted on the isolated surface protein indicated that ß-strands are the dominant higher-order structure. The isolated protein was stained positively by the ß-sheet-specific stain thioflavin T, along with cell surface- and matrix-associated regions of the biofilm. The surface protein has a large unstructured content, including two highly disordered domains at its C terminus. The disordered domains bound to the substratum and thereby facilitated the adhesion of negatively charged latex microspheres, which were used as a proxy for cells. The disordered domains and isolated whole surface protein also underwent liquid-liquid phase separation to form liquid droplets in suspension. Liquid droplets of disordered protein wet the surfaces of microspheres and bacterial cells and facilitated their coalescence. Furthermore, the surface layer protein formed gels as well as ordered crystalline structures. These observations suggest that biophysical remodeling through phase transitions promotes aggregation and biofilm formation.IMPORTANCE By employing biophysical and liquid-liquid phase separation concepts, this study revealed how a highly abundant extracellular protein enhances the key environmental and industrial bioprocess of anaerobic ammonium oxidation (anammox). Extracellular proteins of environmental biofilms are understudied and poorly annotated in public databases. Understanding the function of extracellular proteins is also increasingly important for improving bioprocesses and resource recovery. Here, protein functions were assessed based on theoretical predictions of intrinsically disordered domains, known to promote adhesion and liquid-liquid phase separation, and available surface layer protein properties. A model is thus proposed to explain how the protein promotes aggregation and biofilm formation by extracellular matrix remodeling and phase transitions. This work provides a strong foundation for functional investigations of extracellular proteins involved in biofilm development.

Extracellular protein isolation from the matrix of anammox biofilm using ionic liquid extraction.

Anaerobic ammonium oxidation (anammox)-performing bacteria self-assemble into compact biofilms by expressing extracellular polymeric substances (EPS). Anammox EPS are poorly characterized, largely due to their low solubility in typical aqueous solvents. Pronase digestion achieved 19.5 ± 0.9 and 41.4 ± 1.4% (w/w) more solubilization of laboratory enriched Candidatus Brocadia sinica anammox granules than DNase and amylase, respectively. Nuclear magnetic resonance profiling of the granules confirmed proteins as dominant biopolymer within the EPS. Ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and N,N-dimethylacetamide (EMIM-Ac/DMAc) mixture was applied to extract the major structural proteins. Further treatment by anion exchange chromatography isolated homologous serine (S)- and threonine (T)-rich proteins BROSI_A1236 and UZ01_01563, which were major components of the extracted proteins, and sequentially highly similar to putative anammox extracellular proteins KUSTD1514 and WP_070066018.1 of Ca. Kuenenia stuttgartiensis and Ca. Brocadia sapporoensis, respectively. Six monosaccharides (i.e., arabinose, xylose, rhamnose, fucose, galactose, and mannose) were enriched for BROSI_A1236 against all other major proteins. The sugars, however, contributed < 0.5% (w/w) of total granular biomass and were likely co-enriched as glycoprotein appendages. This study demonstrates that BROSI_A1236 is a major extracellular component of Ca. B. sinica anammox biofilms that is likely a common anammox extracellular polymer, and can be isolated from the matrix following ionic liquid extraction.

Metabolic Traits of Candidatus Accumulibacter clade IIF Strain SCELSE-1 Using Amino Acids As Carbon Sources for Enhanced Biological Phosphorus Removal.

Despite recent evidence from full-scale plants suggesting that Candidatus Accumulibacter may be capable of using amino acids, this metabolic trait has never been confirmed in a bioreactor experiment. Here we show that an enriched culture of Ca. Accumulibacter clade IIF strain SCELSE-1 could metabolize 11 of 20 α-amino acids, with aspartate, glutamate, asparagine, and glutamine resulting in the highest phosphorus removal. The anaerobic uptake of aspartate and glutamate was achieved through a glutamate/aspartate-proton symporter fully powered by the proton motive force (PMF). Under anaerobic conditions aspartate was deaminized and routed into core carbon metabolic pathways to form polyhydroxyalkanoates (PHA). The lack of genes encoding NADH dependent isocitrate dehydrogenase in the Ca. Accumulibacter genome resulted in a kinetic barrier for glutamate to be channelled to the TCA cycle. Glutamate was stored as glutamate polymer. When amino acids (aspartate or glutamate) and acetate were supplied together, Ca. Accumulibacter took up both carbon sources simultaneously, with the uptake rate of each carbon source largely preserved. Overall energy savings (up to 17%) were achieved under mixed carbon scenarios, due to the ability of Ca. Accumulibacter to rearrange its anaerobic carbon metabolism based on the reducing power, PMF and ATP balance.

High Dissolved Oxygen Selection against Nitrospira Sublineage I in Full-Scale Activated Sludge.

A single Nitrospira sublineage I OTU was found to perform nitrite oxidation in full-scale domestic wastewater treatment plants (WWTPs) in the tropics. This taxon had an apparent oxygen affinity constant lower than that of the full-scale domestic activated sludge cohabitating ammonium oxidizing bacteria (AOB) (0.09 ± 0.02 g O2 m-3 versus 0.3 ± 0.03 g O2 m-3). Thus, nitrite oxidizing bacteria (NOB) may in fact thrive under conditions of low oxygen supply. Low dissolved oxygen (DO) conditions selected for and high aeration inhibited the NOB in a long-term lab-scale reactor. The relative abundance of Nitrospira sublineage I gradually decreased with increasing DO until it was washed out. Nitritation was sustained even after the DO was lowered subsequently. The morphologies of AOB and NOB microcolonies responded to DO levels in accordance with their oxygen affinities. NOB formed densely packed spherical clusters with a low surface area-to-volume ratio compared to the Nitrosomonas-like AOB clusters, which maintained a porous and nonspherical morphology. In conclusion, the effect of oxygen on AOB/NOB population dynamics depends on which OTU predominates given that oxygen affinities are species-specific, and this should be elucidated when devising operating strategies to achieve mainstream partial nitritation.

Polyphosphate-accumulating organisms in full-scale tropical wastewater treatment plants use diverse carbon sources.

Enhanced biological phosphorus removal (EBPR) is considered challenging in the tropics, based on a great number of laboratory-based studies showing that the polyphosphate-accumulating organism (PAO) Candidatus Accumulibacter does not compete well with glycogen accumulating organisms (GAOs) at temperatures above 25 °C. Yet limited information is available on the PAO community and the metabolic capabilities in full-scale EBPR systems operating at high temperature. We studied the composition of the key functional PAO communities in three full-scale wastewater treatment plants (WWTPs) with high in-situ EBPR activity in Singapore, their EBPR-associated carbon usage characteristics, and the relationship between carbon usage and community composition. Each plant had a signature community composed of diverse putative PAOs with multiple operational taxonomic units (OTUs) affiliated to Ca. Accumulibacter, Tetrasphaera spp., Dechloromonas and Ca. Obscuribacter. Despite the differences in community composition, ex-situ anaerobic phosphorus (P)-release tests with 24 organic compounds from five categories (including four sugars, three alcohols, three volatile fatty acids (VFAs), eight amino acids and six other carboxylic acids) showed that a wide range of organic compounds could potentially contribute to EBPR. VFAs induced the highest P release (12.0-18.2 mg P/g MLSS for acetate with a P release-to-carbon uptake (P:C) ratio of 0.35-0.66 mol P/mol C, 9.4-18.5 mg P/g MLSS for propionate with a P:C ratio of 0.38-0.60, and 9.5-17.3 mg P/g MLSS for n-butyrate), followed by some carboxylic acids (10.1-18.1 mg P/g MLSS for pyruvate, 4.5-11.7 mg P/g MLSS for lactate and 3.7-12.4 mg P/g MLSS for fumarate) and amino acids (3.66-7.33 mg P/g MLSS for glutamate with a P:C ratio of 0.16-0.43 mol P/mol C, and 4.01-7.37 mg P/g MLSS for aspartate with a P:C ratio of 0.17-0.48 mol P/mol C). P-release profiles (induced by different carbon sources) correlated closely with PAO community composition. High micro-diversity was observed within the Ca. Accumulibacter lineage, which represented the most abundant PAOs. The total population of Ca. Accumulibacter taxa was highly correlated with P-release induced by VFAs, highlighting the latter's importance in tropical EBPR systems. There was a strong link between the relative abundance of individual Ca. Accumulibacter OTUs and the extent of P release induced by distinct carbon sources (e.g., OTU 81 and amino acids, and OTU 246 and ethanol), suggesting niche differentiation among Ca. Accumulibacter taxa. A diverse PAO community and the ability to use numerous organic compounds are considered key factors for stable EBPR in full-scale plants at elevated temperatures.

Draft Genome Sequence of a "Candidatus Brocadia" Bacterium Enriched from Activated Sludge Collected in a Tropical Climate.

Here, we present the draft genome sequence of an anaerobic ammonium-oxidizing bacterium (AnAOB), "Candidatus Brocadia," which was enriched in an anammox reactor. A 3.2-Mb genome sequence comprising 168 contigs was assembled, in which 2,765 protein-coding genes, 47 tRNAs, and one each of 5S, 16S, and 23S rRNAs were annotated. No evidence for the presence of a nitric oxide-forming nitrite reductase was found.

Non-denitrifying polyphosphate accumulating organisms obviate requirement for anaerobic condition.

Enhanced biological phosphorus removal (EBPR) is a widely used process in wastewater treatment that requires anaerobic/aerobic or anaerobic/anoxic cycling. Surprisingly, phosphorus (P) release was observed in the presence of nitrate in the anoxic compartment of the activated sludge tank in a full-scale treatment plant with the Modified Ludzack Ettinger configuration. We therefore studied the potential of this full-scale activated sludge community to perform EBPR under anoxic/aerobic cycling. The polyphosphate accumulating organism (PAO) Candidatus Accumulibacter represented 3.3% of total bacteria based on 16S rRNA gene amplicon sequencing, and metagenome analysis suggested it was likely to be dominated by Clade IIC. Using acetate as the carbon source in batch experiments, active denitrifying organisms (DPAOs) were estimated to comprise 39-44% of the total PAO population in the sludge, with the remaining 56-61% unable to utilize nitrate. When propionate was provided as the organic carbon source, 95% of the PAO population was unable to denitrify. EBPR occurred under defined anoxic/aerobic conditions, despite the presence of DPAOs, when synthetic wastewater was supplemented with either acetate or propionate or when primary effluent was supplied. In addition, the P release and subsequent uptake rates under anoxic/aerobic conditions were comparable to those observed under anaerobic/aerobic conditions. In contrast, a significant reduction in P release rate was observed when acetate was provided under oxic conditions. We postulate that non-DPAOs that recognize the anoxic condition as pseudo-anaerobic were the key players in anoxic/aerobic EBPR.

Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions.

Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

Modeling of nitrous oxide production by autotrophic ammonia-oxidizing bacteria with multiple production pathways.

Autotrophic ammonia oxidizing bacteria (AOB) have been recognized as a major contributor to N2O production in wastewater treatment systems. However, so far N2O models have been proposed based on a single N2O production pathway by AOB, and there is still a lack of effective approach for the integration of these models. In this work, an integrated mathematical model that considers multiple production pathways is developed to describe N2O production by AOB. The pathways considered include the nitrifier denitrification pathway (N2O as the final product of AOB denitrification with NO2(-) as the terminal electron acceptor) and the hydroxylamine (NH2OH) pathway (N2O as a byproduct of incomplete oxidation of NH2OH to NO2(-)). In this model, the oxidation and reduction processes are modeled separately, with intracellular electron carriers introduced to link the two types of processes. The model is calibrated and validated using experimental data obtained with two independent nitrifying cultures. The model satisfactorily describes the N2O data from both systems. The model also predicts shifts of the dominating pathway at various dissolved oxygen (DO) and nitrite levels, consistent with previous hypotheses. This unified model is expected to enhance our ability to predict N2O production by AOB in wastewater treatment systems under varying operational conditions.

A novel methodology to quantify nitrous oxide emissions from full-scale wastewater treatment systems with surface aerators.

The quantification of nitrous oxide (N2O) emissions from open-surface wastewater treatment systems with surface aerators is difficult as emissions from the surface aerator zone cannot be easily captured by floating hoods. In this study, we propose and demonstrate a novel methodology to estimate N2O emissions from such systems through determination of the N2O transfer coefficient (kLa) induced by surface aerators based on oxygen balance for the entire system. The methodology is demonstrated through its application to a full-scale open oxidation ditch wastewater treatment plant with surface aerators. The estimated kLa profile based on a month-long measurement campaign for oxygen balance, intensive monitoring of dissolved N2O profiles along the oxidation ditch over a period of four days, together with mathematical modelling, enabled to determine the N2O emission factor from this treatment plant (0.52 ± 0.16%). Majority of the N2O emission was found to occur in the surface aerator zone, which would be missed if the gas hood method was applied alone.

Fossil organic carbon in wastewater and its fate in treatment plants.

This study reports the presence of fossil organic carbon in wastewater and its fate in wastewater treatment plants. The findings pinpoint the inaccuracy of current greenhouse gas accounting guidelines which defines all organic carbon in wastewater to be of biogenic origin. Stable and radiocarbon isotopes ((13)C and (14)C) were measured throughout the process train in four municipal wastewater treatment plants equipped with secondary activated sludge treatment. Isotopic mass balance analyses indicate that 4-14% of influent total organic carbon (TOC) is of fossil origin with concentrations between 6 and 35 mg/L; 88-98% of this is removed from the wastewater. The TOC mass balance analysis suggests that 39-65% of the fossil organic carbon from the influent is incorporated into the activated sludge through adsorption or from cell assimilation while 29-50% is likely transformed to carbon dioxide (CO2) during secondary treatment. The fossil organic carbon fraction in the sludge undergoes further biodegradation during anaerobic digestion with a 12% decrease in mass. 1.4-6.3% of the influent TOC consists of both biogenic and fossil carbon is estimated to be emitted as fossil CO2 from activated sludge treatment alone. The results suggest that current greenhouse gas accounting guidelines, which assume that all CO2 emission from wastewater is biogenic may lead to underestimation of emissions.

Mathematical modeling of nitrous oxide (N2O) emissions from full-scale wastewater treatment plants.

Mathematical modeling of N2O emissions is of great importance toward understanding the whole environmental impact of wastewater treatment systems. However, information on modeling of N2O emissions from full-scale wastewater treatment plants (WWTP) is still sparse. In this work, a mathematical model based on currently known or hypothesized metabolic pathways for N2O productions by heterotrophic denitrifiers and ammonia-oxidizing bacteria (AOB) is developed and calibrated to describe the N2O emissions from full-scale WWTPs. The model described well the dynamic ammonium, nitrite, nitrate, dissolved oxygen (DO) and N2O data collected from both an open oxidation ditch (OD) system with surface aerators and a sequencing batch reactor (SBR) system with bubbling aeration. The obtained kinetic parameters for N2O production are found to be reasonable as the 95% confidence regions of the estimates are all small with mean values approximately at the center. The model is further validated with independent data sets collected from the same two WWTPs. This is the first time that mathematical modeling of N2O emissions is conducted successfully for full-scale WWTPs. While clearly showing that the NH2OH related pathways could well explain N2O production and emission in the two full-scale plants studied, the modeling results do not prove the dominance of the NH2OH pathways in these plants, nor rule out the possibility of AOB denitrification being a potentially dominating pathway in other WWTPs that are designed or operated differently.

The confounding effect of nitrite on N2O production by an enriched ammonia-oxidizing culture.

The effect of nitrite (NO2(-)) on the nitrous oxide (N2O) production rate of an enriched ammonia-oxidizing bacteria (AOB) culture was characterized over a concentration range of 0-1000 mg N/L. The AOB culture was enriched in a nitritation system fed with synthetic anaerobic digester liquor. The N2O production rate was highest at NO2(-) concentrations of less than 50 mg N/L. At dissolved oxygen (DO) concentration of 0.55 mg O2/L, further increases in NO2(-) concentration from 50 to 500 mg N/L resulted in a gradual decrease in N2O production rate, which maintained at its lowest level of 0.20 mg N2O-N/h/g VSS in the NO2(-) concentration range of 500-1000 mg N/L. The observed NO2(-)-induced decrease in N2O production was even more apparent at increased DO concentration. At DO concentrations of 1.30 and 2.30 mg O2/L, the lowest N2O production rate (0.25 mg N2O-N/h/g VSS) was attained at a lower NO2(-) concentration of 200-250 mg N/L. These observations suggest that N2O production by the culture is diminished by both high NO2(-) and high DO concentrations. Collectively, the findings show that exceedingly high NO2(-) concentrations in nitritation systems could lead to decreased N2O production. Further studies are required to determine the extent to which the same response to NO2(-) is observed across different AOB cultures.

N2O production rate of an enriched ammonia-oxidising bacteria culture exponentially correlates to its ammonia oxidation rate.

The relationship between the ammonia oxidation rate (AOR) and nitrous oxide production rate (N(2)OR) of an enriched ammonia-oxidising bacteria (AOB) culture was investigated. The AOB culture was enriched in a nitritation system fed with synthetic anaerobic digester liquor. The AOR was controlled by adjusting the dissolved oxygen (DO) and pH levels and also by varying the initial ammonium (NH(4)(+)) concentration in batch experiments. Tests were also performed directly on the parent reactor where a stepwise decrease/increase in DO was implemented to alter AOR. The experimental data indicated a clear exponential relationship between the biomass specific N(2)OR and AOR. Four metabolic models were used to analyse the experimental data. The metabolic model formulated based on aerobic N(2)O production from the decomposition of nitrosyl radical (NOH) predicted the exponential correlation observed experimentally. The experimental data could not be reproduced by models developed on the basis of N(2)O production through nitrite (NO(2)(-)) and nitric oxide (NO) reduction by AOB.

Nitrous oxide emissions from wastewater treatment processes.

Nitrous oxide (N(2)O) emissions from wastewater treatment plants vary substantially between plants, ranging from negligible to substantial (a few per cent of the total nitrogen load), probably because of different designs and operational conditions. In general, plants that achieve high levels of nitrogen removal emit less N(2)O, indicating that no compromise is required between high water quality and lower N(2)O emissions. N(2)O emissions primarily occur in aerated zones/compartments/periods owing to active stripping, and ammonia-oxidizing bacteria, rather than heterotrophic denitrifiers, are the main contributors. However, the detailed mechanisms remain to be fully elucidated, despite strong evidence suggesting that both nitrifier denitrification and the chemical breakdown of intermediates of hydroxylamine oxidation are probably involved. With increased understanding of the fundamental reactions responsible for N(2)O production in wastewater treatment systems and the conditions that stimulate their occurrence, reduction of N(2)O emissions from wastewater treatment systems through improved plant design and operation will be achieved in the near future.

The effect of pH on N2O production under aerobic conditions in a partial nitritation system.

Ammonia-oxidising bacteria (AOB) are a major contributor to nitrous oxide (N(2)O) emissions during nitrogen transformation. N(2)O production was observed under both anoxic and aerobic conditions in a lab-scale partial nitritation system operated as a sequencing batch reactor (SBR). The system achieved 55 ± 5% conversion of the 1g NH(4)(+)-N/L contained in a synthetic anaerobic digester liquor to nitrite. The N(2)O emission factor was 1.0 ± 0.1% of the ammonium converted. pH was shown to have a major impact on the N(2)O production rate of the AOB enriched culture. In the investigated pH range of 6.0-8.5, the specific N(2)O production was the lowest between pH 6.0 and 7.0 at a rate of 0.15 ± 0.01 mg N(2)O-N/h/g VSS, but increased with pH to a maximum of 0.53 ± 0.04 mg N(2)O-N/h/g VSS at pH 8.0. The same trend was also observed for the specific ammonium oxidation rate (AOR) with the maximum AOR reached at pH 8.0. A linear relationship between the N(2)O production rate and AOR was observed suggesting that increased ammonium oxidation activity may have promoted N(2)O production. The N(2)O production rate was constant across free ammonia (FA) and free nitrous acid (FNA) concentrations of 5-78 mg NH(3)-N/L and 0.15-4.6 mg HNO(2)-N/L, respectively, indicating that the observed pH effect was not due to changes in FA or FNA concentrations.

Ammonium as a sustainable proton shuttle in bioelectrochemical systems.

This work examines a pH control method using ammonium (NH(4)(+)) as a sustainable proton shuttle in a CEM-equipped BES. Current generation was sustained by adding NH(3) or ammonium hydroxide (NH(4)OH) to the anolyte, controlling its pH at 7. Ammonium ion migration maintained the catholyte pH at approximately 9.25. Such NH(4)(+)/NH(3) migration accounted for 90±10% of the ionic flux in the BES. Reintroducing the volatilized NH(3) from the cathode into the anolyte maintained a suitable anolyte pH for sustained microbial-driven current generation. Hence, NH(4)(+)/NH(3) acted as a proton shuttle that is not consumed in the process.