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1. Integration of EBPR with mainstream anammox process to treat real municipal wastewater: Process performance and microbiology

   Hong, S; Winkler, MKH; Wang, Z; Goel, R (February 2023, Water research)

   The mainstream application of anaerobic ammonium oxidation (anammox) for sustainable N removal remains a challenge. Similarly, with recent additional stringent regulations for P discharges, it is imperative to integrate N with P removal. This research studied integrated fixed film activated sludge (IFAS) technology to simultaneously remove N and P in real municipal wastewater by combining biofilm anammox with flocculent activated sludge for enhanced biological P removal (EBPR). This technology was assessed in a sequencing batch reactor (SBR) operated as a conventional A2O (anaerobic-anoxic-oxic) process with a hydraulic retention time of 8.8 h. After a steady state operation was reached, robust reactor performance was obtained with average TIN and P removal efficiencies of 91.3 ± 4.1% and 98.4 ± 2.4%, respectively. The average TIN removal rate recorded over the last 100 d of reactor operation was 118 mg/L⋅d, which is a reasonable number for mainstream applications. The activity of denitrifying polyphosphate accumulating organisms (DPAOs) accounted for nearly 15.9% of P-uptake during the anoxic phase. DPAOs and canonical denitrifiers removed approximately 5.9 mg TIN/L in the anoxic phase. Batch activity assays, which showed that nearly 44.5% of TIN were removed by the biofilms during the aerobic phase. The functional gene expression data also confirmed anammox activities. The IFAS configuration of the SBR allowed operation at a low solid retention time (SRT) of 5-d without washing out biofilm ammoniumoxidizing and anammox bacteria. The low SRT, combined with low dissolved oxygen and intermittent aeration, provided a selective pressure to washout nitrite-oxidizing bacteria and glycogen-accumulating organisms, as relative abundances. 

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2. All about nitrite: exploring nitrite sources and sinks in the eastern tropical North Pacific oxygen minimum zone

   https://doi.org/10.5194/bg-20-2499-2023  

   Tracey, John C.; Babbin, Andrew R.; Wallace, Elizabeth; Sun, Xin; DuRussel, Katherine L.; Frey, Claudia; Martocello III, Donald E.; Tamasi, Tyler; Oleynik, Sergey; Ward, Bess B. (January 2023, Biogeosciences)

   Abstract. Oxygen minimum zones (OMZs), due to their large volumes of perennially deoxygenated waters, are critical regions for understanding how the interplay between anaerobic and aerobic nitrogen (N) cycling microbial pathways affects the marine N budget. Here, we present a suite of measurements of the most significant OMZ N cycling rates, which all involve nitrite (NO2-) as a product, reactant, or intermediate, in the eastern tropical North Pacific (ETNP) OMZ. These measurements and comparisons to data from previously published OMZ cruisespresent additional evidence that NO3- reduction is the predominant OMZ N flux, followed by NO2- oxidation back to NO3-. The combined rates of both of these N recycling processes were observed to be much greater (up to nearly 200 times) thanthe combined rates of the N loss processes of anammox and denitrification, especially in waters near the anoxic–oxic interface. We also showthat NO2- oxidation can occur when O2 is maintained near 1 nM by a continuous-purge system, NO2-oxidation and O2 measurements that further strengthen the case for truly anaerobic NO2- oxidation. We also evaluate thepossibility that NO2- dismutation provides the oxidative power for anaerobic NO2- oxidation. The partitioning ofN loss between anammox and denitrification differed widely from stoichiometric predictions of at most 29 % anammox; in fact,N loss rates at many depths were entirely due to anammox. Our new NO3- reduction, NO2- oxidation, dismutation, andN loss data shed light on many open questions in OMZ N cycling research, especially the possibility of truly anaerobicNO2- oxidation. 

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3. Ecological dynamics explain modular denitrification in the ocean

   https://doi.org/10.1073/pnas.2417421121  

   Sun, Xin; Buchanan, Pearse J; Zhang, Irene H; San_Roman, Magdalena; Babbin, Andrew R; Zakem, Emily J (December 2024, Proceedings of the National Academy of Sciences)

   Microorganisms in marine oxygen minimum zones (OMZs) drive globally impactful biogeochemical processes. One such process is multistep denitrification (NO₃^–→NO₂^–→NO→N₂O→N₂), which dominates OMZ bioavailable nitrogen (N) loss and nitrous oxide (N₂O) production. Denitrification-derived N loss is typically measured and modeled as a single step, but observations reveal that most denitrifiers in OMZs contain subsets (“modules”) of the complete pathway. Here, we identify the ecological mechanisms sustaining diverse denitrifiers, explain the prevalence of certain modules, and examine the implications for N loss. We describe microbial functional types carrying out diverse denitrification modules by their underlying redox chemistry, constraining their traits with thermodynamics and pathway length penalties, in an idealized OMZ ecosystem model. Biomass yields of single-step modules increase along the denitrification pathway when organic matter (OM) limits growth, which explains the viability of populations respiring NO₂^–and N₂O in a NO₃^–-filled ocean. Results predict denitrifier community succession along environmental gradients: Pathway length increases as the limiting substrate shifts from OM to N, suggesting a niche for the short NO₃^–→NO₂^–module in free-living, OM-limited communities, and for the complete pathway in organic particle-associated communities, consistent with observations. The model captures and mechanistically explains the observed dominance and higher oxygen tolerance of the NO₃^–→NO₂^–module. Results also capture observations that NO₃^–is the dominant source of N₂O. Our framework advances the mechanistic understanding of the relationship between microbial ecology and N loss in the ocean and can be extended to other processes and environments. 

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4. Similar temperature responses suggest future climate warming will not alter partitioning between denitrification and anammox in temperate marine sediments

   https://doi.org/10.1111/gcb.13370  

   Brin, Lindsay D.; Giblin, Anne E.; Rich, Jeremy J. (July 2016, Global Change Biology)

   Abstract Removal of biologically available nitrogen (N) by the microbially mediated processes denitrification and anaerobic ammonium oxidation (anammox) affects ecosystem N availability. Although few studies have examined temperature responses of denitrification and anammox, previous work suggests that denitrification could become more important than anammox in response to climate warming. To test this hypothesis, we determined whether temperature responses of denitrification and anammox differed in shelf and estuarine sediments from coastal Rhode Island over a seasonal cycle. The influence of temperature and organic C availability was further assessed in a 12‐week laboratory microcosm experiment. Temperature responses, as characterized by thermal optima (T_opt) and apparent activation energy (E_a), were determined by measuring potential rates of denitrification and anammox at 31 discrete temperatures ranging from 3 to 59 °C. With a few exceptions,T_optandE_aof denitrification and anammox did not differ in Rhode Island sediments over the seasonal cycle. In microcosm sediments,E_a was somewhat lower for anammox compared to denitrification across all treatments. However,T_opt did not differ between processes, and neither E_a nor T_opt changed with warming or carbon addition. Thus, the two processes behaved similarly in terms of temperature responses, and these responses were not influenced by warming. This led us to reject the hypothesis that anammox is more cold‐adapted than denitrification in our study system. Overall, our study suggests that temperature responses of both processes can be accurately modeled for temperate regions in the future using a single set of parameters, which are likely not to change over the next century as a result of predicted climate warming. We further conclude that climate warming will not directly alter the partitioning of N flow through anammox and denitrification. 

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5. Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired

   https://doi.org/10.1128/aem.01741-23  

   LaSarre, Breah; Morlen, Ryan; Neumann, Gina C; Harwood, Caroline S; McKinlay, James B (January 2024, Applied and Environmental Microbiology)

   Bose, Arpita (Ed.)

   ABSTRACT Denitrification is a form of anaerobic respiration wherein nitrate (NO₃⁻) is sequentially reduced via nitrite (NO₂⁻), nitric oxide, and nitrous oxide (N₂O) to dinitrogen gas (N₂) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria,Rhodopseudomonas palustrisCGA0092 andRhodobacter capsulatusSB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO₂⁻to N₂, and SB1003 reduced N₂O to N₂. For each bacterium, N₂O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N₂O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO₃⁻served as a stable, non-catalyzable molecule that was sufficient to activate N₂O reduction. Using a β-galactosidase reporter, we found that NO₃⁻acted, at least in part, by stimulating N₂O reductase gene expression. In SB1003, NO₂⁻but not NO₃⁻activated N₂O reduction, but NO₂⁻was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use. IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions. 

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