Efficient nitrification and low N2O emission in a weakly acidic bioreactor at low dissolved oxygen levels are due to comammox

Nitrification is an essential process for nutrient removal from wastewater and an important emission source of nitrous-oxide (N2O), which is a powerful greenhouse gas and a dominant ozone-depleting substance. In this study, nitrification and N2O emissions were tested in two weakly acidic (pH = 6.3–6.8) reactors: one with dissolved oxygen (DO) over 2.0 mg/L and the other with DO approximately 0.5 mg/L. Efficient nitrification was achieved in both reactors. Compared to the high-DO reactor, N2O emission in the low-DO reactor decreased slightly by 20% and had insignificant correlation with the fluctuations of DO (P = 0.935) and nitrite (P = 0.713), indicating that N2O might not be mainly produced via nitrifier denitrification. Based on qPCR, qFISH, functional gene amplicon and metagenome sequencing, it was found that complete ammonia oxidizer (comammox) Nitrospira significantly outnumbered canonical ammonia-oxidizing bacteria (AOB) in both weakly acidic reactors, especially in the low DO reactor with the comammox/AOB amoA gene ratio increasing from 6.6 to 17.1. Therefore, it was speculated that the enriched comammox was the primary cause for the slightly decreased N2O emission under long-term low DO in weakly acidic reactor. This study demonstrated that comammox Nitrospira can survive well under the weakly acidic and low-DO conditions, implying that achieving efficient nitrification with low N2O emission as well as low energy and alkalinity consumption is feasible for wastewater treatment. Importance Nitrification in wastewater treatment is an important process for eutrophication control and an emission source for greenhouse gas of N2O. The nitrifying process is usually operated at a slightly alkaline pH and high DO (>2 mg/L) to ensure efficient nitrification. However, it consumes a large amount of energy and chemicals especially for wastewater without sufficient alkalinity. This manuscript demonstrated that comammox can adapt well to the weakly acidic and low-DO bioreactors, with a result of efficient nitrification and low N2O emission. These findings indicate that comammox are significant for sustainable wastewater treatment, which provides an opportunity to achieve efficient nitrification with low N2O production as well as low energy and chemical consumption simultaneously.

Isotope and microbiome analysis indicates variety of N-cycle processes controlling N2O fluxes in a drained peatland forest soil

<p>Nitrous oxide (N2O) is a major greenhouse gas whose presence in atmosphere is continuously increasing. Hence it’s important to understand its production and consumption mechanisms. During the summer of 2020, we conducted lab experiments using heavy nitrogen tracers of Potassium Nitrate 15N 98% atom (Sigma Aldrich) and Ammonium Chloride 15N 98% atom (Sigma Aldrich) under different moisture conditions to get an insight into N2O production mechanisms and on their dependence on soil moisture. We applied the tracer to peat samples (Kärevere, Estonia) placed in 36 (12 control, 12 nitrate treatment & 12 ammonia treatment) plastic buckets (radius-10cm, height-20cm) with soil height of 10 cm and a 10 cm head space left for gas collection. We installed oxygen sensors, water table indicators and temperature sensors on all buckets. We focused on studying physical conditions (soil oxygen, temperature, water table and soil moisture), gas (N2O) emission data, soil chemistry, gas isotope 15N, soil isotope and soil microbiology to get a complete picture of the processes involved in production of N2O gas. Under the ammonia treatment, emissions increased more than ten-fold which could be due to multiple processes of the nitrogen cycle in play. N2O emissions increased as the oxygen conditions shifted from anoxic (Omg/L=0) to sub-oxic (Omg/L=0.5–6) and then decreased as oxygen conditions reached the oxic (Omg/L>6) state. Furthermore, we witnessed negative site preference and 18O values during the nitrate treatment indicating nitrifier-denitrification. Under the ammonia treatment, we recorded both negative as well as high positive site preference values indicating presence of multiple production mechanisms. This was expected as ammonia triggers multiple processes in the nitrogen cycle. In some samples, we observed N2O consumption with little change in site preference as compared to the N2O producing samples. This indicates some bacterial-denitrification along with the prevailing nitrifier-denitrification. We also observed that under both treatments, heavy oxygen increased with increasing site preference. This indicates reduction of N2O (Ostrom et al, 2007) as redox supports 15N and 18O enrichments. After these lab experiments, we conducted the same experiment at a large scale in a drained peatland forest in Agali, Estonia. In this experiment, we established 1m2 triangle-shape mesocosms using experimental draining and flooding to achieve varying oxygen conditions. Preliminary results of qPCR analysis of N-cycle control genes support the domination of ammonia oxidation and denitrification as sources of N2O.</p>

High greenhouse gas fluxes from peatlands under various disturbances in the Peruvian Amazon

Amazonian peat swamp forests remove large amounts of carbon dioxide (CO2) but anaerobic decomposition of the peat produces methane (CH4). Drought or cultivation cuts down on the CH4 production but may increase the CO2 emission. Varying oxygen content in nitrogen-rich peat produces nitrous oxide (N2O). Despite the potentially tremendous changes, greenhouse gas emissions from peatlands under various land uses and environmental conditions have rarely been compared in the Amazon. We measured CO2, CH4 and N2O emissions from the soil surface with manual opaque chambers, and environmental characteristics in three sites around Iquitos, Peru from September 2019 to March 2020: a pristine peat swamp forest, a young forest and a slash-and-burn manioc field. The manioc field showed moderate peat respiration and N2O emission. The swamp forests under slight water table drawdown emitted large amounts of CO2 and N2O while retaining their high CH4 emissions. Most noticeably, a heavy shower after the water-table drawdown in the pristine swamp forest created a hot moment of N2O. Nitrifier denitrification was the likely source mechanism, as we rule out nitrification and heterotrophic denitrification. We base the judgement on the lack of nitrate and oxygen, and the suppressed denitrification potential in the topsoil. Overall, our study shows that even moderate drying in Peruvian palm swamps may create a devastating feedback on climate change through CO2 and N2O emissions.

Minimization of N2O Emission through Intermittent Aeration in a Sequencing Batch Reactor (SBR): Main Behavior and Mechanism

To explore the main behavior and mechanism of minimizing nitrous oxide (N2O) emission through intermittent aeration during wastewater treatment, two lab-scale sequencing batch reactors operated at intermittently aerated mode (SBR1), and continuously aerated mode (SBR2) were established. Compared with SBR2, the intermittently aerated SBR1 reached not only a higher total nitrogen removal efficiency (averaged 93.5%) but also a lower N2O-emission factor (0.01–0.53% of influent ammonia), in which short-cut nitrification and denitrification were promoted. Moreover, less accumulation and consumption of polyhydroxyalkanoates, a potential endogenous carbon source promoting N2O emission, were observed in SBR1. Batch experiments revealed that nitrifier denitrification was the major pathway generating N2O while heterotrophic denitrification played as a sink of N2O, and SBR1 embraced a larger N2O-mitigating capability. Finally, quantitative polymerase chain reaction results suggested that the abundant complete ammonia oxidizer (comammox) elevated in the intermittently aerated environment played a potential role in avoiding N2O generation during wastewater treatment. This work provides an in-depth insight into the utilization of proper management of intermittent aeration to control N2O emission from wastewater treatment plants.

Mineralisation of distinct biogas digestate qualities directly after application to soil

Biogas is an important energy source produced by the anaerobic fermentation of raw faecal slurries and plant residues. Separation of the total digestate increases the fertilizer quality of the liquid fraction and the carbon sequestration potential of the solid fraction. A 12-day incubation study was carried out to investigate the relationships between the chemical composition of different digestate qualities and the immediate response of soil microbial activity and biomass indices. The highest cumulative (Σ) CO2-C efflux was observed after adding the solid fraction and lowest after adding the liquid fraction to soil, which was even lower than that of the control. The ΣCO2-C efflux showed the strongest negative correlation with the raw ash and strong positive correlations with the raw fibre concentration and the C/N ratio of the different digestate qualities. The highest and similar ΣN2O-N efflux was observed after adding the total digestate or the liquid fraction, which were equivalent to approximately 1% of added N. This relatively low percentage indicates a possible origin from nitrifier denitrification. Total digestate and its liquid fraction exhibited considerable net-N mineralisation rates, which could mainly be predicted by the C/N ratios of the different digestate qualities. Microbial biomass C did not respond to the application of any digestate quality, whereas the fungal ergosterol content increased after applying the solid and the composted solid fractions. This raw fibre–induced fungal growth led to strong net-N immobilisation in soil after applying these two digestate qualities.

Isotopic evidence for alteration of nitrous oxide emissions and producing pathways' contribution under nitrifying conditions

Nitrous oxide (N2O) emissions from a nitrifying biofilm reactor were investigated with N2O isotopocules. The nitrogen isotopomer site preference of N2O (15N-SP) indicated the contribution of producing and consuming pathways in response to changes in oxygenation level (from 0 % to 21 % O2 in the gas mix), temperature (from 13.5 to 22.3 ∘C) and ammonium concentrations (from 6.2 to 62.1 mg N L−1). Nitrite reduction, either nitrifier denitrification or heterotrophic denitrification, was the main N2O-producing pathway under the tested conditions. Difference between oxidative and reductive rates of nitrite consumption was discussed in relation to NO2- concentrations and N2O emissions. Hence, nitrite oxidation rates seem to decrease as compared to ammonium oxidation rates at temperatures above 20 ∘C and under oxygen-depleted atmosphere, increasing N2O production by the nitrite reduction pathway. Below 20 ∘C, a difference in temperature sensitivity between hydroxylamine and ammonium oxidation rates is most likely responsible for an increase in N2O production via the hydroxylamine oxidation pathway (nitrification). A negative correlation between the reaction kinetics and the apparent isotope fractionation was additionally shown from the variations of δ15N and δ18O values of N2O produced from ammonium. The approach and results obtained here, for a nitrifying biofilm reactor under variable environmental conditions, should allow for application and extrapolation of N2O emissions from other systems such as lakes, soils and sediments.

Natural Nitrogen Isotope Ratios as a Potential Indicator of N2O Production Pathways in a Floodplain Fen

Nitrous oxide (N2O), a major greenhouse gas and ozone depleter, is emitted from drained organic soils typically developed in floodplains. We investigated the effect of the water table depth and soil oxygen (O2) content on N2O fluxes and their nitrogen isotope composition in a drained floodplain fen in Estonia. Measurements were done at natural water table depth, and we created a temporary anoxic environment by experimental flooding. From the suboxic peat (0.5–6 mg O2/L) N2O emissions peaked at 6 mg O2/L and afterwards decreased with decreasing O2. From the anoxic and oxic peat (0 and >6 mg O2/L, respectively) N2O emissions were low. Under anoxic conditions the δ15N/δ14N ratio of the top 10 cm peat layer was low, gradually decreasing to 30 cm. In the suboxic peat, δ15N/δ14N ratios increased with depth. In samples of peat fluctuating between suboxic and anoxic, the elevated 15N/14N ratios (δ15N = 7–9‰ ambient N2) indicated intensive microbial processing of nitrogen. Low values of site preference (SP; difference between the central and peripheral 15N atoms) and δ18O-N2O in the captured gas samples indicate nitrifier denitrification in the floodplain fen.

Transcriptomic Response of Nitrosomonas europaea Transitioned from Ammonia- to Oxygen-Limited Steady-State Growth

ABSTRACT Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea. Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea’s sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria. IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.