Reactor staging influences microbial community composition and diversity of denitrifying MBBRs- Implications on pharmaceutical removal.

The subdivision of biofilm reactor in two or more stages (i.e., reactor staging) represents an option for process optimisation of biological treatment. In our previous work, we showed that the gradient of influent organic substrate availability (induced by the staging) can influence the microbial activity (i.e., denitrification and pharmaceutical biotransformation kinetics) of a denitrifying three-stage Moving Bed Biofilm Reactor (MBBR) system. However, it is unclear whether staging and thus the long-term exposure to varying organic carbon type and loading influences the microbial community structure and diversity. In this study, we investigated biofilm structure and diversity in the three-stage MBBR system (S) compared to a single-stage configuration (U) and their relationship with microbial functions. Results from 16S rRNA amplicon libraries revealed a significantly higher microbial richness in the staged MBBR (at 99% sequence similarity) compared to single-stage MBBR. A more even and diverse microbial community was selected in the last stage of S (S3), likely due to exposure to carbon limitation during continuous-flow operation. A core of OTUs was shared in both systems, consisting of Burkholderiales, Xanthomonadales, Flavobacteriales and Sphingobacteriales, while MBBR staging selected for specific taxa (i.e., Candidate division WS6 and Deinococcales). Results from quantitative PCR (qPCR) showed that S3 exhibited the lowest abundance of 16S rRNA but the highest abundance of atypical nosZ, suggesting a selection of microbes with more diverse N-metabolism (i.e., incomplete denitrifiers) in the stage exposed to the lowest carbon availability. A positive correlation (p < 0.05) was observed between removal rate constants of several pharmaceuticals with abundance of relevant denitrifying genes, but not with biodiversity. Despite the previously suggested positive relationship between microbial diversity and functionality in macrobial and microbial ecosystems, this was not observed in the current study, indicating a need to further investigate structure-function relationships for denitrifying systems.

Heterotrophs are key contributors to nitrous oxide production in activated sludge under low C-to-N ratios during nitrification-Batch experiments and modeling.

Nitrous oxide (N2 O), a by-product of biological nitrogen removal during wastewater treatment, is produced by ammonia-oxidizing bacteria (AOB) and heterotrophic denitrifying bacteria (HB). Mathematical models are used to predict N2 O emissions, often including AOB as the main N2 O producer. Several model structures have been proposed without consensus calibration procedures. Here, we present a new experimental design that was used to calibrate AOB-driven N2 O dynamics of a mixed culture. Even though AOB activity was favoured with respect to HB, oxygen uptake rates indicated HB activity. Hence, rigorous experimental design for calibration of autotrophic N2 O production from mixed cultures is essential. The proposed N2 O production pathways were examined using five alternative process models confronted with experimental data inferred. Individually, the autotrophic and heterotrophic denitrification pathway could describe the observed data. In the best-fit model, which combined two denitrification pathways, the heterotrophic was stronger than the autotrophic contribution to N2 O production. Importantly, the individual contribution of autotrophic and heterotrophic to the total N2 O pool could not be unambiguously elucidated solely based on bulk N2 O measurements. Data on NO would increase the practical identifiability of N2 O production pathways. Biotechnol. Bioeng. 2017;114: 132-140. © 2016 Wiley Periodicals, Inc.

Aerobic Microbial Respiration In Oceanic Oxygen Minimum Zones.

Oxygen minimum zones are major sites of fixed nitrogen loss in the ocean. Recent studies have highlighted the importance of anaerobic ammonium oxidation, anammox, in pelagic nitrogen removal. Sources of ammonium for the anammox reaction, however, remain controversial, as heterotrophic denitrification and alternative anaerobic pathways of organic matter remineralization cannot account for the ammonium requirements of reported anammox rates. Here, we explore the significance of microaerobic respiration as a source of ammonium during organic matter degradation in the oxygen-deficient waters off Namibia and Peru. Experiments with additions of double-labelled oxygen revealed high aerobic activity in the upper OMZs, likely controlled by surface organic matter export. Consistently observed oxygen consumption in samples retrieved throughout the lower OMZs hints at efficient exploitation of vertically and laterally advected, oxygenated waters in this zone by aerobic microorganisms. In accordance, metagenomic and metatranscriptomic analyses identified genes encoding for aerobic terminal oxidases and demonstrated their expression by diverse microbial communities, even in virtually anoxic waters. Our results suggest that microaerobic respiration is a major mode of organic matter remineralization and source of ammonium (~45-100%) in the upper oxygen minimum zones, and reconcile hitherto observed mismatches between ammonium producing and consuming processes therein.

Aeration strategies to mitigate nitrous oxide emissions from single-stage nitritation/anammox reactors.

Autotrophic nitrogen removal is regarded as a resource efficient process to manage nitrogen-rich residual streams. However, nitrous oxide emissions of these processes are poorly documented and strategies to mitigate emissions unknown. In this study, two sequencing batch reactors performing single-stage nitritation/anammox were operated under different aeration strategies, gradually adjusted over six months. At constant but limiting oxygen loading, synthetic reject water was fed (0.75 g-N/L · d) and high nitrogen removal efficiencies (83 ± 5 and 88 ± 2%) obtained. Dynamics of liquid phase nitrous (N2O) and nitric oxide (NO) concentrations were monitored and N2O emissions calculated. Significant decreases in N2O emissions were obtained when the frequency of aeration was increased while maintaining a constant air flow rate (from >6 to 1.7% ΔN2O/ΔTN). However, no significant effect on the emissions was noted when the duration of aeration was increased while decreasing air flow rate (10.9 ± 3.2% ΔN2O/ΔTN). The extant ammonium oxidation activity (mgNH4(+)-N/gVSS · min) positively correlated with the specific N2O production rate (mgN2O-N/gVSS · min) of the systems. Operating under conditions where anaerobic exceeds aerobic ammonium oxidation activity is proposed to minimize N2O emissions from single-stage nitritation/anammox reactors; increasing the frequency of aeration cycling is an efficient way of obtaining those conditions.

Nitrite oxidation in the Namibian oxygen minimum zone.

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in (15)N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (≤372 nM NO(2)(-) d(-1)) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ~9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO(3)(-) was re-oxidized back to NO(3)(-) via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.

Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium.

A combination of stable isotopes ((15)N) and molecular ecological approaches was used to investigate the vertical distribution and mechanisms of biological N(2) production along a transect from the Omani coast to the central-northeastern (NE) Arabian Sea. The Arabian Sea harbors the thickest oxygen minimum zone (OMZ) in the world's oceans, and is considered to be a major site of oceanic nitrogen (N) loss. Short (<48 h) anoxic incubations with (15)N-labeled substrates and functional gene expression analyses showed that the anammox process was highly active, whereas denitrification was hardly detectable in the OMZ over the Omani shelf at least at the time of our sampling. Anammox was coupled with dissimilatory nitrite reduction to ammonium (DNRA), resulting in the production of double-(15)N-labeled N(2) from (15)NO(2)(-), a signal often taken as the lone evidence for denitrification in the past. Although the central-NE Arabian Sea has conventionally been regarded as the primary N-loss region, low potential N-loss rates at sporadic depths were detected at best. N-loss activities in this region likely experience high spatiotemporal variabilities as linked to the availability of organic matter. Our finding of greater N-loss associated with the more productive Omani upwelling region is consistent with results from other major OMZs. The close reliance of anammox on DNRA also highlights the need to take into account the effects of coupling N-transformations on oceanic N-loss and subsequent N-balance estimates.

15N-labeling experiments to dissect the contributions of heterotrophic denitrification and anammox to nitrogen removal in the OMZ waters of the ocean.

In recent years, (15)N-labeling experiments have become a powerful tool investigating rates and regulations of microbially mediated nitrogen loss processes in the ocean. This chapter introduces the theoretical and practical aspects of (15)N-labeling experiments to dissect the contribution of denitrification and anammox to nitrogen removal in oxygen minimum zones (OMZs). We provide a detailed description of the preparation and realization of the experiments on board. Subsequent measurements of N(2) isotopes using gas chromatography mass spectrometry as well as processing of data and calculation of anammox and denitrification rates are explained. Important supplementary measurements are specified, such as the measurement of nanomolar concentrations of ammonium, nitrite, and nitrate. Nutrient profiles and (15)N-experiments from the Peruvian OMZ are presented and discussed as an example.

Oxygen sensitivity of anammox and coupled N-cycle processes in oxygen minimum zones.

Nutrient measurements indicate that 30-50% of the total nitrogen (N) loss in the ocean occurs in oxygen minimum zones (OMZs). This pelagic N-removal takes place within only ~0.1% of the ocean volume, hence moderate variations in the extent of OMZs due to global warming may have a large impact on the global N-cycle. We examined the effect of oxygen (O(2)) on anammox, NH(3) oxidation and NO(3)(-) reduction in (15)N-labeling experiments with varying O(2) concentrations (0-25 µmol L(-1)) in the Namibian and Peruvian OMZs. Our results show that O(2) is a major controlling factor for anammox activity in OMZ waters. Based on our O(2) assays we estimate the upper limit for anammox to be ~20 µmol L(-1). In contrast, NH(3) oxidation to NO(2)(-) and NO(3)(-) reduction to NO(2)(-) as the main NH(4)(+) and NO(2)(-) sources for anammox were only moderately affected by changing O(2) concentrations. Intriguingly, aerobic NH(3) oxidation was active at non-detectable concentrations of O(2), while anaerobic NO(3)(-) reduction was fully active up to at least 25 µmol L(-1) O(2). Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O(2) concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O(2)-sensitivity of anammox itself, and not by any effects of O(2) on the tightly coupled pathways of aerobic NH(3) oxidation and NO(3)(-) reduction. With anammox bacteria in the marine environment being active at O(2) levels ~20 times higher than those known to inhibit their cultured counterparts, the oceanic volume potentially acting as a N-sink increases tenfold. The predicted expansion of OMZs may enlarge this volume even further. Our study provides the first robust estimates of O(2) sensitivities for processes directly and indirectly connected with N-loss. These are essential to assess the effects of ocean de-oxygenation on oceanic N-cycling.

Aerobic denitrification in permeable Wadden Sea sediments.

Permeable or sandy sediments cover the majority of the seafloor on continental shelves worldwide, but little is known about their role in the coastal nitrogen cycle. We investigated the rates and controls of nitrogen loss at a sand flat (Janssand) in the central German Wadden Sea using multiple experimental approaches, including the nitrogen isotope pairing technique in intact core incubations, slurry incubations, a flow-through stirred retention reactor and microsensor measurements. Results indicate that permeable Janssand sediments are characterized by some of the highest potential denitrification rates (> or =0.19 mmol N m(-2) h(-1)) in the marine environment. Moreover, several lines of evidence showed that denitrification occurred under oxic conditions. In intact cores, microsensor measurements showed that the zones of nitrate/nitrite and O(2) consumption overlapped. In slurry incubations conducted with (15)NO(3)(-) enrichment in gas-impermeable bags, denitrification assays revealed that N(2) production occurred at initial O(2) concentrations of up to approximately 90 microM. Initial denitrification rates were not substantially affected by O(2) in surficial (0-4 cm) sediments, whereas rates increased by twofold with O(2) depletion in the at 4-6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), (29)N(2) and (30)N(2) were produced and O(2) was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O(2) may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.

Revising the nitrogen cycle in the Peruvian oxygen minimum zone.

The oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP) is 1 of the 3 major regions in the world where oceanic nitrogen is lost in the pelagic realm. The recent identification of anammox, instead of denitrification, as the likely prevalent pathway for nitrogen loss in this OMZ raises strong questions about our understanding of nitrogen cycling and organic matter remineralization in these waters. Without detectable denitrification, it is unclear how NH(4)(+) is remineralized from organic matter and sustains anammox or how secondary NO(2)(-) maxima arise within the OMZ. Here we show that in the ETSP-OMZ, anammox obtains 67% or more of NO(2)(-) from nitrate reduction, and 33% or less from aerobic ammonia oxidation, based on stable-isotope pairing experiments corroborated by functional gene expression analyses. Dissimilatory nitrate reduction to ammonium was detected in an open-ocean setting. It occurred throughout the OMZ and could satisfy a substantial part of the NH(4)(+) requirement for anammox. The remaining NH(4)(+) came from remineralization via nitrate reduction and probably from microaerobic respiration. Altogether, deep-sea NO(3)(-) accounted for only approximately 50% of the nitrogen loss in the ETSP, rather than 100% as commonly assumed. Because oceanic OMZs seem to be expanding because of global climate change, it is increasingly imperative to incorporate the correct nitrogen-loss pathways in global biogeochemical models to predict more accurately how the nitrogen cycle in our future ocean may respond.

Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea.

Active expression of putative ammonia monooxygenase gene subunit A (amoA) of marine group I Crenarchaeota has been detected in the Black Sea water column. It reached its maximum, as quantified by reverse-transcription quantitative PCR, exactly at the nitrate maximum or the nitrification zone modeled in the lower oxic zone. Crenarchaeal amoA expression could explain 74.5% of the nitrite variations in the lower oxic zone. In comparison, amoA expression by gamma-proteobacterial ammonia-oxidizing bacteria (AOB) showed two distinct maxima, one in the modeled nitrification zone and one in the suboxic zone. Neither the amoA expression by crenarchaea nor that by beta-proteobacterial AOB was significantly elevated in this latter zone. Nitrification in the suboxic zone, most likely microaerobic in nature, was verified by (15)NO(2)(-) and (15)N(15)N production in (15)NH(4)(+) incubations with no measurable oxygen. It provided a direct local source of nitrite for anammox in the suboxic zone. Both ammonia-oxidizing crenarchaea and gamma-proteobacterial AOB were important nitrifiers in the Black Sea and were likely coupled to anammox in indirect and direct manners respectively. Each process supplied about half of the nitrite required by anammox, based on (15)N-incubation experiments and modeled calculations. Because anammox is a major nitrogen loss in marine suboxic waters, such nitrification-anammox coupling potentially occurring also in oceanic oxygen minimum zones would act as a short circuit connecting regenerated ammonium to direct nitrogen loss, thus reducing the presumed direct contribution from deep-sea nitrate.