Evaluating four mathematical models for nitrous oxide production by autotrophic ammonia‐oxidizing bacteria

There is increasing evidence showing that ammonia‐oxidizing bacteria (AOB) are major contributors to N₂O emissions from wastewater treatment plants (WWTPs). Although the fundamental metabolic pathways for N₂O production by AOB are now coming to light, the mechanisms responsible for N₂O production by AOB in WWTP are not fully understood. Mathematical modeling provides a means for testing hypotheses related to mechanisms and triggers for N₂O emissions in WWTP, and can then also become a tool to support the development of mitigation strategies. This study examined the ability of four mathematical model structures to describe two distinct mechanisms of N₂O production by AOB. The production mechanisms evaluated are (1) N₂O as the final product of nitrifier denitrification with NO as the terminal electron acceptor and (2) N₂O as a byproduct of incomplete oxidation of hydroxylamine (NH₂OH) to NO. The four models were compared based on their ability to predict N₂O dynamics observed in three mixed culture studies. Short‐term batch experimental data were employed to examine model assumptions related to the effects of (1) NH concentration variations, (2) dissolved oxygen (DO) variations, (3) NO accumulations and (4) NH₂OH as an externally provided substrate. The modeling results demonstrate that all these models can generally describe the NH, NO, and NO data. However, none of these models were able to reproduce all measured N₂O data. The results suggest that both the denitrification and NH₂OH pathways may be involved in N₂O production and could be kinetically linked by a competition for intracellular reducing equivalents. A unified model capturing both mechanisms and their potential interactions needs to be developed with consideration of physiological complexity. Biotechnol. Bioeng. 2013; 110: 153–163. © 2012 Wiley Periodicals, Inc.     

Thermodynamic characterization of proton‐ionizable functional groups on the cell surfaces of ammonia‐oxidizing bacteria and archaea

The ammonia‐oxidizing archaeon Nitrosopumilus maritimus strain SCM1 (strain SCM1), a representative of the Thaumarchaeota archaeal phylum, can sustain high specific rates of ammonia oxidation at ammonia concentrations too low to sustain metabolism by ammonia‐oxidizing bacteria (AOB). One structural and biochemical difference between N. maritimus and AOB that might be related to the oligotrophic adaptation of strain SCM1 is the cell surface. A proteinaceous surface layer (S‐layer) comprises the outermost boundary of the strain SCM1 cell envelope, as opposed to the lipopolysaccharide coat of Gram‐negative AOB. In this work, we compared the surface reactivities of two archaea having an S‐layer (strain SCM1 and Sulfolobus acidocaldarius) with those of four representative AOB (Nitrosospira briensis, Nitrosomonas europaea, Nitrosolobus multiformis, and Nitrosococcus oceani) using potentiometric and calorimetric titrations to evaluate differences in proton‐ionizable surface sites. Strain SCM1 and S. acidocaldarius have a wider range of proton buffering (approximately pH 10–3.5) than the AOB (approximately pH 10–4), under the conditions investigated. Thermodynamic parameters describing proton‐ionizable sites (acidity constants, enthalpies, and entropies of protonation) are consistent with these archaea having proton‐ionizable amino acid side chains containing carboxyl, imidazole, thiol, hydroxyl, and amine functional groups. Phosphorous‐bearing acidic functional groups, which might also be present, could be masked by imidazole and thiol functional groups. Parameters for the AOB are consistent with surface structures containing anionic oxygen ligands (carboxyl‐ and phosphorous‐bearing acidic functional groups), thiols, and amines. In addition, our results showed that strain SCM1 has more reactive surface sites than the AOB and a high concentration of sites consistent with aspartic and/or glutamic acid. Because these alternative boundary layers mediate interaction with the local external environment, these data provide the basis for further comparisons of the thermodynamic behavior of surface reactivity toward essential nutrients.     

High diversity of ammonia‐oxidizing archaea in permanent and seasonal oxygen‐deficient waters of the eastern South Pacific

The community structure of putative aerobic ammonia‐oxidizing archaea (AOA) was explored in two oxygen‐deficient ecosystems of the eastern South Pacific: the oxygen minimum zone off Peru and northern Chile (11°S–20°S), where permanent suboxic and low‐ammonium conditions are found at intermediate depths, and the continental shelf off central Chile (36°S), where seasonal oxygen‐deficient and relatively high‐ammonium conditions develop in the water column, particularly during the upwelling season. The AOA community composition based on the ammonia monooxygenase subunit A (amoA) genes changed according to the oxygen concentration in the water column and the ecosystem studied, showing a higher diversity in the seasonal low‐oxygen waters. The majority of the archaeal amoA genotypes was affiliated to the uncultured clusters A (64%) and B (35%), with Cluster A AOA being mainly associated with higher oxygen and ammonium concentrations and Cluster B AOA with permanent oxygen‐ and ammonium‐poor waters. Q‐PCR assays revealed that AOA are an abundant community (up to 10⁵amoA copies ml^?1), while bacterial amoA genes from β proteobacteria were undetected. Our results thus suggest that a diverse uncultured AOA community, for which, therefore, we do not have any physiological information, to date, is an important component of the nitrifying community in oxygen‐deficient marine ecosystems, and particularly in rich coastal upwelling ones.     

Responses of community structure of amoA‐encoding archaea and ammonia‐oxidizing bacteria in ammonia biofilter with rockwool mixtures to the gradual increases in ammonium and nitrate

Aims

To investigate community shifts of amoA‐encoding archaea (AEA) and ammonia‐oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process.

Methods and Results

A laboratory‐scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49–63 NH₃ g m^?3 day^?1. The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real‐time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively.

Conclusions

The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH.

Significance and Impact of the Study

One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.     

Differential photoinhibition of bacterial and archaeal ammonia oxidation

Inhibition by light potentially influences the distribution of ammonia oxidizers in aquatic environments and is one explanation for nitrite maxima near the base of the euphotic zone of oceanic waters. Previous studies of photoinhibition have been restricted to bacterial ammonia oxidizers, rather than archaeal ammonia oxidizers, which dominate in marine environments. To compare the photoinhibition of bacterial and archaeal ammonia oxidizers, specific growth rates of two ammonia-oxidizing archaea (Nitrosopumilus maritimus and Nitrosotalea devanaterra) and bacteria (Nitrosomonas europaea and Nitrosospira multiformis) were determined at different light intensities under continuous illumination and light/dark cycles. All strains were inhibited by continuous illumination at the highest intensity (500 μE m(-2) s(-1)). At lower light intensities, archaeal growth was much more photosensitive than bacterial growth, with greater inhibition at 60 μE m(-2) s(-1) than at 15 μE m(-2) s(-1), where bacteria were unaffected. Archaeal ammonia oxidizers were also more sensitive to cycles of 8-h light/16-h darkness at two light intensities (60 and 15 μE m(-2) s(-1)) and, unlike bacterial strains, showed no evidence of recovery during dark phases. The findings provide evidence for niche differentiation in aquatic environments and reduce support for photoinhibition as an explanation of nitrite maxima in the ocean.     

Myoglobin: a pseudo-enzymatic scavenger of nitric oxide

Myoglobin (Mb) is reported in biochemistry and physiology textbooks to act as an O₂ reservoir and to facilitate O₂ diffusion from capillaries to mitochondria, to sustain cellular respiration. Recently, it has been proposed that Mb is an intracellular scavenger of bioactive nitric oxide (NO), regulating its level in the skeletal and cardiac muscle and thereby protecting mitochondrial respiration, which is impaired by NO. This novel function of Mb is based on the rapid and irreversible reaction of ferrous oxygenated Mb (MbO₂) with NO yielding ferric oxidized Mb (metMb) and nitrate (NO₃^–). The efficiency of this process, which is postulated to depend on the superoxide (O₂^–) character acquired by O₂ once bound to the heme iron, may be enhanced by intramolecular diffusion of NO trapped momentarily into cavities of the protein matrix. O₂ can also react with ferrous nitrosylated Mb (MbNO), albeit very slowly, leading to metMb and NO₃^–. The O₂-dependent NO-detoxification process may be considered to be pseudo-enzymatic given that metMb obtained by the primary reaction of MbO₂ with NO is reduced back to ferrous Mb by a specific metMb-reductase, and can therefore repeat a cycle of NO conversion to harmless nitrate.     

Cycloamphilectenes,a new type of potent marine diterpenes: inhibition of nitric oxide production in murine macrophages

The inhibitory effect of a series of 6 cycloamphilectenes, novel marine diterpenes based on amphilectene skeletons and isolated from the Vanuatu sponge Axinella sp., on NO, PGE(2) and TNFalpha production in murine peritoneal macrophages was studied. These compounds reduced potently nitric oxide production in a concentration-dependent manner with IC(50) values in the submicromolar range (0.1-4.3 microM). Studies on intact cells and Western blot analysis showed that the more potent cycloamphilectenes reduced the expression of inducible nitric oxide synthase without affecting cyclo-oxygenase-2 expression. Among them cycloamphilectene 2, the unique compound bearing an exocyclic methylene group, was able to reduce NO production without affecting TNFalpha release. Cycloamphilectene 2, which is an inhibitor of the nuclear factor-kB pathway, exhibited topical anti-inflammatory activity.     

Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis

Nitrous oxide (N₂O), a greenhouse gas, is emitted during autotrophic and heterotrophic ammonia oxidation. This emission may result from either coupling to aerobic denitrification, or it may be formed in the oxidation of hydroxylamine (NH₂OH) to nitrite (NO₂ ⁻). Therefore, the N₂O production during NH₂OH oxidation was studied with Alcaligenes faecalis strain TUD. Continuous cultures of A. faecalis showed increased N₂O production when supplemented with increasing NH₂OH concentrations. ¹⁵N-labeling experiments showed that this N₂O production was not due to aerobic denitrification of NO₂ ⁻. Addition of ¹⁵N-labeled NH₂OH indicated that N₂O was a direct by-product of NH₂OH oxidation, which was subsequently reduced to N₂. These observations are sustained by the fact that NO₂ ⁻ production was low (0.23 mM maximum) and did not increase significantly with increasing NH₂OH concentration in the feed. The NH₂OH-oxidizing capacity increased with increasing NH₂OH concentrations. The apparent V _max and K _m were 31 nmol min⁻¹ mg dry weight⁻¹ and 1.5 mM respectively. The culture did not increase its growth yield and was not able to use NH₂OH as the sole N source. A non-haem hydroxylamine oxidoreductase was partially purified from A. faecalis strain TUD. The enzyme could only use K₃Fe(CN)₆ as an electron acceptor and reacted with antibodies raised against the hydroxylamine oxidoreductase of Thiosphaera pantotropha. Received: 1 September 1998 / Received revision: 5 November 1998 / Accepted: 7 November 1998     

Community structures of ammonia‐oxidising archaea and bacteria in high‐altitude lakes on the Tibetan Plateau

1. Community structures of planktonic ammonia‐oxidising archaea (AOA) and bacteria (AOB) were investigated for five high‐altitude Tibetan lakes, which could be classified as freshwater, oligosaline or mesosaline, to develop a general view of the AOA and AOB in lakes on the Tibetan Plateau. 2. Based on PCR screening of the ammonia monooxygenase α‐subunit (amoA) gene, AOA were present in 14 out of 17 samples, whereas AOB were detected in only four samples. Phylogenetic analyses indicated that the AOB communities were dominated by a unique monophylogenetic lineage within Nitrosomonas, which may represent a novel cluster of AOB. AOA, on the other hand, were distinct among lakes with different salinities. 3. Multivariate statistical analyses indicated a heterogeneous distribution of the AOA communities among lakes largely caused by lake salinity, whereas the uniform chemical properties within lakes and their geographical isolation may favour relatively homogeneous AOA communities within lakes. 4. Our results suggest a wide occurrence of AOA in Tibetan lakes and provide the first evidence of salinity‐related differentiation of AOA community composition as well as potential geographical isolation of AOA in inland aquatic environments.     

The genome of the ammonia‐oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations

The cohort of the ammonia‐oxidizing archaea (AOA) of the phylum Thaumarchaeota is a diverse, widespread and functionally important group of microorganisms in many ecosystems. However, our understanding of their biology is still very rudimentary in part because all available genome sequences of this phylum are from members of the Nitrosopumilus cluster. Here we report on the complete genome sequence of Candidatus Nitrososphaera gargensis obtained from an enrichment culture, representing a different evolutionary lineage of AOA frequently found in high numbers in many terrestrial environments. With its 2.83 Mb the genome is much larger than that of other AOA. The presence of a high number of (active) IS elements/transposases, genomic islands, gene duplications and a complete CRISPR/Cas defence system testifies to its dynamic evolution consistent with low degree of synteny with other thaumarchaeal genomes. As expected, the repertoire of conserved enzymes proposed to be required for archaeal ammonia oxidation is encoded by N. gargensis, but it can also use urea and possibly cyanate as alternative ammonia sources. Furthermore, its carbon metabolism is more flexible at the central pyruvate switch point, encompasses the ability to take up small organic compounds and might even include an oxidative pentose phosphate pathway. Furthermore, we show that thaumarchaeota produce cofactor F420 as well as polyhydroxyalkanoates. Lateral gene transfer from bacteria and euryarchaeota has contributed to the metabolic versatility of N. gargensis. This organisms is well adapted to its niche in a heavy metal‐containing thermal spring by encoding a multitude of heavy metal resistance genes, chaperones and mannosylglycerate as compatible solute and has the genetic ability to respond to environmental changes by signal transduction via a large number of two‐component systems, by chemotaxis and flagella‐mediated motility and possibly even by gas vacuole formation. These findings extend our understanding of thaumarchaeal evolution and physiology and offer many testable hypotheses for future experimental research on these nitrifiers.     

A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

Inhibition of dynamin‐2 confers endothelial barrier dysfunctions by attenuating nitric oxide production

Hypoxia induces barrier dysfunctions in endothelial cells. Nitric oxide is an autacoid signalling molecule that confers protection against hypoxia‐mediated barrier dysfunctions. Dyn‐2 (dynamin‐2), a large GTPase and a positive modulator of eNOS (endothelial nitric oxide synthase), plays an important role in maintaining vascular homeostasis. The present study aims to elucidate the role of dyn‐2 in hypoxia‐mediated leakiness of the endothelial monolayer in relation to redox milieu. Inhibition of dyn‐2 by transfecting the cells with K44A, a dominant negative construct of dyn‐2, elevated leakiness of the endothelial monolayer under hypoxia. Sodium nitroprusside (nitric oxide donor) and uric acid (peroxynitrite quencher) were used to evaluate the role of nitric oxide and peroxynitrite in maintaining endothelial barrier functions under hypoxia. Administration of nitric oxide and uric acid recovered hypoxia‐mediated leakiness of K44A‐overexpressed endothelial monolayer. Our study confirms that inhibition of dyn‐2 induces leakiness in the endothelial monolayer by increasing the load of peroxynitrite under hypoxia.     

Pathways of dopamine oxidation mediated by nitric oxide

This study was aimed at establishing the interaction between dopamine and nitric oxide and elucidating the mechanistic aspects inherent in this interaction. At high (*) NO concentrations (microM range), dopamine underwent nitrosation with subsequent nitration. Nitrosation is proposed to occur via a nucleophilic attack to N(2)O(3) by dopamine. At low (*) NO concentrations (microM range), dopaminochrome was formed. EPR spin stabilization studies showed the occurrence of two o-semiquinone intermediates during dopaminochrome formation. Heats of formation obtained by AM1 semiempirical calculations supported the formation of the two o-semiquinone species. Hydroxyl radicals were detected by spin trapping EPR, and experiments performed with superoxide dismutase and catalase suggested that peroxynitrite was the source of HO(*). A mechanism is presented that considers the several factors influencing these reactions.     

Targeting spatiotemporal dynamics of planktonic SAGMGC‐1 and segregation of ammonia‐oxidizing thaumarchaeota ecotypes by newly designed primers and quantitative polymerase chain reaction

The annual dynamics of three different ammonia‐oxidizing archaea (AOA) ecotypes (amoA gene) and of the SAGMGC‐1 (Nitrosotalea‐like aquatic Thaumarchaeota) group (16S rRNA gene) were studied by newly designed specific primers and quantitative polymerase chain reaction analysis in a deep oligotrophic high mountain lake (Lake Redon, Limnological Observatory of the Pyrenees, Spain). We observed segregated distributions of the main AOA populations, peaking separately in time and space, and under different ammonia concentrations and irradiance conditions. Strong positive correlation in gene abundances was found along the annual survey between 16S rRNA SAGMAGC‐1 and one of the amoA ecotypes suggesting the potential for ammonia oxidation in the freshwater SAGMAGC‐1 clade. We also observed dominance of Nitrosotalea‐like ecotypes over Nitrosopumilus‐like (Marine Group 1.1a) and not the same annual dynamics for the two thaumarchaeotal clades. The fine scale segregation in space and time of the different AOA ecotypes indicated the presence of phylogenetically close but ecologically segregated AOA species specifically adapted to specific environmental conditions. It remains to be elucidated what would be such environmental drivers.     

Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension

Mathew, Rajamma, Elizabeth S. Gloster, T. Sundararajan, Carl I. Thompson, Guillermo A. Zeballos, andMichael H. Gewitz. Role of inhibition of nitric oxide productionin monocrotaline-induced pulmonary hypertension. J. Appl. Physiol. 82(5): 1493-1498, 1997.Monocrotaline (MCT)-induced pulmonary hypertension (PH) isassociated with impaired endothelium-dependent nitric oxide(NO)-mediated relaxation. To examine the role of NO in PH,Sprague-Dawley rats were given a single subcutaneous injection ofnormal saline [control (C)], 80 mg/kg MCT, or the same doseof MCT and a continuous subcutaneous infusion of 2 mg · kg¹ · day¹of molsidomine, a NO prodrug (MCT+MD). Two weeks later, plasma NO₃ levels, pulmonary arterialpressure (Ppa), ratio of right-to-left ventricular weights (RV/LV) toassess right ventricular hypertrophy, and pulmonary histology wereevaluated. The plasma NO₃ level inthe MCT group was reduced to 9.2 ± 1.5 µM(n = 12) vs. C level of 17.7 ± 1.8 µM (n = 8; P < 0.02). In the MCT+MD group,plasma NO₃ level was 12.3 ± 2.0 µM (n = 8). Ppa and RV/LV in theMCT group were increased compared with C [Ppa, 34 ± 3.4 mmHg(n = 6) vs. 19 ± 0.8 mmHg(n = 8) and 0.41 ± 0.01 (n = 9) vs. 0.25 ± 0.008 (n = 8), respectively;P < 0.001]. In the MCT+MDgroup, Ppa and RV/LV were not different when compared with C [19 ± 0.5 mmHg (n = 5) and 0.27 ± 0.01 (n = 9), respectively;P < 0.001 vs. MCT]. Medial wall thickness of lung vessels in the MCT group was increased comparedwith C [31 ± 1.5% (n = 9)vs. 13 ± 0.66% (n = 9);P < 0.001], and MDpartially prevented MCT-induced pulmonary vascular remodeling [22 ± 1.2% (n = 11);P < 0.001 vs. MCT and C].These results indicate that a defect in the availability of bioactive NO may play an important role in the pathogenesis of MCT-induced PH.