Sulfur-driven autotrophic denitrification of nitric oxide for efficient nitrous oxide recovery

Nitrous oxide (N₂O) was previously deemed as a potent greenhouse gas but is actually an untapped energy source, which can accumulate during the microbial denitrification of nitric oxide (NO). Compared with the organic electron donor required in heterotrophic denitrification, elemental sulfur (S⁰) is a promising electron donor alternative due to its cheap cost and low biomass yield in sulfur-driven autotrophic denitrification. However, no effort has been made to test N₂O recovery from sulfur-driven denitrification of NO so far. Therefore, in this study, batch and continuous experiments were carried out to investigate the NO removal performance and N₂O recovery potential via sulfur-driven NO-based denitrification under various Fe(II)EDTA-NO concentrations. Efficient energy recovery was achieved, as up to 35.5%–40.9% of NO was converted to N₂O under various NO concentrations. N₂O recovery from Fe(II)EDTA-NO could be enhanced by the low bioavailability of sulfur and the acid environment caused by sulfur oxidation. The NO reductase (NOR) and N₂O reductase (N₂OR) were inhibited distinctively at relatively low NO levels, leading to efficient N₂O accumulation, but were suppressed irreversibly at NO level beyond 15 mM in continuous experiments. Such results indicated that the regulation of NO at a relatively low level would benefit the system stability and NO removal capacity during long-term system operation. The continuous operation of the sulfur-driven Fe(II)EDTA-NO-based denitrification reduced the overall microbial diversity but enriched several key microbial community. Thauera, Thermomonas, and Arenimonas that are able to carry out sulfur-driven autotrophic denitrification became the dominant organisms with their relative abundance increased from 25.8% to 68.3%, collectively.     

Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification

Summary The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd ₁) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c ₅₅₂ mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd ₁ lacking heme d ₁, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir^– mutants of another type that had low level synthesis of cytochrome cd ₁, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS ⁺ and nirD ⁺ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.     

Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans

Cytochrome cd ₁-nitrite reductase and nitrous oxide reductase of Thiobacillus denitrificans were purified and characterized by biochemical and immunochemical methods. In contrast to the generally soluble nature of the denitrification enzymes, these two enzymes were isolated from the membrane fraction of T. denitrificans and remained active after solubilization with Triton X-100. The properties of the membrane-derived enzymes were similar to those of their soluble counterparts from the same organism. Nitrous oxide reductase activity was inhibited by acetylene. Nitrite reductase and nitrous oxide reductase cross-reacted with antisera raised against the soluble enzymes from Pseudomonas stutzeri. The nirS, norBC, and nosZ genes encoding the cytochrome cd ₁-nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively, from P. stutzeri hybridized with genomic DNA from T. denitrificans. Cross-reactivity and similar N-terminal amino acid and gene sequences suggest that the primary structures of the Thiobacillus enzymes are homologous to the soluble proteins from P. stutzeri. Received: 18 August 1995 / Accepted: 30 October 1995     

Amperometric method for determining nitrous oxide in denitrification and in nitrogenase-catalyzed nitrous oxide reduction

A conventional Clark-type O₂ probe was used to determine N₂O concentrations in suspensions. At a polarizing voltage of–0.95 V versus the reference Ag/AgCl electrode, the probe is almost half as sensitive for N₂O as for O₂, and the detection limit is less than 1 M N₂O. The probe can also be used to determine NO for which the suitable polarizing voltage is–0.7 V. The method was successfully applied for continuously recording dissimilatory formation or utilization of N₂O by intactAzospirillum brasilense Sp 7, NO production by extracts from this bacterium, and N₂O reduction catalyzed by nitrogenase in intactKlebsiella pneumoniae. It is concluded that the probe is useful for measuring N₂O or NO contents in bacterial suspensions when the O₂ level is zero or kept constant during the assays.     

Role of nitrate and nitrite for production and consumption of nitric oxide during denitrification in soil

Abstract Anaerobic production and consumption of NO was measured in a calcic cambisol (KBE; pH 7.3) and a forest luvisol (PBE; pH 4.4) which were incubated at 80% water-holding capacity and continuously flushed with N₂. Both NO production and NO consumption were negligibly low when nitrate and nitrite concentrations in the soil were exhausted. Addition of glucose alone had no effect, but addition of nitrate ± glucose greatly stimulated both NO production and NO consumption. NO consumption followed an apparent first-order reaction at low NO mixing ratios (1–3 ppmv), but a higher NO mixing ratios it followed Michaelis-Menten kinetics. In PBE the apparent K _m was 980 ppbv NO (1.92 nM in soil water). During reduction of nitrate, nitrite intermediately accumulated and simultaneously, production rates of NO and N₂O were at the maximum. Production rates of NO plus N₂O amounted to 20% and 34% of the nitrate reduction rate in KBE and PBE, respectively. NO production was hyperbolically related to the nitrite concentration, indicating an apparent K_m of 1.6 μg nitrite-N g⁻¹ d.w. soil (equivalent to 172 μM nitrite in soil solution) for the reduction of nitrite to NO in KBE. Under nitrate and nitrite-limiting conditions, 62–76% and 93–97% of the consumed NO-N were recovered as N₂O-N in KBE and PBE, respectively. Gassing of nitrate plus nitrite-depretsu KBE with increasing mixing ratios of NO₂ resulted in increasing rates of NO₂ uptake and presumably in the formation of low concentrations of nitrite and nitrate. This NO₂ uptake resulted in increasing rates of both NO production and NO consumption indicating that nitrite or nitrate was limiting for both reactions.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Studies on the differential inhibition by azide on the nitrite/nitrous oxide level of denitrification.

A gas chromatographic method was used to demonstrate that nitrite can counteract the inhibition by azide of nitrous oxide reductase activity in denitrifiers. This effect explains why azide (and cyanide) can inhibit nitrogen production from nitrous oxide in these organisms but have little effect on nitrogen production from nitrite. Although the physiological basis by which nitrite opposes the action of azide remains unknown, extensive destruction of azide by nitrite can be ruled out as an explanation.     

Studies on the differential inhibition by azide on the nitrite/nitrous oxide level of denitrification.

A gas chromatographic method was used to demonstrate that nitrite can counteract the inhibition by azide of nitrous oxide reductase activity in denitrifiers. This effect explains why azide (and cyanide) can inhibit nitrogen production from nitrous oxide in these organisms but have little effect on nitrogen production from nitrite. Although the physiological basis by which nitrite opposes the action of azide remains unknown, extensive destruction of azide by nitrite can be ruled out as an explanation.     

Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols

NO or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial complex I by several different mechanisms that are not well characterised. There is an inactivation by NO, peroxynitrite and S-nitrosothiols that is reversible by light or reduced thiols, and therefore may be due to S-nitrosation or Fe-nitrosylation of the complex. There is also an irreversible inhibition by peroxynitrite, other oxidants and high levels of NO, which may be due to tyrosine nitration, oxidation of residues or damage of iron sulfur centres. Inactivation of complex I by NO or RNS is seen in cells or tissues expressing iNOS, and may be relevant to inflammatory pathologies, such as septic shock and Parkinson's disease.     

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase)

The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent K_m of 304 μM for PGE₁. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent K_m of 157 μM when PGE₁ is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin.     

Copper-containing nitrite reductase: a DFT study of nitrite and nitric oxide adducts

Copper-containing nitrite reductases (Cu-NIRs) reduce nitrite to NO. Reported here are DFT (density functional theory) results on models of the Cu-NIR active site bound to nitrite and nitric oxide. The Cu-NIR active site appears to have been designed to exclude N-nitrite binding even though N-O bond cleavage would be equally facile in the N- and O-isomers. The active site also appears to force a side-on coordination of the end-product, nitric oxide. The latter feature has to rely on the sterics of the active site to destabilize, thermodynamically speaking, the Cu-NO adduct; under these conditions, the absence of N-nitrite coordination is proposed to be merely a side-effect. For the Cu(II)-NO adduct, sterical crowding appears to also favour the Cu-NO electromer over Cu(I)-NO+, helping to avoid the potentially damaging chemistry associated with an NO+ moiety. These conclusions are in reasonable agreement with previous conclusions drawn from experiment [Science 304 (2004) 867].     

Ornament evolution in dragon lizards: multiple gains and widespread losses reveal a complex history of evolutionary change

The expression in females of ornaments thought to be the target of sexual selection in males is a long-standing puzzle. Two main hypotheses are proposed to account for the existence of conspicuous ornaments in both sexes (mutual ornamentation): genetic correlation between the sexes and sexual selection on females as well as males. We examined the pattern of ornament gains and losses in 240 species of dragon lizards (Agamidae) in order to elucidate the relative contribution of these two factors in the evolution of mutual ornamentation. In addition, we tested whether the type of shelter used by lizards to avoid predators predicts the evolutionary loss or constraint of ornament expression. We found evidence that the origin of female ornaments is broadly consistent with the predictions of the genetic correlation hypothesis. Ornaments appear congruently in both sexes with some lineages subsequently evolving male biased sexual dimorphism, apparently through the process of natural selection for reduced ornamentation in females. Nevertheless, ornaments have also frequently evolved in both sexes independently. This suggests that genetic correlations are potentially weak for several lineages and sexual selection on females is responsible for at least some evolutionary change in this group. Unexpectedly, we found that the evolutionary loss of some ornaments is concentrated more in males than females and this trend cannot be fully explained by our measures of natural selection.     

Phylogenetic endemism: a new approach for identifying geographical concentrations of evolutionary history

We present a new, broadly applicable measure of the spatial restriction of phylogenetic diversity, termed phylogenetic endemism (PE). PE combines the widely used phylogenetic diversity and weighted endemism measures to identify areas where substantial components of phylogenetic diversity are restricted. Such areas are likely to be of considerable importance for conservation. PE has a number of desirable properties not combined in previous approaches. It assesses endemism consistently, independent of taxonomic status or level, and independent of previously defined political or biological regions. The results can be directly compared between areas because they are based on equivalent spatial units. PE builds on previous phylogenetic analyses of endemism, but provides a more general solution for mapping endemism of lineages. We illustrate the broad applicability of PE using examples of Australian organisms having contrasting life histories: pea-flowered shrubs of the genus Daviesia (Fabaceae) and the Australian species of the Australo-Papuan tree frog radiation within the family Hylidae.     

The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Distinction from the nitrite reductase that catalyses synthesis of nitric oxide and evidence from trapping experiments for nitric oxide as a free intermediate during denitrification

1. A Clark-type electrode that responds to nitric oxide has been used to show that cytoplasmic membrane vesicles of Paracoccus denitrificans have a nitric-oxide reductase activity. Nitrous oxide is the reaction product. NADH, succinate or isoascorbate plus 2,3,5,6-tetramethyl-1,4-phenylene diamine can act as reductants. The NADH-dependent activity is resistant to freezing of the vesicles and thus the NADH:nitric-oxide oxidoreductase activity of stored frozen vesicles provides a method for calibrating the electrode by titration of dissolved nitric oxide with NADH. The periplasmic nitrite reductase and nitrous-oxide reductase enzymes are absent from the vesicles which indicates that nitric-oxide reductase is a discrete enzyme associated with the denitrification process. This conclusion was supported by the finding that nitric-oxide reductase activity was absent from both membranes prepared from aerobically grown P. denitrificans and bovine heart submitochondrial particles. 2. The NADH: nitric-oxide oxidoreductase activity was inhibited by concentrations of antimycin or myxothiazol that were just sufficient to inhibit the cytochrome bc1 complex of the ubiquinol--cytochrome-c oxidoreductase. The activity was deduced to be proton translocating by the observations of: (a) up to 3.5-fold stimulation upon addition of an uncoupler; and (b) ATP synthesis with a P:2e ratio of 0.75. 3. Nitrite reductase of cytochrome cd1 type was highly purified from P. denitrificans in a new, high-yield, rapid two- or three-step procedure. This enzyme catalysed stoichiometric synthesis of nitric oxide. This observation, taken together with the finding that the maximum rate of NADH:nitric-oxide oxidoreductase activity catalysed by the vesicles was comparable with that of NADH:nitrate-oxidoreductase, is consistent with a role for nitric-oxide reductase in the physiological conversion of nitrate or nitrite to dinitrogen gas. 4. Intact cells of P. denitrificans also reduced nitric oxide in an antimycin- or myxothiazol-sensitive manner. However, nitric oxide was not detected by the electrode during the reduction of nitrate. Nitric-oxide synthesis from nitrate could be detected with cells in the presence of very low concentrations of Triton X-100 which selectively inhibits nitric-oxide reductase activity. 5. Nitric oxide was detected as an intermediate in denitrification by including haemoglobin with an anaerobic suspension of cells that was reducing nitrate. The characteristic spectrum of the nitric oxide derivative of haemoglobin was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

H218O isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria. Evidence for an electrophilic nitrosyl during reduction of nitric oxide

Reduction of NO and NO2-by whole cells of eight strains of denitrifying bacteria known to contain either heme cd1 or copper-containing nitrite reductases (NiRs) has been examined in the presence of H218O. All organisms containing heme cd1 NiRs exhibited relatively large extents of exchange between NO2- and H218O (39-100%), as monitored by the 18O content of product N2O. Organisms containing copper NiRs gave highly variable results, with Achromobacter cycloclastes and Pseudomonas aureofaciens exhibiting no 18O incorporation and Rhodopseudomonas sphaeroides and Alcaligenes entrophus exhibiting complete exchange between NO2- and H218O. Organisms containing heme cd1 NiRs exhibited significant but lower levels of exchange between NO and H218O than between NO2- and H218O, while organisms containing copper NiRs gave significantly higher amounts of 18O incorporation than observed for the heme cd1 organisms. These results demonstrate the existence of an NO-derived species capable of undergoing O-atom exchange with H218O during the reduction of NO. Trapping experiments with 15NO, 14N3-, and crude extracts of R. sphaeroides support the electrophilic nature of this intermediate and suggest its formulation as an enzyme nitrosyl, E-NO+, analogous to that observed during reduction of NO2-. The observation of lower levels of 18O incorporation with NO2- than with NO as substrate for A. cycloclastes and P. aureofaciens indicates that, for these organisms at least, a sequential pathway involving free NO as an intermediate is significantly less important than a direct pathway in which N2O is formed via reaction of two NO2- ions on a single enzyme.