Denitrification by Azospirillum brasilense Sp 7

Nitrous oxide reduction can consistently be demonstrated with high activities in cells of Azospirillum brasilense Sp 7 which are grown anaerobically in the presence of low amounts of nitrite. Azospirillum can even grow anaerobically with nitrous oxide in the absence of any other respiratory electron acceptor. Nitrous oxide reduction by Azospirillum is inhibited by acetylene, amytal and weakly by carbon monoxide. Azospirillum converts nitrous oxide to molecular nitrogen without the formation of ammonia. The cells must, therefore, be supplied with ammonia from nitrogen fixation during anaerobic growth with nitrous oxide. When no other nitrogen compound besides nitrous oxide is available in the medium, the bacteria synthesize nitrogenase from protein reserves in about 2 h. Nitrogenase synthesis is blocked by chloramphenicol under these conditions. In contrast, the addition of nitrate or nitrite to the medium represses the synthesis of nitrogenase. Nitrous oxide reduction by Azospirillum and other microorganisms is possibly of ecological significance, because the reaction performed by the bacteria may remove nitrous oxide from soils.     

Denitrification and nitrate-reducing bacterial populations in abandoned and reclaimed minesoils

Abstract Microbial populations, nitrogen mineralization potentials, and denitrification enzyme activities were examined in two abandoned carbolithic minesoils. Numbers and activities of bacteria and fungi were lower in nonamended than in lime and/or fly ash amended sites. Rates of aerobic NO₃⁻ production (3 to 38 μg-N kg⁻¹ h⁻¹) and anaerobic NO₃⁻ reduction to N₂O (5 to 68 μg-N kg⁻¹ h⁻¹) were measured. Organisms capable of N₂O production under anaerobic soil conditions were present in low numbers, and their activity was restricted in part by low soil pH. Nondenitrifying nitrate-reducing bacteria were more diverse and in greater numbers than respiratory denitrifiers and may have been responsible for N₂O production in assays measuring denitrification enzyme activity.     

Denitrification and nitrogen fixation by Azospirillum

A model system is described where Azospirillum and germinated wheat seeds were grown in association for a week and then assayed for nitrogen fixation (C₂H₂-reduction) and denitrification (N₂O-formation) activities. The association performed C₂H₂-reduction and N₂O-formation under microaerobic conditions. Both activities were measurable after already 3–5 h of incubation with substantial rates and were strictly dependent on the presence of both plants and bacteria. During the week of the growth of the association, the bacteria had lived exclusively from the carbon compounds supplied by the roots of the plants. C₂H₂-reduction activity by the association was more or less the same with all the Azospirillum brasilense strains, but lower with A. lipoferum and with the A. amazonense strains tested. Two nitrogenase negative mutants of Azospirillum brasilense showed virtually no activity in the association. C₂H₂-reduction activity was strongly dependent on the growth temperature of the association. Denitrification (N₂O-formation) was high also at higher temperatures and at pH-values in the medium around 7.8 but not at neutrality and was strictly dependent on nitrate. The Azospirillum strain used strongly determined the rate of the N₂O-formation in the association. It is suggested that Azospirillum may be beneficial to crops particularly under tropical conditions.Dedicated to Professor Dr. Gerhart Drews, Freiburg, on the occasion of his 60th birthday     

Inhibition of nitrous oxide reduction by a component of Hamilton Harbour sediment

Abstract: A component of Hamilton Harbour sediment prevented nitrous oxide (N₂O) reduction in denitrification assays with a mixed population of endogenous bacteria and a pure culture (HH1) isolated from the sediment. A 5% (v/v) concentration of sediment in nutrient broth caused near maximum inhibition of N₂O reduction. Sediment taken from a site closer to pollution sources (Site 906) was twice as inhibitory (as measured by N₂O accumulation) as sediment from Site 910, further from pollution sources. N₂O persistence was associated with the particulate sediment fraction only. Several heavy metals were tested at in situ concentrations, and ionic cadmium (Cd) and chromium (Cr) caused N₂O accumulation. Ashed sediment did not cause N₂O accumulation, but did decrease initial nitrate reduction rates with HH1.     

Denitrification ability of rhizobial strains isolated from Lotus sp.

Ten rhizobial strains isolated from Lotus sp. have been characterized by their ability to denitrify. Out of the 10 strains, the five slow-growing isolates grew well under oxygen-limiting conditions with nitrate as a sole nitrogen source, and accumulated nitrous oxide in the growth medium when acetylene was used to inhibit nitrous oxide reductase activity. All five strains contained DNA homologous to the Bradyrhizobium japonicum nirK, norBDQ and nosZ genes. In contrast, fast-growing lotus rhizobia were incapable of growing under nitrate-respiring conditions, and did not accumulate nitrous oxide in the growth medium. DNA from each of the five fast-growing strains showed a hybridization band with the B. japonicum nirK gene but not with norBDQ and nosZ genes. Partial 16S rDNA gene sequencing revealed that fast-growing strains could be identified as Mesorhizobium loti species and the slow-growers as Bradyrhizobium sp.     

Denitrification by the mix-culturing of fungi and bacteria with shell

Denitrification by pure and mixed culture of Fusarium oxysporum and Pseudomonas stutzeri in different mineral medium and in synthetic wastewater were examined. The results obtained revealed that a rapid N2 evolution by F. oxysporum and P. stutzeri in mineral medium and synthetic wastewater was observed. In co-cultures of F. oxysporum and P. stutzeri, N2O produced by F. oxysporum was rapidly consumed by P. stutzeri. This indicated that mixed culture of F. oxysporum and P. stutzeri could be used for efficient nitrate and nitrite removal. Using synthetic wastewater, about 87% of nitrate was reduced by co-denitrification of F. oxysporum and P. stutzeri after incubation for 6 days. In the further denitrification tests, the interaction of shell and mixed culture of F. oxysporum and P. stutzeri was investigated. The dinitrogen was rapidly evolved (442.48 micromol N2 produced from 1.0 mmol of NO3(-) in 36 h). These results clearly showed that shell provide a suitable microenvironment for P. stutzeri, which is beneficial to the denitrification.     

Denitrification in field soils

Summary Recent denitrification research is reviewed to answer questions a) how much N is lost from the soil as N₂ and N₂O and b) how do agronomic practices affect this loss? The methods used to quantify denitrification are also discussed. Gaseous losses of inorganic N range between the equivalent of 0 to 20 per cent of the fertilizer N applied to arable soils and 0–7 per cent on grassland soils. Losses are greater on undrained land and also after using direct drilling to establish arable crops.Appendix 1 summarizes reported measurements of gaseous N losses.Introductory lecture     

Membrane-associated, dissimilatory nitrite reductase of the denitrifying fungus Cylindrocarpon tonkinense

Dissimilatory nitrite reductase (Nir) of the fungus Cylindrocarpon tonkinense was isolated and partially characterized. Nir activity was recovered in both the soluble and the membrane fractions, giving a specific activity of 4.74 and 5.80 μmol NO min^–1 (mg protein)^–1, respectively. The soluble and membrane-associated Nir preparations resembled each other in properties, and the results suggested that Nir is a homodimer of a 67-kDa subunit. The absorption spectrum and the inhibition by diethyldithiocarbamate indicated that fungal Nir is a copper-containing Nir (CuNir). Received: 7 September 1998 / Accepted: 1 December 1998     

An 88 amino acid long C-terminal sequence of human lactotransferrin

The terminal oxidoreductase of nitrous oxide respiration in the marine, denitrifying bacterium, Pseudomonas perfectomarinus, was identified as multi-copper protein and purified to electrophoretic homogeneity. The enzyme reduced N₂O to N₂ with hydrogen, clostridial hydrogenase, and methyl viologen as electron-donating system. The copper content of the reductase corresponded to ～ 8 copper atoms/120 000 M_r. The subunit structure was dimeric with two peptides of equal size. Manganese, iron and zinc were absent, or were not found in stoichiometric amounts. The oxidized chromophore had absorption maxima at 350, 480, 530, 620 and 780 nm; addition of dithionite produced a blue protein form with maxima at 470, 635 and 740 nm. Both forms of the enzyme were paramagnetic. The same copper protein was also isolated from Pseudomonas stutzeri.     

Nitrous oxide in brackish Lake Nakaumi, Japan II: the role of nitrification and denitrification in N2O accumulation

In order to understand the role of nitrification and denitrification in the accumulation of nitrous oxide (N₂O) in the hypolimnetic water of brackish Lake Nakaumi, the effects of dissolved oxygen (DO) concentration on these activities were investigated by incubation experiments. N₂O was produced during the oxidation of NH₄ ⁺ to NO₂ ⁻ in nitrification and during the reduction of NO₃ ⁻ to N₂ in denitrification. N₂O-producing activity by nitrification (N₂O_N) increased markedly with decreasing concentrations of DO. Low DO (10%–30% saturation) induced high N₂O_N. In contrast to nitrification, N₂O-producing activity by denitrification (N₂O_D) decreased with decreasing concentrations of DO. Little N₂O was accumulated during denitrification under low-level conditions of DO (10%–30%), because of further reduction of N₂O to N₂. It can therefore be assumed that N₂O produced as the by-product of nitrification is concurrently reduced to N₂ by denitrification under low-DO conditions. This would result in no substantial accumulation of N₂O during active nitrification in the hypolimnetic water of Lake Nakaumi. Received: July 6, 2001 / Accepted: December 10, 2001     

Nitrous oxide in brackish Lakes Shinji and Nakaumi, Japan

Nitrous oxide (N₂O) was measured monthly from September 1997 to August 1998 in the brackish Lakes Shinji and Nakaumi, Japan. N₂O (5–37 μg N l⁻¹) was supersaturated in the overlying water on lake sediments from October 1997 to January 1998. The N₂O concentration in the hypolimnion was higher than that in the epilimnion on 17 October 1997, when N₂O was first observed in a water column of Lake Nakaumi. Afterward, N₂O was almost uniform throughout the water column and then disappeared on 16 February 1998. On the one hand, large amounts of N₂O were found throughout the year in the interstitial water in Lake Shinji, where a high concentration of nitrate was discharged from the Hii River. On the other hand, in Lake Nakaumi, stratified by halocline, a high concentration of N₂O was observed in the interstitial water only from winter to spring. N₂O concentrations in the interstitial water were about 10 to 1000 times as large as those in the overlying water. These results imply that N₂O was mainly produced at the sediment-water interface and was diffused to the overlying water. It was also suggested that the accumulation of N₂O in the sediment-water system was accelerated by a high concentration of hydrogen sulfide. Received: July 6, 2000 / Accepted: November 30, 2000     

Denitrification and nitrous oxide emissions from riparian forests soils exposed to prolonged nitrogen runoff

Compared to upland forests, riparian forest soils have greater potential to remove nitrate (NO₃) from agricultural runoff through denitrification. It is unclear, however, whether prolonged exposure of riparian soils to nitrogen (N) loading will affect the rate of denitrification and its end products. This research assesses the rate of denitrification and nitrous oxide (N₂O) emissions from riparian forest soils exposed to prolonged nutrient runoff from plant nurseries and compares these to similar forest soils not exposed to nutrient runoff. Nursery runoff also contains high levels of phosphate (PO₄). Since there are conflicting reports on the impact of PO₄ on the activity of denitrifying microbes, the impact of PO₄ on such activity was also investigated. Bulk and intact soil cores were collected from N-exposed and non-exposed forests to determine denitrification and N₂O emission rates, whereas denitrification potential was determined using soil slurries. Compared to the non-amended treatment, denitrification rate increased 2.7- and 3.4-fold when soil cores collected from both N-exposed and non-exposed sites were amended with 30 and 60 μg NO₃-N g⁻¹ soil, respectively. Net N₂O emissions were 1.5 and 1.7 times higher from the N-exposed sites compared to the non-exposed sites at 30 and 60 μg NO₃-N g⁻¹ soil amendment rates, respectively. Similarly, denitrification potential increased 17 times in response to addition of 15 μg NO₃-N g⁻¹ in soil slurries. The addition of PO₄ (5 μg PO₄-P g⁻¹) to soil slurries and intact cores did not affect denitrification rates. These observations suggest that prolonged N loading did not affect the denitrification potential of the riparian forest soils; however, it did result in higher N₂O emissions compared to emission rates from non-exposed forest soils.     

Denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO ₃ ^- under anaerobic conditions accumulated NO ₂ ^- in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO ₃ ^- , NO ₂ ^- and N₂O to N₂ gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N₂O reductase was inhibited by NaN₃, C₂H₂ and KCNS. Cells incubated with either K¹⁵NO₃ or K¹⁵NO₂ produced ¹⁵N₂O and ¹⁵N₂. A stoichiometry of 2:1 was recorded for the reduction of either NO ₃ ^- or NO ₂ ^- to N₂O and N₂ and for N₂O to N₂ it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide     

Production of nitric oxide in Nitrosomonas europaea by reduction of nitrite

Nitrosomonas europaea and Nitrosovibrio sp. produced NO and N₂O during nitrification of ammonium. Less then 15% of the produced NO was due to chemical decomposition of nitrite. Production of NO and especially of N₂O increased when the bacteria were incubated under anaerobic conditions at decreasing flow rates of air, or at increasing cell densities. Low concentrations of chlorite (10 M) inhibited the production of NO and N₂, but not of nitrite indicating that NO and N₂O were not produced during the oxidative conversion of ammonium to nitrite. NO and N₂O were produced during reduction of nitrite with hydrazine as electron donor in almost stoichiometric quantities indicating that reduction of nitrite was the main source of NO and N₂O.     

Sedimenticola hydrogenitrophicus sp. nov. a chemolithoautotrophic bacterium isolated from a terrestrial mud volcano,and proposal of Sedimenticolaceae fam. nov. in the order Chromatiales

Chemolithoautotrophic microorganisms can play a significant role in the biogeochemical cycling of elements in deep-subsurface-associated environments. A novel facultatively anaerobic lithoautotrophic bacteria (strains SB48^T and SN1189) were isolated from terrestrial mud volcanoes (Krasnodar Krai, Russia). Cells of the strains were straight motile rods. Growth was observed at temperatures up to 35 °C (optimum at 30 °C), pH 6.0–8.5 (optimum at pH 7.5) and NaCl concentrations of 0.5–4.0% (w/v) (optimum at 1.5–2.0% (w/v)). The isolates grew chemolithoautotrophically with molecular hydrogen or thiosulfate as an electron donor, nitrate as an electron acceptor and CO₂/HCO₃⁻ as a carbon source. They also grew with organic acids, ethanol, yeast extract and peptone. The isolates were capable of either anaerobic respiration with nitrate or nitrous oxide as the electron acceptors or aerobic respiration under microaerobic condition. The total size of the genome of strains SB48^T and SN1189 was 4.71 and 5.13 Mbp, respectively. Based on phenotypic and phylogenetic characteristics, strains SB48^T and SN1189 represent a novel species of the genus Sedimenticola, S. hydrogenitrophicus (the type strain is SB48^T = KCTC 25568 ^T = VKM B-3680 ^T). The new isolates are the first representatives of the genus Sedimenticola isolated from a terrestrial ecosystem. Based on phylogenomic reconstruction we propose to include the genus Sedimenticola and the related genera into a new family Sedimenticolaceae fam. nov. within the order Chromatiales.     

Denitrification by the sessile microbial community of a polluted river

Denitrification by the sessile microbial community of the River Tamagawa was studied in laboratory experiments. Inorganic nitrogen loss was observed when river water was incubated with sessile microbial community of the river in a continuously circulating system. It was confirmed by the ¹⁵N tracer technique that both sessile microbial communities of unpolluted and polluted areas had denitrifying activity, even though they were incubated in oxygenated river water. The denitrification rate of the sessile microbial community taken from a polluted area, measured by the ¹⁵N tracer technique, was 8–16 mg N/m²/day in October and December, 1977, and it was enhanced 10-fold by raising the water temperature from 14 to 30° C. Denitrification in the river was also suggested by determining the N₂: Ar ratio of gases evolved from the river bed.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.