Reevaluation of anaerobic nitrite production as an index for the measurement of metabolic pool of nitrate

The use of anaerobic nitrite production as an index for the measurement of metabolic pool of nitrate was reevaluated using primary leaves of 7-day-old barley and 10-day-old soybean seedlings. The seedlings were grown in nutrient solutions containing 5 to 15 millimolar nitrate. The nitrate-free in vivo assay system of nitrate reductase was used for measuring the production of nitrite. Both the duration and extent of nitrite production were dependent on the level of endogenous nitrate in the tissue. At cessation of nitrite production, 30 to 50% of the endogenous nitrate was reduced to nitrite. Nitrate from the tissue leaked continuously into the surrounding medium so that, at cessation of nitrite production, nitrate supply from the tissue was exhausted. The cessation of nitrite production, therefore, may have been caused by the depletion of endogenous nitrate from the tissue. It is concluded that anaerobic nitrite production is not a valid index for the measurement of the size of the metabolic pool of nitrate.     

The influence of pretreatment with different cations on anaerobic nitrite production by excisedPisum sativum roots

The influence of pretreatment with some cations on anaerobic nitrite production (in an assay medium lacking nitrate) by excised primary roots of pea (Pisum sativum L., ov. Raman), detached from six-day-old seedlings germinated in distilled water, was investigated. When the excised roots were precultivated in one-salt-solutions of KNO3, then these roots produced at 9 mM and 15 mM NO3- concentrations under anaerobic conditions significantly more NO2-, than those precultivated in a nutrient solution containing besides K+ ions also Ca2+ and Mg2+ ions, and they produced nitrite for a longer time. The KNO3 dependent increase in anaerobic NO2- production was counteracted most by Ca2+ and to a lesser extent by Mg2+; Na+ was without effect. NH4+ at higher concentrations (12 and 15 mM) significantly depressed nitrite production both by roots precultivated in a solution containing besides NH4+ only K+, and by roots precultivated in a full nutrient solution containing K+, Ca2+ and Mg2+, however at lower NH4+ concentrations (0.6 and 2mMNH4+; 15mMNO3-) the decrease was more conspicuous in the KNO3 solution than in the full nutrient solution. Nitrate reductase level was not influenced by this pretreatment. When 6% and 7.5% n-propanol, which increases membrane permeability and causes mixing of storage and metabolic nitrate pools in the cells, was added to the assay medium lacking nitrate, anaerobic nitrite production increased and the differences caused by the precultivation disappeared. These results show that higher K+ concentrations in unbalanced one-salt-solutions of KNO3 can cause higher membrane permeability by accentuating Ca8+ deficiency, which results in a faster penetration of NO3- from the storage pool to the sites of its reduction and in an easier penetration of NO2- out of the roots, and that higher NH4+ concentrations can change nitrate compartmentation and diminish the metabolic NO3- pool which results in a slower nitrate reduction. Besides that, lower NH4+ concentrations in KNO3 solutions (15mMNO3-) probably partially counteract the K+ dependent increase in membrane permeability. The results obtained show that there is no simple, direct relationship between the so-called metabolic pool of nitrate (i.e. anaerobic nitrite production) and the level of nitrate reductase, but that the velocity of nitrate reduction can be influenced by nitrate compartmentation in the cell.     

Nitrate reduction and compartmentation in tomato leaves. I. Nitrate accumulation in leaf sections in aqueous and gaseous medium

Nitrate pools in tomato ( Lycopersicon esculentum Mill. cv. Azes) leaf sections were estimated. Nitrite accumulation in aqueous medium was found to be an inadequate estimate of nitrate pools in tomato leaves. The main reason for the cessation of nitrite accumulation was not depletion of nitrate in the metabolic pool but rather a rapid decay of nitrate reductase (NR) activity as measured by nitrite accumulation in vivo and in vitro. Nitrate diffuses out of the tissue into the medium at a rate higher than the accumulation of nitrite in the tissue. Nitrate leakage from the tissue accelerates the loss of NR activity. Nitrite accumulation in leaf sections kept in an anaerobic gaseous atmosphere ceased earlier than in aqueous medium, at a time when NR activity was still relatively high. Measuring nitrite accumulation in gaseous atmosphere is preferable since NR is more stable and movements of nitrate between pools more restricted.     

Factors Controlling Anaerobic Ammonium Oxidation with Nitrite in Marine Sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15°C and a maximum temperature of 37°C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K_m of nitrite consumption was less than 3 μM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

The effect of chloride on nitrate reductase level,on anaerobic nitrite production,and on nitrate content in excisedPisum sativum L. roots

The effect was studied of chloride ions, added in the form of different salts, on nitrate reductase (NR) level in excised pea roots, on anaerobic nitrite production in an assay medium lacking both nitrate and n-propanol, on nitrate content in the roots, and on in vivo NR activity determined in an assay medium containing 5% n-propanol. The presence of Cl in nitrate containing nutrient solutions resulted in lower NR levels, however counterions supplied together with Cl tended to modify slightly this general trend. The negative effect of Cl ions was also apparent, when Cl ions were applied before nitrate ions. Anaerobic nitrite production in the medium lacking both nitrate and n-propanol was not influenced by chloride ions. Nitrate content in the roots was reduced in the presence of chloride both at 3 mM and 15 mM NO₃ in nutrient solutions; however, at 16 mM NO₃, nitrate content in the roots exoeeded even in the presence of 15 mM Cl nitrate content in those root segments which were cultivated in a nutrient solution with 6 mM nitrate, which is the concentration at which NR reaches the level of saturation in excised pea roots. The results obtained suggest that a special induction nitrate pool exists in plant cells besides the storage and metabolic nitrate pools.     

Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation.     

Influence of nitrate starvation on nitrite accumulation during denitrification byPseudomonas stutzeri

Anaerobically denitrifyingPseudomonas stutzeri may transiently accumulate from 0% to 100% of nitrate as nitrite, depending on the nitrate availability during the preceding 24 h. The variations of transient nitrite level were related to the length of previous nitrate starvation. Cells harvested in a late anaerobic growth phase did not accumulate any nitrite during denitrification. Five hours of nitrate starvation caused about 40% (mol/mol) nitrite formation from the further added nitrate and 16 h nearly 80%. Concomitant with this, whereas the nitrate reduction capacity was not significantly affected, the initial nitrite reduction capacity was decreased. These results suggested that nitrate starvation caused a rapid loss of the originally produced nitrite-reducing capacity in the preculture. The lost capacity could be regenerated when nitrate or nitrite was resupplied to the cells. To investigate the nature of the restoration of nitrite-reducing capacity, chloramphenicol was used. The presence of chloramphenicol at 83 μg/ml entirely stopped this restoration, which was otherwise observed in all instances. This indicated that the recovery of nitrite-reducing activity required de novo protein synthesis, which was further confirmed by Western immunoblot assay of cd₁ nitrite reductase.     

Nitrate Reduction Functional Genes and Nitrate Reduction Potentials Persist in Deeper Estuarine Sediments. Why?

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This raises interesting questions as to what the alternative metabolic roles for the various nitrate reductases could be, analogous to the alternative metabolic roles found for nitrite reductases.     

Nitrite utilization by excised Zea mays L. roots under anaerobic conditions

Under both aerobic and anaerobic conditions exogenously supplied nitrite was utilized by sterile excised Zea mays L. root. A slightly greater quantity of nitrite was used under aerobic conditions than under anaerobiosis. The uncoupler of oxidative phosphorylation, pentachlorophenol (PCP), diminished the utilization of nitrite under aerobic and anaerobic conditions resulting in a net accumulation of nitrite rather than a net disappearance of nitrite. Nitrite supplied together with nitrite resulted in a slight reduction in the level of nitrite utilized. Supply of exogenous nitrite had no effect on nitrate reduction under aerobic or anaerobic conditions. A net accumulation of nitrite occurs only when roots are supplied with nitrate in the absence of added nitrite. However, the level of nitrite accumulated under anaerobiosis, when roots were supplied with nitrate only, was found to be a fraction of the quantity of nitrite utilized when roots were supplied with nitrite under anaerobiosis. Nitrate utilization far exceeded the level of nitrite accumulated under anaerobiosis when roots were supplied with nitrate only.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

Anaerobic Nitrate Respiration by Erwinia carotovora subsp. atroseptica during Potato Tuber Invasion

The in planta induction of anaerobic nitrate respiration by Erwinia carotovora subsp. atroseptica in relation to the in situ oxygen status in soft rotting potato tubers has been investigated. In vitro experiments have shown that nitrate was required for the induction of respiratory nitrate reductase activity in E. carotovora. In addition, oxygen was found to repress this activity. Expression of respiratory nitrate reductase was found in E. carotovora cells extracted from soft rotting potato tuber tissue. However, the rate of nitrite production in these cells was approximately 70-fold lower than the rate recorded in fully induced anaerobic cultures. Oxygen measurements in soft rotting potato tubers indicated that the invading bacteria encounter the lowest oxygen concentration at the interphase between healthy and macerated tissue. Consequently, growth of bacteria present in this specific zone will be stimulated by nitrate which is present in sufficient amounts in tuber tissue. A high nitrate content of the tuber will most likely facilitate the proliferation of E. carotovora in the tuber tissue.     

Control of nitrate and nitrite assimilation by carbohydrate reserves,adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N₂ and dark-O₂ were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.     

Heat Production by the Denitrifying Bacterium Pseudomonas fluorescens and the Dissimilatory Ammonium-Producing Bacterium Pseudomonas putrefaciens during Anaerobic Growth with Nitrate as the Electron Acceptor

The heat production rate and the simultaneous nitrate consumption and production and consumption of nitrite and nitrous oxide were monitored during the anaerobic growth of two types of dissimilatory nitrate reducers. Pseudomonas fluorescens, a denitrifier, consumed nitrate and accumulated small amounts of nitrite or nitrous oxide. The heat production rate increased steadily during the course of nitrate consumption and decreased rapidly concomitant with the depletion of the electron acceptors. A mean experimental enthalpy change value of −800 kJ/mol of nitrate and a mean growth yield value of 33 g (dry weight)/mol of nitrate consumed were obtained for different concentrations of nitrate. For Pseudomonas putrefaciens, a dissimilatory ammonium producer, the nitrate consumption resulted in an accumulation of nitrite and nitrous oxide. Nitrite consumption commenced after depletion of the nitrate; consequently, two phases were noted in the heat production rate curve during growth. A mean experimental enthalpy change value of −810 kJ/mol of nitrate was obtained for different concentrations of nitrate.     

Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments

Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15 degrees C and a maximum temperature of 37 degrees C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent K(m) of nitrite consumption was less than 3 microM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.     

Critical evaluation of the in vivo nitrate reductase assay for detection of two nitrate pools in wheat leaves

The usefulness of the nitrate-free in vivo nitrate reductase assay for the study of nitrate pools in wheat leaves was investigated. Leaf sections from 7-day-old wheat seedlings, exposed 24 h before harvest to 1.5, 3.0 or 5.0 m M KNO₃ were used. After 2 to 4 h of incubation nitrite production ceased, reaching a plateau. The time required to reach the plateau and the level of the plateau increased with increasing endogenous nitrate content. At nitrite plateau the amount of nitrate left in the tissue was independent of the original nitrate content in the tissue. Addition of nitrate at plateau caused resumed nitrite production. It is concluded that nitrate was the limiting factor in nitrite production.
Oxygen inhibited nitrate reduction and stimulated further assimilation of nitrite. A considerable initial leakage of nitrate from tissue to the assay medium, followed by a slower continuous leakage, was observed throughout the incubation. N₂-flushing or inclusion of Triton X-100 in the assay medium increased nitrite production by making more nitrate available for reduction. These treatments also increased the leakage of nitrate. At plateau levels the amount of nitrate left in the tissue was dependent on the oxygen tension in the assay medium. Under low oxygen tension nearly all nitrate in the tissue was available for reduction. Nitrite production at plateau is not a useful index for a metabolic nitrate pool and nitrate left in the tissue is not a useful index for a nitrate storage pool because both parameters are highly dependent on the oxygen tension in the assay medium. Further, in view of the considerable leakage, the nitrate-free in vivo nitrate reductase assay cannot be used to detect two separate nitrate pools in wheat leaves.     

Some New Aspects of the in Vivo Assay for Nitrate Reductase in Wheat (Triticum aestivum L.) Leaves: I. REEVALUATION OF NITRATE POOL SIZES

Experiments were carried out to clarify problems encountered in measuring metabolic and storage pool sizes of nitrate in wheat leaf sections with an in vivo nitrate reductase assay. The leaf sections were from seedlings grown on 15 millimolar nitrate. Data obtained show that the cessation of nitrite accumulation, used as an index of the active nitrate pool size, could be caused by lack of anaerobiosis in the assay system, the lack of energy for nitrate reduction, or a loss of nitrate reductase activity. Availability of nitrate was never the limiting factor in this system. It is concluded that pool sizes of nitrate cannot be determined in wheat leaves with the in vivo assays employed.     

Variation in nitrate metabolism in biovars of Pseudomonas solanacearum

A collection of 327 strains of Pseudomonas solanacearum , representing five biovars, was divisible into three groups on the basis of differences in nitrate metabolism. Nine strains (2.8%), of which seven were biovar 2 from bacterial wilt of potato, were nitrate reduction-deficient and failed to produce nitrite from nitrate by either of two methods of detection in five different media. A second group of 231 strains, comprising biovars 1 and 2 and a single biovar 3 strain, produced nitrite from nitrate and grew vigorously in the presence of nitrate under anaerobic conditions but were deficient in ability to denitrify. A third group comprising 57 strains of biovar 3, 28 of biovar 4 and one each of biovar 2 and 5 produced nitrite from nitrate and gave profuse growth and gas production from nitrate under anaerobic conditions. However, production of gas from nitrate (denitrification) was not a consistently reproducible property in some of the media tested. Gas production results were most reproducible when a semi-solid succinate/nitrate or glycerol/nitrate medium was used. Serial passage of four nitrate reduction-deficient isolates in nitrate medium did not restore ability to reduce nitrate.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.