Geochemical Controls on Microbial Nitrate-Dependent U(IV) Oxidation

After reductive immobilization of uranium, the element may be oxidized and remobilized in the presence of nitrate by the activity of dissimilatory nitrate-reducing bacteria. We examined controls on microbially mediated nitrate-dependent U(IV) oxidation in landfill leachate-impacted subsurface sediments. Nitrate-dependent U(IV)-oxidizing bacteria were at least two orders of magnitude less numerous in these sediments than glucose- or Fe(II)-oxidizing nitrate-reducing bacteria and grew more slowly than the latter organisms, suggesting that U(IV) is ultimately oxidized by Fe(III) produced by nitrate-dependent Fe(II)-oxidizing bacteria or by oxidation of Fe(II) by nitrite that accumulates during organotrophic dissimilatory nitrate reduction. We examined the effect of nitrate and reductant concentration on nitrate-dependent U(IV) oxidation in sediment incubations and used the initial reductive capacity (RDC = [reducing equivalents] - [oxidizing equivalents]) of the incubations as a unified measurement of the nitrate or reductant concentration. When we lowered the RDC with progressively higher nitrate concentrations, we observed a corresponding increase in the extent of U(IV) oxidation, but did not observe this relationship between RDC and U(IV) oxidation rate, especially when RDC > 0, suggesting that nitrate concentration strongly controls the extent, but not the rate of nitrate-dependent U(IV) oxidation. On the other hand, when we raised the RDC in sediment incubations with progressively higher reductant (acetate, sulfide, soluble Fe(II), or FeS) concentrations, we observed progressively lower extents and rates of nitrate-dependent U(IV) oxidation. Acetate was a relatively poor inhibitor of nitrate-dependent U(IV) oxidation, while Fe(II) was the most effective inhibitor. Based on these results, we propose that it may be possible to predict the stability of U(IV) in a bioremediated aquifer based on the geochemical characteristics of that aquifer.     

Localization of iron-reducing activity in paddy soilby profile studies

Profiles of iron speciations (porewaterFe(II) and Fe(III), solid-phase Fe(II) andFe(III)) have been studied to localize both ironreduction and oxidation in flooded paddy soil. Sulfateand nitrate were determined to analyze interactions ofredox reactions involved in the iron cycle with thoseof the sulfur and nitrogen cycle. The development ofthe iron(II) and iron(III) profiles was observed inmicroscale over a time period of 11 weeks. After 11weeks the profiles were stable and showed lowestconcentrations of solid-phase iron(II) on the soilsurface with increasing concentrations to a soil depthof 10 mm ( 100 µmol/cm³). Profilesof iron(III) showed a maximum of iron(III) at a depthof 2 to 4 mm ( 100--200 µmol/cm³).Porewater iron(II) concentrations were three orders ofmagnitude lower than extracted iron(II) and indicatedthat most iron(II) was adsorbed to the solid-phase orimmobilized as siderite and vivianite. Diffusive lossof iron from the soil was indicated by iron recovery(0.3 µmol gdw^–1) in the flooding water after12 weeks. The organic content of the soil influencedthe concentrations of solid-phase iron(II) in deepersoil layers (> 6 mm); higher Fe(II) concentrationsin soil with limiting amounts of electron donors mayindicate lower consumption of CO₂ by methanogenicbacteria and therefore a higher sideriteprecipitation. Soil planted with rice showed similariron(II) profiles of fresh paddy soil cores. However,maximal iron(III) concentrations ( 350µmol/cm³) were present in planted soil at adepth of 1 to 2.5 mm where oxygen is provided by a matof fine roots. Sulfate and nitrate concentrations inthe porewater were highest on the soil surface (10µM NO₃ ^–, 40 µM SO₄ ^2–) anddecreased with depth. Similar profiles were detectedfor malate, acetate, lactate, and propionate, theconcentrations decreased gradually from the surface toa depth of 4 mm. Profiles of oxygen showed highestconcentrations at the surface due to photosyntheticproduction and a depletion of oxygen below 3 mm depth.Methane production rates measured from soil layersincubated separately in closed vessels were zero atthe soil surface and increased with depth. In soildepths below 4 mm where iron(III) concentrationsdecreased higher methane production rates werefound.     

Repeated anaerobic microbial redox cycling of iron

Some nitrate- and Fe(III)-reducing microorganisms are capable of oxidizing Fe(II) with nitrate as the electron acceptor. This enzymatic pathway may facilitate the development of anaerobic microbial communities that take advantage of the energy available during Fe-N redox oscillations. We examined this phenomenon in synthetic Fe(III) oxide (nanocrystalline goethite) suspensions inoculated with microflora from freshwater river floodplain sediments. Nitrate and acetate were added at alternate intervals in order to induce repeated cycles of microbial Fe(III) reduction and nitrate-dependent Fe(II) oxidation. Addition of nitrate to reduced, acetate-depleted suspensions resulted in rapid Fe(II) oxidation and accumulation of ammonium. High-resolution transmission electron microscopic analysis of material from Fe redox cycling reactors showed amorphous coatings on the goethite nanocrystals that were not observed in reactors operated under strictly nitrate- or Fe(III)-reducing conditions. Microbial communities associated with N and Fe redox metabolism were assessed using a combination of most-probable-number enumerations and 16S rRNA gene analysis. The nitrate-reducing and Fe(III)-reducing cultures were dominated by denitrifying Betaproteobacteria (e.g., Dechloromonas) and Fe(III)-reducing Deltaproteobacteria (Geobacter), respectively; these same taxa were dominant in the Fe cycling cultures. The combined chemical and microbiological data suggest that both Geobacter and various Betaproteobacteria participated in nitrate-dependent Fe(II) oxidation in the cycling cultures. Microbially driven Fe-N redox cycling may have important consequences for both the fate of N and the abundance and reactivity of Fe(III) oxides in sediments.     

Anaerobic Biooxidation of Fe(II) by Dechlorosoma suillum

Anaerobic microbial oxidation of Fe(II) was only recently discovered and very little is known about this metabolism. We recently demonstrated that several dissimilatory perchlorate-reducing bacteria could utilize Fe(II) as an electron donor under anaerobic conditions. Here we report on a more in-depth analysis of Fe(II) oxidation by one of these organisms, Dechlorosoma suillum. Similarly to most known nitrate-dependent Fe(II) oxidizers, D. suillum did not grow heterotrophically or lithoautotrophically by anaerobic Fe(II) oxidation. In the absence of a suitable organic carbon source, cells rapidly lysed even though nitrate-dependent Fe(II) oxidation was still occurring. The coupling of Fe(II) oxidation to a particular electron acceptor was dependent on the growth conditions of cells of D. suillum. As such, anaerobically grown cultures of D. suillum did not mediate Fe(II) oxidation with oxygen as the electron acceptor, while conversely, aerobically grown cultures did not mediate Fe(II) oxidation with nitrate as the electron acceptor. Anaerobic washed cell suspensions of D. suillum rapidly produced an orange/brown precipitate which X-ray diffraction analysis identified as amorphous ferric oxyhydroxide or ferrihydrite. This is similar to all other identified nitrate-dependent Fe(II) oxidizers but is in contrast to what is observed for growth cultures of D. suillum, which produced a mixed-valence Fe(II)-Fe(III) precipitate known as green rust. D. suillum rapidly oxidized the Fe(II) content of natural sediments. Although the form of ferrous iron in these sediments is unknown, it is probably a component of an insoluble mineral, as previous studies indicated that soluble Fe(II) is a relatively minor form of the total Fe(II) content of anoxic environments. The results of this study further enhance our knowledge of a poorly understood form of microbial metabolism and indicate that anaerobic Fe(II) oxidation by D. suillum is significantly different from previously described forms of nitrate-dependent microbial Fe(II) oxidation.     

Microaerophilic, Fe(II)-dependent growth and Fe(II) oxidation by a Dechlorospirillum species

A species of Dechlorospirillum was isolated from an Fe(II)-oxidizing, opposing-gradient-culture enrichment using an inoculum from a circumneutral, freshwater creek that showed copious amounts of Fe(III) (hydr)oxide precipitation. In gradient cultures amended with a redox indicator to visualize the depth of oxygen penetration, Dechlorospirillum sp. strain M1 showed Fe(II)-dependent growth at the oxic-anoxic interface and was unable to utilize sulfide as an alternate electron donor. The bacterium also grew with acetate as an electron donor under both microaerophilic and nitrate-reducing conditions, but was incapable of organotrophic Fe(III) reduction or nitrate-dependent Fe(II) oxidation. Although members of the genus Dechlorospirillum are primarily known as perchlorate and nitrate reducers, our results suggest that some species are members of the microbial communities involved in iron redox cycling at the oxic-anoxic transition zones in freshwater sediments.     

Effect of oxidation rate and Fe(II) state on microbial nitrate-dependent Fe(III) mineral formation

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to Klebsiella oxytoca. FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II)sol] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II)sol yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II)sol and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.     

Anaerobic nitrate-dependent iron(II) bio-oxidation by a novel lithoautotrophic betaproteobacterium, strain 2002

Microbial nitrate-dependent Fe(II) oxidation is known to contribute to iron biogeochemical cycling; however, the microorganisms responsible are virtually unknown. In an effort to elucidate this microbial metabolic process in the context of an environmental system, a 14-cm sediment core was collected from a freshwater lake and geochemically characterized concurrently with the enumeration of the nitrate-dependent Fe(II)-oxidizing microbial community and subsequent isolation of a nitrate-dependent Fe(II)-oxidizing microorganism. Throughout the sediment core, ambient concentrations of Fe(II) and nitrate were observed to coexist. Concomitant most probable number enumeration revealed the presence of an abundant nitrate-dependent Fe(II)-oxidizing microbial community (2.4 x 10(3) to 1.5 x 10(4) cells g(-1) wet sediment) from which a novel anaerobic, lithoautotrophic, Fe(II)-oxidizing bacterium, strain 2002, was isolated. Analysis of the complete 16S rRNA gene sequence revealed that strain 2002 was a member of the beta subclass of the proteobacteria with 94.8% similarity to Chromobacterium violaceum, a bacterium not previously recognized for the ability to oxidize nitrate-dependent Fe(II). Under nongrowth conditions, both strain 2002 and C. violaceum incompletely reduced nitrate to nitrite with Fe(II) as the electron donor, while under growth conditions nitrate was reduced to gaseous end products (N2 and N2O). Lithoautotrophic metabolism under nitrate-dependent Fe(II)-oxidizing conditions was verified by the requirement of CO2 for growth as well as the assimilation of 14C-labeled CO2 into biomass. The isolation of strain 2002 represents the first example of an anaerobic, mesophilic, neutrophilic Fe(II)-oxidizing lithoautotroph isolated from freshwater samples. Our studies further demonstrate the abundance of nitrate-dependent Fe(II) oxidizers in freshwater lake sediments and provide further evidence for the potential of microbially mediated Fe(II) oxidation in anoxic environments.     

Effect of Oxidation Rate and Fe(II) State on Microbial Nitrate-Dependent Fe(III) Mineral Formation

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to Klebsiella oxytoca. FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II)_sol] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II)_sol yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II)_sol and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.     

Chemolithotrophic nitrate-dependent Fe(II)-oxidizing nature of actinobacterial subdivision lineage TM3

Anaerobic nitrate-dependent Fe(II) oxidation is widespread in various environments and is known to be performed by both heterotrophic and autotrophic microorganisms. Although Fe(II) oxidation is predominantly biological under acidic conditions, to date most of the studies on nitrate-dependent Fe(II) oxidation were from environments of circumneutral pH. The present study was conducted in Lake Grosse Fuchskuhle, a moderately acidic ecosystem receiving humic acids from an adjacent bog, with the objective of identifying, characterizing and enumerating the microorganisms responsible for this process. The incubations of sediment under chemolithotrophic nitrate-dependent Fe(II)-oxidizing conditions have shown the enrichment of TM3 group of uncultured Actinobacteria. A time-course experiment done on these Actinobacteria showed a consumption of Fe(II) and nitrate in accordance with the expected stoichiometry (1:0.2) required for nitrate-dependent Fe(II) oxidation. Quantifications done by most probable number showed the presence of 1 × 10⁴ autotrophic and 1 × 10⁷ heterotrophic nitrate-dependent Fe(II) oxidizers per gram fresh weight of sediment. The analysis of microbial community by 16S rRNA gene amplicon pyrosequencing showed that these actinobacterial sequences correspond to ∼0.6% of bacterial 16S rRNA gene sequences. Stable isotope probing using ¹³CO₂ was performed with the lake sediment and showed labeling of these Actinobacteria. This indicated that they might be important autotrophs in this environment. Although these Actinobacteria are not dominant members of the sediment microbial community, they could be of functional significance due to their contribution to the regeneration of Fe(III), which has a critical role as an electron acceptor for anaerobic microorganisms mineralizing sediment organic matter. To the best of our knowledge this is the first study to show the autotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 group of uncultured Actinobacteria.     

Electric coupling between distant nitrate reduction and sulfide oxidation in marine sediment

Filamentous bacteria of the Desulfobulbaceae family can conduct electrons over centimeter-long distances thereby coupling oxygen reduction at the surface of marine sediment to sulfide oxidation in deeper anoxic layers. The ability of these cable bacteria to use alternative electron acceptors is currently unknown. Here we show that these organisms can use also nitrate or nitrite as an electron acceptor thereby coupling the reduction of nitrate to distant oxidation of sulfide. Sulfidic marine sediment was incubated with overlying nitrate-amended anoxic seawater. Within 2 months, electric coupling of spatially segregated nitrate reduction and sulfide oxidation was evident from: (1) the formation of a 4–6-mm-deep zone separating sulfide oxidation from the associated nitrate reduction, and (2) the presence of pH signatures consistent with proton consumption by cathodic nitrate reduction, and proton production by anodic sulfide oxidation. Filamentous Desulfobulbaceae with the longitudinal structures characteristic of cable bacteria were detected in anoxic, nitrate-amended incubations but not in anoxic, nitrate-free controls. Nitrate reduction by cable bacteria using long-distance electron transport to get privileged access to distant electron donors is a hitherto unknown mechanism in nitrogen and sulfur transformations, and the quantitative importance for elements cycling remains to be addressed.     

Comparison of iron-catalyzed DNA and lipid oxidation

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.     

Ecophysiology and the energetic benefit of mixotrophic Fe(II) oxidation by various strains of nitrate-reducing bacteria

In order to assess the importance of nitrate-dependent Fe(II) oxidation and its impact on the growth physiology of dominant Fe oxidizers, we counted these bacteria in freshwater lake sediments and studied their growth physiology. Most probable number counts of nitrate-reducing Fe(II)-oxidizing bacteria in the sediment of Lake Constance, a freshwater lake in Southern Germany, yielded about 10⁵ cells mL⁻¹ of the total heterotrophic nitrate-reducing bacteria, with about 1% (10³ cells mL⁻¹) of nitrate-reducing Fe(II) oxidizers. We investigated the growth physiology of Acidovorax sp. strain BoFeN1, a dominant nitrate-reducing mixotrophic Fe(II) oxidizer isolated from this sediment. Strain BoFeN1 uses several organic compounds (but no sugars) as substrates for nitrate reduction. It also reduces nitrite, dinitrogen monoxide, and O₂, but cannot reduce Fe(III). Growth experiments with cultures amended either with acetate plus Fe(II) or with acetate alone demonstrated that the simultaneous oxidation of Fe(II) and acetate enhanced growth yields with acetate alone (12.5 g dry mass mol⁻¹ acetate) by about 1.4 g dry mass mol⁻¹ Fe(II). Also, pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans strains can oxidize Fe(II) with nitrate, whereas Pseudomonas fluorescens and Thiobacillus denitrificans strains did not. Our study demonstrates that nitrate-dependent Fe(II) oxidation contributes to the energy metabolism of these bacteria, and that nitrate-dependent Fe(II) oxidation can essentially contribute to anaerobic iron cycling.     

Microbiology of flooded rice paddies

Flooded rice paddies are one of the major biogenic sources of atmospheric methane. Apart from this contribution to the 'greenhouse' effect, rice paddy soil represents a suitable model system to study fundamental aspects of microbial ecology, such as diversity, structure, and dynamics of microbial communities as well as structure-function relationships between microbial groups. Flooded rice paddy soil can be considered as a system with three compartments (oxic surface soil, anoxic bulk soil, and rhizosphere) characterized by different physio-chemical conditions. After flooding, oxygen is rapidly depleted in the bulk soil. Anaerobic microorganisms, such as fermentative bacteria and methanogenic archaea, predominate within the microbial community, and thus methane is the final product of anaerobic degradation of organic matter. In the surface soil and the rhizosphere well-defined microscale chemical gradients can be measured. The oxygen profile seems to govern gradients of other electron acceptors (e.g., nitrate, iron(III), and sulfate) and reduced compounds (e.g., ammonium, iron(II), and sulfide). These gradients provide information about the activity and spatial distribution of functional groups of microorganisms. This review presents the current knowledge about the highly complex microbiology of flooded rice paddies. In Section 2 we describe the predominant microbial groups and their function with particular regard to bacterial populations utilizing polysaccharides and simple sugars, and to the methanogenic archaea. Section 3 describes the spatial and temporal development of microscale chemical gradients measured in experimentally defined model systems, including gradients of oxygen and dissolved and solid-phase iron(III) and iron(II). In Section 4, the results of measurements of microscale gradients of oxygen, pH, nitrate-nitrite, and methane in natural rice fields and natural rice soil cores taken to the laboratory will be presented. Finally, perspectives of future research are discussed (Section 5).     

Microbial Metabolism of Benzene and the Oxidation of Ferrous Iron under Anaerobic Conditions: Implications for Bioremediation

Benzene and toluene were biodegraded when chelated Fe(III) served as the terminal electron acceptor in aquifer sediments contaminated by a petroleum refinery. Benzene biodegradation ceased when Fe(III) was depleted but resumed upon reamendment. Microorganisms from the same sediments degraded toluene, but not benzene, under nitrate reducing conditions. However, the anaerobic oxidation of Fe(II) to Fe(III) was also observed in toluene-degrading incubations. Fe(II) oxidation was dependent on the presence of nitrate and enhanced when organic electron donors were provided. Microbial nitrate-linked Fe(II) oxidation was also documented in other petroleum-contaminated aquifer sediments, sludge from an oil–water separator, a landfill leachate-impacted aquifer and a garden soil. These observations suggest that some of the reported effects of nitrate on hydrocarbon biodegradation may be indirect through the reoxidation of Fe(II).     

Characterization of Microbial Activities and U Reduction in a Shallow Aquifer Contaminated by Uranium Mill Tailings

A characterization of the Shiprock, NM, uranium mill tailing site focused on the geochemical and microbiological factors governing in-situ uranium-redox reactions. Groundwater and aqueous extracts of sediment samples contained a wide concentration range of sulfate, nitrate, and U(VI) with median values of 21.2 mM, 16.1 µM, and 2.7 µM, respectively. Iron(III) was not detected in groundwater, but a median value of 0.3 mM in sediment extracts was measured. Bacterial diversity down gradient from the disposal pile reflected the predominant geochemistry with relatively high numbers of sulfate- and nitrate-reducing microorganisms, and smaller numbers of acetogenic, methanogenic, nitrate-dependent Fe(II)-oxidizing, Fe(III)-reducing, and sulfide-oxidizing bacteria. In aquifer slurry incubations, nitrate reduction was always preferred and had a negative impact on sulfate-, Fe(III)-, and U-reduction rates. We also found that sulfate-reduction rates decreased sharply in the presence of clay, while Fe(III)-reduction increased with no clear impact on U reduction. In the absence of clay, iron and sulfate reduction correlated with concentrations of Fe(III) and sulfate, respectively. Rates of U(VI) loss did not correlate with the concentration of any electron acceptor. With the exception of Fe(III), electron donor amendment was largely unsuccessful in stimulating electron acceptor loss over a 2-week incubation period, suggesting that endogenous forms of organic matter were sufficient to support microbial activity. Our findings suggest that efforts to accelerate biological U reduction should initially focus on stimulating nitrate removal.     

Ammonium Limitation Results in the Loss of Ammonia-Oxidizing Activity in Nitrosomonas europaea

The effects of limiting concentrations of ammonium on the metabolic activity of Nitrosomonas europaea, an obligate ammonia-oxidizing soil bacterium, were investigated. Cells were harvested during late logarithmic growth and were incubated for 24 h in growth medium containing 0, 15, or 50 mM ammonium. The changes in nitrite production and the rates of ammonia- and hydroxylamine-dependent oxygen consumption were monitored. In incubations without ammonium, there was little change in the ammonia oxidation activity after 24 h. With 15 mM ammonium, an amount that was completely consumed, there was an 85% loss of the ammonia oxidation activity after 24 h. In contrast, there was only a 35% loss of the ammonia oxidation activity after 24 h in the presence of 50 mM ammonium, an amount that was not consumed to completion. There was little effect on the hydroxylamine oxidation activity in any of the incubations. The loss of ammonia oxidation activity was not due to differences in steady-state levels of ammonia monooxygenase (AMO) mRNA (amoA) or to degradation of the active site-containing subunit of AMO protein. The incubations were also conducted at a range of pH values to determine whether the loss of ammonia oxidation activity was correlated to the residual ammonium concentration. The loss of ammonia oxidation activity after 24 h was less at lower pH values (where the unoxidized ammonium concentration was higher). When added in conjunction with limiting ammonium, short-chain alkanes, which are alternative substrates for AMO, prevented the loss of ammonia oxidation activity at levels corresponding to their binding affinity for AMO. These results suggest that substrates of AMO can preserve the ammonia-oxidizing activity of N. europaea in batch incubations by protecting either AMO itself or other molecules associated with ammonia oxidation.     

The activities of two pathways of nitrate reduction in Rhodopseudomonas capsulata

1. The disappearance of nitrate from suspensions of intact, washed cells of Rhodopseudomonas capsulata strain N22DNAR⁺ was measured with an ion selective electrode. In samples taken from phototrophic cultures grown to late exponential phase, nitrate disappearance was partially inhibited by light but was not affected by the presence of ammonium. Nitrate disappearance from samples from low density cultures in the early exponential phase of growth was first inhibited and later stimulated by light. In these cells ammonium ions inhibited the light-dependent but not the dark disappearance of nitrate. It is concluded that cells in the early exponential phase of growth possess both an ammonium-sensitive, assimilatory pathway for nitrate reduction (NRI) and an ammonium-insensitive pathway for nitrate reduction (NRII) which is linked to respiratory electron flow and energy conservation. In cells harvested in late exponential phase only the respiratory pathway for pitrate reduction is detectable.
2. Nitrate reduction, as judged by the oxidation of reduced methyl viologen by anaerobic cell suspensions, was measured at high rates in those strains of R. capsulata (AD2, BK5, N22DNAR⁺) which are believed to possess NRII activity but not in those strains (Kbl, R3, N22) which only manifest the ammonium-sensitive NRI pathway. On this basis we have used nitrate-dependent oxidation of reduced methyl viologen as a diagnostic test for the nitrate reductase of NRII in cells harvested from cultures of R. capsulata strain AD2. The activity was readily detectable in cells from cultures grown aerobically in the dark with ammonium nitrate as source of nitrogen. When the oxygen supply to the culture was withdrawn, the level of methyl viologen-dependent nitrate reductase increased considerably and nitrite accumulated in the culture medium. Upon reconnecting the oxygen supply, methyl viologen-dependent nitrate reductase activity decreased and the reduction of nitrate to nitrite in the culture was inhibited. It is concluded that the respiratory nitrate reductase activity is regulated by the availability of electron transport pathways that are linked to the generation of a proton electrochemical gradient.