Inhibition of Chemoautotrophic Nitrification by Sodium Chlorate and Sodium Chlorite: a Reexamination

The oxidation of NH₄⁺ by Nitrosomonas europaea was insensitive to 10 mM NaClO₃ (sodium chlorate) but was strongly inhibited by NaClO₂ (sodium chlorite; K_i, 2 μM). The oxidation of NO₂⁻ by Nitrobacter winogradskyi was inhibited by both ClO₃⁻ and ClO₂⁻ (K_i for ClO₂⁻, 100 μM). N. winogradskyi reduced ClO₃⁻ to ClO₂⁻ under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO₂⁻ was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH₄⁺ and NO₂⁻ was inhibited in the presence of 10 mM ClO₃⁻ after a 2-h lag period, despite the fact that, under these conditions, ClO₂⁻ was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH₄⁺ oxidation is inhibited by ClO₂⁻ produced by reduction of ClO₃⁻ by the NO₂⁻ oxidizer. The use of ClO₃⁻ inhibition of NO₂⁻ oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH₄⁺ is not affected.     

Anaerobic Oxidation of Arsenite Linked to Chlorate Reduction

Microorganisms play a significant role in the speciation and mobility of arsenic in the environment. In this study, the oxidation of arsenite [As(III)] to arsenate [As(V)] linked to chlorate (ClO₃⁻) reduction was shown to be catalyzed by sludge samples, enrichment cultures (ECs), and pure cultures incubated under anaerobic conditions. No activity was observed in treatments lacking inoculum or with heat-killed sludge, or in controls lacking ClO₃⁻. The As(III) oxidation was linked to the complete reduction of ClO₃⁻ to Cl⁻, and the molar ratio of As(V) formed to ClO₃⁻ consumed approached the theoretical value of 3:1 assuming the e⁻ equivalents from As(III) were used to completely reduce ClO₃⁻. In keeping with O₂ as a putative intermediate of ClO₃⁻ reduction, the ECs could also oxidize As(III) to As(V) with O₂ at low concentrations. Low levels of organic carbon were essential in heterotrophic ECs but not in autotrophic ECs. 16S rRNA gene clone libraries indicated that the ECs were dominated by clones of Rhodocyclaceae (including Dechloromonas, Azospira, and Azonexus phylotypes) and Stenotrophomonas under autotrophic conditions. Additional phylotypes (Alicycliphilus, Agrobacterium, and Pseudoxanthomonas) were identified in heterotrophic ECs. Two isolated autotrophic pure cultures, Dechloromonas sp. strain ECC1-pb1 and Azospira sp. strain ECC1-pb2, were able to grow by linking the oxidation of As(III) to As(V) with the reduction of ClO₃⁻. The presence of the arsenite oxidase subunit A (aroA) gene was demonstrated with PCR in the ECs and pure cultures. This study demonstrates that ClO₃⁻ is an alternative electron acceptor to support the microbial oxidation of As(III).The contamination of drinking water with arsenic (As) is a global public health issue. Arsenic is a human carcinogenic compound (2), which poses a risk to millions of people around the world (31). The most common oxidation states of As in aqueous environments are arsenite [As(III), H₃AsO₃] or arsenate [As(V), H₂AsO₄⁻, and HAsO₄²⁻]. Microbial processes play critical roles in controlling the fate and transformation of As in subsurface systems (22). As(V) binds to aluminum oxides more extensively than As(III) under circumneutral pH conditions (12, 16). Both As(III) and As(V) are strongly adsorbed on iron oxides (9). However, As(III) is more rapidly desorbed compared to As(V) (35).Aerobic bacteria can oxidize As(III) forming As(V) (14, 28), which potentially is less mobile in the subsurface environment. Also, in environments with dissolved ferrous iron [Fe(II)] the oxidation of Fe(II) (both abiotic and biotic) would result in formation of Fe(III) (hydr)oxides such as ferrihydrite which adsorb As. Oxidation processes, therefore, can decrease the mobilization of As in groundwater. However, oxygen (O₂) is poorly soluble in groundwater and may become consumed by microbial activity, creating anaerobic zones. Alternative oxidants aside from O₂ also have the potential to support the microbial oxidation of As(III). Recently, several studies have demonstrated that nitrate-dependent As(III) oxidation is carried out by anaerobic microorganisms to gain energy from As(III) oxidation. As(III)-oxidizing denitrifying bacteria have been isolated from various environments including As-contaminated lakes and soil (21, 25), as well as enrichment cultures (ECs), and isolates from pristine sediments and sludge samples (33, 34). 16S rRNA gene clone library characterization of the ECs indicates that the predominant phylotypes were from the genus Azoarcus and the family Comamonadaceae (34).Beside nitrate, chlorate (ClO₃⁻) can also be considered as a possible alternative oxidant for microorganisms to promote the bioremediation of contaminated plumes (6, 17). (Per)chlorate is commonly used as a terminal electron acceptor by anaerobic bacteria; as a result, it is completely degraded to the benign end product, chloride (Cl⁻). Microbial reduction of perchlorate proceeds via a three-step process of ClO₄⁻ → ClO₃⁻→ ClO₂⁻ → O₂ + Cl⁻ (6). Reduction of perchlorate to chlorate, and chlorate to chlorite is catalyzed by respiratory (per)chlorate reductases (3). Subsequent disproportionation of chlorite into Cl⁻ and O₂ is catalyzed by chlorite dismutase, which is the fastest step, and the O₂ produced is immediately consumed for energy of cell synthesis (6). Although organic compounds are the most well studied electron donors for (per)chlorate reduction, Fe(II) oxidation has also been shown to be linked to microbial ClO₃⁻ reduction (36).The main objective of the present study is to explore the potential use of ClO₃⁻ as an electron acceptor for the microbial oxidation of As(III) by anaerobic bacteria. The theoretical stoichiometry of the reaction is presented below: (1) Based on bioenergetic considerations, the reaction is feasible as indicated by the highly exergonic standard change in Gibbs free energy [ΔG⁰′ = −92.4 kJ mol⁻¹ As(III)] calculated from E^0′ values of 0.618 and 0.139 V for ClO₃⁻/Cl⁻ (6) and As(V)/As(III) (18), respectively.     

Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase

Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO₂ ⁻) to Cl⁻ and O₂. The ability of Cld to promote O₂ formation from ClO₂ ⁻ is unusual. Heme enzymes generally utilize ClO₂ ⁻ as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO₂ ⁻) was determined to investigate features responsible for this novel reactivity. The enzyme active site contains a single b-type heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate residue hydrogen-bonded to the heme proximal histidine that may stabilize reactive heme species. A solvent-exposed arginine residue likely gates substrate entry to a tightly confined distal pocket. On the basis of the proposed mechanism of Cld, initial reaction of ClO₂ ⁻ within the distal pocket generates hypochlorite (ClO⁻) and a compound I intermediate. The sterically restrictive distal pocket probably facilitates the rapid rebound of ClO⁻ with compound I forming the Cl⁻ and O₂ products. Common to other heme enzymes, Cld is inactivated after a finite number of turnovers, potentially via the observed formation of an off-pathway tryptophanyl radical species through electron migration to compound I. Three tryptophan residues of Cld have been identified as candidates for this off-pathway radical. Finally, a juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface suggests O₂ may have a preferential direction for exiting the active site.     

Nitrate Uptake into Barley (Hordeum vulgare) Plants : A New Approach Using ClO(3) as an Analog for NO(3)

Evidence is presented that chlorate is an extremely good analog for nitrate during nitrate uptake by intact barley (Hordeum vulgare cv. Fergus) roots. The depletion of ClO₃⁻ or NO₃⁻ from uptake media over 2 to 6 hours by seedlings was found to be dependent on combined NO₃⁻ plus ClO₃⁻ concentrations, and total anion uptake was equivalent at different NO₃⁻/ClO₃⁻ ratios. After loading barley seedlings with ³⁶ClO₃⁻ for 6 hours, kinetic parameters were derived from the analysis of efflux of [³⁶Cl] chlorate into unlabeled solution. On the basis of this analysis, the half times for exchange for the cytoplasmic and vacuolar phases were 17 minutes and 20 hours, respectively.     

Reactivity of lignin-related phenols with haemoglobin and NaClO3

Summary Haemoglobin (Hb), in the presence of sodium chlorate (NaClO₃), promoted the decraboxylation of vanillic acid, the oxidation of guaiacol and the demethoxylation of vanillic and ferulic acids with maximal activity at pH 3.5–4.0. These reactions were also mediated by Hb + chlorine dioxide (ClO₂) with a similar pH profile. However, differences in the activity of sodium chlorite (NaClOP₂) in the presence or absence of Hb were observed, especially at low pH values. It is speculated that ClO₂ is mainly responsible for acitivities observedc. The Hb-dependent activity was more stable in the presence of NaClO₃ than with hydrogen peroxide (H₂O₂). *** DIRECT SUPPORT *** AG903028 00008     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): I. Control of Net Uptake by NO(3) Efflux

A computer-controlled multichannel data acquisition system was employed to obtain continuous measurements of net nitrate or chlorate uptake by roots of intact barley plants (Hordeum vulgare cv Betzes) using nitrate-specific electrodes. Plants, previously grown in solutions maintained at 10 or 200 micromolar NO₃⁻ (low N or high N conditions, respectively), were provided with 200 micromolar NO₃⁻ or ClO₃⁻ during the uptake period. Initial rates of NO₃⁻ uptake were several times higher in low N plants than in high N plants. Within 10 min, uptake in the former plants declined to a new steady rate which was sustained for the remainder of the experiment. No such time-dependent changes were evident in the high N plants. Rates and patterns of net chlorate uptake exhibited almost identical dependence upon previous nitrate provision. NO₃⁻ (³⁶ClO₃⁻) influx, by contrast, appeared to be independent of NO₃⁻ pretreatment prior to influx determination. Nitrate efflux, estimated by several different methods, was strongly correlated with internal nitrate concentration of the roots.     

Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity

Effects of NO₂⁻, ClO₃⁻, and ClO₂⁻ on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO₃⁻ influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m⁻³ NO₂⁻ fully induced NO₃⁻ transport but failed to induce NRA. Similar pretreatments with ClO₃⁻ and ClO₂⁻ induced neither NO₃⁻ transport nor NRA. Net ClO₃⁻ uptake was induced by NO₃⁻ but not by ClO₃⁻ itself, indicating that NO₃⁻ and ClO₃⁻ transport occur via the NO₃⁻ carrier. At the uptake step, NO₂⁻ and ClO₂⁻ strongly inhibited NO₃⁻ influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO₃⁻ proved to be a weak inhibitor of NO₃⁻ influx (K_i = 16 mol m⁻³) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO₃⁻ analogs as screening agents for the isolation of mutants defective in NO₃⁻ transport.     

Effect of Chlorate Treatment on Nitrate Reductase and Nitrite Reductase Gene Expression in Arabidopsis thaliana

The herbicide chlorate has been used extensively to isolate mutants that are defective in nitrate reduction. Chlorate is a substrate for the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Because NR is a substrate (NO₃⁻)-inducible enzyme, we investigated the possibility that chlorate may also act as an inducer. Irrigation of ammonia-grown Arabidopsis plants with chlorate leads to an increase in NR mRNA in the leaves. No such increase was observed for nitrite reductase mRNA following chlorate treatment; thus, the effect seems to be specific to NR. The increase in NR mRNA did not depend on the presence of wild-type levels of NR activity or molybdenum-cofactor, as a molybdenum-cofactor mutant with low levels of NR activity displayed the same increase in NR mRNA following chlorate treatment. Even though NR mRNA levels were found to increase after chlorate treatment, no increase in NR protein was detected and the level of NR activity dropped. The lack of increase in NR protein was not due to inactivation of the cells' translational machinery, as pulse labeling experiments demonstrated that total protein synthesis was unaffected by the chlorate treatment during the time course of the experiment. Chlorate-treated plants still retain the capacity to make functional NR because NR activity could be restored by irrigating the chlorate-treated plants with nitrate. The low levels of NR protein and activity may be due to inactivation of NR by chlorite, leading to rapid degradation of the enzyme. Thus, chlorate treatment stimulates NR gene expression in Arabidopsis that is manifested only at the mRNA level and not at the protein or activity level.     

Identification of Heterotrophic Nitrification in a Sierran Forest Soil

A potential for heterotrophic nitrification was identified in soil from a mature conifer forest and from a clear-cut site. Potential rates of NO₂⁻ production were determined separately from those of NO₃⁻ by using acetylene to block autotrophic NH₄⁺ oxidation and chlorate to block NO₂⁻ oxidation to NO₃⁻ in soil slurries. Rates of NO₂⁻ production were similar in soil from the forest and the clear-cut site and were strongly inhibited by acetylene. The rate of NO₃⁻ production was much greater than that of NO₂⁻ production, and NO₃⁻ production was not significantly affected by acetylene or chlorate. Nitrate production was partially inhibited by cycloheximide, but was not significantly reduced by streptomycin. Neither the addition of ammonium nor the addition of peptone stimulated NO₃⁻ production. ¹⁵N labeling of the NH₄⁺ pool demonstrated that NO₃⁻ was not coming from NH₄⁺. The potential for heterotrophic nitrification in these forest soils was greater than that for autotrophic nitrification.     

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N2O Emissions

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N₂O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg⁻¹. Without added C, peak NO emissions of 4 μg of N kg⁻¹ h⁻¹ were increased to 15 μg of N kg⁻¹ h⁻¹ by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 μg of N kg⁻¹ h⁻¹ in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 μg of N kg⁻¹ h⁻¹ after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N₂O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N₂O produced during nitrification in soil.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.     

Nitrite oxidation in the upper water column and oxygen minimum zone of the eastern tropical North Pacific Ocean

Nitrogen (N) is an essential nutrient in the sea and its distribution is controlled by microorganisms. Within the N cycle, nitrite (NO₂⁻) has a central role because its intermediate redox state allows both oxidation and reduction, and so it may be used by several coupled and/or competing microbial processes. In the upper water column and oxygen minimum zone (OMZ) of the eastern tropical North Pacific Ocean (ETNP), we investigated aerobic NO₂⁻ oxidation, and its relationship to ammonia (NH₃) oxidation, using rate measurements, quantification of NO₂⁻-oxidizing bacteria via quantitative PCR (QPCR), and pyrosequencing. ¹⁵NO₂⁻ oxidation rates typically exhibited two subsurface maxima at six stations sampled: one located below the euphotic zone and beneath NH₃ oxidation rate maxima, and another within the OMZ. ¹⁵NO₂⁻ oxidation rates were highest where dissolved oxygen concentrations were <5 μM, where NO₂⁻ accumulated, and when nitrate (NO₃⁻) reductase genes were expressed; they are likely sustained by NO₃⁻ reduction at these depths. QPCR and pyrosequencing data were strongly correlated (r²=0.79), and indicated that Nitrospina bacteria numbered up to 9.25% of bacterial communities. Different Nitrospina groups were distributed across different depth ranges, suggesting significant ecological diversity within Nitrospina as a whole. Across the data set, ¹⁵NO₂⁻ oxidation rates were decoupled from ¹⁵NH₄⁺ oxidation rates, but correlated with Nitrospina (r²=0.246, P<0.05) and NO₂⁻ concentrations (r²=0.276, P<0.05). Our findings suggest that Nitrospina have a quantitatively important role in NO₂⁻ oxidation and N cycling in the ETNP, and provide new insight into their ecology and interactions with other N-cycling processes in this biogeochemically important region of the ocean.     

Claudin-4 Deficiency Results in Urothelial Hyperplasia and Lethal Hydronephrosis

Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4 ^−/− mice and found that these mice had increased mortality due to hydronephrosis of relatively late onset. While the renal nephrons of Cldn4 ^−/− mice showed a concomitant diminution of Cld8 expression at tight junction (TJ), accumulation of Cld3 at TJ was markedly enhanced in compensation and the overall TJ structure was unaffected. Nonetheless, Cldn4 ^−/− mice showed slightly yet significantly increased fractional excretion of Ca²⁺ and Cl⁻, suggesting a role of Cld4 in the specific reabsorption of these ions via a paracellular route. Although the urine volume tended to be increased concordantly, Cldn4 ^−/− mice were capable of concentrating urine normally on dehydration, with no evidence of diabetes insipidus. In the urothelium, the formation of TJs and uroplaques as well as the gross barrier function were also unaffected. However, intravenous pyelography analysis indicated retarded urine flow prior to hydronephrosis. Histological examination revealed diffuse hyperplasia and a thickening of pelvic and ureteral urothelial layers with markedly increased BrdU uptake in vivo. These results suggest that progressive hydronephrosis in Cldn4 ^−/− mice arises from urinary tract obstruction due to urothelial hyperplasia, and that Cld4 plays an important role in maintaining the homeostatic integrity of normal urothelium.     

Rapid Methane Oxidation in a Landfill Cover Soil

Methane oxidation rates observed in a topsoil covering a retired landfill are the highest reported (45 g m⁻² day⁻¹) for any environment. This microbial community had the capacity to rapidly oxidize CH₄ at concentrations ranging from <1 ppm (microliters per liter) (first-order rate constant [k] = −0.54 h⁻¹) to >10⁴ ppm (k = −2.37 h⁻¹). The physiological characteristics of a methanotroph isolated from the soil (characteristics determined in aqueous medium) and the natural population, however, were similar to those of other natural populations and cultures: the Q₁₀ and optimum temperature were 1.9 and 31°C, respectively, the apparent half-saturation constant was 2.5 to 9.3 μM, and 19 to 69% of oxidized CH₄ was assimilated into biomass. The CH₄ oxidation rate of this soil under waterlogged (41% [wt/vol] H₂O) conditions, 6.1 mg liter⁻¹ day⁻¹, was near rates reported for lake sediment and much lower than the rate of 116 mg liter⁻¹ day⁻¹ in the same soil under moist (11% H₂O) conditions. Since there are no large physiological differences between this microbial community and other CH₄ oxidizers, we attribute the high CH₄ oxidation rate in moist soil to enhanced CH₄ transport to the microorganisms; gas-phase molecular diffusion is 10⁴-fold faster than aqueous diffusion. These high CH₄ oxidation rates in moist soil have implications that are important in global climate change. Soil CH₄ oxidation could become a negative feedback to atmospheric CH₄ increases (and warming) in areas that are presently waterlogged but are projected to undergo a reduction in summer soil moisture.     

Nitrite oxidation in the Namibian oxygen minimum zone

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in ¹⁵N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 nM NO₂⁻ d⁻¹) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO₃⁻ was re-oxidized back to NO₃⁻ via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Isolation,Characterization and Kinetics of Perchlorate Reducing Magnetospirillum Species

Perchlorate reducing bacteria reduce perchlorate to chlorate (ClO₃?), which, in turn, is reduced to chlorite (ClO₂?) and ultimately to chloride (Cl^?). Magnetospirillum strains are reported to use chlorate/perchlorate as electron acceptors. This study describes the perchlorate reducing property of strain VITRJS5, a Magnetopsirillum isolated from freshwater sediment collected from Chelur freshwater lake, Kerala, India. The strain was microaerophile and was phylogenetically related to a Magnetospirillum sp., a member of the α-subclass of the class Proteobacteria. The placement of the isolate in the genus Magnetospirillum has further confirmed the presence of four key magnetosome membrane genes. PCR amplification and phylogenetic analysis of central metabolic genes such as nifH (nitrogenase) and cbbM (type II RubisCo) displayed the highest similarity (97% and 81%, respectively) with Magnetospirillum sp. BB-1 The growth kinetic parameters of the isolate were studied with acetate as the electron donor in batch experiments. Monod's substrate utilization model has been established with oxygen, nitrate and perchlorate as electron acceptors separately. The maximum specific growth rate (µ_max) and half-saturation constant (k_s^conc) for the bacterium varied while utilizing different electron acceptors. The maximum specific growth rate was 0.226, 0.190 and 0.096 per hour and half-velocity constant K_s was 25.09, 33.36 and 65.37 mg acetate/l for oxygen, nitrate and perchlorate, respectively. The reduction of perchlorate has been analyzed using kinetic studies of the substrate uptake by the bacteria and the half-velocity constant K_s was found to be 52.8 mg/l. The results indicate that the strain VITRJS5 effectively reduces perchlorate by using it as an electron acceptor.     

Anaerobic Ammonium Oxidation Measured in Sediments along the Thames Estuary, United Kingdom

Until recently, denitrification was thought to be the only significant pathway for N₂ formation and, in turn, the removal of nitrogen in aquatic sediments. The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment. By using a combination of ¹⁵N-labeled NH₄⁺, NO₃⁻, and NO₂⁻ (and ¹⁴N analogues), production of ²⁹N₂ and ³⁰N₂ was measured in anaerobic sediment slurries from six sites along the Thames estuary. The production of ²⁹N₂ in the presence of ¹⁵NH₄⁺ and either ¹⁴NO₃⁻ or ¹⁴NO₂⁻ confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO₂⁻. Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO₂⁻ or indirect reduction, following the initial reduction of NO₃⁻. Whether NO₂⁻ was directly present at 800 μM or it accumulated at 3 to 20 μM (from the reduction of NO₃⁻), the rate of ²⁹N₂ formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO₂⁻. We observed a shift in the significance of anaerobic ammonium oxidation to N₂ formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast. The relative importance of anaerobic ammonium oxidation was positively correlated (P < 0.05) with sediment organic content. This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system.