Nitrogen retention in the hyporheic zone of a glacial river in interior Alaska

We examined the hydrologic controls on nitrogen biogeochemistry in the hyporheic zone of the Tanana River, a glacially-fed river, in interior Alaska. We measured hyporheic solute concentrations, gas partial pressures, water table height, and flow rates along subsurface flowpaths on two islands for three summers. Denitrification was quantified using an in situ ¹⁵NO₃⁻ push–pull technique. Hyporheic water level responded rapidly to change in river stage, with the sites flooding periodically in mid−July to early−August. Nitrate concentration was nearly 3-fold greater in river (ca. 100 μg NO₃⁻–N l⁻¹) than hyporheic water (ca. 38 μg NO₃⁻–N l⁻¹), but approximately 60–80% of river nitrate was removed during the first 50 m of hyporheic flowpath. Denitrification during high river stage ranged from 1.9 to 29.4 mg N kg sediment⁻¹ day⁻¹. Hotspots of methane partial pressure, averaging 50,000 ppmv, occurred in densely vegetated sites in conjunction with mean oxygen concentration below 0.5 mgO₂ l⁻¹. Hyporheic flow was an important mechanism of nitrogen supply to microbes and plant roots, transporting on average 0.41 gNO₃⁻–N m⁻² day⁻¹, 0.22 g NH₄⁺–N m⁻² day⁻¹, and 3.6 g DON m⁻² day⁻¹ through surface sediment (top 2 m). Our results suggest that denitrification can be a major sink for river nitrate in boreal forest floodplain soils, particularly at the river-sediment interface. The stability of the river hydrograph and the resulting duration of soil saturation are key factors regulating the redox environment and anaerobic metabolism in the hyporheic zone.     

Biomass Production with Willow and Poplar Short Rotation Coppices on Sensitive Areas—the Impact on Nitrate Leaching and Groundwater Recharge in a Drinking Water Catchment near Hanover,Germany

In a lowland drinking water catchment area, nitrate leaching as well as groundwater recharge (GWR) was investigated in willow and poplar short rotation coppice (SRC) plantations of different ages, soil preparation measures prior to planting and harvesting intervals. Significantly increased nitrate concentrations of 16.6 ± 1.6 mg NO₃-N L⁻¹ were measured in winter/spring 2010 on a poplar site, established in 2009 after deep plowing (90 cm) but then, subsequently decreased strongly to below 2 mg NO₃-N L⁻¹ in spring 2011. The fallow ground reference plot showed nitrate concentrations consistently below 1 mg L⁻¹ and estimated annual seepage output loss was only 1.36 ± 1.1 kg ha⁻¹ a⁻¹. Leaching loss from a neighboring willow plot from 2005 was 14.3 ± 6.6 kg NO₃-N ha⁻¹ during spring 2010 but decreased to 2.0 ± 1.5 kg NO₃-N ha⁻¹ during the subsequent drainage period. A second willow plot, not harvested since its establishment in 1994, showed continuously higher nitrate concentrations (10.2 ± 1.7 NO₃-N L⁻¹), while a neighboring poplar plot, twice harvested since 1994 showed significantly reduced nitrate concentrations. Water balance simulations, referenced by soil water tension and throughfall measurements, showed that at 655 mm annual rainfall, GWR from the reference plot (300 mm a⁻¹) was reduced by 40 % (to 180 mm a⁻¹) on the 2005 willow stand, mainly due to doubled rainfall interception losses. However, transpiration was limited by low soil water storage capacities, which in turn led to a moderate impact on GWR. We conclude that well-managed SRC on sensitive areas can prevent nitrate leaching, while impacts on GWR may be mitigated by management options.     

Vadose Zone Microbial Biobarriers Remove Nitrate from Percolating Groundwater

Microbial biobarriers are an established technique for cleansing contaminants from aquifers. This study evaluated their use under well-drained conditions within the vadose or unsaturated zone. Three sets of sand filled columns, the positive control, field-capacity, and sub-field-capacity groups, contained biobarriers formed by mixing sand with sawdust and soybean oil. The biobarriers were positioned 1 m from the top of the 145 cm columns. A fourth set of column, the negative control, contained no biobarrier. The positive control group’s biobarriers were saturated while biobarriers in the other groups were allowed to drain. At intervals water containing 20 mg l⁻¹ NO₃⁻–N was applied to the columns, the water was allowed to percolate through the columns, and the effluents were collected and analyzed. The biobarriers were highly effective at removing NO₃⁻. NO₃⁻–N in the effluents from the field-capacity, sub-field-capacity, and positive control groups averaged 0.4 ± 0.1, 0.6 ± 0.1, and 0.8 ± 0.1 mg l⁻¹, respectively, during the final weeks of the study while effluents from the negative control group averaged 17.9 ± 0.4 mg l⁻¹. The barriers removed NO₃⁻ even when the water content was in the 20–40% pore filled space range. During the 12-week study the field-capacity barriers lost 5.6% of their organic content while those in the sub-field-capacity group lost no detectable organic matter indicating that the barriers contained sufficient substrate to last for several years. Vadose zone biobarriers could provide a useful means of protecting surface waters and aquifers from NO₃⁻.     

Accelerated root growth induced by nitrate deficit is related to apoplast acidification

We measured the rate of growth, osmotic pressure, hydraulic conductance, longitudinal and transverse extensibility of barley (Hordeum vulgare L.) roots in Knop solution with nitrate and at substitution of NO₃⁻ with Cl⁻. During the first three days after NO₃⁻ removal, root growth acceleration was related to the increase in their longitudinal extensibility. It was shown that root exposure to buffer with pH 4.5 and also activation of H⁺ pump with naphthyl acetate imitated changes in extensibility induced by NO₃⁻ deficit. Earlier, we have demonstrated medium acidification near root surface and calculated its expected level (pH 4.5). This permits a supposition that the cause for changes in extensibility and root growth acceleration at NO₃⁻ deficit was apoplast acidification, evidently related to the ceasing of NO₃⁻ symport with H⁺ and activation of the plasmalemmal H⁺ pump. ABA did not affect root extensibility at pH 4.5; however, at pH 6.0, it was similar to the action of diethylstilbestrol, an inhibitor of H⁺ pump, and opposite to the action of NO₃⁻ deficit. Thus, the absence of ABA effects on root growth, in spite of its accumulation at NO₃⁻ deficit, could be explained by apoplast acidification as well.     

Coral calcification responds to seawater acidification: a working hypothesis towards a physiological mechanism

The decrease in the saturation state of seawater, Ω, following seawater acidification, is believed to be the main factor leading to a decrease in the calcification of marine organisms. To provide a physiological explanation for this phenomenon, the effect of seawater acidification was studied on the calcification and photosynthesis of the scleractinian tropical coral Stylophora pistillata. Coral nubbins were incubated for 8 days at three different pH (7.6, 8.0, and 8.2). To differentiate between the effects of the various components of the carbonate chemistry (pH, CO₃²⁻, HCO₃⁻, CO₂, Ω), tanks were also maintained under similar pH, but with 2-mM HCO₃⁻ added to the seawater. The addition of 2-mM bicarbonate significantly increased the photosynthesis in S. pistillata, suggesting carbon-limited conditions. Conversely, photosynthesis was insensitive to changes in pH and pCO₂. Seawater acidification decreased coral calcification by ca. 0.1-mg CaCO₃ g⁻¹ d⁻¹ for a decrease of 0.1 pH units. This correlation suggested that seawater acidification affected coral calcification by decreasing the availability of the CO₃²⁻ substrate for calcification. However, the decrease in coral calcification could also be attributed either to a decrease in extra- or intracellular pH or to a change in the buffering capacity of the medium, impairing supply of CO₃²⁻ from HCO₃⁻.     

Effects of NO2? and NO3? on the Fe(III)EDTA reduction in a chemical absorption–biological reduction integrated NOx removal system

The biological reduction of Fe(III) ethylenediaminetetraacetic acid (EDTA) is a key step for NO removal in a chemical absorption–biological reduction integrated process. Since typical flue gas contain oxygen, NO₂ ⁻ and NO₃ ⁻ would be present in the absorption solution after NO absorption. In this paper, the interaction of NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA reduction was investigated. The experimental results indicate that the Fe(III)EDTA reduction rate decrease with the increase of NO₂ ⁻ or NO₃ ⁻ addition. In the presence of 10 mM NO₂ ⁻ or NO₃ ⁻, the average reduction rate of Fe(III)EDTA during the first 6-h reaction was 0.076 and 0.17 mM h⁻¹, respectively, compared with 1.07 mM h⁻¹ in the absence of NO₂ ⁻ and NO₃ ⁻. Fe(III)EDTA and either NO₂ ⁻ or NO₃ ⁻ reduction occurred simultaneously. Interestingly, the reduction rate of NO₂ ⁻ or NO₃ ⁻ was enhanced in presence of Fe(III)EDTA. The inhibition patterns observed during the effect of NO₂ ⁻ and NO₃ ⁻ on the Fe(III)EDTA reduction experiments suggest that Escherichia coli can utilize NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA as terminal electron acceptors.     

Nitrate and nitrite inhibition of methanogenesis during denitrification in granular biofilms and digested domestic sludges

Anaerobic bioreactors that can support simultaneous microbial processes of denitrification and methanogenesis are of interest to nutrient nitrogen removal. However, an important concern is the potential toxicity of nitrate (NO₃ ⁻) and nitrite (NO₂ ⁻) to methanogenesis. The methanogenic toxicity of the NO_x⁻ compounds to anaerobic granular biofilms and municipal anaerobic digested sludge with two types of substrates, acetate and hydrogen, was studied. The inhibition was the severest when the NO_x⁻ compounds were still present in the media (exposure period). During this period, 95% or greater inhibition of methanogenesis was evident at the lowest concentrations of added NO₂ ⁻ tested (7.6–10.2 mg NO₂ ⁻-N l⁻¹) or 8.3–121 mg NO₃ ⁻-N l⁻¹ of added NO₃ ⁻, depending on substrate and inoculum source. The inhibition imparted by NO₃ ⁻ was not due directly to NO₃ ⁻ itself, but instead due to reduced intermediates (e.g., NO₂ ⁻) formed during the denitrification process. The toxicity of NO_x⁻ was found to be reversible after the exposure period. The recovery of activity was nearly complete at low added NO_x⁻ concentrations; whereas the recovery was only partial at high added NO_x⁻ concentrations. The recovery is attributed to the metabolism of the NO_x⁻ compounds. The assay substrate had a large impact on the rate of NO₂ ⁻ metabolism. Hydrogen reduced NO₂ ⁻ slowly such that NO₂ ⁻ accumulated more and as a result, the toxicity was greater compared to acetate as a substrate. The final methane yield was inversely proportional to the amount of NO_x⁻ compounds added indicating that they were the preferred electron acceptors compared to methanogenesis.     

Soil-mixing effects on inorganic nitrogen production and consumption in forest and shrubland soils

Soils that are physically disturbed are often reported to show net nitrification and NO₃⁻ loss. To investigate the response of soil N cycling rates to soil mixing, we assayed gross rates of mineralization, nitrification, NH₄⁺ consumption, and NO₃⁻ consumption in a suite of soils from eleven woody plant communities in Oregon, New Mexico, and Utah. Results suggest that the common response of net NO₃⁻ flux from disturbed soils is not a straightforward response of increased gross nitrification, but instead may be due to the balance of several factors. While mineralization and NH₄⁺ assimilation were higher in mixed than intact cores, NO₃⁻ consumption declined. Mean net nitrification was 0.12 mg N kg⁻¹ d⁻¹ in disturbed cores, which was significantly higher than in intact cores (−0.19 mg N kg⁻¹ d⁻¹). However, higher net nitrification rates in disturbed soils were due to the suppression of NO₃⁻ consumption, rather than an increase in nitrification. Our results suggest that at least in the short term, disturbance may significantly increase NO₃⁻ flux at the ecosystem level, and that N cycling rates measured in core studies employing mixed soils may not be representative of rates in undisturbed soils.     

Effects of macroelements and nitrogen source on biomass accumulation and withanolide-A production from cell suspension cultures of <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

Withania somnifera is an important medicinal plant that contains withanolides and withaferins, both bioactive compounds. We have tested the effects of macroelements and nitrogen source in W. somnifera cell suspension cultures with the aim of optimizing the production of biomass and withanolide A. The effects of the macroelements NH₄NO_3, KNO_3, CaCl_2, MgSO₄ and KH₂PO₄ at concentrations of 0.0, 0.5, 1.0, 1.5 and 2.0× strength and of the nitrogen source [NH₄ ⁺/NO₃ ⁻ (mM/mM) ratio of: 0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20, and 14.38/37.60 (mM)] in Murashige and Skoog medium were tested for biomass and withanolide A production. The highest accumulation of biomass [147.81 g l⁻¹ fresh weight (FW) and 14.02 g l⁻¹ (dry weight (DW)] was recorded in the medium containing a 0.5× concentration of NH₄NO₃, and the highest production of withanolide A content was recorded in the medium with 2.0× KNO₃ (4.36 mg g⁻¹ DW). The NH₄ ⁺/NO₃ ⁻ ratio also influenced cell growth and withanolide A production, with both parameters being larger when the NO₃ ⁻ concentration was higher than that of NH₄ ⁺. Maximum biomass growth (110.45 g l⁻¹ FW and 9.29 g l⁻¹ DW) was achieved at an NH₄ ⁺/NO₃ ⁻ ratio of 7.19/18.80, while withanolide A production was greatest (3.96 mg g⁻¹ DW) when the NH₄ ⁺/NO₃ ⁻ ratio was 14.38/37.60 mM.     

The effects of gap disturbance on nitrogen cycling and retention in late-successional northern hardwood–hemlock forests

Late-successional forests in the upper Great Lakes region are susceptible to nitrogen (N) saturation and subsequent nitrate (NO₃⁻) leaching loss. Endemic wind disturbances (i.e., treefall gaps) alter tree uptake and soil N dynamics; and, gaps are particular susceptible to NO₃⁻ leaching loss. Inorganic N was measured throughout two snow-free periods in throughfall, forest floor leachates, and mineral soil leachates in gaps (300–2,000 m², 6–9 years old), gap-edges, and closed forest plots in late-successional northern hardwood, hemlock, and northern hardwood–hemlock stands. Differences in forest water inorganic N among gaps, edges, and closed forest plots were consistent across these cover types: NO₃⁻ inputs in throughfall were significantly greater in undisturbed forest plots compared with gaps and edges; forest floor leachate NO₃⁻ was significantly greater in gaps compared to edges and closed forest plots; and soil leachate NO₃⁻ was significantly greater in gaps compared to the closed forest. Significant differences in forest water ammonium and pH were not detected. Compared to suspected N-saturated forests with high soil NO₃⁻ leaching, undisturbed forest plots in these late-successional forests are not losing NO₃⁻ (net annual gain of 2.8 kg ha⁻¹) and are likely not N-saturated. Net annual NO₃⁻ losses were observed in gaps (1.3 kg ha⁻¹) and gap-edges (0.2 kg ha⁻¹), but we suspect these N leaching losses are a result of decreased plant uptake and increased soil N mineralization associated with disturbance, and not N-saturation.     

Nitrate reductase activity and nitrogen compounds in xylem exudate of Juglans nigra seedlings: relation to nitrogen source and supply

Nitrogen (N) limits plant productivity and its uptake and assimilation may be regulated by N source, N availability, and nitrate reductase activity (NRA). Knowledge of how these factors interact to affect N uptake and assimilation processes in woody angiosperms is limited. We fertilized 1-year-old, half-sib black walnut (Juglans nigra L.) seedlings with ammonium (NH₄ ⁺) [as (NH₄)₂SO₄], nitrate (NO₃ ⁻) (as NaNO₃), or a mixed N source (NH₄NO₃) at 0, 800, or 1,600 mg N plant⁻¹ season⁻¹. Two months following final fertilization, growth, in vivo NRA, plant N status, and xylem exudate N composition were assessed. Specific leaf NRA was higher in NO₃ ⁻-fed and NH₄NO₃-fed plants compared to observed responses in NH₄ ⁺-fed seedlings. Regardless of N source, N addition increased the proportion of amino acids (AA) in xylem exudate, inferring greater NRA in roots, which suggests higher energy cost to plants. Root total NRA was 37% higher in NO₃ ⁻-fed than in NH₄ ⁺-fed plants. Exogenous NO₃ ⁻ was assimilated in roots or stored, so no difference was observed in NO₃ ⁻ levels transported in xylem. Black walnut seedling growth and physiology were generally favored by the mixed N source over NO₃ ⁻ or NH₄ ⁺ alone, suggesting NH₄NO₃ is required to maximize productivity in black walnut. Our findings indicate that black walnut seedling responses to N source and level contrast markedly with results noted for woody gymnosperms or herbaceous angiosperms.     

Nitrogen Transformations in Flowpaths Leading from Soils to Streams in Amazon Forest and Pasture

The modification of large areas of tropical forest to agricultural uses has consequences for the movement of inorganic nitrogen (N) from land to water. Various biogeochemical pathways in soils and riparian zones can influence the movement and retention of N within watersheds and affect the quantity exported in streams. We used the concentrations of NO₃ ⁻ and NH₄ ⁺ in different hydrological flowpaths leading from upland soils to streams to investigate inorganic N transformations in adjacent watersheds containing tropical forest and established cattle pasture in the southwestern Brazilian Amazon Basin. High NO₃ ⁻ concentrations in forest soil solution relative to groundwater indicated a large removal of N mostly as NO₃ ⁻ in flowpaths leading from soil to groundwater. Forest groundwater NO₃ ⁻ concentrations were lower than in other Amazon sites where riparian zones have been implicated as important N sinks. Based on water budgets for these watersheds, we estimated that 7.3–10.3 kg N ha⁻¹ y⁻¹ was removed from flowpaths between 20 and 100 cm, and 7.1–10.2 kg N ha⁻¹ y⁻¹ was removed below 100 cm and the top of the groundwater. N removal from vertical flowpaths in forest exceeded previously measured N₂O emissions of 3.0 kg N ha⁻¹ y⁻¹ and estimated emissions of NO of 1.4 kg N ha⁻¹ y⁻¹. Potential fates for this large amount of nitrate removal in forest soils include plant uptake, denitrification, and abiotic N retention. Conversion to pasture shifted the system from dominance by processes producing and consuming NO₃ ⁻ to one dominated by NH₄ ⁺, presumably the product of lower rates of net N mineralization and net nitrification in pasture compared with forest. In pasture, no hydrological flowpaths contained substantial amounts of NO₃ ⁻ and estimated N removal from soil vertical flowpaths was 0.2 kg N ha⁻¹ y⁻¹ below the depth of 100 cm. This contrasts with the extent to which agricultural sources dominate N inputs to groundwater and stream water in many temperate regions. This could change, however, if pasture agriculture in the tropics shifts toward intensive crop cultivation.     

Denitrification of a landfill leachate with high nitrate concentration in an anoxic rotating biological contactor

The denitrification performance of a lab-scale anoxic rotating biological contactor (RBC) using landfill leachate with high nitrate concentration was evaluated. Under a carbon to nitrogen ratio (C/N) of 2, the reactor achieved N-NO₃ ⁻ removal efficiencies above 95% for concentrations up to 100 mg N-NO₃ ⁻ l⁻¹. The highest observed denitrification rate was 55 mg N-NO₃ ⁻ l⁻¹ h⁻¹ (15 g N-NO₃ ⁻ m⁻² d⁻¹) at a nitrate concentration of 560 mg N-NO₃ ⁻ l⁻¹. Although the reactor has revealed a very good performance in terms of denitrification, effluent chemical oxygen demand (COD) concentrations were still high for direct discharge. The results obtained in a subsequent experiment at constant nitrate concentration (220 mg N-NO₃ ⁻ l⁻¹) and lower C/N ratios (1.2 and 1.5) evidenced that the organic matter present in the leachate was non-biodegradable. A phosphorus concentration of 10 mg P-PO₄ ³⁻ l⁻¹ promoted autotrophic denitrification, revealing the importance of phosphorus concentration on biological denitrification processes.     

Abiotic immobilization of nitrate in two soils of relic <Emphasis Type="Italic">Abies pinsapo</Emphasis>-fir forests under Mediterranean climate

Evidence for abiotic immobilization of nitrogen (N) in soil is accumulating, but remains controversial. Identifying the fate of N from atmospheric deposition is important for understanding the N cycle of forest ecosystems. We studied soils of two Abies pinsapo fir forests under Mediterranean climate seasonality in southern Spain—one with low N availability and the other with symptoms of N saturation. We hypothesized that biotic and abiotic immobilization of nitrate (NO₃ ⁻) would be lower in soils under these forests compared to more mesic temperate forests, and that the N saturated stand would have the lowest rates of NO₃ ⁻ immobilization. Live and autoclaved soils were incubated with added ¹⁵NO₃ ⁻ (10 μg N g⁻¹ dry soil; 99% enriched) for 24 h, and the label was recovered as total dissolved-N, NO₃ ⁻, ammonium (NH₄ ⁺), or dissolved organic-N (DON). To evaluate concerns about possible iron interference in analysis of NO₃ ⁻ concentrations, both flow injection analysis (FIA) and ion chromatography (IC) were applied to water extracts, soluble iron was measured in both water and salt extracts, and standard additions of NO₃ ⁻ to salt extracts were analyzed. Good agreement between FIA and IC analysis, low concentrations of soluble Fe, and 100% (±3%) recovery of NO₃ ⁻ standard additions all pointed to absence of an interference problem for NO₃ ⁻ quantification. On average, 85% of the added ¹⁵NO₃ ⁻ label was recovered as ¹⁵NO₃ ⁻, which supports our hypothesis that rates of immobilization were generally low in these soils. A small amount (mean = 0.06 μg N g⁻¹ dry soil) was recovered as ¹⁵NH₄ ⁺ in live soils and none in sterilized soils. Mean recovery as DO¹⁵N ranged from 0.6 to 1.5 μg N g⁻¹ dry soil, with no statistically significant effect of sterilization or soil type, indicating that this was an abiotic process that occurred at similar rates in both soils. These results demonstrate a detectable, but modest rate of abiotic immobilization of NO₃ ⁻ to DON, supporting our first hypothesis. These mineral soils may not have adequate carbon availability to support the regeneration of reducing microsites needed for high rates of NO₃ ⁻ reduction. Our second hypothesis regarding lower expected abiotic immobilization in soils from the N-saturated site was not supported. The rates of N deposition in this region may not be high enough to have swamped the capacity for soil NO₃ ⁻ immobilization, even in the stand showing some symptoms of N saturation. A growing body of evidence suggests that soil abiotic NO₃ ⁻ immobilization is common, but that rates are influenced by a combination of factors, including the presence of plentiful available carbon, reduced minerals in anaerobic microsites and adequate NO₃ ⁻ supply.     

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (<Emphasis Type="Italic">cnorB</Emphasis>) gene abundance and mRNA levels in <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis> inoculated into anoxic soil

The effect of glucose addition (0 and 500 μg C g⁻¹ soil) and nitrate (NO₃) addition (0, 10, 50 and 500 μg NO₃–N g⁻¹ soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB _p (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO₃ addition treatments. Without glucose addition, cnorB _p mRNA levels were higher when 500 μg NO₃–N g⁻¹ soil was added compared with other NO₃ additions. In treatments with glucose added, addition of 50 μg NO₃–N g⁻¹ soil resulted in higher cnorB _p mRNA levels than soil without NO₃ but was not different from the 10 and 500 μg NO₃–N g⁻¹ treatments. cnorB _p abundance in soils without glucose addition was significantly higher in soils with 500 μg NO₃–N g⁻¹ soil compared to lower N-treated soils. Conversely, addition of 500 μg NO₃–N g⁻¹ soil resulted in lower cnorB _p abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 μg glucose-C g⁻¹ soil, and 50 or 500 μg NO₃–N g⁻¹ was higher than all other treatments. There was a positive correlation between cnorB _p abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB _p abundance and mRNA levels than soils without glucose added, however response of cnorB _p abundance and mRNA levels to NO₃ supply depended on carbon availability.     

Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor

High-rate biological conversion of sulfide and nitrate in synthetic wastewater to, respectively, elemental sulfur (S⁰) and nitrogen-containing gas (such as N₂) was achieved in an expanded granular sludge bed (EGSB) reactor. A novel strategy was adopted to first cultivate mature granules using anaerobic sludge as seed sludge in sulfate-laden medium. The cultivated granules were then incubated in sulfide-laden medium to acclimate autotrophic denitrifiers. The incubated granules converted sulfide, nitrate, and acetate simultaneously in the same EGSB reactor to S⁰, N-containing gases and CO₂ at loading rates of 3.0 kg S m⁻³ d⁻¹, 1.45 kg N m⁻³ d⁻¹, and 2.77 kg Ac m⁻¹ d⁻¹, respectively, and was not inhibited by sulfide concentrations up to 800 mg l⁻¹. Effects of the C/N ratio on granule performance were identified. The granules cultivated in the sulfide-laden medium have Pseudomonas spp. and Azoarcus sp. presenting the heterotrophs and autotrophs that co-work in the high-rate EGSB-SDD (simultaneous desulfurization and denitrification) reactor.     

High-Affinity NO− 3-H+ Cotransport in the Fungus Neurospora: Induction and Control by pH and Membrane Voltage

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO⁻ ₃ challenge and to quantify transport activity. The NO⁻ ₃-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO⁻ ₃-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 μm NO⁻ ₃. In the latter, induction showed a latency of 40–80 min and rose in scalar fashion with full transport activity measurable approx. 100 min after first exposure to NO⁻ ₃; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO⁻ ₃ additions which, after induction, resulted in reversible membrane depolarizations of (+)54–85 mV in the presence of 50 μm NO⁻ ₃; and it was suppressed when NH₄ ⁺ was present during the first, inductive exposure to NO⁻ ₃. Voltage clamp measurements carried out immediately before and following NO⁻ ₃ additions showed that the NO⁻ ₃-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages (−400 to +100 mV). Measurements of NO⁻ ₃ uptake using NO⁻ ₃-selective macroelectrodes indicated a charge stoichiometry for NO⁻ ₃ transport of 1(+):1(NO⁻ ₃) with common K _m and J _max values around 25 μm and 75 pmol NO⁻ ₃ cm⁻²sec⁻¹, respectively, and combined measurements of pH _o and [NO⁻ ₃] _o showed a net uptake of approx. 1 H⁺ with each NO⁻ ₃ anion. Analysis of the NO⁻ ₃ current demonstrated a pronounced voltage sensitivity within the normal physiological range between −300 and −100 mV as well as interactions between the kinetic parameters of membrane voltage, pH _o and [NO⁻ ₃] _o . Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pH _o of 6.1, driving the membrane voltage from −350 to −150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO⁻ ₃. By contrast, the same depolarization effected an approx. 20% fall in the K _m for transport as a function in [H⁺] _o . These, and additional results are consistent with a charge-coupling stoichiometry of 2(H⁺) per NO⁻ ₃ anion transported across the membrane, and implicate a carrier cycle in which NO⁻ ₃ binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO⁻ ₃ transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO⁻ ₃ transport; finally, they distinguish metabolite repression of NO⁻ ₃ transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate. Received: 17 March 1997/Revised: 20 June 1997     

Iron interference in the quantification of nitrate in soil extracts and its effect on hypothesized abiotic immobilization of nitrate

Human alteration of the nitrogen cycle has stimulated research on nitrogen cycling in many aquatic and terrestrial ecosystems, where analyses of nitrate (NO₃ ⁻) by standard laboratory methods are common. A recent study by Colman et al. (Biogeochemistry 84:161–169, 2007) identified a potential analytical interference of soluble iron (Fe) with NO₃ ⁻ quantification by standard flow-injection analysis of soil extracts, and suggested that this interference may have led Dail et al. (Biogeochemistry 54:131–146, 2001) to make an erroneous assessment of abiotic nitrate immobilization in prior ¹⁵N pool dilution studies of Harvard Forest soils. In this paper, we reproduce the Fe interference problem systematically and show that it is likely related to dissolved, complexed-Fe interfering with the colorimetric analysis of NO₂ ⁻. We also show how standard additions of NO₃ ⁻ and NO₂ ⁻ to soil extracts at native dissolved Fe concentrations reveal when the Fe interference problem occurs, and permit the assessment of its significance for past, present, and future analyses. We demonstrate low soluble Fe concentrations and good recovery of standard additions of NO₃ ⁻ and NO₂ ⁻ in extracts of sterilized Harvard Forest soils. Hence, we maintain that rapid NO₃ ⁻ immobilization occurred in sterilized samples of the Harvard Forest O horizon in the study by Dail et al. (2001). Furthermore, additional evidence is accumulating in the literature for rapid disappearance of NO₃ ⁻ added to soils, suggesting that our observations were not the result of an isolated analytical artifact. The conditions for NO₃ ⁻ reduction are likely to be highly dependent on microsite properties, both in situ and in the laboratory. The so-called “ferrous wheel hypothesis” (Davidson et al., Glob Chang Biol 9:228–236, 2003) remains an unproven, viable explanation for published observations.     

Nitrogen dynamics and soil nitrate retention in a <Emphasis Type="Italic">Coffea arabica</Emphasis>—<Emphasis Type="Italic">Eucalyptus deglupta</Emphasis> agroforestry system in Southern Costa Rica

Nitrogen fertilization is a key factor for coffee production but creates a risk of water contamination through nitrate (NO₃⁻) leaching in heavily fertilized plantations under high rainfall. The inclusion of fast growing timber trees in these coffee plantations may increase total biomass and reduce nutrient leaching. Potential controls of N loss were measured in an unshaded coffee (Coffea arabica L.) plot and in an adjacent coffee plot shaded with the timber species Eucalyptus deglupta Blume (110 trees ha⁻¹), established on an Acrisol that received 180 kg N ha⁻¹ as ammonium-nitrate and 2,700 mm yr⁻¹ rainfall. Results of the one year study showed that these trees had little effect on the N budget although some N fluxes were modified. Soil N mineralization and nitrification rates in the 0–20 cm soil layer were similar in both systems (≈280 kg N ha⁻¹ yr⁻¹). N export in coffee harvest (2002) was 34 and 25 kg N ha⁻¹ yr⁻¹ in unshaded and shaded coffee, and N accumulation in permanent biomass and litter was 25 and 45 kg N ha⁻¹ yr⁻¹, respectively. The losses in surface runoff (≈0.8 kg mineral N ha⁻¹ yr⁻¹) and N₂O emissions (1.9 kg N ha⁻¹ yr⁻¹) were low in both cases. Lysimeters located at 60, 120, and 200 cm depths in shaded coffee, detected average concentrations of 12.9, 6.1 and 1.2 mg NO₃⁻-N l⁻¹, respectively. Drainage was slightly reduced in the coffee-timber plantation. NO₃⁻ leaching at 200 cm depth was about 27 ± 10 and 16 ± 7 kg N ha⁻¹ yr⁻¹ in unshaded and shaded coffee, respectively. In both plots, very low NO₃⁻ concentrations in soil solution at 200 cm depth (and in groundwater) were apparently due to NO₃⁻ adsorption in the subsoil but the duration of this process is not presently known. In these conventional coffee plantations, fertilization and agroforestry practices must be refined to match plant needs and limit potential NO₃⁻ contamination of subsoil and shallow soil water.     

Production of d-arabitol by a newly isolated Kodamaea ohmeri

A new yeast, isolated from natural osmophilic sources, produces d-arabitol as the main metabolic product from glucose. According to 18S rRNA analysis, the NH-9 strain belongs to the genus Kodamaea. The optimal culture conditions for inducing production of d-arabitol were 37 °C, neutral pH, 220 rpm shaking, and 5% inoculum. The yeast produced 81.2 ± 0.67 g L⁻¹ d-arabitol from 200 g L⁻¹ d-glucose in 72 h with a yield of 0.406 g g⁻¹ glucose and volumetric productivity Q_\textP Q_{\text{P}} of 1.128 g L⁻¹ h⁻¹. Semi-continuous repeated-batch fermentation was performed in shaker-flasks to enhance the process of d-arabitol production by Kodamaea ohmeri NH-9 from d-glucose. Under repeated-batch culture conditions, the highest volumetric productivity was 1.380 g L⁻¹ h⁻¹.