Regulation of nitrate metabolism through anion polycompartmentation in plant roots

A new concept illustrated by a corresponding mathematical model of nitrate metabolism regulation is proposed. The model is based on root nitrate compartmentation in several functional pools: storage, metabolic and a mobile pool which is intended for translocation to shoots. Data on nitrate uptake, compartmentation, reduction in intact roots and translocation to shoots were obtained on steady-state wheat seedlings grown at 25 and 12 degrees C in the root zone. The net uptake, influx/efflux ratio, mobile pool size and translocation changed depending on the medium temperature. The oscillations of the net uptake rate, nitrate tissue concentration were revealed and the effect of temperature on these changes was demonstrated. The scheme of regulation is based on the idea that net uptake through nitrate influx/efflux is under the control of the nitrate the mobile pool whose size was dependent on the nitrate translocation into shoots. The mathematical model is represented by a system of ordinary differential equations simplified according to the time hierarchy of reactions. It has a limit cycle at definite values of the parameters. The model postulates the mechanism of a positive feed-back regulation of the transfer of newly absorbed nitrate into translocated pool formed in the root cortex. Theoretical results are verified experimentally.     

Graphite electrodes as electron donors for anaerobic respiration

It has been demonstrated previously that Geobacter species can transfer electrons directly to electrodes. In order to determine whether electrodes could serve as electron donors for microbial respiration, enrichment cultures were established from a sediment inoculum with a potentiostat-poised graphite electrode as the sole electron donor and nitrate as the electron acceptor. Nitrate was reduced to nitrite with the consumption of electrical current. The stoichiometry of electron and nitrate consumption and nitrite accumulation were consistent with the electrode serving as the sole electron donor for nitrate reduction. Analysis of 16 rRNA gene sequences demonstrated that the electrodes supplied with current were specifically enriched in microorganisms with sequences most closely related to the sequences of known Geobacter species. A pure culture of Geobacter metallireducens was shown to reduce nitrate to nitrite with the electrode as the sole electron donor with the expected stoichiometry of electron consumption. Cells attached to the electrode appeared to be responsible for the nitrate reduction. Attached cells of Geobacter sulfurreducens reduced fumarate to succinate with the electrode as an electron donor. These results demonstrate for the first time that electrodes may serve as a direct electron donor for anaerobic respiration. This finding has implications for the harvesting of electricity from anaerobic sediments and the bioremediation of oxidized contaminants.     

Reduction of nitrate and nitrite in a cyclically operated continuous biological reactor

Biological reduction of nitrate and nitrite was studied with a continuously operated cyclic reactor. The medium was fed to the reactor during the first phase of the cycle, and the effluent was drawn from the reactor during the third phase of the cycle; reaction occurred throughout the cycle. The process was described mathematically based on kinetic expressions revealed in an independent study. The model equations were subjected to detailed analysis with numerical codes based on the bifurcation theory for forced systems. The analysis has shown that in the operating parameter space there are extensive regions where the system can reach up to three different periodic states. The results of this analysis are shown in the form of two-dimensional operating diagrams. Numerical results have also shown that under certain operating conditions nitrate can be completely eliminated, while nitrite remains practically untreated. An experimental unit was designed, constructed, and used in experiments with a strain of Pseudomonas denitrificans [American Type Culture Collection (ATCC) 13867] under different operating conditions. The experimental results confirmed the theoretical predictions both qualitatively and quantitatively. Conditions under which complete reduction of both nitrate and nitrite is achieved, were found and experimentally verified. The results of this study suggest a methodology for analysis and design of cyclically operated bioreactors employed in denitrification of wastewaters. (c) 1995 John Wiley & Sons, Inc.     

Modelling the interactions between water and nutrient uptake and root growth

A model of three-dimensional root growth has been developed to simulate the interactions between root systems, water and nitrate in the rooting environment. This interactive behaviour was achieved by using an external-supply/internal-demand regulation system for the allocation of endogenous plant resources. Data from pot experiments on lupins heterogeneously supplied with nitrate were used to test and parameterise the model for future simulation work. The model reproduced the experimental results well (R ² = 0.98), simulating both the root proliferation and enhanced nitrate uptake responses of the lupins to differential nitrate supply. These results support the use of the supply/demand regulation system for modelling nitrate uptake by lupins. Further simulation work investigated the local uptake response of lupins when nitrate was supplied to a decreasing fraction of the root system. The model predicted that the nitrate uptake activity of lupin roots will increase as the fraction of root system with access to nitrate decreases, but is limited to an increase of around twice that of a uniformly supplied control. This work is the first example of a modelled root system responding plastically to external nutrient supply. This model will have a broad range of applications in the study of the interactions between root systems and their spatially and temporally heterogeneous environment.     

Reduction of Nitrate in Agricultural Soils by Bio-electrokinetics

High nitrate concentration in surface soils is a serious concern for the agricultural industry throughout the world. Nitrate reduction can be achieved by chemical or biological processes; however, these processes are difficult to achieve in-situ because of the low permeability of clays. This study evaluates bio-electrokinetic processes for nitrate treatment in low-permeability soils. The concept is based on using iron electrodes to generate an electric field and a reducing environment in the soil to facilitate nitrate reduction by existing bacteria. Experiments were conducted using starch as an additive for microbial activity in the anolyte. Three sets of experiments were conducted under 0.5, 1.0, and 2.0 V cm^?1 voltage gradient, and using starch as an anolyte for enhancing existing microbial activity in the soil. Initial nitrate concentration in the soil (agricultural soil collected from Jinju, Korea) was between 782 and 800 mg kg^?1. Removal of 100% nitrate was achieved in the soil under 0.5 and 1.0 V cm^?1 due to the combined effect of biological and iron reduction. A control experiment with iron electrodes, but without starch, was not as effective. Ammonium was also effectively removed by the combined action of starch and iron under 0.5 and 1.0 V cm^?1. The role of starch with iron on the nitrate and ammonium removal process is evaluated along with the role of transport by electro-osmosis and electro-migration. The bacterial action on denitrification and nitrification is assessed and the relationship between pH and the efficiency of nitrate reduction in the bio-EK systems is evaluated.     

A biofilm model to understand the onset of sulfate reduction in denitrifying membrane biofilm reactors

This work presents a multispecies biofilm model that describes the co‐existence of nitrate‐ and sulfate‐reducing bacteria in the H₂‐based membrane biofilm reactor (MBfR). The new model adapts the framework of a biofilm model for simultaneous nitrate and perchlorate removal by considering the unique metabolic and physiological characteristics of autotrophic sulfate‐reducing bacteria that use H₂ as their electron donor. To evaluate the model, the simulated effluent H₂, UAP (substrate‐utilization‐associated products), and BAP (biomass‐associated products) concentrations are compared to experimental results, and the simulated biomass distributions are compared to real‐time quantitative polymerase chain reaction (qPCR) data in the experiments for parameter optimization. Model outputs and experimental results match for all major trends and explain when sulfate reduction does or does not occur in parallel with denitrification. The onset of sulfate reduction occurs only when the nitrate concentration at the fiber's outer surface is low enough so that the growth rate of the denitrifying bacteria is equal to that of the sulfate‐reducing bacteria. An example shows how to use the model to design an MBfR that achieves satisfactory nitrate reduction, but suppresses sulfate reduction. Biotechnol. Bioeng. 2013; 110: 763–772. © 2012 Wiley Periodicals, Inc.     

Sensory gating and its modulation by cannabinoids: electrophysiological, computational and mathematical analysis

Gating of sensory information can be assessed using an auditory conditioning-test paradigm which measures the reduction in the auditory evoked response to a test stimulus following an initial conditioning stimulus. Recording brainwaves from specific areas of the brain using multiple electrodes is helpful in the study of the neurobiology of sensory gating. In this paper, we use such technology to investigate the role of cannabinoids in sensory gating in the CA3 region of the rat hippocampus. Our experimental results show that application of the exogenous cannabinoid agonist WIN55,212-2 can abolish sensory gating. We have developed a phenomenological model of cannabinoid dynamics incorporated within a spiking neural network model of CA3 with synaptically interacting pyramidal and basket cells. Direct numerical simulations of this model suggest that the basic mechanism for this effect can be traced to the suppression of inhibition of slow GABA_B synapses. Furthermore, by working with a simpler mathematical firing rate model we are able to show the robustness of this mechanism for the abolition of sensory gating.     

Material recycling in a closed aquatic ecosystem. I. Nitrogen transformation cycle and preferential utilization of ammonium to nitrate by phytoplankton as an optimal control problem

A mathematical model of the nitrogen transformation cycle in an aquatic environment is studied. Using Pontryagin's maximum principle, a preferential utilization of ammonium to nitrate by phytoplankton is explained and verified by experimental data. A multiparameter bifurcation is given. The model was found to have four types of equilibrium sets. It is shown that a Hopf bifurcation may occur.     

Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture

The performance of Haematococcus pluvialis in continuous photoautotrophic culture has been analyzed, especially from the viewpoint of astaxanthin production. To this end, chemostat cultures of Haematococcus pluvialis were carried out at constant light irradiance, 1,220 microE/m2.s, and dilution rate, 0.9/d, but varying the nitrate concentration in the feed medium reaching the reactor, from 1.7 to 20.7 mM. Both growth and biomass composition were affected by the nitrate supply. With saturating nitrate, the biomass productivity was high, 1.2 g/L.d, but astaxanthin accumulation did not take place, the C/N ratio of the biomass being 5.7. Under moderate nitrate limitation, biomass productivity was decreased, as also did biomass concentration at steady state, whereas accumulation of astaxanthin developed and the C/N ratio of the biomass increased markedly. Astaxanthin accumulation took place in cells growing and dividing actively, and its extent was enhanced in response to the limitation in nitrate availability, with a recorded maximum for astaxanthin cellular level of 0.8% of dry biomass and of 5.6 mg/L.d for astaxanthin productivity. The viability of a significant continued generation of astaxanthin-rich H. pluvialis cells becomes thus demonstrated, as also does the continuous culture option as an alternative to current procedures for the production of astaxanthin using this microalga. The intensive variable controlling the behavior of the system has been identified as the specific nitrate input, and a mathematical model developed that links growth rate with both irradiance and specific nitrate input. Moreover, a second model for astaxanthin accumulation, also as a function of irradiance and specific nitrate input, was derived. The latter model takes into account that accumulation of astaxanthin is only partially linked to growth, being besides inhibited by excess nitrate. Simulations performed fit experimental data and emphasize the contention that astaxanthin can be efficiently produced under continuous mode by adjustment of the specific nitrate input, predicting even higher values for astaxanthin productivity. The developed models represent a powerful tool for management of such an astaxanthin-generating continuous process, and could allow the development of improved systems for the production of astaxanthin-rich Haematococcus cells.     

Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K_m values for nitrate and nitrite reduction were 15 μM or less for each isolate. The K_m value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from ¹³NO₃⁻ by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.     

Autotrophic nitrite removal in the cathode of microbial fuel cells

Nitrification to nitrite (nitritation process) followed by reduction to dinitrogen gas decreases the energy demand and the carbon requirements of the overall process of nitrogen removal. This work studies autotrophic nitrite removal in the cathode of microbial fuel cells (MFCs). Special attention was paid to determining whether nitrite is used as the electron acceptor by exoelectrogenic bacteria (biologic reaction) or by graphite electrodes (abiotic reaction). The results demonstrated that, after a nitrate pulse at the cathode, nitrite was initially accumulated; subsequently, nitrite was removed. Nitrite and nitrate can be used interchangeably as an electron acceptor by exoelectrogenic bacteria for nitrogen reduction from wastewater while producing bioelectricity. However, if oxygen is present in the cathode chamber, nitrite is oxidised via biological or electrochemical processes. The identification of a dominant bacterial member similar to Oligotropha carboxidovorans confirms that autotrophic denitrification is the main metabolism mechanism in the cathode of an MFC.     

Effect of nitrate on acetylene reduction activity and carbohydrate composition of Phaseolus vulgaris nodules

When Phaseolus vulgaris L. cv. Kentucky Wonder plants were supplied with various levels of nitrate for 34 days, nodule weight (plant)⁻¹, acetylene reduction activity (g nodule)⁻¹, and sugar concentration in nodules were depressed >60% (7.5 m M nitrate vs nil nitrate). Starch concentration in nodules was more than double the sugar concentration and declined only slightly in response to nitrate level. At the highest level of nitrate, sugar concentration in nodules was 50% greater than that in roots and nodule starch was about 6-fold greater than root starch on a fresh weight basis. When plants were grown with 1 m M nitrate and then supplied with 12 m M nitrate for 7 days, the rapid decline in acetylene reduction activity coincided with a decline in sucrose concentration. However, glucose and fructose concentrations declined only after the largest decrease in acetylene reduction had occurred, and the quantitative decrease in glucose and fructose in nodules was small relative to sucrose. Other results showed that the magnitude of the effect of nitrate on some nodule carbohydrate compounds depends on Rhizobium phaseoli strain and on whether plants were grown with or without nitrate prior to experimental treatments. Some of the results are consistent with the carbohydrate-deprivation hypothesis for inhibition of legume nodules by nitrate. However, there are several complications involved in the interpretation of results of this type, and other possible explanations for the results are suggested.     

Quantification of denitrification by strain T1 during anaerobic degradation of toluene

Summary Strain T1 is a denitrifying bacterium that is capable of toluene degradation under anaerobic conditions. During anaerobic growth on toluene, the specific growth rate of strain T1 was 0.14 h^–1. Nitrite accumulated in the medium stoichiometrically with the depletion of nitrate. When nitrate was nearly depleted from the medium nitrite reduction and dinitrogen formation began. A non-kinetic model was formulated that was based on a hypothesis of non-simultaneous nitrate and nitrite reduction, independent of the concentrations of nitrate and nitrite. The model was verified experimentally over a wide range of conditions that included nitrate and nitrite limitation, toluene limitation, and various ratios of nitrate to nitrite. The model and its experimental verification demonstrated that strain T1 reduces nitrate and nitrite non-simultaneously, even if nitrite is initially present in the medium in addition to nitrate. Offprint requests to: L. Y. Young     

Analysis of nitrate in food extracts using a thermostable formate linked nitrate reductase enzyme system

Summary A method for reduction of nitrate to nitrite for determination of nitrate is described, using a thermostable formate linked nitrate reductase enzyme system (FLNR). The reduction of nitrate to nitrite was found to be quantitative in water and in various food samples containing nitrate. The method is suggested as an alternative for the cadmium reduction method.     

Dissimilatory nitrate reduction by Pseudomonas alcaliphila with an electrode as the sole electron donor

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were considered two alternative pathways of dissimilatory nitrate reduction. In this study, we firstly reported that both denitrification and DNRA occurred in Pseudomonas alcaliphila strain MBR with an electrode as the sole electron donor in a double chamber bio‐electrochemical system (BES). The initial concentration of nitrate appeared as a factor determining the type of nitrate reduction with electrode as the sole electron donor at the same potential (?500 mV). As the initial concentration of nitrate increased, the fraction of nitrate reduced through denitrification also increased. While nitrite (1.38 ± 0.04 mM) was used as electron acceptor instead of nitrate, the electrons recovery via DNRA and denitrification were 43.06 ± 1.02% and 50.51 ± 1.37%, respectively. The electrochemical activities and surface topography of the working electrode catalyzed by strain MBR were evaluated by cyclic voltammetry and scanning electron microscopy. The results suggested that cells of strain MBR were adhered to the electrode, playing the role of electron transfer media for nitrate and nitrite reduction. Thus, for the first time, the results that DNRA and denitrification occurred simultaneously were confirmed by powering the strain with electricity. The study further expanded the range of metabolic reactions and had potential value for the recognization of dissimilatory nitrate reduction in various ecosystems. Biotechnol. Bioeng. 2012; 109: 2904–2910. © 2012 Wiley Periodicals, Inc.     

A model on liquid penetration into soft material with application to needle-free jet injection

A mathematical model has been presented for a high speed liquid jet penetration into soft solid by a needle-free injection system. The model consists of a cylindrical column formed by the initial jet penetration and an expansion sphere due to continuous deposition of the liquid. By solving the equations of energy conservation and volume conservation, the penetration depth and the radius of the expansion sphere can be predicted. As an example, the calculation results were presented for a typical needle-free injection system into which a silicon rubber was injected into. The calculation results were compared with the experimental results.     

Competition between nitrate and nitrite reduction in denitrification by Pseudomonas fluorescens

A pure culture of Pseudomonas fluorescens was used as a model system to study the kinetics of denitrification. An exponentially growing culture was harvested and resuspended in an anoxic acetate solution buffered with K/Na phosphate at pH values of 6.6, 7.0, 7.4, and 7.8. The temperature was kept at 28 degrees C in all assays. Nitrate pulses of approximately 0.2 mg N/L caused nitrite to accumulate due to a faster rate of nitrate reduction over nitrite reduction. The rate of nitrate reduction was observed to depend on its concentration as predicted by the Michaelis-Menten equation. At nonlimiting nitrate concentrations, nitrite reduction was described by the same equation. Otherwise, nitrite reduction also depended on nitrate concentration. Consequently, nitrate and nitrite reductions compete with each other for the oxidation of common electron donors. A kinetic model for nitrate competitive inhibition of nitrite reduction is proposed. The model was used to interpret the nitrate and nitrite profiles observed at the four pH values: the optimum pH value was 7.0 in both cases; the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(3) ) = 0.04 mg N/L); the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(2) ) = 0.06 mg N/L), but it decreased sharply for the pH value of 7.8. Although the ratio between the two maximum reduction rates (V(max NO(2) )/V(max NO(3) )) is constant, nitrite accumulation depends on the medium pH value. Therefore, the regulation mechanism that shifts the electron flow between the two terminal reductases is readily reversible and does not change their relative maximum reduction rates. (c) 1995 John Wiley & Sons, Inc.