Patterns in the release of gaseous ammonia by terrestrial isopods

Summary In the fall and in early spring P. scaber and O. asellus released gaseous ammonia in the form of more or less regularly spaced bursts. In the spring about twice as much ammonia was released by O. asellus than in the fall. In late spring and summer, however, both species released ammonia in a rhythmic fashion, with a maximum at noon and early in the afternoon, and a minimum early at night. Sometimes a second maximum occurred late at night.In O. asellus the addition of a moist substrate to the reaction chamber shifted the maximum of the release of ammonia from noon to late night and early morning.Fed specimens of P. scaber released only about one-third as much NH₃ as fasting animals and—at least in constant darkness—with a period of much reduced amplitude.It is concluded that the rhythmical release of ammonia is inversely related to the pattern of locomotory activity of these animals. This would implicate mechanisms that regulate either the production or the release of ammonia in such a way that the maximum occurs at a time when the animals' production of energy is at a minimum and when they are protected against loss of water by sitting in their moist retreats.The work at Innsbruck was supported by the Fonds zur Förderung der wissenschaftlichen Forschung of Austria.     

Significance of gaseous NO for ammonia oxidation by Nitrosomonas eutropha

Nitrification by the obligately lithoautotrophic ammonia oxidizer Nitrosomonas eutropha was significantly inhibited when nitric oxide was removed from the culture medium by means of intensive aeration and turbulence. Nearly complete recovery of ammonia oxidation could be achieved by adding 100 ppm NO to the supplied air. Inhibition of ammonia oxidation occurred also upon addition of the NO binding agens 2,3-Dimercapto-1-propane-sulfonic acid (DMPS). Recovery of ammonia oxidation occurred within 3 h in the presence of 100 ppm NO and within 76 h in the absence of externally added NO. In co-cultures of N. eutropha and the NO detoxifying bacterium Pseudomonas PS88, hardly any nitrification was detectable and release of NO was extremely low when the heterotroph was provided with an organic substrate. When cells of Pseudomonas PS88 were added to a mixotrophically nitrifying culture of N. eutropha the release of NO decreased drastically upon the addition and ammonia oxidation ceased. These results confirm for the first time the significance of NO in the course of ammonia oxidation by N. eutropha.     

Ion movement through gramicidin A channels. Interfacial polarization effects on single-channel current measurements.

Gramicidin A single-channel current-voltage characteristics were studied at low permeant ion concentrations and very high applied potentials. The purpose of these experiments was to elucidate the basis for the small, but definite, voltage dependence observed under these circumstances. It was found that this residual voltage dependence is a reflection of interfacial polarization effects, similar to those proposed by Walz et al. (Biophys. J. 9:1150-1159). It will be concluded that there exists an effectively voltage-independent step in the association reaction between a gramicidin A channel and the permeating ion. Some consequences of interfacial polarization effects for the analysis of conductance vs. activity relations will be discussed.     

Design of laboratory continuous-culture equipment for accurate gaseous metabolism measurements

The accuracy of kinetic and stoichiometric data obtained from most laboratory-scale continuous-culture equipment, particularly involving gaseous measurements, may be much lower than many workers realize, despite the use of good quality instruments. For example, errors in specific oxygen uptake measurements (QO(2)) easily can be as high as +/-100%. This article assesses the accuracies of individual instruments and of the overall system in greater detail than has previously been reported and suggestions are made as to how the errors can be reduced to acceptable levels.     

Assimilation of gaseous ammonia and the transport of its products in barley and spinach leaves

The interactions between the assimilation and transport of nitrogenand carbon were investigated in barley and spinach leaves. Bothplants were fumigated with NH₃ (1 mg m^–3 and the contentof amino acids, sucrose and carbon intermediates of amino acidmetabolism were analysed in the leaves, apoplast and phloemsap. The following changes took place in the C- and N-metabolismof barley leaves during 5 h of fumigation with NH₃ (a) The contentsof amino acids, especially glutamine, largely increased andthe contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate,and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvatecontent was accompanied by an increased activity of phosphoenolpyruvatecarboxylase. (c) The altered cytosolic concentrations of aminoacids and sucrose during NH₃ fumigation correlated with similarchanges in the apoplast and phloem sap. The altered percentageof each amino acid relative to the total amino acid concentrationin the cytosol, caused by NH₃ fumigation, is reflected in theapoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrationsin the phloem. Key words: Amino acids, ammonia fumigation, barley leaves, C: N partitioning, phosphoenolpyruvate carboxylase, phloem sap, spinach leaves     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Acquisition and assimilation of gaseous ammonia as revealed by intracellular pH changes in leaves of higher plants

Atmospheric ammonia (NH₃) from various anthropogenic sources has become a serious problem for natural vegetation. Ammonia not only causes changes in plant nitrogen metabolism, but also affects the acid-base balance of plants. Using the pH-sensitive fluorescent dyes pyranine and esculin, cytosolic and vacuolar pH changes were measured in leaves of C₃ and C₄ plants exposed for brief periods to concentrations of NH₃ in air ranging from 1.33 to 8.29 mol NH₃ · mol^-1 gas (0.94–5.86 mg · m^-3). After a lag phase, uptake of NH₃ from air at a rate of 200 nmol NH₃ · m ^{- 2} leaf area · s^{- 1} into leaves of Zea mays L. increased pyranine fluorescence indicating cytosolic alkalinisation. The increase was much larger in the dark than in the light. In illuminated leaves of the C₃ plant Pelargonium zonale L. and the C₄ plants Z. mays and Amaranthus caudatus L., NH₃-dependent cytosolic alkalinisation was particularly pronounced when CO₂ was supplied at very low levels (16 or 20 mol CO₂ · mol^{- 1} gas, containing 210 mmol O₂ · mol^{- 1} gas). An increase in esculin fluorescence, which was smaller than that of pyranine, was indicative of trapping of some of the NH₃ in the vacuoles of leaves of Spinacia oleracea L. and Z. mays. Photosynthesis and transpiration remained unchanged during exposure of illuminated leaves to NH₃, yielding an influx of 200 nmol NH₃ · m^-2 leaf area · s^-1 for up to 30 min, the longest exposure time used. Both CO₂ and O₂ influenced the extent of cytosolic alkalinisation. At 500 mol CO₂ · mol^-1 gas the cytosolic alkalinisation was suppressed more than at 16 or 20 mol CO₂ · mol^-1 gas. The suppressing effect of CO₂ on the NH₃induced alkalinisation was larger in illuminated leaves of the C₄ plants Z. mays and A. caudatus than in leaves of the C₃ plant P. zonale. A reduction of the O₂ concentration from 210 to 10 mmol O₂ · mol ^-1 gas, which inhibits photorespiration, increased the NH₃induced cytosolic alkalinisation in C₃ plants. Suppression by CO₂ or O₂ of the alkaline pH shift caused by the dissolution and protonation of NH₃ in queous leaf compartments, and possibly by the production of organic compounds synthesised from atmospheric NH₃, indicates that NH₃ which enters leaves is rapidly assimilated if photosynthesis or photorespiration provide nitrogen acceptor molecules.This work was supported by the Biotechnology and Biological Sciences Research Council and the Deutsche Forschungsgemein-schaft within the framework of the research of Sonderforschun-gsbreich 251 of the University of Würzburg. We are grateful to Dr. B. Wollenweber (The Royal Veterinary and Agricultural University, Denmark) for discussions.     

Membrane current and noise measurements in voltage-clamped node ofRanvier

Summary In voltage-clamp configurations for nodes ofRanvier the axoplasm resistance functions as a voltage-current converter. In existing configurations this resistance cannot be measured directly. In the present arrangement the electrical resistances of the preparation (axoplasm, membrane and seals) can be measured only from two measurements. This allows us to: 1. calibrate the ionic current under voltage-clamp conditions, and 2. calculate the intensity of the current fluctuations, not arising from the membrane (background noise). The measured axoplasm resistances are considerably higher than the values calculated on the basis of fiber geometry and axoplasm resistivity. The difference is due to the presence of constrictions in the nerve fiber. Membrane current estimation based on geometrical parameters in the presence of wide seals may contain large errors. Variations in the axoplasm resistance for voltage-membrane current conversion were observed within 1.5 hr. In 68% of the fibers this resistance decreased with 30% of the original value. With our current calibration the values for the maximum sodium conductance (at 0 mV membrane potential), maximum potassium conductance and leakage conductance are 49.5×10^–8, 6.66×10^–8 and 1.71×10^–8 S, respectively. The contribution of the different noise sources to the total background noise was calculated at the holding potential. For frequencies below 10³ Hz there is an excellent agreement between measured and calculated noise levels.     

Amplifier spurious input current components in electrode-electrolyte interface impedance measurements

Background  

In Impedance Microbiology, the time during which the measuring equipment is connected to the bipolar cells is rather long, usually between 6 to 24 hrs for microorganisms with duplication times in the order of less than one hour and concentrations ranging from 10¹ to 10⁷ [CFU/ml]. Under these conditions, the electrode-electrolyte interface impedance may show a slow drift of about 2%/hr. By and large, growth curves superimposed on such drift do not stabilize, are less reproducible, and keep on distorting all over the measurement of the temporal reactive or resistive records due to interface changes, in turn originated in bacterial activity. This problem has been found when growth curves were obtained by means of impedance analyzers or with impedance bridges using different types of operational amplifiers.     

On measurements of higher harmonics of transmembrane current

Measurements of higher harmonics of transmembrane current in bilayer lipid membranes from diphytanoyl phosphatidylcholine (DPhPC) in n-decane and n-tetradecane, caused by alternating voltage applied to the membrane, have been conducted. A universal relation between the amplitudes of harmonics was suggested and experimentally checked. This allowed one to calculate the coefficients of expansion of membrane capacity in series with even powers of membrane voltage and to compare the inhomogeneity of membranes from diphytanoyl phosphatidylcholine in n-decane and n-tetradecane with respect to thickness.     

Start-up and the effect of gaseous ammonia additions on a biofilter for the elimination of toluene vapors

Biotechnological techniques, including biofilters and biotrickling filters are increasingly used to treat air polluted with VOCs (Volatile Organic Compounds). In this work, the start-up, the effect of the gaseous ammonia addition on the toluene removal rate, and the problems of the heat accumulation on the performance of a laboratory scale biofilter were studied. The packing material was sterilized peat enriched with a mineral medium and inoculated with an adapted consortium (two yeast and five bacteria). Start-up showed a short adaptation period and an increased toluene elimination capacity (EC) up to a maximum of 190 g/m3/h. This was related to increased CO2 outlet concentration and temperature gradients between the packed bed and the inlet (Tm-Tin). These events were associated with the growth of the microbial population. The biofilter EC decreased thereafter, to attain a steady state of 8 g/m3/h. At this point, gaseous ammonia was added. EC increased up to 80 g/m3/h, with simultaneous increases on the CO2 concentration and (Tm-Tin). Two weeks after the ammonia addition, the new steady state was 30 g/m3/h. In a second ammonia addition, the maximum EC attained was 40 g/m3/h, and the biofilter was in steady state at 25 g/m3/h. Carbon, heat, and water balances were made through 88 d of biofilter operation. Emitted CO2 was about 44.5% of the theoretical value relative to the total toluene oxidation, but accumulated carbon was found as biomass, easily biodegradable material, and carbonates. Heat and water balances showed strong variations depending on EC. For 88 d the total metabolic heat was -181.2 x 10(3) Kcal/m3, and water evaporation was found to be 56.5 kg/m3. Evidence of nitrogen limitation, drying, and heterogeneities were found in this study.     

Urease gene‐containing Archaea dominate autotrophic ammonia oxidation in two acid soils

The metabolic traits of ammonia‐oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA‐based stable isotope probing (SIP) and high‐throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea‐amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water‐amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time‐course incubations indicated that archaeal amoA genes were increasingly labelled by ¹³CO₂ in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the ¹³C‐DNA, and acetylene inhibition suggests that autotrophic growth of urease‐containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a‐associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.     

Possible consequences of containing microplankton for physiological rate measurements

Ten experiments were done in oligotrophic North Pacific waters to examine the effects of small-volume, short-period containment (6–24 h) on plankton and their implications for productivity measurements. Effects of pre-filtering samples were also considered. Chlorophyll α and adenosine triphosphate were measured, but the major emphasis was on changes in the taxonomic composition and abundance of the microplankton.Chlorophyll α measurements indicate the variability of replicate samples increased significantly during incubation; the coefficient of variation of duplicate productivity measurements ranged between 3 % and 40 %. A large fraction of this may be due to increasing heterogeneity of the phytoplankton during incubation.Pre-filtering samples through 202, 102, 35 and 15μm mesh filters removed a small but consistent fraction of phytoplankton (as estimated by chlorophyll α) but a highly variable amount of total biomass (as estimated by ATP). The effect of pre-filtration on primary production was very inconsistent between experiments, varying both in magnitude and direction.The composition of the contained microplankton changed markedly during incubation. Nearly all components decreased in abundance regardless of pre-filtration treatment and some ciliate groups completely disappeared.     

Phase-resolved spectral measurements with several two tryptophan containing proteins

We have used frequency domain fluorescence techniques to resolve the component emission spectra for several two tryptophan containing proteins (e.g., horse liver alcohol dehydrogenase, sperm whale apomyoglobin, yeast 3-phosphoglycerate kinase, apoazurin from Alcaligenes denitricans). We have first performed multifrequency phase/modulation measurements and have found the fluorescence of each of these proteins to be described by a double exponential. Then, using phase-sensitive detection and the algorithm of Gratton and Jameson [Gratton, E., & Jameson, D. M. (1985) Anal. Chem. 57, 1694-1697], we have determined the emission spectrum associated with each decay time for these proteins. We have compared these phase-resolved spectra with the fractional contributions of the component fluorophores determined by selective solute quenching experiments. Reasonably good agreement is seen in most cases, which argues that the individual Trp residues emit independently. In the case of apoazurin, however, a negative amplitude is seen for the phase-resolved spectrum of the short-lifetime component. This pattern is consistent with the occurrence of energy transfer from the internal Trp residue to the surface Trp of this protein. We also present multifrequency lifetime measurements, phase-resolved spectra, and solute quenching data for a few protein-ligand complexes, to illustrate the utility of this approach for the study of changes in the fluorescence of proteins.