Ion-molecule condensation reactions: A mechanism for organic synthesis in ionized reducing atmospheres

The CH₃ ⁺ ion, formed in ionized methane, undergoes consecutive eliminative condensation reactions with methane to form the carbonium ions C₂H₅ ⁺, i-C₃H₇ ⁺ and t-C₄H₉ ⁺. AtT<500°K, \(N_{CH_4 } \) ?10¹⁶ cm^?3 these ions react with NH₃ in competitive condensation-H⁺ transfer reactions, e.g. $$\begin{gathered} C_2 H_5 ^ + + NH_3 \xrightarrow{M} C_2 H_5 NH_3 ^ + \hfill \\ - - - \to NH_4 ^ + + C_2 H_4 \hfill \\ \end{gathered} $$ At particle densities of \(N_{CH_4 } \) <10¹⁶ cm^?3 proton transfer is the only significant reaction channel. At \(N_{CH_4 } \) >10¹⁷ cm^?3 condensation constitutes 5–20% of the overall reactions. The product of the condensation reaction further associates with CO₂ to form C₂H₅NH₃ ⁺·CO₂; the atomic composition of this cluster ion is identical with the protonated amino acid alanine. The carbonium ions i-C₃H₇ ⁺ and t-C₄H₉ ⁺ condense also with HCN to yield protonated isocyanides. HCNH⁺ also appears to condense with HCN atT>570°K, and form cluster ions with HCN at lower temperatures. The rate constants of the condensation reactions vary with temperature and pressure in a complex manner. Under conditions similar to those on Titan at an altitude of 100 km (T=100–150°K, \(N_{CH_4 } \) ≈10¹⁸ cm^?3), with a methane atmosphere containing 1% H₂ and traces of NH₃ and H₂O, ion-molecule condensation reactions followed by H⁺ transfer are expected to lead to the atmospheric synthesis of C₂H₆, C₃H₈, CH₃OH, C₂H₅OH and the terminal ions NH₄ ⁺, CH₃NH₃ ⁺ and C₂H₅NH₃ ⁺. At higher temperatures (250°K<T<400°K), the synthesis of i-C₄H₁₀, i-C₃H₇OH and t-C₄H₉OH and of the ions i-C₃H₇NH₃ ⁺ and t-C₄H₉NH₃ ⁺ is also expected. Electron recombination of the terminal ions may yield amines, imines and nitriles. Cycles of protonation and dissociative recombination of the alkanes and alcohols produced in condensation reactions will also produce unsaturated hydrocarbons, ketones and aldehydes in the ionized atmosphere.     

Photochemistry of NH3, CH4 and PH3. Possible applications to the Jovian planets

Photolysis of NH₃ at 185 nm in the presence of a two-fold excess of CH₄ results in the loss of about 0.25 mole of CH₄ per mole of NH₃ decomposed (ΔCH₄/ΔNH₃). The loss arises from the abstraction of hydrogen atoms from CH₄ by photolytically generated hot hydrogen atoms, the presence of which is established by the constancy of ΔCH₄/ΔNH₃ between 298 and 156 K and by the quenching of the abstraction reaction when either H₂ or SF₆ is added. From the latter result, it can be concluded that NH₃ photolysis in the H₂-abundant atmosphere of Jupiter is not responsible for the presence of the carbon compounds observed there such as ethane, acetylene, and hydrogen cyanide, but may have had a role in the early atmosphere of Titan. Photolysis of PH₃ with a 206 nm light source gives P₂H₄, which in turn is converted to a red-brown solid (P₄?). The course of the photolysis is not changed appreciably when the temperature is lowered to 157 K except that the concentration of P₂H₄ increases. The presence of H₂ has no effect on the P₂H₄ yield. Photolysis of 9∶1 NH₃∶PH₃ gives a rate of decomposition of PH₃ that is comparable with that observed by the direct photolysis of PH₃. Comparable amounts of P₂H₄ and the red-brown solid are also observed. The mechanisms of these photochemical reactions together with their implications to the atmospheric chemistry of Jupiter are discussed. The structures of the compounds responsible for the wide array of colorse.g., brown, red and white, observed in the atmosphere of Jupiter have been the subject of extensive speculation. One theory suggests that these colors are due to organic materials formed by the action of either solar ultraviolet light or electric discharges on mixtures of CH₄, NH₃ and NH₄HS in the Jovian atmosphere (Ponnamperuma, 1976; Khareet al., 1978). An alternative hypothesis is that the colors are due to inorganic compounds resulting from the photolysis of NH₄HS and PH₃ (Lewis and Prinn, 1970; Prinn and Lewis, 1975). In this paper we will summarize our experiments which were designed to test some of these hypotheses.     

Carbon and energy yields in prebiotic syntheses using atmospheres containing CH4, CO and CO2

Yields based on carbon are usually reported in prebiotic experiments, while energy yields (moles cal^–1) are more useful in estimating the yields of products that would have been obtained from the primitive atmosphere of the earth. Energy yields for the synthesis of HCN and H₂CO from a spark discharge were determined for various mixtures of CH₄, CO, CO₂, H₂, H₂O, N₂ and NH₃. The maximum yields of HCN and H₂CO from CH₄, CO, and CO₂ as carbon sources are about 4×10^–8 moles cal^–1.     

Plasma membrane H+-ATPase in sorghum roots as affected by potassium deficiency and nitrogen sources

We studied the influence of inorganic nitrogen sources (NO₃ ^? or NH₄ ⁺) and potassium deficiency on expression and activity of plasma membrane (PM) H⁺-ATPase in sorghum roots. After 15 d of cultivation at 0.2 mM K⁺, the plants were transferred to solutions lacking K⁺ for 2 d. Then, K⁺ depletion assays were performed in the presence or absence of vanadate. Further, PMs from K⁺-starved roots were extracted and used for the kinetic characterization of ATP hydrolytic activity and the immunodetection of PM H⁺-ATPase. Two major genes coding PM H⁺-ATPase (SBA1 and SBA2) were analyzed by real-time PCR. PM H⁺-ATPase exhibited a higher V_max and K_m in NH₄ ⁺-fed roots compared with NO₃ ^? -fed roots. The optimum pH of the enzyme was slightly lower in NO₃ ^? -fed roots than in NH₄ ⁺-fed roots. The vanadate sensitivity was similar. The expressions of SBA1 and SBA2 increased in roots grown under NH₄ ⁺. Concomitantly, an increased content of the enzyme in PM was observed. The initial rate of K⁺ uptake did not differ between plants grown with NO₃ ^? or NH₄ ⁺, but it was significantly reduced by vanadate in NH₄ ⁺-grown plants.     

Mechanism for the Coupled Photochemistry of Ammonia and Acetylene: Implications for Giant Planets,Comets and Interstellar Organic Synthesis

Laboratory studies provide a fundamental understanding of photochemical processes in planetary atmospheres. Photochemical reactions taking place on giant planets like Jupiter and possibly comets and the interstellar medium are the subject of this research. Reaction pathways are proposed for the coupled photochemistry of NH₃ (ammonia) and C₂H₂ (acetylene) within the context Jupiter’s atmosphere. We then extend the discussion to the Great Red Spot, Extra-Solar Giant Planets, Comets and Interstellar Organic Synthesis. Reaction rates in the form of quantum yields were measured for the decomposition of reactants and the formation of products and stable intermediates: HCN (hydrogen cyanide), CH₃CN (acetonitrile), CH₃CH = N-N = CHCH₃ (acetaldazine), CH₃CH = N-NH₂ (acetaldehyde hydrazone), C₂H₅NH₂ (ethylamine), CH₃NH₂ (methylamine) and C₂H₄ (ethene) in the photolysis of NH₃/C₂H₂ mixtures. Some of these compounds, formed in our investigation of pathways for HCN synthesis, were not encountered previously in observational, theoretical or laboratory photochemical studies. The quantum yields obtained allowed for the formulation of a reaction mechanism that attempts to explain the observed results under varying experimental conditions. In general, the results of this work are consistent with the initial observations of Ferris and Ishikawa (1988). However, their proposed reaction pathway which centers on the photolysis of CH₃CH = N-N = CHCH₃ does not explain all of the results obtained in this study. The formation of CH₃CH = N-N = CHCH₃ by a radical combination reaction of CH₃CH = N? was shown in this work to be inconsistent with other experiments where the CH₃CH = N? radical is thought to form but where no CH₃CH = N-N = CHCH₃ was detected. The importance of the role of H atom abstraction reactions was demonstrated and an alternative pathway for CH₃CH = N-N = CHCH₃ formation involving nucleophilic reaction between N₂H₄ and CH₃CH = NH is advanced.     

Real time monitoring of a biogas digester with gas chromatography, near-infrared spectroscopy, and membrane-inlet mass spectrometry

Four methods of monitoring the anaerobic digestion process were studied at pilot scale. The methods employed were Micro Gas Chromatography (μ-GC) and Membrane Inlet Mass Spectrometry (MIMS) for measurements in the gas phase, Near Infrared Spectroscopy (NIRS) and pH in the liquid phase. Micro Gas Chromatography accurately measured H₂, CH₄, H₂S, N₂ and O₂ in the headspace whereas the MIMS accurately measured CH₄, CO₂, H₂S, reduced organic sulfur compounds and p-cresol, also in the headspace. In the liquid phase, NIRS was found to be suitable for estimating the concentrations of acetate, propionate and total volatile fatty acids (VFA) but the error of prediction was too large for accurate quantification. Both the μ-GC and NIRS were low maintenance methods whereas the MIMS required frequent cleaning and background measurements.     

Protein modeling and molecular dynamics simulation of the two novel surfactant proteins SP-G and SP-H

The prospects of a control for a novel gallium nitride pseudo-halide vapor phase epitaxy (PHVPE) with HCN were thoroughly analyzed for hydrocarbons–NH₃–Ga gas phase on the basis of quantum chemical investigation with DFT (B3LYP, B3LYP with D3 empirical correction on dispersion interaction) and ab-initio (CASSCF, coupled clusters, and multireference configuration interaction including MRCI+Q) methods. The computational screening of reactions for different hydrocarbons (CH₄, C₂H₆, C₃H₈, C₂H₄, and C₂H₂) as readily available carbon precursors for HCN formation, potential chemical transport agents, and for controlled carbon doping of deposited GaN was carried out with the B3LYP method in conjunction with basis sets up to aug-cc-pVTZ. The gas phase intermediates for the reactions in the Ga-hydrocarbon systems were predicted at different theory levels. The located π-complexes Ga…C₂H₂ and Ga…C₂H₄ were studied to determine a probable catalytic activity in reactions with NH₃. A limited influence of the carbon-containing atmosphere was exhibited for the carbon doping of GaN crystal in the conventional GaN chemical vapor deposition (CVD) process with hydrocarbons injected in the gas phase. Our results provide a basis for experimental studies of GaN crystal growth with C₂H₄ and C₂H₂ as auxiliary carbon reagents for the Ga-NH₃ and Ga-C-NH₃ CVD systems and prerequisites for reactor design to enhance and control the PHVPE process through the HCN synthesis.     

Mineral nutrition and in vitro growth of Gerbera hybrida (Asteraceae)

The effects of mineral nutrient were examined on in vitro growth of Gerbera hybrida (G. jamesonii?×?G. viridifolia), specifically Gerbera hybrida cv. Pasadena. Four types of experiments were conducted to quantify the effects of mineral nutrients on four in vitro growth responses (quality, shoot number, leaf number, and shoot height) of gerbera and included groups of mineral nutrients (macros/mesos, micros, and Fe), individual salts (CuSO₄·5H₂O, MnSO₄·4H₂O, ZnSO₄·7H₂O, and Fe/EDTA), and the specific ions NO₃ ^?, NH₄ ⁺, and K⁺. Experiments included mixture-amount designs that are essential for separating the effects of proportion and concentration. Highly significant effects were observed in all experiments, but the mineral nutrients with the largest effects varied among the four growth responses. For example, leaf number was strongly affected by the macronutrient group in one experiment and by NH₄ ⁺ and K⁺, which were in the macronutrient group, in the NO₃ ^?/NH₄ ⁺/K⁺ ion-specific experiment, whereas quality was strongly affected by the micronutrients ZnSO₄ and Fe/EDTA. Because mineral nutrient effects varied significantly with the response measured, defining an appropriate formulation requires a clear definition of “optimal” growth.     

Kinetics investigation of the hydrogen abstraction reaction between CH<Subscript>3</Subscript>SS and CN radicals

The reaction mechanisms and rates for the H abstraction reactions between CH₃SS and CN radicals in the gas phase were investigated with density functional theory (DFT) methods. The geometries, harmonic vibrational frequencies, and energies of all stationary points were obtained at B3PW91/6-311G(d,p) level of theory. Relationships between the reactants, intermediates, transition states and products were confirmed, with the frequency and the intrinsic reaction coordinate (IRC) analysis at the same theoretical level. High accurate energy information was provided by the G3(MP2) method combined with the standard statistical thermodynamics. Gibbs free energies at 298.15 K for all of the reaction steps were reported, and were used to describe the profile diagrams of the potential energy surface. The rate constants were evaluated with both the classical transition state theory and the canonical variational transition state theory, in which the small-curvature tunneling correction was included. A total number of 9 intermediates (IMs) and 17 transition states (TSs) were obtained. It is shown that IM1 is the most stable intermediate by the largest energy release, and the channel of CH₃SS?+?CN?→?IM3?→?TS10?→?P1(CH₂SS?+?HCN) is the dominant reaction with the lowest energy barrier of 144.7 kJ mol^?1. The fitted Arrhenius expressions of the calculated CVT/SCT rate constants for the rate-determining step of the favorable channel is k =7.73?×?10⁶? T ^1.40exp(?14,423.8/T) s^?1 in the temperature range of 200–2000 K. The apparent activation energy E _a(app.) for the main channel is ?102.5 kJ mol^?1, which is comparable with the G3(MP2) energy barrier of ?91.8 kJ mol^?1 of TS10 (relative to the reactants).     

Structure characterization of a methylated ester biosurfactant produced by a newly isolated <Emphasis Type="Italic">Dietzia cinnamea</Emphasis> KA1

Biosurfactans are amphiphilic compounds synthesized by a wide group of microorganisms and tend to interact with surfaces of different polarities. In the present study we purified and characterized a biosurfactant produced by Dietzia cinnamea KA-1 when cultured by n-hexadecane as sole carbon source. The crude biosurfactant was extracted with ethyl acetate and purified by freezing at–20°C and then silica Gel column chromatography. The purified biosurfactant applied for more characterization using Elemental analysis (CHNS), Fourier Transform Infrared Spectroscopy (FTIR), Mass Spectroscopy (MS) and Nuclear Magnetic Resonance (¹H and ¹³C-NMR) analysis. CHNS analysis showed the presence of C (74.92%) and H (11.63%) but not N or S. Functional groups of OH, CH₂, CH₃, C=O and aliphatic C?O revealed by FTIR analysis. The presence and position of these groups were confirmed by NMR analysis, and molecular mass of biosurfactant calculated using MS analysis. Finally, the product characterized as a methylated ester compound with molecular formula of C₂₁H₄₂O₄. This is the first report of biosurfactant of species D. cinnamea identified as ester, furthermore the ester was found to be in the methylated form.     

Syntheses and structural analyses of chiral rhenium containing amines of the formula (η-C5H5)Re(NO)(PPh3)((CH2)nNRR′) (n = 0, 1)

The reaction of the racemic chiral methyl complex (η⁵-C₅H₅)Re(NO)(PPh₃)(CH₃) (1) with CF₃SO₃H and then NH₂CH₂C₆H₅ gives [(η⁵-C₅H₅)Re(NO)(PPh₃)(NH₂CH₂C₆H₅)]⁺ ([4a-H]⁺; 73%), and deprotonation with t-BuOK affords the amido complex (η⁵-C₅H₅)Re(NO)(PPh₃)(NHCH₂C₆H₅) (76%). Reactions of 1 with Ph₃C⁺ X⁻ and then primary or secondary amines give [(η⁵-C₅H₅)Re(NO)(PPh₃)(CH₂NHRR′)]⁺ X⁻ ([6-H]⁺ X⁻; R/R′/X = a, H/NH₂CH₂C₆H₅/BF₄; a′, H/NH₂CH₂C₆H₅/PF₆; b, H/NH₂CH₂(CH₂)₂CH₃/PF₆; c, H/(S)-NH₂CH(CH₃)C₆H₅/BF₄); d, CH₂CH₃/CH₂CH₃/PF₆; e, CH₂(CH₂)₂CH₃/CH₂(CH₂)₂CH₃/PF₆; f, CH₂C₆H₅/CH₂C₆H₅/PF₆; g, -CH₂(CH₂)₂CH₂-/PF₆; h, -CH₂(CH₂)₃CH₂-/PF₆; i, CH₃/CH₂CH₂OH/PF₆ (62-99%). Deprotonations with t-BuOK afford the amines (η⁵-C₅H₅)Re(NO)(PPh₃)(CH₂NRR′) (6a-i; 99-40%), which are more stable and isolated in analytically pure form when R ≠ H. Enantiopure 1 is used to prepare (R_ReS_C)-[6c-H]⁺, (R_ReS_C)-6c, (S)-[6g-H]⁺, and (S)-6g. The crystal structures of [4a-H]⁺, a previously prepared NH₂CH₂Si(CH₃)₃ analog, [6a′,d,f,h-H]⁺, (R_ReS_C)-6c, and 6f are determined and analyzed in detail, particularly with respect to cation/anion hydrogen bonding and conformation. In contrast to analogous rhenium containing phosphines, 6a-i show poor activities in reactions that are catalyzed by organic amines.     

Study on the photochemical reaction of HCN and its polymer products relating to primary chemical evolution.

The photochemical reaction of HCN at 184.9 nm is studied in the gas phase. (CN)2, H2, CH4, NH3, N2H4, C2H6, and CH3NH2 are identified as gas phase products, and a reaction mechanism is proposed. HCN polymers are also obtained as solid reaction products, and their structure is investigated by Infrared Spectorscopy, UV-Visible Spectroscopy, Mass Spectrometry, and Amino Acid Analysis. The process and nature of the formation of the polymers are discussed.     

Solvatothermal chemistry of organically-templated vanadium fluorides and oxyfluorides

The solvatothermal reactions of V₂O₅, the appropriate organoamine and HF in the temperature range 100-180 °C yielded a series of vanadium fluorides and oxyfluorides. The compounds [NH₄][H₃N(CH₂)₂NH₃][VF₆] (1) and [H₃N(CH₂)₂NH₃][VF₅(H₂O)] (2) contain mononuclear V(III) anions, while [H₃N(CH₂)₂NH₂(CH₂)₂NH₃]₂ [VF₅(H₂O)]₂[VOF₄(H₂O)] (3) exhibits both V(IV) and V(III) mononuclear anions. Both compound 4, [H₃NCH₂(C₆H₄)CH₂NH₃][VOF₄]·H₂O (4·H₂O) and compound 5, [HN(C₂H₄)₃NH][V₂O₂F₆ (H₂O)₂] (5) contain binuclear anions constructed from edge-sharing V(IV) octahedra. In contrast, [H₃N(CH₂)₂NH₂(CH₂)₂NH₃]₂[V₄O₄F₁₄(H₂O)₂], (6) exhibits a tetranuclear unit of edge- and corner-sharing V(IV) octahedra. Compound 7, [H₃N(CH₂)₂NH₂][VF₅], contains chains of corner-sharing {V^IVF₆} octahedra, while [H₂N(C₂H₄)₂NH₂]₃[V₄F₁₇O]·1.5H₂O (8·1.5H₂O) is two-dimensional with a layer of V(III) and V(IV) octahedra in an edge- and corner-sharing arrangement. In the case of [H₃N(CH₂)₂NH₃][V₂O₆] (9), there was no fluoride incorporation, and the anion is a one-dimensional chain of corner-sharing V(V) tetrahedra.     

Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity

The combined effects of arbuscular mycorrhizal fungi (AMF) and low temperature (LT) on cucumber plants were investigated with respect to biomass production, H₂O₂ accumulation, NADPH oxidase, ATPase activity and related gene expression. Mycorrhizal colonization ratio was gradually increased after AMF-inoculation. However, LT significantly decreased mycorrhizal colonization ability and mycorrhizal dependency. Regardless of temperature, the total fresh and dry mass, and root activity of AMF-inoculated plants were significantly higher than that of the non-AMF control. The H₂O₂ accumulation in AMF-inoculated roots was decreased by 42.44 % compared with the control under LT. H₂O₂ predominantly accumulated on the cell walls of apoplast but was hardly detectable in the cytosol or organelles of roots. Again, NADPH oxidase activity involved in H₂O₂ production was significantly reduced by AMF inoculation under LT. AMF-inoculation remarkably increased the activities of P-type H⁺-ATPase, P-Ca²⁺-ATPase, V-type H⁺-ATPase, total ATPase activity, ATP concentration and plasma membrane protein content in the roots under LT. Additionally, ATP concentration and expression of plasma membrane ATPase genes were increased by AMF-inoculation. These results indicate that NADPH oxidase and ATPase might play an important role in AMF-mediated tolerance to chilling stress, thereby maintaining a lower H₂O₂ accumulation in the roots of cucumber.     

Synthesis and characterization of triaminecobalt(III) complexes and acid hydrolysis kinetics of fac-[Codpt(H2O)nX3−n]n+ (X = Cl,Br; n = 0,1,2)

The complexes CodptX₃ and [Codpt(H₂O)X₂]ClO₄ (X = Cl, Br; dpt = dipropylenetriamine = NH(CH₂CH₂CH₂NH₂)₂) have been prepared and characterized. Rate constants (s⁻¹) for aqueous solution at 25 °C and μ = 0.5 M (NaClO₄), for the acid-independent sequential ractions.

have been measured spectrophotometrically. For X = Cl: k₁ ⋍ 2 × 10⁻², k₂ = 1.7 × 10⁻⁴ and k₃ = 4.8 × 10⁻⁶, and for X = Br: k₁ ⋍ 2 × 10⁻², k₂ = 5.25 × 10⁻⁴ and k₃ = 2.5 × 10⁻⁵ The primary equation was found to be acid independent, while the secondary and tertiary aquations were acid-inhibited reactions. For the second step, the rate of the reaction was given by the rate equation

where C_t is the complex concentration in the aqua-and hydroxodihalo species, k′₂ is the rate constant for the acid-dependent pathway and K_a is the equilibrium constant between the hydroxo and aqua complex ions. The activation parameters were evaluated, for X = Cl: ΔH^≠₂ = 106.3 ± 0.4 kJ mol⁻¹ and ΔS^≠₂ = 40.2 ± 1.7 J K⁻¹ mol⁻, and for X = Br: ΔH^≠₂ = 91.6 ± 0.4 kJ mol⁻¹ and ΔS^≠₂ = 0.4 ± 1.7 J K⁻¹ mol⁻¹. The results are discussed and detailed comparisons of the reactivities of these complexes with other haloaminecobalt(III) species are presented.     

Theoretical study on the functionalization of BC2N nanotube with amino groups

Using density functional theory calculations, we investigated properties of a functionalized BC₂N nanotube with NH₃ and five other NH₂-X molecules in which one of the hydrogen atoms of NH₃ is substituted by X = ?CH₃, ?CH₂CH₃, ?COOH, ?CH₂COOH and ?CH₂CN functional groups. It was found that NH₃ can be preferentially adsorbed on top of the boron atom, with adsorption energy of ?12.0 kcal mol^?1. The trend of adsorption-energy change can be correlated with the trend of relative electron-withdrawing or -donating capability of the functional groups. The adsorption energies are calculated to be in the range of ?1.8 to ?14.2 kcal mol^?1, and their relative magnitude order is found as follows: H₂N(CH₂CH₃) > H₂N(CH₃) > NH₃ > H₂N(CH₂COOH) > H₂N(CH₂CN) > H₂N(COOH). Overall, the functionalization of BC₂N nanotube with the amino groups results in little change in its electronic properties. The preservation of electronic properties of BC₂N coupled with the enhancement of solubility renders their chemical modification with either NH₃ or amino functional groups to be a way for the purification of BC₂N nanotubes.     

NH4 + induces antioxidant cellular machinery and provides resistance to salt stress in citrus plants

Key message

NH ₄ ⁺ acts as a mild oxidative stressor, which triggers antioxidant cellular machinery and provide resistance to salinity.

Abstract

NH₄ ⁺ nutrition in Carrizo citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L) plants acts as an inducer of resistance against salinity conditions. NH₄ ⁺ treatment triggers mild chronic stress that primes plant defence responses by stress imprinting and confers protection against subsequent salt stress. In this work, we studied the influence of NH₄ ⁺ nutrition on antioxidant enzymatic activities and metabolites involved in detoxification of reactive oxygen species (ROS) to clarify their involvement in NH₄ ⁺-mediated salt resistance. Our results showed that NH₄ ⁺ nutrition induces in citrus plants high levels of H₂O₂, strongly inhibits superoxide dismutase (SOD) and glutathione reductase (GR) activities, and leads to higher content of oxidised glutathione (GSSG) than in control plants in the absence of salt, thus providing evidence to confirm mild stress induced by NH₄ ⁺ nutrition. However, upon salinity, plants grown with NH₄ ⁺ (N-NH₄ ⁺ plants) showed a reduction of H₂O₂ levels in parallel to an increase of catalase (CAT), SOD, and GR activities compared with the control plants. Moreover, N-NH₄ ⁺ plants were able to keep high levels of reduced glutathione (GSH) upon salinity and were able to induce glutathione-S-transferase (GST) and phospholipid hydroperoxide glutathione peroxidise (PHGPx) mRNA accumulation. Based on this evidence, we confirm that sublethal concentrations of NH₄ ⁺ might act as a mild oxidative stressor, which triggers antioxidant cellular machinery that can provide resistance to subsequent salt stress.     

Factors affecting the determination of total mercury in biological samples by continuous-flow cold vapor atomic absorption spectrophotometry

The acidic reduction of Hg using a continuous-flow analytical system was evaluated. With 25% SnCl₂ as the reductant, characteristic concentrations (sensitivities) of 0.44 μg/L (open cell) and 0.29 μg/L (flow-through cell) were obtained using inorganic Hg²⁺ standards in 1.5% HCl. When CH₃Hg⁺ standards were used, absorption signals were an order of magnitude lower, indicating that Sn(II) is incapable of producing Hg° from organic Hg in this acidic reduction system. Addition of CdCl₂ to the SnCl₂ reductant, as suggested by Magos (1) for the reduction of organomercurials under alkaline conditions, was without beneficial effect. Similarly, combining Sn, with another reducing agent (hydroxylamine hydrochloride), or a strong alkaline solution (40% NaOH), in the reaction coil of the flow-through system did not significantly enhance the Hg absorption signal for either inorganic or organic Hg. Recovery of Hg from spiked liver homogenates digested at 70–80°C using a HNO₃/H₂SO₄/HCl procedure and stabilized with 0.5 mM K₂Cr₂O₇ was >85%, using either inorganic Hg²⁺ or CH₃Hg⁺, indicating that this digestion procedure successfully breaks the C-Hg bond to form readily reducible Hg species. Usingl-cysteine to stabilize standards of inorganic Hg²⁺ in HCl caused significant depressions of the Hg absorption signal atl-cysteine concentrations >0.001% (≈0.5 mM); 0.1%l-cysteine caused total suppression of the Hg signal. These results indicate that: (1) acidic reduction of Hg by Sn in this continuous-flow system requires breakdown of organomercurials prior to analysis; (2) tissue digestion using HNO₃/H₂SO₄/HCl followed by the addition of K₂Cr₂O₇ to stabilize Hg²⁺ achieves this breakdown and allows good recovery of total Hg; and (3) use ofl-cysteine to complex and prevent losses of Hg should be avoided in systems using acidic reduction of Hg. Concentrations of endogenous tissue sulfhydryls are generally lower than those associated with depressed absorbance signals during the acidic reduction of Hg.     

Low-Concentration Kinetics of Atmospheric CH4 Oxidation in Soil and Mechanism of NH4+ Inhibition

NH₄⁺ inhibition kinetics for CH₄ oxidation were examined at near-atmospheric CH₄ concentrations in three upland forest soils. Whether NH₄⁺-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH₄⁺ were very low in the three soils, desorption of endogenous NH₄⁺ was not a significant factor in this study. The K_m(app) values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH₄ concentrations of ≤15 μl liter⁻¹, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the K_m(app) values in the temperate soils increased in the presence of NH₄⁺ salts, whereas the V_max(app) values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH₄⁺ salts. Compared to the corresponding K⁺ salt controls, the K_m(app) values for NH₄⁺ salts increased substantially, whereas the V_max(app) values remained virtually unchanged, indicating that NH₄⁺ acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH₄ concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH₄⁺ and K⁺ salts, and the slope of the increase was not affected by the addition of NH₄⁺. Hence, the increase in inhibition resulted from an NH₄⁺-independent mechanism. These results show that NH₄⁺ inhibition of atmospheric CH₄ oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH₄⁺.Atmospheric CH₄ contributes substantially to the greenhouse effect, and the concentration of atmospheric CH₄ has increased dramatically in the past century because of human activity associated with agriculture, land use changes, and industry (34, 35). Bacterial oxidation of atmospheric CH₄ in well-drained soils is an important regulator of atmospheric CH₄ concentration, yet the organisms responsible remain unidentified and the physiology of the process is poorly understood (9, 35, 36). Although soil CH₄ consumption is inhibited by a wide variety of anthropogenic disturbances, such as agriculture, N deposition, and forestry (12, 17, 22, 23, 32, 43, 44), predictable inhibition patterns have failed to emerge, which has made it difficult to predict the effects of disturbance on soil CH₄ flux in various ecosystems. The most commonly reported disturbance effect is that of NH₄⁺ fertilizers, which can suppress soil CH₄ consumption by up to 70% (1, 8, 10, 17, 22, 32, 33, 37, 38, 43). In the field, inhibition may occur immediately following fertilization, may be delayed for months to years, or may never occur despite years of chronic fertilization (9, 17). This variety of responses may stem at least in part from the distribution of physiologically diverse methane oxidizer populations across sites (17, 18, 20).Of the various NH₄⁺ inhibition patterns, immediate inhibition is the best documented. As in field studies, however, physiological laboratory studies have produced variable results, suggesting that there may be multiple inhibition mechanisms (15, 17, 26–28, 36, 39). Physicochemical similarities between CH₄ and NH₃ may permit these two compounds to compete for enzyme active sites so that fortuitous NH₃ oxidation competitively inhibits CH₄ oxidation (38). Although this mechanism has been demonstrated to occur in pure cultures of methanotrophic bacteria (6) and in a CH₄-producing agricultural soil (15), it has not been demonstrated to occur in well-drained, nonagricultural mineral soils, which comprise the dominant terrestrial sink for atmospheric CH₄ (14, 38, 45), nor has it been demonstrated to occur at near-atmospheric CH₄ concentrations. In many cases, the kinetics of immediate NH₄⁺ inhibition in soil cannot be reconciled easily with substrate competition (15, 16, 26–28, 39). An alternative mechanism has been proposed, whereby the toxicity of NO₂⁻ or NH₂OH produced by fortuitous NH₄⁺ oxidation suppresses methanotrophic activity (26, 27, 39). Hence, multiple inhibition mechanisms may be involved, and these mechanisms may vary with the physiology of different CH₄ oxidizer populations (17).Two physiologically distinct communities of CH₄ oxidizers apparently exist in soil. One group, generally associated with atmospheric CH₄ consumption, exhibits an extremely high affinity for CH₄. Representatives of this group have yet to be cultivated or otherwise identified (9). The second group exhibits a much lower affinity for CH₄ and is generally associated with common methanotrophs, such as those that have been studied in pure culture for many years (2, 9). In upland mineral soils, only high-affinity activity is usually detectable without artificial enrichment with high CH₄ concentrations in the laboratory. However, the only prior study in which kinetic constants for NH₄⁺ inhibition of soil CH₄ oxidation were reported was conducted in a periodically moist, organic matter-rich agricultural soil with demonstrable methanogenesis (15, 16). Such a soil potentially harbors a rich community of CH₄ oxidizers representing a continuum from low-affinity organisms to high-affinity organisms. Although this important investigation demonstrated that NH₄⁺ inhibits CH₄ oxidation via enzymatic substrate competition in an agricultural humisol, it is unclear to what extent its results apply to well-drained mineral soils lacking endogenous CH₄ sources. Physiological studies of soil CH₄ oxidation typically derive kinetic constants from oxidation rates at CH₄ concentrations ranging from atmospheric levels (∼1.7 μl liter⁻¹) to ≫K_m for high-affinity CH₄ oxidizers. Even in soil in which only high-affinity organisms are active, the CH₄-oxidizing enzyme(s) could respond differently to NH₄⁺ at high CH₄ concentrations than at near-atmospheric concentrations (15, 39). Thus, to study NH₄⁺ inhibition of high-affinity CH₄ oxidizers per se, it would be preferable to examine inhibition kinetics at near-atmospheric CH₄ concentrations in a soil with no apparent endogenous CH₄ source.A common shortcoming of NH₄⁺ inhibition studies, regardless of the organisms involved, has been a lack of attention to nonammoniacal salt effects despite numerous reports of substantial inhibition by such salts (1, 10, 15, 17, 24). King and Schnell (28) examined the effects of several Cl⁻ and SO₄²⁻ salts and concluded that nonammoniacal salts indirectly inhibit CH₄ oxidation by desorbing endogenous NH₄⁺ from cation exchange sites in the soil, which then directly inhibit CH₄ oxidation. Many N-limited soils, however, have extremely low concentrations of exchangeable NH₄⁺, yet are substantially inhibited by nonammoniacal salts (17), suggesting that these salts have NH₄⁺-independent effects on atmospheric CH₄ oxidizers. Additional mechanisms may alter inhibition kinetics, thus hindering the diagnosis of NH₄⁺-specific inhibition.With the limitations described above in mind, we used a simple steady-state kinetics approach to assess the mechanism of NH₄⁺ inhibition of CH₄ oxidation at near-atmospheric concentrations (1.8 to 15 μl liter⁻¹) in three well-drained, N-limited forest soils that lack known endogenous CH₄ sources. In addition, we examined the effects of nonammoniacal salts in parallel samples to judge the utility of these salts as experimental controls for neutralizing NH₄⁺-independent salt effects.