Quantification of theobromine and caffeine in saliva,plasma and urine via liquid chromatography–tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies

Targeted analyses of clinically relevant metabolites in human biofluids often require extensive sample preparation (e.g., desalting, protein removal and/or preconcentration) prior to quantitation. In this report, a single ultra-centrifugation based sample pretreatment combined with a designed liquid chromatography–tandem mass spectrometry (LC–MS/MS) protocol provides selective quantification of 3,7-dimethylxanthine (theobromine) and 1,3,7-trimethylxanthine (caffeine) in human saliva, plasma and urine samples. The optimized chromatography permitted elution of both analytes within 1.3 min of the applied gradient. Positive-mode electrospray ionization and a triple quadruple MS/MS instrument operated in multiple reaction mode were used for detection. ¹³C₃ isotopically labeled caffeine was included as an internal standard to improve accuracy and precision. Implementing a 20-fold dilution of the isolated low MW biofluid fraction prior to injection effectively minimized the deleterious contributions of all three matrices to quantitation. The assay was linear over a 160-fold concentration range from 2.5 to 400 μmol L^?1 for both theobromine (average R² 0.9968) and caffeine (average R² 0.9997) respectively. Analyte peak area variations for 2.5 μmol L^?1 caffeine and theobromine in saliva, plasma and urine ranged from 5 and 10% (intra-day, N = 10) to 9 and 13% (inter-day, N = 25) respectively. The intra- and inter-day precision of theobromine and caffeine elution times were 3 and <1% for all biofluids and concentrations tested. Recoveries for caffeine and theobromine ranged from 114 to 118% and 99 to 105% at concentration levels of 10 and 300 μmol L^?1. This validated protocol also permitted the relative saliva, plasma and urine distribution of both theobromine and caffeine to be quantified following a cocoa intervention.     

Long-term effects of exogenous silicon on cadmium translocation and toxicity in rice (Oryza sativa L.)

Most nutrient solution studies on the interactions between silicon (Si) and cadmium (Cd) are short term. Here we reported a long-term experiment in which rice (Oryza sativa L.) was cultured for 105 days and harvested at four different growth stages to measure biomass accumulation and Cd uptake and distribution in shoots and roots. Exogenous Si increased shoot biomass by 61–238% and root biomass by 48–173% when the culture solution was free of Cd. When 2 μmol L^?1 Cd was added, Si supply increased shoot and root biomass by 125–171% and by 100–106% compared to the zero-Si treatment. Increasing the Cd concentration to 4 μmol L^?1 decreased the beneficial effects of Si on root and shoot biomass. Silicon supply decreased shoot Cd concentrations by 30–50% and Cd distribution ratio in shoot by 25.3–46%, compared to the treatment without Si supply. Additionally, lower Si supply or more serious Cd stress would lead to roots with bigger biomass and higher Si concentration. Energy-dispersive X-ray microanalysis showed that both Si and Cd accumulated synchronously in the border and middle of phytoliths of the shoots. We conclude that Si enhances plant growth and decreases Cd accumulation in shoots and thereby helps to lower the potential risks of food contamination.     

Importance of the core structure of flavones in promoting inhibition of the mitochondrial respiratory chain

Flavonoids are a large group of polyphenolic compounds that have received considerable attention because of their biological and physiological importance. The flavone (2-phenyl-4H-1-benzopyran-4one) used in this work is found in some cereal grains and generates several biological activities, including: apoptosis induction, cell cycle arrest, caspase activation and inhibition of tumor cell proliferation. However, its effects on the hepatic mitochondrial metabolism are still unknown. We evaluated the effect of flavone on the metabolism of mitochondria isolated from rat liver. Polarographic experiments using 200 μmol L^?1 flavone and rat liver mitochondria oxidizing glutamate or succinate indicated that both substrates underwent: (i) reduction of state 3 respiration; (ii) stimulation of state 4 respiration; (iii) reduction of the respiratory control coefficient; and (iv) reduction of the ADP/O ratio. An analysis of the activity of enzymatic complexes in the respiratory chain showed that flavone acts between complexes I and III. Flavone reduced the membrane electric potential at doses of 100, 150 and 200 μmol L^?1. Flavone at certain doses (75–200 μmol L^?1) reduced mitochondrial swelling in the presence of valinomycin and KNO₃, suggesting that flavone could induce changes in mitochondrial membrane properties. These results demonstrate that the inhibition of mitochondrial enzymes in the respiratory chain coupled with the effects on membrane properties are promoted by the core structure of flavones, and these effects may be in part responsible for the cytotoxic effects of flavones.     

Urea loading from a spring storm—Knysna estuary,South Africa

Urea concentrations were tracked in the Knysna estuary, South Africa, following a strong initial storm of the austral summer rainy season in 2000. These post-storm urea concentrations are compared against a 1-year survey of these concentrations and demonstrate the initial and longer-term impacts of urea loading on a near-pristine estuary. Fifteen stations along the main channel of the estuary were sampled four times each, 2–4 weeks before the storm, providing and average urea concentration of 1.4 μmol N L⁻¹. When these same stations were sampled 12 h after the storm the average urea concentration had increased to 9.4 μmol N L⁻¹. The average urea concentration at these 15 stations remained high, averaging 10.2 μmol N L⁻¹ and 10.0 μmol N L⁻¹ 36 and 84 h post-storm, respectively. The urea concentrations in the rivers supplying fresh water to the Knysna estuary, one draining pasturelands and the other draining informal housing settlements that relied on pit toilets and open sewers are also compared before and after this storm. These data suggest that spring storms significantly influence nutrient availability, which in turn, may be related to the large dinoflagellates blooms witnessed in the Knysna estuary during the later portion of the wet summer season.     

Effects of Zn2+ and niflumic acid on photosynthesis in Glycine soja and Glycine max seedlings under NaCl stress

The effects of two anion/Cl^? channel inhibitors, Zn²⁺ and niflumic acid (NA), on seedling photosynthetic and fluorescent parameters of two Glycine soja populations (salt-tolerant BB52; salt-sensitive N23227) and Glycine max cultivar (salt-tolerant Lee68) were studied and compared under salt stress. Treatments with Zn²⁺ and NA only (10, 20 μmol L^?1) were also imposed for comparisons. Results showed that, there were non-toxic and non-nutritional effects of Zn²⁺ and NA treatments alone on seed germination and seedling growth of soybeans. Under 150 mmol L^?1 NaCl for 6 d, leaf chlorophyll and carotenoid contents, net photosynthetic rate (P_n), stomatal conductance (G_s), intercellular CO₂ concentration (C_i), transpiration rate (Tr), and the maximum photochemical efficiency of photosystem II (PS II) (F_v/F_m) except the stomatal limitation (Ls) significantly decreased in three kinds of soybean seedlings when compared with their control plants. The NaCl stress plus additional 20 μmol L^?1 Zn showed an obvious enhancement of leaf chlorophyll and carotenoid contents, P_n, G_s, C_i and Tr, especially for the G. max cultivar Lee68, but the supplementation of 20 μmol L^?1 NA showed the reverse effects.     

Evaluation of the effects of light intensity on growth of the benthic dinoflagellate Ostreopsis sp. 1 using a newly developed photoirradiation-culture system and a novel regression analytical method

Benthic dinoflagellates of the genus Ostreopsis are found all over the world in temperate, subtropical, and tropical coastal regions. Our recent studies revealed that a putative “cryptic” species of Ostreopsis ovata is present widely along Japanese coasts. This organism, Ostreopsis sp. 1, possesses palytoxin analogs and thus its toxic blooms may be responsible for potential toxification of marine organisms. To evaluate the bloom dynamics of Ostreopsis sp. 1, the present study examined the growth responses of Ostreopsis sp. 1 strain s0716 to various light intensities (photon flux densities: μmol photons m⁻² s⁻¹) using a newly devised photoirradiation-culture system. This novel system has white light-emitting diodes (LEDs) capable of more closely simulating the wavelength spectrum of light entering the oceanic water column than do fluorescent tubes and halogen lamps. In this system, the light intensity of the white LEDs was reduced through two polarizing filters by varying the rotation angles of the filters. Thereby, the new system was capable of culturing microalgae under well-controlled light intensity conditions. Ostreopsis sp. 1 grew proportionally when light intensity was increased from 49.5 to 199 μmol photons m⁻² s⁻¹, but its growth appeared to be inhibited slightly at ≥263 μmol photons m⁻² s⁻¹. The relationship between observed growth rates and light intensity was calculated at R > 0.99 (P < 0.01) using a regression analysis with a modified equation of the photosynthesis-light intensity (P-L) model. The equation determined the critical light intensities for growth of Ostreopsis sp. 1 and the organism's growth potential as follows: (1) the threshold light intensity for growth: 29.8 μmol photons m⁻² s⁻¹; (2) the optimum light intensity (L_m) giving the maximum growth rate (μ_max = 0.659 divisions day⁻¹): 196 μmol photons m⁻² s⁻¹; (3) the optimum light intensity range (L_opt) giving ≥95% μ_max: 130–330 μmol photons m⁻² s⁻¹; (4) the semi-optimum range (L_sopt) giving ≥80% μ_max: 90 to over 460 μmol photons m⁻² s⁻¹. The L_sopt represents 4.5–23% ambient light intensity present in surface waters off of a temperate region of the Japanese coast, Tosa Bay; putatively, this semi-optimum range of light intensity appears at depth of 12.9–27.8 m. Considering these issues, our data indicate that Ostreopsis sp. 1 in coastal environments may form blooms at ca. ∼28 m depth in regions along Japanese coasts.     

Impact of in vitro CO2 enrichment and sugar deprivation on acclimatory responses of Phalaenopsis plantlets to ex vitro conditions

We assessed the effect of growth at either 400 μmol mol^?1 (ambient) or 1000 μmol mol^?1 (elevated) CO₂ and 0 g L^?1 (deprivation) or 30 g L^?1 (supplementation) sugar on morphological traits, photosynthetic attributes and intrinsic elements of the CAM pathway using the CAM orchid Phalaenopsis ‘Amaglade’. The growth of shoot (retarded) and root (induced) was differently affected by CO₂ enrichment and mixotrophic regime (+sugar). The Fv/Fm ratio was 14% more in CO₂-enriched treatment than at ambient level during in vitro growth. At elevated level of CO₂ and sugar treatment, the content of Chl(a + b), Chl a/b and Chl/Car was enhanced while carotenoid content remained unaltered. During in vitro growth, gas-exchange analysis indicated that increased uptake of CO₂ accorded with the increased rate of transpiration and unchanged stomatal conductance at elevated level of CO₂ under both photo- and mixotrophic growth condition. At elevated level of CO₂ and sugar deprivation, activities of Rubisco (26.4%) and PEPC (74.5%) was up-regulated. Among metabolites, the content of sucrose and starch was always higher under CO₂ enrichment during both in vitro and ex vitro growth. Our results indicate that plantlets grown under CO₂ enrichment developed completely viable photosynthetic apparatus ready to be efficiently transferred to ex vitro condition that has far-reaching implications in micropropagation of Phalaenopsis.     

Cultivation impacts soil microbial dynamics in dry tropical forest ecosystem in India

We studied for two years the seasonal changes in plant available nitrate and ammonium nitrogen (N), nitrification, N-mineralization, microbial biomass carbon (MBC), nitrogen (MBN) and phosphorus (MBP) in two forest and three cropland sites, derived from a tropical forest ecosystem of India. Results indicated that seasonal values of nitrate N, ammonium N and phosphate P ranged from 7.33–12.99, 5.1–10.22 and 4.0–7.8 μg g^?1 in forest and 4.13–9.26, 9.35–14.46 and 2.8–5.8 μg g^?1 in cropland ecosystems, respectively, with maximum values in summer and minimum in rainy seasons. Nitrification and N-mineralization values varied from 6–28 and 4–26 μg g^?1 mo^?1 in forest and 3–14 μg g^?1 mo^?1 and 4–17 μg g^?1 mo^?1 in cropland, with maximum values in rainy season and minimum in summer season.MBC, MBN MBP ranged from 393–753, 34–80 and 16–36 μg g^?1 in forests and 186–414, 21–41 and 11–22 μg g^?1 in croplands, being maximum in summer and minimum in rainy seasons. There was gradual increase in the values of inorganic N, nitrification, N-mineralization and MBC, MBN and MBP along the age of cropland. Analysis of variance indicated significant difference in the concentration of inorganic N, nitrification and N-mineralization and MBC, MBN and MBP due to sites and seasons.Cultivation caused decline in the mean annual organic C, N and P by 42%, 29% and 13%. The values of nitrate N were decreased by 23–38%, while ammonium N was increased by 39–74%. Nitrification and N-mineralization values were reduced by 39–63% and 40–60%, respectively. Microbial C, N and P were reduced by 44–54%, 41–50% and 28–44%, respectively. Nonetheless, the contribution of soil microbial biomass reflected in total N was enhanced from 4.76% in forest to 5.03% in cropland ecosystem. Enhancement of plant available ammonium-N and microbial contribution in total N are an indicator of natural conserving mechanism to check the nitrogen loss from the nutrient poor agro-ecosystem.     

Effect of light quality on Bacillus amyloliquefaciens JBC36 and its biocontrol efficacy

Light is one of the most important environmental signals regulating physiological processes of many microorganisms. However, very few studies have been reported on the qualitative or quantitative effects of light on control of postharvest spoilage using antagonistic bacteria. In this study, we investigated the effects of white, red, green, and blue light at photon flux densities of 40, 240, and 360 μmol m^?2 s^?1 on Bacillus amyloliquefaciens JBC36 (JBC36), which has been reported as a promising candidate for biocontrol of green and blue mold on mandarin fruit. With the exception of blue light at 240 and 360 μmol m^?2 s^?1, light generally stimulated growth of JBC36 compared to the controls grown in the dark. Red light increased swarming motility irrespective of intensity and significantly enhanced biofilm formation at 240 μmol m^?2 s^?1. Production of antifungal metabolites and antifungal activity on Penicillium digitatum was also affected by light quality. Interestingly, antifungal activity was significantly increased when JBC36 and P. digitatum was co-incubated under red and green light at an intensity of 240 μmol m^?2 s^?1. We also demonstrated that the quality of light resulted in changes in colonization of JBC36 on mandarin fruit and control of green mold. In particular, red light increased the population level on mandarin fruit and biocontrol efficacy against green mold. These results represent the first report on the effect of light quality on an antagonistic bacterium for the control of postharvest spoilage. We believe that an improved understanding of the JBC36 response to light quality may help in the development of strategies to increase biocontrol efficacy of postharvest spoilage.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Effects of shade treatments on the photosynthetic capacity,chlorophyll fluorescence,and chlorophyll content of Tetrastigma hemsleyanum Diels et Gilg

Tetrastigma hemsleyanum Diels et Gilg was grown under full sunlight and moderate and high levels of shade for one month to evaluate its photosynthetic and chlorophyll fluorescence response to different light conditions. The results showed that T. hemsleyanum attained greatest leaf size and P_n when cultivated with 67% shade. Leaves of seedlings grown with 90% shade were the smallest. Leaf color of plants grown under full sunlight and 50% shade was yellowish-green. The P_n value increased rapidly as PPFD increased to 200 μmol m^?2 s^?1 and then increased slowly to a maximum, followed by a slow decrease as PPFD was increased to 1000 μmol m^?2 s^?1. P_n was highest for the 67% shade treatment and the LSP for this shade treatment was 600 μmol m^?2 s^?1. Full sunlight and 50% shade treatments resulted in significant reduction of ETR and qP and increased NPQ. Chl a, Chl b and total chlorophyll content increased and Chl a/b values decreased with increased shading. Results showed that light intensity greater than that of 50% shade depressed photosynthetic activity and T. hemsleyanum growth. Irradiance less than that of 75% shade limited carbon assimilation and led to decreased plant growth. Approximately 67% shade is suggested to be the optimum light irradiance condition for T. hemsleyanum cultivation.     

Carbonic anhydrase activators. Activation of the membrane-associated isoform XV with amino acids and amines

An activation study of the membrane-associated carbonic anhydrase (CA, EC 4.2.1.1) isoform XV with a series of natural and non-natural amino acids and aromatic/heterocyclic amines is reported. Murine CA XV was strongly activated by some amino acids (d-Phe, l-/d-DOPA, d-Trp, l-Tyr) and amines (dopamine, serotonin, l-adrenaline and 4-(2-aminoethyl)-morpholine) with activation constants in the range of 4.0–9.5 μM. l-/d-His, l-Phe, histamine and several other heterocyclic amines showed less efficient activation (K_As in the range of 11.6–33.4 μM). The activation profile of CA XV is quite different from that of the cytosolic isoforms CA I and II or the membrane-associated CA IV. All mammalian isoforms CA I–XV are thus characterized for their interaction with this set of amino acid and amine activators, some of which are biogenic amines or neurotransmitters present in sufficiently high amounts in various tissues for exerting significant biologic responses.     

Criteria to distinguish between natural situations and illegal use of boldenone,boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap?). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L^?1 and 0.4 μg L^?1 on triple quadrupole and 0.1 μg L^?1 and 0.2 μg L^?1 on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

Management scenario evaluation for a large treatment wetland using a spatio-temporal phosphorus transport and cycling model

This paper describes the development of a two-dimensional, spatially distributed model to simulate coupled hydrologic and phosphorus (P) biogeochemical processes in a 147-ha cell of a 1544-ha stormwater treatment wetland designed to help protect the greater Everglades, FL, USA. The model was used to assess the effects of a suite of feasible management alternatives on the long-term ability of the wetland to sustain total P (TP) removal. The spatial and temporal dynamics of TP retention were simulated under historical (1995–2000) conditions, and under assumptions of removal of short-circuiting channels and ditches, changes in external hydraulic and TP loading, and long-term (>20 years) impacts on soil and water column TP dynamics under current and reduced load conditions. Internal hydrology and transport processes were calibrated against measured tracer concentrations, and subsequently validated against outflow discharge and spatial chloride concentration data. Cycling of P was simulated as first-order uptake and release, with different uptake coefficients for open water/sparse submerged aquatic vegetation (SAV) areas (0.2 day^?1) and dense SAV areas (0.4 day^?1), and a much lower, uniform release coefficient (1.97 × 10^?4 day^?1). The calibration and validation of the P model showed good agreement with field measurements of water column TP concentrations measured at the wetland outlet (calibration RMSE = 10.5 μg L^?1; validation RMSE = 15.6 μg L^?1). Under simulated conditions of preferential channels eliminated, average annual TP treatment effectiveness increased by 25%. When inflow TP loads were assumed to be eliminated after 6 years of loading, the release of accumulated soil P sustained predicted annual average outlet concentrations above 6.7 μg L^?1 for 18 years, decreasing at a rate of 0.16 μg L^?1 yr^?1. Sensitivity analyses indicate that the most critical model input factors include flow resistance parameters, initial soil TP content, and P cycling parameters compared to initial water level, initial TP concentration in water column, ET and transport parameters.     

Nitrate removal and greenhouse gas production in a stream-bed denitrifying bioreactor

Denitrifying bioreactors are currently being tested as an option for treating nitrate (NO₃^?) contamination in groundwater and surface waters. However, a possible side effect of this technology is the production of greenhouse gases (GHG) including nitrous oxide (N₂O) and methane (CH₄). This study examines NO₃^? removal and GHG production in a stream-bed denitrifying bioreactor currently operating in Southern Ontario, Canada. The reactor contains organic carbon material (pine woodchips) intended to promote denitrification. Over a 1 year period, monthly averaged removal of influent (stream water) NO₃^? ranged from 18 to 100% (0.3–2.5 mg N L^?1). Concomitantly, reactor dissolved N₂O and CH₄ production, averaged 6.4 μg N L^?1 (2.4 mg N m^?2 d^?1), and 974 μg C L^?1 (297 mg C m^?2 d^?1) respectively, where production is calculated as the difference between inflow and effluent concentrations. Gas bubbles entrapped in sediments overlying the reactor had a composition ranging from 19 to 64% CH₄, 1 to 6% CO₂, and 0.5 to 2 ppmv N₂O; however, gas bubble emission rates were not quantified in this study. Dissolved N₂O production rates from the bioreactor were similar to emission rates reported for some agricultural croplands (e.g. 0.1–15 mg N m^?2 d^?1) and remained less than the highest rates observed in some N-polluted streams and rivers (e.g. 110 mg N m^?2 d^?1, Grand R., ON). Dissolved N₂O production represented only a small fraction (0.6%) of the observed NO₃^? removal over the monitoring period. Dissolved CH₄ production during summer months (up to 1236 mg C m^?2 d^?1), was higher than reported for some rivers and reservoirs (e.g. 6–66 mg C m^?2 d^?1) but remained lower than rates reported for some wastewater treatment facilities (e.g. sewage treatment plants and constructed wetlands, 19,500–38,000 mg C m^?2 d^?1).     

Environmental indicators for estimating the potential soil respiration rate in alpine zone

Soil respiration is the main form of carbon flux from soil to atmosphere in the global carbon cycle. The effect of temperature on soil respiration rate is important in evaluating the potential feedback of soil organic carbon to global warming. We incubated soils from the alpine meadow zone and upper rocky zone along an altitudinal gradient (4400–5500 m a.s.l.) on the Tibetan Plateau under various temperature and soil moisture conditions. We evaluated the potential effects of temperature and soil moisture on soil respiration and its variation across altitudes. Soil respiration rates increased as the temperature increased. At 60% of soil water content, they averaged 0.21–5.33 μmol g soil⁻¹ day⁻¹ in the alpine meadow zone and 0.11–0.50 μmol g soil⁻¹ day⁻¹ in the rocky zone over the experimental temperature range. Soil respiration rates in the rocky zone did not increase between 25 and 35 °C, probably because of heat stress. Rates of decomposition of organic matter were high in the rocky zone, where the CN ratio was smaller than in the middle altitudes. Soil respiration rates also increased with increasing soil water content from 10% to 80% at 15 °C, averaging 0.04–2.00 μmol g soil⁻¹ day⁻¹ in the alpine meadow zone and 0.03–0.35 μmol g soil⁻¹ day⁻¹ in the rocky zone. Maximum respiration rates were obtained in the middle part of the alpine slope in any case of experimental temperature and soil moisture. The change patterns in soil respiration rate along altitude showed similar change pattern in soil carbon content. Although the altitude is a variable including various environmental factors, it might be used as a surrogate parameter of soil carbon content in alpine zone. Results suggest that temperature, soil moisture and altitude are used as appropriate environmental indicators for estimating the spatial distribution of potential soil respiration in alpine zone.     

Catalytic removal of sulfide by an immobilized sulfide-oxidase bioreactor

A bioreactor packed with chitosan immobilized sulfide-oxidase from Streptomyces species LD048 was developed to treat a liquid stream of sulfide. The inoculation system was composed of glass with a 0.7 L working volume and enzyme activity of 2 mmol S g^?1 carrier. The sulfide removal efficiency was almost 100% when the volumetric loading was increased up to 3.9 mmol S L^?1 h^?1 at a space velocity of 18 h^?1. The maximal elimination capacity was 22.1 mmol S L^?1 h^?1 with a space velocity of 72 h^?1. When the aeration was increased from 0.05 to 0.1 L min^?1, the average removal efficiency improved from 81% to 94%. A removal efficiency of 90% was obtained after 15 days of operation with a load rate of 8.9 mmol S L^?1 h^?1 and a space velocity of 14.28 h^?1. An operational equation based on the ideal plug flow bioreactor and the Michaelis–Menten model predicted the performance of this bioreactor.     

Elevated CO2 and water-availability effect on gas exchange and nodule development in N2-fixing alfalfa plants

N₂-fixing alfalfa plants were grown in controlled conditions at different CO₂ levels (350 μmol mol^?1 versus 700 μmol mol^?1) and water-availability conditions (WW, watered at maximum pot water capacity versus WD, watered at 50% of control treatments) in order to determine the CO₂ effect (and applied at two water regimes) on plant growth and nodule activity in alfalfa plants. The CO₂ stimulatory effect (26% enhancement) on plant growth was limited to WW plants, whereas no CO₂ effect was observed in WD plants. Exposure to elevated CO₂ decreased Rubisco carboxylation capacity of plants, caused by a specific reduction in Rubisco (EC 4.1.1.39) concentration (11% in WW and 43% in WD) probably explained by an increase in the leaf carbohydrate levels. Plants grown at 700 μmol mol^?1 CO₂ maintained control photosynthetic rates (at growth conditions) by diminishing Rubisco content and by increasing nitrogen use efficiency. Interestingly, our data also suggest that reduction in shoot N demand (reflected by the TSP and especially Rubisco depletion) affected negatively nodule activity (malate dehydrogenase, EC 1.1.1.37, and glutamate-oxaloacetate transaminase, EC 2.6.1.1, activities) particularly in water-limited conditions. Furthermore, nodule DM and TSS data revealed that those nodules were not capable to overcome C sink strength limitations.     

Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna

The efficacy of an essential oil obtained from Pimpinella anisum fruits and its major compound, trans-Anethole, was tested on the eggs, larvae and adults of Culex quinquefasciatus. While causing no significant mortality on eggs, other tested stages were very sensitive to the essential oil and trans-Anethole. LC₅₀ for the 2nd to 4th instar larvae was estimated as 26–27 μL·L^− 1 and 15–19 μL·L^− 1 for the essential oil and trans-Anethole, respectively. As for the essential oil applied on adults, LC(LD)₅₀ was estimated as 9.3 μL mL^− 1 (spray test), 1.9 μL L^− 1 (fumigation test) and 0.6 μg cm^− 2 (tarsal test), and for trans-Anethole as 8.1 μL mL^− 1 (spray test), 2.1 μL L^− 1 (fumigation test) and 0.4 μg cm^− 2 (tarsal test). The time needed to achieve 50% mortality after application of LC(LD)₉₉ of the essential oil was significantly different; for example, in larvicidal assays it ranged from 15 to 235 min depending on the larval instar, and from 9 to 180 min when applied to adults, depending on the mode of application. It was also found that temperature had an important effect on the larvicidal efficacy of the essential oil, and oviposition deterrent activity was studied.The essential oil and trans-Anethole were toxic for Daphnia magna (62–92% mortality) and significantly reduced its fertility at high concentrations (35–50 μL mL^− 1) and long exposure (48 h). However, no negative effect on Daphnia mortality or fertility was found at shorter exposure times (6 h) and/or lower concentrations (20 μL mL^− 1).Based on the results of this study, we can recommend the essential oil from P. anisum as a suitable active substance for potential botanical insecticides.     

Continuous production of selenomethionine-enriched Chlorella sorokiniana biomass in a photobioreactor

This article describes the enrichment of the fresh-water green microalga Chlorella sorokiniana in selenomethionine (SeMet). The microalga was cultivated in a 2.2 L glass-vessel photobioreactor, in a culture medium supplemented with selenate (SeO₄^2?) concentrations ranging from 5 to 50 mg L^?1. Although selenate exposure lowered culture viability, C. sorokiniana grew well at all tested selenate concentrations, however cultures supplemented with 50 mg L^?1 selenate did not remain stable at steady state. A suitable selenate concentration in fresh culture medium for continuous operation was determined, which allowed stable long-term cultivation at steady state and maximal SeMet productivity. In order to do that, the effect of dilution rate on biomass productivity, viability and SeMet content of C. sorokiniana at several selenate concentrations were determined in the photobioreactor. A maximal SeMet productivity of 21 μg L^?1 day^?1 was obtained with 40 mg L^?1 selenate in the culture medium. Then a continuous cultivation process at several dilution rates was performed at 40 mg L^?1 selenate obtaining a maximum of 246 μg L^?1 day^?1 SeMet at a low dilution rate of 0.49 day^?1, calculated on total daily effluent volume. This paper describes for the first time an efficient long-term continuous cultivation of C. sorokiniana for the production of biomass enriched in the high value amino acid SeMet, at laboratory scale.