Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.     

Oxidation of isoeugenol and coniferyl alcohol catalyzed by laccases isolated from Rhus vernicifera Stokes and Pycnoporus coccineus

Laccases isolated from Rhus vernicifera Stokes (tree) and Pycnoporus coccineus (fungus) catalyzed the oxidation of isoeugenol (1) and coniferyl alcohol (5) in acetone–water (1:1, v/v). These oxidations follow a first order rate law. In general, the rates of Pycnoporus laccase-catalyzed oxidation of 1 and 5 are approximately three and seven times faster than the corresponding rates of Rhus laccase-catalyzed oxidation, respectively. Thus, synthesis for 2-(4-hydroxyphenyl)coumaran type compounds, such as dehydrodiconiferyl alcohol, can be accomplished by Rhus laccase-catalyzed dehydrogenative polymerization of the corresponding 1-(4-hydroxyphenyl)–1-propene derivatives. The reaction proceeds under very mild reaction conditions. The resulting reaction mixtures are chromatographed on a silica gel column to isolate the products in approximately 30–40% yield.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.     

Resolution of 1,3-dioxolane derivatives using the lipase from an Acinetobacter junii SY-01

Acinetobacter junii SY-01 producing a lipase enantioselectively hydrolyzing 1,3-dioxolane derivatives was isolated from water sludge sample and the effect of solvent, acyl donor, vinyl acetate concentration, substrate concentration, operating temperature and immobilization on activity and enantioselectivity was studied for the resolution of 1,3-dioxolane derivatives through transesterification reaction using a lipase from the isolated strain. Best selectivity was obtained at lower substrate concentration (3–5 mM), higher vinyl acetate concentration (500–1000 mM) and lower temperature (30–40 °C) in the reaction mixture. Lipase immobilized onto Accurel MP-1000 (micro-porous polypropylene) gave the best results and the reactivity was about 29-fold higher than the free enzyme without the decrease of enantioselectivity. Resolution of 1,3-dioxolane derivatives was carried out in flask scale containing 100 ml solvents using the lipase immobilized onto Accurel MP-1000. In this reaction, the yield and enantiomeric excess of the remaining (2R, 4S)-alcohol were 31.2% and 98.2%, respectively. This result suggests that it can be used as an alternative method, compared to the present synthetic method, for the production of optically pure (2R, 4S)-itraconazole.     

Effects of cellulose derivatives and carrageenans on the pasting, paste, and gel properties of rice starches

Effects of different cellulose derivatives and carrageenans on the pasting, rheological, and textural properties of normal (NRS) and waxy (WRS) rice starches were investigated. When suspensions of both NRS and WRS were heated in a Rapid Visco Analyser (RVA) in the presence of the hydrocolloids, increases in apparent pasting temperatures and peak and final viscosities in the following decreasing order were observed: methylcellulose (MC) > carboxymethylcellulose (CMC) for cellulose derivatives and λ- > í- > κ-carregeenan for carrageenans. Slight decreases in peak and final viscosities were observed when hydroxypropylmethylcellulose (HPMC) was the hydrocolloid. Dynamic viscoelasticity measurements indicated that NRS–hydrocolloid pastes were less solid-like than the control, as evidenced by their higher tan δ values, whereas tan δ values of WRS–hydrocolloid pastes were the same as that of the control. Steady shear rheological measurements showed that addition of the different hydrocolloids used increased the apparent viscosity (η_a,100) and consistency coefficient (K) values of both starches with the same trend as that observed during pasting, whereas the opposite trend was observed for the flow behavior index (n) values. The hardness and adhesiveness of NRS pastes were significantly increased by addition of κ- and í-carrageenans, but were unaffected by the other hydrocolloids. A similar effect was observed for WRS, with the exception of κ-carregeenan, in which the hardness of the mixed paste was decreased. The starch–hydrocolloid pastes exhibited a phase-separated microstructure in which amylose- and amylopectin-rich domains were dispersed in a hydrocolloid-rich continuous phase.     

Determination of sodium cromoglycate in human plasma by liquid chromatography–mass spectrometry in the turbo ion spray mode

A highly sensitivity liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100–50.0 ng/ml, using ¹³C₄ sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C₁₈ solid-phase extraction cartridges. The dried residue was reconstituted with 100 μl of 0.01 N HCl, and 30 μl was injected onto the LC–MS–MS system. Chromatographic separation was achieved on a C8 (3.5 μm) column with an isocratic mobile phase of methanol–water–0.5 M ammonium acetate (35:64.8:0.2, v/v/v). The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 469.2 (precursor ion) to m/z 245.1 (product ion) for SCG and m/z 473.2 (precursor ion) to m/z 247.1 (product ion) for ¹³C₄ SCG (I.S.). The average recoveries of SCG and the I.S. from human plasma were 91 and 87%, respectively. The low limit of quantitation was 0.100 ng/ml. Results from a 4-day validation study demonstrated excellent precision (C.V.% values were between 1.9 and 6.5%) and accuracy (−5.4 to −1.2%) across the calibration range of 0.100–50.0 ng/ml.     

Chromosomal aberrations in tire plant workers and interaction with polymorphisms of biotransformation and DNA repair genes

We evaluated chromosomal aberrations in lymphocytes of 177 workers exposed to xenobiotics in a tire plant and in 172 controls, in relation to their genetic background. Nine polymorphisms in genes encoding biotransformation enzymes and nine polymorphisms in genes involved in main DNA repair pathways were investigated for possible modulation of chromosomal damage. Chromosomal aberration frequencies were the highest among exposed smokers and the lowest in non-smoking unexposed individuals (2.5 ± 1.8% vs. 1.7 ± 1.2%, respectively). The differences between groups (ANOVA) were borderline significant (F = 2.6, P = 0.055). Chromosomal aberrations were higher in subjects with GSTT1-null (2.4 ± 1.7%) than in those with GSTT1-plus genotype (1.8 ± 1.4%; F = 7.2, P = 0.008). Considering individual groups, this association was significant in smoking exposed workers (F = 4.4, P = 0.040). Individuals with low activity EPHX1 genotype exhibited significantly higher chromosomal aberrations (2.3 ± 1.6%) in comparison with those bearing medium (1.7 ± 1.2%) and high activity genotype (1.5 ± 1.2%; F = 4.7, P = 0.010). Both chromatid- and chromosome-type aberration frequencies were mainly affected by exposure and smoking status. Binary logistic regression analysis revealed that frequencies of chromatid-type aberrations were modulated by NBS1 Glu185Gln (OR 4.26, 95%CI 1.38–13.14, P = 0.012), and to a moderate extent, by XPD Lys751Gln (OR 0.16, 95%CI 0.02–1.25, P = 0.081) polymorphisms. Chromosome-type aberrations were lowest in individuals bearing the EPHX1 genotype conferring the high activity (OR 0.38, 95%CI 0.15–0.98, P = 0.045). Present results show that exposed individuals in the tire production, who smoke, exhibit higher chromosomal aberrations frequencies, and the extent of chromosomal damage may additionally be modified by relevant polymorphisms.     

Cultivation and preservation of vinegar bacteria

Ten strains of acetic acid bacteria were investigated for their characteristics of growth and metabolism. The strains were identified as those presently in use for industrial vinegar production in southern Germany. At the time of isolation from industrial acetators the total concentrations, i.e. acetic acid (w/v) plus ethanol (v/v), of the fermenting vinegars were 6.1–14.9%. The applicability of a previously described method for starter preparation was examined for the various isolates as well as for the type strains of species of the genera Gluconobacter and Acetobacter. Isolates from cider or wine vinegar fermentations grew readily in RAE-medium to total counts of >1×10⁹ cells ml⁻¹. For the cultivation of strains isolated from spirit vinegar fermentations AE-medium proved most suitable. Cultures of these strains exhibited lag phases of 2–5 days and grew up to total counts of <1×10⁹ cells ml⁻¹. All type strains could be grown on RAE-agar. The use of 20% malt extract as cryo-protectant was effective for the preservation of all strains. Upon revitalization the cultures were suitable as inoculum for starting fermentations in pilot acetators. 16S rRNA-targeted oligonucleotide probes were constructed which were species specific for Gluconobacter oxydans or Acetobacter aceti or group specific for Acetobacter europaeus/Acetobacter xylinum. The probes hybridized with the DNA of the respective type strains. Four isolates were allotted to A. europaeus/A. xylinum applying the group specific probe. The DNA of six of the Acetobacter sp. hybridized with none of the probes.     

Effects of geometry on transmission and sensing potential of tapered fiber sensors

Geometry of tapered fiber sensors critically affects the response of an evanescent field sensor to cell suspensions. Single-mode fibers (nominally at 1300 nm) were tapered to symmetric or asymmetric tapers with diameters in the range of 3–20 μm, and overall lengths of 1–7 mm. Their transmission characteristics in air, water and in the presence of Escherichia coli (JM101 strain) at concentrations of 100, 1000, 7000 and 7 million cells/mL were measured in the 400–800 nm range and gave rich spectral data that lead to the following conclusions. (1) No change in transmission was observed due to E. coli with tapers that showed no relative change in transmission in water compared to air. (2) Tapers that exhibited a significant difference in transmission in water compared to air gave weak response to the presence of the E. coli. Of these, tapers with low waist diameters (6 μm) showed sensitivity to E. coli at 7000 cells/mL and higher concentration. (3) Tapers that showed modest difference in water transmission compared to air, and those that had small waist diameters gave excellent response to E. coli at 100–7000 cells/mL. In addition, mathematical modeling showed that: (1) at low wavelength (470 nm) and small waist diameter (6 μm), transmission with water in the waist region is higher than in air. (2) Small changes in waist diameter (0.05 μm) can cause larger changes in transmission at 470 nm than at 550 nm at waist diameter of 6 μm. (3) For the same overall geometry, a 5.5 μm diameter taper showed larger refractive index sensitivity compared to a 6.25 μm taper at 470 nm.     

Hydrophilization of immobilized model enzymes suggests a widely applicable method for enhancing protein stability in polar organic co-solvents

β-Galactosidases from Escherichia coli, Kluyveromyces lactis and Aspergillus oryzae were used to characterize the potential for enzyme stabilization of a two-step strategy: (i) immobilization on glutaraldehyde-agarose (Glut90), (ii) subsequent generation of a hydrophilic nano-environment by reaction with polyaldehyde-dextran polymer (Glut90-Pal), followed by polyamine-dextran polymer (Glut90-Pal-Pam). The derivatives were characterized by kinetics parameters, co-solvent (ethanol and acetone) and temperature stability. Hydrophilization achieved important co-solvent stabilization in all cases. One of the most remarkable results obtained was a 25-fold increase in the half-life of the A. oryzae Glut90-Pal-Pam derivative in 50% (v/v) acetone. Stabilization achieved in very drastic co-solvent concentrations is directly related to the hydrophilization of the nano-environment. The K_M values show that the hydrophilic shell appears to behave as an open structure and may create a “partition effect” that protects the enzymes from denaturation. These results show the potential of hydrophilization for building up additional stabilization of immobilized enzymes which would make possible the development of industrial applications.     

The stereoisomerism of bacterial, reaction-center-bound carotenoids revisited: An electronic absorption, resonance Raman and H-NMR study

In order to solve discrepancies between earlier assignments we have reinvestigated the stereoisomerism of the spheroidene molecule bound to reaction centers (RC) of Rhodobacter sphaeroides. A stable cis isomer could be extracted and purified from the reaction centres by working at very low ambient light. Resonance Raman spectroscopy showed that this cis isomer assumed the same configuration as that of the RC-bound molecule. Proton-NMR spectroscopy of the extracted isomer permitted to assign it the 15–15′ mono cis configuration. Comparisons between resonance Raman spectra of the native form and of the 15 cis extract showed that, in the reaction center, 15 cis spheroidene is in addition twisted into a non-planar conformation. Comparisons of extraction-induced changes in relative intensities of Raman bands of the 760–1060 cm⁻¹ regions, which largely correspond to out-of-plane modes, further indicated that the out-of-plane twist of RC-bound spheroidene should predominantly affect C₈–C₁₂ and/or C_8′–C_12′ regions of the molecule rather than the central region. Comparisons between difference electronic absorption spectra of RC-bound spheroidene and of RC-bound methoxyneurosporene showed that the out-of-plane twisting of both these native forms results in a drastic weakening of their ¹C ← ¹A electronic transitions, compared with those of the planar, 15 cis forms. Finally, it is proposed, on the basis of their resonance Raman spectra, that spirilloxanthin bound to RCs of Rhodospirillum rubrum as well as dihydroneurosporene or dihydrolycopene bound to RCs of Rhodopseudomonas viridis shares 15 cis configurations and out-of-plane twisting with carotenoids bound to RCs of various strains of Rb. sphaeroides.     

Determination of the substance P receptor antagonist CP-122, 721 in plasma by narrow-bore high-performance liquid chromatography-ionspray tandem mass spectrometry

A simple, highly sensitive and specific LC-MS-MS assay was developed for the determination of CP-122,721 (I) in rat and human plasma. I and a structural analog, CP-129,943 (II, internal standard), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile-20 mM ammonium acetate, pH 5 (50:50, v/v). Column effluents were monitored by ionspray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ion combinations of m/z 381 → 205 and 395 → 219 was used to quantitate I and II, respectively. The assay exhibited a linear dynamic range of 0.2–100 ng/ml. Absolute recoveries from plasma were above 80% for both I and II. The precision and accuracy values for the method were within ±3 and ±9%, respectively. Sample analysis times were less than 5 min from one injection to the next. The assay has proved to be applicable to the pharmacokinetic study of I in rats.     

Efficient plant regeneration from seedling explants of two commercially important temperate eucalypt species–Eucalyptus nitens and E. globulus

A reliable and reproducible method for plant regeneration in vitro of two important temperate eucalypts, Eucalyptus nitens and E. globulus, has been developed which utilises seedling explants. Highly regenerative callus was obtained from individual cotyledon and hypocotyledon explants of both species following cultivation on Murashige and Skoog’s (MS) basal nutrient medium supplemented with 30 g l⁻¹ sucrose, 5–10% (v/v) coconut water, 0.8% agar, 1 mg l⁻¹ -naphthalene-acetic acid (NAA) and 0.5 mg l⁻¹ N⁶ benzylaminopurine (BAP). Shoot differentiation was observed 7–8 weeks after transfer of callus onto regeneration medium containing 0.5 mg l⁻¹ NAA and 1 mg l⁻¹ BAP. In a few instances, direct shoot regeneration occurred without an intervening callus phase in both species. The frequency of plant regeneration was higher for callus derived from hypocotyl segments (30–35%) compared to cotyledonary explants (20–25%) though the average number of shoots per cotyledonary explant was generally higher than for hypocotyl explants. Somatic embryos were observed occasionally in E. nitens, arising from the surface of organogenic callus. Organised structures closely resembling somatic embryos were also observed in E. globulus. Regenerated shoots (30–40%) of both species could be rooted in modified MS media containing indole-3-butyric acid (IBA) and plantlets were successfully transferred to soil.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Analysis of the fixed oils of the genus Nigella L. (Ranunculaceae) in Turkey

The fatty acid composition of fixed oils obtained from the seeds of 10 species of Nigella (Nigella orientalis L., Nigella oxypetala L., Nigella latisecta P.H. Davis, Nigella segetalis Bieb., Nigella arvensis L., Nigella damascena L., Nigella elata Boiss., Nigella nigellastrum (L.) Willk., Nigella unguicularis (Lam.) Spenner, and Nigella lancifolia Hub.-Mor.) from Turkey have been investigated. The seeds contained 17.6–41.3% fixed oils. Linoleic (31.21–69.5%) and oleic acids (15.79–36.03%) were the major fatty acids in the oils. Eicosenoic acid was found in high amounts in the oils of N. nigellastrum and N. unguicularis seeds (23.12 and 17.47%, respectively). N. nigellastrum, N. elata and N. unguicularis seed oils showed the highest concentration of eicosadienoic acid (9.40, 8.39 and 7.17%, respectively). In all fixed oils, the quantities of unsaturated fatty acids were higher than those of the saturated analogues.     

Determination of Guan-Fu Base A, a new anti-arrhythmic, in human plasma by gas chromatography and electron–capture detection

A gas chromatographic–electron capture detection (GC–ECD) method has been developed for determining Guan-Fu Base A (GFA), an experimental anti-arrhythmic, in human plasma. The method was based on one-step liquid–liquid extraction with toluene and chemical derivatization with pentafluoropropionic anhydride followed by GC–ECD. The derivatives of GFA and metoprolol (Met, internal standard) were confirmed by gas chromatography–mass spectrometry (GC–MS) to be dipentafluoropropionyl-GFA and dipentafluoropropionyl-Met. The method was linear over the concentration ranges of 0.1–20.0 and 1.0–30.0 μg/ml with the detection limit of 0.05 μg/ml at S/N=5. The intra- and inter-assay precisions were less than 6 and 10%, and accuracy 99.70±3.30 and 97.60±5.99%, respectively. The absolute recoveries were 81.88, 77.35, 80.79 and 83.85% for GFA at concentrations of 0.5, 1.0, 5.0 and 14.0 μg/ml and 88.24% for Met at 3.0 μg/ml, respectively.     

Composition and infraspecific variability of essential oil from Thymus herba barona Lois

The composition of the essential oils of individual plants of Thymus herba barona Lois. growing wild in Corsica was investigated by GC, GC/MS an carbon-13 NMR. Eight groups of essential oil were distinguished: (i) thymol, (ii) carvacrol, (iii) linalool, (iv) geraniol, (v) -terpenyl acetate, (vi) terpinen-4-ol, (vii) carvone and cis-dihydrocarvone. Three chemotypes -thymol, carvacrol and linalool – are common in the genus Thymus, two others – geraniol, -terpenyl acetate – are scarce, while the latest three ones – terpinen-4-ol, carvone and cis-dihydrocarvone are quite original. It is the first time that the cis-dihydrocarvone chemotype is described in the genus Thymus     

Zooplankton feeding ecology: bacterivory by metazoan microzooplankton

Bacterivory by the rotifer Brachionus plicatilis Müller, nauplii and copepodites of the copepods Centropages Krøyer sp. and Acartia tonsa Dana, and the tintinnid Favella panamensis Kofoid & Campbell was examined using fluorescently labelled bacteria (FLB) and epifluorescence microscopy. FLB were < 1 μm in diameter, and were offered at environmental concentrations (1.47−9.08 × 10⁶ cells·ml⁻¹). FLB were visible within rotifers, nauplii, copepodites, and tintinnids, confirming ingestion. Rotifer clearance rates (32–418 μl·animal⁻¹·h⁻¹) exhibited no relation with FLB concentration. In some cases rates of clearance of FLB by rotifers were different with alternative phytoplankton food (Nanochloris Naumann sp.) than in replicates with FLB alone, whereas in other cases presence of alternative food exhibited no clear effects on rates of ingestion of FLB. Clearance rates for all six naupliar stages of A. tonsa nauplii (0–320 μl·animal⁻¹·h⁻¹) were stage-related, with higher rates by NIII-VI nauplii than NI-II nauplii. Nauplii had higher rates of clearance of FLB in the absence of alternative phytoplankton food (Isochrysis Parke sp.). Clearance rates of FLB by a single stage of Centropages sp. nauplii, A. tonsa CI copepodites and F. panamensis (each obtained at only a single food concentration of either 1.5 or 5.0 × 10⁶ cells·ml⁻¹) were within the range of 85–142 μl·animal⁻¹·h⁻¹. These ranges were similar to those of rotifers and A. tonsa nauplii. This is the first report of FLB ingestion by metazoan marine microzooplankton. Although rotifers and ciliates might be expected to ingest small particles such as FLB using ciliary induced feeding currents, the means by which nauplii and copepodites eat FLB is less clear. We propose that they may “eat” bacteria as they “drink” to osmoregulate.