Jan Tříska

Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C═C stretching mode) and 1,445 cm−1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.

Fungal bioremediation of the creosote-contaminated soil: influence of Pleurotus ostreatus and Irpex lacteus on polycyclic aromatic hydrocarbons removal and soil microbial community composition in the laboratory-scale study.

The aim of this study was to determine the efficacy of selected basidiomycetes in the removing of polycyclic aromatic hydrocarbons (PAH) from the creosote-contaminated soil. Fungi Pleurotus ostreatus and Irpex lacteus were supplemented with creosote-contaminated (50-200 mg kg(-1) PAH) soil originating from a wood-preserving plant and incubated at 15 °C for 120 d. Either fungus degraded PAH with 4-6 aromatic rings more efficiently than the microbial community present initially in the soil. PAH removal was higher in P. ostreatus treatments (55-67%) than in I. lacteus treatments (27-36%) in general. P. ostreatus (respectively, I. lacteus) removed 86-96% (47-59%) of 2-rings PAH, 63-72% (33-45%) of 3-rings PAH, 32-49% (9-14%) of 4-rings PAH and 31-38% (11-13%) of 5-6-rings PAH. MIS (Microbial Identification System) Sherlock analysis of the bacterial community determined the presence of dominant Gram-negative bacteria (G-) Pseudomonas in the inoculated soil before the application of fungi. Complex soil microbial community was characterized by phospholipid fatty acids analysis followed by GC-MS/MS. Either fungus induced the decrease of bacterial biomass (G- bacteria in particular), but the soil microbial community was influenced by P. ostreatus in a different way than by I. lacteus. The bacterial community was stressed more by the presence of I. lacteus than P. ostreatus (as proved by the ratio of the fungal/bacterial markers and by the ratio of trans/cis mono-unsaturated fatty acids). Moreover, P. ostreatus stimulated the growth of Gram-positive bacteria (G+), especially actinobacteria and these results indicate the potential of the positive synergistic interaction of this fungus and actinobacteria in creosote biodegradation.

Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food, gut and excrement of saprophagous larvae ofPenthetria holosericea (Diptera: Bibionidae): a pilot study

Amplified ribosomal DNA restriction analysis (ARDRA) was used to compare the bacterial communities of the food, the gut sections (ceca, anterior and posterior midgut, hindgut) and the excrement of the litter feeding bibionid larvae ofPenthetria holosericea. For universal eubacterial primers ARDRA patterns were complex with only minor differences among samples. Taxon specific primers were also applied to characterize the samples. Fragment composition was transformed to presence/absence binary data and further analyzed. Cluster analysis revealed that bacterial communities of gut highly resembled each other with the exception of the ceca. ARDRA patterns of consumed leaves clustered together with the intact leaves but differed from those of the excrement. ARDRA results were compared with microbial community structure based on phospholipid fatty acid (PLFA) fingerprints. The cluster analysis of PLFA (presence/absence binary) data resulted in a pattern similar to the ARDRA data. The PCA analysis of PLFA relative content separated microbial communities into five groups: (1) anterior and posterior midgut, (2) hindgut, (3) ceca, (4) consumed and intact litter, (5) excrement. Both methods indicated that conditions in the larval gut result in formation of a specific microbial community which differs from both the food and excrement ones. Particularly ceca — (blind appendages, harbor very specific microbial community) are divided from the rest of the gut by perithropic membrane.

Separation of α-amino acid enantiomers by reversed phase high-performance liquid chromatography after derivatization with o-phthaldialdehyde and a sodium salt of 1-thio-β-d-glucose

Summary A sensitive system for d,l -amino acid analysis has been developed, using fluorescence derivatization with o -phthaldialdehyde in the presence of sodium salt of 1-thio- β - d -glucose. The reagents rapidly form fluorescent diastereoisomeric derivatives with primary amino acids. These derivatives are efficiently separated on a conventional reversed-phase column with an analysis time of 60 min. Simultaneous determination of enantiomers of various amino acids was achieved by a simple binary gradient elution with methanol in 0.05 M aqueous sodium acetate.

Possible ecological risk of two pharmaceuticals diclofenac and paracetamol demonstrated on a model plant Lemna minor.

Lemna minor is often used in environmental risk assessment and it can be supposed that usually evaluated parameters will be reliable even for assessing the risk of pharmaceuticals. Subtle changes in duckweed plant number, biomass production, and leaf area size induced by 10-day-exposure to diclofenac (DCF) and paracetamol (PCT) (0.1, 10, and 100 μg/L), excepting 100 μg/L DCF, are in contrast with considerable changes on biochemical and histochemical level. Both drugs caused a decrease in content of photosynthetic pigments (by up to 50%), an increase in non-photochemical quenching (by 65%) and decrease in relative chlorophyll fluorescence decay values (by up to 90% with DCF). Both DCF and especially PCT increased amount of reactive nitrogen and oxygen species in roots. DCF-induced effects included mainly increased lipid peroxidation (by 78%), disturbation in membrane integrity and lowering both oxidoreductase and dehydrogenase activities (by 30%). PCT increased the content of soluble proteins and phenolics. Higher concentrations of both DCF and PCT increased the levels of oxidised ascorbate (by 30%) and oxidised thiols (by up to 84% with DCF). Glutathion-reductase activity was elevated by both pharmaceuticals (nearly by 90%), glutathion-S-transferase activity increased mainly with PCT (by 22%). The early and sensitive indicators of DCF and PCT phytotoxicity stress in duckweed are mainly the changes in biochemical processes, connected with activation of defense mechanisms against oxidative stress.

Various Extraction Methods for Obtaining Stilbenes from Grape Cane of Vitis vinifera L.

Grape cane, leaves and grape marc are waste products from viticulture, which can be used to obtain secondary stilbene derivatives with high antioxidant value. The presented work compares several extraction methods: maceration at laboratory temperature, extraction at elevated temperature, fluidized-bed extraction, Soxhlet extraction, microwave-assisted extraction, and accelerated solvent extraction. To obtain trans-resveratrol, trans-ε-viniferin and r2-viniferin from grape cane of the V. vinifera variety Cabernet Moravia, various conditions were studied: different solvents, using powdered versus cut cane material, different extraction times, and one-step or multiple extractions. The largest concentrations found were 6030 ± 680 µg/g dry weight (d.w.) for trans-resveratrol, 2260 ± 90 µg/g d.w. for trans-ε-viniferin, and 510 ± 40 µg/g d.w. for r2-viniferin. The highest amounts of stilbenes (8500 ± 1100 µg/g d.w.) were obtained using accelerated solvent extraction in methanol.

The effects of polycyclic aromatic hydrocarbons and heavy metals on terrestrial annelids in urban soils

The effect of soil contamination by polycyclic aromatic hydrocarbons (PAH) and heavy metals on earthworms and enchytraeids was studied in urban parks, in Brno, Czech Republic. In spring and autumn 2007, annelids were collected and soil samples taken in lawns along transects, at three different distances (1, 5 and 30 m) from streets with heavy traffi c. In both seasons, two parks with two transects each were sampled. Earthworms were collected using the electrical octet method. Enchytraeids were extracted by the wet funnel method from soil cores. All collected annelids were counted and identifi ed. Basic chemical parameters and concentrations of 16 PAH, Cd, Cu, Pb, and Zn were analysed from soil from each sampling point. PAH concentrations were rather low, decreasing with the distance from the street in spring but not in autumn. Heavy metal concentrations did not decrease signifi cantly with increasing distance. Annelid densities did not signifi cantly differ between distances, although there was a trend of increase in the number of earthworms with increasing distance. There were no signifi cant correlations between soil content of PAH or heavy metals and earthworm or enchytraeid densities. Earthworm density and biomass were negatively correlated with soil pH; and enchytraeid density was positively correlated with soil phosphorus.

Disturbing Impact of Outdoor Cattle Husbandry on Community of Arbuscular Mycorrhizal Fungi in Upland Pasture Soil

The aim of the pilot study was to describe the impact of outdoor cattle husbandry on the communities of arbuscular mycorrhizal fungi on the overwintering upland pasture (South Bohemia, Czech Republic). We selected three sites with severe, moderate, and light (control) impact intensity. Roots of plant species with a plant cover area >5% were collected at each site. The ratio of nonmycorrhizal plants species decreased with the decreasing impact of cattle. The highest mycorrhizal colonization was found at the control site on a level of the screened plant community (78.57% of root length) as well as on a species level (97.78% of root length of Plantago major L.). At the severely impacted site, 31.67% and 35.56% of root colonization was found on community and species level (Plantago major L.), respectively. Similar results were found also in the length of extraradical mycelium and arbuscules numbers.

Determination of atrazine and simazine in drinking and surface waters by solid-phase extraction and high performance thin layer chromatography

Abstract A simple high-performance thin layer Chromatographie (HPTLC) method has been developed for the determination of atrazine and simazine herbicides in drinking and surface waters. The method involves solid-phase extraction on C18 Bakerbond cartridges followed by development of the concentrated extracts on HPTLC silica plates with a nitromethane-tetrachloromethane (1:1, v/v) mobile phase and quantitation by UV scanning densitometry. Using the proposed mobile phase composition, pronounced background suppression on the chromatograms of the real water samples was accomplished. The detection limits of the method were 30 and 60 ng l for atrazine and simazine, respectively, at the 80–400 ng l fortification level in the surface waters. The method was successfully applied to the analyses of tap and surface waters with overall recoveries between 58 and 93% and a relative standard deviation below 12%. The results show, that the HPTLC method is sufficiently selective and sensitive to be employed in screening of contaminated waters containing the triazines below the maximum residues limits of the European Community.

Separation and Identification of 1,2,4-Trihydroxynaphthalene-1-O-glucoside in Impatiens glandulifera Royle

Methanolic extract from lyophilized roots of Impatiens glandulifera Royle was analyzed by high performance liquid chromatography using DAD and FLD detection and this revealed one dominant highly fluorescent very unstable substance. The stability of this derivative is strongly dependent on the plant material drying procedure and extraction procedure used. The structure of the substance was established as 1,2,4-trihydroxynaphthalene-1-O-glucoside (THNG) according LC-MS and NMR measurements. When lyophilized plant material was extracted with methanol an almost four times higher amount of THNG was found in the extract, compared to the amount of 2-hydroxy-1,4-naphthoquinone obtained, while in the case of the same lyophilized plant material extracted with water there was no THNG in the extract. The main compounds in this case was 2-hydroxy-1,4-naphthoquinone. In the plant material dried at the laboratory temperature and extracted by methanol there are only traces of THNG.