Michael Raessler

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth

BACKGROUND AND AIMS Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated. METHODS Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma-optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes. KEY RESULTS The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes. CONCLUSIONS The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.

Determination of Water-Extractable Nonstructural Carbohydrates, including Inulin, in Grass Samples with High-Performance Anion Exchange Chromatography and Pulsed Amperometric Detection

The exact and reliable determination of carbohydrates in plant samples of different origin is of great importance with respect to plant physiology. Additionally, the identification and quantification of carbohydrates are necessary for the evaluation of the impact of these compounds on the biogeochemistry of carbon. To attain this goal, it is necessary to analyze a great number of samples with both high sensitivity and selectivity within a limited time frame. This paper presents a rugged and easy method that allows the isocratic chromatographic determination of 12 carbohydrates and sugar alcohols from one sample within 30 min. The method was successfully applied to a variety of plant materials with particular emphasis on perennial ryegrass samples of the species Lolium perenne. The method was easily extended to the analysis of the polysaccharide inulin after its acidic hydrolysis into the corresponding monomers without the need for substantial change of chromatographic conditions or even the use of enzymes. It therefore offers a fundamental advantage for the analysis of the complex mixture of nonstructural carbohydrates often found in plant samples.

A novel single-run dual temperature combustion (SRDTC) method for the determination of organic, in-organic and total carbon in soil samples

The quantification of organic (OC) and inorganic carbon (IC) in soils provides an essential tool for understanding biogeochemical processes. Examples of its potential application are the assessment of the humification degree of soil organic matter, the calculation of carbon fluxes and budgets in terrestrial systems on a regional and global scale and the investigation of the carbon storage potential of soils. The verification of changes in carbon stocks requires an extensive number of samples as well as precise and reliable analyses. Due to the wide variation in the concentrations of the two forms of carbon in solid samples, the exact distinction is very difficult. We present the advantages of a single-run dual temperature combustion method (SRDTC) at 515 degrees C for OC and 925 degrees C for IC, which allows the determination of OC, IC and total carbon (TC) within one single analytical run. The three parameters are analyzed in less than 30 min. Additionally, the method is characterized by a significantly reduced variability and low operator bias, as there is no need of chemical sample pre-treatment. It is applicable to a broad range of varying OC and IC contents, which is demonstrated by the use of numerous synthetic soil mixtures that have been analyzed. Furthermore, SRDTC indicates the presence of thermally instable carbonates, like magnesite, in the sample. Use of silver boats as a catalytic agent results in an improved distinction between OC and IC in this case. To examine the accuracy and reliability of the SRDTC method, it was compared to other techniques frequently used for carbon determination in soil samples: total combustion by elemental analysis to determine TC and acidification of the sample prior to combustion to determine OC. We will show that the rugged SRDTC method offers a substantial progress for both the reliable and rapid OC and IC determination in soil samples where elemental carbon is negligible.

Speciation of Inorganic Arsenic and Selenium in Contaminated Ground Water Samples—Distribution and Long-Term Stability of Species

Abstract This paper presents the results of speciation studies of arsenic and selenium in contaminated ground water samples from Kelheim / Germany. Results are based on separation of ions by HPLC using a phosphate buffer. The separated species were collected in fractions and analysed element-specifically by HG-AAS. Dominating species were arsenate and selenate, while arsenite and selenite were less important. Long-term studies revealed that As (V), Se (IV) and Se (VI) remained stable for up to 12 months, whereas oxidation of As (III) was observed after 3 months.

Determination of trace metals in woodlice and their skins with particular emphasis on quality control

AI, Fe, Mn and Zn were determined in two ditTerent species of woodlouse: Porcellio scaber and Porcellio dilalalus. Both species were cultivated under standardized conditions in a climatic chamber. Moreover, skins of the cultivated animals were collected and analyzed separately to examine a possible way of decontamination by moulting. To obtain enough sample material for each species, IS animals of the same age and size were pooled. For skin analysis, 10–12 skins were collected and pooled. The animals and their skins were dried, ground and digested in pure concentrated nitric acid using multiwave-assisted high-pressure digestion and. finally, analyzed by ICP-OES. Special emphasis was given to quality control: the reference materials Dorm-2, Dogfish Muscle (Squalus acanthias) and SRM 1577b Bovine Liver, were used to evaluate the whole analytical process including sample digestion. These reference materials of animal origin were selected to match the matrix of the samples as closely as possible. While concentrations of Fe. Mn and Zn were traced to both reference materials, the concentration of AI was checked by Dorm-2 only. Analyses of the elements in the reference materials were carried out using four ditTerent wavelengths for each element simultaneously. aiming at the determination of the best suited wavelength for each element. Analyses of woodlouse samples and their skins were finally carried out using the wavelengths with the highest sensitivities after absence of spectral interferences had been demonstrated.

Determination of trace amounts of nitrate in potassium chloride-extracts by HPLC with ultra-violet-detection

Abstract A method is presented that allows the determination of trace amounts of nitrate in 1 M potassium chloride extracts of environmental soil samples from Northern Europe. To avoid the interference of bromine ions on the evaluation of the nitrate peak, 50 µL of a 50 mg/L nitrate standard solution are added to a 5 mL soil extract. This makes sure that the overall nitrate concentration in the spike is >500 µg/L which allows an exact and reliable determination of the nitrate concentration. Analyses of the soil extracts are carried out by anion-exchange HPLC with ultra-violet detection at λ = 210 nm using 0.1 M KCl as eluent. Concentrations down to 25 µg/L of nitrate can be analyzed.

A method for detection and quantification of hydroxyethyl starch in plasma

The use of hydroxyethylstarch (HES) is a controversial issue due to increasing evidence that HES accumulates in plasma and various tissues and therefore leads to unfavourable outcome in critically ill patients [1,2]. No simple methods are available for monitoring HES plasma levels; present technologies to measure HES are based on gas chromatography-mass spectrometry, which are time-consuming and need advanced equipment [3]. Here, we applied Lugols iodine solution (LUGOL; Sigma-Aldrich, Steinheim, Germany) to determinate HES concentrations in plasma and compare the results with those obtained by high-performance liquid chromatography (HPLC). The study was approved by the institutional ethical committee (Friedrich-Schiller-University, Jena) as well as by the animal welfare committee (Thuringer Landesamt fur Lebensmittelsicherheit und Verbraucherschutz, Germany). Blood samples from healthy volunteers were diluted with balanced 6% HES 130/0.4 (Fresenius Kabi, Bad Homburg, Germany) to get concentrations up to 30 mg/ml. Samples were centrifuged (10 minutes, 4,700 × g, 4°C), and plasma aliquots were mixed with 10% trichloroacetic acid and re-centrifuged (5 minutes, 4,700 × g, 4°C). The supernatant was mixed (4:1) with LUGOL and optical density was measured at 530 nm. For in vivo experiments, 5 ml or 10 ml of 6% HES 130/0.4 was infused into wistar rats over 1 hour via a central venous catheter. Blood samples were obtained prior and up to 24 hours after infusion. Plasma samples were prepared as described. For HPLC analysis, plasma samples were diluted 1:10 with water, then mixed with 60% perchloric acid, heated at 90°C for 60 minutes, diluted again (1:50) and subjected to HPLC [4]. For correlation analysis the Pearson correlation coefficient was calculated. Using the LUGOL method, we found a linear correlation (r2 > 0.99) between calculated and measured plasma HES concentrations after serial dilutions of HES 130/0.4 in human whole blood (Figure ​(Figure1a).1a). At a concentration of 6 mg/ml the half life of HES 130/0.4 was estimated to be 1.8 hours (Figure ​(Figure1b).1b). In rats, plasma levels of 2.9 mg/ml and 4.6 mg/ml were measured immediately after infusion of 5 or 10 ml HES 130/0.4. Two hours after infusion the measured levels were only 10% of the initial levels (Figure ​(Figure2).2). Comparing the plasma concentrations measured by LUGOL and HPLC revealed a correlation coefficient of r2 > 0.90 (P < 0.001). Figure 1 Quantification of HES by Lugols iodine solution. (a) Measurement of HES concentration using LUGOLs iodine solution in samples of human whole blood incubated with different HES 130/0.4 concentrations. (b) Time course of HES concentration in human whole ... Figure 2 Time course of HES plasma levels after infusion of 5 or 10 ml HES 130/0.4 solution over 1 hour. Significance was tested using ANOVA (*P < 0.05). The new method described here allows the rapid and simple detection of HES in plasma samples. LUGOL is known to stain polysaccharides, including amylose, amylopectin and glycogen. Amylose and amylopectin, due to their herbal origin, should not be found in blood. However, glycogen might interfere with the measurements, but it is present in only negligible amounts in plasma [5]. Measurement of HES plasma levels in patients could help to gain new insights into HES degradation and plasma accumulation after infusion.