Detection of bioavailable phosphorus forms for the alga Pseudokirchneriella subcapitata

Phosphorus (P) is generally the limiting nutrient for primary production in freshwater systems. Elevated P concentrations can cause eutrophication, potentially resulting in adverse effects on the quality of water systems due to excessive algal blooms. Algal growth is not only related to the total P concentration in water but also to the P speciation i.e. the chemical form of P such as ionic orthophosphate (Pi), organic P forms (mainly phosphate esters) and inorganic colloidal P forms such as iron colloids loaded with P. Current analytical methods detect different proportions of these P forms and simple detection techniques, like colorimetric methods, are not sufficiently successful in predicting the bioavailable P fraction. The general objective of this study was to quantify the bioavailability of dissolved (<0.45 µm) P species in freshwater in relation to P detected by most frequently used analytical methods. Previous studies, mostly neglecting colloidal P forms, report that the long term bioavailable P fraction of organic P range from 0 to 100%. The long term availability is generally defined as the P that becomes ultimately bioavailable by naturally occurring processes and is higher than the short term (readily) P bioavailability.Four analytical methods to measure P in solution were compared: ion chromatography (IC), malachite green colorimetric method (CM), diffusive gradient in thin films technique (DGT) and, for total dissolved P (TDP), inductively coupled plasma with optical emission spectroscopy (ICP-OES). These methods were compared on three sets of solutions in the dissolved (filtered over < 0.45 µm) fraction: solutions with model organic P compounds, suspensions of synthesized inorganic Fe and Al colloids loaded with P, and environmental P containing samples. Model organic P compounds were found to be generally rather refractory towards both colorimetry and ion chromatography (typical P detection < 2% of TDP) while synthetic inorganic colloids were detected almost completely by colorimetry and partially by chromatography. The DGT technique is the best method to exclude inorganic colloidal P from in measurement of Pi. It is hypothesized that colloidal particles are too large to diffuse through the diffusive DGT layer. However, substantial fractions of certain organic P compounds might contribute to the DGT measurement since colorimetric detection of the DGT eluates was lower than TDP detection for three out of the five tested organic P compounds. Detection of organic P by DGT could be reduced by (i) using ion chromatographic or colorimetric detection of the DGT eluates instead of TDP detection, (ii) decreasing the acidity of the eluents and (iii) decreasing the elution time. From a wide set of 271 samples of soil pore water, groundwater and surface water, data suggest that surface water P is largely present as orthophosphate and phosphate sorbed onto inorganic colloids, whereas organic P forms contribute more importantly in groundwaters and soil pore waters.It is well established that inorganic colloidal and dissolved organic forms of P are bioavailable to freshwater algae but analytical P measurements have rarely been compared with bioavailability for both model systems and environmental samples. In a second part, the P bioavailability of similar sample sets as described above (organic, colloidal and environmental) was examined in relation to analytical measurements of initial P concentrations. The P bioavailability test used the 14 day growth response of an axenic culture of Pseudokirchneriella subcapitata in solution with P as limiting nutrient. Growth on these P forms was referenced to supplies of Pi as operational definition of the relative potential bioavailability. The 14 environmental samples were standardized to equal concentrations of dissolved organic C and other nutrients. The bioavailability of organic P compounds ranged from 1 to 70% of Pi while these forms were generally not detected by colorimetry or ion chromatography The bioavailability of colloidal P ranged from 55 to 85% of Pi and these forms were completely detected by colorimetry and partially by ion chromatography. The bioavailability of total dissolved P in the environmental samples ranged from 7 to 85% (mean 43%) of Pi. The P detected by ion chromatography underestimates largely, while colorimetric detection and TDP overestimate the bioavailable P fraction by, on average 44% (colorimetric detected P) or 57% (TDP) in the environmental samples. We conclude that colorimetric P detection is the best index among the three tested for predicting long term availability of environmental dissolved P in which colloidal P contributes more importantly than organic P. Phosphorus starved algae have a capacity to rapidly take up P when resupplied with P. In the third part of this research, an experiment to measure to what extent P starvation enhances the potential of algae to utilize organic P forms was carried out. The initial (< 0.5 h) Pi uptake rates of cells of Pseudokirchneriella subcapitata increased up to 18-fold with increasing starvation. Algae from different levels of P starvation were subsequently exposed to different model organic P forms and carrier-free 33Pi. Uptake (1h) of P from organic P increased up to 5-fold with increasing P starvation. The bioavailability of organic P, relative to Pi, was calculated from uptake of 31P and 33P isotopes assuming no isotopic exchange with organic P forms. This relative bioavailability ranged from 0 to 57% and remained generally unaffected by the extent of P-starvation. This result was found for cells that were either or not treated by a wash method to remove extracellular phosphatases. Short term P uptake rate sharply increases with decreasing internal P content of the algal cells but the bioavailability of organic P, relative to PO4, is not enhanced. Such finding suggests that P-starvation enhances the PO4 uptake capacity and the hydrolysis capacity of organic P forms to about the same extent.Overall, this study illustrates that colloidal P forms may contribute importantly in environmental water samples. These P forms contribute importantly to P detection by ion chromatography and colorimetry measurement but its bioavailability would remain rather limited. It is not expected that organic P forms in environmental waters contribute significantly to ion chromatographical or colorimetrical detection but these forms may contribute to P bioavailability, with increasing importance in function of time (minutes versus weeks). Future research should test the DGT technique for P bioavailability estimation in environmental samples as indirect evidence in this work suggest that DGT may be superior than colorimetric P detection. Subsequently, the short term P bioavailability of environmental inorganic colloidal P may be examined as well as the presence of other algal and bacterial species on this P bioavailability.Table of contents Dankwoord I Summary III Samenvatting VII List of abbreviations XI Table of contents XIII Chapter 1. Phosphorus in fresh-water systems: environmental context, speciation and bioavailability for algae 1 Chapter 2. Effect of organic P forms and P present in inorganic colloids on the determination of dissolved P in environmental samples 21 Chapter 3. The bioavailability of colloidal and dissolved organic phosphorus for the alga Pseudokirchneriella subcapitata in relation to analytical phosphorus measurements 41 Chapter 4. Bioavailability of organic phosphorus to Pseudokirchneriella subcapitata as affected by phosphorus starvation: an isotope dilution study 63 Chapter 5. General conclusions 83 Appendices 95 References 107 List of publications 117nrpages: 135status: publishe

DAX-8 fractionation of dissolved organic matter (DOM) from soils: calibration with test components and application to contrasting soil solutions

Most methods to fractionate natural dissolved organic matter (DOM) rely on sorption of acidified DOM samples onto XAD-8 or DAX-8 resin. Procedural differences among methods are large and their interpretation is limited because there is a lack of calibration with DOM model molecules. An automated column-based DOM fractionation method was set up for 10-ml DOM samples, dividing DOM into hydrophilic (HPI), hydrophobic acid (HPOA) and hydrophobic neutral (HPON) fractions. Fifteen DOM model components were tested in isolation and in combination. Three reference DOM samples of the International Humic Substances Society were included to facilitate comparison with other methods. Aliphatic low-molecular-weight acids (LMWAs) and carbohydrates were classified as HPI DOM, but some LMWAs showed also a partial HPO character. Aromatic LMWAs and polyphenols partitioned in the HPOA fraction, menadione (quinone) and geraniol (terpenoid) in HPON DOM. Molecules with log K-ow > 0.5 had negligible HPI fractions. The HPO molecules except geraniol had specific UV absorbance (SUVA, measure for aromaticity) > 3 litres g-1 cm-1 while HPI molecules had SUVA values < 3 litres g-1 cm-1. Distributions of DOM from eight soils ranged from 31 to 72% HPI, 25 to 46% HPOA and 2 to 28% HPON of total dissolved organic carbon. The SUVA of the HPI DOM was consistently smaller compared with the HPOA DOM. The SUVA of the natural DOM samples was not explained statistically by fractionation and the variation coefficient of SUVA among samples was not reduced by fractionation. Hence, fractionation did not reduce the variability in this DOM property, which casts some doubts on the practical role of DOM fractionation in predicting DOM properties.status: publishe

Iron colloids reduce the bioavailability of phosphorus to the green alga Raphidocelis subcapitata

Phosphorus (P) is a limiting nutrient in many aquatic systems. The bioavailability of P in natural waters strongly depends on its speciation. In this study, structural properties of iron colloids were determined and related to their effect on P sorption and P bioavailability. The freshwater green alga Raphidocelis subcapitata was exposed to media spiked with radiolabelled 33-PO4, and the uptake of 33-P was monitored for 1 h. The media contained various concentrations of synthetic iron colloids with a size between 10 kDa and 0.45 μm. The iron colloids were stabilised by natural organic matter. EXAFS spectroscopy showed that these colloids predominantly consisted of ferrihydrite with small amounts of organically complexed Fe. In colloid-free treatments, the P uptake flux by the algae obeyed Michaelis-Menten kinetics. In the presence of iron colloids at 9 or 90 μM Fe, corresponding to molar P:Fe ratios between 0.02 and 0.17, the truly dissolved P (<10 kDa) was between 4 and 60% of the total dissolved P (<0.45 μm). These colloids reduced the P uptake flux by R. subcapitata compared to colloid-free treatments at the same total dissolved P concentration. However, the P uptake flux from colloid containing solutions equalled that from colloid-free ones when expressed as truly dissolved P. This demonstrates that colloidal P did not contribute to the P uptake flux. It is concluded that, on the short term, phosphate adsorbed to ferrihydrite colloids is not available to the green alga R. subcapitata.publisher: Elsevier articletitle: Iron colloids reduce the bioavailability of phosphorus to the green alga Raphidocelis subcapitata journaltitle: Water Research articlelink: http://dx.doi.org/10.1016/j.watres.2014.04.010 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved.status: publishe

The dissociation kinetics of Cu-dissolved organic matter complexes from soil and soil amendments

Complexes between dissolved organic matter (DOM) and copper (Cu) that dissociate very slowly can theoretically facilitate Cu leaching to the groundwater. Data on dissociation kinetics of Cu-DOM complexes present in soil and in soil amendments are limited. The dissociation kinetics of different Cu-DOM complexes from soil, wastewater, pig manure and sewage sludge was measured with the Competitive Ligand Exchange Method (CLEM) and Diffusive Gradient in Thin films (DGT) technique. The solutions were set at constant pH, Ca concentration and free Cu2+ activity to allow comparison between the different samples. The average dissociation rate constant kd of the complexes, as measured by CLEM, was about 10-3 s-1 and the fractions of dissolved Cu that were undissociated after 8 hours ranged from <1 to 25%. These fractions determined by CLEM were significantly correlated with the non-labile fractions (0–82%) determined in the DGT tests and data analysis show that DGT data can be predicted from CLEM data. The dissociation rates decreased when Cu-DOM complexes had been equilibrated at lower Cu2+ activities. Increasing the Cu-DOM contact time (7–297 days) decreased the dissociation rate. The non-labile fractions were positively correlated with the specific UV absorbance suggesting that aromatic moieties in DOM hold non-labile Cu. All natural Cu-DOM complexes contained a detectable fraction with a dissociation rate constant kd lower than 10-5 s-1 which can theoretically lead to non-equilibrium conditions and leaching risks in soil.status: publishe

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance