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Title: Detection of bioavailable phosphorus forms for the alga Pseudokirchneriella subcapitata
Other Titles: Detectie van biobeschikbare fosforvormen voor de alg Pseudokirchneriella subcapitata
Authors: Van Moorleghem, Christoff; S0109725
Issue Date: 19-Apr-2013
Abstract: 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: 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 117
ISBN: 978-90-8826-300-2
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Division Soil and Water Management

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