Water quality in a shallow eutrophic lake is unaffected by extensive thinning of planktivorous and benthivorous fish species.

Ambitious to fulfill the European Water Framework Directive obligations, the European governments support projects to rehabilitate lakes with poor water quality. However, most lake restorations having relied on biomanipulation by fish thinning have failed to improve or even maintain water quality. Previous attempts removed all target fish species simultaneously, thus making it impossible to assess the specific impact of each feeding group on water chemistry. Lake Bromme was selected for extensive, time-selective fish biomanipulation to improve water clarity and promote submerged macrophytes and piscivorous fish stocks over a three-year monitoring period. Thinning of adult benthivorous bream (Abramis brama) and tench (Tinca tinca) was conducted throughout year one while thinning in years two and three targeted planktivorous roach (Rutilus rutilus), juvenile bream, and small perch (Perca fluviatilis). Yearly fish surveys assessed changes in fish population structure and biomass. Water quality parameters were monitored continually, and the cover of submerged macrophytes was surveyed annually via sonar. We found no improvement in water clarity or reductions of nutrients, organic particles, chlorophyll concentrations, or watercolor, despite a 6-fold thinning of total estimated fish biomass, from 112 to 19 kg ha-1. Over the period, the macrophyte cover increased from 0.8 to 13.5 %, but no recruitment of large piscivorous fish (perch and pike (Esox lucius) > 10 cm) was detected. We found higher correlations of particle concentration and water clarity to water temperature than to wind speed, which indicates sediment particle resuspension by the remaining fish community (mostly carp Cyprinus carpio) that forage on benthos in shallow lakes. Further system-ecological research in Lake Bromme should evaluate whether thinning the stock of carp and increasing plant cover may improve water quality and test which optical properties sustain high water turbidity and prevent shallow, eutrophic lakes like Lake Bromme from responding to intense fish thinning.

Synthetic and biopolymers for lake restoration - An evaluation of flocculation mechanism and dewatering performance.

In the frame of the global phosphorus (P) crisis and ongoing eutrophication issues in the environmental sector, lake sediment can be considered as an alternative P source after its removal from eutrophic lakes. However, high water contents make sediment dewatering a crucial step towards the efficient reusability of remaining solids. The application of polymeric substances facilitates solid-liquid separation by flocculation of suspended particles. To lower the environmental risk of contamination with toxic, non-biodegradable monomeric residues during and after the application of synthetic polyacrylamide(PAM)-based polymers, switching to natural polymeric substances (biopolymers), e.g., starch- or chitosan-based, is increasingly emphasized. The dewatering performance of four conventional PAM-based polymers was compared to two starch- and one chitosan-based biopolymer. Laboratory experiments were conducted to determine the dewatering rate, floc size and strength, and reject water quality. Biopolymers generally caused the formation of smaller but less shear-sensitive flocs, and lower P levels in the reject water compared to synthetic polymers. Dewatering performance was correlated to the most important functioning influencing polymer-specific properties intrinsic viscosity (polymer extension) and surface charge density (CD). Due to the high CD and low intrinsic viscosity of the biopolymers, electrostatic patch flocculation seems to be the favored flocculation mechanism, while for synthetic polymers bridging seems to be dominating. Solid-liquid separation technologies should be adjusted to the resulting floc size and structure, while surface CD and intrinsic viscosity are important properties for the choice of biopolymer. Overall, biopolymers can function as a more environmentally friendly alternative to synthetic products for lake sediment dewatering accompanied by the potential for P recovery.

Characterization of biochar produced from sewage sludge and its potential use as a substrate and plant growth improver.

Biochar is a product rich in carbon produced by pyrolysis of different kinds of biomass and it modifies the physical, chemical, and biological properties of soil. In this study, biochar, produced at different pyrolysis temperatures (590 °C, 665 °C, and 765 °C), was physico-chemically characterized. It was explored whether biochar made from sewage sludge can become an alternative solution for future water and phosphorus management in agricultural production. A pot experiment was conducted using Chinese cabbage (Brassica rapa subsp. pekinensis) to investigate the effect of applying different biochars to the substrate, taking into account different growth parameters and the biochemical composition of the plants, as well as the physico-chemical properties of the substrate. According to the results, pyrolysis temperature influences the content of elements in biochar and their availability to plants, with total phosphorus contents in biochar ranging from 4.6% to 4.9%. In addition, applying biochar to the substrate significantly increases the volumetric water content up to 4.5 fold more compared to the control, which indicates a promising application in drought stress conditions and, at the same time, is a source of nutrients and can help to reduce the amount of mineral fertilizer application.

Variation in Phosphorus Speciation of Sewage Sludge throughout Three Wastewater Treatment Plants: Determined by Sequential Extraction Combined with Microscopy, NMR Spectroscopy, and Powder X-ray Diffraction.

The variation in phosphorus (P) speciation of sewage sludge throughout three wastewater treatment plants (WWTPs) was obtained by combining sequential P extraction with optical and scanning electron microscopy (SEM), chemical analyses, powder X-ray diffraction (PXRD), and 27Al and 31P nuclear magnetic resonance (NMR) spectroscopy. The WWTPs combine chemical P removal (CPR) and enhanced biological P removal (EBPR) and were compared to understand the effect of iron (Fe) dosing with and without codosing of aluminum (Al) and thermal hydrolysis on the P speciation. 31P NMR showed comparable inorganic orthophosphate (ortho-P, 53-60% of total P) and organophosphate (organic-P, 37-45%) in primary sludge, whereas polyphosphate (poly-P, 23-44%) from poly-P accumulating organisms (PAOs) was mainly observed in the secondary sludge. Inorganic ortho-P (90-98%) dominated after anaerobic digestion, which degraded poly-P and most organic-P. The inorganic ortho-P was mainly Fe bound P (Fe-P), especially after anaerobic digestion (71%). Codosing of Fe and Al led to two comparable fractions: Fe-P (38%) and P sorbed on amorphous Al (hydr)oxides (38%). Vivianite was identified in all samples by microscopy and chemical extraction but was PXRD amorphous in 12 out of 17 samples. Thus, vivianite may be more common in sewage sludge than previously known.

Quantification of Biologically and Chemically Bound Phosphorus in Activated Sludge from Full-Scale Plants with Biological P-Removal.

Phosphorus (P) is present in activated sludge from wastewater treatment plants in the form of metal salt precipitates, extracellular polymeric substances, or bound into the biomass, for example, as intracellular polyphosphate (poly-P). Several methods for a reliable quantification of the different P-fractions have recently been developed, and this study combines them to obtain a comprehensive P mass-balance of activated sludge from four enhanced biological phosphate removal (EBPR) plants. Chemical characterization by ICP-OES and sequential P fractionation showed that chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals. Raman microspectroscopy, solution state 31P NMR, and 31P MAS NMR spectroscopy applied before and after anaerobic P-release experiments, were used to quantify poly-P, which constituted 22-54% of total P and was found in approximately 25% of all bacterial cells. Raman microspectroscopy in combination with fluorescence in situ hybridization was used to quantify poly-P in known polyphosphate-accumulating organisms (PAO) (Tetrasphaera, Candidatus Accumulibacter, and Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. Interestingly, only 1-13% of total P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are known.

Speciation of inositol phosphates in lake sediments by ion-exchange chromatography coupled with mass spectrometry, inductively coupled plasma atomic emission spectroscopy, and 31P NMR spectroscopy.

A method for the detection and speciation of inositol phosphates (InsP(n)) in sediment samples was tested, utilizing oxalate-oxalic acid extraction followed by determination by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) using electrospray ionization (ESI) in negative mode. The chromatographic separation was carried out using water and ammonium bicarbonate as mobile phase in gradient mode. Data acquisition under MS/MS was attained by multiple reaction monitoring. The technique provided a sensitive and selective detection of InsP(n) in sediment samples. Several forms of InsP(n) in the oxalate-oxalic acid extracted sediment were identified. InsP6 was the dominating form constituting 0.250 mg P/g DW (dry weight); InsP5 and InsP4 constituted 0.045 and 0.014 mg P/g DW, respectively. The detection limit of the LC-ESI-MS/MS method was 0.03 µM InsP(n), which is superior to the currently used method for the identification of InsP(n), (31)P nuclear magnetic resonance spectroscopy ((31)P NMR). Additionally sample handling time was significantly reduced.

Characterization of phosphate sequestration by a lanthanum modified bentonite clay: a solid-state NMR, EXAFS, and PXRD study.

Phosphate (Pi) sequestration by a lanthanum (La) exchanged clay mineral (La-Bentonite), which is extensively used in chemical lake restoration, was investigated on the molecular level using a combination of (31)P and (139)La solid state NMR spectroscopy (SSNMR), extended X-ray absorption spectroscopy (EXAFS), powder X-ray diffraction (PXRD) and sorption studies. (31)P SSNMR show that all Pi was immobilized as rhabdophane (LaPO4·n H2O, n ≤ 3), which was further supported by (139)La SSNMR and EXAFS. However, PXRD results were ambiguous with respect to rhabdophane and monazite (LaPO4). Adsorption studies showed that at dissolved organic carbon (DOC) concentration above ca. 250 µM the binding capacity was only 50% of the theoretical value or even less. No other La or Pi phases were detected by SSNMR and EXAFS indicating the effect of DOC is kinetic. Moreover, (31)P SSNMR showed that rhabdophane formed upon Pi sequestration is in close proximity to the clay matrix.

Do new lakes behave like natural lakes regarding sediment composition and phosphorus fluxes?

Numerous new lakes have been established during the last few decades. Lakes established on former agricultural soils often have high legacy phosphorus (P)-content, which constitutes a risk for potential internal P-loading after the lake is formed. In this study, we compared the P release and sediment P-pools from 31 new lakes and 31 natural lakes, to assess their similarities and differences. A suite of other sediment characteristics was identified and compared for both natural and new lakes; catchment characteristics of the new lakes also were analyzed. P release from the sediment of new lakes was significantly lower than from natural lakes (13.2 mg P m-2 d-1) compared to new lakes (6.9 mg P m-2 d-1). The P release was found to be low when molar Fe:P ratios were above 10. A significant correlation was found between the content of mobile-P (loosely adsorbed P, iron-bound P, and leachable organic P) and TP in the sediment, irrespective of lake type. The composition of the mobile P-pool also differed, with the new non-excavated lakes showing a higher proportion of RP-BD; both new lake types had significantly (p = 0.021) lower proportions of nrP, compared to natural lakes in the uppermost 10 cm sediment. In addition, variance in P release and mobile-P content of new lakes could be explained in terms of the land use of the catchments. Most sediment characteristics of new lakes established without topsoil excavation reached the average levels of natural Danish lakes with respect to density, organic content and P content within 20-30 years, while excavated lakes showed no such tendencies.

Flocculating and dewatering of lake sediment: An in-situ pilot study comparing synthetic polymers and biopolymers for restoring lake water quality and reusing phosphorus.

Dredging of lake sediment is a method to remove accumulated phosphorus and nitrogen in lakes and thereby reducing the risk of eutrophication. After dredging, the sediment is dewatered to reduce the volume. It is important to get a high dry matter content and ensure that the filtrate does not contain harmful compounds so it can be returned to the lake. A pilot-scale belt filter and flexible intermediate bulk containers (FIBC) were used for dewatering lake sediment with the sediment treated with a synthetic polymer or three different biopolymers. The goal of the study was to retain the phosphorus in the filter cake while returning the filtrate to the lake with a minimal phosphorus content. Results showed dry matter content of up to 16 % in the dewatered sediment and the sediment retained 96-99 % of the phosphorus. Furthermore, nitrogen was reduced by 27-71 % in the filtrate water. Toxicity tests found low ecotoxicity for most biopolymer filtrates, whereas synthetic polymer showed the highest potential ecotoxicity. Consequently, biopolymers provided satisfactory results, proving more environmentally friendly despite requiring longer filtration time.

Methane and carbon dioxide fluxes at high spatiotemporal resolution from a small temperate lake.

Lakes are hotspots for CH4 and CO2 effluxes, but their magnitude and underlying drivers are still uncertain due to high spatiotemporal variation within and between lakes. We measured CH4 and CO2 fluxes at high temporal (hourly) and spatial resolution (approx. 13 m) using 24 automatic floating chambers equipped with continuously recording sensors that enabled the determination of diffusive and ebullitive gas fluxes. Additionally, we measured potential drivers such as weather patterns, water temperature, and O2 above the sediment. During five days in autumn 2021, we conducted measurements at 88 sites in a small, shallow eutrophic Danish Lake. CH4 ebullition was intense (mean 54.8 µmol m-2 h-1) and showed pronounced spatiotemporal variation. Ebullition rates were highest in deeper, hypoxic water (5-7 m). Diffusive CH4 fluxes were 4-fold lower (mean 15.0 µmol m-2 h-1) and spatially less variable than ebullitive fluxes, and significantly lower above hard sediments and submerged macrophyte stands. CO2 concentration in surface waters was permanently supersaturated at the mid-lake station, and diffusive fluxes (mean 919 µmol m-2 h-1) tended to be higher from deeper waters and increased with wind speed. To obtain mean whole-lake fluxes within an uncertainty of 20 %, we estimated that 72 sites for CH4 ebullition, 39 sites for diffusive CH4 fluxes and 27 sites for diffusive CO2 fluxes would be required. Thus, accurate whole-lake quantification of the dominant ebullitive CH4 flux requires simultaneous operation of many automated floating chambers. High spatiotemporal variability challenges the identification of essential drivers and current methods for upscaling lake CH4 and CO2 fluxes. We successfully overcame this challenge by using automatic floating chambers, which offer continuous CH4 and CO2 flux measurements at high temporal resolution and, thus, are an improvement over existing approaches.

Crushed concrete as a phosphate binding material: a potential new management tool.

To avoid eutrophication of receiving waters, effective methods to remove P in urban and agricultural runoff are needed. Crushed concrete may be an effective filter material to remove dissolved and particulate P. Five types of crushed concrete were tested in the laboratory to evaluate the retention capacity of dissolved P. All types removed P very effectively (5.1-19.6 g P kg(-1) concrete), while the possible release of bound P varied between 0.4 and 4.6%. The retention rate was positively related to a decreasing concrete grain size due to an increasing surface area for binding. The P retention was also related to a marked increase in pH (up to pH 12), and the highest retention was observed when pH was high. Under these circumstances, column experiments showed outlet P concentrations <0.0075 mg P L(-1). Furthermore, experiments revealed that release of heavy metals is of no importance for the treated water. We demonstrate that crushed concrete can be an effective tool to remove P in urban and agricultural runoff as filter material in sedimentation/infiltration ponds provided that pH in the treated water is neutralized or the water is diluted before outlet to avoid undesired effects caused by the high pH.

Diagenesis of settling seston: identity and transformations of organic phosphorus.

Solution (31)phosphorus NMR spectroscopy and sequential fractionation were used to follow diagenetic changes in phosphorus forms during decomposition of settling seston in Lake Nordborg, a shallow eutrophic lake in Denmark. In a decomposition experiment, seston released >60% of their total phosphorus during ~50 days incubation, although seston collected during summer contained more phosphorus and released it over a longer period compared to seston collected during spring. Seston decomposition increased concentrations of potentially bioavailable polyphosphate and phosphodiesters, but also promoted the formation of refractory phosphorus forms that might be buried permanently in the sediment. Combining these results with in situ measurements of phosphorus concentrations in lake water and sediment traps revealed that the release from settling seston plays only a minor role in the accumulation of phosphorus in the hypolimnion of Lake Nordborg.

The impact of climate change and eutrophication on phosphorus forms in sediment: Results from a long-term lake mesocosm experiment.

Characteristics of bottom sediments in lake mesocosms 11 years after starting the experiment were studied in order to determine the effects of nutrient loading, temperature increase and vegetation type on concentration and vertical distribution of phosphorus (P) forms. The experimental setup consisted of 24 outdoor flow-through mesocosms with two nutrient treatments - low (L) and high (H) and 3 temperature levels - ambient (T0), heated by 2-4 °C (T1) and 3-6 °C (T2) in four replicates. Thickness of the organic sediment was measured and the sediment analysed for dry weight, organic matter, and P fractions (according to a sequential extraction scheme) and organic P compounds (by 31P nuclear magnetic resonance spectroscopy). Higher nutrient loading led to increased sediment accumulation and higher concentration of total P and most P fractions, except P bound to aluminium and humic matter. The dominant vegetation type covaried with nutrient levels. Vertical gradients in Ca bound P and mobile P in low nutrient mesocosms was perhaps a result of P coprecipitation with calcite on macrophytes and P uptake by roots indicating that in macrophyte-rich lakes, plants can be important modifiers of early P diagenesis. Temperature alone did not significantly affect sediment accumulation rate but the interaction effect between nutrient and temperature treatments was significant. At high nutrient loading, sediment thickness decreased with increasing temperature, but at low nutrient loading, it increased with warming. The effect of warming on sediment composition became obvious only in nutrient enriched mesocosms showing that eutrophication makes shallow lake ecosystems more susceptible to climate change.

Occurrence of orthophosphate monoesters in lake sediments: significance of myo- and scyllo-inositol hexakisphosphate.

Orthophosphate monoesters often constitute a significant fraction of total phosphorus in lake sediments. The knowledge on the specific composition and recalcitrance of these compounds is however limited. The main aim was therefore to identify and quantify specific orthophosphate monoesters in sediment from 15 Danish lakes by solution (31)P NMR spectroscopy. The four most quantitatively important orthophosphate monoesters were myo-inositol hexakisphosphate (myo-IP(6)), scyllo-inositol hexakisphosphate (scyllo-IP(6)) α-glycerophosphate (α-GP) and ß-glycerophosphate (ß-GP). The compounds were identified in 9, 4, 8 and in all 15 lakes, respectively. In total these four components made up 46-100% of the orthophosphate monoester pool. The glycerophosphates (GPs) are most likely degradation products of phospholipids, created as an artifact by the alkaline extraction procedure used for (31)P NMR spectroscopy, while the inositol hexakisphosphates (IPs) are naturally occurring compounds. There was a significant positive correlation between myo-IP(6) and total aluminium in the sediment and a negative correlation between myo-IP(6) and lake water pH, suggesting that myo-IP(6) is stabilized in the sediment by adsorption at slightly acidic or neutral conditions. In three lakes, the depth distribution of the orthophosphate monoesters was investigated. The content of scyllo-IP(6) and myo-IP(6) was constant with sediment depth in two of the lakes while the content of myo-IP(6) decreased with depth in one of the lakes. In all cases the IPs seem to be preserved with sediment depth to a higher extent than the orthophosphate diesters and especially the GPs suggesting that IPs can be a sink for phosphorus in the lake ecosystem or at least delay P-recycling for years.

The combined effects of macrophytes (Vallisneria denseserrulata) and a lanthanum-modified bentonite on water quality of shallow eutrophic lakes: A mesocosm study.

Establishment of submerged macrophyte beds and application of chemical phosphorus inactivation are common lake restoration methods for reducing internal phosphorus loading. The two methods operate via different mechanisms and may potentially supplement each other, especially when internal phosphorous loading is continuously high. However, their combined effects have so far not been elucidated. Here, we investigated the combined impact of the submerged macrophyte Vallisneria denseserrulata and a lanthanum-modified bentonite (Phoslock®) on water quality in a 12-week mesocosm experiment. The combined treatment led to stronger improvement of water quality and a more pronounced reduction of porewater soluble reactive phosphorus than each of the two measures. In the combined treatment, total porewater soluble reactive phosphorus in the top 10 cm sediment layers decreased by 78% compared with the control group without Phoslock® and submerged macrophytes. Besides, in the upper 0-1 cm sediment layer, mobile phosphorus was transformed into recalcitrant forms (e.g. the proportion of HCl-P increased to 64%), while in the deeper layers, (hydr)oxides-bound phosphorus species increased 17-28%. Phoslock®, however, reduced the clonal growth of V. denseserrulata by 35% of biomass (dry weight) and 27% of plant density. Our study indicated that Phoslock® and submerged macrophytes may complement each other in the early stage of lake restoration following external nutrient loading reduction in eutrophic lakes, potentially accelerating the restoration process, especially in those lakes where the internal phosphorus loading is high.

Quantitative determination of vivianite in sewage sludge by a phosphate extraction protocol validated by PXRD, SEM-EDS, and 31P NMR spectroscopy towards efficient vivianite recovery.

Vivianite (Fe3(PO4)2⋅8H2O) is a potential phosphorus (P) recovery product from wastewater treatment plants (WWTPs). However, routine methods for quantification of vivianite bound P (vivianite-P) are needed to establish the link between vivianite formation and operating conditions, as current approaches require specialized instrumentation (Mössbauer or synchrotron). This study modified a conventional sequential P extraction protocol by insertion of an extraction step (0.2% 2,2'-bipyridine + 0.1 M KCl) targeting vivianite-P (Gu et al., Water Research, 2016, 103, 352-361). This protocol was tested on digested and dewatered sludge from two WWTPs, in which vivianite (molar Fe:P ratios of 1.0-1.6) was unambiguously identified by optical microscopy, powder X-ray diffraction, and scanning electron microscopy with energy dispersive X-ray spectroscopy. The results showed that vivianite-P was separated from iron(III)-bound P (Fe(III)-P) in the sludge. Vivianite-P constituted about half of the total P (TP) in the sludge from a Fe dosing chemical P removal (CPR) WWTP, but only 16-26% of TP in the sludge from a WWTP using a combination of Fe dosing CPR and enhanced biological P removal (EBPR). The modified protocol revealed that Fe-bound P (Fe-P, i.e., vivianite-P + Fe(III)-P) was the dominant P fraction, in agreement with quantitative 31P nuclear magnetic resonance (NMR) experiments. Moreover, it was shown that the conventional P extraction protocol underestimated the Fe-P content by 6-35%. The established protocol represents a reliable in-house analytical method that can distinguish and quantify vivianite-P and Fe(III)-P in sludge, i.e. facilitate optimized vivianite production at WWTPs.

Calcium Affects Polyphosphate and Lipid Accumulation in Mucoromycota Fungi.

Calcium controls important processes in fungal metabolism, such as hyphae growth, cell wall synthesis, and stress tolerance. Recently, it was reported that calcium affects polyphosphate and lipid accumulation in fungi. The purpose of this study was to assess the effect of calcium on the accumulation of lipids and polyphosphate for six oleaginous Mucoromycota fungi grown under different phosphorus/pH conditions. A Duetz microtiter plate system (Duetz MTPS) was used for the cultivation. The compositional profile of the microbial biomass was recorded using Fourier-transform infrared spectroscopy, the high throughput screening extension (FTIR-HTS). Lipid content and fatty acid profiles were determined using gas chromatography (GC). Cellular phosphorus was determined using assay-based UV-Vis spectroscopy, and accumulated phosphates were characterized using solid-state 31P nuclear magnetic resonance spectroscopy. Glucose consumption was estimated by FTIR-attenuated total reflection (FTIR-ATR). Overall, the data indicated that calcium availability enhances polyphosphate accumulation in Mucoromycota fungi, while calcium deficiency increases lipid production, especially under acidic conditions (pH 2-3) caused by the phosphorus limitation. In addition, it was observed that under acidic conditions, calcium deficiency leads to increase in carotenoid production. It can be concluded that calcium availability can be used as an optimization parameter in fungal fermentation processes to enhance the production of lipids or polyphosphates.

Extraction and quantification of polyphosphates in activated sludge from waste water treatment plants by 31P NMR spectroscopy.

Polyphosphate (poly-P) is a major constituent in activated sludge from wastewater treatment plants with enhanced biological phosphorus removal due to poly-P synthesis by poly-P accumulating organisms where it plays an important role for recovery of phosphorus from waste water. Our aim was to develop a reliable protocol for poly-P quantification by 31P NMR spectroscopy. This has so far been complicated by the risks of inefficient extraction and poly-P hydrolysis in the extracts. A protocol for complete extraction, identification and quantification of poly-P in activated sludge from a waste water treatment plant was identified based on test and evaluation of existing extraction protocols in combination with poly-P determination and quantification by solution and solid state 31P NMR spectroscopy. The total poly-P middle group content was quantified by solid state NMR for comparison with the poly-P middle groups quantified by solution NMR, which is novel. Three different extraction protocols previously used in literature were compared: 1) a single 0.25 M NaOH-0.05 M EDTA extraction, 2) a 0.05 M EDTA pre-extraction followed by a 0.25 M NaOH main extraction and 3) a 0.05 M EDTA pre-extraction followed by a 0.25 M NaOH-0.05 M EDTA main extraction. The results showed that the extraction protocol 2 was optimal for fresh activated sludge, extracting 10.8 ±â€¯0.4 to 11.4 ±â€¯1.2 mgP/gDW poly-P. Extraction protocols 1 and 3 extracted less than 9.4 ±â€¯0.5 mgP/gDW poly-P. A comparison of the quantification of poly-P by 31P solution NMR and by 31P solid state NMR spectroscopy of lyophilised activated sludge showed 86 ±â€¯9% extraction efficiency of poly-P, which confirms that the extraction protocol recovered most of the poly-P from the samples without pronounced poly-P degradation.