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

Staal, Line Boisen; Petersen, Anne Boisen; Jørgensen, Charlotte Adam; Nielsen, Ulla Gro; Nielsen, Per Halkjær; Reitzel, Kasper

doi:10.1016/j.watres.2019.03.065

Cited by 37 publications

(43 citation statements)

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“…The Aalborg West sample was different from the other three plants as it had the highest concentration of P (14 mg/gSS). We note that 31 P present in magnetic phases such as iron phosphate minerals formed e.g., by addition of Fe(III) salts to the plant, will not be observed under the experimental conditions used, as recently discussed (Staal et al, 2019). There was a small (2-4 ppm) shift in the centre of gravity of the ortho-P resonance, which contains contributions from both inorganic and organic species after 3 h, implying that the speciation of ortho-P in the sludge changed during the release experiment.…”

Section: Quantification Of Total Poly-p By Different Methodsmentioning

confidence: 59%

“…Two different, but complementary, NMR-based approaches were used to calculate the total amount of poly-P in the four activated sludge samples. While 31 P MAS NMR is used to quantify all poly-P middle groups, solution state 31 P NMR is also showing other organic-bound P forms, as well as poly-P end groups (Staal et al, 2019). Their combined application ensures a higher accuracy in the quantification and allows the retrieval of information regarding important organic P species, such as DNA and phospholipids.…”

Section: Quantification Of Total Poly-p By Different Methodsmentioning

confidence: 99%

“…To identify and quantify poly-P in the sludge, solid state (see below) and solution state 31 P NMR spectroscopy was carried out as recently described in Staal et al (2019). Briefly, 30 mL of activated sludge were centrifuged for 10 min at 2000 rpm and the sludge pellet was used for a two-step extraction, with a 1 h pre-extraction in 40 mL of a 0.05 M EDTA solution followed by a main extraction in 40 mL of 0.25 M NaOH for 16 h. Quantitative 31 P solution NMR spectra were recorded on a JEOL ECZ 500R 500 MHz spectrometer, using a 90° pulse (12 µs), 2.16 s acquisition time, 25 s relaxation delay, 512 scans, and proton decoupling.…”

Section: Poly-p Quantification By Solution State 31 P Nmr Spectroscopymentioning

confidence: 99%

“…VnmrJ and MestrNova were used for data analyses. A detailed description of the protocol for data analyses was recently reported (Staal et al, 2019).…”

Section: Poly-p Quantification By Solid-state 31 P Mas Nmr Spectroscopymentioning

confidence: 99%

“…In contrast, common methods used for detection and quantification of intracellular poly-P in environmental samples are not completely reliable, as they suffer from several limitations such as inefficient extraction or degradation of poly-P (Majed et al, 2012). One of the most used techniques is staining with DNA-binding dyes followed by fluorometry, which may be unsuccessful because of the failure of the dye to penetrate cell membranes or the degradation of storage compounds during alkaline extraction (Staal et al, 2019). Raman microspectroscopy in combination with fluorescence in situ hybridization (FISH) provides a reliable single-cell level identification and quantification of intracellular poly-P in activated sludge (Majed et al, 2012), and it was recently used to successfully quantify poly-P content and other storage compounds in PAO cells in situ (Fernando et al, 2019;Petriglieri et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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Quantification of biologically and chemically bound phosphorus in activated sludge from full-scale plants with biological P-removal

Petriglieri¹,

Petersen²,

Gomez³

et al. 2021

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Large amounts of phosphorus (P) are present in activated sludge from municipal wastewater treatment plants, where it exists in the form of metal salt precipitates or biologically bound into the biomass as nucleic acids, cell membrane components, and the extracellular polymeric substances or, in special polyphosphate-accumulating organisms (PAOs), as intracellular polyphosphate. Only recently, methods that reliably allow an absolute quantification of the different P-fractions, such as sequential extraction, Raman microspectroscopy, solid-state 31P magic angle spinning (MAS) NMR, and solution state 31P NMR have been developed. This study combines these techniques to obtain a comprehensive P mass-balance of activated sludge from four wastewater treatment plants with enhanced biological phosphate removal (EBPR). The total content of P and various cations was measured by chemical analysis (ICP-OES), and different P fractions were extracted for chemical characterization. Chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals, while organically bound P constituted 7-9%. By using Raman microspectroscopy and solution state 31P NMR and 31P MAS NMR spectroscopy before and after anaerobic P-release experiments, poly-P was quantified and constituted 22-54% of total P in the activated sludges and was found in approx. 25% of all bacterial cells. Moreover, Raman microspectroscopy in combination with fluorescence in situ hybridization (FISH) was used to quantify the species-specific intracellular poly-P of known PAO genera (Tetrasphaera, Ca. Accumulibacter, Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. They were all abundant, as measured by quantitative-FISH and amplicon sequencing, and accumulated large amount of poly-P, depending on their cell-size, contributing substantially to the P-removal. Interestingly, in all four EBPR plants investigated, only 1-13% of total poly-P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are now known.HighlightsExhaustive P mass-balance of main organic and inorganic P-species in four EBPR plantsQuantification of poly-P of FISH-defined PAO and other species with high P contentTotal P content was 36-50 mgP/gSS of which 31-62% was in biomass and as poly-PA high fraction of all cells (25-30%) contained a high content of poly-PKnown PAOs contained almost all poly-P in the EBPR plants investigated

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Section: Quantification Of Total Poly-p By Different Methodsmentioning

confidence: 59%

Section: Quantification Of Total Poly-p By Different Methodsmentioning

confidence: 99%

Section: Poly-p Quantification By Solution State 31 P Nmr Spectroscopymentioning

confidence: 99%

“…VnmrJ and MestrNova were used for data analyses. A detailed description of the protocol for data analyses was recently reported (Staal et al, 2019).…”

Section: Poly-p Quantification By Solid-state 31 P Mas Nmr Spectroscopymentioning

confidence: 99%