Jun 22, 2026
A Chain-Length-Independent Method for Particulate Polyphosphate Quantification from Phytoplankton
- Yingyu Hu1,
- Zoe Finkel1
- 1Dalhousie University
- Marine Microbial Macroecology LabTech. support email: [email protected]
Protocol Citation: Yingyu Hu, Zoe Finkel 2026. A Chain-Length-Independent Method for Particulate Polyphosphate Quantification from Phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mow7l25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 21, 2026
Last Modified: June 22, 2026
Protocol Integer ID: 315444
Keywords: phytoplankton accurate quantification of polyphosphate, independent method for particulate polyphosphate quantification, particulate polyphosphate quantification, phytoplankton accurate quantification, phosphorus cycling in marine system, balance framework across diverse phytoplankton taxa, measured particulate phosphorus, diverse phytoplankton taxa, resolving phosphorus cycling, particulate phosphorus, cellular phosphorus, phytoplankton culture, polyphosphate, independent quantification of particulate polyp, hydrolysable phosphate, phosphate glass, sodium phosphate glass, particulate polyp, conversion of polyp
Funders Acknowledgements:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
Accurate quantification of polyphosphate (polyP) is critical for resolving phosphorus cycling in marine systems, yet existing fluorescence-based approaches are limited by incomplete extraction and chain-length-dependent bias. Here, we present a precipitation–high-temperature dry combustion (Prec-HTDC) method for direct, chain-length-independent quantification of particulate polyP in phytoplankton cultures and natural samples. The method combines bead-milling extraction, enzymatic removal of nucleic acids, quantitative precipitation, and conversion of polyP to hydrolysable phosphate via high-temperature combustion, followed by low-volume acid hydrolysis and colorimetric detection. Method performance was validated using sodium phosphate glass (type 45) standards and a cellular phosphorus mass-balance framework across diverse phytoplankton taxa, demonstrating high recovery, negligible systematic bias, and agreement with independently measured particulate phosphorus. Compared to fluorescence-based approaches, the Prec-HTDC method yielded consistently higher and more reproducible polyP estimates, reflecting improved extraction efficiency and elimination of chain-length effects.
Protocol materials
Sodium phosphate glass type 45MilliporeSigmaCatalog #S4379
N-Lauroylsarcosine sodium salt solutionMilliporeSigmaCatalog #61747
EDTA, Disodium Salt, Dihydrate, 0.5 Molar SolutionMilliporeSigmaCatalog #4055-100ML
Tris(hydroxymethyl)aminomethane hydrochloride, 1M soln., pH 8.0, RNase freeThermofisherCatalog #J60080.AK
Reagent AlcoholMilliporeSigmaCatalog #676829
Sodium Acetate Anhydrous (White Crystals or Granular Powder)Fisher ScientificCatalog #BP3331
Acetic AcidMilliporeSigmaCatalog #A6283
Potassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
Magnesium Chloride Hexahydrate (Crystalline/Certified ACS)Fisher ScientificCatalog #M33500
Ribonuclease A from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #R6513-50MG
DEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
12 N Hydrochloric acid
Ammonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Sample collection
Biomass requirement
Field samples from surface water
| Chlorophyll before acidified (ug/L) | L | |
| < 0.5 | 5 | |
| 0.5 ~ 2 | 2 | |
| > 2 | 1 |
Lab culture at repleted condition, same biomass as total particulate phosphorus
Where, 1.5 is the L-LOD of phosphorus in the assay (µM), 0.005 is the 5 mL hydrochloric acid used in the assay for measuring total particulate phosphorus,
For lab culture under nutrient starvation or other stress, it is strongly recommended to collect samples for assay test during acclimation, particularly for cultures under phosphate starvation.
Filter type:
polycarbonate filter or pre-combusted 25 mm GF/F fitler
Multiple filters for one sample are not recommended, due to the limited space in Matrix D tube.
Filter culture or field seawater onto the filters, using gentle vacuum pressure (130 mmHg).
Rinse the inside of filter funnel with saline (35 g NaCl in 1 L Milli-Q water) to avoid sample loss.
Use filter forceps, fold filter with sample in half along its diameter, creating a semicircular shape
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Fold once more in the same direction, resulting in a long strip.
Place sample filters in 2 mL Cryogenic Vials.
Flash freeze filters and store at -80 °C .
Blanks
Culture blank
Blank is prepared by filtering cell-free culture medium through filter at the same volume as the sample.
Field sample blank
Seawater is first filtered through a 0.2 μm, 142 mm PC filter (Isophore‱, GTTP14250) using a Millipore‱ filter holder (YY3014236). Then blank is prepared by filtering filtered seawater through filter at the same volume as the sample.
Use filter forceps, fold filter with sample in half along its diameter, creating a semicircular shape
Fold once more in the same direction, resulting in a long strip.
Place sample filters in 2 mL Cryogenic Vials.
Flash freeze filters and store at -80 °C .
Freeze-dry before extraction.
Primary polyP-45 standard stock solution
Weigh Sodium phosphate glass (Type 45), log the mass
Sodium phosphate glass type 45MilliporeSigmaCatalog #S4379
Transfer the pellet into a 100 mL graduated cylinder, top to 100 mL with Milli-Q water.
| Mass (mg) | Volume (mL) | C | |
| 100 |
Aliquot into 50 µL per microcentrifuge tube, and store at -20 °C
Primary phosphate stock solution
Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
Potassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
Place the beaker into an oven, dry KH2PO4 at 110 °C for at least 02:00:00
Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
Dissolve around 0.136 g dried KH2PO4 in 1 L milliQ water.
- Use 1 L volumetric flask
- Take notes of the actual weight of KH2PO4 for final concentration of standard stock solution
Aliquot the stock solution into 50 mL Falcon tubes, store in the fridge.
Polyphosphate extraction buffer
Tris buffer5 millimolar (mM) 8.0
In a 1 L volumetric flask, add 5 mL Tris buffer (1 Molarity (M) 8.0 ) and then top to 1 L with Milli-Q water
Tris(hydroxymethyl)aminomethane hydrochloride, 1M soln., pH 8.0, RNase freeThermofisherCatalog #J60080.AK
In a 1 L volumetric flask, add 16.5 mL 30% sarcosine solution and 500 µL 0.5 Molarity (M) EDTA, and then top to 1 L with Tris buffer5 millimolar (mM) 8.0
N-Lauroylsarcosine sodium salt solutionMilliporeSigmaCatalog #61747
EDTA, Disodium Salt, Dihydrate, 0.5 Molar SolutionMilliporeSigmaCatalog #4055-100ML
Enzyme treatment reagents
1 Molarity (M) MgCl2 solution
In a 50 mL volumetric flask, add 10.2 g MgCl2, and then top to 50 mL with Tris buffer5 millimolar (mM) 8.0
Magnesium Chloride Hexahydrate (Crystalline/Certified ACS)Fisher ScientificCatalog #M33500
10 mg/ml RNase
Uncap the original package of Ribonuclease A from bovin pancreas and add 5 mL Tris buffer5 millimolar (mM) 8.0
Ribonuclease A from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #R6513-50MG
Cap the package and invert for a thorough mix.
Aliquot 30 µL to 600 µL microcentrifuge tubes.
Store at -20 °C
5 mg/mL DNase
Uncap the original package of Deoxyribonuclease1 and add 1 mL Tris buffer5 millimolar (mM) 8.0
DEOXYRIBONUCLEASE1 RNase and Protease FreeBioshopCatalog # DRB002.10
Cap the package and invert for a thorough mix.
Aliquot 60 µL to 600 µL microcentrifuge tubes.
Store at -20 °C
Precipitation reagents
-20ºC reagent ethanol
Safety information
Store ethanol in Flammable Material Storage Freezer.
Reagent AlcoholMilliporeSigmaCatalog #676829
70% ethanol
In a 1 L volumetric flask, add 700 mL reagent ethanol, and then top to 1 L with Milli-Q water
Aliquot into 50 mL Falcon tubes, store at 0~4ºC.
Safety information
Store ethanol in Flammable Material Storage Fridge.
3 Molarity (M) sodium acetate/acetic acid solution (pH 5.3)
Add the following chemicals into a 100 mL beaker
19.1 g sodium acetate
3.8 mL acetic acid
70 mL Milli-Q water
Sodium Acetate Anhydrous (White Crystals or Granular Powder)Fisher ScientificCatalog #BP3331
Acetic AcidMilliporeSigmaCatalog #A6283
Equipment
Orion Star™ A211 Benchtop pH Meter
NAME
pH Meter
TYPE
Thermo Scientific
BRAND
STARA2119
SKU
LINK
Equipment
Universal pH Electrodes
NAME
Single junction, ceramic diaphram, SP 223 (spear), Glass, 12/6 mm, BNC, 1 m cable, KCl 3 mol/L
TYPE
VWR®
BRAND
76460-482
SKU
Acetic acid is added dropwise, with continuous stirring, until the solution reaches pH 5.3 (approximately 11 mL required).
Transfer the solution to a 100 mL volumetric flask.
Rinse the beaker with Milli-Q water, and combine the rinsate with the solution in the flask
Bring the final volume to 100 mL with Milli-Q water
Aliquot the solution into 15 mL Falcon tubes and store in the fridge.
Polyphosphate extraction
47m
Rinse forceps with 95% ethanol and air dry.
Use reverse pipetting, add 400 µL Polyphosphate extraction buffer into Matrix D tube.
Equipment
Lysing tube matrix D
NAME
2 mL
TYPE
MP biomedical
BRAND
116913500
SKU
Transfer filter into each tube, vortex and then keep On ice .
Turn on 37 °C incubator
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU
Turn on refrigerated centrifuge, setup temperature at 4 °C
Equipment
CENTRIFUGE 5430 R
NAME
Eppendorf
BRAND
MP2231000510
SKU
Disrupt samples on the bead mill at 6.5 m/s for 00:00:30
Equipment
FastPrep-24™ 5G bead beating grinder and lysis system
NAME
Sample homogenizer
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK
30s
Keep tubes On ice for 00:01:30 . Check the label on each tube, restore the label if it fades.
1m 30s
Disrupt samples on the bead mill at 6.5 m/s for 00:00:30
Keep tubes On ice for 00:01:30 . Check the label on each tube, restore the label if it fades.
Disrupt samples on the bead mill at 6.5 m/s for 00:00:30
Keep tubes On ice for 00:01:30 . Check the label on each tube, restore the label if it fades.
Disrupt samples on the bead mill at 6.5 m/s for 00:00:30
Keep tubes On ice for 00:01:30 .
Centrifuge extracted samples 20000 rcf, 4°C, 00:05:00
5m
Add 400 µL HPLC grade chloroform into each tube, vortex.
Centrifuge 20000 rcf, 4°C, 00:10:00
10m
Using a 100 µL pipette tip, perform reverse pipetting to transfer three 100 µL aliquots of the supernatant into a new conical tube.
About 30 min for processing 24 samples
30m
Enzyme treatment
50m
In each tube, add 1 µL 1 M MgCl2, 2 µL 10 mg/ml RNase and 4 µL 5 mg/mL DNase
About 30 min for 24 samples
30m
Vortex
Incubate at 200 rpm, 37°C, 00:20:00
20m
Remove tubes from the incubator, freeze for 15 min, then keep tubes On ice
Precipitation
36m
Add reagent ethanol 1100 µL and 3 M sodium acetate 160 µL into each tube.
Upright sit at -20 °C Overnight
Safety information
The precipitation is in Flammable Material Storage Freezer.
Centrifuge 20000 rcf, 4°C, 00:30:00
30m
Attach a 100 µL tip to a 1000 µL pipette tip and remove as much liquid as possible while avoiding disturbance of the precipitate.
Place tubes On ice , add 500 µL 70% ethanol (from the fridge), wash for 00:01:00 .
1m
Centrifuge 20000 rcf, 4°C, 00:05:00
5m
Attach a 100 µL tip to a 1000 µL pipette tip and remove as much liquid as possible while avoiding disturbance of the precipitate.
Add 500 µL Milli-Q water into each tube, vortex to loosen the pellet. Work on the next tube.
Note
This gives the pellet more time to redissolve.
Label 12 mL glass vials with oil-based white sharpie.
Vortex and transfer the suspension to a 12 mL glass vial.
Note
PolyP pellets from polycarbonate filters are more difficult to redissolve than those with GF/F filters. Transfer all materials into the glass vial to ensure complete recovery.
Rinse the tube with 200 µL Milli-Q water, vortex, combine the rinsate into the glass vial.
Rinse the tube with 200 µL Milli-Q water, vortex, combine the rinsate into the glass vial.
Recovery reference standard
5 µL Primary polyp-45 standard stock solution in glass vial, add 500 µL Milli-Q water
2 M MgSO4 solution
Dissolve 24 g Magnesium sulfate anhydrous (CAS 7487-88-9) in MilliQ water and top it to 100 mL .
Note
The dissolving process releases heat, which may cause the water to boil.
Add 17 µL of MgSO4 solution into each vial
Reagent blank
In a glass vial, add 17 µL of MgSO4 solution .
Hight-temperature-dry-combustion
6h
Keep vials uncovered in an oven at 90 °C until completely dry.
Combust at 500 °C for 06:00:00 without ramp rate, slowly cool down in the muffle furnace.
6h
Molybdate assay
3h
Preheat oven to 90 °C
0.2 M HCl reagent:
In a reagent bottle, dissolve one part of 12 N HCl in 59 parts of MilliQ water
12 N Hydrochloric acid
Note
Volume of HCl_0.2M_mL = (0.3_mL) X #Sample
Add 300 µL 0.2 M HCl to each vial.
Tightly cap the vial and vortex.
Place vials in the oven for 00:30:00
Cool samples down to Room temperature
Preheat shaker/incubator to 37 °C
Standard working solutions and reagents are prepared during hydrolysis.
Standard working solution
| Code | Primary (µL) | MilliQ (µL) | |
| S1 | 0 | 1000 | |
| S2 | 5 | 995 | |
| S3 | 10 | 990 | |
| S4 | 20 | 980 | |
| S5 | 50 | 950 | |
| S6 | 100 | 900 | |
| S7 | 150 | 850 | |
| S8 | 200 | 800 |
Transfer 500 µL of each standard working solution to a new 2 mL microtube.
2.5 % ammonium molybdate reagent:
Weigh 0.25 g ammonium molybdate in a Falcon tube and top to 10 g with MilliQ water.
Cap and shake until totally dissolved.
Ammonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
10 % ascorbic acid reagent (avoid light exposure):
Weigh 1 g ascorbic acid in a Falcon tube and top to 10 g with MilliQ water;
Cap and shake until all dissolved.
Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Assay reagent
In a 50 mL Falcon tube, add 17 mL Milli-Q water.
Add 1 mL of concentrated sulfuric acid (18 M). Aspirate and dispense the solution using the pipette three times to ensure thorough mixing of the acid.
Add 6 mL of 2.5% ammonium molybdate
Add 6 mL of 10% ascorbic acid
Note
The same pipette tip can be used to transfer Milli-Q water, ammonium molybdate and ascorbic acid, as they are mixed into the final reagent.
Add 500 µL assay reagent to each standard working solution
Add 300 µL assay reagent to each sample
Incubate 150 rpm, 37°C, 03:00:00
3h
Load microplate with 250 ul reactant from each tube, duplicate.
Read plate in microplate reader
| A | B | |
| Shake duration | 00:00:05 | |
| Shaking type | Continuous | |
| Shaking force | High | |
| Shaking speed [rpm] | 600 | |
| Wavelength [nm] | 820 | |
| Use transmittance | No | |
| Pathlength correction | No | |
| Measurement Time [ms] | 100 |
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU
Calculation
Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.
Subtract the average absorbance at 820 nm of the blank sample (i.e. blank filter) replicates from the absorbance at 820 nm of all other individual samples.
Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in µM.
Molar Mass of KH2PO4: 136.086 g/mol
Use the standard curve to determine the orthophosphate concentration (P_uM) of each unknown sample by using its blank-corrected 820 nm absorbance.
Recovery of polyP-45
polyP-45: Sodium phosphate glass Nan+2PnO3n+1
Formula weight: 4650.033143
Conc: Concentration of polyp-45 primary, mg per 100 mL
%P in polyP-45: 0.2997075 (mass in mass, Hu et al. 2022)
Actual phosphorus in recovery reference standard (µmol per vial):
P_act = (conc X 1000 X 10 X 0.2997075 / 30.97) X 5 X 1e-6
Measured phosphorus in recovery reference standard (µmol per vial):
P_measured = P_uM X 300 X 1e-6
Recovery = P_measured / P_act
P in polyP (µmol per sample)
Ppolyp = P_µM X 300 X 1e-6 X (400/300) / Recovery