Jan 28, 2025

Public workspaceExtraction and Measurement of Polyphosphate and Inorganic Phosphorus from Microalgae Samples

 Forked from DAPI-Based Polyphosphate Estimation with Extraction Sufficiency Validation: A Method for Quantifying Polyphosphate from Microalgae Samples
DOI
dx.doi.org/10.17504/protocols.io.q26g7pzdqgwz/v1
  • 1Dalhousie University
  • Marine Microbial Macroecology Lab
    Tech. support email: ruby.hu@dal.ca
Ying-Yu Hu
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DOI: dx.doi.org/10.17504/protocols.io.q26g7pzdqgwz/v1
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2025. Extraction and Measurement of Polyphosphate and Inorganic Phosphorus from Microalgae Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pzdqgwz/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: June 12, 2023
Last Modified: January 28, 2025
Protocol Integer ID: 83255
Keywords: DAPI, polyphosphate, microtiter plate, microplate, microalgae, fluorescence
Funders Acknowledgements:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
Polyphosphate and inorganic phosphate are extracted by hot Tris buffer. After centrifugation, 500 uL of extract is used for inorganic phosphate measurement, 200 uL of extract is used for polyP extraction sufficiency test.

Testing the number of treatment rounds necessary to extract all polyP is crucial. However, even when collecting samples from the same field location or cultivation condition, there can be high variability in treatment rounds among replicates, leading to significant background fluorescence and rendering the polyP from the sample undetectable. This challenge is especially prominent when measuring polyP from field samples. Limited sample availability and insufficient polyP extraction, combined with high background fluorescence, make the laborious measurement unpredictable and hinder accurate polyP measurement. This obstacle is a significant hurdle in polyP measurement. In our assay, we overcome the challenge by validating the sufficiency of extraction for each sample and then measuring the polyP values.

The utilization of DAPI-based fluorometric estimation for polyphosphate (polyP) analysis from microalgae involves evaluating the fluorescence of DAPI-stained samples in quartz cuvettes using a spectrofluorometer. To reduce the consumption of reagent, time, and labor while minimizing DAPI photobleaching, we have adapted this method to a 96-well black microtiter plate with a black film-covered lid. Additionally, the calculation method has been modified to account for matrix effects in microplates.

To conduct the polyP assay, roughly 400 uL RNase, 400 uL DNase, and 700 uL proteinase are required for four samples.

Inorganic P is measured by molybdate/ascorbic acid/sulphuric acid reagent following Chen et al. (1956).

CITATION
P. S. Chen, T. Y. Toribara, and Huber. Warner (2022). Microdetermination of Phosphorus. Anal. Chem..
LINK
https://doi.org/10.1021/ac60119a033


CITATION
Martin, Patrick & Van Mooy, Benjamin. Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols, Matrix Effects, and Nucleic Acid Interference. Applied and Environmental Microbiology.
LINK
http://doi.org/10.1128/AEM.02592-12


Guidelines

  1. Extracted polyphosphate must be measured on the same day. Polyphosphate loss has been observed if the extraction is processed days after.
  2. The polyphosphate standard aliquot can only be thawed and used once. Do not refrozen and thawed multiple times.
  3. Inorganic phosphate extract can be stored in the fridge and measured the next day.

Materials
Chemicals

ReagentTris Buffer 1M pH 7.0Fisher ScientificCatalog #BP1756-500
ReagentSodium phosphate glass type 45Sigma AldrichCatalog #S4379-500MG
ReagentProteinase-K Fisher ScientificCatalog #BP1700-500
ReagentRNase A: 500 U/mL; RNase T1: 20000 U/mL Fisher ScientificCatalog #AM2288
ReagentTURBO DNase 2 U/uLFisher ScientificCatalog #AM2239
ReagentDAPI: 4′6-Diamidino-2-phenylindole dihydrochlorideFisher ScientificCatalog #D1306


Protocol materials
ReagentRNase A: 500 U/mL; RNase T1: 20000 U/mL Fisher ScientificCatalog #AM2288
ReagentTURBO DNase 2 U/uLFisher ScientificCatalog #AM2239
ReagentDAPI: 4′6-Diamidino-2-phenylindole dihydrochlorideFisher ScientificCatalog #D1306
ReagentTris Buffer 1M pH 7.0Fisher ScientificCatalog #BP1756-500
ReagentSodium phosphate glass type 45Merck MilliporeSigma (Sigma-Aldrich)Catalog #S4379-500MG
ReagentProteinase-K Fisher ScientificCatalog #BP1700-500
ReagentPotassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
ReagentAmmonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
ReagentAscorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Sample collection
Sample collection
Filter microalgae in liquid media onto GF/F or PC filters, using gentle vacuum pressure (130 mmHg).
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU

Rinse sample with filtered saline (no nutrients)
Place sample filters in cryogenic vials
Filter same volume of the blank media (without cells) through GF/F or PC filter as blank.
Flash freeze filters and stored at Temperature-80 °C

Freeze dry before measurement.
Equipment
FreeZone® 2.5 L Benchtop Freeze Dryers
NAME
Labconco®
BRAND
700202000
SKU

Preparation of reagents
Preparation of reagents
Tris buffer Concentration20 Mass Percent Ph7.0
Note
Budget:
About 250 mL per four samples

In a 1 L volumetric flask, top Amount20 mL Concentration1 Mass Percent Ph7.0 Tris buffer to 1 L with MilliQ
Store at TemperatureRoom temperature
PolyP primary standard stock
Weigh one glass pellet of polyP (45) and write down the weight.

Equipment
Microbalance
NAME
Cubis series
TYPE
Sartorius
BRAND
MSE6.6S-000-DM
SKU

Transfer the pellet into a 100 mL graduated cylinder.
Dilute to 100 mL with Tris Concentration20 Mass Percent Ph7.0
Aliquot primary stock into 10~50 uL per microtube with Stepper and store at Temperature-20 °C
PolyP secondary standard stock
If the pellet is far more than 10 mg, dilute primary to secondary to bring down the concentration before preparing working standard
Proteinase K Concentration20 Mass Percent
Add Amount25 mL MilliQ directly into the original package of Proteinase K, vortex to mix
Aliquot 700 uL to microtubes and keep frozen at Temperature-20 °C
DAPI primary stock Concentration14.3 Mass Percent
Add Amount2 mL MilliQ directly into the original package and keep frozen at Temperature-20 °C
Preliminary extraction efficiency test
Preliminary extraction efficiency test
Prepare boiling bath.

Equipment
VWR® Advanced Hot Plates
NAME
VWR
BRAND
97042-658
SKU

Equipment
Hollow Polypropylene (PP) Ball Bath Covers, 20 mm
NAME
Cole-Parmer
BRAND
UZ-06821-04
SKU

Equipment
Tube rack
NAME
Simport MultiRack™
BRAND
CA48648-606
SKU

Prepare Temperature37 °C incubator/shaker.

Transfer sample into glass centrifuge tube
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU

Label centrifuge tube for different samples, place one Pasteur pipet into the tube for transferring extract from the same sample
Label 15 mL Falcon tube from 1 to 15 for each one sample.
Add Amount4 mL Tris buffer Concentration20 Mass Percent Ph7.0 , vortex and then sonicate.
Equipment
Specific Pipette Tips 5mL
NAME
Thermo Scientific™ Finntip™
BRAND
21-377-304
SKU

15s
Keep in boiling bath.

Note
Make sure the tube rack is in the middle of the boiling bath and covered with PP balls. Tris solution in the tube should be boiling during the 5 minutes' incubation.

5m
Sonicate
15s
Vortex and then transfer extract to 15 mL Falcon tube, according to the extract number.
Note
Do not push filter to the bottom. Use Pasteur pipet, gently lift the filter upwards, and then transfer as much extract as possible. Gently press the extract out of the filter.

Equipment
Disposable Soda-Lime Glass Pasteur Pipets
NAME
5 3/4"
TYPE
Fisherbrand
BRAND
13-678-6A
SKU

Repeat Step 17 to Step 20 until complete 15 times' extraction in total.
Centrifuge the extract
Centrifigation3200 rpm, Room temperature, 00:05:00
5m
Transfer Amount500 µL supernatant to a 2 mL microtube for Inorganic P measurement.
Label each tube as
1-1, 1-2, ..., 1-N
2-1, 2-2, ..., 2-N
3-1, 3-2, ..., 3-N
4-1, 4-2, ..., 4-N
Use forward pipetting, load black microtitre plate with Amount200 µL supernatant from the extract (one well for one extract, no need to load replicates).
Tris buffer Concentration20 Mass Percent Ph7.0 is used as blank.

Equipment
96-Well Black Microplates
NAME
Polystyrene
TYPE
Greiner Bio-One
BRAND
655076
SKU


Prepare DAPI working solution Concentration100 Mass Percent
Dilute Amount12.6 µL of Concentration14.3 Mass Percent DAPI stock with Amount1800 µL MilliQ in a foil wrapped microtube and vortex.
In the dimmed room with only red light bulb on add Amount24 µL Concentration100 Mass Percent DAPI to each sample in the plate.
Adhere black film on the top of a microplate lid and cover the plate with this lid.
Equipment
Black Vinyl Films for Fluorescence and Photoprotection
NAME
VWR
BRAND
89087-692
SKU

Shake at room temperature for Duration00:07:00
7m
Read fluorescence: excitation at 410 nm and emission at 550 nm
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

Plot fluorescence intensity versus number of extraction.
The number of extract (N) is the stationary point where the fluorescence of stained extract stops decreasing or the derivative of the fluorescence after that point is close to zero.

If , proceed to extract five additional times. And then measure the stained extract following the previous steps.
Combine Extraction 1 to Extraction N into a falcon tube.
Note
Try to transfer all solution including debris from each tube.
If the total volume is over 50 mL, use a beaker instead.

Sample codeNV(Tris) per extract (mL)


Enzyme treated extract
Enzyme treated extract
Well mix 1~N extract, transfer 12 mL into 15 mL falcon tube, centrifuge Centrifigation3200 rpm, Room temperature, 00:05:00
5m
Transfer Amount1.8 mL supernatant to a 2 mL tube (Set S).
Note
Sample is triplicated into S1a, S1b and S1c; S2a, S2b, S2c...etc.

Centrifuge extract "N+1" Centrifigation3200 rpm, Room temperature, 00:05:00
Note
Blank is duplicated into B1a and B1b; B2a and B2b... etc.

5m
Transfer Amount1.5 mL supernatant into a 2 mL tube (Set B).
In Set S, add Amount18 µL RNase and Amount18 µL DNase
Note
RNase tends to leave residue in the tip. However one package has only 1 mL RNase, it will be a waste to use reverse pipetting. After dispensing RNase into the vial, use the same tip to draw the solution and gently dispense it back into the solution for about three time, so that there is no residue remaining in the tip. Replace a new tip for the next vial.

Note
Require ~400 uL RNase and ~400 uL DNase.

In Set B, add Amount15 µL RNase and Amount15 µL DNase
Incubate at Temperature37 °C , shake continuously
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU

Note
Start the timer when temperature reaches Temperature37 °C


10m
Thaw proteinase (~700uL)

In Set S, add Amount36 µL Proteinase
In Set B, add Amount30 µL Proteinase
Incubate at Temperature37 °C , shake continuously.
Note
Start the timer when temperature reaches Temperature37 °C


30m
Enzyme treated standard amended extract
Enzyme treated standard amended extract
Prepare PolyP working standard [PO3]~Concentration7.6 Mass Percent
Based on the actual concentration of PolyP (45) primary or secondary standard stock, dilute a certain volume of stock with Tris buffer Concentration20 Mass Percent Ph7.0
For a final concentration Concentration7.6 Mass Percent
Note
Total volume = 160 X N (ul), where N = sample number

Note
FW(45Na2O.55P2O5)=10600
Mol of PO3 per mol of PolyP (45) = 110


Transfer Amount840 µL of enzyme treated extract (1~N) into 2 mL tubes (Set A).
Note
Forward pipetting, aspire and dispense for three times to mix.


Add Amount160 µL Concentration7.6 Mass Percent polyP working standard to Amount840 µL of enzyme treated extract, vortex.
Prepare DAPI working solution Concentration100 Mass Percent

Dilute Amount12.6 µL of Concentration14.3 Mass Percent DAPI stock with Amount1800 µL MilliQ in a foil wrapped microtube and vortex.
Load microtiter plate
Load microtiter plate
7m
7m
Load Amount200 µL blanks (B: N+1), samples (S: 1~N) and amended samples (A: Amended 1~N) to the microplate. Organize samples as shown in the following scheme:


Note
Reverse pipetting


In a dimmed room with only red bulb on, add Amount24 µL DAPI working solution Concentration100 Mass Percent to each sample in the microplate except for those labelled with (UN).
Note
Forward pipetting


Adhere black film on the top of a microplate lid and cover the plate with this lid.
Shake at room temperature for Duration00:07:00
7m
Shake duration: 1 min
Shaking type: continuous
Shaking speed and force: 600 rpm/High
Fluorescence: excitation at 410 nm and emission at 550 nm
Measurement time: 300 ms
Excitation bandwidth: 5 nm
Calculation
Calculation


Inorganic P measurement
Inorganic P measurement
5h
5h
KH2PO4 primary standard stock solution (≈ 1 mM)
ReagentPotassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550

Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
Place the beaker into an oven, dry KH2PO4 at Temperature110 °C for at least Duration02:00:00

2h
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
Transfer standard stock solution into a 1 L bottle and store in the fridge.
Note
This stock solution lasts quite a long time, unless there is evidence for growth of algae or other extraneous biotic material.

Standard working solution
KH2PO4Primary (uL)MilliQ (uL)
S101000
S25995
S310990
S420980
S550950
S6100900
S7150850
S8200800

Transfer 500 uL of each standard working solution to 2 mL microtube.
Remove extract from the fridge and warm up to room temperature.
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. ReagentAmmonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G

10 % ascorbic acid reagent:
Weigh 1 g ascorbic acid in a Falcon tube and top to 10 g with MilliQ water;
Cap and shake until all dissolved. ReagentAscorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G

6 N (3 M) sulfuric acid reagent:
Carefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water

Note
Save the pipet tip for final reagent preparation.

Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
Mix the reagents into Falcon tube:
ReagentParts in volume
MilliQ2
6N sulphuric acid1
2.5% ammonium molybdate1
10% ascorbic acid1

Preheat incubator/shaker to Temperature37 °C

Add Amount500 µL reagent to each standard, sample and blank, starting from blanks, including blank for standards and blank for samples.
Equipment
Finntip Stepper Tips
NAME
5 mL
TYPE
Thermo Scientific
BRAND
9404200
SKU

Note
Before dispensing the reagent, wipe or dab the liquid drop on the outside of the tip, avoid wiping the open tip.


Vortex each tube
Incubate at Temperature37 °C for Duration03:00:00 while shaking at 200 rpm

3h
Load microplate with 250 uL reactant from each tube, duplicate.
Example of loading the microplate
Read plate in microplate reader

AB
Shake duration00:00:05
Shaking typeContinuous
Shaking forceHigh
Shaking speed [rpm]600
Wavelength [nm]820
Use transmittanceNo
Pathlength correctionNo
Measurement Time [ms]100

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 uM.
Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
22.5% polyP can be measured by molybdate assay, possibly due to the partial hydrolysis in acid condition
Where, Piumol is the concentration of inorganic P, PjuM is inorganic P from each extract, Vj is the volume of Tris, n is the total extraction times, polyPumol is the polyP concentration in the sample.
Citations
P. S. Chen, T. Y. Toribara, and Huber. Warner. Microdetermination of Phosphorus
https://doi.org/10.1021/ac60119a033
Martin, Patrick & Van Mooy, Benjamin. Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols, Matrix Effects, and Nucleic Acid Interference
http://doi.org/10.1128/AEM.02592-12