TITLE: Extraction and Measurement of Polyphosphate and Inorganic Phosphorus from Microalgae Samples
AUTHORS: Ying-Yu Hu, Zoe V. Finkel

[DESCRIPTION]
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).

 


 



[GUIDELINES]

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


[STEPS]
SECTION: Sample collection
1. Filter microalgae in liquid media onto GF/F or PC filters, using gentle vacuum pressure (130 mmHg).
 

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

SECTION: Sample collection
6. Freeze dry before measurement.
 

SECTION: Preparation of reagents
7. Tris buffer  20 mM pH 7.0 
 

SECTION: Preparation of reagents
7.1. In a 1 L volumetric flask, top 20 mL 1 M pH 7.0 Tris buffer to 1 L with MilliQ
SECTION: Preparation of reagents
7.2. Store at Room temperature
SECTION: Preparation of reagents
8. PolyP primary standard stock
SECTION: Preparation of reagents
8.1. Weigh one glass pellet of polyP (45) and write down the weight.

 

SECTION: Preparation of reagents
8.2. Transfer the pellet into a 100 mL graduated cylinder.
SECTION: Preparation of reagents
8.3. Dilute to 100 mL with Tris 20 mM pH 7.0 
SECTION: Preparation of reagents
8.4. Aliquot primary stock into 10~50 uL per microtube with Stepper and store at -20 °C
SECTION: Preparation of reagents
9. 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
SECTION: Preparation of reagents
10. Proteinase K 20 mg/ml 
SECTION: Preparation of reagents
10.1. Add 25 mL MilliQ directly into the original package of Proteinase K, vortex to mix
SECTION: Preparation of reagents
10.2. Aliquot 700 uL to microtubes and keep frozen at -20 °C
SECTION: Preparation of reagents
11. DAPI primary stock 14.3 mM  
Add 2 mL MilliQ directly into the original package and keep frozen at -20 °C
SECTION: Preliminary extraction efficiency test
12. Prepare boiling bath.

 

 

 

SECTION: Preliminary extraction efficiency test
14. Transfer sample into glass centrifuge tube 
 

SECTION: Preliminary extraction efficiency test
17. Add 4 mL Tris buffer 20 mM pH 7.0 , vortex and then sonicate.
 

SECTION: Preliminary extraction efficiency test
18. Keep in boiling bath. 

 

SECTION: Preliminary extraction efficiency test
19. Sonicate
SECTION: Preliminary extraction efficiency test
20. Vortex and then transfer extract to 15 mL Falcon tube, according to the extract number.
 

 

SECTION: Preliminary extraction efficiency test
21. Repeat Step 17 to Step 20 until complete 15 times' extraction in total. 
SECTION: Preliminary extraction efficiency test
24. Use forward pipetting, load black microtitre plate with 200 µL supernatant from the extract (one well for one extract, no need to load replicates).
Tris buffer 20 mM pH 7.0 is used as blank.
 
 
 


SECTION: Preliminary extraction efficiency test
25. Prepare DAPI working solution 100 uM
Dilute 12.6 µL of 14.3 mM DAPI stock with 1800 µL MilliQ in a foil wrapped microtube and vortex.
SECTION: Preliminary extraction efficiency test
26.  In the dimmed room with only red light bulb on add 24 µL 100 uM DAPI to each sample in the plate.
SECTION: Preliminary extraction efficiency test
27. Adhere black film on the top of a microplate lid and cover the plate with this lid. 
 

SECTION: Preliminary extraction efficiency test
28. Shake at room temperature for 7 min 
SECTION: Preliminary extraction efficiency test
29. Read fluorescence: excitation at 410 nm and emission at 550 nm
 

SECTION: Preliminary extraction efficiency test
30. 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. 
SECTION: Enzyme treated extract
33. Well mix 1~N extract, transfer 12 mL into 15 mL falcon tube, centrifuge 3200 rpm, 5 min 
SECTION: Enzyme treated extract
37. In Set S, add 18 µL RNase and 18 µL DNase
 

 

SECTION: Enzyme treated extract
39. Incubate at 37 °C, shake continuously
 

 

SECTION: Enzyme treated extract
41. In Set S, add 36 µL Proteinase
SECTION: Enzyme treated extract
43. Incubate at 37 °C, shake continuously.
 

SECTION: Enzyme treated standard amended extract
47. Prepare DAPI working solution 100 uM

Dilute 12.6 µL of 14.3 mM DAPI stock with 1800 µL MilliQ in a foil wrapped microtube and vortex.
SECTION: Enzyme treated standard amended extract
44. Prepare PolyP working standard [PO3]~7.6 uM 
Based on the actual concentration of PolyP (45) primary or secondary standard stock, dilute a certain volume of stock with Tris buffer 20 mM pH 7.0
For a final concentration  7.6 uM
 

 


SECTION: Enzyme treated standard amended extract
45. Transfer 840 µL of enzyme treated extract (1~N) into 2 mL tubes (Set A).
 


SECTION: Enzyme treated standard amended extract
46. Add 160 µL  7.6 uM  polyP working standard to 840 µL of enzyme treated extract, vortex.
SECTION: Load microtiter plate
48. Load 200 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:
 
     

 


SECTION: Load microtiter plate
49. In a dimmed room with only red bulb on, add 24 µL DAPI working solution 100 uM to each sample in the microplate except for those labelled with (UN).
        
 


SECTION: Load microtiter plate
50. Adhere black film on the top of a microplate lid and cover the plate with this lid. 
SECTION: Load microtiter plate
51. Shake at room temperature for 7 min 
SECTION: Load microtiter plate
52. 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
SECTION: Preliminary extraction efficiency test
22. Centrifuge the extract
3200 rpm, 5 min
SECTION: Enzyme treated extract
35. Centrifuge extract "N+1" 3200 rpm, 5 min 
 

SECTION: Enzyme treated extract
34. Transfer 1.8 mL supernatant to a 2 mL tube (Set S).
 

SECTION: Enzyme treated extract
36. Transfer  1.5 mL supernatant into a 2 mL tube (Set B).
SECTION: Enzyme treated extract
38. In Set B, add 15 µL RNase and 15 µL DNase
SECTION: Enzyme treated extract
40. Thaw proteinase (~700uL)

SECTION: Enzyme treated extract
42. In Set B, add 30 µL Proteinase
SECTION: Calculation
53.    
 
 
SECTION: Calculation
54.  
 
SECTION: Preliminary extraction efficiency test
31. Combine Extraction 1 to Extraction N into a falcon tube. 
 

 

SECTION: Preliminary extraction efficiency test
15. Label centrifuge tube for different samples, place one Pasteur pipet into the tube for transferring extract from the same sample
SECTION: Preliminary extraction efficiency test
16. Label 15 mL Falcon tube from 1 to 15 for each one sample. 
SECTION: Preliminary extraction efficiency test
13. Prepare 37 °C incubator/shaker.

SECTION: Preliminary extraction efficiency test
32.  
SECTION: Preliminary extraction efficiency test
23. Transfer 500 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
SECTION: Inorganic P measurement
55. KH2PO4 primary standard stock solution (≈ 1 mM)
  

SECTION: Inorganic P measurement
55.1. Transfer about 1 g KH2PO4  into a beaker, cover the beaker with foil
SECTION: Inorganic P measurement
55.2. Place the beaker into an oven, dry KH2PO4  at 110 °C for at least 120 min

SECTION: Inorganic P measurement
55.3. Move KH2PO4  into a vacuum desiccator, allow KH2PO4 to cool to room temperature
SECTION: Inorganic P measurement
55.4. 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
SECTION: Inorganic P measurement
55.5. Transfer standard stock solution into a 1 L bottle and store in the fridge.
 

SECTION: Inorganic P measurement
56. Standard working solution
 

SECTION: Inorganic P measurement
57. Transfer 500 uL of each standard working solution to 2 mL microtube. 
SECTION: Inorganic P measurement
58. Remove extract from the fridge and warm up to room temperature.
SECTION: Inorganic P measurement
59. 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.    

SECTION: Inorganic P measurement
60. 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.   

SECTION: Inorganic P measurement
61. 6 N (3 M) sulfuric acid reagent:
Carefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water 

 

SECTION: Inorganic P measurement
62. Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
SECTION: Inorganic P measurement
63. Mix the reagents into Falcon tube: 
 

SECTION: Inorganic P measurement
64. Preheat incubator/shaker to 37 °C

SECTION: Inorganic P measurement
65. Add 500 µL reagent to each standard, sample and blank, starting from blanks, including blank for standards and blank for samples.
 

 


SECTION: Inorganic P measurement
66. Vortex each tube
SECTION: Inorganic P measurement
67. Incubate at 37 °C  for 180 min  while shaking at 200 rpm

SECTION: Inorganic P measurement
68. Load microplate with 250 uL reactant from each tube, duplicate.
 
SECTION: Inorganic P measurement
69. Read plate in microplate reader

 

SECTION: Inorganic P measurement
70. Calculation
SECTION: Inorganic P measurement
70.1. Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.
SECTION: Inorganic P measurement
70.2. 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.
SECTION: Inorganic P measurement
70.3. Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
SECTION: Inorganic P measurement
70.4. Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
SECTION: Inorganic P measurement
70.5. 22.5% polyP can be measured by molybdate assay, possibly due to the partial hydrolysis in acid condition