TITLE: An X-HTDC method for estimating particulate phosphorus from microalgae
AUTHORS: Yingyu  Hu, Zoe V. Finkel

[DESCRIPTION]
Here we describe a protocol to estimate particulate phosphorus associated with microalgae using an extra-high temperature dry combustion method (X-HTDC). An 800°C-combustion can completely decompose intracellular macromolecules, improving estimates of particulate phosphorus relative to methods that use lower combustion temperatures. 
We recommend microalgae be collected on polycarbonate filters and then combusted at 800 °C before digestion by 0.2 M HCl for 30 minutes at 90°C. The resulting orthophosphate is detected by mixing the digested sample with a mixture of molybdate and ascorbic acid to produce molybdenum blue (Chen 1956). 
The working range of this assay is 0.074 to 200 uM orthophosphate. Minimum sampling volume (mL)=130/Chl-a_ug/L. 
In order to assess the intracellular phosphorus in microalgae, we recommend an oxalate reagent (Tovar-Sanchez 2003) to wash the microalgae collected on the filter to remove surface adsorbed phosphorus.

 

 


[BEFORE_START]
(1) Wash
For new crucibles: Gently brush new crucibles with phosphate free detergent and then rinsed with reverse osmosis water. Do not soak crucibles in detergent due to the low resistance to alkaline solution.
For crucibles after hydrolysis: Dump hydrolysate to soda solution for neutralization, rinse crucible with reverse osmosis water.
(2) Fill crucibles with 0.2 M HCl (about 80% full), put lids on, keep in the oven at 90 °C for 30 min. 
(3) Collect acid in a bottle to reuse, rinse crucibles with reverse osmosis water and milliQ water for the last rinse.
(4) Try to drain water as much as possible, then cover the crucibles, dry in the oven completely. 
(5) Combust dry crucibles  at 500 °C for 360 min at ramp-rate 150 °C/h. Some crucibles may shatter, but the remainders can intact after being combusted with samples at 800 °C. Crucibles are numbered with pencil at the bottom of crucible cups. 


[STEPS]
SECTION: Sampling
1. Sampling microalgae for total particulate phosphorus (i.e. intracellular phosphorus and adsorbed phosphorus)
SECTION: Sampling
1.1. Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (5 inches Hg).
 

SECTION: Sampling
1.2. Rinse samples with filtered seawater
SECTION: Sampling
1.3. Place sample filters in 2 mL Cryogenic Vials.
 

SECTION: Sampling
1.4. Filter blank media (without cells) through polycarbonate filter as blank. 
SECTION: Sampling
1.5. Flash freeze filters and store at -20 °C.
SECTION: Sampling
2. Sampling microalgae for intracellular particulate phosphorus 
SECTION: Sampling
2.1. Filter microalgae in liquid media onto polycarbonate filters , using gentle vacuum pressure (5 inches Hg).
 

SECTION: Sampling
2.2. Add 5 mL Preparation of oxalate reagent oxalate reagent onto the filter, and let oxalate reagent sit in the filter funnel for 5 min

 


SECTION: Sampling
2.3. Drain and then rinse the sample with filtered seawater once
SECTION: Sampling
2.4. Place sample filters in 2 mL Cryogenic Vials.
 

SECTION: Sampling
2.5. Filter blank media (without cells) through polycarbonate filter as blank. 
SECTION: Sampling
2.6. Flash freeze filters and store at -20 °C.
SECTION: X-HTDC-ing
3. Mark number at the bottom of each crucible with pencil, log the following information:
(1) The number of crucible
(2) The code of sample in the crucible
 

 
    
SECTION: X-HTDC-ing
4. Transfer sample to crucible with clean filter forceps and lay filter at the bottom.

SECTION: X-HTDC-ing
5.  0.17 M MgSO4 reagent:
Dissolve 1.023 g MgSO4  in 50 mL MilliQ water 
 
SECTION: X-HTDC-ing
6. Add 200 µL 0.17 M MgSO­4 directly onto each sample and blank filter.
 

SECTION: X-HTDC-ing
7. Cover the crucibles and place in the oven  at 90 °C  until samples are completely dry. 
 

 

SECTION: X-HTDC-ing
8. Combust dried samples at 800 °C for  540 min
 

 

 

SECTION: X-HTDC-ing
9. Allow samples to gradually cool down in the muffle furnace.
SECTION: X-HTDC-ing
10. Pencil mark on crucibles should be still visible, however, it can be easily removed by water. Therefore, when removing samples out of the furnace, label the lid and crucible with sharpie immediately.  
SECTION: Digesting
11. 0.2 M HCl reagent:
In a reagent bottle, dissolve one part of 12 N HCl in 59 parts of MilliQ water 
 
 

SECTION: Digesting
12. Preheat oven to 90 °C 

SECTION: Digesting
13. Add 5 mL 0.2 M HCl to each crucible. 
SECTION: Digesting
14. Gently swirl the crucible. 
SECTION: Digesting
15. Cover the crucibles and place crucibles in the oven for 30 min 

SECTION: Digesting
16. Cool samples down to Room temperature 
SECTION: Digesting
17. Gently swirl the crucible and then transfer 500 ul solution to 2 mL microtube. Duplicate each sample and blank.
 

SECTION: Preparing standard working solutions
18. Standard working solutions and reagents can be prepared during sample digestion.
SECTION: Preparing standard working solutions
19. KH2PO4 primary standard stock solution (≈ 1 mM)
 

SECTION: Preparing standard working solutions
19.1. Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
SECTION: Preparing standard working solutions
19.2. Place the beaker into an oven, dry KH2PO4 at 110 °C for at least 120 min 

SECTION: Preparing standard working solutions
19.3. Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
SECTION: Preparing standard working solutions
19.4. 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: Preparing standard working solutions
19.5. Transfer standard stock solution into a 1 L bottle and store in the fridge.
 

SECTION: Preparing standard working solutions
20. Standard working solution

 

SECTION: Preparing standard working solutions
21. Transfer 500 ul of each standard working solution to 2 mL microtube. 
SECTION: Preparing working reagents
22. 
 

SECTION: Preparing working reagents
23.  6 N (3 M) sulfuric acid reagent:
Carefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water 
 

SECTION: Preparing working reagents
24.  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: Preparing working reagents
25. 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: Preparing working reagents
26. Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
SECTION: Preparing working reagents
27. Mix the reagents into Falcon tube:

 

SECTION: Colorimetric measurement
28. Preheat incubator/shaker to 37 °C 
 

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

 


SECTION: Colorimetric measurement
30. Vortex each tube.
SECTION: Colorimetric measurement
31. Incubate at 37 °C  for 180 min  while shaking at 200 rpm
SECTION: Colorimetric measurement
32. Load microplate with 250 ul reactant from each tube, duplicate.
  

 

SECTION: Colorimetric measurement
33. Read plate in microplate reader

 

 

SECTION: Calculating
34. 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: Calculating
35. 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: Calculating
36. Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
SECTION: Calculating
37. Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
SECTION: Calculating
38. (P per sample)_ug = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)