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

[DESCRIPTION]
Total particulate phosphorus (TPP) is often determined using the High Temperature Dry Combustion (HTDC) method followed by hydrolysis of the ash and then molybdenum colorimetry. Here we show that a higher than traditionally-used combustion temperature, 800 °C vs. 450 - 550 °C, improves phosphorus recovery from several organic phosphorus compounds, marine phytoplankton cultures and particulate samples from the field. The ashing auxiliary MgSO4 further improves P recovery by improving decomposition, reducing volatilization during combustion, and increasing the efficiency of hydrolysis. A 0.2 M HCl hydrolysis, under 90 °C for 30 minutes yields a higher P recovery compared with hydrolysis at room temperature or 60 °C. In aggregate these improvements to the method double the P recovery from phospholipids to 97%. TPP recovery from laboratory phytoplankton cultures and field samples increased an average of 11%, primarily due to the improvements in P recovery from phospholipids, polyphosphates, and nucleic acids. We refer to this new method as the eXtra high temperature dry combustion ash/hydrol method (X-HTDC) and recommend its application for measuring particulate phosphorus from organic compounds in aquatic systems. 
The working range of this assay is 1.22 to 500 uM orthophosphate. Minimum sampling biomass is 0.19 ug P/filter.
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]
We have found that crucibles may lose their temperature resistance after acid-washing or long soaks in alkaline detergent. Crucibles tended to shatter in the oven during the initial increase in temperature from room temperature to 500 °C, even when the ramp rate was carefully controlled at 150 °C/h. We recommend not soaking crucibles in acid but instead we suggest the crucibles be filled with 0.2 M HCl and then incubated at 90 °C for 30 minutes as the acid-washing step. It is necessary to inspect the temperature resistance of newly acquired crucibles by combusting them at 500 °C for 6 h (ramp rate: 150 °C/h) after acid-washing. We found that crucibles that pass this inspection do not usually shatter when heated to 800 °C.  

[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 (130 mmHg).
 

SECTION: Sampling
1.2. Rinse filter tunnel with filtered artificial seawater (phosphate free) to avoid sample loss.
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 filter funnel with filtered artificial seawater (phosphate free).
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 muffin tin pan for easier-handling. 

SECTION: Digesting
16. Incubate in the oven for 30 min 
SECTION: Digesting
17. Cool samples down to Room temperature 
SECTION: Digesting
18. Gently swirl the crucible and then transfer 500 uL solution to 2 mL microtube. Duplicate each sample and blank.
 

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

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

SECTION: Preparing standard working solutions
20.3. Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
SECTION: Preparing standard working solutions
20.4. 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
20.5. Transfer standard stock solution into a 1 L bottle and store in the fridge.
 

SECTION: Preparing standard working solutions
21. Standard working solution

 

SECTION: Preparing standard working solutions
22. Transfer 500 uL of each standard working solution to 2 mL microtube. 
SECTION: Preparing working reagents
23. 
 

SECTION: Preparing working reagents
24.  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
25.  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
26. 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
27. 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
28. Mix the reagents into Falcon tube:

 

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

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

 


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

 

SECTION: Colorimetric measurement
34. Read plate in microplate reader

 

 

SECTION: Calculating
35. 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
36. 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
37. 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
38. Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
SECTION: Calculating
39. (P per sample)_ug = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)