TITLE: X-HTDC method for better estimation of particulate phosphorus in microalgae
AUTHORS: Yingyu Hu, Zoe V. Finkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we describe a protocol using extra-high temperature dry combustion method (X-HTDC) to estimate particulate phosphorus in microalgae. The 800°C-combustion can completely decompose intracellular macromolecules. Thus the estimation of intracellular particulate phosphorus can be increased 10% compared with traditional 500 °C-combustion; while the estimation of total particulate phosphorus can be increased about 5%.</div><div class = "text-block">Microalgae is collected on PC filter and then combusted at 800 °C. Phosphate is then digested by 0.2 M HCl for 30 minutes incubated at 90°C. The resulted orthophosphate reacts with the mixture of molybdate and ascorbic acid to produce molybdenum blue (Chen 1956). </div><div class = "text-block">The working range of this assay is 0 ~ 200 uM orthophosphate. The recommended maximum phosphorus per sample is about 30 ug. </div><div class = "text-block">In order to assess the intracellular phosphorus quotas in microalgae, an oxalate reagent (Tovar-Sanchez 2003) is used to wash the sample collected on the filter to remove surface adsorbed phosphorus.</div></div>

[STEPS]
?. [Prepare standard working solutions]
KH2PO4 primary standard stock solution (≈ 1 mM)
?. [Prepare standard working solutions]
Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
?. [Prepare standard working solutions]
Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
?. [Prepare standard working solutions]
Dissolve around 0.136 g dried KH2PO4 in 1 L milliQ water.Use 1 L volumetric flaskTake notes of the actual weight of KH2PO4 for final concentration of standard stock solution
?. [Prepare standard working solutions]
Transfer standard stock solution into a 1 L bottle and store in the fridge.
This stock solution lasts quite a long time, unless there is evidence for growth of algae or other extraneous biotic material.
?. [X-HTDC]
Add  MgSO­4 to each sample and blank filter.
200 µl
?. [X-HTDC]
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
?. [X-HTDC]
Partly cover the crucibles and place in the oven  at   until samples are completely dry.
90 °C
?. [X-HTDC]
Combust dried samples at  for
800 °C
Map the location of crucibles in the oven, in case pencil mark disappears under 800°C.
Ramp rate should be controlled at   /hour, otherwise the crucibles will shatter.SP.RAT: 150PAMPU: hour
?. [Hydrolysis]
Cap the vial tightly and vortex
?. [Hydrolysis]
Place in a drying oven at  for
90 °C
?. [Hydrolysis]
Cool down to
90 Room temperature
?. [Hydrolysis]
Transfer samples to centrifuge tubes
?. [Hydrolysis]
Total particulate or intracellular phosphorus sample and their blank Transfer 1 mL to 1.7~2 mL microtubeLipid phosphorus sample and the blank: Transfer all liquid to 0.6 mL microtubeStandards: transfer the same volume as samplesCentrifuge at around 14K rpm for 5 minTransfer supernatant to microtubes, avoid particulate ash or other residue
?. [Quantitate orthophosphate]
Working reagent
?. [Quantitate orthophosphate]
Calculate the volume of molybdate-ascorbic reagent:Total volume of reagent (mL) = (0.5 mL) X (# standard working solutions + # samples + # blank) X 2
?. [Quantitate orthophosphate]
Mix the reagents into Falcon tube:2 part MilliQ1 part 6 N sulphuric acid1 part 2.5% ammonium molybdate1 part 10% ascorbic acid
?. [Quantitate orthophosphate]
Preheat incubator/shaker to
37 °C
?. [Quantitate orthophosphate]
Add 500 ul reagent to each sample, standard and blank.
Wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open before dispensing the liquid.
?. [Quantitate orthophosphate]
Incubate at  for while shaking at 200 rpm
37 °C
?. [Quantitate orthophosphate]
Read plate in microplate reader:  AB1Duration [hh:mm:ss]:00:00:052Shaking typeContinuous3Shaking forceHigh4Shaking speed [rpm]600  AB1Wavelength [nm]8202Use transmittanceNo3Pathlength correctionNo4Measurement Time [ms]100
AB1Duration [hh:mm:ss]:00:00:052Shaking typeContinuous3Shaking forceHigh4Shaking speed [rpm]600
AB1Wavelength [nm]8202Use transmittanceNo3Pathlength correctionNo4Measurement Time [ms]100
?. [Calculate]
Subtract the average 820 nm absorbance measurement of the blank standard replicates from the 820 nm measurements of all other individual standard working solution.
?. [Hydrolysis]
?. [Collect sample]
Total particulate phosphorus (i.e. intracellular phosphorus and adsorbed phosphorus)
?. [Collect sample]
Intracellular particulate phosphorus
?. [Collect sample]
Filter sample onto PC filters, using gentle vacuum pressure (5 inches Hg).
?. [Collect sample]
Rinse samples with filtered seawater
?. [Collect sample]
Place sample filters in 2 mL Cryogenic Vials.
?. [Collect sample]
Filter blank media through PC filter as blank.
?. [Collect sample]
Flash freeze and store at .
-20 °C
?. [Collect sample]
Filter sample onto PC filters , using gentle vacuum pressure (5 inches Hg).
?. [Collect sample]
Add  oxalate reagent onto the filter, and let oxalate reagent sit in the filter funnel for
5 mL
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?. [Collect sample]
Drain and then rinse the sample with filtered seawater once
?. [Collect sample]
Place sample filters in 2 mL Cryogenic Vials.
?. [Collect sample]
Filter blank media through PC filter as blank.
?. [Collect sample]
Flash freeze and store at .
-20 °C
?. [Prepare standard working solutions]
Place the beaker into an oven, dry KH2PO4 at  for at least
110 °C
?. [Prepare standard working solutions]
ABC1KH2PO4Primary (ul)MQ (uL)2S1010003S259954S3109905S4209806S5509507S61009008S71508509S8200800
ABC1KH2PO4Primary (ul)MQ (uL)2S1010003S259954S3109905S4209806S5509507S61009008S71508509S8200800
?. [Prepare standard working solutions]
Transfer 500 ul standard working solution to 2 mL microtube. Duplicate each standard.
?. [Quantitate orthophosphate]
Load microplate with 250 ul reactant from each tube, in duplicate
?. [Calculate]
Subtract the average 820 nm absorbance measurement of the blank sample (filter) replicates from the 820 nm measurements of all other individual sample.
?. [Calculate]
Prepare a standard curve by plotting the average Blank-corrected 820 nm measurement for eash standard versus its concentration in uM.
?. [Calculate]
Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 absorbance.
?. [Calculate]
P/sample_ug = (Orthophosphate_uM) X (HCl_mL X0.001) X 30.97
?. [X-HTDC]
MgSO4 reagent:Dissolve  MgSO4  in 50 mL MilliQ water
1.023 g
?. [X-HTDC]
Pencil mark 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.
?. [X-HTDC]
Allow samples to gradually cool down in the muffle furnace.
?. [Prepare standard working solutions]
Standard working solution  ABC1KH2PO4Primary (ul)MilliQ (ul)2S1010003S259954S3109905S4209806S5509507S61009008S71508509S8200800
ABC1KH2PO4Primary (ul)MilliQ (ul)2S1010003S259954S3109905S4209806S5509507S61009008S71508509S8200800
?. [Prepare standard working solutions]
Transfer 500 ul of each standard working solution to 2 mL microtube.
?. [Prepare working reagents]
(3 M) sulfuric acid reagent:Carefully add 1 part  concentrated sulfuric acid into 5 part MilliQ water
?. [Prepare working reagents]
ammonium molybdate reagent:Weigh  ammonium molybdate in a Falcon tube and top to  with MilliQ water.Cap and shake until totally dissolved.
0.25 g
10 g
?. [Prepare working reagents]
ascorbic acid reagent:Weigh  ascorbic acid in a Falcon tube and top to  with MilliQ water;Cap and shake until all dissolved.
1 g
10 g
Wrap the tube with foil if the reagent is not used right after prepared.
?. [Digest]
HCl reagent:Dissolve one part of  HCl in 59 parts of MilliQ water
Volume of HCl_0.2M_mL = (5_mL) X (#Sample + #Blank)
?. [Digest]
Preheat oven to
90 °C
?. [Digest]
Add 2.5 mL 0.2 M HCl to each crucible.
?. [Digest]
Gently swirl the crucible.
?. [Digest]
Cover the crucibles and place crucibles in the oven for
?. [Digest]
Cool samples down to
0 Room temperature
?. [Digest]
Gently swirl the crucible and then transfer 500 ul solution to 2 mL microtube. Duplicate each sample and blank.
?. [Quantitate orthophosphate]
6 N Sulfuric acid reagentCarefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water
?. [Quantitate orthophosphate]
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.
?. [Quantitate orthophosphate]
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.
Wrap the tube with foil if the reagent is not used right after prepared.
?. [X-HTDC]
Transfer sample to crucible with clean filter forceps.
?. [Prepare standard working solutions]
?. [Prepare standard working solutions]
Standard working solutions and reagents can be prepared during sample digestion.
?. [Prepare working reagents]
Calculate the volume of molybdate-ascorbic reagent:Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
?. [Prepare working reagents]
Mix the reagents into Falcon tube:  AB1ReagentPart(s) as in volume2MilliQ236N sulphuric acid142.5% ammonium molybdate1510% ascorbic acid1
AB1ReagentPart(s) as in volume2MilliQ236N sulphuric acid142.5% ammonium molybdate1510% ascorbic acid1
?. [Prepare working reagents]
Preheat incubator/shaker to
37 °C
?. [Prepare working reagents]
Add  reagent to each standard, sample and blank
500 µl
Before dispensing the reagent, wipe or dab the liquid drop on the outside of the tip, avoid wiping the tip open.
?. [Prepare working reagents]
Vortex each tube.
?. [Prepare working reagents]
Incubate at   for   while shaking at 200 rpm
37 °C
?. [Prepare working reagents]
Load microplate with 250 ul reactant from each tube, duplicate.
?. [Prepare working reagents]
Read plate in microplate reader  AB1Shake duration00:00:052Shaking typeContinuous3Shaking forceHigh4Shaking speed [rpm]6005Wavelength [nm]8206Use transmittanceNo7Pathlength correctionNo8Measurement Time [ms]100
AB1Shake duration00:00:052Shaking typeContinuous3Shaking forceHigh4Shaking speed [rpm]6005Wavelength [nm]8206Use transmittanceNo7Pathlength correctionNo8Measurement Time [ms]100
?. [Calculate]
Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other individual standard working solution.
?. [Calculate]
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 sample.
?. [Calculate]
Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
?. [Calculate]
Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
?. [Calculate]
(P per sample)_ug = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)