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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. Based on tests on Prorocentrum triestinum in exponential growth in f/2 medium, estimates of intracellular particulate phosphorus are increased by 10% compared with the traditional 500 °C combustion method.</div><div class = "text-block">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). </div><div class = "text-block">The working range of this assay is 0.074 to 200 uM orthophosphate. Minimum sampling volume (mL)=130/Chl-a_ug/L. </div><div class = "text-block">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.</div><br/><br/><br/></div>

[STEPS]
?. [Sampling]
Sampling microalgae for total particulate phosphorus (i.e. intracellular phosphorus and adsorbed phosphorus)
?. [Sampling]
Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (5 inches Hg).
?. [Sampling]
Rinse samples with filtered seawater
?. [Sampling]
Place sample filters in 2 mL Cryogenic Vials.
?. [Sampling]
Filter blank media (without cells) through polycarbonate filter as blank.
?. [Sampling]
Flash freeze filters and store at .
-20 °C
?. [Sampling]
Sampling microalgae for intracellular particulate phosphorus
?. [Sampling]
Filter microalgae in liquid media onto polycarbonate filters , using gentle vacuum pressure (5 inches Hg).
?. [Sampling]
Add  oxalate reagent onto the filter, and let oxalate reagent sit in the filter funnel for
5 mL
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?. [Sampling]
Drain and then rinse the sample with filtered seawater once
?. [Sampling]
Place sample filters in 2 mL Cryogenic Vials.
?. [Sampling]
Filter blank media (without cells) through polycarbonate filter as blank.
?. [Sampling]
Flash freeze filters and store at .
-20 °C
?. [X-HTDC-ing]
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-ing]
Transfer sample to crucible with clean filter forceps and lay filter at the bottom.
?. [X-HTDC-ing]
MgSO4 reagent:Dissolve  MgSO4  in 50 mL MilliQ water
1.023 g
?. [X-HTDC-ing]
Add  MgSO­4 directly onto each sample and blank filter.
200 µl
Sing-use pipet tip to avoid cross-contamination.
?. [X-HTDC-ing]
Cover the crucibles and place in the oven  at   until samples are completely dry.
90 °C
Remove samples out of the oven as soon as they are dried. If muffle furnace is not available, remove crucibles to vacuum desiccator.
?. [X-HTDC-ing]
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 or follow the instruction provided by manufacture, otherwise the crucibles might shatter.SP.RAT: 150/PAMPU: hourOr SP.RAT: 2.5/PAMPU: minute
?. [X-HTDC-ing]
Allow samples to gradually cool down in the muffle furnace.
?. [X-HTDC-ing]
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.
?. [Digesting]
HCl reagent:In a reagent bottle, dissolve one part of  HCl in 59 parts of MilliQ water
Volume of HCl_0.2M_mL = (5_mL) X (#Sample + #Blank)
?. [Digesting]
Preheat oven to
90 °C
?. [Digesting]
Add 5 mL 0.2 M HCl to each crucible.
?. [Digesting]
Gently swirl the crucible.
?. [Digesting]
Cover the crucibles and place crucibles in the oven for
?. [Digesting]
Cool samples down to
0 Room temperature
?. [Digesting]
Gently swirl the crucible and then transfer 500 ul solution to 2 mL microtube. Duplicate each sample and blank.
?. [Preparing standard working solutions]
Standard working solutions and reagents can be prepared during sample digestion.
?. [Preparing standard working solutions]
KH2PO4 primary standard stock solution (≈ 1 mM)
?. [Preparing standard working solutions]
Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
?. [Preparing standard working solutions]
Place the beaker into an oven, dry KH2PO4 at  for at least
110 °C
?. [Preparing standard working solutions]
Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
?. [Preparing 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
?. [Preparing 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.
?. [Preparing standard working solutions]
Standard working solution  ABC1KH2PO4Primary (ul)MilliQ (ul)2S1010003S259954S3109905S4209806S5509507S61009008S71508509S8200800
ABC1KH2PO4Primary (ul)MilliQ (ul)2S1010003S259954S3109905S4209806S5509507S61009008S71508509S8200800
?. [Preparing standard working solutions]
Transfer 500 ul of each standard working solution to 2 mL microtube.
?. [Preparing working reagents]
All reagents are freshly prepared before colorimetric measurement.
?. [Preparing working reagents]
(3 M) sulfuric acid reagent:Carefully add 1 part  concentrated sulfuric acid into 5 part MilliQ water
?. [Preparing 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
?. [Preparing 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.
?. [Preparing working reagents]
Calculate the volume of molybdate-ascorbic reagent:Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
?. [Preparing 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
?. [Colorimetric measurement]
Preheat incubator/shaker to
37 °C
?. [Colorimetric measurement]
Add  reagent to each standard, sample and blank, starting from blanks, including blank for standards and blank for samples.
500 µl
Before dispensing the reagent, wipe or dab the liquid drop on the outside of the tip, avoid wiping the open tip.
?. [Colorimetric measurement]
Vortex each tube.
?. [Colorimetric measurement]
Incubate at   for   while shaking at 200 rpm
37 °C
?. [Colorimetric measurement]
Load microplate with 250 ul reactant from each tube, duplicate.
?. [Colorimetric measurement]
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
?. [Calculating]
Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.
?. [Calculating]
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.
?. [Calculating]
Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
?. [Calculating]
Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
?. [Calculating]
(P per sample)_ug = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)