Aug 12, 2022
Version 6
An X-HTDC method for estimating particulate phosphorus from microalgae V.6
- Ying-Yu Hu1,
- Zoe V. Finkel1
- 1Dalhousie University
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2022. An X-HTDC method for estimating particulate phosphorus from microalgae. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg35dq7v25/v6Version created by Ying-Yu Hu
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2022
Last Modified: August 12, 2022
Protocol Integer ID: 68589
Keywords: particulate phosphorus, intracellular phosphorus, phosphomolybdenum-ascorbic reduction, orthophosphate, oxalate reagent, adsorbed phosphorus, X-HTDC, High temperature dry combustion
Abstract
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.
CITATION
CITATION
Protocol materials
12 N Hydrochloric acid
Step 11
Potassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
Step 20
18M sulfuric acid
Step 24
Ammonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
Step 25
Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Step 26
Magnesium sulfate anhydrousFisher ScientificCatalog #M65500
Step 5
Safety warnings
Polycarbonate filter can release toxic gas and smoke during combustion. An exhaust system is required for muffle furnace while using the X-HTDC method.
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.
Sampling
Sampling
Sampling microalgae for total particulate phosphorus (i.e. intracellular phosphorus and adsorbed phosphorus)
Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg).
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Rinse filter tunnel with filtered artificial seawater (phosphate free) to avoid sample loss.
Place sample filters in 2 mL Cryogenic Vials.
Equipment
Cryogenic Vials with Closures
NAME
Polypropylene, 2 mL
TYPE
Corning®
BRAND
66021-974
SKU
Filter blank media (without cells) through polycarbonate filter as blank.
Flash freeze filters and store at -20 °C .
Sampling microalgae for intracellular particulate phosphorus
Filter microalgae in liquid media onto polycarbonate filters , using gentle vacuum pressure (5 inches Hg).
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Add 5 mL oxalate reagent onto the filter, and let oxalate reagent sit in the filter funnel for 00:05:00
Protocol
NAME
Preparation of oxalate reagent
CREATED BY
Ying-Yu Hu
5m
Add 50 mL MilliQ water in a 250 mL beaker.
Weigh 40 g NaOH and slowly pour into the beaker.
Use squeeze bottle to rinse the weighing boat and transfer rinse water into the same beaker.
Use glass rod to gently stir and fully dissolve NaOH.
Note
The solution is very hot and corrosive. It can cause skin burns and eye damage.
Carefully transfer NaOH solution into 100 mL volumetric flask by using glass rod.
Rinse beaker with small amount of MilliQ water three times, transfer rinse water into the flask.
Mix the solution by gently shaking the capped volumetric flask and top to 100 mL with MilliQ water.
Transfer the prepared reagent into a 250 mL PP bottle.
Label the bottle with SDS pictogram.
In a 1000 mL beaker with stir bar, add 600 mL MilliQ water.
Add 18.6 g EDTA, 14.7 g sodium citrate, 0.74 g KCl and 5 g NaCl into the beaker, stir until all ingredients are dissolved. 5.7
10 Molarity (M) NaOH is added dropwise to bring pH in between 6 to 7 by using a transfer pipet
Add 12.6 g oxalic acid to the solution, stir the mixture while heating.
After oxalic acid is completely dissolved, stop heating and let it cool to room temperature. A water bath filled with tap water can be used to speed up cooling. 3.3
Add 10 Molarity (M) NaOH dropwise to bring pH to 8
Top to 1 L in volumetric flask with MilliQ water.
Filter oxalate reagent by rapid flow to a 1 L PP bottle.
Equipment
Sterile Disposable Filter Units with PES Membrane
NAME
Thermo Scientific™ Nalgene™ Rapid-Flow™
BRAND
5964520
SKU
Label the bottle and keep it at Room temperature .
Drain and then rinse filter funnel with filtered artificial seawater (phosphate free).
Place sample filters in 2 mL Cryogenic Vials.
Equipment
Cryogenic Vials with Closures
NAME
Polypropylene, 2 mL
TYPE
Corning®
BRAND
66021-974
SKU
Filter blank media (without cells) through polycarbonate filter as blank.
Flash freeze filters and store at -20 °C .
X-HTDC-ing
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
Equipment
Porcelain crucibles
NAME
40 mL
TYPE
VWR
BRAND
89037-996
SKU
Equipment
Crucible cover
NAME
VWR
BRAND
71000-146
SKU
Transfer sample to crucible with clean filter forceps and lay filter at the bottom.
0.17 M MgSO4 reagent:
Dissolve 1.023 g MgSO4 in 50 mL MilliQ water
Magnesium sulfate anhydrousVWR InternationalCatalog #M65500
Add 200 µL 0.17 M MgSO4 directly onto each sample and blank filter.
Note
Sing-use pipet tip to avoid cross-contamination.
Cover the crucibles and place in the oven at 90 °C until samples are completely dry.
Equipment
Forced air oven
NAME
VWR
BRAND
89511-410
SKU
Note
Remove samples out of the oven as soon as they are dried. If muffle furnace is not available, keep crucibles in the vacuum desiccator.
Combust dried samples at 800 °C for 09:00:00
Equipment
Muffle furnace
NAME
F30428C
TYPE
Thermo
BRAND
10-505-13
SKU
Note
Map the location of crucibles in the oven, in case pencil mark disappears under 800°C.
Note
Ramp rate should be controlled at < 200 °C /hour or follow the instruction provided by manufacture, otherwise the crucibles might shatter.
SP.RAT: 150/PAMPU: hour
Or
SP.RAT: 2.5/PAMPU: minute
9h
Allow samples to gradually cool down in the muffle furnace.
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
Digesting
0.2 M HCl reagent:
In a reagent bottle, dissolve one part of 12 N HCl in 59 parts of MilliQ water
12 N Hydrochloric acidVWR International
Note
Volume of HCl_0.2M_mL = (5_mL) X (#Sample + #Blank)
Preheat oven to 90 °C
Add 5 mL 0.2 M HCl to each crucible.
Gently swirl the crucible.
Cover the crucibles and place crucibles in the muffin tin pan for easier-handling.
Incubate in the oven for 00:30:00
30m
Cool samples down to Room temperature
Gently swirl the crucible and then transfer 500 uL solution to 2 mL microtube. Duplicate each sample and blank.
Equipment
Maxymum Recovery® Snaplock Microcentrifuge Tube
NAME
2.0 mL, Polypropylene, Clear, Nonsterile,
TYPE
Axygen®
BRAND
MCT-200-L-C
SKU
Preparing standard working solutions
Preparing standard working solutions
2h
2h
Standard working solutions and reagents can be prepared during sample digestion.
KH2PO4 primary standard stock solution (≈ 1 mM)
Potassium dihydrogen orthophosphateVWR InternationalCatalog #P-4550
Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
Place the beaker into an oven, dry KH2PO4 at 110 °C for at least 02:00:00
2h
Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
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
Transfer standard stock solution into a 1 L bottle and store in the fridge.
Note
This stock solution lasts quite a long time, unless there is evidence for growth of algae or other extraneous biotic material.
Standard working solution
| KH2PO4 | Primary (ul) | MilliQ (ul) | |
| S1 | 0 | 1000 | |
| S2 | 5 | 995 | |
| S3 | 10 | 990 | |
| S4 | 20 | 980 | |
| S5 | 50 | 950 | |
| S6 | 100 | 900 | |
| S7 | 150 | 850 | |
| S8 | 200 | 800 |
Transfer 500 uL of each standard working solution to 2 mL microtube.
Preparing working reagents
Preparing working reagents
2h
2h
Note
All reagents are freshly prepared before colorimetric measurement.
6 N (3 M) sulfuric acid reagent:
Carefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water
18M sulfuric acidVWR International
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.
Ammonium molybdateVWR InternationalCatalog #09878-100G
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.
Ascorbic acidVWR InternationalCatalog #A5960-100G
Note
Wrap the tube with foil if the reagent is not used right after prepared.
Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
Mix the reagents into Falcon tube:
| A | B | |
| Reagent | Part(s) as in volume | |
| MilliQ | 2 | |
| 6N sulphuric acid | 1 | |
| 2.5% ammonium molybdate | 1 | |
| 10% ascorbic acid | 1 |
Colorimetric measurement
Colorimetric measurement
2h
2h
Preheat incubator/shaker to 37 °C
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU
Add 500 µL reagent to each standard, sample and blank, starting from blanks, including blank for standards and blank for samples.
Equipment
Finntip Stepper Tips
NAME
5 mL
TYPE
Thermo Scientific
BRAND
9404200
SKU
Note
Before dispensing the reagent, wipe or dab the liquid drop on the outside of the tip, avoid wiping the open tip.
Vortex each tube.
Incubate at 37 °C for 03:00:00 while shaking at 200 rpm
3h
Load microplate with 250 uL reactant from each tube, duplicate.
Example of loading the microplate
Equipment
96-Well Microplates, Polystyrene, Clear,
NAME
Greiner Bio-One
BRAND
655101
SKU
Read plate in microplate reader
| A | B | |
| Shake duration | 00:00:05 | |
| Shaking type | Continuous | |
| Shaking force | High | |
| Shaking speed [rpm] | 600 | |
| Wavelength [nm] | 820 | |
| Use transmittance | No | |
| Pathlength correction | No | |
| Measurement Time [ms] | 100 |
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU
Calculating
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.
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.
Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
(P per sample)_ug = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)
Citations
AntonioTovar-Sanchez, Sergio A Sañudo-Wilhelmy, Manuel Garcia-Vargas, Richard S Weaver, Linda C Popels, David A Hutchins. A trace metal clean reagent to remove surface-bound iron from marine phytoplankton
https://doi.org/10.1016/S0304-4203(03)00054-9P.S. Chen, T.Y. Toribara and Huber Warner. Microdetermination of Phosphorus
https://doi.org/10.1021/ac60119a033