An X-HTDC method for estimating particulate phosphorus from microalgae

Ying-Yu Hu, Zoe V. Finkel

Published: 2022-08-13 DOI: 10.17504/protocols.io.kqdg35dq7v25/v6

phosphomolybdenum-ascorbic reduction

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

P.S. Chen, T.Y. Toribara and Huber Warner Microdetermination of Phosphorus Anal. Chem. https://doi.org/10.1021/ac60119a033

Citation

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 Marine Chemistry https://doi.org/10.1016/S0304-4203(03)00054-9

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

Sampling

Sampling microalgae for total particulate phosphorus (i.e. intracellular phosphorus and adsorbed phosphorus)

1.1.

Filter microalgae in liquid media onto polycarbonate filters, using gentle vacuum pressure (130 mmHg). Equipment

Value	Label
Filter forceps	NAME
blunt end, stainless steel	TYPE
Millipore	BRAND
XX6200006P	SKU
http://www.emdmillipore.com/	LINK

1.2.

Rinse filter tunnel with filtered artificial seawater (phosphate free) to avoid sample loss.

1.3.

Place sample filters in 2 mL Cryogenic Vials.

Equipment

Value	Label
Cryogenic Vials with Closures	NAME
Polypropylene, 2 mL	TYPE
Corning®	BRAND
66021-974	SKU

1.4.

Filter blank media (without cells) through polycarbonate filter as blank.

1.5.

Flash freeze filters and store at -20°C.

Sampling microalgae for intracellular particulate phosphorus

2.1.

Filter microalgae in liquid media onto polycarbonate filters , using gentle vacuum pressure (5 inches Hg). Equipment

Value	Label
Filter forceps	NAME
blunt end, stainless steel	TYPE
Millipore	BRAND
XX6200006P	SKU
http://www.emdmillipore.com/	LINK

2.2.

Add 5mL oxalate reagent onto the filter, and let oxalate reagent sit in the filter funnel for 0h 5m 0s

Preparation of oxalate reagent

2.3.

Drain and then rinse filter funnel with filtered artificial seawater (phosphate free).

2.4.

Place sample filters in 2 mL Cryogenic Vials.

Equipment

Value	Label
Cryogenic Vials with Closures	NAME
Polypropylene, 2 mL	TYPE
Corning®	BRAND
66021-974	SKU

2.5.

Filter blank media (without cells) through polycarbonate filter as blank.

2.6.

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 Equipment

Value	Label
Porcelain crucibles	NAME
40 mL	TYPE
VWR	BRAND
89037-996	SKU

Equipment

Value	Label
Crucible cover	NAME
VWR	BRAND
71000-146	SKU

Transfer sample to crucible with clean filter forceps and lay filter at the bottom.

0.17M MgSO₄reagent:Dissolve 1.023g MgSO₄ in 50 mL MilliQ water

Add 200µL 0.17M 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

Value	Label
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 9h 0m 0s

Equipment

Value	Label
Muffle furnace	NAME
F30428C	TYPE
Thermo	BRAND
10-505-13	SKU
https://www.fishersci.com/us/en/home.html	LINK

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: hourOr SP.RAT: 2.5/PAMPU: minute

Allow samples to gradually cool down in the muffle furnace.

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.

Digesting

11.

0.2M HCl reagent:In a reagent bottle, dissolve one part of 12N HCl in 59 parts of MilliQ water

Note

Volume of HCl_0.2M_mL = (5_mL) X (#Sample + #Blank)

12.

Preheat oven to 90°C

13.

Add 5 mL 0.2 M HCl to each crucible.

14.

Gently swirl the crucible.

15.

Cover the crucibles and place crucibles in the muffin tin pan for easier-handling.

16.

Incubate in the oven for 0h 30m 0s

17.

Cool samples down to Room temperature

18.

Gently swirl the crucible and then transfer 500 uL solution to 2 mL microtube. Duplicate each sample and blank.

Equipment

Value	Label
Maxymum Recovery® Snaplock Microcentrifuge Tube	NAME
2.0 mL, Polypropylene, Clear, Nonsterile,	TYPE
Axygen®	BRAND
MCT-200-L-C	SKU

Preparing standard working solutions

19.

Standard working solutions and reagents can be prepared during sample digestion.

20.

KH₂PO₄ primary standard stock solution (≈ 1 mM)

20.1.

Transfer about 1 g KH₂PO₄ into a beaker, cover the beaker with foil

20.2.

Place the beaker into an oven, dry KH₂PO₄ at 110°C for at least 2h 0m 0s

20.3.

Move KH₂PO₄ into a vacuum desiccator, allow KH₂PO₄ to cool to room temperature

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

20.5.

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.

21.

Standard working solution

A	B	C
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

22.

Transfer 500 uL of each standard working solution to 2 mL microtube.

Preparing working reagents

23.

Note

All reagents are freshly prepared before colorimetric measurement.

24.

6N (3 M) sulfuric acid reagent:Carefully add 1 part 18M concentrated sulfuric acid into 5 part MilliQ water

25.

2.5% ammonium molybdate reagent:

Weigh 0.25g ammonium molybdate in a Falcon tube and top to 10g with MilliQ water.

Cap and shake until totally dissolved.

26.

10% ascorbic acid reagent:Weigh 1g ascorbic acid in a Falcon tube and top to 10g with MilliQ water;Cap and shake until all dissolved.

Note

Wrap the tube with foil if the reagent is not used right after prepared.

27.

Calculate the volume of molybdate-ascorbic reagent:

Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)

28.

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

29.

Preheat incubator/shaker to 37°C

Equipment

Value	Label
SHAKING INCUBATOR	NAME
71L	TYPE
Corning® LSE™	BRAND
6753	SKU

30.

Add 500µL reagent to each standard, sample and blank, starting from blanks, including blank for standards and blank for samples.

Equipment

Value	Label
Finntip Stepper Tips	NAME
5 mL	TYPE
Thermo Scientific	BRAND
9404200	SKU
https://www.fishersci.com/us/en/home.html	LINK

Note

Before dispensing the reagent, wipe or dab the liquid drop on the outside of the tip, avoid wiping the open tip.

31.

Vortex each tube.

32.

Incubate at 37°C for 3h 0m 0s while shaking at 200 rpm

33.

Load microplate with 250 uL reactant from each tube, duplicate.

Equipment

Value	Label
96-Well Microplates, Polystyrene, Clear,	NAME
Greiner Bio-One	BRAND
655101	SKU

34.

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

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU

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.

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.

37.

Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.

38.

Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.

39.

(P per sample)_ug = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)

An X-HTDC method for estimating particulate phosphorus from microalgae

Abstract

Before start

Steps

Sampling

X-HTDC-ing

Digesting

Preparing standard working solutions

Preparing working reagents

Colorimetric measurement

Calculating

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