Jan 28, 2025

Public workspaceTotal particulate carbohydrate from microalgae V.2

DOI
dx.doi.org/10.17504/protocols.io.yxmvmk24ng3p/v2
  • 1Dalhousie University
  • Marine Microbial Macroecology Lab
    Tech. support email: ruby.hu@dal.ca
Ying-Yu Hu
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DOI: dx.doi.org/10.17504/protocols.io.yxmvmk24ng3p/v2
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2025. Total particulate carbohydrate from microalgae. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmk24ng3p/v2Version created by Ying-Yu Hu
Manuscript citation:
Ying-Yu Hu, Andrew J. Irwin and Zoe V. Finkel. 2024.  An improved method to quantify bulk carbohydrate in marine planktonic samples. https://doi.org/10.1002/lom3.10614
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: June 30, 2023
Last Modified: January 28, 2025
Protocol Integer ID: 84319
Keywords: TPTZ method, ferricyanide, hydrolysis, Total particulate carbohydrate
Funders Acknowledgements:
Simons Foundation
Grant ID: 723789
Simons Foundation
Grant ID: 549937
Abstract
Here we describe a protocol to estimate the total particulate carbohydrate from microalgae. Carbohydrate samples are initially vortexed in 9 M H2SO4 for 15 s. The solution is diluted for a final H2SO4 molarity of 1.6 M and hydrolyzed for 3 hours at 90 °C. The hydrolysate is alkalinized by adding 12 M NaOH to the hydrolysate, the ratio of [H+] from the hydrolysate to [OH-] from NaOH is 0.82. The alkalinized hydrolysate is oxidized by ferricyanide solution. The absorbance of TPTZ-Fe2+ complex is measured in microtiter plate at 595 nm. Our method has shown high reproducibility in aldohexoses, ketohexoses, deoxysugars, aldopentoses, uronic acid and amino sugars. The linear range of response is between 5 ng to 10 µg C/mL.







Protocol materials
ReagentNa2CO3VWR International (Avantor)Catalog #97061-972
ReagentNaOHFisher ScientificCatalog #BP359-500
ReagentTPTZMerck MilliporeSigma (Sigma-Aldrich)Catalog #T253-5G
ReagentD-glucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8270-100G
ReagentK3[Fe(CN)6] Fisher ScientificCatalog #AC424120050
ReagentSodium acetate anhydrous Fisher ScientificCatalog #BP333-500
ReagentCitric acid Merck MilliporeSigma (Sigma-Aldrich)Catalog # 251275-500G
ReagentAcetic acid Fisher ScientificCatalog #M1000632500
ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34860-4X2L-R
Safety warnings
Ferric waste should be disposed into trace metal waste container.
Waste acid should be neutralized before disposed into sink.
Before start
  • Combust pasteur pipets at Temperature500 °C Duration02:00:00
  • Combust vials at Temperature500 °C Duration06:00:00
  • Combust glassware to hold 18 M H2SO4 at Temperature500 °C Duration06:00:00
  • Combust glass centrifuge tubes at Temperature500 °C Duration06:00:00
  • Centrifuge caps are acid washed and oven-dried.
  • If lipids extraction is performed after the carbohydrate hydrolysis:
(1) Centrifuge caps for samples and blanks are 95% ethanol washed and air-dried
(2) 5 mL glass serological pipet for dispensing chloroform and methanol is 95% ethanol washed and air-dried
Sample collection
Sample collection
2h
2h
Combust GF/F filter for Duration04:00:00 at Temperature450 °C
4h
Filter microalgae in liquid media onto precombusted GF/F filters, using gentle vacuum pressure (130 mm Hg).
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU

Rinse filtration funnel with filtered saline to avoid sample loss.
Fold the filter with two tweezers:
(1) Fold in half along its diameter, creating a semicircular shape;
(2) Fold once more in the same direction, resulting in a long strip
Place sample filters in Cryogenic Vials.
Filter same volume of blank media (without cells) through filter as blank.
Fold the filter with two tweezers:
(1) Fold in half along its diameter, creating a semicircular shape;
(2) Fold once more in the same direction, resulting in a long strip
Place filter in Cryogenic Vials.
Flash freeze filters and store at Temperature-80 °C
Freeze-dry before processed.
Day 1- Preparation
Day 1- Preparation
30m
30m
Prepare water bath Temperature95 °C
Equipment
Digital General Purpose Water Baths
NAME
20 L
TYPE
VWR®
BRAND
76308-900
SKU

30m
Day 1- Glucose standard solutions
Day 1- Glucose standard solutions
Primary standard
In a 2 mL microtube, weigh ~ 2 mg D-glucoseReagentD-glucoseVWR InternationalCatalog #G8270-100G
Add Milli-Q for a final concentration of 1 mg/mL
Note
Volume requirement for preparing standard working solutions: >1400 µL

Prepare eight 10 mL precombusted centrifuge tubes, label tubes from SD1 to SD8.
Caps for the standard solutions: acid-washed and dried
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU

Equipment
Polypropylene Screw Caps
NAME
Linerless, 15-415
TYPE
Kimble Chase
BRAND
73805-15415
SKU

Follow the sheet to add primary standard and Milli-Q into the tube for working standard solutions.
StandardsPrimary (uL)MilliQ (uL)
SD10500
SD225475
SD350450
SD4100400
SD5150350
SD6250250
SD7350150
SD845050

Day 1 - Samples
Day 1 - Samples
2h
2h
Considering the working hours from 9 am to 4 pm, suggested sample number is:
# of blank + # of samples = 24
Label 10 mL centrifuge tubes, log sample information.

Note
Caps for the samples: 95% ethanol rinsed and dried (if lipids are measured from the same filter)

Rinse forceps with 95% ethanol and air dry.
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU

Transfer each filter strip into its centrifuge tube, starting from blank.
Add Amount500 µL Milli-Q directly to the bottom of the tube, , avoiding contact with the filter.
Note
Do not vortex!

GF/F, especially 47 mm, quickly absorbs all Milli-Q after vortexing and leaves no Milli-Q to dilute 18 M H2SO4. Besides, only 500 uL 18 M H2SO4 is not enough to soak the entire filter, which might result in insufficient pre-treatment.

Day 1- Hydrolysis
Day 1- Hydrolysis
3m
3m
Transfer 18 M H2SO4 into a precombusted glassware (such as, scintillation vial, beaker... etc)
Using the reverse pipetting technique, carefully dispense Amount500 µL 18 M H2SO4 into the MilliQ solution, ensuring it does not touch the filter. Immediately vortex the mixture for Duration00:00:15 using a timer or stopwatch.
Note
Do not cap the centrifuge tube!


15s
Add 4.5 mL MilliQ, tightly cap the centrifuge tube, and vortex for Duration00:00:05 .
5s
Place tube into water bath, log the time for each tube.
Note
Three-hour hydrolysis duration for each sample/blank/standard should be accurately monitored.

3h
After all samples are placed in the water bath, reduce temperature to Temperature90 °C .
Label pre-combusted 12 mL clear vials for supernatant.

# of vials = # of samples + # of blanks
Label pre-combusted 12 mL amber vials for TPTZ measurement with white oil based sharpie.

# of vials = # of samples + # of blanks + # of standards
Equipment
Storage Vials and Closures
NAME
12 mL amber
TYPE
Thermo Scientific
BRAND
B7800-12A
SKU
VWR 66030-686
SPECIFICATIONS

As soon as hydrolysis duration reaches 3 hours, remove the tubes from water bath, cool in the tap water bath with ice to quickly stop hydrolysis.
Day 1- Prepare for lipids extraction
Day 1- Prepare for lipids extraction
3m
3m

Note
The carbohydrate hydrolysis procedure breaks the bond between lipids and non-lipid components, releasing bound lipids into an easily extractable form.
  1. The acid in lipids can charge phospholipids to optimize extraction.
  2. The acid facilitates the separation of the lipid fraction from extraneous material such as protein.
  3. Hydrolysis removes most pigments (including chlorophyll and carotenoids), carbohydrates, and proteins from lipids.


Use glass serological pipet, add 2 mL chloroform into the hydrolysate of the samples (not the standard solutions). Vortex well.

ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
Note
  • Glucose is insoluble in chloroform in the presence of water, and the concentration of glucose in the hydrolysate is no higher than 0.5 mM. While phospholipids can induce the migration of glucose into chloroform, this process doesn't occur instantly; the attainment of equilibrium is substantially delayed. Even when glucose reaches about 5 mM in the aqueous layer and phospholipids are present at concentrations of at least 8.5 mM, the molar ratio of solubilized glucose to phospholipid content remains approximately 0.0025. Therefore, under our conditions, glucose is unlikely to migrate into the lipid extract.

CITATION
CHAN Y. JUNG, JAMES E. CHANEY, AND PAUL G. LEFEVRE. Enhanced Migration of Glucose from Water into Chloroform in Presence of Phospholipids. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS.
LINK
10.1016/0003-9861(68)90454-2

Centrifuge Centrifigation3200 rpm, 00:05:00
5m
Use pre-combusted Pasteur pipets, transfer supernatant to 12 mL clear or amber vial by avoiding disturbing organic layer, leaving around 1 mL of hydrolysate for phase separation in lipids extraction.
Equipment
Disposable Soda-Lime Glass Pasteur Pipets
NAME
5 3/4"
TYPE
Fisherbrand
BRAND
13-678-6A
SKU

Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU

Keep all hydrolysate (standards, blank and samples) in a dark cabinet at TemperatureRoom temperature .
Use glass serological pipet, add 1 mL methanol into the organic layer, vortex well, freeze at Temperature-80 °C until lipids extraction.ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34860-4X2L-R
Prepare reagents
Prepare reagents
12 M NaOH
Add Amount15 mL Milli-Q water into a 50 mL Falcon tube.
Add Amount12 g NaOH pellet into the water, swirl and have the pellets completely dissolved, let it cool down to TemperatureRoom temperature .
Transfer the solution into a 25 mL PP volumetric flask, rinse the tube three times by small amount of Milli-Q and combine the rinsed water into flask, top with Milli-Q water to 25 mL.
Alkaline solution for potassium ferricyanide
Dissolve Amount400 mg NaOH and Amount20 g Na2CO3 in volumetric flask and top to 1 L by Milli-Q. Store at room temperature.
ReagentNaOHVWR InternationalCatalog #BP359-500
ReagentNa2CO3VWR InternationalCatalog #97061-972
Sodium acetate solution
Dissolve Amount164 g sodium acetate, Amount42 g citric acid and Amount300 g acetic acid in a 1 L volumetric flask and top to1 L with Mill-Q water.
Note
1. In this solution, sodium acetate, citric acid and acetic acid is 2 M, 0.2 M and 5 M respectively.
2. Add sodium acetate into the dry volumetric flask first. Sodium acetate is highly hygroscopic, the absorbance of moist hardens the powder into a bulk and clogs the neck of flask.
ReagentSodium acetate anhydrous VWR InternationalCatalog #BP333-500 ReagentCitric acid VWR InternationalCatalog # 251275-500G ReagentAcetic acid VWR InternationalCatalog #M1000632500

Store at room temperature.
Dispense solution by serological pipet to avoid having salt precipitated around sealing surface of the bottle.
3 M acetic acid
Weigh Amount180 g acetic acid in fumehood, transfer the acid into volumetric flask, top to 1 L with Milli-Q water. Store at room temperature.

Day 2 Preparation
Day 2 Preparation
Boiling bath
Day 2 TPTZ reagents
Day 2 TPTZ reagents
Potassium ferricyanide (Reagent A)
Weigh Amount23 mg potassium ferricyanide and transfer into a 100 mL amber reagent bottle. Add Amount100 mL alkaline solution, vortex until powder is completely dissolved. It is stable for two weeks at room temperature.
ReagentK3[Fe(CN)6] VWR InternationalCatalog #AC424120050
Equipment
Reagent bottle
NAME
100 mL, amber
TYPE
VWR
BRAND
14216-240
SKU

Ferric chloride (Reagent B)
Ferric chloride hexahydrate is in spherical shape. It is hard to weigh exact 54 mg for a 100 mL solution. Pick a very small ferric chloride ball and log the weight. Transfer the ball into a 100 mL amber reagent bottle. Calculate the acetate solution required.
Add acetate solution into the amber bottle, vortex until the ball is completely dissolved.

V_acetate = 100 X W_actual/54
Note
This reagent needs to be prepared right prior to analysis. It can only be stable for no more than two days.

TPTZ (Reagent C)

Estimate the total volume required for the assay: 2 mL X (standard # + blank # + sample #)

For each 100 mL TPTZ reagent, weigh and transfer 78 mg TPTZ into an amber reagent bottle, add 100 mL acetic acid solution, vortex until the powder is completely dissolved.
ReagentTPTZVWR InternationalCatalog #T253-5G
Note
This solution is stored at room temperature and stable for one week.

Day 2- Alkalinization of standard solutions
Day 2- Alkalinization of standard solutions
3m
3m
Forward pipetting: transfer Amount270 µL of hydrolysate of standard working solutions to amber vial.
Equipment
Storage Vials and Closures
NAME
12 mL amber
TYPE
Thermo Scientific
BRAND
B7800-12A
SKU
VWR 66030-686
SPECIFICATIONS

Add Amount640 µL Milli-Q and vortex.

Add Amount90 µL 12 M NaOH and vortex.
Note
12 M NaOH: reverse pipetting

Day 2- Alkalinization of samples
Day 2- Alkalinization of samples
3m
3m
Transfer 2 mL of hydrolysate into 2 mL microtube, centrifuge Centrifigation13000 rpm, 00:05:00
5m
Use reverse pipetting to transfer Amount750 µL hydrolysate to a 12 mL amber vial.

Note
If using forward pipetting, the hydrolysate of samples tends to retain a small volume of liquid at the tip, which reduces the chance of error due to incomplete dispensing.

Add Amount250 µL 12 M NaOH , vortex.
Note
12 M NaOH: reverse pipetting


TPTZ method
TPTZ method
10m
10m
In a room with dim light, add Amount1 mL Reagent A into blanks, standards and samples.
Tightly cap the vial and vortex.
Keep in a boiling water bath for Duration00:10:00
10m
Remove boiling bath from the heat, keep all vials in the hot water and move them into the room with dim light.
Add Amount1 mL Reagent B and Amount2 mL Reagent C into the vial and vortex.
Shake at TemperatureRoom temperature for Duration00:30:00 .
30m
Under dim light, using reverse pipetting, load 250 uL of blanks, standards, and samples into the microplate (duplicate).
Load column by column. After one column has been loaded, immediately cover the column with a lid, which has a black membrane on the top to protect sample from light.

Read in microplate reader:

Shake for 5 s at 600 rpm in a continuous and high force mode
Read endpoint 595 nm with a measurement time 100 ms
If the absorbance is higher than the absorbance of SD7, calculate the volume of sample to be loaded into the microplate.


Where, is the volume of sample with absorbance higher than the absorbance of SD7,

is the absorbance of SD7, is the absorbance of sample after TPTZ assay.

Transfer of sample into microplate in duplicate

Transfer of sample blank into microplate in duplicate
Add into the sample
Read in microplate reader:

Shake for 5 s at 600 rpm in a continuous and high force mode
Read endpoint 595 nm with a measurement time 100 ms
Spectra of hydrolysate (optional step)
Spectra of hydrolysate (optional step)
10m
10m
Load 250 ul hydrolysate into microplate.
Scan UV/VIS spectra from 200 to 850 nm at a step of 2 nm.
Waste disposal
Waste disposal
All hydrolysate and TPTZ reagents need to be neutralized by soda before disposed into the sink.
TPTZ reagent B is collected in trace metal waste container.
Citations
Step 29
CHAN Y. JUNG, JAMES E. CHANEY, AND PAUL G. LEFEVRE. Enhanced Migration of Glucose from Water into Chloroform in Presence of Phospholipids
10.1016/0003-9861(68)90454-2