Dec 28, 2022

Public workspaceTotal particulate carbohydrate from microalgae V.1

DOI
dx.doi.org/10.17504/protocols.io.yxmvmk24ng3p/v1
  • 1Dalhousie University
Ying-Yu Hu
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DOI: dx.doi.org/10.17504/protocols.io.yxmvmk24ng3p/v1
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2022. Total particulate carbohydrate from microalgae. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmk24ng3p/v1
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: November 23, 2020
Last Modified: December 28, 2022
Protocol Integer ID: 44812
Keywords: TPTZ method, ferricyanide, hydrolysis, Total particulate carbohydrate
Funders Acknowledgement:
Simons Collaboration on Computational Biogeochemical Modeling of Marine Ecosystems
Grant ID: 549937
Simons Collaborative on Ocean Processes and Ecology
Grant ID: 723789
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 0.18 to 10 µg C/mL.







Protocol materials
ReagentD-glucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8270-100G
Step 9.1
ReagentTPTZMerck MilliporeSigma (Sigma-Aldrich)Catalog #T253-5G
Step 41
ReagentNaOHFisher ScientificCatalog #BP359-500
Step 35
ReagentNa2CO3VWR InternationalCatalog #97061-972
Step 35
ReagentK3[Fe(CN)6] Fisher ScientificCatalog #AC424120050
Step 39
ReagentSodium acetate anhydrous Fisher ScientificCatalog #BP333-500
Step 36.1
ReagentCitric acid Merck MilliporeSigma (Sigma-Aldrich)Catalog # 251275-500G
Step 36.1
ReagentAcetic acid Fisher ScientificCatalog #M1000632500
Step 36.1
Safety warnings
Ferric waste should be disposed into trace metal waste container.
Waste acid should be neutralized before disposed into sink.
Sample collection
Sample collection
2h
2h
Combust GFF filter for Duration04:00:00 at Temperature450 °C
4h
Filter microalgae in liquid media onto precombusted GFF 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 seawater to avoid sample loss.
Place sample filters in 2 mL Cryogenic Vials.
Filter blank media (without cells) through filter as blank.
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
30m
Day 1- Glucose standard solutions
Day 1- Glucose standard solutions
Primary standard
In a 2 mL microtube, weigh 1 ~ 2 mg D-glucoseReagentD-glucoseVWR InternationalCatalog #G8270-100G
Add Milli-Q for a final concentration of 1 mg/mL (Volume requirement for preparing standard working solutions: >1800 µL).
Prepare eight 10 mL precombusted (Duration06:00:00 Temperature500 °C ) centrifuge tubes, label tubes from SD1 to SD8.
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU

Caps for the standard working solutions are acid-washed.
Equipment
Polypropylene Screw Caps
NAME
Linerless, 15-415
TYPE
Kimble Chase
BRAND
73805-15415
SKU


6h
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:
# blank + # samples = 24
Label 10 mL centrifuge tubes, log sample information.
Rinse forceps with 95% ethanol and air dry.
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU

Transfer each filter into its centrifuge tube, starting from blank.
Add Amount500 µL Milli-Q into each tube, vortex.

Day 1- Hydrolysis
Day 1- Hydrolysis
3m
3m
Transfer 18 M H2SO4 into a 30 mL precombusted glassware (scint vial, beaker... etc)
Vortex sample.
Use reverse pipetting technique, add Amount500 µL 18 M H2SO4 into the suspension instead of onto the filter, immediately vortex for Duration00:00:15 (Critical step: monitored by 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
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 5 mL centrifuge tubes for supernatant.

# of vials = # of samples + # of blanks
Label 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 tube from water bath, let it sit 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
1. The procedure of carbohydrate hydrolysis can break the bond between lipids and non-lipid component, which releases bound lipids into easily extractable form.
2. The acid in lipids can charge phospholipids to optimize extraction.
3. The acid can faciliate the separation of the lipid fraction from extraneous material such as protein.
4. Hydrolysis helps to remove most of the pigment (including chlorophyll and carotenoids), carbohydrate and protein from lipids.


Add 2 mL chloroform into hydrolysate. Vortex.
Note
  • Glucose is insoluble in chloroform in the presence of water.
  • Glucose in hydrolysate is no higher than 0.5 mM.
  • Although phospholipids can induce the migration of glucose into chloroform, it doesn't instantly take place. The attainment of equilibrium is substantially delayed.
  • The molar ratio of glucose solubilized to the phospholipid content remains approximately 0.0025 when glucose is about 5 mM level in the aqueous layer while phospholipids is up to at least 8.5 mM.
  • Therefore, glucose is unlikely to migrate into lipids extract under our condition.

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
Transfer supernatant to 12 mLamber vial by avoiding disturbing organic layer. Keep all hydrolysate in a dark cabinet at TemperatureRoom temperature .
Equipment
Disposable Soda-Lime Glass Pasteur Pipets
NAME
5 3/4"
TYPE
Fisherbrand
BRAND
13-678-6A
SKU

Note
Precombust pasteur pipets at Temperature500 °C Duration02:00:00
Precombust vials at Temperature500 °C Duration06:00:00

Add 1 mL MeOH into the organic layer, mix well, freeze at Temperature-80 °C until lipids extraction.
Estimation
Estimation
1d
1d
Estimate carbohydrate content on the filter for each sample:

[Carbohydrate]ug/filter= [Chl-a]ug/L X (15/1.1) X VolumeL
Cassay= 0.4 * Chl * (15/1.1) * V * (Hy/1000)/5.5

Where Cassay is Carbon in total particulate carbohydrate (ug/mL) in TPTZ assay, 0.4 is the median content of carbon in carbohydrate, Chl is the concentration of chlorophyll-a (ug/L), 15 and 1.1 are the median content of carbohydrate and chlorophyll-a in microalgae dry mass, V is sampling volume (L), Hy is the volume of hydrolysate (ul), 1000 is the total volume of neutralized hydrolysate, 5.5 is the total volume of MilliQ and H2SO4 in hydrolysis (mL).

Linear range in TPTZ assay: 0~10 ug C/mL
LOD in TPTZ assay: 0.02 ug C/mL
Use the following sheet to calculate the final concentration of carbon in total particulate carbohydrate , choose the suitable volume of hydrolysate so that the final concentration of estimated carbon of all the samples in TPTZ assay is about Concentration1 Mass / % volume ug C/mL

Note
Estimated carbon is much lower than actual carbon in microalgae under nutrient stress condition or high light level.


MilliQ (uL)H2SO4 (mL)H2SO4 (M)MilliQ (uL)Hydrolysate (uL)MilliQ (uL)12 M NaOH (uL)[H+]/[OH-]
5000.518.00450090880300.82
5000.518.004500180760600.82
5000.518.004500270640900.82
5000.518.0045003605201200.82
5000.518.0045004504001500.82
5000.518.0045005402801800.82
5000.518.0045006301602100.82
5000.518.004500720402400.82
5000.518.00450075002500.82

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
In this solution, sodium acetate, citric acid and acetic acid is 2 M, 0.2 M and 5 M respectively.
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 standards
Day 2- Alkalinization of standards
3m
3m
Transfer Amount270 µL of hydrolysate of standard working solution 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
Based on the estimation at Go to , transfer a certain volume of hydrolysate to a 12 mL amber vial.

Add MilliQ and 12 M NaOH based on the sheet Go to , 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.

123456789101112
ASD1SD1
BSD2SD2
CSD3SD3
DSD4SD4
ESD5SD5
FSD6SD6
GSD7SD7
HSD8SD8
Microplate layout

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 27
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